Nicolae Simionescu

Structural basis of permeability in sequential segments of the microvasculature of the diaphragm: II. Pathways followed by microperoxidase across the endothelium

Abstract Bipolar microvascular fields of mouse diaphragm were used to investigate the existence of characteristic modulations in the structural basis of permeability along successive segments of the microcirculation. Using hemeundecapeptide (microperoxidase; MW, ∼1900; mol diam, ∼20 A) as a probe molecule, the observations indicate that the timing and pathways followed by this tracer across the endothelium were different in each microvascular segment. The first permeated by these molecules were the pericytic venules at the level of their endothelial junctions. Among the latter, ∼25 to 30% are normally open to a space of ∼30 to 60 A. The vesicular transport is particularly extensive in capillaries and pericytic venules, and the transition from phase I to phase III is faster in the venular segments of capillaries, probably due to their high frequency in transendothelial channels formed by single vesicles. In the arteriolar and middle segments of capillaries, the H11P passed via vesicular transport and transendothelial channels. Endothelial junctions in arterioles and along the entire length of capillaries appeared to be impermeable to molecules of ≥20 A in diameter.

Structural basis of permeability in sequential segments of the microvasculature of the diaphragm: I. Bipolar microvascular fields

Abstract A new method is described for the reliable identification of successive segments in the microvasculature of skeletal muscle. The procedure relies on the existence in the postero-lateral regions of the mouse diaphragm of bipolar microvascular fields (BMFs) in which the supplying arteriole enters and the draining venules leave the capillary bed from opposite ends. In such fields the different segments of the microvasculature were initially recognized at the light microscope level in whole mounts of the muscle; their identity was confirmed by successive examination under the light and the electron microscope. In these BMFs, the average capillary path length is ∼580 μm, the average inner diameter increases progressively from ∼3 μm for the arteriolar, to ∼3.5 μm for the venular segments of capillaries; from this level, there is a sharp increase to the inner diameters of the postcapillary venules (∼11.5 μm). The new procedure has the advantage of allowing precise sampling in the muscle microvasculature and can be used to study regional differences in the structure and function of the small vessels of the peripheral vasculature.

Prelesional events in atherogenesis: Colocalization of apolipoprotein B, unesterified cholesterol and extracellular phospholipid liposomes in the aorta of hyperlipidemic rabbit

The appearance and accumulation of apolipoprotein B and unesterified cholesterol in the lesion-prone areas of the aorta in rabbits with diet-induced hyperlipidemia were investigated by histo-, and cytochemical techniques. Apolipoprotein B was detected by an indirect immunoperoxidase procedure both in the light and electron microscopy. Unesterified cholesterol was revealed using filipin and tomatine as specific probes. In the prelesional stages of atherogenesis, before the appearance of any structurally detectable lesions, as demonstrated by bright-field and fluorescence microscopy, apolipoprotein B and free cholesterol accumulated progressively in the extracellular matrix of the subendothelial space. At ultrastructural level, extracellular phospholipid liposomes, unesterified cholesterol and apolipoprotein B concomitantly appeared and accumulated focally in the same areas. Apolipoprotein B was preferentially located on the outer surface of the free cholesterol-containing phospholipid lamellae of the extracellular liposomes. In the lesional stages leading to fatty streak formation, the extracellular liposomes, apolipoprotein B and unesterified cholesterol had also topographically a superimposed localization pattern. Intracellular apolipoprotein B and unesterified cholesterol were also colocalized in some intimal lipid-laden cells. In the prelesional stages of hyperlipidemia the prevalent localization of apolipoprotein B around individual unesterified cholesterol-rich extracellular phospholipid liposomes, progressively accumulating in the subendothelial space, suggests their possible origin from serum-derived lipoproteins.

Development of intracellular lipid deposits in the lipid-laden cells of atherosclerotic lesions: A cytochemical and ultrastructural study

In atherosclerotic lesions of rabbits fed a cholesterol-rich diet, the lipid deposits of foam cells derived from monocytes, smooth muscle and endothelial cells were studied by physical, cytochemical and ultrastructural methods. Beginning with the third week of diet, the lipid material that could be visualized at the light microscope level by Oil red O and Nile red staining was progressively accumulated in the intimal cells of the atherosclerotic lesions. In the early stages of foam cell formation, the deposits occurred especially as intracytoplasmic non-membrane bound lipid inclusions (lipid droplets). In polarizing microscopy these appeared as a mixture of iso-, and anisotropic material. The latter were birefringent and showed an axial symmetry with a black cross image, suggesting that the lipids were in a liquid crystalline state. In chemically-fixed specimens, the content of lipid inclusions was preserved in various degrees. In freeze-fractured preparations they displayed a layered onion-like arrangement with smooth cleavage faces surrounding an amorphous core. Upon incubation with filipin, that specifically binds to 3 beta-hydroxysterols, the peripheral layers of the inclusions were labeled, revealing the existence of unesterified cholesterol. In the advanced stages of foam cell formation, lipids were additionally accumulated in the lysosomal compartment as polymorphic multilamellar structures concentrically arranged, with cleavage faces devoid of intralamellar particles. The presence of acid phosphatase showed that these features were modified lysosomes and were tentatively named lysosomal lipid bodies. In the latest stages examined cholesterol crystals developed within lysosomal lipid bodies usually enclosed in multilamellar structures. This lipid coat may represent the place of crystal formation and presumably acts as barrier for the turnover of the crystalline cholesterol, thus impeding plaque regression.

Endothelial cell dysfunctions

General: Endothelial Cell Response to Normal and Abnormal Stimuli (M. Simionescu). Endothelial Cell Regrowth (M.A. Reidy, V. Lindner). Endothelial Cell in Inflammation and Immunity: LeukocyteMediated Endothelial Injury (R.K. Winn et al.). Endothelial Cell Adhesive Interactions (E. Dejana et al.). Endothelial Cells in the Viral Infection: Response of Human Vascular Cells to Viral Infection (N.A. Kefalides). Endothelial Cells in Hypertension, Hyperlipidemia, and Atherosclerosis: Endothelial Dysfunction and Atherosclerosis (R. Ross). Endothelial Cells in Diabetes: Vascular Endothelium and Diabetes Mellitus (R.S. Bar). Endothelial Cells in Neoplasia and Metastasis: The Microvascular Phases of Metastasis (L. Weiss, F.W. Orr). Endothelial Cells in Other Disturbances Twentythree additional articles. Index

Isolation and characterization of endothelial cells from the heart microvasculature

Abstract A population of viable endothelial cells has been obtained from rabbit hearts by a method which disrupts muscle cells and eliminates their dispersed constituents and concomitantly isolates and preserves intact endothelial cells primarily of microvascular origin. The procedure employs as major steps: removal of large superficial vessels of ventricles, tissue mincing, enzymatic dissociation with crude collagenase (200 U/ml), washes with Ca 2+ -free Dulbeccos phosphate-buffered saline, homogenization of muscle fragments, and separation by velocity sedimentation through a 6% bovine serum albumin cushion. Cell isolation is monitored by phase contrast microscopy and subsequent electron microscopic examination. The final cell suspension consists of 85 to 90% endothelial cells predominantly organized in sheets of two to six cells or small vessel fragments formed by one to three endothelial cells linked by characteristic tight junctions. These features together with the absence of communicating (gap) junctions and the extremely rare occurrence of Weibel-Palade bodies indicate that the isolated endothelial cells are derived mostly from capillaries. The cells have highly irregular folded profiles presumably because they are no longer subject to intravascular pressure and are detached from the basal laminae. Isolated endothelial cells preserve their characteristic vesicles, the great majority of which (∼80 to 90%) are apparently internalized. The cells preserve anionic sites (detected with cationized ferritin) on their cell coat, retain tissue factor antigen (revealed immunocytochemically), are also capable of taking up macromolecular tracers (ferritin and hemeundecapeptide), and attach to collagen in culture conditions. As indicated by hemocytometer counting, packed cell volume, and DNA values, a yield of ∼ 1.0 to 1.5 × 10 7 endothelial cells/g tissue can be obtained by this procedure.

In vitro formation of oxidatively-modified and reassembled human low-density lipoproteins: antioxidant effect of albumin.

In early atherogenesis, excess plasma lipoproteins accumulate into the arterial lesion-prone areas as modified and reassembled lipoproteins (MRLp) appearing mostly as lipid droplets and vesicles. In the present study we produced such MRLp, in a cell-free system, devoid of any component of extracellular matrix, by subjecting in vitro human low-density lipoproteins (LDL) to autoxidation or copper-induced oxidation, for up to 96 h. As visualized by negative staining electron microscopy, a large number of lipoprotein particles (Lp) were progressively transformed into aggregates (ALp), fused particles (FLp) and vesicles (VLp). These modifications were paralleled by peroxidation of the samples as revealed by chemical analysis of each MRLp fraction isolated by a three-step purification procedure. LDL peroxidation in the above conditions was inhibited by the presence of albumin as assessed by TBARS and lipid analysis, and by the lack of MRLp formation. This protective effect was independent of albumin source (bovine, human, rabbit) and occurs at an albumin/LDL ratio of 1 when Cu2+ was present, and at a ratio of 0.25 in autoxidative conditions. The results show that: (i) in vitro LDL autoxidation or copper-induced peroxidation in a cell-free system can generate modified and reassembled lipoproteins similar to those detected in vivo in the arterial intima at the inception of atherogenesis; (ii) Lp particles appear to be sequentially transformed in self-aggregates, droplets and vesicles; (iii) serum albumin can completely prevent these LDL alterations.

Partial chemical characterization of the anionic sites in the basal lamina of fenestrated capillaries

The distribution of anionic sites in the basal laminae of the blood capillaries of the murine pancreas was studied in specimens fixed in ruthenium red (RR)-glutaraldehyde mixtures. The sites appeared as discrete, small (6 to 18 nm) particles distributed throughout the three laminae but concentrated primarily in the lamina rara externa, in which--spaced 80-100 nm apart--they formed a planar, partially ordered lattice comparable to that revealed by cationized ferritin in previous studies (M. Simionescu, N. Simionescu, and G. E. Palade, 1982, J. Cell Biol. 95, 425-434). The chemical nature of the anionic sites was explored by incubating fresh tissue specimens in solutions of selected enzymes before fixation in RR-glutaraldehyde mixtures. Pronase P and papain removed completely the anionic sites and left behind an extensively degraded and disorganized basal lamina. Trypsin caused the removal of anionic sites only, did not degrade the rest of the basal lamina, but detached it completely from the endothelium. Chondroitinase ABC reduced slightly the size and the surface density of RR-stainable particles, and detached focally the rest of the basal lamina from the endothelium and pericytes. Crude heparinase caused a nearly complete removal of anionic sites, and pure heparitinase gave comparable but less extensive results. Similar effects were recorded on the basal laminae of smooth muscle fibers and pancreatic acini and ducts. The results indicate that the anionic sites of all basal laminae examined are contributed primarily by heparin sulfate proteoglycans and trace amounts of chondroitin sulfate proteoglycans.

RECENT STUDIES ON VASCULAR ENDOTHELIUM

There is increasing evidence that processes that lead to vascular diseases, atherosclerosis included, are closely related to perturbations in endothelial permeability. Information has been accumulated that under various stress conditions, focal separations of endothelial junctions may occur preferentially in certain vascular segments. Such separations are involved in inflammatory reactions, hemorrhage, hypertension, thrombosis, and atheros~lerosis.~.~.’~.~~.~~.~~ A deeper knowledge of the normal structure and function of the endothelium is a prerequisite for a better understanding of its implication in vascular diseases. The vascular endothelium represents a special type of continuous, unistratified epithelium that behaves as a semipermeable partition across which, especially at the level of capillaries and venules, massive exchanges of small and large molecules occur between plasma and interstitial fluid. Like other cell membranes, the endothelial plasmalemma is assumed to be readily permeated by lipid-soluble molecules, but the endothelium as a whole has an unusually high permeability for water and solutes (much higher than that of other cells and of other epithelia). Moreover, unlike other epithelia, the endothelium is also permeable to large water-soluble molecules, which are assumed to pass through some water-filled channels or “pores” across the capillary W ~ I I . ~ . ’ ~ Physiologic experiments with graded molecules (dextrans) have indicated the presence of two categories of “pores”: small (diam., 90 A; frequency, 15-20/pm2; aggregate a r e a , 0.1%) and, large (diam., 500-700 A; frequency, 1/15-20 pm2). These figures have frequently been revised, the trend being toward smaller aggregate areas for the small-pore For the small pores, diffusion is restricted with increasing molecular size. Electron microscopic investigations of the vascular wallL.18.20 have revealed that blood vessels are provided with two basic types of endothelium: continuous endothehum, which is the most common type and is primarily encountered in all large vessels and in muscular capillaries, and fenestrated endothelium, which is found as several variants in visceral capillaries. Several peculiar features have been detected in the fine structure of the endothelial cells, the most characteristic being a high population of plasmalemmal vesicle^^^^^^ [outer diam., 600-700 A; frequency, 60-100/pm2 of endothelial surface; volume, 16% o f the endothelial cytoplasmic volume (in the peripheral zone)], about 60% of which are opened on one front o r the other of the C ~ I I S . ~ . ~ ~ I n addition, the visceral capillaries display transcellular round openings, the fenestrae (diam., 600-700 A; frequency, 15-25/pm2), usually closed by a thin single-layered diaphragm of unknown porosity and chemical n a t ~ r e . ~ . ’ ~ . ~ ~ * ~ * In thinsectioned specimens, the junctions of the endothelium appear to be of a relatively simple occludiag type with various degrees of membrane fusion, but the existence of open (20 40 A) junctions has been recorded.’” In fact, it is difficult to ascertain whether the endothelial tight junctions form complete sealing belts around each cell. However, none of the endothelial structures revealed by electron microscopy (vesicles, fenestrae, diaphragms, junctions) display the exact geometric dimensions and frequency postulated by the pore theory of capillary permeability.

Intimal thickenings of human aorta contain modified reassembled lipoproteins

The aim of this study was to determine whether in human aortas early minute changes such as minimal intimal thickenings (MIT), developed in areas known to have a predilection to atherosclerosis, contain modified reassembled lipoproteins (MRLp) such as extracellular liposomes (EL) and lipid droplets (LD). These features have been previously detected in the aortic lesion-prone areas of rabbits and hamsters fed a fat-rich diet. Tissue samples of the aortic arch and thoracic aorta from 12 young subjects who died in accidents were selectively collected from grossly normal regions. By light microscopy, some of these regions were found to contain MIT. The normal areas and the MIT were separately examined by electron microscopy or subjected to fractionation and partial biochemical characterization. The MIT (approximately 25-100 microns thick) were constituted by a pronounced proliferation of extracellular matrix, especially elastin and microfibrils, with interspersed lipid deposits appearing as EL and LD. Commonly, MIT did not contain smooth muscle cells, macrophages, foam cells or cytolytic debris. Such components were only occasionally found in specimens excised from the vicinity of fatty streaks. Saline extracts of MIT or grossly normal aortic regions were subjected to a four-step purification procedure consisting of gel filtration, affinity chromatography on anti-apo B and anti-albumin Sepharose, followed by density gradient ultracentrifugation. The entire procedure was monitored by negative staining, lipid assays, SDS PAGE and immunoblotting. From the initial MRLp mixture, two fractions were obtained: fraction 1 containing multilamellar EL and LD, and fraction 2 composed mostly of unilamellar EL. As compared with serum LDL, the cholesteryl ester/unesterified cholesterol ratio was 4-6-fold lower in fraction 1 and 15-19-fold lower in fraction 2. On SDS-PAGE the fraction 2 displayed a single protein band of 66 kDa, immunochemically identified as albumin. The MRLp isolated from human aortas with minimal intimal thickenings appeared to be similar to those purified from the prelesional stage aorta of hyperlipidemic rabbits and hamsters.