Analysis of glycosaminoglycans in bovine retinal microvessel basement membrane.

Glycosaminoglycans (GAG) were isolated from bovine retinal microvessel basement membrane (RMV-BM) and quantitatively analyzed using a recently described competitive binding assay that is specific for and sensitive to nanogram amounts of heparan and chondroitin sulfates. Treatment of osmotically lysed retinal microvessels with the ionic detergent deoxycholate (DOC), required for liberation of the extracellular matrix for plasma membrane lipoproteins and purification of the insoluble matrix, solubilized less than 5% of the GAG in the water-insoluble material. Total GAG content in the DOC-insoluble basement membranes was approx. 0.52 micrograms/mg dry weight; about 70% of the measurable GAG was resistant to both chondroitinase ABC and chondroitinase AC digestion and was sensitive to nitrous acid treatment, indicating its heparan sulfate nature. Cellulose acetate electrophoresis revealed two bands, one of which had an electrophoretic mobility similar to heparan sulfate standard and was sensitive to nitrous acid; the other migrated in the same position as chondroitin sulfate standard and was sensitive to chondroitinase ABC and chondroitinase AC digestion. These results provide evidence that RMV-BM contains chondroitin sulfate(s) as well as heparan sulfate, and offer the first quantitative analysis of GAG in this extracellular matrix.

Expression of fibronectin, laminin, and factor VIII-related antigen during development of the human cutaneous microvasculature.

The regulation of angiogenesis during human skin development is poorly understood. Since fibronectin is involved in cell movement and organization during embryogenesis and morphogenesis in a variety of species, we investigated the expression of fibronectin and factor VIII-related antigen, a marker for endothelial cells, at various stages in the development of the human cutaneous microvasculature. Skin specimens were obtained from 4 human fetuses during the second trimester (14-18 weeks), from newborn foreskins, and from consenting normal adults. Cryostat sections were stained with both fluorescein-conjugated rabbit antihuman fibronectin and rhodamine-conjugated goat antihuman factor VIII-related antigen. Expression of fibronectin in the microvasculature was striking in fetal skin but became progressively less prominent with maturation. Fibronectin appeared in fetal blood vessels as a bright continuous linear array, in neonatal blood vessels as a bright interrupted linear and speckled array, and in adult blood vessels as a sparse interrupted linear and speckled array. In contrast, expression of factor VIII-related antigen by the endothelium became more prominent with the degree of maturation of the microvasculature. Granular factor VIII-related antigen staining was scant in the newly forming blood vessels of fetal skin, bright but focal in the microvasculature of newborn skin, and intense and almost confluent in the blood vessels of adult skin. Although expression of fibronectin and factor VIII-related antigen changed, expression of laminin was consistent throughout development. Staining of the same skin specimens with fluorescein-conjugated sheep antihuman laminin produced a bright continuous linear pattern in all blood vessels. The reciprocal relationship manifested by intense fibronectin staining during human blood vessel development and prominent factor VIII-related antigen staining in mature blood vessels supports the hypotheses that fibronectin plays a role in human blood vessel modulation and morphogenesis, and that factor VIII-related antigen is a marker for endothelial cell differentiation.

Adrenergic receptors in bovine retinal microvessels: presence of alpha 2- and beta- but not alpha 1-receptors.

In bovine retinal microvessels, alpha 1, alpha 2- and beta-adrenergic receptors were characterized by binding assay, using [3H]prazosin, [3H]para-aminoclonidine and [125I]iodocyanopindolol as radioligands, respectively. The microvessels were purified from bovine eyes by differential centrifugation through a high concentration of bovine serum albumin followed by use of a glass bead filtration technique. In the preparation, specific binding sites for [3H]para-aminoclonidine and [125I]iodocyanopindolol were observed, whereas [3H]prazosin binding was not detected. The [3H]para-aminoclonidine binding sites localized to the microvessels were characterized by high affinity and saturability (KD: 173 +/- 9 pM; Bmax: 394 +/- 11 fmol/mg protein) as well as the [125I]iodocyanopindolol binding sites (KD: 20 +/- 3 pM; Bmax: 43 +/- 4 fmol/mg protein). Furthermore, the specificity of both binding sites was pharmacologically evaluated by measuring the inhibitory effects of various adrenergic reagents on binding. The existence of alpha 2- and beta-adrenergic receptors which were characterized by high affinity, saturability and stereospecificity, leads to the hypothesis that the retinal microcirculation is under neuronal control.

Ultrastructural localization of lectin receptors on the luminal and abluminal aspects of brain micro-blood vessels.

Lectin- or glycoprotein-colloidal gold complexes were used for detection of specific monosaccharide residues in mouse brain micro-blood vessels (MBVs). The lectins tested recognize the following residues: beta-D-galactosyl (Ricinus communis agglutinin-120, RCA-1), alpha-N-acetylgalactosaminyl (Helix pomatia agglutinin, HPA), alpha-D-mannosyl and alpha-D-glucosyl (Concanavalin A, Con A), sialoglycoconjugates (Limax flavus agglutinin, LFA), N-acetylglucosaminyl and sialyl (wheat germ agglutinin, WGA), and alpha-L-fucosyl (Ulex europeus agglutinin, UEA-1). Use of these lectin-gold complexes and ultrathin sections of Lowicryl K4M-embedded tissue makes it possible to gain insights into localization of lectin receptors in the entire cross-section of MBV walls. Receptors for all lectins, except UEA-1, were found on both luminal and abluminal fronts of the endothelial cells (ECs). Differential labeling of luminal and abluminal fronts of ECs with some lectins (Con A, HPL) is considered to reflect the polarity of the endothelium. Some differences noted in the distribution of lectin receptors in the wall of representatives of three types of MBVs (capillaries, arterioles, and venules) are thought to be associated with different functions performed by the above-mentioned segments of the microvasculature in maintenance of the blood-brain barrier.

Retinal microvessel extracellular matrix: an immunofluorescent study.

The vasculature of the retina functions within a sheath of extracellular matrix (ECM). Unfortunately, little is known about the biochemical composition of this matrix. Abnormalities in the ECM of the retinal microvasculature are important in diabetic retinopathy as well as vasculopathies associated with connective tissue disorders. The ECM of unfixed frozen human retinal blood vessels was examined by indirect immunofluorescence using antibodies raised against collagen types I, II, III, IV, and V as well as the structural glycoproteins laminin and fibronectin. Antisera against collagen types I and IV as well as laminin and fibronectin stained a broad spectrum of retinal vessels, from large thick-walled vessels down to microvessels less than 10 micron in diameter. In contrast, antibodies against types III and V collagen were seen to stain primarily the walls of the larger vessels. Antibodies against type II collagen did not react with retinal vessels. Preincubation with the appropriate antigen or preimmune serum eliminated staining of the vessels by the antisera.

A monoclonal antibody to the endothelium of rat brain microvessels.

A rat brain fraction enriched with microvessels was used as the immunogen to produce mouse hybridoma cell lines secreting monoclonal antibodies. One of these antibodies, selected from 156 supernatants by enzyme-linked immunosorbent and immunofluorescent assays, reacted only with the endothelium of microvessels in the brain. The endothelium-specific antibody labelled the cytoplasm of microvascular endothelial cells, their luminal membranes, and an extracellular layer, the endocapillary coat, which covered the luminal surface of these cells. In the kidney, the antibody specifically stained the brush border of the proximal tubuli, and in the liver, the antibody specifically stained bile canaliculi. This demonstrates that 3 morphological structures with important transport functions, cerebral microvascular endothelium, brush border of kidney proximal tubuli, and liver bile canaliculi, express the same epitope.

Functional identification of adenylate cyclase-coupled adenosine receptors in rat brain microvessels.

It was investigated whether adenosine affects the adenylate cyclase system of microvessels, isolated from the rat cerebral cortex, via an external receptor recently classified as R-site receptor. Several adenosine analogs caused GTP-dependent stimulation of adenylate cyclase. The rank order of potency: NECA (5'-(N-ethylcarboxamido)-adenosine, EC50 0.2 microM) greater than adenosine (0.71 microM) = 2-chloroadenosine (0.72 microM) greater than L-PIA (N6-(L-phenylisopropyl)-adenosine, 1.03 microM) greater than D-PIA (N6-(D-phenylisopropyl)-adenosine, 5.27 microM) is consistent with that for agonism at activatory (Ra-site) adenosine receptors in other tissues. Adenylate cyclase stimulation by PIA displayed stereoselectivity. The action of NECA was competitively antagonized by 8-phenyltheophylline. These findings provide functional evidence for Ra-site adenosine receptors in rat brain microvessels. However, direct identification of these receptors by binding studies was not possible. Binding of [3H]NECA to rat brain microvessels displayed rapid on-off kinetics and saturability, but equivocal specificity.

The effect of locus ceruleus lesion on water, sodium and potassium content of the rat cerebral cortex.

Locus ceruleus lesion decreases the density of ouabain binding sites, and presumably Na+, K+-ATPase, in brain microvessels. To determine if this decrease affects the transport of Na+, K+ or water across the blood-brain barrier, we studied the influence of unilateral locus ceruleus lesion on Na+, K+ and water content of the ipsilateral cerebral cortex. Unilateral locus ceruleus lesion depleted norepinephrine in the ipsilateral cerebral cortex but had no effect on tissue Na+, K+ or water under steady-state conditions. When the Na+/K+ exchange pump of the blood-brain barrier was stressed by hyperkalaemia, K+ content in the ipsilateral cerebral cortex rose to higher levels than in the contralateral cortex, but the difference did not reach statistical significance. Locus ceruleus lesion also did not cause significant differences in the cerebral cortical content of water, Na+ or K+ in hyponatraemia. The results suggest that brain water and ion homeostasis are tightly controlled, probably by multiple mechanisms with biological redundancies, so that even a 50% decrease in the density of ouabain binding sites in brain endothelium does not result in significant alterations in brain water, Na+ or K+ content.

An intravital microscopy model for studies of immune complex induced inflammation at the microvascular level.

The hamster cheek pouch model was used for studies of immune complex induced inflammation. Vascular leakage and leukocyte accumulation were observed after topical application of antigen. This occurred at the post-capillary venules, where also antigen localization could be seen, indicating immune complex deposition at these sites.

[Identification of 11-HETE in the rat brain and its relevancy to ischemic cerebral edema].

Eicosanoids are thought to be important in the pathogenetic mechanism of ischemic brain damage. But little is known about lipoxygenase products and their roles in brain. In the present study, lipoxygenase metabolism in the brain and its relevancy to ischemic brain edema were studied using high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The rat middle cerebral artery (MCA) occlusion model was used because the time course of ischemic edema formation has been well known. The rat brain was fixed by in situ freezing 24 and 72 hours after MCA occlusion, and removed. Identification and quantitative analysis of hydroxyeicosatetraenoic acids (HETEs) in normal and ischemic brain homogenates was performed by HPLC and GC-MS. The rat brain was divided into the microvessel (MV) fraction and the rest. Then the same analysis was carried out in the both fractions obtained from the normal rat brain. Only 11-HETE was detected in the normal and ischemic brain. Quantitatively, normal brains contained 1288 +/- 66 (mean +/- SE) of 11-HETE ng/g wet weight, while the hemispheres rendered ischemic for 24 and 72 hours after MCA occlusion contained 1101 +/- 48, and 1663 +/- 147, respectively. The 11-HETE content was significantly increased 72 hours after MCA occlusion (p less than 0.05). The MV fraction of rat brain contained 11-HETE (653 ng/mg protein) ten times as much as the other fraction. The identification of 11-HETE in the rat brain is a new finding.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteoglycans in the microvasculature. I. Histochemical localization in microvessels of the rabbit eye.

The ultrastructural organization of ruthenium red (RR) stainable material within small blood vessels located in the limbus of the rabbit eye was studied. Proteoglycans were identified in this material by digesting tissues with Streptomyces hyaluronidase, testicular hyaluronidase, chondroitinase ABC, or heparinase before ruthenium red staining. Neuraminidase digestion enabled separate identification of sialoglycoprotein. The luminal surface of endothelial cells demonstrates an RR-stained glycocalyx containing both sialoglycoprotein and proteoglycans, which are removed by testicular hyaluronidase and crude heparinase. The basal coat of endothelial cells and small granules (10-20 nm in diameter) located within the basal lamina stain with RR and are removed only by crude heparinase. The surface coat of smooth muscle cells and small granules (10-20 nm) within their basal laminas are also digested by crude heparinase. Large proteoglycan granules (20-50 nm), which are completely removed by testicular hyaluronidase and partially digested by Streptomyces hyaluronidase, are deposited between the connective tissue fibers of the media and adventitia. Other large granules that are attached to collagen fibers contain enzyme-resistant anionic materials. The surface coat of adventitial fibroblasts is removed only by crude heparinase. Thin filaments (3-5 nm in diameter) interconnect the cell coat material, basal lamina granules, and large connective tissue granules, to form a network of proteoglycans that traverses the intima, media, and adventitia. The highly ordered arrangement of proteoglycans in the microvascular wall suggests that these macromolecules play several roles in microvascular function.

Isolated brain microvessels: preparation, morphology, histamine and catecholamine contents.

A method for the rapid preparation of parenchymal microvessels from rat, rabbit and bovine brains is described. Light and electron microscopic examination of the isolated microvessels showed the smooth muscle and endothelial cells to be intact and substantially free of neutrophil contamination. The observation of 0.2 to 2.0 micron electron dense granules in the adventitial space associated with some of the isolated arteriolar elements and the identification of histamine in microvessel extracts is suggestive of the presence of mast cells in the microvessel preparations. Age-dependent changes in histamine content and an increased histamine content in microvessels from hypertensive rats were noted. Using a sensitive radioenzymatic assay, the catecholamine (CA) contents of microvessels isolated from cow, rat and rabbit were measured. The total CA contents of all microvessel preparations were small (< 0.35 microgram g-1) with norepinephrine being the predominant CA present. A large ratio of dopamine to norepinephrine was found in microvessels from rat and bovine brain. The possible origins of the CAs present in the isolated microvessels are discussed.

Proteoglycans in the microvascular. II. Histochemical localization in proliferating capillaries of the rabbit cornea.

The ultrastructural distribution of proteoglycans around capillaries growing in the cornea of the rabbit eye was determined after staining with ruthenium red (RR). Proteoglycans were identified by digesting tissues with glycosaminoglycan-degradative enzymes. Sialoglycoproteins were differentiated from proteoglycans by neuraminidase digestion. The capillary sprouts demonstrated a luminal glycocalyx containing testicular hyaluronidase-sensitive proteoglycans but little or no sialoglycoprotein. At the capillary tips, where mitosing and migrating endothelial cells are located, the basal cell surface displayed a network of small RR-stained granules (8 nm in diameter), which was partially removed by streptomyces hyaluronidase but not by testicular hyaluronidase. Thin filaments connected the granules to the endothelial cell plasmalemma and to a similar network of granules that is normally present in the corneal stroma. The stroma granules were partially digested by testicular hyaluronidase. In older capillary regions, where endothelial cells ceased proliferation, the basal network of proteoglycan granules was gradually infiltrated by fibrillar material until a basal lamina was formed. The proteoglycan granules were then arranged on both sides of the lamina densa, and a thin glycocalyx covered the basal endothelial cell surface. Thus, proteoglycans and anionic materials associated with growing capillaries serve to link proliferating and migrating endothelial cells to the extracellular matrix, help to organize the capillary basal lamina, form an anionic surface along the luminal front of capillaries, and probably help stabilize the structure of the capillary wall after proliferation ceases.

Sulfated glucuronyl paragloboside in rat brain microvessels.

In patients with neuropathy associated with paraproteinemia, there are monoclonal immunoglobulin M antibodies reacting with myelin-associated glycoprotein and sulfated glucuronyl glycolipids. There are indications that the monoclonal antibodies may be responsible for these neuropathies. However, the mechanism by which the antibodies gain access to the nervous tissue, which is separated by the blood-brain barrier or blood-nerve barrier, is still unknown. In this study, we examined the presence of the sulfated glucuronyl glycolipid antigens on brain endothelial cells. Microvessels were isolated from adult Lewis rat brain cortex. Sulfated glucuronyl paragloboside (SGPG) was detected in the acidic lipid fraction by a TLC immunostaining method. Immunofluorescence studies showed positive staining on the surface of microvessels. In addition, SGPG could be detected in the cultured endothelial cells of human umbilical vein. These findings suggest that the endothelial cells contain antigenic sites for interaction with the autoantibodies. This type of interaction may result in damages to the endothelial cell function and may be responsible for changes in the blood-brain barrier permeability and the ensuing penetration of large molecules, such as immunoglobulins, into the endoneurial space.