Impact of The Protective Renin-Angiotensin System (RAS) on The Vasoreparative
Function of CD34+ CACs in Diabetic Retinopathy
Yaqian Duan1, Leni Moldovan2, Rehae C. Miller2, Eleni Beli2, Tatiana Salazar2, Sugata Hazra3, Jude Al-Sabah2, KV Chalam4, Sneha Raghunandan5, Ruchi J. Vyas5, Patricia Parsons-Wingerter5, Gavin Y. Oudit6, and Maria B. Grant1, 2
1. Department of Integrative and Cellular Physiology, Indiana University School of Medicine, Indianapolis2. Department of Ophthalmology, Indiana University School of Medicine, Indianapolis
3. Department of Internal Medicine, University of Utah, Salt Lake City4. Department of Ophthalmology, University of Florida, Jacksonville, Florida
5. Space Life Sciences Research Branch, NASA Ames Research Center, Moffett Field CA6. Department of Medicine, University of Alberta, Canada
Abstract
Purpose: In diabetes, the impaired vasoreparative function of circulating angiogenic cells (CACs) is believed
to contribute to the progression of diabetic retinopathy (DR). Accumulating evidence suggests that the
protective arm of renin-angiotensin system (RAS) “ACE2/Angiotensin-(1-7)/Mas” plays an important role in
restoring the function of diabetic CACs. We examined the protective RAS in CACs in diabetic individuals with
different stages of retinopathy.
Methods: Study subjects (n=43) were recruited as controls or diabetics with either no DR, mild non-
proliferative DR (NPDR), moderate NPDR, severe NPDR or proliferative DR (PDR). Fundus photography and
fluorescein angiograms were analyzed using Vessel Generation Analysis (VESGEN) software in a cohort of
subjects. CD34+ CACs were isolated from peripheral blood of diabetics and control subjects. RAS gene
expression in CACs were measured by qPCR. The vasoreparative function of CACs was assessed by their
migration ability toward CXCL12 using the QCM 5μM 96-well chemotaxis cell migration assay.
Results: ACE2 gene is a key enzyme converting the deleterious Angiotensin II to the beneficial Angiotensin-
(1-7). ACE2 expression in CACs from diabetic subjects without DR was increased compared to controls,
suggestive of compensation (p=0.0437). The expression of Mas (Angiotensin-(1-7) receptor) in CACs was also
increased in diabetics without DR, while being reduced in NPDR compared to controls (p=0.0002). This
indicates a possible loss of compensation of the protective RAS at this stage of DR. The presence of even
mild NPDR was associated with CD34+ CAC migratory dysfunction. When pretreating CACs of DR subjects
with Angiotensin-(1-7) migratory ability to CXCL12 was restored (p=0.0008). By VESGEN analysis, an
increase in small vessel density was observed in NPDR subjects when compared with the controls.
Conclusions These data suggest the protective RAS axis within diabetic CACs may help maintain their
vasoreparative potential. The VESGEN analysis supports the presence of retinal repair in small vessels. The
loss of the protective arm of RAS may predict the progression of DR.
Results
Background
Endothelial dysfunction is an essential pathological change in the process of diabetic retinopathy
CACs play a vital role in endothelial repair and new vessel growth by homing to the injured vasculature and
providing paracrine factors
In diabetes with microvascular complications, CD34+ CACs are dysfunctional
The protective renin-angiotensin system (RAS) plays an important role in restoring the function of diabetic
CACs
Methods
Conclusions
Study subjects (n=43) were recruited as controls or diabetics with either no DR, mild non-proliferative DR
(NPDR), moderate NPDR, severe NPDR or proliferative DR (PDR)
CD34+ CACs were isolated from the peripheral blood mononuclear cells by using the EasySep™ human
CD34 positive selection kit
RAS gene expression levels were measured by qPCR
Migration function of CACs was analyzed by measuring their ability to migrate towards CXCL12 using the
QCM 5μM 96-well chemotaxis cell migration assay.
Fundus photography and fluorescein angiograms were analyzed using Vessel Generation Analysis
(VESGEN) software in a cohort of subjects
The protective RAS axis is activated within CACs from diabetic patients with no microvascular complications. However, a possible loss of compensation of
the protective RAS is observed at the stage of NPDR.
CACs from the diabetic patients with moderate and severe NPDR have decreased migration toward CXCL12 compared with healthy subjects.
Angiotensin 1-7 treatment improved the migration function of CACs from severe NPDR.
The VESGEN analysis helps to interpretate the presence of retinal repair in small vessels.
Ang 1-7 Treatment Enhanced The Migration of Impaired CACs toward
CXCL12 in Severe NPDR
Figure2. Effects of
Ang-(1-7) on migration
of CD34+ cells toward
CXCL12. (*p<0.05-
compared to control,
**p<0.05 compared to
CXCL12 group, #p<
0.05 compared to no
treatment group)
Figure1. mRNA levels of RAS genes within CACs. * P < 0.05 Compared to control; ** P<0.05
Compared to DM-NC
Presentation No.:
2721
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7Mas
Control DM-NC PDRMild Moderate Severe
NPDR
*
****
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ela
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e c
op
y n
um
be
r
(2^(
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1
2
3
4
5
6
7
MRGPRD
Control DM-NC PDR
*
Re
lati
ve
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py
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0.01
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1
10
ACE2ACE
**
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ve
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py
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(2^(
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*1
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) lo
g s
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1
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(2^(
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0.0
0.5
1.0
1.5
2.0
2.5
no treatment
CXCL12
CXCL12 + Ang 1-7 100nM
*****#
Mig
rati
on
of
CD
34
+ C
ell
s
(fo
ld c
ha
ng
e)
Control Diabetes/N
C
Mild
NPDRModerate
NPDR
Severe
NPDR
PDR
Alamandine Improves The Migratory Ability of CACs from Healthy
Control
0
0.5
1
1.5
2
2.5
Control Diabetic
no treatment
CXCL12
Alamandine 100nM/CXCL12
*
#
Mig
rati
on
of
CD
34
+ C
ell
s
(fo
ld c
ha
ng
e)
Vascular Changes of Retinopathy by Vessel Generation (VESGEN)
AnalysisVESGEN
Arteries
VESGEN
Venous
Mild
NPDR
Moderate
NPDR
Control
Fluorescein
Angiography
Adapted from S.H.S. Santos , J.M.O. Andrade Peptides 59 (2014) 34-41
Characteristics of Control and Diabetic Individuals
Control Diabetes
Number 13 30
Gender (M/F) 6/7 13/17
Age 39+13 60+11
HbA1C 4.8 8.6+2.0
Retinopathy -
25Mild
NPDR
7
Moderate
NPDR
12
Severe
NPDR
3
PDR
3
Neuropathy - 7
Nephropathy - 5
Hypertension 1 25
Hypercholesterolemia 1 16
Activation of Protective RAS Genes in CACs from Diabetic Individuals with No Diabetic Retinopathy
Results
Figure3. Effects of Alamandine on migration of CD34+ cells toward CXCL12. *p<0.05
compared to no treatment group; #p<0.05 compared to CXCL12 group.
Figure4. Vascular Generation Analysis of Different Stages of Retinopathy.
0
0.001
0.002
0.003
0.004
0.005
0.006
Control Mild
NPDR
Moderate
NPDR
Sm
all V
essel L
en
gth
Den
sit
y
(VE
SG
EN
Art
eri
al T
ree
)
00.0010.0020.0030.0040.0050.0060.0070.0080.009
0.01
Sm
all V
essel L
en
gth
Den
sit
y
(VE
SG
EN
Ven
ou
s T
ree)
Control Mild
NPDR
Moderate
NPDR
https://ntrs.nasa.gov/search.jsp?R=20160006388 2018-06-16T02:34:43+00:00Z