The epicardium makes essential cellular and paracrine contributions to the growth of the fetal myocardium and the formation of the coronary vasculature. However, whether the epicardium has similar roles postnatally in the normal and injured heart remains enigmatic. Here, we have investigated this question using genetic fate-mapping approaches in mice. In uninjured postnatal heart, epicardial cells were quiescent. Myocardial infarction increased epicardial cell proliferation and stimulated formation of epicardium-derived cells (EPDCs), which remained in a thickened layer on the surface of the heart. EPDCs did not adopt cardiomyocyte or coronary EC fates, but rather differentiated into mesenchymal cells expressing fibroblast and smooth muscle cell markers. In vitro and in vivo assays demonstrated that EPDCs secreted paracrine factors that strongly promoted angiogenesis. In a myocardial infarction model, EPDC-conditioned medium reduced infarct size and improved heart function. Our findings indicate that epicardium modulates the cardiac injury response by conditioning the subepicardial environment, potentially offering a new therapeutic strategy for cardiac protection.

Acetylated-low density lipoprotein (Ac-LDL) is taken up by macrophages and endothelial cells via the "scavenger cell pathway" of LDL metabolism. In this report, aortic and microvascular endothelial cells internalized and degraded 7-15 times more [1251]-Ac-LDL than did smooth muscle cells or pericytes. Bound [12Sl]-Ac-LDL was displaced by unlabeled Ac-LDL, but not unmodified LDL. The ability to identify endothelial cells based on their increased metabolism of Ac-LDL was examined using Ac-LDL labeled with the fluorescent probe 1,1 '-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). When cells were incubated with 10 pg/ml DiI-Ac-LDL for 4 h at 37°C and subsequently examined by fluorescence microscopy, capillary and aortic endothelial cells were brilliantly fluorescent whereas the fluorescent intensity of retinal pericytes and smooth muscle cells was only slightly above background levels. DiI-Ac-LDL at the concentration used for labeling cells had no effect on endothelial cell growth rate. When primary cultures of bovine adrenal capillary cells were labeled with 10 pg/ml of DiI-Ac-LDL for 4 h at 37°C, then trypsinized and subjected to fluorescence-activated cell sorting, pure cultures of capillary endothelial cells could be obtained. Utilizing this method, large numbers of early passage microvascular endothelial cells can be obtained in significantly less time than with conventional methods.A major problem in the study of microvascular endothelial cells is the identification of the desired cell population and the subsequent isolation of pure cultures. Our currently used method of establishing pure cultures of capillary endothelial cells involves many weeks of "weeding out" nonendothelial cells (l). The weeding technique involves the assumption that the morphology of capillary endothelial cells is similar to other endothelial cells and is thus directed at isolating colonies with those characteristics. Several markers for endothelial cells are routinely used for confirmation that established cell lines are of endothelial origin. These include the presence of factor VIII related antigen (2, 3) and angiotensin converting enzyme (4, 5). Microvascular endothelial cells differ from large vessel endothelial cells in their requirement for additional growth factors and modified surfaces for optimal growth (l), and their response to tumor factors (l, 6).The receptor-mediated uptake of low density lipoprotein (LDL ~) by cells has been studied in detail (for a review see reference 7). An alternative pathway for the metabolism of chemically modified lipoproteins has also been described (8) and has been termed the "scavenger cell pathway" of LDL metabolism, due to its occurrence in rodent and canine macrophages (9-11) and human monocytes (12). Various chemical methods for modification of LDL have been used to modify the charge of amino groups on LDL including acetylation (8), acetoacetylation (9), and malondialdehyde treatment (12). These modified lipoproteins are taken up by ~Abbreviations us...

Plaque angiogenesis promotes the growth of atheromas, but the functions of plaque capillaries are not fully determined. Neovascularization may act as a conduit for the entry of leukocytes into sites of chronic inflammation. We observe vasa vasorum density correlates highly with the extent of inflammatory cells, not the size of atheromas in apolipoprotein E-deficient mice. We show atherosclerotic aortas contain activities that promote angiogenesis. The angiogenesis inhibitor angiostatin reduces plaque angiogenesis and inhibits atherosclerosis. Macrophages in the plaque and around vasa vasorum are reduced, but we detect no direct effect of angiostatin on monocytes. After angiogenesis blockade in vivo, the angiogenic potential of atherosclerotic tissue is suppressed. Activated macrophages stimulate angiogenesis that can further recruit inflammatory cells and more angiogenesis. Our findings demonstrate that late-stage inhibition of angiogenesis can interrupt this positive feedback cycle. Inhibition of plaque angiogenesis and the secondary reduction of macrophages may have beneficial effects on plaque stability.angiogenesis ͉ inflammation ͉ vasa vasorum ͉ endothelium