Differential analyses of angiogenesis and expression of growth factors in micro- and macrovascular endothelial cells of type 2 diabetic rats

Abstract

Aims

This study observed the relationship of angiogenesis and differential expression of growth factors and their receptors in micro- and macrovascular endothelial cells of diabetic and normal rats.

Main methods

Myocardial microvascular endothelial cells (MMVEC) and aortic endothelial cells (AEC) were isolated from type 2 diabetic-Goto–Kakizaki (GK) rats and age-matched normal Wistar rats. In vitro and in vivo Angiogenesis assay were used to observe the difference between GK rats and Wistar rats. mRNA and protein expression were analyzed by Real-time RT-PCR and Western blotting.

Key findings

MMVEC but not AEC of diabetic rats had reduced abilities of angiogenesis in vitro. Real-time RT-PCR showed increased mRNA levels of VEGF, fms-like tyrosine kinase (Flt-1) and kinase insert domain containing receptor (Flk-1) in GK-MMVEC, but not the hypoxia-inducible factor-1α (Hif-1α), basic fibroblast growth factor (bFGF), fibroblast growth factor receptor 1 (FGFR1), Angiopoietin-1(Ang-1), Angiopoietin-2(Ang-2), Tie-1 and Tie-2. In contrast, Western blotting showed decreased protein levels of VEGF and receptors, including the phosphorylation of receptors. No significant differences in the expression of theses genes were observed between AEC from diabetic and control rats. Anti-rat VEGF antibodies inhibited MMVEC angiogenic function including cell proliferation, adhesion, migration, scratch wound healing and capillary-like tube formation. The in vivo angiogenesis assay had similar results.

Significance

These result indicated that decreased expression of VEGF and its receptors caused by post-transcription disorder in MMVEC may be responsible for diabetic impaired cardiac angiogenesis.

Introduction

Diabetes mellitus is associated with angiopathy that leads to increased risk of peripheral ischemia (Emanueli et al., 2007). Development of collateral vessels of large arteries is considered an attempt to minimize the degree of ischemic damage(Sasso et al., 2005). Microvascular rarefaction and insufficient angiogenic response to myocardial ischemia in diabetic patients contribute to increased cardiovascular morbidity and mortality (Marfella et al., 2004). Clinical retrospective and experimental studies found lower capillary density and less coronary artery collateral vessel formation in ischemic cardiovascular diseases of diabetics than non-diabetics (Abaci et al., 1999, Melidonis et al., 1999, Toblli et al., 2004, Waltenberger, 2001). Although the impairment of collateral vessel development is demonstrated in diabetic patients (Abaci et al., 1999, Barzilay et al., 1994, Marfella et al., 2004, Melidonis et al., 1999, Yarom et al., 1992), further investigation is necessary to determine bioactive factors responsible for diabetic endothelial dysfunction and insufficient angiogenesis.

Angiogenesis is a regulated balance between stimulatory and inhibitory factors. It was found that the expression of VEGF, Angiopoietin-1, and Tie-2 was reduced, whereas antiangiogenic proteins, angiostatin and endostatin were significantly increased in the diabetic myocardium (Boodhwani et al., 2007). In addition, previous studies indicated that the glycated-bFGF was detected in diabetic individuals. It strongly affected bFGF structure and caused a significant reduction in the ability of bFGF to activate angiogenesis (Duraisamy et al., 2001, Facchiano et al., 2006). Therefore, abnormal expression of proangiogenic factors and their receptors as well as signal transduction disorder are the leading causes of diabetes-induced angiogenesis impairment.

Cultured endothelial cells (EC) are commonly used to study mechanisms of diabetic cardiovascular complications because they play an essential role in the abnormal angiogenesis process of many diseases including diabetes mellitus. Endothelial characteristics depend on their organ and tissue origin (Giannini et al., 2006). Micro- and macrovascular endothelial cells response differently to insulin (King et al., 1983) and glucose (Duffy et al., 2006, Rymaszewski et al., 1992). Therefore, the characteristics of EC of different origins may determine the different angiogenesis impairments of micro- and macrovasculatures. In the present study, we used cultured myocardial microvascular endothelial cells (MMVEC) and aortic endothelial cells (AEC) in same individual from age-matched diabetic and normal rats to study the relationship of in vitro angiogenesis and growth factors.