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

To test the hypotheses that cyclic stretch of 1) cardiac myocytes produces factors that trigger angiogenic events in coronary microvascular endothelia l cells (CMEC) and 2) CMEC enhances the expression of growth factors, cardi ac myocytes and CMEC were subjected to cyclic stretch in a Flexercell Strai n Unit. Vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor mRNA and protein levels increased approximately twofold in my ocytes after 1 h of stretch. CMEC DNA synthesis increased approximately two fold when conditioned medium from stretched myocytes or VEGF protein was ad ded, and addition of VEGF neutralizing antibody blocked the increase. CMEC migration and tube formation increased with the addition of conditioned med ia but were markedly attenuated by VEGF neutralizing antibody. Myocyte tumo r growth factor-beta (TGF-beta) increased 2.5-fold after 1 h of stretch, an d the addition of TGF-beta neutralizing antibodies inhibited the stretch-in duced upregulation of VEGF. Stretch of CMEC increased VEGF mRNA in these ce lls (determined by Northern blot and RT-PCR) and increased the levels of VE GF protein (determined by ELISA analysis) in the conditioned media. Therefo re, cyclic stretch of cardiac myocytes and CMEC appears to be an important primary stimulus for coronary angiogenesis through both paracrine and autoc rine VEGF pathways. These data indicate that 1) CMEC DNA synthesis, migrati on, and tube formation are increased in response to VEGF secreted from stre tched cardiac myocytes; 2) VEGF in CMEC subjected to stretch is upregulated and secreted; and 3) TGF-beta signaling may regulate VEGF expression in ca rdiac myocytes.