Development of tissues in vitro with dimensions larger than 150 to 200 microm requires the presence of a functional vascular network. Therefore, we have studied capillary morphogenesis under controlled biological and biophysical conditions with the aim of promoting vascular structures in tissue constructs. We and others have previously demonstrated that physiological values of interstitial fluid flow normal to an endothelial monolayer in combination with vascular endothelial growth factor play a critical role during capillary morphogenesis by promoting cell sprouting. In the present work, we studied the effect that a range of interstitial flow velocities (0-50 microm/min) has in promoting the amount, length, and branching of developing sprouts during capillary morphogenesis. The number of capillary-like structures developed from human umbilical vein endothelial cell monolayers across the interstitial flow values tested was not significantly affected. Instead, the length and branching degree of the sprouts presented a significant maximum at flow velocities of 10 to 20 microm/min. More-over, at these same flow values, the phosphorylation level of Src also showed its peak. We discovered that capillary morphogenesis is restricted to patches of Src-activated cells (phosphorylated Src (pSrc)) at the monolayer, suggesting that the transduction pathway in charge of sensing the mechanical stimulus induced by flow is promoting predetermined mechanically sensitive areas (pSrc) to undergo capillary morphogenesis
Summary. Background: Prompt coronary thrombus resolution, reducing time of ischemia, improves cardiac recovery. The factors triggered by ischemia that contribute to the clinical outcome are not fully known. We hypothesize that unabated inflammation due to cardiac ischemia may be a contributing factor. Aims: As a proof-of-concept, we evaluated the effect of short-term myocardial ischemia on the local and systemic inflammatory response. Methods: Pigs underwent either 90-min mid-left anterior descending (LAD) coronary artery balloon occlusion (infarct size 25% ± 1% left ventricle; 29% heart function deterioration) or a sham-operation procedure. Periinfarcted and non-ischemic cardiac tissue was obtained for histopathologic, molecular and immunohistochemical analysis of inflammatory markers [interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a), modified C-reactive protein (mCRP), and human alveolar macrophage-56 (HAM-56)]. Blood (femoral vein) was withdrawn prior to myocardial infarction (MI) induction (t = 0) and at 30 and 90 min to evaluate: (i) systemic cytokine levels (IL-6, TNF-a, CRP); (ii) proinflammatory gene and protein expression in peripheral blood mononuclear cells (PBMCs) of tissue factor (TF), cyclo-oxygenase-2 (Cox-2), monocyte chemoattractant protein-1 (MCP-1), and CRP; and (iii) platelet activation (assessed by perfusion studies and RhoA activation). Results: Short-term ischemia triggered cardiac IL-6 and TNF-a expression, recruitment of inflammatory cells, and mCRP expression in infiltrated macrophages (P < 0.05 vs. t = 0 and sham). PBMC mRNA and protein expression of MCP-1, Cox-2 and TF was significantly increased by ischemia, whereas no differences were detected in CRP. Ischemia increased cardiac troponin-I, IL-6 and TNF-a systemic levels, and was associated with higher platelet deposition and RhoA activation (P < 0.001 vs. t = 0 and sham). Conclusion: Shortterm myocardial ischemia, even without atherosclerosis, induces an inflammatory phenotype by inducing local recruitment of macrophages and systemic activation of mononuclear cells, and renders platelets more susceptible to activation.
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