Research ArticleAster koraiensis Extract and Chlorogenic Acid Inhibit RetinalAngiogenesis in a Mouse Model of Oxygen-Induced Retinopathy
Junghyun Kim 12 YunMi Lee1 Wookwon Jung2 Su-Bin Park2
Chan-Sik Kim 1 and Jin Sook Kim 1
1Korean Medicine Convergence Research Division Korea Institute of Oriental Medicine Daejeon Republic of Korea2Department of Oral Pathology School of Dentistry Chonbuk National University Jeonju 54896 Republic of Korea
Correspondence should be addressed to Chan-Sik Kim chskimkiomrekr and Jin Sook Kim jskimkiomrekr
Received 28 July 2017 Accepted 4 February 2018 Published 22 April 2018
Academic Editor Gabino Garrido
Copyright copy 2018 Junghyun Kim et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Aster koraiensis extract (AKE) is a standard dietary herbal supplement Chlorogenic acid (CA) is the major compound present inAKE Retinal neovascularization is a common pathophysiology of retinopathy of prematurity diabetic retinopathy and wet formage-related macular degeneration In this study we aimed to evaluate the effects of AKE and CA on retinal neovascularization ina mouse model of oxygen-induced retinopathy (OIR) Vascular endothelial growth factor- (VEGF-) induced tube formation wasassayed in human vascular endothelial cells Experimental retinal neovascularization was induced by exposing C57BL6 mice to75 oxygen on postnatal day 7 (P7) and then returning them to normal oxygen pressure on P12 AKE (25 and 50mgkgday) andCA (25 and 50mgkgday) were administered intraperitoneally for 5 days (P12ndashP16) Retinal flat mounts were prepared to measurethe extent of retinal neovascularization at P17 The incubation of human vascular endothelial cells with AKE and CA (1ndash10 120583gmL)resulted in the inhibition of VEGF-mediated tube formation in a dose-dependent manner The neovascular area was significantlysmaller in AKE or CA-treatedmice than in the vehicle-treatedmiceThese results suggest that AKE is a potent antiangiogenic agentand that its antiangiogenic activity may in part be attributable to the bioactive component CA
1 Introduction
Retinal neovascularization is themost common cause of irre-versible vision loss in individuals older than 65 years [1] and isa severe complication of retinopathy of prematurity diabeticretinopathy and wet form age-related macular degeneration(AMD) [2]
Vascular endothelial growth factor (VEGF) is a well-known proangiogenic and vascular permeability factor andis a key mediator in the pathogenesis of these retinal diseases[3] Recently the use of VEGF antagonists to inhibit theVEGF signaling pathway has successfully diminished retinalneovascularization in several experimental animal models[4] and human subjects [5] In numerous clinical trialsintravitreally injected anti-VEGF agents including beva-cizumab ranibizumab and aflibercept notably suppressedneovascularization and stabilized vision loss [6ndash8] Howeverthe intravitreal injection of anti-VEGF agents presents the
risk of adverse events [9 10] Repeated intravitreal injectionsincreased the incidence of ocular complications includingendophthalmitis ocular inflammation traumatic cataractsintraocular pressure elevation retinal detachment and vit-reous hemorrhage [11]Thus interest in the use of oral agentshas been increasing [12ndash14]
Aster koraiensis (Korean starwort) is a valuable peren-nial plant native to Korea This herb has been used asfood such as appetizers and side dishes and in traditionalmedicine to treat several diseases including pneumoniachronic bronchitis diabetes and pertussis [15 16] In ourprior studies we reported that the extract of A koraiensis(AKE) prevented podocyte apoptosis in the renal tissuesof streptozotocin- (STZ-) induced diabetic rats [17] andalso inhibited retinal pericyte apoptosis in these rats [18]Although various effects of AKE on retinal injury in ananimal model of diabetes have been reported its effect onretinal pathogenic neovascularization remains unknown To
HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 6402650 8 pageshttpsdoiorg10115520186402650
2 Evidence-Based Complementary and Alternative Medicine
elucidate this we investigated the inhibitory effect of AKEand its major compound chlorogenic acid (CA) on retinalneovascularization in a mouse model of oxygen-inducedretinopathy (OIR) We also investigated the inhibitory effectof AKE and CA on the VEGF-induced tube formation ofhuman vascular endothelial cells
2 Materials and Methods
21 Preparation of AKE The aerial parts which include theflowers leaves and stems of A koraiensis were purchasedfrom Gongju (Chungcheongnam South Korea) in August2007 The AKE was prepared according to a previouslyreported method [17] Briefly 25 kg A koraiensis was accu-rately weighed and extracted with EtOH (3 times 20 L) via mac-eration at room temperature for 3 daysThe extracted solutionwas concentrated in vacuo at 40∘C to provide anAKE powder(303 g) Voucher specimens ofA koraiensiswere deposited atthe Herbarium of the Korea Institute of Oriental Medicine(Daejeon Korea Herbarium number KIOM-83A) AKE wasstandardized using CA as a reference compound (Sigma-Aldrich Merck Millipore Darmstadt Germany) by high-performance liquid chromatography (HPLC) The HPLCfingerprint and the CA content of AKE are described in ourprevious report [18]
22 Cell Viability Assay Cell viability was examined usingan MTS assay kit (CellTiter 96 AQueous One Solution CellProliferation Assay Promega Corporation Madison WIUSA) Human umbilical vein endothelial cells (Korean CellLine Bank Seoul Korea) were plated (1 times 104 cellswell) inquadruplicate into 96-well plates containing various doses ofAKE or CA (1ndash100 120583gmL) Cell viability was measured at24 h following incubation The results of the MTS assay wereobtained by measuring absorbance using a microplate reader(Tecan Group Ltd Mannedorf Switzerland) at 490 nm Allexperiments were repeated three times
23 Tube Formation Assay Tissue culture plates (96 wells)were coated with 400 120583L growth factor reduced basementmembrane matrix (Matrigel BD Biosciences FranklinLakes NJ USA) Human umbilical vein endothelial cellswere seeded at a density of 1 times 106 cellswell and treatedwith serum-free EGM-2 media (WelGENE Inc DaeguKorea) containing AKE or CA (0ndash10 120583gmL) and recombi-nant humanVEGF (20 ngmL) for 17 h at 37∘C Capillary-liketube structures formed by human umbilical vein endothe-lial cells on the Matrigel were photographed with a DP71digital camera (Olympus Corporation) Tube formation wasquantified by counting the number of branching points ofthe capillary-like structures per visual field The experimentswere repeated three times independently
24 Experimental Retinal Neovascularization in OIR Exper-imental retinal neovascularization was induced in C57BL6mice as previously described [19] Mice were exposed to75 oxygen on postnatal day 7 (P7) and then returned tonormal oxygen pressure on P12 The mice were divided intofive groups of seven mice each as follows (1) OIR mice (2)
Cel
l Via
bilit
y(
of c
ontro
l)
Con 1 10 100 1 10 100
AKE (gmL) CA (gmL)
0
20
40
60
80
100
120
140
Figure 1 Effects of AKE and CA on the viability of human vascularendothelial cells The viability of human vascular endothelial cellswas determined by MTS assay Data are expressed as percentage ofcontrol Data are expressed as mean plusmn SEM 119899 = 4
OIR mice treated with AKE (25mgkg body weight) (3) OIRmice treatedwithAKE (50mgkg bodyweight) (4) OIRmicetreated with CA (25mgkg body weight) and (5) OIR micetreated with CA (50mgkg body weight) AKE and CA wereadministered intraperitoneally for 5 days (P12ndashP16)Themicein the OIR group received an equal volume of the vehiclefor 5 days All procedures were approved by the InstitutionalAnimal Care and Use Committee (IACUC approval number14-053)
25 Fluorescein-Dextran Microscopy and Lectin Staining forNeovascular Area Analysis At necropsy (P17) all mice wereanesthetized by isoflurane inhalation Fluorescein-dextran(FD40 Sigma-Aldrich Merck Millipore) in PBS at a concen-tration of 50mgml was injected into the left ventricle Thetracer dye was allowed to perfuse for 15min and the eyeballswere then placed in 4 paraformaldehyde for 15 hoursThe retinas were dissected and then mounted on micro-scope slides The whole-mount retinas were observed usinga fluorescence microscope (Olympus Corporation TokyoJapan) The nonperfusion area in the retina was determinedby ImageJ software (National Institutes of Health BethesdaMD USA) The neovascular tufts in the retina were stainedwith rhodamine-labeled Bandeiraea simplicifolia isolectinB4 (Vector Laboratories Ltd Burlingame CA USA) Theneovascular areas labeled with lectin were examined using afluorescence microscope The sizes of the neovascular tuftswere calculated using the ImageJ software
26 Real-Time PCR Analysis Frozen retinal samples wereweighed and the total RNA was isolated using TRIzolreagent (InvitrogenThermo Fisher Scientific Inc WalthamMA USA) Real-time PCR was conducted according to apreviously described protocol [20] The primer sequenceswere as follows VEGF 51015840-TCC TCC TAT CTC CAC CACCTA TCC-31015840 and 51015840-GAC CCA GCC AGC CAT ACC C-31015840and GAPDH 51015840-AAC GAC CCC TTC ATT GAC-31015840 and 51015840-TCC ACG ACA TAC TCA GCA C-31015840 The mRNA levels ofVEGFwere determined using the iQ5 optical system software(Bio-Rad Laboratories Inc Hercules CA USA)
Evidence-Based Complementary and Alternative Medicine 3
Con AKE (01 gmL) AKE (1 gmL) AKE (10 gmL)
CA (01 gmL) CA (1 gmL) CA (10 gmL)
(a)
Con 01 1 10 01 1 10
AKE (gmL) CA (gmL)
0
20
40
60
80
100
120
Mea
n nu
mbe
r of b
ranc
hes
field
lowastlowast lowast
lowast
(b)
Figure 2 AKE inhibits tube formation in human vascular endothelial cells (a) Human vascular endothelial cells were treated with serum-freemedia containing AKE or CA (0ndash10120583gmL) with recombinant human VEGF (20 ngmL) for 17 h Tube formation on Matrigel was observedwith a microscope (b) The bar graph represents the quantification of tube formation Data are expressed as mean plusmn SEM 119899 = 4 lowast119901 lt 001versus control
27 Statistical Analysis Group data were analyzed by one-way analysis of variance followed by Tukeyrsquos multiple com-parison test or an unpaired Studentrsquos t-test using GraphPadPrism v60 software (GraphPad Software Inc La Jolla CAUSA) A 119901 value of lt005 was considered to indicate astatistically significant difference
3 Results
31 HPLC Analysis of AKE The content of the majorconstituent compound in AKE was determined via HPLCanalysis CA (124 plusmn 002) was found to be the majorcomponent of AKE (Table 1)
32 AKE and CA Inhibit VEGF-Induced Tube Formationin Human Vascular Endothelial Cells To investigate the
Table 1 Chlorogenic acid content in AKE
Compound Content (mean plusmn SD 119899 = 3)mgg
Chlorogenic acid 1235 plusmn 022
cytotoxic effect of AKE on human vascular endothelial cellswe performed an MTS assay using various concentrationsof AKE or CA (1ndash100 120583gmL) The viability of AKE or CA-treated human vascular endothelial cells was not affectedup to concentrations of 100 120583gmL (Figure 1) Next weexamined whether AKE or CA could inhibit tube formationan endothelial function crucial to angiogenesis in humanvascular endothelial cells VEGF was used as an angiogenicfactor Treatment with AKE or CA inhibited the formation
4 Evidence-Based Complementary and Alternative Medicine
OIR AKE (25 mgkg) AKE (50mgkg)
CA (25 mgkg) CA (50mgkg)
(a)
Avas
cula
r are
aTo
tal a
rea o
f ret
ina (
)
OIR 25 50 25 50AKE (mgkg) CA (mgkg)
0
10
20
30
40
(b)
Figure 3The effect of AKE and CA on vascular obliteration of the central retina in OIR mice (a) The retinal blood vessels were visualized viafluorescein angiography using FITC-dextran (b) The quantification results are expressed as the percentage of the central nonperfused areawithin the total retinal area The bar graph values represent the mean plusmn SEM 119899 = 7 lowast119901 lt 005 versus OIR mice
of the extensive capillary-like networks of human vascularendothelial cells in a dose-dependent manner (Figure 2)Theinhibitory activity of CAwasmore potent comparedwith thatof AKE
321 AKE and CA Inhibit Retinal Neovascularization in OIRThemice subjected to ischemic retinopathy showed vascularobliteration of the central retina and pathogenic retinalneovascularization Newly formed neovascular tufts werevisualized by immunofluorescence staining with isolectin B4OIR mice treated with AKE or CA exhibited a significantdecrease in these retinal vascular changes that occur duringischemic retinopathy As presented in Figure 3 treatmentwith AKE or CA failed to induce significant changes in the
vascular obliteration of the central retina However AKEinhibited the formation of neovascular tufts by 2627 plusmn424 and 3875 plusmn 404 at doses of 25 and 50mgkgdayrespectively CA also inhibited retinal neovascularizationby 2968 plusmn 235 and 5024 plusmn 277 at doses of 25 and50mgkgday respectively (Figure 4) These results indicatedthat AKE and CA treatments significantly reduce the size ofneovascular tufts demonstrating thatCA is an antiangiogenicbioactive compound of AKE
322 AKE and CA Downregulate VEGF mRNA ExpressionTo examine the changes in VEGF expression in the retinawe measured the expression levels of VEGF mRNA usingreal-time PCR As predicted we observed a marked increase
Evidence-Based Complementary and Alternative Medicine 5
OIR AKE (25 mgkg) AKE (50mgkg)
CA (25 mgkg) CA (50mgkg)
(a)
OIR 25 50 25 50AKE (mgkg) CA (mgkg)
0
5
10
15
20
Neo
vasc
ular
tufts
area
()
lowast
lowastlowast
lowast
(b)
Figure 4The effect of AKE and CA on retinal neovascularization in OIR mice (a)The retinal neovascular tufts were visualized using isolectinB4 staining (b) Quantification results are expressed as neovascular tufts on the retina surface The bar graph values represent the mean plusmnSEM 119899 = 7 lowast119901 lt 005 versus OIR mice
in VEGF mRNA during ischemic retinopathy However theVEGFmRNA levels markedly decreased following treatmentwith AKE or CA in the OIR mice (Figure 5)
4 Discussion
Pathogenic angiogenesis is a primary cause of severe visionloss in several retinal degenerative diseases including dia-betic retinopathy and wet form AMD [21] VEGF and itsreceptors play an important role in the development of theseretinal disorders [3] and inhibiting angiogenesis by targetingVEGF has become a major focus in drug development [22]In the present study we aimed to evaluate the effect of AKE
on retinal neovascularization in a mouse model of OIRTo the best of our knowledge this study demonstrated forthe first time that AKE inhibits tube formation in humanvascular endothelial cells in vitro through a VEGF-mediatedmechanism In addition AKE significantly suppressed retinalneovascularization and VEGF mRNA expression in a mousemodel of experimentalOIRMoreover CA is one of themajorcompounds present in AKE CA also exhibited a preventiveeffect against pathological retinal neovascularization Takentogether these results suggest that the inhibitory effect ofAKE on retinal neovascularization primarily stems from itspotent anti-VEGF activity and that its antiangiogenic activitymay in part be due to the bioactive compound CA
6 Evidence-Based Complementary and Alternative Medicine
00
05
10
15
Relat
ive V
EGF
mRN
A le
vels
(AU
)
OIR 25 50 25 50AKE (mgkg) CA (mgkg)
lowast
lowastlowast
lowast
Figure 5 The effect of AKE and CA on VEGF mRNA expressionin OIR mice Real-time PCR analysis of VEGF mRNA levels inOIR mice VEGF mRNAs were markedly reduced after AKE or CAtreatment The data are shown as the mean plusmn SEM 119899 = 7 lowast119901 lt 005versus OIR mice
VEGF is a potent angiogenic and vascular permeabilityfactor [23] that stimulates endothelial cell proliferation andangiogenesis In OIR mice VEGF expression was suppressedduring the hyperoxic phase (P7ndashP12) [24] Once hyper-oxia was terminated (P12ndashP17) hypoxia-driven upregulationof VEGF was observed even under normoxic conditions[25 26] Furthermore in ischemic retinopathy such asthat present in diabetic retinopathy and neovascular AMDthe robust upregulation of proangiogenic VEGF expressionleads to the activation of angiogenic signaling pathwaysand triggers neovascularization [27] Numerous studies havesuggested that VEGF has a key role in retinal vasculopa-thy and its inhibition significantly blocks the pathogenicalterations of retinal vasculature [28 29] Anti-VEGF agentswere recently reported to exhibit beneficial effects in patientswith proliferative diabetic retinopathy and neovascular AMD[2 30]
Medicinal herbs are rich sources of potential preven-tive and therapeutic agents AKE is a standardized dietaryherbal supplement To the best of our knowledge our studydemonstrated the antiangiogenic effects of AKE in vitro andin vivo for the first time Several studies have reported thatcertain crude herbal extracts and phytochemicals can inhibitpathogenic neovascularization in tumorigenesis [31 32] andretinal neovascular diseases [33ndash37] CA is the ester of caffeicacid with quinic acid and a major phenolic compound incoffee and various fruits [38] CA is also a major compoundpresent inAKE [18]This phytocompoundhas potent antioxi-dant [39] anti-inflammatory [40] and anticancer effects [41]Kim et al [42] reported that CA inhibited laser-inducedchoroidal neovascularization in a rat model of neovascularAMD More recently Park et al [43] showed that CAblocks hypoxia-stimulated angiogenesis in human vascularendothelial cells through inhibition of the hypoxia-induciblefactor-1120572AKT signaling pathway In the present study CAalso inhibited the VEGF-mediated tube formation of human
vascular endothelial cells and retinal neovascularization inmice with ischemic retinopathy Although the underlyingmechanism of action of AKE as a VEGF inhibitor remainsunclear it is hypothesized that the antiangiogenic activity ofAKE may be due to the antiantigenic effect of the bioactivecomponent CA
In conclusion this is the first study to provide evidencethat AKE and its bioactive compound CA inhibit exper-imental retinal neovascularization in ischemic retinopathyin vivo In addition in vitro studies showed that AKEand CA inhibit VEGF-induced tube formation in humanvascular endothelial cells Further studies may be required todetermine the feasibility of using AKE for the treatment ofpatients with ischemic retinopathy
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
Chan-Sik Kim and Jin Sook Kim contributed equally to thispaper as cocorrespondence authors
Acknowledgments
This research was supported by the Korea Institute of Plan-ning and Evaluation for Technology in Food AgricultureForestry and Fisheries (IPET) funded by the Ministry ofAgriculture Food and Rural Affairs (316023-05-2-CG000)and the Korea Institute of Oriental Medicine (K17270)
References
[1] R D Jager W F Mieler and J W Miller ldquoAge-related maculardegenerationrdquo The New England Journal of Medicine vol 358no 24 pp 2606ndash2617 2008
[2] P A Campochiaro ldquoOcular neovascularizationrdquo Journal ofMolecular Medicine vol 91 no 3 pp 311ndash321 2013
[3] L P Aiello ldquoVascular endothelial growth factor and the eyeBiochemical mechanisms of action and implications for noveltherapiesrdquoOphthalmic Research vol 29 no 5 pp 354ndash362 1997
[4] K Muranaka Y Yanagi Y Tamaki et al ldquoSuppression of laser-induced choroidal neovascularization by oral administration ofSA3443 in micerdquo FEBS Letters vol 579 no 27 pp 6084ndash60882005
[5] D RGuyer G Fish J AHaller et al ldquoAnti-vascular endothelialgrowth factor therapy for subfoveal choroidal neovasculariza-tion secondary to age-related macular degeneration Phase IIstudy resultsrdquoOphthalmology vol 110 no 5 pp 979ndash986 2003
[6] J E Frampton ldquoRanibizumab a review of its use in thetreatment of neovascular age-related macular degenerationrdquoDrugs amp Aging vol 30 no 5 pp 331ndash358 2013
[7] A Garcıa-Layana M S Figueroa J Araiz et al ldquoTreatmentof Exudative Age-related Macular Degeneration Focus onAfliberceptrdquo Drugs amp Aging vol 32 no 10 pp 797ndash807 2015
[8] C Campa and S P Harding ldquoAnti-VEGF compounds in thetreatment of neovascular age related macular degenerationrdquoCurrent Drug Targets vol 12 no 2 pp 173ndash181 2012
Evidence-Based Complementary and Alternative Medicine 7
[9] T Diago C AMcCannel S J Bakri J S Pulido A O Edwardsand J M Pach ldquoInfectious endophthalmitis after intravitrealinjection of antiangiogenic agentsrdquo Retina vol 29 no 5 pp601ndash605 2009
[10] D R Fintak G K Shah K J Blinder et al ldquoIncidenceof endophthalmitis related to intravitreal injection of beva-cizumab and ranibizumabrdquoRetina vol 28 no 10 pp 1395ndash13992008
[11] K Ghasemi Falavarjani and Q D Nguyen ldquoAdverse eventsand complications associated with intravitreal injection of anti-VEGF agents a review of literaturerdquo Eye vol 27 no 7 pp 787ndash794 2013
[12] H Takahashi Y Tamaki N Ishii et al ldquoIdentification of anovel vascular endothelial growth factor receptor 2 inhibitorand its effect for choroidal neovascularization in vivordquo CurrentEye Research vol 33 no 11-12 pp 1002ndash1010 2008
[13] S Honda T Nagai N Kondo et al ldquoTherapeutic effect of oralbisphosphonates on choroidal neovascularization in the humaneyerdquo Journal of Ophthalmology vol 2010 Article ID 206837 7pages 2010
[14] E L Meredith N Mainolfi S Poor et al ldquoDiscovery of oralVEGFR-2 inhibitors with prolonged ocular retention that areefficacious in models of wet age-related macular degenerationrdquoJournal of Medicinal Chemistry vol 58 no 23 pp 9273ndash92852015
[15] D K Ahn Illustrated Book of KoreanMedicinal Herbs Kyo-HakPublishing Seoul Republic of Korea 1998
[16] J Y KO and K K Lee ldquoEffect of plant growth regulators ongrowth and flowering of potted Lychnis cognata Aster koraien-sis and Campanula takesimanardquo RDA Journal of AgriculturalScience vol 38 pp 627ndash632 1996
[17] E Sohn J Kim C-S Kim Y S Kim D S Jang and J SKim ldquoExtract of the aerial parts of Aster koraiensis reduceddevelopment of diabetic nephropathy via anti-apoptosis ofpodocytes in streptozotocin-induced diabetic ratsrdquo Biochemicaland Biophysical Research Communications vol 391 no 1 pp733ndash738 2010
[18] J Kim K Jo I-S Lee C-S Kim and J S Kim ldquoThe extract ofaster koraiensis prevents retinal pericyte apoptosis in diabeticrats and its active compound chlorogenic acid inhibits ageformation and agerage interactionrdquo Nutrients vol 8 no 9article no 585 2016
[19] Y M Lee J Kim K Jo et al ldquoEthyl pyruvate inhibits reti-nal pathogenic neovascularization by downregulating HMGB1expressionrdquo Journal of Diabetes Research vol 2013 Article ID245271 8 pages 2013
[20] Y M Lee Y-R Lee C-S Kim et al ldquoCnidium officinaleextract and butylidenephthalide inhibits retinal neovasculariza-tion in vitro and in vivordquo BMC Complementary and AlternativeMedicine vol 16 no 1 article no 231 2016
[21] KMGehrs DHAnderson L V Johnson andG SHagemanldquoAge-related macular degenerationmdashemerging pathogeneticand therapeutic conceptsrdquo Annals of Medicine vol 38 no 7 pp450ndash471 2006
[22] P Van Wijngaarden and S H Qureshi ldquoInhibitors of vascularendothelial growth factor (VEGF) in the management of neo-vascular age-related macular degeneration A review of currentpracticerdquo Clinical and Experimental Optometry vol 91 no 5pp 427ndash437 2008
[23] D Shweiki A Itin D Soffer and E Keshet ldquoVascular endothe-lial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesisrdquo Nature vol 359 no 6398 pp 843ndash8451992
[24] J Stone T Chan-Ling J Persquoer A Itin H Gnessin and E KeshetldquoRoles of vascular endothelial growth factor and astrocytedegeneration in the genesis of retinopathy of prematurityrdquoInvestigative Ophthalmology amp Visual Science vol 37 no 2 pp290ndash299 1996
[25] N Ashton ldquoOxygen and the growth and development of retinalvessels In vivo and in vitro studies The XX Francis I ProctorLecturerdquo American Journal of Ophthalmology vol 62 no 3 pp412ndash435 1966
[26] N Ashton ldquoRetinal vascularization in health and diseaseproctor award lecture of the association for research in ophthal-mologyrdquo American Journal of Ophthalmology vol 44 no 4 pp7ndash17 1957
[27] A Hoeben B Landuyt M S Highley H Wildiers A T vanOosterom and E A de Bruijn ldquoVascular endothelial growthfactor and angiogenesisrdquo Pharmacological Reviews vol 56 no4 pp 549ndash580 2004
[28] L P Aiello R L Avery P G Arrigg et al ldquoVascular endothelialgrowth factor in ocular fluid of patients with diabetic retinopa-thy and other retinal disordersrdquo The New England Journal ofMedicine vol 331 no 22 pp 1480ndash1487 1994
[29] C K Dorey S Aouididi X Reynaud H F Dvorak and LF Brown ldquoCorrelation of vascular permeability factorvascularendothelial growth factor with extraretinal neovascularizationin the ratrdquo JAMA Ophtalmology vol 114 no 10 pp 1210ndash12171996
[30] D S Dhoot andR L Avery ldquoVascular endothelial growth factorinhibitors for diabetic retinopathyrdquo Current Diabetes Reportsvol 16 no 12 article no 122 2016
[31] I M W Ruma E W Putranto E Kondo et al ldquoExtract ofCordyceps militaris inhibits angiogenesis and suppresses tumorgrowth of human malignant melanoma cellsrdquo InternationalJournal of Oncology vol 45 no 1 pp 209ndash218 2014
[32] D R Yance Jr and S M Sagar ldquoTargeting angiogenesis withintegrative cancer therapiesrdquo Integrative Cancer Therapies vol5 no 1 pp 9ndash29 2006
[33] S K Gupta B Kumar B P Srinivasan et al ldquoRetinoprotectiveeffects of Moringa oleifera via antioxidant anti-inflammatoryand anti-angiogenic mechanisms in streptozotocin-induceddiabetic ratsrdquo Journal of Ocular Pharmacology andTherapeuticsvol 29 no 4 pp 419ndash426 2013
[34] J Tanaka S Nakamura K Tsuruma M Shimazawa H Shi-moda and H Hara ldquoPurple Rice (Oryza sativa L) extract andits constituents inhibit VEGF-induced angiogenesisrdquoPhytother-apy Research vol 26 no 2 pp 214ndash222 2012
[35] J Hua K I Guerin J Chen et al ldquoResveratrol inhibits patho-logic retinal neovascularization in Vldlr(minusminus) micerdquo Investiga-tive Ophthalmology amp Visual Science vol 52 no 5 pp 2809ndash2816 2011
[36] L Cao H Liu D S-C Lam G H-F Yam and C-P Pang ldquoInvitro screening for angiostatic potential of herbal chemicalsrdquoInvestigative Ophthalmology amp Visual Science vol 51 no 12 pp6658ndash6664 2010
[37] E Jung W Jung S Park C Kim J S Kim and J KimldquoEGHB010 a Standardized Extract of Paeoniae Radix andGlycyrrhizae Radix Inhibits VEGF-Induced Tube Formation
8 Evidence-Based Complementary and Alternative Medicine
In Vitro and Retinal Vascular Leakage and Choroidal Neo-vascularization In Vivordquo Evidence-Based Complementary andAlternative Medicine vol 2017 pp 1ndash7 2017
[38] M N Clifford K L Johnston S Knight and N KuhnertldquoHierarchical scheme for LC-MSn identification of chlorogenicacidsrdquo Journal of Agricultural and Food Chemistry vol 51 no10 pp 2900ndash2911 2003
[39] W-J Yen B-SWang L-WChang andP-DDuh ldquoAntioxidantproperties of roasted coffee residuesrdquo Journal of Agricultural andFood Chemistry vol 53 no 7 pp 2658ndash2663 2005
[40] T Krakauer ldquoThe polyphenol chlorogenic acid inhibitsstaphylococcal exotoxin-induced inflammatory cytokines andchemokinesrdquo Immunopharmacology and Immunotoxicologyvol 24 no 1 pp 113ndash119 2002
[41] C Kandaswami L T Lee P P Lee et al ldquoThe antitumoractivities of flavonoidsrdquo In Vivo vol 19 pp 895ndash909 2005
[42] C Kim H G Yu and J Sohn ldquoThe anti-angiogenic effectof chlorogenic acid on choroidal neovascularizationrdquo KoreanJournal of Ophthalmology vol 24 no 3 pp 163ndash168 2010
[43] J J Park S J Hwang J-H Park and H-J Lee ldquoChloro-genic acid inhibits hypoxia-induced angiogenesis via down-regulation of the HIF-1120572AKT pathwayrdquo Cellular Oncology vol38 no 2 pp 111ndash118 2015
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
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Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
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Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
2 Evidence-Based Complementary and Alternative Medicine
elucidate this we investigated the inhibitory effect of AKEand its major compound chlorogenic acid (CA) on retinalneovascularization in a mouse model of oxygen-inducedretinopathy (OIR) We also investigated the inhibitory effectof AKE and CA on the VEGF-induced tube formation ofhuman vascular endothelial cells
2 Materials and Methods
21 Preparation of AKE The aerial parts which include theflowers leaves and stems of A koraiensis were purchasedfrom Gongju (Chungcheongnam South Korea) in August2007 The AKE was prepared according to a previouslyreported method [17] Briefly 25 kg A koraiensis was accu-rately weighed and extracted with EtOH (3 times 20 L) via mac-eration at room temperature for 3 daysThe extracted solutionwas concentrated in vacuo at 40∘C to provide anAKE powder(303 g) Voucher specimens ofA koraiensiswere deposited atthe Herbarium of the Korea Institute of Oriental Medicine(Daejeon Korea Herbarium number KIOM-83A) AKE wasstandardized using CA as a reference compound (Sigma-Aldrich Merck Millipore Darmstadt Germany) by high-performance liquid chromatography (HPLC) The HPLCfingerprint and the CA content of AKE are described in ourprevious report [18]
22 Cell Viability Assay Cell viability was examined usingan MTS assay kit (CellTiter 96 AQueous One Solution CellProliferation Assay Promega Corporation Madison WIUSA) Human umbilical vein endothelial cells (Korean CellLine Bank Seoul Korea) were plated (1 times 104 cellswell) inquadruplicate into 96-well plates containing various doses ofAKE or CA (1ndash100 120583gmL) Cell viability was measured at24 h following incubation The results of the MTS assay wereobtained by measuring absorbance using a microplate reader(Tecan Group Ltd Mannedorf Switzerland) at 490 nm Allexperiments were repeated three times
23 Tube Formation Assay Tissue culture plates (96 wells)were coated with 400 120583L growth factor reduced basementmembrane matrix (Matrigel BD Biosciences FranklinLakes NJ USA) Human umbilical vein endothelial cellswere seeded at a density of 1 times 106 cellswell and treatedwith serum-free EGM-2 media (WelGENE Inc DaeguKorea) containing AKE or CA (0ndash10 120583gmL) and recombi-nant humanVEGF (20 ngmL) for 17 h at 37∘C Capillary-liketube structures formed by human umbilical vein endothe-lial cells on the Matrigel were photographed with a DP71digital camera (Olympus Corporation) Tube formation wasquantified by counting the number of branching points ofthe capillary-like structures per visual field The experimentswere repeated three times independently
24 Experimental Retinal Neovascularization in OIR Exper-imental retinal neovascularization was induced in C57BL6mice as previously described [19] Mice were exposed to75 oxygen on postnatal day 7 (P7) and then returned tonormal oxygen pressure on P12 The mice were divided intofive groups of seven mice each as follows (1) OIR mice (2)
Cel
l Via
bilit
y(
of c
ontro
l)
Con 1 10 100 1 10 100
AKE (gmL) CA (gmL)
0
20
40
60
80
100
120
140
Figure 1 Effects of AKE and CA on the viability of human vascularendothelial cells The viability of human vascular endothelial cellswas determined by MTS assay Data are expressed as percentage ofcontrol Data are expressed as mean plusmn SEM 119899 = 4
OIR mice treated with AKE (25mgkg body weight) (3) OIRmice treatedwithAKE (50mgkg bodyweight) (4) OIRmicetreated with CA (25mgkg body weight) and (5) OIR micetreated with CA (50mgkg body weight) AKE and CA wereadministered intraperitoneally for 5 days (P12ndashP16)Themicein the OIR group received an equal volume of the vehiclefor 5 days All procedures were approved by the InstitutionalAnimal Care and Use Committee (IACUC approval number14-053)
25 Fluorescein-Dextran Microscopy and Lectin Staining forNeovascular Area Analysis At necropsy (P17) all mice wereanesthetized by isoflurane inhalation Fluorescein-dextran(FD40 Sigma-Aldrich Merck Millipore) in PBS at a concen-tration of 50mgml was injected into the left ventricle Thetracer dye was allowed to perfuse for 15min and the eyeballswere then placed in 4 paraformaldehyde for 15 hoursThe retinas were dissected and then mounted on micro-scope slides The whole-mount retinas were observed usinga fluorescence microscope (Olympus Corporation TokyoJapan) The nonperfusion area in the retina was determinedby ImageJ software (National Institutes of Health BethesdaMD USA) The neovascular tufts in the retina were stainedwith rhodamine-labeled Bandeiraea simplicifolia isolectinB4 (Vector Laboratories Ltd Burlingame CA USA) Theneovascular areas labeled with lectin were examined using afluorescence microscope The sizes of the neovascular tuftswere calculated using the ImageJ software
26 Real-Time PCR Analysis Frozen retinal samples wereweighed and the total RNA was isolated using TRIzolreagent (InvitrogenThermo Fisher Scientific Inc WalthamMA USA) Real-time PCR was conducted according to apreviously described protocol [20] The primer sequenceswere as follows VEGF 51015840-TCC TCC TAT CTC CAC CACCTA TCC-31015840 and 51015840-GAC CCA GCC AGC CAT ACC C-31015840and GAPDH 51015840-AAC GAC CCC TTC ATT GAC-31015840 and 51015840-TCC ACG ACA TAC TCA GCA C-31015840 The mRNA levels ofVEGFwere determined using the iQ5 optical system software(Bio-Rad Laboratories Inc Hercules CA USA)
Evidence-Based Complementary and Alternative Medicine 3
Con AKE (01 gmL) AKE (1 gmL) AKE (10 gmL)
CA (01 gmL) CA (1 gmL) CA (10 gmL)
(a)
Con 01 1 10 01 1 10
AKE (gmL) CA (gmL)
0
20
40
60
80
100
120
Mea
n nu
mbe
r of b
ranc
hes
field
lowastlowast lowast
lowast
(b)
Figure 2 AKE inhibits tube formation in human vascular endothelial cells (a) Human vascular endothelial cells were treated with serum-freemedia containing AKE or CA (0ndash10120583gmL) with recombinant human VEGF (20 ngmL) for 17 h Tube formation on Matrigel was observedwith a microscope (b) The bar graph represents the quantification of tube formation Data are expressed as mean plusmn SEM 119899 = 4 lowast119901 lt 001versus control
27 Statistical Analysis Group data were analyzed by one-way analysis of variance followed by Tukeyrsquos multiple com-parison test or an unpaired Studentrsquos t-test using GraphPadPrism v60 software (GraphPad Software Inc La Jolla CAUSA) A 119901 value of lt005 was considered to indicate astatistically significant difference
3 Results
31 HPLC Analysis of AKE The content of the majorconstituent compound in AKE was determined via HPLCanalysis CA (124 plusmn 002) was found to be the majorcomponent of AKE (Table 1)
32 AKE and CA Inhibit VEGF-Induced Tube Formationin Human Vascular Endothelial Cells To investigate the
Table 1 Chlorogenic acid content in AKE
Compound Content (mean plusmn SD 119899 = 3)mgg
Chlorogenic acid 1235 plusmn 022
cytotoxic effect of AKE on human vascular endothelial cellswe performed an MTS assay using various concentrationsof AKE or CA (1ndash100 120583gmL) The viability of AKE or CA-treated human vascular endothelial cells was not affectedup to concentrations of 100 120583gmL (Figure 1) Next weexamined whether AKE or CA could inhibit tube formationan endothelial function crucial to angiogenesis in humanvascular endothelial cells VEGF was used as an angiogenicfactor Treatment with AKE or CA inhibited the formation
4 Evidence-Based Complementary and Alternative Medicine
OIR AKE (25 mgkg) AKE (50mgkg)
CA (25 mgkg) CA (50mgkg)
(a)
Avas
cula
r are
aTo
tal a
rea o
f ret
ina (
)
OIR 25 50 25 50AKE (mgkg) CA (mgkg)
0
10
20
30
40
(b)
Figure 3The effect of AKE and CA on vascular obliteration of the central retina in OIR mice (a) The retinal blood vessels were visualized viafluorescein angiography using FITC-dextran (b) The quantification results are expressed as the percentage of the central nonperfused areawithin the total retinal area The bar graph values represent the mean plusmn SEM 119899 = 7 lowast119901 lt 005 versus OIR mice
of the extensive capillary-like networks of human vascularendothelial cells in a dose-dependent manner (Figure 2)Theinhibitory activity of CAwasmore potent comparedwith thatof AKE
321 AKE and CA Inhibit Retinal Neovascularization in OIRThemice subjected to ischemic retinopathy showed vascularobliteration of the central retina and pathogenic retinalneovascularization Newly formed neovascular tufts werevisualized by immunofluorescence staining with isolectin B4OIR mice treated with AKE or CA exhibited a significantdecrease in these retinal vascular changes that occur duringischemic retinopathy As presented in Figure 3 treatmentwith AKE or CA failed to induce significant changes in the
vascular obliteration of the central retina However AKEinhibited the formation of neovascular tufts by 2627 plusmn424 and 3875 plusmn 404 at doses of 25 and 50mgkgdayrespectively CA also inhibited retinal neovascularizationby 2968 plusmn 235 and 5024 plusmn 277 at doses of 25 and50mgkgday respectively (Figure 4) These results indicatedthat AKE and CA treatments significantly reduce the size ofneovascular tufts demonstrating thatCA is an antiangiogenicbioactive compound of AKE
322 AKE and CA Downregulate VEGF mRNA ExpressionTo examine the changes in VEGF expression in the retinawe measured the expression levels of VEGF mRNA usingreal-time PCR As predicted we observed a marked increase
Evidence-Based Complementary and Alternative Medicine 5
OIR AKE (25 mgkg) AKE (50mgkg)
CA (25 mgkg) CA (50mgkg)
(a)
OIR 25 50 25 50AKE (mgkg) CA (mgkg)
0
5
10
15
20
Neo
vasc
ular
tufts
area
()
lowast
lowastlowast
lowast
(b)
Figure 4The effect of AKE and CA on retinal neovascularization in OIR mice (a)The retinal neovascular tufts were visualized using isolectinB4 staining (b) Quantification results are expressed as neovascular tufts on the retina surface The bar graph values represent the mean plusmnSEM 119899 = 7 lowast119901 lt 005 versus OIR mice
in VEGF mRNA during ischemic retinopathy However theVEGFmRNA levels markedly decreased following treatmentwith AKE or CA in the OIR mice (Figure 5)
4 Discussion
Pathogenic angiogenesis is a primary cause of severe visionloss in several retinal degenerative diseases including dia-betic retinopathy and wet form AMD [21] VEGF and itsreceptors play an important role in the development of theseretinal disorders [3] and inhibiting angiogenesis by targetingVEGF has become a major focus in drug development [22]In the present study we aimed to evaluate the effect of AKE
on retinal neovascularization in a mouse model of OIRTo the best of our knowledge this study demonstrated forthe first time that AKE inhibits tube formation in humanvascular endothelial cells in vitro through a VEGF-mediatedmechanism In addition AKE significantly suppressed retinalneovascularization and VEGF mRNA expression in a mousemodel of experimentalOIRMoreover CA is one of themajorcompounds present in AKE CA also exhibited a preventiveeffect against pathological retinal neovascularization Takentogether these results suggest that the inhibitory effect ofAKE on retinal neovascularization primarily stems from itspotent anti-VEGF activity and that its antiangiogenic activitymay in part be due to the bioactive compound CA
6 Evidence-Based Complementary and Alternative Medicine
00
05
10
15
Relat
ive V
EGF
mRN
A le
vels
(AU
)
OIR 25 50 25 50AKE (mgkg) CA (mgkg)
lowast
lowastlowast
lowast
Figure 5 The effect of AKE and CA on VEGF mRNA expressionin OIR mice Real-time PCR analysis of VEGF mRNA levels inOIR mice VEGF mRNAs were markedly reduced after AKE or CAtreatment The data are shown as the mean plusmn SEM 119899 = 7 lowast119901 lt 005versus OIR mice
VEGF is a potent angiogenic and vascular permeabilityfactor [23] that stimulates endothelial cell proliferation andangiogenesis In OIR mice VEGF expression was suppressedduring the hyperoxic phase (P7ndashP12) [24] Once hyper-oxia was terminated (P12ndashP17) hypoxia-driven upregulationof VEGF was observed even under normoxic conditions[25 26] Furthermore in ischemic retinopathy such asthat present in diabetic retinopathy and neovascular AMDthe robust upregulation of proangiogenic VEGF expressionleads to the activation of angiogenic signaling pathwaysand triggers neovascularization [27] Numerous studies havesuggested that VEGF has a key role in retinal vasculopa-thy and its inhibition significantly blocks the pathogenicalterations of retinal vasculature [28 29] Anti-VEGF agentswere recently reported to exhibit beneficial effects in patientswith proliferative diabetic retinopathy and neovascular AMD[2 30]
Medicinal herbs are rich sources of potential preven-tive and therapeutic agents AKE is a standardized dietaryherbal supplement To the best of our knowledge our studydemonstrated the antiangiogenic effects of AKE in vitro andin vivo for the first time Several studies have reported thatcertain crude herbal extracts and phytochemicals can inhibitpathogenic neovascularization in tumorigenesis [31 32] andretinal neovascular diseases [33ndash37] CA is the ester of caffeicacid with quinic acid and a major phenolic compound incoffee and various fruits [38] CA is also a major compoundpresent inAKE [18]This phytocompoundhas potent antioxi-dant [39] anti-inflammatory [40] and anticancer effects [41]Kim et al [42] reported that CA inhibited laser-inducedchoroidal neovascularization in a rat model of neovascularAMD More recently Park et al [43] showed that CAblocks hypoxia-stimulated angiogenesis in human vascularendothelial cells through inhibition of the hypoxia-induciblefactor-1120572AKT signaling pathway In the present study CAalso inhibited the VEGF-mediated tube formation of human
vascular endothelial cells and retinal neovascularization inmice with ischemic retinopathy Although the underlyingmechanism of action of AKE as a VEGF inhibitor remainsunclear it is hypothesized that the antiangiogenic activity ofAKE may be due to the antiantigenic effect of the bioactivecomponent CA
In conclusion this is the first study to provide evidencethat AKE and its bioactive compound CA inhibit exper-imental retinal neovascularization in ischemic retinopathyin vivo In addition in vitro studies showed that AKEand CA inhibit VEGF-induced tube formation in humanvascular endothelial cells Further studies may be required todetermine the feasibility of using AKE for the treatment ofpatients with ischemic retinopathy
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
Chan-Sik Kim and Jin Sook Kim contributed equally to thispaper as cocorrespondence authors
Acknowledgments
This research was supported by the Korea Institute of Plan-ning and Evaluation for Technology in Food AgricultureForestry and Fisheries (IPET) funded by the Ministry ofAgriculture Food and Rural Affairs (316023-05-2-CG000)and the Korea Institute of Oriental Medicine (K17270)
References
[1] R D Jager W F Mieler and J W Miller ldquoAge-related maculardegenerationrdquo The New England Journal of Medicine vol 358no 24 pp 2606ndash2617 2008
[2] P A Campochiaro ldquoOcular neovascularizationrdquo Journal ofMolecular Medicine vol 91 no 3 pp 311ndash321 2013
[3] L P Aiello ldquoVascular endothelial growth factor and the eyeBiochemical mechanisms of action and implications for noveltherapiesrdquoOphthalmic Research vol 29 no 5 pp 354ndash362 1997
[4] K Muranaka Y Yanagi Y Tamaki et al ldquoSuppression of laser-induced choroidal neovascularization by oral administration ofSA3443 in micerdquo FEBS Letters vol 579 no 27 pp 6084ndash60882005
[5] D RGuyer G Fish J AHaller et al ldquoAnti-vascular endothelialgrowth factor therapy for subfoveal choroidal neovasculariza-tion secondary to age-related macular degeneration Phase IIstudy resultsrdquoOphthalmology vol 110 no 5 pp 979ndash986 2003
[6] J E Frampton ldquoRanibizumab a review of its use in thetreatment of neovascular age-related macular degenerationrdquoDrugs amp Aging vol 30 no 5 pp 331ndash358 2013
[7] A Garcıa-Layana M S Figueroa J Araiz et al ldquoTreatmentof Exudative Age-related Macular Degeneration Focus onAfliberceptrdquo Drugs amp Aging vol 32 no 10 pp 797ndash807 2015
[8] C Campa and S P Harding ldquoAnti-VEGF compounds in thetreatment of neovascular age related macular degenerationrdquoCurrent Drug Targets vol 12 no 2 pp 173ndash181 2012
Evidence-Based Complementary and Alternative Medicine 7
[9] T Diago C AMcCannel S J Bakri J S Pulido A O Edwardsand J M Pach ldquoInfectious endophthalmitis after intravitrealinjection of antiangiogenic agentsrdquo Retina vol 29 no 5 pp601ndash605 2009
[10] D R Fintak G K Shah K J Blinder et al ldquoIncidenceof endophthalmitis related to intravitreal injection of beva-cizumab and ranibizumabrdquoRetina vol 28 no 10 pp 1395ndash13992008
[11] K Ghasemi Falavarjani and Q D Nguyen ldquoAdverse eventsand complications associated with intravitreal injection of anti-VEGF agents a review of literaturerdquo Eye vol 27 no 7 pp 787ndash794 2013
[12] H Takahashi Y Tamaki N Ishii et al ldquoIdentification of anovel vascular endothelial growth factor receptor 2 inhibitorand its effect for choroidal neovascularization in vivordquo CurrentEye Research vol 33 no 11-12 pp 1002ndash1010 2008
[13] S Honda T Nagai N Kondo et al ldquoTherapeutic effect of oralbisphosphonates on choroidal neovascularization in the humaneyerdquo Journal of Ophthalmology vol 2010 Article ID 206837 7pages 2010
[14] E L Meredith N Mainolfi S Poor et al ldquoDiscovery of oralVEGFR-2 inhibitors with prolonged ocular retention that areefficacious in models of wet age-related macular degenerationrdquoJournal of Medicinal Chemistry vol 58 no 23 pp 9273ndash92852015
[15] D K Ahn Illustrated Book of KoreanMedicinal Herbs Kyo-HakPublishing Seoul Republic of Korea 1998
[16] J Y KO and K K Lee ldquoEffect of plant growth regulators ongrowth and flowering of potted Lychnis cognata Aster koraien-sis and Campanula takesimanardquo RDA Journal of AgriculturalScience vol 38 pp 627ndash632 1996
[17] E Sohn J Kim C-S Kim Y S Kim D S Jang and J SKim ldquoExtract of the aerial parts of Aster koraiensis reduceddevelopment of diabetic nephropathy via anti-apoptosis ofpodocytes in streptozotocin-induced diabetic ratsrdquo Biochemicaland Biophysical Research Communications vol 391 no 1 pp733ndash738 2010
[18] J Kim K Jo I-S Lee C-S Kim and J S Kim ldquoThe extract ofaster koraiensis prevents retinal pericyte apoptosis in diabeticrats and its active compound chlorogenic acid inhibits ageformation and agerage interactionrdquo Nutrients vol 8 no 9article no 585 2016
[19] Y M Lee J Kim K Jo et al ldquoEthyl pyruvate inhibits reti-nal pathogenic neovascularization by downregulating HMGB1expressionrdquo Journal of Diabetes Research vol 2013 Article ID245271 8 pages 2013
[20] Y M Lee Y-R Lee C-S Kim et al ldquoCnidium officinaleextract and butylidenephthalide inhibits retinal neovasculariza-tion in vitro and in vivordquo BMC Complementary and AlternativeMedicine vol 16 no 1 article no 231 2016
[21] KMGehrs DHAnderson L V Johnson andG SHagemanldquoAge-related macular degenerationmdashemerging pathogeneticand therapeutic conceptsrdquo Annals of Medicine vol 38 no 7 pp450ndash471 2006
[22] P Van Wijngaarden and S H Qureshi ldquoInhibitors of vascularendothelial growth factor (VEGF) in the management of neo-vascular age-related macular degeneration A review of currentpracticerdquo Clinical and Experimental Optometry vol 91 no 5pp 427ndash437 2008
[23] D Shweiki A Itin D Soffer and E Keshet ldquoVascular endothe-lial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesisrdquo Nature vol 359 no 6398 pp 843ndash8451992
[24] J Stone T Chan-Ling J Persquoer A Itin H Gnessin and E KeshetldquoRoles of vascular endothelial growth factor and astrocytedegeneration in the genesis of retinopathy of prematurityrdquoInvestigative Ophthalmology amp Visual Science vol 37 no 2 pp290ndash299 1996
[25] N Ashton ldquoOxygen and the growth and development of retinalvessels In vivo and in vitro studies The XX Francis I ProctorLecturerdquo American Journal of Ophthalmology vol 62 no 3 pp412ndash435 1966
[26] N Ashton ldquoRetinal vascularization in health and diseaseproctor award lecture of the association for research in ophthal-mologyrdquo American Journal of Ophthalmology vol 44 no 4 pp7ndash17 1957
[27] A Hoeben B Landuyt M S Highley H Wildiers A T vanOosterom and E A de Bruijn ldquoVascular endothelial growthfactor and angiogenesisrdquo Pharmacological Reviews vol 56 no4 pp 549ndash580 2004
[28] L P Aiello R L Avery P G Arrigg et al ldquoVascular endothelialgrowth factor in ocular fluid of patients with diabetic retinopa-thy and other retinal disordersrdquo The New England Journal ofMedicine vol 331 no 22 pp 1480ndash1487 1994
[29] C K Dorey S Aouididi X Reynaud H F Dvorak and LF Brown ldquoCorrelation of vascular permeability factorvascularendothelial growth factor with extraretinal neovascularizationin the ratrdquo JAMA Ophtalmology vol 114 no 10 pp 1210ndash12171996
[30] D S Dhoot andR L Avery ldquoVascular endothelial growth factorinhibitors for diabetic retinopathyrdquo Current Diabetes Reportsvol 16 no 12 article no 122 2016
[31] I M W Ruma E W Putranto E Kondo et al ldquoExtract ofCordyceps militaris inhibits angiogenesis and suppresses tumorgrowth of human malignant melanoma cellsrdquo InternationalJournal of Oncology vol 45 no 1 pp 209ndash218 2014
[32] D R Yance Jr and S M Sagar ldquoTargeting angiogenesis withintegrative cancer therapiesrdquo Integrative Cancer Therapies vol5 no 1 pp 9ndash29 2006
[33] S K Gupta B Kumar B P Srinivasan et al ldquoRetinoprotectiveeffects of Moringa oleifera via antioxidant anti-inflammatoryand anti-angiogenic mechanisms in streptozotocin-induceddiabetic ratsrdquo Journal of Ocular Pharmacology andTherapeuticsvol 29 no 4 pp 419ndash426 2013
[34] J Tanaka S Nakamura K Tsuruma M Shimazawa H Shi-moda and H Hara ldquoPurple Rice (Oryza sativa L) extract andits constituents inhibit VEGF-induced angiogenesisrdquoPhytother-apy Research vol 26 no 2 pp 214ndash222 2012
[35] J Hua K I Guerin J Chen et al ldquoResveratrol inhibits patho-logic retinal neovascularization in Vldlr(minusminus) micerdquo Investiga-tive Ophthalmology amp Visual Science vol 52 no 5 pp 2809ndash2816 2011
[36] L Cao H Liu D S-C Lam G H-F Yam and C-P Pang ldquoInvitro screening for angiostatic potential of herbal chemicalsrdquoInvestigative Ophthalmology amp Visual Science vol 51 no 12 pp6658ndash6664 2010
[37] E Jung W Jung S Park C Kim J S Kim and J KimldquoEGHB010 a Standardized Extract of Paeoniae Radix andGlycyrrhizae Radix Inhibits VEGF-Induced Tube Formation
8 Evidence-Based Complementary and Alternative Medicine
In Vitro and Retinal Vascular Leakage and Choroidal Neo-vascularization In Vivordquo Evidence-Based Complementary andAlternative Medicine vol 2017 pp 1ndash7 2017
[38] M N Clifford K L Johnston S Knight and N KuhnertldquoHierarchical scheme for LC-MSn identification of chlorogenicacidsrdquo Journal of Agricultural and Food Chemistry vol 51 no10 pp 2900ndash2911 2003
[39] W-J Yen B-SWang L-WChang andP-DDuh ldquoAntioxidantproperties of roasted coffee residuesrdquo Journal of Agricultural andFood Chemistry vol 53 no 7 pp 2658ndash2663 2005
[40] T Krakauer ldquoThe polyphenol chlorogenic acid inhibitsstaphylococcal exotoxin-induced inflammatory cytokines andchemokinesrdquo Immunopharmacology and Immunotoxicologyvol 24 no 1 pp 113ndash119 2002
[41] C Kandaswami L T Lee P P Lee et al ldquoThe antitumoractivities of flavonoidsrdquo In Vivo vol 19 pp 895ndash909 2005
[42] C Kim H G Yu and J Sohn ldquoThe anti-angiogenic effectof chlorogenic acid on choroidal neovascularizationrdquo KoreanJournal of Ophthalmology vol 24 no 3 pp 163ndash168 2010
[43] J J Park S J Hwang J-H Park and H-J Lee ldquoChloro-genic acid inhibits hypoxia-induced angiogenesis via down-regulation of the HIF-1120572AKT pathwayrdquo Cellular Oncology vol38 no 2 pp 111ndash118 2015
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 3
Con AKE (01 gmL) AKE (1 gmL) AKE (10 gmL)
CA (01 gmL) CA (1 gmL) CA (10 gmL)
(a)
Con 01 1 10 01 1 10
AKE (gmL) CA (gmL)
0
20
40
60
80
100
120
Mea
n nu
mbe
r of b
ranc
hes
field
lowastlowast lowast
lowast
(b)
Figure 2 AKE inhibits tube formation in human vascular endothelial cells (a) Human vascular endothelial cells were treated with serum-freemedia containing AKE or CA (0ndash10120583gmL) with recombinant human VEGF (20 ngmL) for 17 h Tube formation on Matrigel was observedwith a microscope (b) The bar graph represents the quantification of tube formation Data are expressed as mean plusmn SEM 119899 = 4 lowast119901 lt 001versus control
27 Statistical Analysis Group data were analyzed by one-way analysis of variance followed by Tukeyrsquos multiple com-parison test or an unpaired Studentrsquos t-test using GraphPadPrism v60 software (GraphPad Software Inc La Jolla CAUSA) A 119901 value of lt005 was considered to indicate astatistically significant difference
3 Results
31 HPLC Analysis of AKE The content of the majorconstituent compound in AKE was determined via HPLCanalysis CA (124 plusmn 002) was found to be the majorcomponent of AKE (Table 1)
32 AKE and CA Inhibit VEGF-Induced Tube Formationin Human Vascular Endothelial Cells To investigate the
Table 1 Chlorogenic acid content in AKE
Compound Content (mean plusmn SD 119899 = 3)mgg
Chlorogenic acid 1235 plusmn 022
cytotoxic effect of AKE on human vascular endothelial cellswe performed an MTS assay using various concentrationsof AKE or CA (1ndash100 120583gmL) The viability of AKE or CA-treated human vascular endothelial cells was not affectedup to concentrations of 100 120583gmL (Figure 1) Next weexamined whether AKE or CA could inhibit tube formationan endothelial function crucial to angiogenesis in humanvascular endothelial cells VEGF was used as an angiogenicfactor Treatment with AKE or CA inhibited the formation
4 Evidence-Based Complementary and Alternative Medicine
OIR AKE (25 mgkg) AKE (50mgkg)
CA (25 mgkg) CA (50mgkg)
(a)
Avas
cula
r are
aTo
tal a
rea o
f ret
ina (
)
OIR 25 50 25 50AKE (mgkg) CA (mgkg)
0
10
20
30
40
(b)
Figure 3The effect of AKE and CA on vascular obliteration of the central retina in OIR mice (a) The retinal blood vessels were visualized viafluorescein angiography using FITC-dextran (b) The quantification results are expressed as the percentage of the central nonperfused areawithin the total retinal area The bar graph values represent the mean plusmn SEM 119899 = 7 lowast119901 lt 005 versus OIR mice
of the extensive capillary-like networks of human vascularendothelial cells in a dose-dependent manner (Figure 2)Theinhibitory activity of CAwasmore potent comparedwith thatof AKE
321 AKE and CA Inhibit Retinal Neovascularization in OIRThemice subjected to ischemic retinopathy showed vascularobliteration of the central retina and pathogenic retinalneovascularization Newly formed neovascular tufts werevisualized by immunofluorescence staining with isolectin B4OIR mice treated with AKE or CA exhibited a significantdecrease in these retinal vascular changes that occur duringischemic retinopathy As presented in Figure 3 treatmentwith AKE or CA failed to induce significant changes in the
vascular obliteration of the central retina However AKEinhibited the formation of neovascular tufts by 2627 plusmn424 and 3875 plusmn 404 at doses of 25 and 50mgkgdayrespectively CA also inhibited retinal neovascularizationby 2968 plusmn 235 and 5024 plusmn 277 at doses of 25 and50mgkgday respectively (Figure 4) These results indicatedthat AKE and CA treatments significantly reduce the size ofneovascular tufts demonstrating thatCA is an antiangiogenicbioactive compound of AKE
322 AKE and CA Downregulate VEGF mRNA ExpressionTo examine the changes in VEGF expression in the retinawe measured the expression levels of VEGF mRNA usingreal-time PCR As predicted we observed a marked increase
Evidence-Based Complementary and Alternative Medicine 5
OIR AKE (25 mgkg) AKE (50mgkg)
CA (25 mgkg) CA (50mgkg)
(a)
OIR 25 50 25 50AKE (mgkg) CA (mgkg)
0
5
10
15
20
Neo
vasc
ular
tufts
area
()
lowast
lowastlowast
lowast
(b)
Figure 4The effect of AKE and CA on retinal neovascularization in OIR mice (a)The retinal neovascular tufts were visualized using isolectinB4 staining (b) Quantification results are expressed as neovascular tufts on the retina surface The bar graph values represent the mean plusmnSEM 119899 = 7 lowast119901 lt 005 versus OIR mice
in VEGF mRNA during ischemic retinopathy However theVEGFmRNA levels markedly decreased following treatmentwith AKE or CA in the OIR mice (Figure 5)
4 Discussion
Pathogenic angiogenesis is a primary cause of severe visionloss in several retinal degenerative diseases including dia-betic retinopathy and wet form AMD [21] VEGF and itsreceptors play an important role in the development of theseretinal disorders [3] and inhibiting angiogenesis by targetingVEGF has become a major focus in drug development [22]In the present study we aimed to evaluate the effect of AKE
on retinal neovascularization in a mouse model of OIRTo the best of our knowledge this study demonstrated forthe first time that AKE inhibits tube formation in humanvascular endothelial cells in vitro through a VEGF-mediatedmechanism In addition AKE significantly suppressed retinalneovascularization and VEGF mRNA expression in a mousemodel of experimentalOIRMoreover CA is one of themajorcompounds present in AKE CA also exhibited a preventiveeffect against pathological retinal neovascularization Takentogether these results suggest that the inhibitory effect ofAKE on retinal neovascularization primarily stems from itspotent anti-VEGF activity and that its antiangiogenic activitymay in part be due to the bioactive compound CA
6 Evidence-Based Complementary and Alternative Medicine
00
05
10
15
Relat
ive V
EGF
mRN
A le
vels
(AU
)
OIR 25 50 25 50AKE (mgkg) CA (mgkg)
lowast
lowastlowast
lowast
Figure 5 The effect of AKE and CA on VEGF mRNA expressionin OIR mice Real-time PCR analysis of VEGF mRNA levels inOIR mice VEGF mRNAs were markedly reduced after AKE or CAtreatment The data are shown as the mean plusmn SEM 119899 = 7 lowast119901 lt 005versus OIR mice
VEGF is a potent angiogenic and vascular permeabilityfactor [23] that stimulates endothelial cell proliferation andangiogenesis In OIR mice VEGF expression was suppressedduring the hyperoxic phase (P7ndashP12) [24] Once hyper-oxia was terminated (P12ndashP17) hypoxia-driven upregulationof VEGF was observed even under normoxic conditions[25 26] Furthermore in ischemic retinopathy such asthat present in diabetic retinopathy and neovascular AMDthe robust upregulation of proangiogenic VEGF expressionleads to the activation of angiogenic signaling pathwaysand triggers neovascularization [27] Numerous studies havesuggested that VEGF has a key role in retinal vasculopa-thy and its inhibition significantly blocks the pathogenicalterations of retinal vasculature [28 29] Anti-VEGF agentswere recently reported to exhibit beneficial effects in patientswith proliferative diabetic retinopathy and neovascular AMD[2 30]
Medicinal herbs are rich sources of potential preven-tive and therapeutic agents AKE is a standardized dietaryherbal supplement To the best of our knowledge our studydemonstrated the antiangiogenic effects of AKE in vitro andin vivo for the first time Several studies have reported thatcertain crude herbal extracts and phytochemicals can inhibitpathogenic neovascularization in tumorigenesis [31 32] andretinal neovascular diseases [33ndash37] CA is the ester of caffeicacid with quinic acid and a major phenolic compound incoffee and various fruits [38] CA is also a major compoundpresent inAKE [18]This phytocompoundhas potent antioxi-dant [39] anti-inflammatory [40] and anticancer effects [41]Kim et al [42] reported that CA inhibited laser-inducedchoroidal neovascularization in a rat model of neovascularAMD More recently Park et al [43] showed that CAblocks hypoxia-stimulated angiogenesis in human vascularendothelial cells through inhibition of the hypoxia-induciblefactor-1120572AKT signaling pathway In the present study CAalso inhibited the VEGF-mediated tube formation of human
vascular endothelial cells and retinal neovascularization inmice with ischemic retinopathy Although the underlyingmechanism of action of AKE as a VEGF inhibitor remainsunclear it is hypothesized that the antiangiogenic activity ofAKE may be due to the antiantigenic effect of the bioactivecomponent CA
In conclusion this is the first study to provide evidencethat AKE and its bioactive compound CA inhibit exper-imental retinal neovascularization in ischemic retinopathyin vivo In addition in vitro studies showed that AKEand CA inhibit VEGF-induced tube formation in humanvascular endothelial cells Further studies may be required todetermine the feasibility of using AKE for the treatment ofpatients with ischemic retinopathy
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
Chan-Sik Kim and Jin Sook Kim contributed equally to thispaper as cocorrespondence authors
Acknowledgments
This research was supported by the Korea Institute of Plan-ning and Evaluation for Technology in Food AgricultureForestry and Fisheries (IPET) funded by the Ministry ofAgriculture Food and Rural Affairs (316023-05-2-CG000)and the Korea Institute of Oriental Medicine (K17270)
References
[1] R D Jager W F Mieler and J W Miller ldquoAge-related maculardegenerationrdquo The New England Journal of Medicine vol 358no 24 pp 2606ndash2617 2008
[2] P A Campochiaro ldquoOcular neovascularizationrdquo Journal ofMolecular Medicine vol 91 no 3 pp 311ndash321 2013
[3] L P Aiello ldquoVascular endothelial growth factor and the eyeBiochemical mechanisms of action and implications for noveltherapiesrdquoOphthalmic Research vol 29 no 5 pp 354ndash362 1997
[4] K Muranaka Y Yanagi Y Tamaki et al ldquoSuppression of laser-induced choroidal neovascularization by oral administration ofSA3443 in micerdquo FEBS Letters vol 579 no 27 pp 6084ndash60882005
[5] D RGuyer G Fish J AHaller et al ldquoAnti-vascular endothelialgrowth factor therapy for subfoveal choroidal neovasculariza-tion secondary to age-related macular degeneration Phase IIstudy resultsrdquoOphthalmology vol 110 no 5 pp 979ndash986 2003
[6] J E Frampton ldquoRanibizumab a review of its use in thetreatment of neovascular age-related macular degenerationrdquoDrugs amp Aging vol 30 no 5 pp 331ndash358 2013
[7] A Garcıa-Layana M S Figueroa J Araiz et al ldquoTreatmentof Exudative Age-related Macular Degeneration Focus onAfliberceptrdquo Drugs amp Aging vol 32 no 10 pp 797ndash807 2015
[8] C Campa and S P Harding ldquoAnti-VEGF compounds in thetreatment of neovascular age related macular degenerationrdquoCurrent Drug Targets vol 12 no 2 pp 173ndash181 2012
Evidence-Based Complementary and Alternative Medicine 7
[9] T Diago C AMcCannel S J Bakri J S Pulido A O Edwardsand J M Pach ldquoInfectious endophthalmitis after intravitrealinjection of antiangiogenic agentsrdquo Retina vol 29 no 5 pp601ndash605 2009
[10] D R Fintak G K Shah K J Blinder et al ldquoIncidenceof endophthalmitis related to intravitreal injection of beva-cizumab and ranibizumabrdquoRetina vol 28 no 10 pp 1395ndash13992008
[11] K Ghasemi Falavarjani and Q D Nguyen ldquoAdverse eventsand complications associated with intravitreal injection of anti-VEGF agents a review of literaturerdquo Eye vol 27 no 7 pp 787ndash794 2013
[12] H Takahashi Y Tamaki N Ishii et al ldquoIdentification of anovel vascular endothelial growth factor receptor 2 inhibitorand its effect for choroidal neovascularization in vivordquo CurrentEye Research vol 33 no 11-12 pp 1002ndash1010 2008
[13] S Honda T Nagai N Kondo et al ldquoTherapeutic effect of oralbisphosphonates on choroidal neovascularization in the humaneyerdquo Journal of Ophthalmology vol 2010 Article ID 206837 7pages 2010
[14] E L Meredith N Mainolfi S Poor et al ldquoDiscovery of oralVEGFR-2 inhibitors with prolonged ocular retention that areefficacious in models of wet age-related macular degenerationrdquoJournal of Medicinal Chemistry vol 58 no 23 pp 9273ndash92852015
[15] D K Ahn Illustrated Book of KoreanMedicinal Herbs Kyo-HakPublishing Seoul Republic of Korea 1998
[16] J Y KO and K K Lee ldquoEffect of plant growth regulators ongrowth and flowering of potted Lychnis cognata Aster koraien-sis and Campanula takesimanardquo RDA Journal of AgriculturalScience vol 38 pp 627ndash632 1996
[17] E Sohn J Kim C-S Kim Y S Kim D S Jang and J SKim ldquoExtract of the aerial parts of Aster koraiensis reduceddevelopment of diabetic nephropathy via anti-apoptosis ofpodocytes in streptozotocin-induced diabetic ratsrdquo Biochemicaland Biophysical Research Communications vol 391 no 1 pp733ndash738 2010
[18] J Kim K Jo I-S Lee C-S Kim and J S Kim ldquoThe extract ofaster koraiensis prevents retinal pericyte apoptosis in diabeticrats and its active compound chlorogenic acid inhibits ageformation and agerage interactionrdquo Nutrients vol 8 no 9article no 585 2016
[19] Y M Lee J Kim K Jo et al ldquoEthyl pyruvate inhibits reti-nal pathogenic neovascularization by downregulating HMGB1expressionrdquo Journal of Diabetes Research vol 2013 Article ID245271 8 pages 2013
[20] Y M Lee Y-R Lee C-S Kim et al ldquoCnidium officinaleextract and butylidenephthalide inhibits retinal neovasculariza-tion in vitro and in vivordquo BMC Complementary and AlternativeMedicine vol 16 no 1 article no 231 2016
[21] KMGehrs DHAnderson L V Johnson andG SHagemanldquoAge-related macular degenerationmdashemerging pathogeneticand therapeutic conceptsrdquo Annals of Medicine vol 38 no 7 pp450ndash471 2006
[22] P Van Wijngaarden and S H Qureshi ldquoInhibitors of vascularendothelial growth factor (VEGF) in the management of neo-vascular age-related macular degeneration A review of currentpracticerdquo Clinical and Experimental Optometry vol 91 no 5pp 427ndash437 2008
[23] D Shweiki A Itin D Soffer and E Keshet ldquoVascular endothe-lial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesisrdquo Nature vol 359 no 6398 pp 843ndash8451992
[24] J Stone T Chan-Ling J Persquoer A Itin H Gnessin and E KeshetldquoRoles of vascular endothelial growth factor and astrocytedegeneration in the genesis of retinopathy of prematurityrdquoInvestigative Ophthalmology amp Visual Science vol 37 no 2 pp290ndash299 1996
[25] N Ashton ldquoOxygen and the growth and development of retinalvessels In vivo and in vitro studies The XX Francis I ProctorLecturerdquo American Journal of Ophthalmology vol 62 no 3 pp412ndash435 1966
[26] N Ashton ldquoRetinal vascularization in health and diseaseproctor award lecture of the association for research in ophthal-mologyrdquo American Journal of Ophthalmology vol 44 no 4 pp7ndash17 1957
[27] A Hoeben B Landuyt M S Highley H Wildiers A T vanOosterom and E A de Bruijn ldquoVascular endothelial growthfactor and angiogenesisrdquo Pharmacological Reviews vol 56 no4 pp 549ndash580 2004
[28] L P Aiello R L Avery P G Arrigg et al ldquoVascular endothelialgrowth factor in ocular fluid of patients with diabetic retinopa-thy and other retinal disordersrdquo The New England Journal ofMedicine vol 331 no 22 pp 1480ndash1487 1994
[29] C K Dorey S Aouididi X Reynaud H F Dvorak and LF Brown ldquoCorrelation of vascular permeability factorvascularendothelial growth factor with extraretinal neovascularizationin the ratrdquo JAMA Ophtalmology vol 114 no 10 pp 1210ndash12171996
[30] D S Dhoot andR L Avery ldquoVascular endothelial growth factorinhibitors for diabetic retinopathyrdquo Current Diabetes Reportsvol 16 no 12 article no 122 2016
[31] I M W Ruma E W Putranto E Kondo et al ldquoExtract ofCordyceps militaris inhibits angiogenesis and suppresses tumorgrowth of human malignant melanoma cellsrdquo InternationalJournal of Oncology vol 45 no 1 pp 209ndash218 2014
[32] D R Yance Jr and S M Sagar ldquoTargeting angiogenesis withintegrative cancer therapiesrdquo Integrative Cancer Therapies vol5 no 1 pp 9ndash29 2006
[33] S K Gupta B Kumar B P Srinivasan et al ldquoRetinoprotectiveeffects of Moringa oleifera via antioxidant anti-inflammatoryand anti-angiogenic mechanisms in streptozotocin-induceddiabetic ratsrdquo Journal of Ocular Pharmacology andTherapeuticsvol 29 no 4 pp 419ndash426 2013
[34] J Tanaka S Nakamura K Tsuruma M Shimazawa H Shi-moda and H Hara ldquoPurple Rice (Oryza sativa L) extract andits constituents inhibit VEGF-induced angiogenesisrdquoPhytother-apy Research vol 26 no 2 pp 214ndash222 2012
[35] J Hua K I Guerin J Chen et al ldquoResveratrol inhibits patho-logic retinal neovascularization in Vldlr(minusminus) micerdquo Investiga-tive Ophthalmology amp Visual Science vol 52 no 5 pp 2809ndash2816 2011
[36] L Cao H Liu D S-C Lam G H-F Yam and C-P Pang ldquoInvitro screening for angiostatic potential of herbal chemicalsrdquoInvestigative Ophthalmology amp Visual Science vol 51 no 12 pp6658ndash6664 2010
[37] E Jung W Jung S Park C Kim J S Kim and J KimldquoEGHB010 a Standardized Extract of Paeoniae Radix andGlycyrrhizae Radix Inhibits VEGF-Induced Tube Formation
8 Evidence-Based Complementary and Alternative Medicine
In Vitro and Retinal Vascular Leakage and Choroidal Neo-vascularization In Vivordquo Evidence-Based Complementary andAlternative Medicine vol 2017 pp 1ndash7 2017
[38] M N Clifford K L Johnston S Knight and N KuhnertldquoHierarchical scheme for LC-MSn identification of chlorogenicacidsrdquo Journal of Agricultural and Food Chemistry vol 51 no10 pp 2900ndash2911 2003
[39] W-J Yen B-SWang L-WChang andP-DDuh ldquoAntioxidantproperties of roasted coffee residuesrdquo Journal of Agricultural andFood Chemistry vol 53 no 7 pp 2658ndash2663 2005
[40] T Krakauer ldquoThe polyphenol chlorogenic acid inhibitsstaphylococcal exotoxin-induced inflammatory cytokines andchemokinesrdquo Immunopharmacology and Immunotoxicologyvol 24 no 1 pp 113ndash119 2002
[41] C Kandaswami L T Lee P P Lee et al ldquoThe antitumoractivities of flavonoidsrdquo In Vivo vol 19 pp 895ndash909 2005
[42] C Kim H G Yu and J Sohn ldquoThe anti-angiogenic effectof chlorogenic acid on choroidal neovascularizationrdquo KoreanJournal of Ophthalmology vol 24 no 3 pp 163ndash168 2010
[43] J J Park S J Hwang J-H Park and H-J Lee ldquoChloro-genic acid inhibits hypoxia-induced angiogenesis via down-regulation of the HIF-1120572AKT pathwayrdquo Cellular Oncology vol38 no 2 pp 111ndash118 2015
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
4 Evidence-Based Complementary and Alternative Medicine
OIR AKE (25 mgkg) AKE (50mgkg)
CA (25 mgkg) CA (50mgkg)
(a)
Avas
cula
r are
aTo
tal a
rea o
f ret
ina (
)
OIR 25 50 25 50AKE (mgkg) CA (mgkg)
0
10
20
30
40
(b)
Figure 3The effect of AKE and CA on vascular obliteration of the central retina in OIR mice (a) The retinal blood vessels were visualized viafluorescein angiography using FITC-dextran (b) The quantification results are expressed as the percentage of the central nonperfused areawithin the total retinal area The bar graph values represent the mean plusmn SEM 119899 = 7 lowast119901 lt 005 versus OIR mice
of the extensive capillary-like networks of human vascularendothelial cells in a dose-dependent manner (Figure 2)Theinhibitory activity of CAwasmore potent comparedwith thatof AKE
321 AKE and CA Inhibit Retinal Neovascularization in OIRThemice subjected to ischemic retinopathy showed vascularobliteration of the central retina and pathogenic retinalneovascularization Newly formed neovascular tufts werevisualized by immunofluorescence staining with isolectin B4OIR mice treated with AKE or CA exhibited a significantdecrease in these retinal vascular changes that occur duringischemic retinopathy As presented in Figure 3 treatmentwith AKE or CA failed to induce significant changes in the
vascular obliteration of the central retina However AKEinhibited the formation of neovascular tufts by 2627 plusmn424 and 3875 plusmn 404 at doses of 25 and 50mgkgdayrespectively CA also inhibited retinal neovascularizationby 2968 plusmn 235 and 5024 plusmn 277 at doses of 25 and50mgkgday respectively (Figure 4) These results indicatedthat AKE and CA treatments significantly reduce the size ofneovascular tufts demonstrating thatCA is an antiangiogenicbioactive compound of AKE
322 AKE and CA Downregulate VEGF mRNA ExpressionTo examine the changes in VEGF expression in the retinawe measured the expression levels of VEGF mRNA usingreal-time PCR As predicted we observed a marked increase
Evidence-Based Complementary and Alternative Medicine 5
OIR AKE (25 mgkg) AKE (50mgkg)
CA (25 mgkg) CA (50mgkg)
(a)
OIR 25 50 25 50AKE (mgkg) CA (mgkg)
0
5
10
15
20
Neo
vasc
ular
tufts
area
()
lowast
lowastlowast
lowast
(b)
Figure 4The effect of AKE and CA on retinal neovascularization in OIR mice (a)The retinal neovascular tufts were visualized using isolectinB4 staining (b) Quantification results are expressed as neovascular tufts on the retina surface The bar graph values represent the mean plusmnSEM 119899 = 7 lowast119901 lt 005 versus OIR mice
in VEGF mRNA during ischemic retinopathy However theVEGFmRNA levels markedly decreased following treatmentwith AKE or CA in the OIR mice (Figure 5)
4 Discussion
Pathogenic angiogenesis is a primary cause of severe visionloss in several retinal degenerative diseases including dia-betic retinopathy and wet form AMD [21] VEGF and itsreceptors play an important role in the development of theseretinal disorders [3] and inhibiting angiogenesis by targetingVEGF has become a major focus in drug development [22]In the present study we aimed to evaluate the effect of AKE
on retinal neovascularization in a mouse model of OIRTo the best of our knowledge this study demonstrated forthe first time that AKE inhibits tube formation in humanvascular endothelial cells in vitro through a VEGF-mediatedmechanism In addition AKE significantly suppressed retinalneovascularization and VEGF mRNA expression in a mousemodel of experimentalOIRMoreover CA is one of themajorcompounds present in AKE CA also exhibited a preventiveeffect against pathological retinal neovascularization Takentogether these results suggest that the inhibitory effect ofAKE on retinal neovascularization primarily stems from itspotent anti-VEGF activity and that its antiangiogenic activitymay in part be due to the bioactive compound CA
6 Evidence-Based Complementary and Alternative Medicine
00
05
10
15
Relat
ive V
EGF
mRN
A le
vels
(AU
)
OIR 25 50 25 50AKE (mgkg) CA (mgkg)
lowast
lowastlowast
lowast
Figure 5 The effect of AKE and CA on VEGF mRNA expressionin OIR mice Real-time PCR analysis of VEGF mRNA levels inOIR mice VEGF mRNAs were markedly reduced after AKE or CAtreatment The data are shown as the mean plusmn SEM 119899 = 7 lowast119901 lt 005versus OIR mice
VEGF is a potent angiogenic and vascular permeabilityfactor [23] that stimulates endothelial cell proliferation andangiogenesis In OIR mice VEGF expression was suppressedduring the hyperoxic phase (P7ndashP12) [24] Once hyper-oxia was terminated (P12ndashP17) hypoxia-driven upregulationof VEGF was observed even under normoxic conditions[25 26] Furthermore in ischemic retinopathy such asthat present in diabetic retinopathy and neovascular AMDthe robust upregulation of proangiogenic VEGF expressionleads to the activation of angiogenic signaling pathwaysand triggers neovascularization [27] Numerous studies havesuggested that VEGF has a key role in retinal vasculopa-thy and its inhibition significantly blocks the pathogenicalterations of retinal vasculature [28 29] Anti-VEGF agentswere recently reported to exhibit beneficial effects in patientswith proliferative diabetic retinopathy and neovascular AMD[2 30]
Medicinal herbs are rich sources of potential preven-tive and therapeutic agents AKE is a standardized dietaryherbal supplement To the best of our knowledge our studydemonstrated the antiangiogenic effects of AKE in vitro andin vivo for the first time Several studies have reported thatcertain crude herbal extracts and phytochemicals can inhibitpathogenic neovascularization in tumorigenesis [31 32] andretinal neovascular diseases [33ndash37] CA is the ester of caffeicacid with quinic acid and a major phenolic compound incoffee and various fruits [38] CA is also a major compoundpresent inAKE [18]This phytocompoundhas potent antioxi-dant [39] anti-inflammatory [40] and anticancer effects [41]Kim et al [42] reported that CA inhibited laser-inducedchoroidal neovascularization in a rat model of neovascularAMD More recently Park et al [43] showed that CAblocks hypoxia-stimulated angiogenesis in human vascularendothelial cells through inhibition of the hypoxia-induciblefactor-1120572AKT signaling pathway In the present study CAalso inhibited the VEGF-mediated tube formation of human
vascular endothelial cells and retinal neovascularization inmice with ischemic retinopathy Although the underlyingmechanism of action of AKE as a VEGF inhibitor remainsunclear it is hypothesized that the antiangiogenic activity ofAKE may be due to the antiantigenic effect of the bioactivecomponent CA
In conclusion this is the first study to provide evidencethat AKE and its bioactive compound CA inhibit exper-imental retinal neovascularization in ischemic retinopathyin vivo In addition in vitro studies showed that AKEand CA inhibit VEGF-induced tube formation in humanvascular endothelial cells Further studies may be required todetermine the feasibility of using AKE for the treatment ofpatients with ischemic retinopathy
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
Chan-Sik Kim and Jin Sook Kim contributed equally to thispaper as cocorrespondence authors
Acknowledgments
This research was supported by the Korea Institute of Plan-ning and Evaluation for Technology in Food AgricultureForestry and Fisheries (IPET) funded by the Ministry ofAgriculture Food and Rural Affairs (316023-05-2-CG000)and the Korea Institute of Oriental Medicine (K17270)
References
[1] R D Jager W F Mieler and J W Miller ldquoAge-related maculardegenerationrdquo The New England Journal of Medicine vol 358no 24 pp 2606ndash2617 2008
[2] P A Campochiaro ldquoOcular neovascularizationrdquo Journal ofMolecular Medicine vol 91 no 3 pp 311ndash321 2013
[3] L P Aiello ldquoVascular endothelial growth factor and the eyeBiochemical mechanisms of action and implications for noveltherapiesrdquoOphthalmic Research vol 29 no 5 pp 354ndash362 1997
[4] K Muranaka Y Yanagi Y Tamaki et al ldquoSuppression of laser-induced choroidal neovascularization by oral administration ofSA3443 in micerdquo FEBS Letters vol 579 no 27 pp 6084ndash60882005
[5] D RGuyer G Fish J AHaller et al ldquoAnti-vascular endothelialgrowth factor therapy for subfoveal choroidal neovasculariza-tion secondary to age-related macular degeneration Phase IIstudy resultsrdquoOphthalmology vol 110 no 5 pp 979ndash986 2003
[6] J E Frampton ldquoRanibizumab a review of its use in thetreatment of neovascular age-related macular degenerationrdquoDrugs amp Aging vol 30 no 5 pp 331ndash358 2013
[7] A Garcıa-Layana M S Figueroa J Araiz et al ldquoTreatmentof Exudative Age-related Macular Degeneration Focus onAfliberceptrdquo Drugs amp Aging vol 32 no 10 pp 797ndash807 2015
[8] C Campa and S P Harding ldquoAnti-VEGF compounds in thetreatment of neovascular age related macular degenerationrdquoCurrent Drug Targets vol 12 no 2 pp 173ndash181 2012
Evidence-Based Complementary and Alternative Medicine 7
[9] T Diago C AMcCannel S J Bakri J S Pulido A O Edwardsand J M Pach ldquoInfectious endophthalmitis after intravitrealinjection of antiangiogenic agentsrdquo Retina vol 29 no 5 pp601ndash605 2009
[10] D R Fintak G K Shah K J Blinder et al ldquoIncidenceof endophthalmitis related to intravitreal injection of beva-cizumab and ranibizumabrdquoRetina vol 28 no 10 pp 1395ndash13992008
[11] K Ghasemi Falavarjani and Q D Nguyen ldquoAdverse eventsand complications associated with intravitreal injection of anti-VEGF agents a review of literaturerdquo Eye vol 27 no 7 pp 787ndash794 2013
[12] H Takahashi Y Tamaki N Ishii et al ldquoIdentification of anovel vascular endothelial growth factor receptor 2 inhibitorand its effect for choroidal neovascularization in vivordquo CurrentEye Research vol 33 no 11-12 pp 1002ndash1010 2008
[13] S Honda T Nagai N Kondo et al ldquoTherapeutic effect of oralbisphosphonates on choroidal neovascularization in the humaneyerdquo Journal of Ophthalmology vol 2010 Article ID 206837 7pages 2010
[14] E L Meredith N Mainolfi S Poor et al ldquoDiscovery of oralVEGFR-2 inhibitors with prolonged ocular retention that areefficacious in models of wet age-related macular degenerationrdquoJournal of Medicinal Chemistry vol 58 no 23 pp 9273ndash92852015
[15] D K Ahn Illustrated Book of KoreanMedicinal Herbs Kyo-HakPublishing Seoul Republic of Korea 1998
[16] J Y KO and K K Lee ldquoEffect of plant growth regulators ongrowth and flowering of potted Lychnis cognata Aster koraien-sis and Campanula takesimanardquo RDA Journal of AgriculturalScience vol 38 pp 627ndash632 1996
[17] E Sohn J Kim C-S Kim Y S Kim D S Jang and J SKim ldquoExtract of the aerial parts of Aster koraiensis reduceddevelopment of diabetic nephropathy via anti-apoptosis ofpodocytes in streptozotocin-induced diabetic ratsrdquo Biochemicaland Biophysical Research Communications vol 391 no 1 pp733ndash738 2010
[18] J Kim K Jo I-S Lee C-S Kim and J S Kim ldquoThe extract ofaster koraiensis prevents retinal pericyte apoptosis in diabeticrats and its active compound chlorogenic acid inhibits ageformation and agerage interactionrdquo Nutrients vol 8 no 9article no 585 2016
[19] Y M Lee J Kim K Jo et al ldquoEthyl pyruvate inhibits reti-nal pathogenic neovascularization by downregulating HMGB1expressionrdquo Journal of Diabetes Research vol 2013 Article ID245271 8 pages 2013
[20] Y M Lee Y-R Lee C-S Kim et al ldquoCnidium officinaleextract and butylidenephthalide inhibits retinal neovasculariza-tion in vitro and in vivordquo BMC Complementary and AlternativeMedicine vol 16 no 1 article no 231 2016
[21] KMGehrs DHAnderson L V Johnson andG SHagemanldquoAge-related macular degenerationmdashemerging pathogeneticand therapeutic conceptsrdquo Annals of Medicine vol 38 no 7 pp450ndash471 2006
[22] P Van Wijngaarden and S H Qureshi ldquoInhibitors of vascularendothelial growth factor (VEGF) in the management of neo-vascular age-related macular degeneration A review of currentpracticerdquo Clinical and Experimental Optometry vol 91 no 5pp 427ndash437 2008
[23] D Shweiki A Itin D Soffer and E Keshet ldquoVascular endothe-lial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesisrdquo Nature vol 359 no 6398 pp 843ndash8451992
[24] J Stone T Chan-Ling J Persquoer A Itin H Gnessin and E KeshetldquoRoles of vascular endothelial growth factor and astrocytedegeneration in the genesis of retinopathy of prematurityrdquoInvestigative Ophthalmology amp Visual Science vol 37 no 2 pp290ndash299 1996
[25] N Ashton ldquoOxygen and the growth and development of retinalvessels In vivo and in vitro studies The XX Francis I ProctorLecturerdquo American Journal of Ophthalmology vol 62 no 3 pp412ndash435 1966
[26] N Ashton ldquoRetinal vascularization in health and diseaseproctor award lecture of the association for research in ophthal-mologyrdquo American Journal of Ophthalmology vol 44 no 4 pp7ndash17 1957
[27] A Hoeben B Landuyt M S Highley H Wildiers A T vanOosterom and E A de Bruijn ldquoVascular endothelial growthfactor and angiogenesisrdquo Pharmacological Reviews vol 56 no4 pp 549ndash580 2004
[28] L P Aiello R L Avery P G Arrigg et al ldquoVascular endothelialgrowth factor in ocular fluid of patients with diabetic retinopa-thy and other retinal disordersrdquo The New England Journal ofMedicine vol 331 no 22 pp 1480ndash1487 1994
[29] C K Dorey S Aouididi X Reynaud H F Dvorak and LF Brown ldquoCorrelation of vascular permeability factorvascularendothelial growth factor with extraretinal neovascularizationin the ratrdquo JAMA Ophtalmology vol 114 no 10 pp 1210ndash12171996
[30] D S Dhoot andR L Avery ldquoVascular endothelial growth factorinhibitors for diabetic retinopathyrdquo Current Diabetes Reportsvol 16 no 12 article no 122 2016
[31] I M W Ruma E W Putranto E Kondo et al ldquoExtract ofCordyceps militaris inhibits angiogenesis and suppresses tumorgrowth of human malignant melanoma cellsrdquo InternationalJournal of Oncology vol 45 no 1 pp 209ndash218 2014
[32] D R Yance Jr and S M Sagar ldquoTargeting angiogenesis withintegrative cancer therapiesrdquo Integrative Cancer Therapies vol5 no 1 pp 9ndash29 2006
[33] S K Gupta B Kumar B P Srinivasan et al ldquoRetinoprotectiveeffects of Moringa oleifera via antioxidant anti-inflammatoryand anti-angiogenic mechanisms in streptozotocin-induceddiabetic ratsrdquo Journal of Ocular Pharmacology andTherapeuticsvol 29 no 4 pp 419ndash426 2013
[34] J Tanaka S Nakamura K Tsuruma M Shimazawa H Shi-moda and H Hara ldquoPurple Rice (Oryza sativa L) extract andits constituents inhibit VEGF-induced angiogenesisrdquoPhytother-apy Research vol 26 no 2 pp 214ndash222 2012
[35] J Hua K I Guerin J Chen et al ldquoResveratrol inhibits patho-logic retinal neovascularization in Vldlr(minusminus) micerdquo Investiga-tive Ophthalmology amp Visual Science vol 52 no 5 pp 2809ndash2816 2011
[36] L Cao H Liu D S-C Lam G H-F Yam and C-P Pang ldquoInvitro screening for angiostatic potential of herbal chemicalsrdquoInvestigative Ophthalmology amp Visual Science vol 51 no 12 pp6658ndash6664 2010
[37] E Jung W Jung S Park C Kim J S Kim and J KimldquoEGHB010 a Standardized Extract of Paeoniae Radix andGlycyrrhizae Radix Inhibits VEGF-Induced Tube Formation
8 Evidence-Based Complementary and Alternative Medicine
In Vitro and Retinal Vascular Leakage and Choroidal Neo-vascularization In Vivordquo Evidence-Based Complementary andAlternative Medicine vol 2017 pp 1ndash7 2017
[38] M N Clifford K L Johnston S Knight and N KuhnertldquoHierarchical scheme for LC-MSn identification of chlorogenicacidsrdquo Journal of Agricultural and Food Chemistry vol 51 no10 pp 2900ndash2911 2003
[39] W-J Yen B-SWang L-WChang andP-DDuh ldquoAntioxidantproperties of roasted coffee residuesrdquo Journal of Agricultural andFood Chemistry vol 53 no 7 pp 2658ndash2663 2005
[40] T Krakauer ldquoThe polyphenol chlorogenic acid inhibitsstaphylococcal exotoxin-induced inflammatory cytokines andchemokinesrdquo Immunopharmacology and Immunotoxicologyvol 24 no 1 pp 113ndash119 2002
[41] C Kandaswami L T Lee P P Lee et al ldquoThe antitumoractivities of flavonoidsrdquo In Vivo vol 19 pp 895ndash909 2005
[42] C Kim H G Yu and J Sohn ldquoThe anti-angiogenic effectof chlorogenic acid on choroidal neovascularizationrdquo KoreanJournal of Ophthalmology vol 24 no 3 pp 163ndash168 2010
[43] J J Park S J Hwang J-H Park and H-J Lee ldquoChloro-genic acid inhibits hypoxia-induced angiogenesis via down-regulation of the HIF-1120572AKT pathwayrdquo Cellular Oncology vol38 no 2 pp 111ndash118 2015
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 5
OIR AKE (25 mgkg) AKE (50mgkg)
CA (25 mgkg) CA (50mgkg)
(a)
OIR 25 50 25 50AKE (mgkg) CA (mgkg)
0
5
10
15
20
Neo
vasc
ular
tufts
area
()
lowast
lowastlowast
lowast
(b)
Figure 4The effect of AKE and CA on retinal neovascularization in OIR mice (a)The retinal neovascular tufts were visualized using isolectinB4 staining (b) Quantification results are expressed as neovascular tufts on the retina surface The bar graph values represent the mean plusmnSEM 119899 = 7 lowast119901 lt 005 versus OIR mice
in VEGF mRNA during ischemic retinopathy However theVEGFmRNA levels markedly decreased following treatmentwith AKE or CA in the OIR mice (Figure 5)
4 Discussion
Pathogenic angiogenesis is a primary cause of severe visionloss in several retinal degenerative diseases including dia-betic retinopathy and wet form AMD [21] VEGF and itsreceptors play an important role in the development of theseretinal disorders [3] and inhibiting angiogenesis by targetingVEGF has become a major focus in drug development [22]In the present study we aimed to evaluate the effect of AKE
on retinal neovascularization in a mouse model of OIRTo the best of our knowledge this study demonstrated forthe first time that AKE inhibits tube formation in humanvascular endothelial cells in vitro through a VEGF-mediatedmechanism In addition AKE significantly suppressed retinalneovascularization and VEGF mRNA expression in a mousemodel of experimentalOIRMoreover CA is one of themajorcompounds present in AKE CA also exhibited a preventiveeffect against pathological retinal neovascularization Takentogether these results suggest that the inhibitory effect ofAKE on retinal neovascularization primarily stems from itspotent anti-VEGF activity and that its antiangiogenic activitymay in part be due to the bioactive compound CA
6 Evidence-Based Complementary and Alternative Medicine
00
05
10
15
Relat
ive V
EGF
mRN
A le
vels
(AU
)
OIR 25 50 25 50AKE (mgkg) CA (mgkg)
lowast
lowastlowast
lowast
Figure 5 The effect of AKE and CA on VEGF mRNA expressionin OIR mice Real-time PCR analysis of VEGF mRNA levels inOIR mice VEGF mRNAs were markedly reduced after AKE or CAtreatment The data are shown as the mean plusmn SEM 119899 = 7 lowast119901 lt 005versus OIR mice
VEGF is a potent angiogenic and vascular permeabilityfactor [23] that stimulates endothelial cell proliferation andangiogenesis In OIR mice VEGF expression was suppressedduring the hyperoxic phase (P7ndashP12) [24] Once hyper-oxia was terminated (P12ndashP17) hypoxia-driven upregulationof VEGF was observed even under normoxic conditions[25 26] Furthermore in ischemic retinopathy such asthat present in diabetic retinopathy and neovascular AMDthe robust upregulation of proangiogenic VEGF expressionleads to the activation of angiogenic signaling pathwaysand triggers neovascularization [27] Numerous studies havesuggested that VEGF has a key role in retinal vasculopa-thy and its inhibition significantly blocks the pathogenicalterations of retinal vasculature [28 29] Anti-VEGF agentswere recently reported to exhibit beneficial effects in patientswith proliferative diabetic retinopathy and neovascular AMD[2 30]
Medicinal herbs are rich sources of potential preven-tive and therapeutic agents AKE is a standardized dietaryherbal supplement To the best of our knowledge our studydemonstrated the antiangiogenic effects of AKE in vitro andin vivo for the first time Several studies have reported thatcertain crude herbal extracts and phytochemicals can inhibitpathogenic neovascularization in tumorigenesis [31 32] andretinal neovascular diseases [33ndash37] CA is the ester of caffeicacid with quinic acid and a major phenolic compound incoffee and various fruits [38] CA is also a major compoundpresent inAKE [18]This phytocompoundhas potent antioxi-dant [39] anti-inflammatory [40] and anticancer effects [41]Kim et al [42] reported that CA inhibited laser-inducedchoroidal neovascularization in a rat model of neovascularAMD More recently Park et al [43] showed that CAblocks hypoxia-stimulated angiogenesis in human vascularendothelial cells through inhibition of the hypoxia-induciblefactor-1120572AKT signaling pathway In the present study CAalso inhibited the VEGF-mediated tube formation of human
vascular endothelial cells and retinal neovascularization inmice with ischemic retinopathy Although the underlyingmechanism of action of AKE as a VEGF inhibitor remainsunclear it is hypothesized that the antiangiogenic activity ofAKE may be due to the antiantigenic effect of the bioactivecomponent CA
In conclusion this is the first study to provide evidencethat AKE and its bioactive compound CA inhibit exper-imental retinal neovascularization in ischemic retinopathyin vivo In addition in vitro studies showed that AKEand CA inhibit VEGF-induced tube formation in humanvascular endothelial cells Further studies may be required todetermine the feasibility of using AKE for the treatment ofpatients with ischemic retinopathy
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
Chan-Sik Kim and Jin Sook Kim contributed equally to thispaper as cocorrespondence authors
Acknowledgments
This research was supported by the Korea Institute of Plan-ning and Evaluation for Technology in Food AgricultureForestry and Fisheries (IPET) funded by the Ministry ofAgriculture Food and Rural Affairs (316023-05-2-CG000)and the Korea Institute of Oriental Medicine (K17270)
References
[1] R D Jager W F Mieler and J W Miller ldquoAge-related maculardegenerationrdquo The New England Journal of Medicine vol 358no 24 pp 2606ndash2617 2008
[2] P A Campochiaro ldquoOcular neovascularizationrdquo Journal ofMolecular Medicine vol 91 no 3 pp 311ndash321 2013
[3] L P Aiello ldquoVascular endothelial growth factor and the eyeBiochemical mechanisms of action and implications for noveltherapiesrdquoOphthalmic Research vol 29 no 5 pp 354ndash362 1997
[4] K Muranaka Y Yanagi Y Tamaki et al ldquoSuppression of laser-induced choroidal neovascularization by oral administration ofSA3443 in micerdquo FEBS Letters vol 579 no 27 pp 6084ndash60882005
[5] D RGuyer G Fish J AHaller et al ldquoAnti-vascular endothelialgrowth factor therapy for subfoveal choroidal neovasculariza-tion secondary to age-related macular degeneration Phase IIstudy resultsrdquoOphthalmology vol 110 no 5 pp 979ndash986 2003
[6] J E Frampton ldquoRanibizumab a review of its use in thetreatment of neovascular age-related macular degenerationrdquoDrugs amp Aging vol 30 no 5 pp 331ndash358 2013
[7] A Garcıa-Layana M S Figueroa J Araiz et al ldquoTreatmentof Exudative Age-related Macular Degeneration Focus onAfliberceptrdquo Drugs amp Aging vol 32 no 10 pp 797ndash807 2015
[8] C Campa and S P Harding ldquoAnti-VEGF compounds in thetreatment of neovascular age related macular degenerationrdquoCurrent Drug Targets vol 12 no 2 pp 173ndash181 2012
Evidence-Based Complementary and Alternative Medicine 7
[9] T Diago C AMcCannel S J Bakri J S Pulido A O Edwardsand J M Pach ldquoInfectious endophthalmitis after intravitrealinjection of antiangiogenic agentsrdquo Retina vol 29 no 5 pp601ndash605 2009
[10] D R Fintak G K Shah K J Blinder et al ldquoIncidenceof endophthalmitis related to intravitreal injection of beva-cizumab and ranibizumabrdquoRetina vol 28 no 10 pp 1395ndash13992008
[11] K Ghasemi Falavarjani and Q D Nguyen ldquoAdverse eventsand complications associated with intravitreal injection of anti-VEGF agents a review of literaturerdquo Eye vol 27 no 7 pp 787ndash794 2013
[12] H Takahashi Y Tamaki N Ishii et al ldquoIdentification of anovel vascular endothelial growth factor receptor 2 inhibitorand its effect for choroidal neovascularization in vivordquo CurrentEye Research vol 33 no 11-12 pp 1002ndash1010 2008
[13] S Honda T Nagai N Kondo et al ldquoTherapeutic effect of oralbisphosphonates on choroidal neovascularization in the humaneyerdquo Journal of Ophthalmology vol 2010 Article ID 206837 7pages 2010
[14] E L Meredith N Mainolfi S Poor et al ldquoDiscovery of oralVEGFR-2 inhibitors with prolonged ocular retention that areefficacious in models of wet age-related macular degenerationrdquoJournal of Medicinal Chemistry vol 58 no 23 pp 9273ndash92852015
[15] D K Ahn Illustrated Book of KoreanMedicinal Herbs Kyo-HakPublishing Seoul Republic of Korea 1998
[16] J Y KO and K K Lee ldquoEffect of plant growth regulators ongrowth and flowering of potted Lychnis cognata Aster koraien-sis and Campanula takesimanardquo RDA Journal of AgriculturalScience vol 38 pp 627ndash632 1996
[17] E Sohn J Kim C-S Kim Y S Kim D S Jang and J SKim ldquoExtract of the aerial parts of Aster koraiensis reduceddevelopment of diabetic nephropathy via anti-apoptosis ofpodocytes in streptozotocin-induced diabetic ratsrdquo Biochemicaland Biophysical Research Communications vol 391 no 1 pp733ndash738 2010
[18] J Kim K Jo I-S Lee C-S Kim and J S Kim ldquoThe extract ofaster koraiensis prevents retinal pericyte apoptosis in diabeticrats and its active compound chlorogenic acid inhibits ageformation and agerage interactionrdquo Nutrients vol 8 no 9article no 585 2016
[19] Y M Lee J Kim K Jo et al ldquoEthyl pyruvate inhibits reti-nal pathogenic neovascularization by downregulating HMGB1expressionrdquo Journal of Diabetes Research vol 2013 Article ID245271 8 pages 2013
[20] Y M Lee Y-R Lee C-S Kim et al ldquoCnidium officinaleextract and butylidenephthalide inhibits retinal neovasculariza-tion in vitro and in vivordquo BMC Complementary and AlternativeMedicine vol 16 no 1 article no 231 2016
[21] KMGehrs DHAnderson L V Johnson andG SHagemanldquoAge-related macular degenerationmdashemerging pathogeneticand therapeutic conceptsrdquo Annals of Medicine vol 38 no 7 pp450ndash471 2006
[22] P Van Wijngaarden and S H Qureshi ldquoInhibitors of vascularendothelial growth factor (VEGF) in the management of neo-vascular age-related macular degeneration A review of currentpracticerdquo Clinical and Experimental Optometry vol 91 no 5pp 427ndash437 2008
[23] D Shweiki A Itin D Soffer and E Keshet ldquoVascular endothe-lial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesisrdquo Nature vol 359 no 6398 pp 843ndash8451992
[24] J Stone T Chan-Ling J Persquoer A Itin H Gnessin and E KeshetldquoRoles of vascular endothelial growth factor and astrocytedegeneration in the genesis of retinopathy of prematurityrdquoInvestigative Ophthalmology amp Visual Science vol 37 no 2 pp290ndash299 1996
[25] N Ashton ldquoOxygen and the growth and development of retinalvessels In vivo and in vitro studies The XX Francis I ProctorLecturerdquo American Journal of Ophthalmology vol 62 no 3 pp412ndash435 1966
[26] N Ashton ldquoRetinal vascularization in health and diseaseproctor award lecture of the association for research in ophthal-mologyrdquo American Journal of Ophthalmology vol 44 no 4 pp7ndash17 1957
[27] A Hoeben B Landuyt M S Highley H Wildiers A T vanOosterom and E A de Bruijn ldquoVascular endothelial growthfactor and angiogenesisrdquo Pharmacological Reviews vol 56 no4 pp 549ndash580 2004
[28] L P Aiello R L Avery P G Arrigg et al ldquoVascular endothelialgrowth factor in ocular fluid of patients with diabetic retinopa-thy and other retinal disordersrdquo The New England Journal ofMedicine vol 331 no 22 pp 1480ndash1487 1994
[29] C K Dorey S Aouididi X Reynaud H F Dvorak and LF Brown ldquoCorrelation of vascular permeability factorvascularendothelial growth factor with extraretinal neovascularizationin the ratrdquo JAMA Ophtalmology vol 114 no 10 pp 1210ndash12171996
[30] D S Dhoot andR L Avery ldquoVascular endothelial growth factorinhibitors for diabetic retinopathyrdquo Current Diabetes Reportsvol 16 no 12 article no 122 2016
[31] I M W Ruma E W Putranto E Kondo et al ldquoExtract ofCordyceps militaris inhibits angiogenesis and suppresses tumorgrowth of human malignant melanoma cellsrdquo InternationalJournal of Oncology vol 45 no 1 pp 209ndash218 2014
[32] D R Yance Jr and S M Sagar ldquoTargeting angiogenesis withintegrative cancer therapiesrdquo Integrative Cancer Therapies vol5 no 1 pp 9ndash29 2006
[33] S K Gupta B Kumar B P Srinivasan et al ldquoRetinoprotectiveeffects of Moringa oleifera via antioxidant anti-inflammatoryand anti-angiogenic mechanisms in streptozotocin-induceddiabetic ratsrdquo Journal of Ocular Pharmacology andTherapeuticsvol 29 no 4 pp 419ndash426 2013
[34] J Tanaka S Nakamura K Tsuruma M Shimazawa H Shi-moda and H Hara ldquoPurple Rice (Oryza sativa L) extract andits constituents inhibit VEGF-induced angiogenesisrdquoPhytother-apy Research vol 26 no 2 pp 214ndash222 2012
[35] J Hua K I Guerin J Chen et al ldquoResveratrol inhibits patho-logic retinal neovascularization in Vldlr(minusminus) micerdquo Investiga-tive Ophthalmology amp Visual Science vol 52 no 5 pp 2809ndash2816 2011
[36] L Cao H Liu D S-C Lam G H-F Yam and C-P Pang ldquoInvitro screening for angiostatic potential of herbal chemicalsrdquoInvestigative Ophthalmology amp Visual Science vol 51 no 12 pp6658ndash6664 2010
[37] E Jung W Jung S Park C Kim J S Kim and J KimldquoEGHB010 a Standardized Extract of Paeoniae Radix andGlycyrrhizae Radix Inhibits VEGF-Induced Tube Formation
8 Evidence-Based Complementary and Alternative Medicine
In Vitro and Retinal Vascular Leakage and Choroidal Neo-vascularization In Vivordquo Evidence-Based Complementary andAlternative Medicine vol 2017 pp 1ndash7 2017
[38] M N Clifford K L Johnston S Knight and N KuhnertldquoHierarchical scheme for LC-MSn identification of chlorogenicacidsrdquo Journal of Agricultural and Food Chemistry vol 51 no10 pp 2900ndash2911 2003
[39] W-J Yen B-SWang L-WChang andP-DDuh ldquoAntioxidantproperties of roasted coffee residuesrdquo Journal of Agricultural andFood Chemistry vol 53 no 7 pp 2658ndash2663 2005
[40] T Krakauer ldquoThe polyphenol chlorogenic acid inhibitsstaphylococcal exotoxin-induced inflammatory cytokines andchemokinesrdquo Immunopharmacology and Immunotoxicologyvol 24 no 1 pp 113ndash119 2002
[41] C Kandaswami L T Lee P P Lee et al ldquoThe antitumoractivities of flavonoidsrdquo In Vivo vol 19 pp 895ndash909 2005
[42] C Kim H G Yu and J Sohn ldquoThe anti-angiogenic effectof chlorogenic acid on choroidal neovascularizationrdquo KoreanJournal of Ophthalmology vol 24 no 3 pp 163ndash168 2010
[43] J J Park S J Hwang J-H Park and H-J Lee ldquoChloro-genic acid inhibits hypoxia-induced angiogenesis via down-regulation of the HIF-1120572AKT pathwayrdquo Cellular Oncology vol38 no 2 pp 111ndash118 2015
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
6 Evidence-Based Complementary and Alternative Medicine
00
05
10
15
Relat
ive V
EGF
mRN
A le
vels
(AU
)
OIR 25 50 25 50AKE (mgkg) CA (mgkg)
lowast
lowastlowast
lowast
Figure 5 The effect of AKE and CA on VEGF mRNA expressionin OIR mice Real-time PCR analysis of VEGF mRNA levels inOIR mice VEGF mRNAs were markedly reduced after AKE or CAtreatment The data are shown as the mean plusmn SEM 119899 = 7 lowast119901 lt 005versus OIR mice
VEGF is a potent angiogenic and vascular permeabilityfactor [23] that stimulates endothelial cell proliferation andangiogenesis In OIR mice VEGF expression was suppressedduring the hyperoxic phase (P7ndashP12) [24] Once hyper-oxia was terminated (P12ndashP17) hypoxia-driven upregulationof VEGF was observed even under normoxic conditions[25 26] Furthermore in ischemic retinopathy such asthat present in diabetic retinopathy and neovascular AMDthe robust upregulation of proangiogenic VEGF expressionleads to the activation of angiogenic signaling pathwaysand triggers neovascularization [27] Numerous studies havesuggested that VEGF has a key role in retinal vasculopa-thy and its inhibition significantly blocks the pathogenicalterations of retinal vasculature [28 29] Anti-VEGF agentswere recently reported to exhibit beneficial effects in patientswith proliferative diabetic retinopathy and neovascular AMD[2 30]
Medicinal herbs are rich sources of potential preven-tive and therapeutic agents AKE is a standardized dietaryherbal supplement To the best of our knowledge our studydemonstrated the antiangiogenic effects of AKE in vitro andin vivo for the first time Several studies have reported thatcertain crude herbal extracts and phytochemicals can inhibitpathogenic neovascularization in tumorigenesis [31 32] andretinal neovascular diseases [33ndash37] CA is the ester of caffeicacid with quinic acid and a major phenolic compound incoffee and various fruits [38] CA is also a major compoundpresent inAKE [18]This phytocompoundhas potent antioxi-dant [39] anti-inflammatory [40] and anticancer effects [41]Kim et al [42] reported that CA inhibited laser-inducedchoroidal neovascularization in a rat model of neovascularAMD More recently Park et al [43] showed that CAblocks hypoxia-stimulated angiogenesis in human vascularendothelial cells through inhibition of the hypoxia-induciblefactor-1120572AKT signaling pathway In the present study CAalso inhibited the VEGF-mediated tube formation of human
vascular endothelial cells and retinal neovascularization inmice with ischemic retinopathy Although the underlyingmechanism of action of AKE as a VEGF inhibitor remainsunclear it is hypothesized that the antiangiogenic activity ofAKE may be due to the antiantigenic effect of the bioactivecomponent CA
In conclusion this is the first study to provide evidencethat AKE and its bioactive compound CA inhibit exper-imental retinal neovascularization in ischemic retinopathyin vivo In addition in vitro studies showed that AKEand CA inhibit VEGF-induced tube formation in humanvascular endothelial cells Further studies may be required todetermine the feasibility of using AKE for the treatment ofpatients with ischemic retinopathy
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
Chan-Sik Kim and Jin Sook Kim contributed equally to thispaper as cocorrespondence authors
Acknowledgments
This research was supported by the Korea Institute of Plan-ning and Evaluation for Technology in Food AgricultureForestry and Fisheries (IPET) funded by the Ministry ofAgriculture Food and Rural Affairs (316023-05-2-CG000)and the Korea Institute of Oriental Medicine (K17270)
References
[1] R D Jager W F Mieler and J W Miller ldquoAge-related maculardegenerationrdquo The New England Journal of Medicine vol 358no 24 pp 2606ndash2617 2008
[2] P A Campochiaro ldquoOcular neovascularizationrdquo Journal ofMolecular Medicine vol 91 no 3 pp 311ndash321 2013
[3] L P Aiello ldquoVascular endothelial growth factor and the eyeBiochemical mechanisms of action and implications for noveltherapiesrdquoOphthalmic Research vol 29 no 5 pp 354ndash362 1997
[4] K Muranaka Y Yanagi Y Tamaki et al ldquoSuppression of laser-induced choroidal neovascularization by oral administration ofSA3443 in micerdquo FEBS Letters vol 579 no 27 pp 6084ndash60882005
[5] D RGuyer G Fish J AHaller et al ldquoAnti-vascular endothelialgrowth factor therapy for subfoveal choroidal neovasculariza-tion secondary to age-related macular degeneration Phase IIstudy resultsrdquoOphthalmology vol 110 no 5 pp 979ndash986 2003
[6] J E Frampton ldquoRanibizumab a review of its use in thetreatment of neovascular age-related macular degenerationrdquoDrugs amp Aging vol 30 no 5 pp 331ndash358 2013
[7] A Garcıa-Layana M S Figueroa J Araiz et al ldquoTreatmentof Exudative Age-related Macular Degeneration Focus onAfliberceptrdquo Drugs amp Aging vol 32 no 10 pp 797ndash807 2015
[8] C Campa and S P Harding ldquoAnti-VEGF compounds in thetreatment of neovascular age related macular degenerationrdquoCurrent Drug Targets vol 12 no 2 pp 173ndash181 2012
Evidence-Based Complementary and Alternative Medicine 7
[9] T Diago C AMcCannel S J Bakri J S Pulido A O Edwardsand J M Pach ldquoInfectious endophthalmitis after intravitrealinjection of antiangiogenic agentsrdquo Retina vol 29 no 5 pp601ndash605 2009
[10] D R Fintak G K Shah K J Blinder et al ldquoIncidenceof endophthalmitis related to intravitreal injection of beva-cizumab and ranibizumabrdquoRetina vol 28 no 10 pp 1395ndash13992008
[11] K Ghasemi Falavarjani and Q D Nguyen ldquoAdverse eventsand complications associated with intravitreal injection of anti-VEGF agents a review of literaturerdquo Eye vol 27 no 7 pp 787ndash794 2013
[12] H Takahashi Y Tamaki N Ishii et al ldquoIdentification of anovel vascular endothelial growth factor receptor 2 inhibitorand its effect for choroidal neovascularization in vivordquo CurrentEye Research vol 33 no 11-12 pp 1002ndash1010 2008
[13] S Honda T Nagai N Kondo et al ldquoTherapeutic effect of oralbisphosphonates on choroidal neovascularization in the humaneyerdquo Journal of Ophthalmology vol 2010 Article ID 206837 7pages 2010
[14] E L Meredith N Mainolfi S Poor et al ldquoDiscovery of oralVEGFR-2 inhibitors with prolonged ocular retention that areefficacious in models of wet age-related macular degenerationrdquoJournal of Medicinal Chemistry vol 58 no 23 pp 9273ndash92852015
[15] D K Ahn Illustrated Book of KoreanMedicinal Herbs Kyo-HakPublishing Seoul Republic of Korea 1998
[16] J Y KO and K K Lee ldquoEffect of plant growth regulators ongrowth and flowering of potted Lychnis cognata Aster koraien-sis and Campanula takesimanardquo RDA Journal of AgriculturalScience vol 38 pp 627ndash632 1996
[17] E Sohn J Kim C-S Kim Y S Kim D S Jang and J SKim ldquoExtract of the aerial parts of Aster koraiensis reduceddevelopment of diabetic nephropathy via anti-apoptosis ofpodocytes in streptozotocin-induced diabetic ratsrdquo Biochemicaland Biophysical Research Communications vol 391 no 1 pp733ndash738 2010
[18] J Kim K Jo I-S Lee C-S Kim and J S Kim ldquoThe extract ofaster koraiensis prevents retinal pericyte apoptosis in diabeticrats and its active compound chlorogenic acid inhibits ageformation and agerage interactionrdquo Nutrients vol 8 no 9article no 585 2016
[19] Y M Lee J Kim K Jo et al ldquoEthyl pyruvate inhibits reti-nal pathogenic neovascularization by downregulating HMGB1expressionrdquo Journal of Diabetes Research vol 2013 Article ID245271 8 pages 2013
[20] Y M Lee Y-R Lee C-S Kim et al ldquoCnidium officinaleextract and butylidenephthalide inhibits retinal neovasculariza-tion in vitro and in vivordquo BMC Complementary and AlternativeMedicine vol 16 no 1 article no 231 2016
[21] KMGehrs DHAnderson L V Johnson andG SHagemanldquoAge-related macular degenerationmdashemerging pathogeneticand therapeutic conceptsrdquo Annals of Medicine vol 38 no 7 pp450ndash471 2006
[22] P Van Wijngaarden and S H Qureshi ldquoInhibitors of vascularendothelial growth factor (VEGF) in the management of neo-vascular age-related macular degeneration A review of currentpracticerdquo Clinical and Experimental Optometry vol 91 no 5pp 427ndash437 2008
[23] D Shweiki A Itin D Soffer and E Keshet ldquoVascular endothe-lial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesisrdquo Nature vol 359 no 6398 pp 843ndash8451992
[24] J Stone T Chan-Ling J Persquoer A Itin H Gnessin and E KeshetldquoRoles of vascular endothelial growth factor and astrocytedegeneration in the genesis of retinopathy of prematurityrdquoInvestigative Ophthalmology amp Visual Science vol 37 no 2 pp290ndash299 1996
[25] N Ashton ldquoOxygen and the growth and development of retinalvessels In vivo and in vitro studies The XX Francis I ProctorLecturerdquo American Journal of Ophthalmology vol 62 no 3 pp412ndash435 1966
[26] N Ashton ldquoRetinal vascularization in health and diseaseproctor award lecture of the association for research in ophthal-mologyrdquo American Journal of Ophthalmology vol 44 no 4 pp7ndash17 1957
[27] A Hoeben B Landuyt M S Highley H Wildiers A T vanOosterom and E A de Bruijn ldquoVascular endothelial growthfactor and angiogenesisrdquo Pharmacological Reviews vol 56 no4 pp 549ndash580 2004
[28] L P Aiello R L Avery P G Arrigg et al ldquoVascular endothelialgrowth factor in ocular fluid of patients with diabetic retinopa-thy and other retinal disordersrdquo The New England Journal ofMedicine vol 331 no 22 pp 1480ndash1487 1994
[29] C K Dorey S Aouididi X Reynaud H F Dvorak and LF Brown ldquoCorrelation of vascular permeability factorvascularendothelial growth factor with extraretinal neovascularizationin the ratrdquo JAMA Ophtalmology vol 114 no 10 pp 1210ndash12171996
[30] D S Dhoot andR L Avery ldquoVascular endothelial growth factorinhibitors for diabetic retinopathyrdquo Current Diabetes Reportsvol 16 no 12 article no 122 2016
[31] I M W Ruma E W Putranto E Kondo et al ldquoExtract ofCordyceps militaris inhibits angiogenesis and suppresses tumorgrowth of human malignant melanoma cellsrdquo InternationalJournal of Oncology vol 45 no 1 pp 209ndash218 2014
[32] D R Yance Jr and S M Sagar ldquoTargeting angiogenesis withintegrative cancer therapiesrdquo Integrative Cancer Therapies vol5 no 1 pp 9ndash29 2006
[33] S K Gupta B Kumar B P Srinivasan et al ldquoRetinoprotectiveeffects of Moringa oleifera via antioxidant anti-inflammatoryand anti-angiogenic mechanisms in streptozotocin-induceddiabetic ratsrdquo Journal of Ocular Pharmacology andTherapeuticsvol 29 no 4 pp 419ndash426 2013
[34] J Tanaka S Nakamura K Tsuruma M Shimazawa H Shi-moda and H Hara ldquoPurple Rice (Oryza sativa L) extract andits constituents inhibit VEGF-induced angiogenesisrdquoPhytother-apy Research vol 26 no 2 pp 214ndash222 2012
[35] J Hua K I Guerin J Chen et al ldquoResveratrol inhibits patho-logic retinal neovascularization in Vldlr(minusminus) micerdquo Investiga-tive Ophthalmology amp Visual Science vol 52 no 5 pp 2809ndash2816 2011
[36] L Cao H Liu D S-C Lam G H-F Yam and C-P Pang ldquoInvitro screening for angiostatic potential of herbal chemicalsrdquoInvestigative Ophthalmology amp Visual Science vol 51 no 12 pp6658ndash6664 2010
[37] E Jung W Jung S Park C Kim J S Kim and J KimldquoEGHB010 a Standardized Extract of Paeoniae Radix andGlycyrrhizae Radix Inhibits VEGF-Induced Tube Formation
8 Evidence-Based Complementary and Alternative Medicine
In Vitro and Retinal Vascular Leakage and Choroidal Neo-vascularization In Vivordquo Evidence-Based Complementary andAlternative Medicine vol 2017 pp 1ndash7 2017
[38] M N Clifford K L Johnston S Knight and N KuhnertldquoHierarchical scheme for LC-MSn identification of chlorogenicacidsrdquo Journal of Agricultural and Food Chemistry vol 51 no10 pp 2900ndash2911 2003
[39] W-J Yen B-SWang L-WChang andP-DDuh ldquoAntioxidantproperties of roasted coffee residuesrdquo Journal of Agricultural andFood Chemistry vol 53 no 7 pp 2658ndash2663 2005
[40] T Krakauer ldquoThe polyphenol chlorogenic acid inhibitsstaphylococcal exotoxin-induced inflammatory cytokines andchemokinesrdquo Immunopharmacology and Immunotoxicologyvol 24 no 1 pp 113ndash119 2002
[41] C Kandaswami L T Lee P P Lee et al ldquoThe antitumoractivities of flavonoidsrdquo In Vivo vol 19 pp 895ndash909 2005
[42] C Kim H G Yu and J Sohn ldquoThe anti-angiogenic effectof chlorogenic acid on choroidal neovascularizationrdquo KoreanJournal of Ophthalmology vol 24 no 3 pp 163ndash168 2010
[43] J J Park S J Hwang J-H Park and H-J Lee ldquoChloro-genic acid inhibits hypoxia-induced angiogenesis via down-regulation of the HIF-1120572AKT pathwayrdquo Cellular Oncology vol38 no 2 pp 111ndash118 2015
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 7
[9] T Diago C AMcCannel S J Bakri J S Pulido A O Edwardsand J M Pach ldquoInfectious endophthalmitis after intravitrealinjection of antiangiogenic agentsrdquo Retina vol 29 no 5 pp601ndash605 2009
[10] D R Fintak G K Shah K J Blinder et al ldquoIncidenceof endophthalmitis related to intravitreal injection of beva-cizumab and ranibizumabrdquoRetina vol 28 no 10 pp 1395ndash13992008
[11] K Ghasemi Falavarjani and Q D Nguyen ldquoAdverse eventsand complications associated with intravitreal injection of anti-VEGF agents a review of literaturerdquo Eye vol 27 no 7 pp 787ndash794 2013
[12] H Takahashi Y Tamaki N Ishii et al ldquoIdentification of anovel vascular endothelial growth factor receptor 2 inhibitorand its effect for choroidal neovascularization in vivordquo CurrentEye Research vol 33 no 11-12 pp 1002ndash1010 2008
[13] S Honda T Nagai N Kondo et al ldquoTherapeutic effect of oralbisphosphonates on choroidal neovascularization in the humaneyerdquo Journal of Ophthalmology vol 2010 Article ID 206837 7pages 2010
[14] E L Meredith N Mainolfi S Poor et al ldquoDiscovery of oralVEGFR-2 inhibitors with prolonged ocular retention that areefficacious in models of wet age-related macular degenerationrdquoJournal of Medicinal Chemistry vol 58 no 23 pp 9273ndash92852015
[15] D K Ahn Illustrated Book of KoreanMedicinal Herbs Kyo-HakPublishing Seoul Republic of Korea 1998
[16] J Y KO and K K Lee ldquoEffect of plant growth regulators ongrowth and flowering of potted Lychnis cognata Aster koraien-sis and Campanula takesimanardquo RDA Journal of AgriculturalScience vol 38 pp 627ndash632 1996
[17] E Sohn J Kim C-S Kim Y S Kim D S Jang and J SKim ldquoExtract of the aerial parts of Aster koraiensis reduceddevelopment of diabetic nephropathy via anti-apoptosis ofpodocytes in streptozotocin-induced diabetic ratsrdquo Biochemicaland Biophysical Research Communications vol 391 no 1 pp733ndash738 2010
[18] J Kim K Jo I-S Lee C-S Kim and J S Kim ldquoThe extract ofaster koraiensis prevents retinal pericyte apoptosis in diabeticrats and its active compound chlorogenic acid inhibits ageformation and agerage interactionrdquo Nutrients vol 8 no 9article no 585 2016
[19] Y M Lee J Kim K Jo et al ldquoEthyl pyruvate inhibits reti-nal pathogenic neovascularization by downregulating HMGB1expressionrdquo Journal of Diabetes Research vol 2013 Article ID245271 8 pages 2013
[20] Y M Lee Y-R Lee C-S Kim et al ldquoCnidium officinaleextract and butylidenephthalide inhibits retinal neovasculariza-tion in vitro and in vivordquo BMC Complementary and AlternativeMedicine vol 16 no 1 article no 231 2016
[21] KMGehrs DHAnderson L V Johnson andG SHagemanldquoAge-related macular degenerationmdashemerging pathogeneticand therapeutic conceptsrdquo Annals of Medicine vol 38 no 7 pp450ndash471 2006
[22] P Van Wijngaarden and S H Qureshi ldquoInhibitors of vascularendothelial growth factor (VEGF) in the management of neo-vascular age-related macular degeneration A review of currentpracticerdquo Clinical and Experimental Optometry vol 91 no 5pp 427ndash437 2008
[23] D Shweiki A Itin D Soffer and E Keshet ldquoVascular endothe-lial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesisrdquo Nature vol 359 no 6398 pp 843ndash8451992
[24] J Stone T Chan-Ling J Persquoer A Itin H Gnessin and E KeshetldquoRoles of vascular endothelial growth factor and astrocytedegeneration in the genesis of retinopathy of prematurityrdquoInvestigative Ophthalmology amp Visual Science vol 37 no 2 pp290ndash299 1996
[25] N Ashton ldquoOxygen and the growth and development of retinalvessels In vivo and in vitro studies The XX Francis I ProctorLecturerdquo American Journal of Ophthalmology vol 62 no 3 pp412ndash435 1966
[26] N Ashton ldquoRetinal vascularization in health and diseaseproctor award lecture of the association for research in ophthal-mologyrdquo American Journal of Ophthalmology vol 44 no 4 pp7ndash17 1957
[27] A Hoeben B Landuyt M S Highley H Wildiers A T vanOosterom and E A de Bruijn ldquoVascular endothelial growthfactor and angiogenesisrdquo Pharmacological Reviews vol 56 no4 pp 549ndash580 2004
[28] L P Aiello R L Avery P G Arrigg et al ldquoVascular endothelialgrowth factor in ocular fluid of patients with diabetic retinopa-thy and other retinal disordersrdquo The New England Journal ofMedicine vol 331 no 22 pp 1480ndash1487 1994
[29] C K Dorey S Aouididi X Reynaud H F Dvorak and LF Brown ldquoCorrelation of vascular permeability factorvascularendothelial growth factor with extraretinal neovascularizationin the ratrdquo JAMA Ophtalmology vol 114 no 10 pp 1210ndash12171996
[30] D S Dhoot andR L Avery ldquoVascular endothelial growth factorinhibitors for diabetic retinopathyrdquo Current Diabetes Reportsvol 16 no 12 article no 122 2016
[31] I M W Ruma E W Putranto E Kondo et al ldquoExtract ofCordyceps militaris inhibits angiogenesis and suppresses tumorgrowth of human malignant melanoma cellsrdquo InternationalJournal of Oncology vol 45 no 1 pp 209ndash218 2014
[32] D R Yance Jr and S M Sagar ldquoTargeting angiogenesis withintegrative cancer therapiesrdquo Integrative Cancer Therapies vol5 no 1 pp 9ndash29 2006
[33] S K Gupta B Kumar B P Srinivasan et al ldquoRetinoprotectiveeffects of Moringa oleifera via antioxidant anti-inflammatoryand anti-angiogenic mechanisms in streptozotocin-induceddiabetic ratsrdquo Journal of Ocular Pharmacology andTherapeuticsvol 29 no 4 pp 419ndash426 2013
[34] J Tanaka S Nakamura K Tsuruma M Shimazawa H Shi-moda and H Hara ldquoPurple Rice (Oryza sativa L) extract andits constituents inhibit VEGF-induced angiogenesisrdquoPhytother-apy Research vol 26 no 2 pp 214ndash222 2012
[35] J Hua K I Guerin J Chen et al ldquoResveratrol inhibits patho-logic retinal neovascularization in Vldlr(minusminus) micerdquo Investiga-tive Ophthalmology amp Visual Science vol 52 no 5 pp 2809ndash2816 2011
[36] L Cao H Liu D S-C Lam G H-F Yam and C-P Pang ldquoInvitro screening for angiostatic potential of herbal chemicalsrdquoInvestigative Ophthalmology amp Visual Science vol 51 no 12 pp6658ndash6664 2010
[37] E Jung W Jung S Park C Kim J S Kim and J KimldquoEGHB010 a Standardized Extract of Paeoniae Radix andGlycyrrhizae Radix Inhibits VEGF-Induced Tube Formation
8 Evidence-Based Complementary and Alternative Medicine
In Vitro and Retinal Vascular Leakage and Choroidal Neo-vascularization In Vivordquo Evidence-Based Complementary andAlternative Medicine vol 2017 pp 1ndash7 2017
[38] M N Clifford K L Johnston S Knight and N KuhnertldquoHierarchical scheme for LC-MSn identification of chlorogenicacidsrdquo Journal of Agricultural and Food Chemistry vol 51 no10 pp 2900ndash2911 2003
[39] W-J Yen B-SWang L-WChang andP-DDuh ldquoAntioxidantproperties of roasted coffee residuesrdquo Journal of Agricultural andFood Chemistry vol 53 no 7 pp 2658ndash2663 2005
[40] T Krakauer ldquoThe polyphenol chlorogenic acid inhibitsstaphylococcal exotoxin-induced inflammatory cytokines andchemokinesrdquo Immunopharmacology and Immunotoxicologyvol 24 no 1 pp 113ndash119 2002
[41] C Kandaswami L T Lee P P Lee et al ldquoThe antitumoractivities of flavonoidsrdquo In Vivo vol 19 pp 895ndash909 2005
[42] C Kim H G Yu and J Sohn ldquoThe anti-angiogenic effectof chlorogenic acid on choroidal neovascularizationrdquo KoreanJournal of Ophthalmology vol 24 no 3 pp 163ndash168 2010
[43] J J Park S J Hwang J-H Park and H-J Lee ldquoChloro-genic acid inhibits hypoxia-induced angiogenesis via down-regulation of the HIF-1120572AKT pathwayrdquo Cellular Oncology vol38 no 2 pp 111ndash118 2015
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
8 Evidence-Based Complementary and Alternative Medicine
In Vitro and Retinal Vascular Leakage and Choroidal Neo-vascularization In Vivordquo Evidence-Based Complementary andAlternative Medicine vol 2017 pp 1ndash7 2017
[38] M N Clifford K L Johnston S Knight and N KuhnertldquoHierarchical scheme for LC-MSn identification of chlorogenicacidsrdquo Journal of Agricultural and Food Chemistry vol 51 no10 pp 2900ndash2911 2003
[39] W-J Yen B-SWang L-WChang andP-DDuh ldquoAntioxidantproperties of roasted coffee residuesrdquo Journal of Agricultural andFood Chemistry vol 53 no 7 pp 2658ndash2663 2005
[40] T Krakauer ldquoThe polyphenol chlorogenic acid inhibitsstaphylococcal exotoxin-induced inflammatory cytokines andchemokinesrdquo Immunopharmacology and Immunotoxicologyvol 24 no 1 pp 113ndash119 2002
[41] C Kandaswami L T Lee P P Lee et al ldquoThe antitumoractivities of flavonoidsrdquo In Vivo vol 19 pp 895ndash909 2005
[42] C Kim H G Yu and J Sohn ldquoThe anti-angiogenic effectof chlorogenic acid on choroidal neovascularizationrdquo KoreanJournal of Ophthalmology vol 24 no 3 pp 163ndash168 2010
[43] J J Park S J Hwang J-H Park and H-J Lee ldquoChloro-genic acid inhibits hypoxia-induced angiogenesis via down-regulation of the HIF-1120572AKT pathwayrdquo Cellular Oncology vol38 no 2 pp 111ndash118 2015
![Page 1: ResearchArticle Aster koraiensis Extract and Chlorogenic ...downloads.hindawi.com/journals/ecam/2018/6402650.pdf · chronic bronchitis, diabetes, and pertussis [, ]. In our priorstudies,wereportedthattheextractof](https://reader033.documents.pub/reader033/viewer/2022060815/609484b22c4be851be11f4e3/html5/thumbnails/1.jpg)
![Page 2: ResearchArticle Aster koraiensis Extract and Chlorogenic ...downloads.hindawi.com/journals/ecam/2018/6402650.pdf · chronic bronchitis, diabetes, and pertussis [, ]. In our priorstudies,wereportedthattheextractof](https://reader033.documents.pub/reader033/viewer/2022060815/609484b22c4be851be11f4e3/html5/thumbnails/2.jpg)
![Page 3: ResearchArticle Aster koraiensis Extract and Chlorogenic ...downloads.hindawi.com/journals/ecam/2018/6402650.pdf · chronic bronchitis, diabetes, and pertussis [, ]. In our priorstudies,wereportedthattheextractof](https://reader033.documents.pub/reader033/viewer/2022060815/609484b22c4be851be11f4e3/html5/thumbnails/3.jpg)
![Page 4: ResearchArticle Aster koraiensis Extract and Chlorogenic ...downloads.hindawi.com/journals/ecam/2018/6402650.pdf · chronic bronchitis, diabetes, and pertussis [, ]. In our priorstudies,wereportedthattheextractof](https://reader033.documents.pub/reader033/viewer/2022060815/609484b22c4be851be11f4e3/html5/thumbnails/4.jpg)
![Page 5: ResearchArticle Aster koraiensis Extract and Chlorogenic ...downloads.hindawi.com/journals/ecam/2018/6402650.pdf · chronic bronchitis, diabetes, and pertussis [, ]. In our priorstudies,wereportedthattheextractof](https://reader033.documents.pub/reader033/viewer/2022060815/609484b22c4be851be11f4e3/html5/thumbnails/5.jpg)
![Page 6: ResearchArticle Aster koraiensis Extract and Chlorogenic ...downloads.hindawi.com/journals/ecam/2018/6402650.pdf · chronic bronchitis, diabetes, and pertussis [, ]. In our priorstudies,wereportedthattheextractof](https://reader033.documents.pub/reader033/viewer/2022060815/609484b22c4be851be11f4e3/html5/thumbnails/6.jpg)
![Page 7: ResearchArticle Aster koraiensis Extract and Chlorogenic ...downloads.hindawi.com/journals/ecam/2018/6402650.pdf · chronic bronchitis, diabetes, and pertussis [, ]. In our priorstudies,wereportedthattheextractof](https://reader033.documents.pub/reader033/viewer/2022060815/609484b22c4be851be11f4e3/html5/thumbnails/7.jpg)
![Page 8: ResearchArticle Aster koraiensis Extract and Chlorogenic ...downloads.hindawi.com/journals/ecam/2018/6402650.pdf · chronic bronchitis, diabetes, and pertussis [, ]. In our priorstudies,wereportedthattheextractof](https://reader033.documents.pub/reader033/viewer/2022060815/609484b22c4be851be11f4e3/html5/thumbnails/8.jpg)
![Page 9: ResearchArticle Aster koraiensis Extract and Chlorogenic ...downloads.hindawi.com/journals/ecam/2018/6402650.pdf · chronic bronchitis, diabetes, and pertussis [, ]. In our priorstudies,wereportedthattheextractof](https://reader033.documents.pub/reader033/viewer/2022060815/609484b22c4be851be11f4e3/html5/thumbnails/9.jpg)
