Focus on cardiac pericytes.

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.

Wall structures of myocardial precapillary arterioles and postcapillary venules reexamined and reconstructed in vitro for studies on barrier functions.

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, microparticle-containing 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 (L(P) = 3.24 ± 0.52·10(-8)cm·s(-1)·cmH(2)O(-1)) and a high selectivity index for transmural passage of albumin (SI(Alb) = 0.95 ± 0.02). In contrast, L(P) and SI(Alb) in the PCV model (almost no ECM) were 2.55 ± 0.32·10(-7)cm·s(-1)·cmH(2)O(-1) and 0.88 ± 0.03, respectively, in PLM, and 1.39 ± 0.10·10(-6)cm·s(-1)·cmH(2)O(-1) and 0.49 ± 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.

Regulation of coronary venular barrier function by blood borne inflammatory mediators and pharmacological tools: insights from novel microvascular wall models.

We hypothesized that postcapillary venules play a central role in the control of the tightness of the coronary system as a whole, particularly under inflammatory conditions. Sandwich cultures of endothelial cells and pericytes of precapillary arteriolar or postcapillary venular origin from human myocardium as models of the respective vascular walls (sandwich cultures of precapillary arteriolar or postcapillary venular origin) were exposed to thrombin and components of the acutely activatable inflammatory system, and their hydraulic conductivity (L(P)) was registered. L(P) of SC-PAO remained low under all conditions (3.24 ± 0.52·10(-8)cm·s(-1)·cmH(2)O(-1)). In contrast, in the venular wall model, PGE(2), platelet-activating factor (PAF), leukotriene B(4) (LTB(4)), IL-6, and IL-8 induced a prompt, concentration-dependent, up to 10-fold increase in L(P) with synergistic support when combined. PAF and LTB(4) released by metabolically cooperating platelets, and polymorphonuclear leucocytes (PMNs) caused selectively venular endothelial cells to contract and to open their clefts widely. This breakdown of the barrier function was preventable and even reversible within 6-8 h by the presence of 50 µM quercetin glucuronide (QG). LTB(4) synthesis was facilitated by biochemical involvement of erythrocytes. Platelets segregated in the arterioles and PMNs in the venules of blood-perfused human myocardium (histological studies on donor hearts refused for heart transplantation). Extrapolating these findings to the coronary microcirculation in vivo would imply that the latter's complex functionality after accumulation of blood borne inflammatory mediators can change rapidly due to selective breakdown of the postcapillary venular barrier. The resulting inflammatory edema and venulo-thrombosis will severely impair myocardial performance. The protection afforded by QG could be of particular relevance in the context of cardiosurgical intervention.

Isolation, bulk cultivation, and characterization of coronary microvascular pericytes: the second most frequent myocardial cell type in vitro.

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.

Search for optimized conditions for sealing and storage of bypass vessels: influence of preservation solution and filling pressure on the degree of endothelialization.

The aim of the present study was to develop methods for the rapid assessment of intimal quality of coronary bypass segments of venous origin, and to prevent endothelial damage by improved intraoperative handling of graft segments. Particular attention was paid to the influence of the composition of the preservation solution and the intravasal filling pressure on the degree of endothelialization. Intrava-sal exposure to Alcian blue at pH<3 resulted in highly specific staining of intimal regions with functionally or structurally damaged endothelium. Standardization of preparation, staining and image acquisition of the intimal surface of graft remnants and subsequent computer-aided planimetry of these images made it possible for the first time to perform rapid serial investigations for quality control of bypass grafts. Using saline as the rinsing and intraoperative storage medium resulted in the loss of more than 50% of the endothelium at intravasal pressures of 0-100 mmHg. Increasing the pressure resulted eventually in complete de-endothelialization. In contrast, grafts incubated in a customized plasma derivative tolerated pressures of up to 200 mmHg with no significant endothelial loss; and even after exposure to 1,000 mmHg (10 times the average mean arterial pressure!) more than 70% of the endothelium were intact and vital. These findings imply strongly that the quality of aortocoronary bypass grafts of venous origin can be improved substantially by the use of a plasma derivative solution for intraoperative preservation and by monitoring and controlling the intravasal pressures reached during sealing and storage.

Pericytes in the macrovascular intima: possible physiological and pathogenetic impact.

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.10(6) 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.

Extensive deendothelialization and thrombogenicity in routinely prepared vein grafts for coronary bypass operations: facts and remedy.

The objective of this study was to gain deeper insight into the early reasons for saphenous vein graft disease and to find a practical approach to obviate it. Intraoperative storage of freshly explanted venous grafts (45 min, 20 degrees C; n=25 in each case) in saline, saline + 5% albumin, or HTK-solution and also in heparinized autologous blood was poorly tolerated by the endothelium. Large endothelial areas (mostly >75% of total surface) were detached already during brief non-pulsatile flushing just before the transplantation. Contact of deendothelialized areas in graft remnants with defined mixtures of coagulation factors or blood (n=11-17) caused rapid coagulatory processes via expression of tissue factor and assembly of prothrombinase in the subendothelium. Attached platelets and leukocytes accelerated the procoagulatory processes further, and endothelium-dependent anticoagulatory activities were significantly abolished. During pulsatile arterial flow, the resulting blood clots exacerbated the damage of the intima markedly, because they were flushed away tearing off further endothelium. In contrast, storage of venous grafts in a plasma preparation freed from isoagglutinins and coagulation factors preserved the endothelium, which resisted arterial flow and revealed anticoagulatory activity in the presence of antithrombin III and/or protein C. We conclude that gentle preparation and preservation of the vascular endothelium with a suitable storage solution during bypass surgery is a decisive first step to obviate saphenous vein graft disease.

Protective effects of flavonoids contained in the red vine leaf on venular endothelium against the attack of activated blood components in vitro.

New methods are described that allow the selective isolation of venular endothelial cells and their cultivation on porous filters to confluent monolayers. These filters with the attached endothelial cell layer can be mounted in a specially adapted apparatus allowing not only blood filtration studies, but now also the continuous registration of hydraulic conductivity (Lp) of tissue layers. This preparation responds dramatically to certain release products from simultaneously activated blood platelets and polymorphonuclear granulocytes (PMN) with a rise in Lp that, in situ, would lead rapidly to local oedema, arteriolar constriction and venular thrombosis. Selectively activated PMN alone induced only a modest increase in endothelial Lp that could be prevented by uric acid, an antioxidant. ASA prevented the activation of the blood cells, but not the effect of the release products per se, implying that the release products are probably eicosanoids. A standardized extract from red vine leaves (AS 195, active ingredient of Antistax Venenkapseln), containing in particular the flavonoids quercetin-3-O-beta-D-glucuronide and isoquercitrin (quercetin-3-O-beta-D-glucoside), not only prevented the deleterious effect of the release products on the venular endothelial monolayers but, applied promptly to an endothelium damaged by prior exposure to these release products, resulted in the repair of the endothelium. These findings identify for the first time the venular endothelium as a possible important therapeutic target in certain vascular diseases, chronic venous insufficiency being perhaps the most prominent example.