In situ distribution of the beta-subunit of platelet-derived growth factor receptor in nonneoplastic tissue and in soft tissue tumors.

The distribution of the beta-subunit of platelet-derived growth factor receptor (PDGFR-beta) was assessed by a sensitive immunoalkaline phosphatase technique using the monoclonal antibody PR7212. Frozen tissue sections of several nonneoplastic human tissues were stained along with 42 soft tissue sarcomas, 16 benign soft tissue proliferations, and 7 epithelial tumors. In all nonneoplastic tissue, there was intense labeling of cell processes of perivascular fibroblasts or pericytes in and about the walls of muscular blood vessels and of fibroblast cell processes around some glandular and ductal epithelia. No PDGFR-beta was found in the endothelial cells of muscular arteries and veins, but cells of uncertain identity within some capillaries were immunoreactive and the possibility that endothelial cells of some small capillaries express PDGFR-beta could not be excluded. In kidney there was strong labeling of glomerular mesangial cells and interstitial fibroblasts. Some histological types of soft tissue sarcomas were uniformly and strongly labeled with anti-PDGFR-beta, but other types were infrequently labeled or unreactive. The order of decreasing frequency and strength of labeling of the various types of benign and malignant soft tissue proliferations was as follows: benign fibromatosis and neurofibroma greater than malignant fibrous histiocytoma greater than liposarcoma greater than leiomyosarcoma greater than rhabdomyosarcoma. No tumor cell labeling was detected in epithelioid, synovial or clear cell sarcomas, leiomyomas, or carcinomas, but there was usually strong labeling of fibroblast and/or pericyte cell processes within tumor, especially around blood vessels. We conclude that PDGFR-beta is strongly expressed by vascular and stromal tissues of most tumors and normal organs and by tumor cells of several types of soft tissue tumors and proliferations, most notably those of fibroblastic origin.

Immunohistochemical study of cerebral amyloid angiopathy. III. Widespread Alzheimer A4 peptide in cerebral microvessel walls colocalizes with gamma trace in patients with leukoencephalopathy.

Brain tissue from 11 patients with cerebral amyloid angiopathy, changes of Alzheimer's disease, and variable degrees of subcortical leukoencephalopathy was examined by immunohistochemical methods, using primary antibodies to peptide segments representing portions of the Alzheimer A4 (beta-) peptide or gamma-trace peptide (seen most commonly in Icelandic patients with cerebral hemorrhage (hereditary cerebral hemorrhage with amyloidosis [HCHWA-I]). Variable A4 immunostaining was seen within cortical (and rarely white matter) parenchyma in the form of senile plaques (with or without central cores), and within capillary and arteriolar walls. Within individual patients, A4 deposits were often primarily parenchymal or vascular, and when they were vascular they tended to be more prominent in arteriolar than in capillary wall segments. Perivascular A4 deposits were often detected around strongly immunoreactive microvessels. Gamma-trace immunoreactivity was noted in many A4-positive microvessel walls, but staining was always less intense than with the anti-A4 antibody. We conclude that patients with severe cerebral amyloid angiopathy may show wide variation in the severity and topography of A4 deposits within brain parenchyma. A4 may colocalize with gamma-trace peptide, suggesting that A4 and gamma-trace forms of cerebral amyloid angiopathy may not be as biochemically distinctive as has been suggested. Other proteases or protease inhibitors may contribute to the pathogenesis of cerebral amyloid angiopathy or cerebral amyloid angiopathy-related stroke syndromes.

Massive vascular AA-amyloidosis: a histologically and biochemically distinctive subtype of reactive systemic amyloidosis.

Amyloid protein AA consists of several subspecies which mainly arise through proteolytic cleavage at various sites of the precursor, serum protein AA. The most common protein AA subspecies (the protein AA prototype) contains 76 amino-acid residues. In previous studies we have shown that distinctive patterns of amyloid infiltration occur in AA-amyloidosis. The amyloid in different patterns of infiltration seems to consist of distinctive protein AA subspecies. In the present study we have analysed protein AA in three patients with a form of AA-amyloidosis with heavy vascular infiltration and show that the amyloid fibrils contain two groups of protein AA subspecies. One, quantitatively predominating, group contains large subspecies of up to 94 amino-acid residues and a second group of protein AA-molecules contains around 50 amino-acid residues. The AA molecules lack the N-terminal arginine residue. It is concluded that AA-amyloidosis with massive vascular infiltration is a distinctive subform with typical clinical and histological appearance and with fibrils containing characteristic protein AA subspecies.

Immunohistochemical localization of chondroitin sulfate and dermatan sulfate proteoglycan in human gingival connective tissue.

This study investigated the immunohistochemical localization of chondroitin sulfate (chondroitin, 4-sulfate and 6-sulfate) and dermatan sulfate proteoglycan (PG) in human gingival connective tissue, using monoclonal antibodies. Dermatan sulfate was found to be widespread in connective tissue, with an especially strong response shown in collagen fiber bundles under the epithelial basement membrane. Chondroitin 4-sulfate occurred widely in connective tissue but showed only a weak response. Chondroitin 6-sulfate was located in peripheral blood vessels. Chondroitin was not detected in gingival connective tissue.

Vasopressin-mediated forearm vasodilation in normal humans. Evidence for a vascular vasopressin V2 receptor.

Arginine vasopressin (AVP) is a potent vasopressor and antidiuretic neurohormone. However, when administered intravenously to humans, AVP causes forearm vasodilation. This effect has been attributed to sympathetic withdrawal, secondary to AVP-induced sensitization of baroreceptors. The possibility that AVP also causes forearm vasodilation directly has not been examined. Accordingly, the direct effect of AVP was determined by studying the forearm blood flow (FBF) response to intraarterial (IA) AVP infusion (0.01-1.0 ng/kg per min). Infusion of IA AVP increased FBF (96%) in the infused arm, but not the control arm, in a dose-dependent manner. The role of specific AVP V1 receptors in mediating this FBF response was determined before and after pretreatment with a V1 antagonist (AVP-A). AVP-A alone had no effect on FBF, but coadministration of AVP and AVP-A potentiated the vasodilatory response (223%). IA infusion of the V2 agonist, 1-desamino[8-D-arginine] vasopressin, caused a dose-dependent increase in FBF. These findings suggest that AVP causes direct, dose-dependent vasodilation in the human forearm that may be mediated by V2 vasopressinergic receptors. In contrast, AVP infusion caused digital vasoconstriction that was blocked by AVP-A, whereas dDAVP did not affect digital blood flow. Thus, AVP induces regionally selective vascular effects, with concurrent forearm vasodilation and digital vasoconstriction.

The localisation of fibrin, fibronectin and class II major histocompatibility complex antigen in the spinal cord in chronic relapsing experimental allergic encephalomyelitis.

Light and electron immunocytochemistry using antibodies recognising a class II major histocompatibility complex antigen, fibrin, fibronectin, albumin and factor VIII related antigen has been used to stain sections of spinal cord from normal guinea pigs and those with chronic relapsing experimental allergic encephalomyelitis (CREAE). It was found that class II MHC antigens, fibrin and fibronectin were present in normal blood vessels and at high levels in lesions from animals at all stages of the disease. The possible immunological roles of these antigens suggest their participation in the initiation and maintenance of disease state.

Cationic distribution in vessels, heart, skeletal muscle, and blood of normotensive (NWR) and spontaneously hypertensive rats (SHR).

Electrolytic analysis carried out with the atomic absorption spectrophotometer permitted to compare the cationic (Ca2+, Mg2+, Zn2+, K+ and Na+) tissue content of spontaneously hypertensive male rats (SHR) and normotensive male Wistar rats (NWR). In all SHR-tissues, Zn2+ is augmented, but mainly in the atria (left atrium), inferior vena cava, left ventricle, and skeletal muscle. The inferior cava vein and the right atrium have a similar, accentuated high bivalent (Ca2+, Mg2+, and Zn2+) cation content; Ca2+ and Mg2+ is present in a minor content in the right ventricle and type II "pale" skeletal muscle while only Mg2+ was also reduced in the left atrium, aorta, and the whole blood. A higher K+ concentration is seen in the right atrium, aorta, and type I "red" skeletal muscle. In the aortic wall and the whole blood a higher Na+- tissue content is found, confirming earlier observations. There is a discret water retention in the tissues from the left ventricle and skeletal muscle, simultaneously with a small depletion in the whole blood. We have concluded, that there is a specific cationic profile in SHR structural and functional different cardiovascular tissues, including skeletal muscle. The cationic tissue distribution is related to underlying genetic and adaptative factors and may be involved into specific drug effects.

Immunohistochemical differentiation between lymphangiographically verified lymphatic vessels and blood vessels.

In order to investigate the antigen profile in human lymphatic vessels when compared with blood vessels, postmortem retrograde lymphangiography was done via the thoracic duct on six patients. Formalin fixed, paraffin embedded tissue was stained immunohistochemically for Factor VIII-Related antigen (F VIII R:Ag), with Ulex Europaeus 1 lectin (UEA-1) and for laminin. The results show that the endothelium of blood vessels and lymphatics at all levels of the lymphatic system react positively following staining for Factor VIII-R:Ag and with UEA-1 lectin. The staining for F VIII R:Ag was generally weaker in the endothelial cells lining lymphatic vessels. Staining for the basement membrane component laminin can be used to distinguish lymphatic capillaries and smaller lymphatic collecting vessels from blood vessels.

Identity of ED2-positive perivascular cells in rat brain.

A controversial, though fundamental, issue in neurobiology concerns the nature, origin, and function of brain macrophages. By immunocytochemical analysis using monoclonal antibodies directed against rat macrophage antigens, i.e., ED1-3, Ox-41, Ox-42, and Ki-M2R, we show that a group of perivascular cells located within the basal membrane of CNS blood vessels are immunoreactive. These cells, which resemble pericytes in terms of their anatomical distribution, are distinct from resting parenchymal microglia immunologically as well as morphologically. Our results demonstrate considerable heterogeneity in the immunophenotype of resident brain macrophages, which may be part of the immune-nervous system interface.

Absence of N-methyl-D-aspartate receptors on ovine cerebral microvessels.

Radioreceptor methods were used to quantitate the N-methyl-D-aspartate (NMDA) receptor-complex of ovine cerebral microvessels and cerebral gray matter. Specific binding of D[3H]2-amino-5-phosphono-pentanoate and [3H]1-[1-(2-thienyl)cyclohexyl]piperidine, ligands for the NMDA primary acceptor site and ionophore, respectively, was found in cerebral gray matter but was not detectable in membranes prepared from brain microvessels enriched in capillaries. Sigma receptors, another locus of action for phencyclidine congeners, were also not present on microvessels but were found in cortical homogenates. On the other hand, cerebral microvessels and gray matter contained significant numbers of beta-adrenoceptors. Our results indicate the NMDA receptors and NMDA antagonists are unlikely to regulate the function of the cerebral microvasculature.

Determination of fatty acids as phenacyl esters in rat adipose tissue and blood vessel walls by high-performance liquid chromatography.

Twenty-two biologically relevant (6:0-22:6) saturated, monounsaturated and polyunsaturated fatty acids were separated by reversed-phase high-performance liquid chromatography after derivatization with phenacyl bromide. An optimal resolution of the critical combinations linolenic-myristic, docosahexaenoic-palmitoleic-arachidonic and palmitic-oleic acids and cis and trans isomers of octadecenoic (n9) and octadecadienoic (n9, 12) acids was achieved by continuous gradient elution with methanol-acetonitrile-water. Elution of mixtures of 6:0-22:1 fatty acids was completed within 80 min at a flow-rate of 1 ml/min. By the use of UV detection at 242 nm the detection limits for short- and long-chain fatty acids were found to be about 0.8 and 12 ng per injection, respectively. Linearity was tested up to 100 ng. The method was applied to the determination of fatty acids in rat adipose tissue and blood vessel walls of animals fed hydrogenated fat diets. The results are comparable to those obtained by gas chromatography and surpass the latter for the resolution of oleic and elaidic acids.

Cerebral microvascular and parenchymal phospholipid composition in the mouse.

Cerebral microvessels consisting predominantly of capillaries and small arterioles (less than 30 micron dia.) were isolated from the cerebral cortex and cerebellum of 3-month-old mice. Lipids were extracted from both microvascular and brain parenchymal fractions and the major phospholipid classes (choline phosphoglyceride, ethanolamine phosphoglyceride, inositol phosphoglyceride, serine phosphoglyceride, and sphingomyelin) separated by 2-dimensional TLC. Comparison of mol % determined by phosphate analysis of each phospholipid revealed significant differences in membrane composition of ethanolamine phosphoglyceride, inositol phosphoglyceride, and sphingomyelin between microvascular and parenchymal components of the central nervous system. Moreover, the choline phosphoglyceride/sphingomyelin mol ratio, one of three determinants of membrane fluidity, is significantly lower for microvessel membrane than for membranes of the brain parenchyma.

Effects of the novel dopamine beta-hydroxylase inhibitor SK&F 102698 on catecholamines and blood pressure in spontaneously hypertensive rats.

The novel dopamine beta-hydroxylase (D beta H) inhibitor SK&F 102698 was characterized in vitro with soluble enzyme from bovine adrenal medulla and in vivo by measuring the dopamine/norepinephrine (DA/NE) ratio in the mesenteric artery, heart and brains of spontaneously hypertensive rats (SHR). SK&F 102698 was a potent D beta H inhibitor with an IC50 of 1.2 microM on crude enzyme and had a Ki value of 40 nM on purified enzyme. SK&F 102698 produced a dose-dependent fall in NE and a dose-dependent increase in DA in the vasculature of SHR after p.o. administration. Elevation of the vascular DA/NE ratio was observed within 0.5 hr after administration. Peak effects were observed at 12 hr and values were still significantly increased at 18 hr. The rise in the DA/NE ratio of the blood vessels correlated with the fall in blood pressure following the first 4 hr after SK&F 102698. SK&F 102698 inhibited SHR heart D beta H and elevated the myocardial DA/NE ratio approximately 2.4-fold. SK&F 102698 also caused a dose-dependent increase in the whole brain DA/NE ratio of SHR. Catecholamine levels were also studied in six discrete brain regions and SK&F 102698 produced the greatest increase in the DA/NE ratio in the cerebellum, brain stem and midbrain regions, whereas the striatum was the region least affected. No overt sedation was observed in the rats. Further study with SK&F 102698 is warranted to better explore the role of DA and D beta H in pathophysiology, and to determine whether this drug or a congener D beta H inhibitor will be a useful therapeutic agent in humans.

Occurrence and distribution of glycoconjugates in human tissues as detected by the Erythrina cristagalli lectin.

We applied a horseradish peroxidase-Erythrina cristagalli agglutinin (HRP-ECA) conjugate for histochemical staining of tissue sections from various formalin-fixed, paraffin-embedded human tissue specimens. The HRP-ECA conjugate showed broad reactivity, but there was a distinct distribution of native (not masked by sialic acid) and sialic acid-masked ECA binding sites in the various organs. Free ECA binding sites could be detected on red blood cells, lymphocytes of thymus, tonsil, lymph node, and in mucous substances of different organs. Independent of blood group type, the vascular endothelium exhibited strong ECA reactivity. Free ECA binding sites occurred in the cytoplasm of Kupffer's cells in liver, in histiocytic cells of thymus, lymph node, tonsil, and in bone marrow. Podocytes of kidney glomerulus, syncytiotrophoblasts of placenta, megakaryocytes in bone marrow, myelin sheath of nerve, medullary thymocytes, and hepatocytes, as well as islet cells of pancreas, contained only sialic acid-capped ECA binding sites. Inhibiting studies with galactose, lactose, and N-acetyl-lactosamine, as well as other sugars, revealed that this lectin is specific for galactosyl residues. In comparison to galactose and lactose, N-acetyl-lactosamine exhibited the highest inhibitory activity on lectin binding, supporting the concept that this lectin is most reactive with N-acetyl-lactosamine-type (type 2 chain) glycoconjugates.

Ultrastructural studies of glycoconjugates in brain micro-blood vessels and amyloid plaques of scrapie-infected mice.

Lectin or glycoprotein-gold complexes and samples of scrapie-infected mouse brain embedded in Lowicryl K4M were used for ultrastructural localization of glycoconjugates. The lectins tested recognize the following residues: beta-D-galactosyl [RCA, Ricinus communis agglutinin (aggl.) 120], N-acetyl and N-glycolyl neuraminic acid (LFA, Limax flavus aggl.), N-acetyl-D-glucosaminyl and sialyl (WGA, Wheat germ aggl.), N-acetyl-D-galactosaminyl (HPA, Helix pomatia aggl., and DBA, Dolichos biflorus aggl.), alpha-D-mannosyl/alpha-D-glucosyl (Con A, Concanavalin A), alpha-D-galactosyl and alpha-D-galactopyranoside (BSA, Bandeirea simplicifolia aggl., izolectin B4). Labeling of the majority of micro-blood vessels (MBVs) located outside the plaque area and in the remaining cerebral cortex was similar to that which has been previously observed in non-infected animals. Some MBVs, however, located inside the plaque area and surrounded directly by amyloid fibers showed attenuation of the endothelium, the surface of which was scarcely and irregularly decorated with RCA, LFA, WGA and Con A. These abnormalities in the composition of glycoconjugates can be associated with previously noted increased permeability of some MBVs in the brains of scrapie-infected mice. Some vessels in the plaque area were encapsulated by perivascular deposits of homogeneous or flocculogranular material containing several glycoconjugates. A very intimate structural relation between reactive (microglial-like) cells and amyloid fibers suggests the participation of these cells in elaboration of plaque material. Labeling of the cell surface and adjacent amyloid fibers with the same lectins (RCA, WGA, DBA, Con A) suggests the possibility that the glycosylation of these fibers occurs extracellularly. Only WGA and DBA were occasionally labeling some Golgi elements of the reactive cells.