Mechanisms of coronary angiogenesis in response to stretch: role of VEGF and TGF-β

To test the hypotheses that cyclic stretch of 1) cardiac myocytes produces factors that trigger angiogenic events in coronary microvascular endothelial cells (CMEC) and 2) CMEC enhances the expression of growth factors, cardiac myocytes and CMEC were subjected to cyclic stretch in a Flexercell Strain Unit. Vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor mRNA and protein levels increased approximately twofold in myocytes after 1 h of stretch. CMEC DNA synthesis increased approximately twofold when conditioned medium from stretched myocytes or VEGF protein was added, and addition of VEGF neutralizing antibody blocked the increase. CMEC migration and tube formation increased with the addition of conditioned media but were markedly attenuated by VEGF neutralizing antibody. Myocyte transforming growth factor-beta corrected (TGF-beta) increased 2.5-fold after 1 h of stretch, and the addition of TGF-beta neutralizing antibodies inhibited the stretch-induced upregulation of VEGF. Stretch of CMEC increased VEGF mRNA in these cells (determined by Northern blot and RT-PCR) and increased the levels of VEGF protein (determined by ELISA analysis) in the conditioned media. Therefore, cyclic stretch of cardiac myocytes and CMEC appears to be an important primary stimulus for coronary angiogenesis through both paracrine and autocrine VEGF pathways. These data indicate that 1) CMEC DNA synthesis, migration, and tube formation are increased in response to VEGF secreted from stretched cardiac myocytes; 2) VEGF in CMEC subjected to stretch is upregulated and secreted; and 3) TGF-beta signaling may regulate VEGF expression in cardiac myocytes.