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

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Alamandine Improves The Migratory Ability of CACs from Healthy

Control

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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.

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https://ntrs.nasa.gov/search.jsp?R=20160006388 2018-06-16T02:34:43+00:00Z