Basic Research—Biology

Iloprost Up-regulates Vascular Endothelial Growth FactorExpression in Human Dental Pulp Cells In Vitro andEnhances Pulpal Blood Flow In VivoChalida Nakalekha Limjeerajarus, DDS, PhD,*† Thanaphum Osathanon, DDS, PhD,†‡

Jeeranan Manokawinchoke, MSc,‡ and Prasit Pavasant, DDS, PhD†‡

Abstract

Introduction: Prostacyclin (PGI2) is a biomoleculecapable of enhancing angiogenesis and cellular prolifera-tion. Methods: We investigated the influence of a PGI2analogue (iloprost) on dental pulp revascularizationin vitro and in vivo by using human dental pulp cells(HDPCs) and a rat tooth injury model, respectively. Ilo-prost stimulated the human dental pulp cell mRNAexpression of vascular endothelial growth factor(VEGF), fibroblast growth factor-2 (FGF-2), andplatelet-derived growth factor (PDGF) in a significantdose-dependent manner. This mRNA up-regulation wassignificantly inhibited by pretreatment with a PGI2 recep-tor antagonist and forskolin (a protein kinase A activator).In contrast, a protein kinase A inhibitor significantlyenhanced the iloprost-induced mRNA expression ofVEGF, FGF-2, and PDGF. Pretreatment with a fibro-blast growth factor receptor inhibitor attenuated theVEGF, FGF-2, and PDGF mRNA expression, indicatingopposing regulatory mechanisms. Results: The effect ofiloprost on the dental pulp was investigated in vivo byusing a rat molar pulp injury model. The iloprost-treatedgroup exhibited a significant increase in pulpal bloodflow at 72 hours compared with control. Conclusions:The present study indicates that iloprost may be a candi-date agent to promote neovascularization in dental pulptissue, suggesting the potential clinical use of iloprost invital pulp therapy. (J Endod 2014;-:1–6)

Key WordsDental pulp, prostacyclin, pulpal blood flow, vascularendothelial growth factor

From the *Department of Physiology, †Mineralized TissueResearch Unit, and ‡Department of Anatomy, Faculty ofDentistry, Chulalongkorn University, Bangkok, Thailand.

Address requests for reprints to Dr Chalida Nakalekha Lim-jeerajarus, Department of Physiology, Faculty of Dentistry, Chu-lalongkorn University, Henri-Dunant Road, Bangkok 10330,Thailand. E-mail address: kandychula@yahoo.com0099-2399/$ - see front matter

Copyright ª 2014 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2013.10.025

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Prostacyclin (PGI2), a powerful physiologic vasodilating agent, is known for its abilityto enhance angiogenesis and cell proliferation. PGI2 is also involved in bone remod-

eling (1, 2). Prostacyclin synthase-knockout animals exhibited an increase in bonemass (1). Osteoblasts produce PGI2 in response to growth factors, and both osteoblastsand osteocytes release PGI2 in response to mechanical loading (3, 4). Several studieshave reported that PGI2 induced vascular endothelial growth factor (VEGF) expressionin many cell types (5, 6). PGI2 stimulated VEGF release from fibroblasts and increasedthe proliferation of endothelial cells. In addition, the exogenous administration of PGI2resulted in an increase in VEGF production by monocytes, dendritic cells, andmacrophages (5, 6). Thus, the stimulation of VEGF expression by PGI2 could lead toan increase in endothelial cell proliferation and angiogenesis.

PGI2 is unstable; thus a synthetic PGI2 with greater chemical stability has been intro-duced and used clinically. Iloprost, a long-acting analogue of PGI2, is widely prescribedto treat pulmonary hypertension (7, 8). Iloprost is also used clinically to prevent bonenecrosis (9). It has also been shown that the levels of circulating endothelial cells andtheir progenitors were increased after iloprost infusion in patients (10). In addition,iloprost treatment enhanced ICAM-1, BCL2, and VEGF expression in these cells (10).Together, these data indicate the promotive effect of iloprost on angiogenesis.

Maintaining tooth vitality is extremely important in dental pulp treatment. Recently,advances in tissue engineering and stem cell research have led to the discovery of apotential treatment for pulp/dentin revascularization and regeneration (11). The keyto success in vital pulp therapy is to ensure that the dental pulp is in a healthy environmentwith excellent blood supply (11). We hypothesized that iloprost might be used to pro-mote angiogenesis in the dental pulp. The aims of the present study were to identifythe effects and mechanisms of exogenously administered iloprost on the expression ofangiogenic factors in vitro and to evaluate the effect of iloprost on dental pulpal bloodflow in vivo.

Materials and MethodsHuman Dental Pulp Cell Isolation and Culture

The human dental pulp cell (HDPC) isolation protocol was approved by the EthicsCommittee of the Faculty of Dentistry, Chulalongkorn University (HREC-DCU2012-024). Freshly extracted teeth were collected from healthy adults (age, 18–30years). The HDPCs were prepared as previously described (12, 13). Cells frompassages 3–5 were used in the study. All experiments were performed in triplicatewith cells obtained from 3 different donors.

PGI2 Analogue TreatmentHDPCs (3 � 105 cells) were seeded in 6-well plates and maintained in normal

growth medium for 24 hours. The cells were then cultured in serum-free mediumand subsequently treated with various dosages of iloprost (Ilomedin; Bayer AG, Leverku-sen, Germany) (7, 14). Cell viability was determined by using methyl-thiazol-diphenyltetrazolium assay (Sigma-Aldrich, St Louis, MO). The mRNA expression ofVEGF, fibroblast growth factor-2 (FGF-2), and platelet-derived growth factor (PDGF)was determined by real-time quantitative polymerase chain reaction (qPCR). The culture

loprost Up-regulates VEGF Expression in HDPCs, Enhances Pulpal Blood Flow 1

Figure 1. HDPC morphology (A) and viability (B) after iloprost treatment for 24 hours. VEGF protein levels were determined by ELISA (C). The mRNA expression ofVEGF, FGF-2, and PDGF was evaluated by using real-time qPCR at 24 hours after iloprost treatment (D–F). The time-dependent mRNA expression was examined at 30minutes and 1 hour of exposure to iloprost at a concentration of 10�6 mol/L (G–I). Asterisks indicate statistically significant difference compared with the control.

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medium was collected and evaluated for VEGF and cyclic adenosinemonophosphate (cAMP) protein secretion by using enzyme-linkedimmunosorbent assay (ELISA) after 24 hours of iloprost treatment.

HDPCs were pretreated for 30 minutes before iloprost treatmentwith the following chemical inhibitors: prostacyclin receptor (IP)antagonist CAY10449 (Cayman Chemical Company, Ann Arbor, MI),fibroblast growth factor receptor (FGFR) inhibitor SU5402 (Sigma-Aldrich) protein kinase A (PKA) inhibitor (Calbiochem, Darmstadt,Germany), or forskolin (Santa Cruz Biotechnology, Santa Cruz, CA).The concentrations of inhibitors were 3 nmol/L, 20 mmol/L, 10mmol/L, and 10mmol/L for IP antagonist, FGFR inhibitor, PKA inhibitor,and forskolin, respectively.

Real-time qPCR and ELISATotal cellular RNA was extracted with Trizol reagent (Roche Diag-

nostics, Indianapolis, IN). RNA samples (1mg) were converted to cDNAby using reverse transcriptase (Promega, Madison, WI) with a LightCy-cler 480 SYBR Green I Master kit (Roche Diagnostics). The amplifica-tion profile was 95�C/10 s, 60�C/10 s, and 72�C/20 s for 40 cycles. Thereaction product was quantified by using GAPDH as the referencegene. The primer sequences used were VEGF forward 50-atgagga-

2 Limjeerajarus et al.

caccggctctgacca-30, reverse 50-aggctcctgaatcttccaggca-30; FGF-2 for-ward 50-ggcttcttcctgcgcatccac-30, reverse 50-ggtaacggttagcacacactcctt-30; PDGF forward 50-tcaggtgggttagagatggagt-30, reverse 50-gaaaggaacca-gaggaagaggt-30; GAPDH forward 50-cactgccaacgtgtcagtggtg-30, reverse50-gtagcccaggatgcccttgag-30.

The culture medium was collected and assayed to quantify theVEGF and cAMP protein levels by using ELISA kits (R & D Systems, Min-neapolis, MN).

Rat Molar Mechanical Pulp Injury ModelThe protocol was approved by the Animal Ethics Committee of the

Faculty of Dentistry, Chulalongkorn University (Protocol number1132005). Twenty-eight–week–old, male Wistar rats were used inthe present study. To create the mechanical injury, the upper first molarwas prepared (15). In brief, the cavity was made on the mesial surfaceby slow-speed motor, followed by mechanical exposure into the pulpchamber. The upper left first molars received iloprost treatment afterpulp exposure. The treatment group was divided into 4 subgroups (n= 4 each group): iloprost at concentrations of 10�6, 10�7, or 10�8

mol/L and calcium hydroxide (Ca[OH]2). After application of iloprost,the cavity was restored by using glass ionomer cement (Fuji, GC, Tokyo,

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Figure 2. Iloprost-induced VEGF, FGF-2, and PDGFmRNA expression was abolished after IP receptor antagonist (CAY 10449) pretreatment (A–C). The cAMP levelswere determined by ELISA after treatment with iloprost or combination of iloprost and IP receptor antagonist (D). Effect of iloprost (10�6 mol/L) on proliferation ofHDPCs was determined at 24 and 72 hours with or without the presence of CAY10449. Data are presented as fold increase normalized to the control (no treatment) at 24hours (E). Asterisks indicate statistically significant difference compared with the control. Bars indicate statistically significant difference between those 2 groups.

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Japan). The upper right first molars served as controls (exposurewithout treatment).

Measuring Levels of Blood FlowAfter the operation, the blood flow rate of each sample was

measured for 5minutes by using laser Doppler flowmetry (Moor Instru-ments, Axminster, UK). Mean pulpal blood flow (PBF) was recorded asblood perfusion units immediately after restoration and at 24 hours and72 hours. The laser probe was positioned at the mesial surface of thetooth. The probe was stabilized by using a clear polyvinyl tube to main-tain the probe-tooth distance at 2 mm.

Statistical AnalysesThe data are shown as mean � standard deviation. Independent

Student t test was performed for 2 independent samples comparison.A 1-way analysis of variance followed by Tukey honestly significant dif-ference test was used to compare each experimental group with the con-trol group in the experiments containing 3 or more groups. Differencesat P < .05 were considered to be statistically significant.

ResultsIloprost Promoted VEGF, FGF-2, and PDGF mRNAExpression

Cell viability was evaluated after treating HDPCs for 24 hours withiloprost. The iloprost-treated groups exhibited HDPCmorphology similarto that of the control group (Fig. 1A). A significant dose-dependent in-crease in cell number was seen in the iloprost-treated groups(Fig. 1B). After 24 hours, iloprost treatment dramatically up-regulatedthe mRNA levels of VEGF, FGF-2, and PDGF in a dose-dependent manner(Fig. 1D–F). The time-course study revealed a significant up-regulation ofthese genes as early as 1 hour after iloprost treatment (Fig. 1G–I). VEGFprotein levels increased in a dose-dependent manner, corresponding tothe mRNA expression pattern (Fig. 1C).

The effect of iloprost on gene expression was further investigatedby using an IP receptor antagonist (CAY10449). The results indicated

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that pretreating HDPCs with CAY10449 inhibited the iloprost-inducedVEGF, FGF-2, and PDGF mRNA expression (Fig. 2A–C), confirmingthe effect of iloprost on the regulation of these genes. Furthermore,the effect of iloprost on cell proliferation at 24 and 72 hours wasalso suppressed by CAY10449 (Fig. 2E).

Iloprost Induced VEGF, FGF-2, and PDGF via cAMP,but not the PKA Signaling Pathway

After exposing the HDPCs to iloprost for 24 hours, cAMP levelswere markedly increased (Fig. 2D). IP antagonist attenuated thecAMP increase, suggesting that iloprost binding to the IP receptorregulates cAMP levels (Fig. 2D).

To further dissect the downstream pathway of iloprost-inducedgene expression, HDPCs were pretreated with a PKA inhibitor. ThePKA inhibitor significantly enhanced the inductive effect of iloproston VEGF, FGF-2, and PDGF mRNA expression (Fig. 3A–C). After pre-treating the HDPCs with forskolin, which is a known PKA activator, theiloprost-induced VEGF, FGF-2, and PDGF mRNA expression wasmarkedly reduced (Fig. 3D–F). To explore the participation of theFGFR in the expression of these genes, an FGFR inhibitor was used.Pretreating the HDPCs with FGFR resulted in a significant reductionof VEGF, FGF-2, and PDGF mRNA levels compared with the control(Fig. 3G–I).

Iloprost Increased Blood Flow in a Dental Pulp InjuryModel In Vivo

A rat molar mechanical pulp exposure model was used to investi-gate the effect of iloprost on blood flow in vivo. Iloprost at concentra-tions of 10�6, 10�7, or 10�8 M was used in the in vivo study. Theiloprost-treated groups showed a significant increase in PBF in adose-dependent manner at 72 hours after injury, indicating the effectof iloprost on PBF as compared with Ca(OH)2 (Fig. 4B). No significantdifferences were found in PBF when assayed immediately and 24 hoursafter iloprost application.

loprost Up-regulates VEGF Expression in HDPCs, Enhances Pulpal Blood Flow 3

Figure 3. VEGF, FGF-2, and PDGF mRNA expression was determined by using real-time qPCR with PKA inhibitor (A–C), forskolin (D–F), and FGFR inhibitor(G–I) pretreatment before iloprost exposure. Bars indicate statistically significant difference between those 2 groups.

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DiscussionIn the present study, we investigated the influence of iloprost

(a PGI2 analogue) on angiogenic gene expression by HDPCs and bloodflow in the dental pulp. We found that iloprost induced VEGF, FGF-2,and PDGF mRNA expression via the IP receptor and cAMP signalingpathway. In contrast, PKA activation reduced the iloprost-inducedVEGF, FGF-2, and PDGF mRNA expression in HDPCs. Moreover, theresults illustrated that iloprost could enhance PBF after a mechanicalinjury in a rat molar model.

VEGF, FGF-2, and PDGF are known stimulators of angiogenesisand neovascularization in damaged or traumatized tissues (16, 17).These molecules were found in the dentin matrix and pulp tissue andmay promote angiogenesis in the dental pulp by enhancingchemotaxis, cell proliferation, and cell differentiation in dental pulpcells (18, 19). Previously, it was demonstrated that the VEGFexpression in human dental pulp was up-regulated on stimulating thecells with various agents, for example, hypoxia mimicking chemicalagents and histone deacetylase (20, 21). In the present study, weshowed that iloprost could up-regulate these proangiogenic moleculesin HDPCs, which could further lead to the enhancement of PBF in theinjured dental pulp area. Increased angiogenesis and blood flow wouldbe beneficial to the dentin/pulp healing process.

4 Limjeerajarus et al.

When considering the intracellular signaling mechanisms underly-ing our observations, it has been previously shown that PGI2up-regulated cAMP-dependent PKA through the IP receptor. PGI2-induced cAMP also activated phospholipase Cg and diacylglycerollipase, resulting in the generation of arachidonic acid and intracellularcalcium elevation (22, 23). However, our data indicated that iloprost-induced VEGF, FGF-2, and PDGF mRNA expression was positively regu-lated through IP receptor/cAMP signaling and negatively controlled bythe PKA signaling pathway. We demonstrated that forskolin could inhibitthe iloprost-induced VEGF, FGF-2, and PDGFmRNA expression, whereaspretreatment with a PKA inhibitor enhanced their expression. Incontrast, a previous study showed that in dendritic cells, forskolin treat-ment resulted in an interleukin-10 expression pattern similar to that ofiloprost treatment (5). The conflicting results could be due to the use ofdifferent cell types and may imply that iloprost uses different signalingpathways in regulating the expression of different cytokines or growthfactors.

In the present study, we found that VEGF, FGF-2, and PDGF expres-sion was also regulated via FGFR signaling. A more detailed study of thecrosstalk between PGI2 signaling and the FGF receptor pathway shouldbe performed. One potential mechanism is PGI2 mediated peroxisomeproliferator-activated receptor (PPAR) activation. Therefore, the

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Figure 4. Mechanical dental pulp exposure in the rat molar model (A). PBF was measured after iloprost application for 72 hours (B). Asterisks indicate statisticallysignificant difference compared with the control. The potential intracellular mechanism of iloprost-induced mRNA expression (C). BPU, blood perfusion units.

Basic Research—Biology

response to PGI2 may occur via both IP and PPAR in certain cell types(24–27). Our proposed intracellular signaling pathway in iloprost-treated HDPCs is seen in Figure 4C.

The process of dental pulp repair begins with neovascularizationand proceeds with the proliferation, migration, and subsequent differ-entiation of precursor cells into odontoblast-like cells (19, 28). In thepresent study, we determined the influence of iloprost on PBF in vivo.The PBF in the iloprost-treated group was significantly increased 72hours after iloprost application compared with the control, whereasno significant difference was noted at the earlier time points. Thetime lapse between the application of iloprost and the PBF response im-plies that the increase in PBF resulted from iloprost-induced angiogen-esis, which requires longer time (29).

On the basis of our results, we propose that iloprost-induced up-regulation of VEGF, FGF-2, and PDGF can increase vascularization in thedental pulp and may lead to the enhancement of dental pulp tissue heal-ing. VEGF is a crucial factor in enhancing angiogenesis, inducing pro-genitor cell migration, and increasing nutrient supply (18). It was notedthat VEGF had a strong correlation with the expression of bonemorpho-genetic protein-2, implying the potential involvement of proangiogenicfactor on the hard tissue formation, ie, reparative dentin (30). In addi-tion, as the influence of VEGF on endothelial cell migration, the osteo-genic/odontogenic differentiation might be enhanced. It has beenshown that the direct co-culture of endothelial cells and human dentalpulp stem cells resulted in promoting osteogenic/odontogenic differen-tiation of human dental pulp stem cells in vitro (31). Thus, by up-regulating VEGF, the reparative dentin formation might be further pro-moted.

Progenitor cell migration and proliferation are also enhanced byFGF-2 and PDGF (32). FGF-2 also facilitates progenitor cell differenti-ation toward the odontogenic lineage (33). New vessels could beformed not only by the endothelial cells residing in the dental pulpbut also by dental pulp cells. A recent study has shown that human adultdental pulp cells exhibited the endothelial cell marker CD 146 (34, 35).In addition, these cells highly expressed CD44 (34, 35), which is knownto regulate angiogenesis. Thus, subpopulations of the cells residing inthe dental pulp might also participate in the neovascularization process.

Besides the role of angiogenesis, iloprost has been shown toinfluence platelet behaviors by attenuating platelet aggregation. Localdelivery of iloprost reduced the platelet aggregation and deposition ininjured cardiovascular structures (36–38). The platelets normally

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participated in healing process of dental pulp at the early homeostasisphase. The platelets also released various cytokines and growthfactors that could promote dental pulp healing. In this regard, the useof platelet-rich plasma in human premolars resulted in the formationof vital pulp-like structures (39), implying the influence of platelets indental pulp regeneration. However, the inhibiting effect of iloprost onplatelet aggregation may not be relevant to our findings because the in-crease of PBF by iloprost in vivo could be observed at the later time point(72 hours).

Currently, the revascularization concept has become widelyaccepted as a regenerative endodontic procedure (40). Dentin matrixprotein could enhance angiogenesis by enhancing endothelial cell pro-liferation, new vessel formation, as well as promoting the expression ofthe proangiogenic factor and their receptors (41). The proper use ofscaffolds, cells, and growth factors could enhance the clinical outcomeof dental stem cell therapy. Thus, the in vivomechanism(s) of iloprost-induced neovascularization should be further examined. It would beinteresting to investigate its effects on promoting angiogenesis whenused in a well-designed delivery system as a direct pulp-capping agentin vivo.

In conclusion, the current study suggests that the prostacyclinanalogue iloprost is part of a signaling network between vascularizationand proliferation of the dental pulp via the up-regulation of VEGF, FGF-2, and PDGF. Our in vivo study of PBF at early time points of dental pulphealing suggested that neovascularization was promoted by iloprost.Thus, we propose that iloprost might be used as an adjuvant for promot-ing the regeneration of dentin/pulp tissues, especially when an increasein blood flow in the dental pulp is required; such as the treatment of anecrotic pulp or in an avulsed immature tooth.

AcknowledgmentsSupported by the Thailand Research Fund MRG5680165, the

Ratchadapiseksomphot Endowment Fund of Chulalongkorn Uni-versity (RES560530156-HR), and Research Chair Grant of the Na-tional Science and Technology Development Agency (NSTDA). Theauthors also received funding from the DRU in Molecular pain aswell as DRU of Genetic and Anatomical Analyses of CraniofacialStructures, Faculty of Dentistry, Chulalongkorn University,Thailand.

The authors deny any conflicts of interest related to this study.

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Basic Research—Biology

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