![]() Briefly, minced ventricular myocytes were placed into potassium glutamate solution, which was obtained from the Hybridoma Facility at the University of Iowa. Primary cardiac myocyte cultures were prepared from ventricles of 2-day-old Sprague-Dawley rats, with modifications of the protocol described previously ( 1). Second, we tested the hypothesis that stretch of endothelial cells serves to trigger autocrine signaling by growth factors. First, we tested the hypothesis that specific angiogenic events in coronary microvascular endothelial cells (CMEC) are triggered by stretch-activated growth factors in cardiomyocytes, which provide paracrine signals for CMEC. The current study was designed to test two hypotheses based on these findings. The demonstration that VEGF secretion in rat cardiomyocytes occurs in response to cyclic stretch has provided direct evidence that stretch per se provides a trigger for VEGF secretion in this cell type ( 28). Cyclic stretch triggers TGF-β secretion in mesangial ( 25) and smooth muscle ( 19) cells. When VEGF neutralizing antibodies were administered to the bradycardia group, angiogenesis was completely prevented.Įvidence that stretch is a stimulus for growth factors has emerged from in vitro studies. Our data documented an increase in VEGF mRNA associated with the myocardial angiogenesis, i.e., a 23% increase in capillary length density. To test the hypothesis that myocardial angiogenesis is a response to enhanced diastolic filling in a nonhypertrophic model, we administered the bradycardia drug alinidine to young rats for a period of 3 wk ( 37). They also showed that this increase was mediated, at least in part, by tumor growth factor-β (TGF-β). These findings provided the rationale for the hypothesis that stretch of the ventricular wall might trigger angiogenesis.Įvidence that vascular endothelial growth factor (VEGF), a key angiogenic factor, is upregulated by stretch of the ventricular wall was provided by Li and colleagues ( 18), who found a marked increase in VEGF mRNA after diastolic pressure had been increased to 35 mmHg for 30 min in an isolated Langendorff preparation. Taken together, data from these studies indicate that coronary angiogenesis is an adaptation to either increased coronary blood flow or increased diastolic filling. Previous work in our laboratory and reports by others suggest that mechanical factors play a major role in coronary angiogenesis (reviewed in Ref. ![]() Although the primary stimuli that activate angiogenic molecules have not been established for all models of angiogenesis, metabolic and mechanical factors are implicated as stimuli for both angiogenesis and vascular remodeling (reviewed in Ref. Our knowledge concerning angiogenesis has been advanced by identifying growth factors and other molecules that initiate and regulate a cascade of events leading to neovascularization. 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-β signaling may regulate VEGF expression in cardiac myocytes. 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. 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. Myocyte tumor growth factor-β (TGF-β) increased 2.5-fold after 1 h of stretch, and the addition of TGF-β neutralizing antibodies inhibited the stretch-induced upregulation of VEGF. CMEC migration and tube formation increased with the addition of conditioned media but were markedly attenuated by VEGF neutralizing antibody. ![]() 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. 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. ![]() 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.
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