Erythropoietin attenuates cardiac dysfunction by increasing myocardial angiogenesis and inhibiting interstitial fibrosis in diabetic rats

Abstract

Background: Recent studies revealed that erythropoietin (EPO) has tissue-protective effects in the heart by increasing vascular endothelial growth factor (VEGF) expression and attenuating myocardial fibrosis in ischemia models. In this study, we investigated the effect of EPO on ventricular remodeling and blood vessel growth in diabetic rats.

Methods: Male SD rats were randomly divided into 3 groups: control rats, streptozotocin (STZ)-induced diabetic rats, and diabetic rats treated with 1000 U/kg EPO by subcutaneous injection once per week. Twelve weeks later, echocardiography was conducted, and blood samples were collected for counting of peripheral blood endothelial progenitor cells (EPCs). Myocardial tissues were collected, quantitative real-time PCR (RT-PCR) was used to detect the mRNA expression of VEGF and EPO-receptor (EPOR), and Western blotting was used to detect the protein expression of VEGF and EPOR. VEGF, EPOR, transforming growth factor beta (TGF-β), and CD31 levels in the myocardium were determined by immunohistochemistry. To detect cardiac hypertrophy, immunohistochemistry of collagen type I, collagen type III, and Picrosirius Red staining were performed, and cardiomyocyte cross-sectional area was measured.

Results: After 12 weeks STZ injection, blood glucose increased significantly and remained consistently elevated. EPO treatment significantly improved cardiac contractility and reduced diastolic dysfunction. Rats receiving the EPO injection showed a significant increase in circulating EPCs (27.85 ± 3.43%, P < 0.01) compared with diabetic untreated animals. EPO injection significantly increased capillary density as well as EPOR and VEGF expression in left ventricular myocardial tissue from diabetic rats. Moreover, EPO inhibited interstitial collagen deposition and reduced TGF-β expression.

Conclusions: Treatment with EPO protects cardiac tissue in diabetic animals by increasing VEGF and EPOR expression levels, leading to improved revascularization and the inhibition of cardiac fibrosis.

Figure 1

EPO administration increased the number of CD34⁺Flk-1⁺ progenitors in diabetic rats. (A) Representative histograms of Control rats, DM rats, and EPO rats. (B): Tabulated data. ^#P <0.01 vs control, ^▵P <0.01 vs diabetes.

Figure 2

EPO administration increased angiogenesis in diabetic rats and was dependent on VEGF and EPOR up-regulation. (A) Representative micrographs of heart sections stained with antibodies against CD31, EPOR and VEGF (400x magnification). (B) Capillary density in number of capillaries per mm². Quantitative data showed EPOR and VEGF staining. (C) Representative western blots of myocardial VEGF and EPOR levels. (D) Real-time PCR analysis of VEGF and EPOR mRNA levels. Data are expressed as the mean ± SEM (n = 5). *P < 0.05 vs control, ^#P <0.01 vs control, ^▴P <0.05 vs diabetes, ^▵P <0.01 vs diabetes.

Figure 3

EPO inhibited myocardial fibrosis and cardiomyocyte hypertrophy in diabetic rats and was dependent on the reduction of TGF-β expression. (A) Picrosirius Red staining showing the collagen content in LV sections from control, diabetic, and diabetic EPO–treated rats. The arrowhead indicates elevated collagen content that was stained red in the extracellular matrix in the LV myocardium of diabetic rats and is significantly higher than control and EPO–treated group (200×magnification). (B) Collagen content was quantified from Picrosirius Red staining using Adobe Photoshop. (C) Immunohistochemical staining of TGF-β: quantitative data of TGF-β staining (400× magnification). (D) Immunohistochemical staining of collagen type I: quantitative data of collagen type I staining (400× magnification). (E) Immunohistochemical staining of collagen type III: quantitative data of collagen type III staining (400× magnification). (F) Caridomyocyte size was measured by hematoxylin-eosin staining in cross-sectional areas. (400× magnification). Bar graph shows quantitative analysis of cardiomyocyte cross-sectional area. Data are expressed as the mean ± SEM (n = 5).*P < 0.05 vs control, ^#P <0.01 vs control, ^▴P <0.05 vs diabetes, ^▵P <0.01 vs diabetes.