Densely arranged pericytes engird the endothelial tube of all coronary microvessels. Since the experimental access to these abundant cells in situ is difficult, a prerequisite for broader investigation is the availability of sufficient numbers of fully differentiated pericytes in homogenous culture. To reach this goal, we applied strictly standardized cell isolation techniques, optimized culture methods and specific histological staining. Approximately 1,000-fold enriched pericytes were proteolytically detached from highly purified coronary microvascular networks (density gradient centrifugation) of eight mammalian species including human. Addition of species-autologous fetal or neonatal serum (10-20% vol/vol) was a precondition for longer term survival of homogenous pericyte cultures. This ensured optimal growth (doubling time <14 h) and full expression of pericyte-specific markers. In 3-mo, 10(10) pericytes (15 g) could be cultivated from 1 bovine heart. Pericytes could be stored in liquid N(2), recultured, and passaged repeatedly without loss of typical features. In cocultures with EC or vascular smooth muscle cells, pericytes transferred fluorescent calcein to each other and to EC via their antler-like extensions, organized angiogenetic sprouting of vessels, and rapidly activated coagulation factors X and II via tissue factor and prothrombinase. The interconnected pericytes of the coronary system are functionally closely correlated with the vascular endothelium and may play key roles in the adjustment of local blood flow, the regulation of angiogenic processes, and the induction of procoagulatory processes. Their successful bulk cultivation enables direct experimental access under defined in vitro conditions and the isolation of pericyte specific antigens for the production of specific antibodies.

The frequently observed de-endothelialization of venous coronary bypass grafts prepared using standard methods exposes subendothelial prothrombotic cells to blood components, thus endangering patients by inducing acute thromboembolic infarction or long-term proliferative stenosis. Our aim was to gain deeper histological and physiological insight into these relations. An intricate network of subendothelial cells, characterized by histological features specific for true pericytes, was detected even in healthy vessels and forms, coupled to the luminal endothelium, a second leaflet of the macrovascular intima. These cells, and particularly those in the venous intima, express enormous concentrations of tissue factor and can recruit additional amounts of up to the 25-fold concentration within 1 h during preincubation with serum (intimal pericytes of venous origin activate 30.71 ± 4.07 pmol coagulation factor x·min−1·10−6 cells; n = 15). Moreover, decoupled from the endothelium, they proliferate rapidly (generation time, 15 ± 2.1 h, n = 8). Central regions of atherosclerotic plaques, as well as of those of restenosed areas of coronary vein grafts, consist almost completely of these cells. In stark contrast with the prothrombogenicity of the intimal pericytes, intact luminal endothelium recruits high concentrations of thrombomodulin (CD 141) specifically within its intercellular junctions, activates Protein C rapidly (42 ± 5.1 pmol/min·106 venous endothelial cells at thrombin saturation; n = 15), can thus actively prevent coagulatory processes, and never expresses histologically detectable and functionally active tissue factor. Given this strongly prothrombotic potential of the intimal pericytes and their overshooting growth behavior in endothelium-denuded vascular regions, they may play important roles in the development of atherosclerosis, thrombosis, and saphenous vein graft disease.

The barrier functions of myocardial precapillary arteriolar and postcapillary venular walls (PCA or PCV, respectively) are of considerable scientific and clinical interest (regulation of blood flow and recruitment of immune defense). Using enzyme histochemistry combined with confocal microscopy, we reexamined the cell architecture of human PCA and PVC and reconstructed appropriate in vitro models for studies of their barrier functions. Contrary to current opinion, the PCA endothelial tube is encompassed not by smooth muscle cells but rather by a concentric layer of pericytes cocooned in a thick, microparticlecontaining extracellular matrix (ECM) that contributes substantially to the tightness of the arteriolar wall. This core tube extends upstream into the larger arterioles, there additionally enwrapped by smooth muscle. PCV consist of an inner layer of large, contractile endothelial cells encompassed by a fragile, wide-meshed pericyte network with a weakly developed ECM. Pure pericyte and endothelial cell preparations were isolated from PCA and PCV and grown in sandwich cultures. These in vitro models of the PCA and PCV walls exhibited typical histological and functional features. In both plasma-like (PLM) and serum-containing (SCM) media, the PCA model (including ECM) maintained its low hydraulic conductivity (LP ϭ 3.24 Ϯ 0.52 · 10 Ϫ8 cm·s Ϫ1 · cmH2O Ϫ1 ) and a high selectivity index for transmural passage of albumin (SIAlb ϭ 0.95 Ϯ 0.02). In contrast, LP and SIAlb in the PCV model (almost no ECM) were 2.55 Ϯ 0.32 · 10 Ϫ7 cm·s Ϫ1 · cmH2O Ϫ1 and 0.88 Ϯ 0.03, respectively, in PLM, and 1.39 Ϯ 0.10 · 10 Ϫ6 cm·s Ϫ1 · cmH2O Ϫ1 and 0.49 Ϯ 0.04 in SCM. With the use of these models, systematic, detailed studies on the regulation of microvascular barrier properties now appear to be feasible.

The wall of myocardial terminal vessels, consisting of a continuous endothelial tube with an adventitial coat of pericytes in their extracellular matrix, constitutes a remarkably tight barrier to solute transport between the blood and the parenchyma. This constructional principle of precapillary arterioles, capillaries and postcapillary venules extends both up- and downstream into the arterial and venous limbs, where the original microvessel tube widens and becomes the innermost layer-the intima-of all the larger coronary vessels. In the myocardium's smallest functional units and in the intima of the coronaries, the pericytes play key roles by virtue of both their central histological localization and their physiological functions. Recognition and integration of these properties has led to new pathogenetic models for diverse heart diseases and suggests that current therapeutic concepts need to be revised.

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