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Transport of 2-deoxy-D-[3H]glucose in microvessels isolated from bovine cerebral cortex.

Microvessels were isolated from a bovine cortex and the transport of glucose was investigated by using 2-deoxy-D-[3H]glucose (2-DG). The apparent Km for 2-DG transport was 118 microM and therefore indicates a significant high affinity for the substrate. The inhibition of 2-DG uptake by D-glucose showed an apparent Ki of 222 microM. Other sugars, e.g., 3-methyl-D glucose and D-fructose, also inhibited the 2-DG uptake by 60.6 and 36.0%, respectively. Phloretin (1 X 10(-3) M) inhibited the 2-DG transport more than phlorizin (83.7 vs. 53.8%). Ouabain (1 and 5 X 10(-4) M) did not inhibit the uptake of 2-DG but 2,4-dinitrophenol (1 X 10(-4) M) did (78.0%). The uptake of 2-DG could not be demonstrated in homogenized microvessels. Adenine nucleotides (conc. 2 mM) had various effects on the 2-DG uptake by microvessels. ATP inhibited the uptake by 20.7%, ADP was virtually without effect, and AMP stimulated the uptake of 2-DG by 8.5%. It was also found that the decrease of adenylate energy charge favors the uptake of 2-DG. All these findings suggest that, in cerebral microvessels of a bovine cortex, 2-DG is apparently transported by a specific, carrier-mediated transport system.

2,4-Dinitrophenol↗

Beta-adrenergic receptor activity of cerebral microvessels is reduced in aged rats.

The effect of age on beta- (beta) adrenergic receptor number (Bmax) and adenylate cyclase (AC) activity was determined in microvessels isolated from male F-344 rats at 3, 18, and 24 months of age. Scatchard analysis of [125I]iodocyanopindolol (ICYP) binding indicated reduced Bmax (fmol/mg) of microvessels isolated from 24 month old rats (27.2 +/- 4.9) compared with 3 month old (50.4 +/- 5.2) and 18 month old rats (p less than 0.01) (61.4 +/- 7.6). The basal AC activity (pmol cAMP/mg) in 24 month old rats (32.0 +/- 6.7) and in 18 month old rats (30.4 +/- 2.1) were significantly reduced compared to the basal activity in the young (50.1 +/- 4.2). The net isoproterenol or NaF stimulated AC activity in 24 month old rats (zero and 15.6 +/- 8.5 respectively) was also reduced compared to young rats (10.1 +/- 3.9 and 166.0 +/- 21.2 respectively). It is concluded that aging is associated with reduced isoproterenol stimulated AC activity of cerebral microvessels. This reduction is the product of reduced beta-adrenergic receptor number and reduced activity of AC in aged rat cerebral microvessels.

Adenylyl Cyclases↗

Effects of gamma irradiation on cultured rat and mouse microvessel endothelial cells: metastatic tumor cell adhesion, subendothelial matrix degradation, and secretion of tumor cell growth factors.

The effects of gamma-irradiation on the properties of microvessel endothelial cells were studied in vitro. After incubating confluent endothelial cell monolayers in low serum-containing medium for 24 h, the monolayers were irradiated with 137Cs. Survival of rat lung microvessel endothelial (RLE) and mouse brain microvessel endothelial (MBE) cells were similar after irradiation (Do = 2.17 and 1.75 Gy, Dq = 4.44 and 5.67 Gy, and n = 7.8 and 25 for RLE and MBE cells, respectively). We examined the effects of gamma-irradiation on endothelial cell morphology, adhesion of syngeneic rat lung or mouse brain metastasizing tumor cells, release of the subendothelial matrix-degrading enzyme heparanase, and secretion of soluble mitogenic factors that stimulated the growth of syngeneic metastatic tumor cells. The effects of gamma-irradiation were not apparent until several hours after irradiation, and by 24 h doses of greater than or equal to 10 Gy caused limited endothelial cell retraction and reorganization of the endothelial monolayer. By 24 h after irradiation there was also increased adhesion of metastatic tumor cells to RLE but not MBE cells. We also examined the effects of gamma-irradiation on the release from endothelial cells of enzymes that solubilize the subendothelial matrix. Radiation resulted in a significant increase in the release of matrix-degrading enzyme (heparanase) that solubilized [35S]-labeled heparan sulfate from subendothelial matrix. This was most pronounced in the 24 h sample from gamma-irradiated endothelial cells. Finally, we examined the gamma-irradiation-induced release of mitogenic factors from endothelial cells that could stimulate the growth of metastatic cells in serum-limiting medium. The medium from RLE but not MBE cells stimulated the growth of a rat mammary carcinoma cell line. The results suggest that gamma-irradiation of microvessel endothelial cells can affect the interactions of tumor cells with endothelial cells and their subendothelial matrix; these processes could facilitate metastasis formation in irradiated tissues such as the lung.

Adenocarcinoma↗

Monoamine oxidase activities in human brain microvessels.

Microvessels can be easily isolated from human brain samples obtained at autopsy. Human frontal cortex MAO type A and B activities are similar in microvessel and microvessel-free preparations. In microvessels, enzyme activities and the ratio of MAO type A to type B vary among the areas studied and could selectively regulate the passage of certain amines though the blood vessel wall.

Animals↗

Endothelin receptor in microvessels isolated from human meningiomas: quantification with radioluminography.

1. We characterized specific 125I-endothelin-1 (125I-ET-1) binding sites in microvessels isolated from human meningiomas, using an in vitro quantitative receptor autoradiographic technique coupled to a radioluminographic imaging plate system. 2. This newly developed and highly sensitive method revealed high-affinity ET receptors present in pellet sections of the microvessels from all the meningiomas studied, regardless of histological subtypes (dissociation constant, 1.2 +/- 0.3 nM; maximum binding capacity, 185 +/- 56 fmol/mg; means +/- SE for nine tumors). 3. In five cases of meningiomas, ET-3 competed for 125I-ET-1 binding to microvessels from those tumors with a low affinity [50% inhibiting concentration (IC50) of 1.6 +/- 0.4 x 10(-6) M], and a selective ETB receptor agonist, sarafotoxin S6c, up to 10(-6) M, did not displace ET binding from the sections. 4. In the sections of microvessels from four other tumors, biphasic competition curves were obtained in the case of incubation in the presence of increasing concentrations of ET-3, with an IC50 of 1.1 +/- 0.2 x 10(-9) M for the high-affinity component and 1.6 +/- 0.3 x 10(-6) M for the low-affinity component, respectively. In addition, S6c competed for ET binding to those sections (IC50 = 2.3 +/- 0.2 x 10(-10) M) and 10(-6) M S6c displaced 30% of the control, corresponding to the high-affinity component of competition curves obtained in the presence of ET-3. 5. Our results suggest that (a) capillaries in human meningiomas express a large number of high-affinity ETA (non-ETB) receptors with a small proportion of ETB receptors, and (b) ET may have a role in neovascularization, tumor blood flow, and/or function of the blood-tumor barrier in meningioma tissues by interacting with specific receptors present on the surface of the endothelium.

Adolescent↗

Alpha 1-adrenergic receptor binding sites in post-mortal human cerebral microvessel preparations: preservation in multi-infarct dementia and dementia of Alzheimer type.

Cerebral cortical microvessels were prepared from control, dementia of Alzheimer type (AD/SDAT) and multi-infarct dementia (MID) autopsy cases. The microvessel yields were approx. 200 micrograms protein/g starting material, and did not differ significantly between control, MID and AD/SDAT groups. The purity of the preparations was confirmed both by light and electron microscopy and by measurement of enrichment of the endothelial markers gamma-glutamyltranspeptidase and alkaline phosphatase. Higher microvessel alkaline phosphatase activities and higher microvessel/homogenate ratios of activities of both enzymes in the MID and the AD/SDAT samples than in the control samples were found, which may be consistent with previous findings of structural abnormalities of the cerebral endothelial cells in AD/SDAT. The levels of [3H]prazosin binding did not differ significantly between control. MID and AD/SDAT samples at any [ligand] tested (0.05, 0.1, and 0.5 nM), suggesting conservation of microvessel alpha 1-adrenoceptors in MID and AD/SDAT.

Aged↗

Ultrastructure of microvessels and tumor cell processes on central neurocytoma.

A case of central neurocytoma that was confirmed with ultrastructural and immunohistochemical studies has been reported. Ultrastructurally, thin cytoplasmic processes of tumor cells showed differentiation into neuronal cells containing parallel bundles of microtubules and abortive synapses with dense-core vesicles and/or clear vesicles. It was frequently found that the clusters of tumor cell processes were close to or around the microvessels. Microvessels were composed of endothelial cells without fenestrations and had tight junctions in the endothelial clefts. Neurosecretory granules in thin cell processes appeared close to microvessels and may have been secreted around microvessels.

Adult↗

Dynamic MR Imaging and Tumor Angiogenesis in DMBA-Induced Rat Breast Cancer: Three Dimensional (3D) Reconstructed Image Analysis of Tumor Microvessels by Confocal Laser Scanning Microscopy.

We investigated the correlation between dynamic magnetic resonance imaging (MRI) findings in breast cancer and tumor angiogenesis in a dimethylbenz(a)anthracene(DMBA)-induced rat breast cancer model. In this study, we clearly demonstrated the three-dimensional (3D) architecture of tumor microvessel networks(MN)by confocal laser scanning microscopy (CLSM)and also investigated the hyperpermeability of tumor microvessels. Dynamic MRI findings were closely related to tumor angiogenesis. Three-dimensional reconstructed image analysis by CLSM revealed that the depicted images were dependent on microvessel density as well as microvessel permeability. It should be emphasized that dynamic MRI may have the potential to evaluate the tumor angiogenic activity in human breast carcinoma.

Journal Article↗

Impact of c-Met expression on angiogenesis in soft tissue sarcomas: correlation to microvessel-density.

PURPOSE AND METHODS: The c-Met protein is significant for oncogenesis and angiogenesis within the c-Met/HGF/SF-mediator system. The aim of this study was the analysis of c-Met immunoexpression/-synthesis and microvessel density as parameters for angiogenesis and prognosis in 115 soft tissue sarcomas. RESULTS: C-Met could be detected by immunohistochemistry in 87% of sarcomas. In all, 60.9% of cases exhibited absent or faint expression of c-Met protein, and 39.1% high expression of c-Met protein with a correlation between tumor grading and c-Met immunoexpression. Using in situ hybridization with detection of c-met-mRNA-transcripts, c-Met protein synthesis within tumor cells could be demonstrated. A statistically significant correlation between c-Met immunoexpression and tumor microvessel density was found. No prognostic value of c-Met expression and microvessel density could be detected in 56 patients with clinical follow-up ( P=0.8506 and P=0.9329 for disease-free survival). CONCLUSIONS: The results underline a role of c-Met as an oncoprotein in soft tissue sarcomas with correlation between immunoexpression and grading. The statistically significant correlation between c-Met expression and microvessel density (as a parameter of tumor angiogenesis) suggests an angiogenic function of the c-Met/HGF/SF mediator system in malignant mesenchymal tumors.

Adult↗

Angiogenesis of glioma: evaluation of ultrastructural characteristics of microvessels and tubular bodies (Weibel-Palade) in endothelial cells and immunohistochemical findings with VEGF and p53 protein.

Capillary endothelial proliferation is often a prominent feature of malignant gliomas. The understanding of structural and functional characteristics of the vascular microenvironment in gliomas is essential for the design of future therapeutic strategies against this tumor. Electron microscopic analysis of the capillary endothelial proliferation in malignant gliomas indicated that the complex vascular structures within the tumor were composed essentially of immature capillaries. Immature capillaries had a narrow slitlike lumen composed of endothelial cells with their high nuclear : cytoplasmic ratio and the relative paucity of organelles. They resembled capillary buds seen in normal repair tissue. Immature microvessels caused by angiogenesis were found more frequently in marginal zone of the tumors with increased microvessels. The tubular body was an organelle observed in vascular endothelial cells and was used frequently as a marker of the endothelial cell. Tubular bodies were evaluated by quantitative measurement of the mean percent (%) ratio of the number of endothelial cells with tubular bodies to all endothelial cells in microvessels of tumors. In glioblastomas it yielded a value of 32.4% in the margin, about two times as high as that in the center of the tumors. However, it was lower in all locations of astrocytomas. Tubular bodies in endothelial cells could be increased in proportion to neovascularization, and they might serve as a marker for increasing microvessels in astrocytic tumors. Tumor angiogenesis may be regulated by growth factors with angiogenic activities that are secreted by tumor cells. Vascular endothelial growth factor (VEGF) may play a critical role in the regulation of vascular endothelial proliferation. We found that 86% of 29 glioblastomas and 79% of 14 anaplastic astrocytomas demonstrated immunoreactivity for VEGF in their tumor cells. There tended to be a correlation between VEGF and vascularity. A correlation existed between the grade of immunoreactivity for VEGF and the grade of p53 protein expression in the malignant gliomas. However, the MIB-1 indices did not increase in correlation with increase in the extent of immunoreactivity for VEGF.

Brain↗

Barrier function of microvessels and roles of glial cell line-derived neurotrophic factor in the rat testis.

The expression and localization of glial cell line-derived neurotrophic factor (GDNF) and its receptor GFR alpha1 in the testis were examined, because the blood-testis barrier is a well-known tissue barrier and we previously reported that GDNF reduced the endothelial permeability of the blood-brain barrier (BBB). Five minutes after intravenous injection of Evans blue (molecular weight, 960.6) or fluorescent dextran (molecular weight 10000 and 70000), Evans blue was observed outside microvessels of the testis, whereas the fluorescent dextran was not. Immunohistochemically, GDNF was detected in alpha-smooth muscle actin-positive cells around the seminiferous tubules and in microvessels. On the other hand, GFR alpha1 was detected in endothelial cells in the interstitial space, as well as in spermatocytes. Although occludin was positive in Sertoli cells and endothelium, claudin-5 was localized only in the endothelium of the microvessels. Thus, it became very clear that the microvessels in the testis possessed relatively impermeable tight junctions, and that the alpha-smooth muscle actin-positive cells secreted GDNF, which receptor was expressed in endothelial cells. Because this relation between GDNF and GFR alpha1 is similar to that observed in the BBB, we hypothesize that GDNF is a general regulator of tight junctions of the endothelium forming a blood-tissue barrier in a paracrine fashion.

Amino Acid Sequence↗

JSR photolithography based microvessel scaffold fabrication and cell seeding.

A simple and inexpensive lithograph approach, in which the PMMA polymer was selected to be the substrate, the negative photoresist JSR was employed to form the microchannel structure, was adopted to fabricate the microvessel scaffold. In addition, a soft PDMS based microvessel scaffold was built by using a mold that was made up of the negative photoresist JSR. With O(2) plasma treatment, the PDMS based microvessel scaffold became more hydrophilic such that the cell culture could be easier to conduct. During cell culture, it was found that the fabricated scaffold enabled the bovine endothelial cells (BEC) to statically grow. However, the overall exchange of nutrient and oxygen was inefficient. Dynamic seeding by a novel apparatus was further conducted to have better circulation of culture medium. The bovine endothelial cells could successfully be cultivated in the microvessel scaffold by dynamic seeding.

Animals↗

Modeling, design and validation of a novel microfluidic sensor for in-vitro isotonic measurement of microvessel contraction/dilation.

The paper presents an innovative fluidic microdevice for in-vitro isotonic measurements of microvessel contraction/dilation. The proposed microdevice is based on the well-known fluid dynamic principle which correlates a geometrical change of a solid body immersed in a constant flow, with the differential pressure change induced in the flow itself. Indeed, a biochemically-induced change of microvessel diameter lead to a change of its hydraulic resistance which can be in-vitro measured by a differential pressure sensor. The novel microfluidic sensor has been modeled, designed and fabricated in order to properly implement this working principle. The fluidic scheme of the sensor consists of two pressure-sensorized chambers which are connected by a calibrated channel where the microvessel is placed. Experimental tests have been performed by using three microvessel passive simulators with different inner diameter (300, 600 and 800 microm), in order to validate the expected sensor functioning characterized by two sensitivities: the pressure drop S (PL) and the overall S 2-3 ones. Their measured values (S (PL,m)=2.132 mV/V/Pa, S (2-3,m)=0.787 mV/V/(mm3/s)/mm) confirm the validity of the proposed working principle and permit to be confident in a future application of the sensor in a clinical context.

Blood Flow Velocity↗

Transforming growth factor beta activates protein kinase C in microvessels isolated from immature rat brain.

We investigated the activation of protein kinase C in microvessels isolated from rat brain. We found that unstimulated kinase activity in microvessels from immature animals is soluble while that from adults is particulate. The tumor promoter, phorbol 12-myristate 13-acetate, and the diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol, caused the redistribution of protein kinase C activity to the membrane fraction in microvessels from immature rats. Exposure to transforming growth factor beta resulted in similar redistribution of kinase activity. To our knowledge, this is the first report of an effect of transforming growth factor beta on protein kinase C. The kinase activity in microvessels from adult animals was unaffected by exposure to these agonists. We suggest that protein kinase C activation promotes differentiation of the brain microvasculature. Transforming growth factor beta may mediate this process.

Aging↗

C-type natriuretic peptide is a potent activator of guanylate cyclase in endothelial cells from brain microvessels.

An exposure of endothelial cells from rat brain microvessels to C-type natriuretic peptide (CNP) resulted in a rapid and large increase in cGMP formation. The action of CNP did not require inhibitors of phosphodiesterases to be observed and occurred at nanomolar concentrations. Other natriuretic peptides (ANP and BNP) also stimulated cGMP formation in endothelial cells from brain microvessels but with a potency that was at least 100 times less than that of CNP. In contrast, endothelial cells from the aorta showed large cGMP responses to low concentrations of ANP and BNP but were unresponsive to CNP up to concentrations as large as 100 nM. It is concluded that endothelial cells from brain microvessels and from aorta express different receptors subtypes for natriuretic peptides. Endothelial cells from brain microvessels express CNP specific ANPB receptors; aortic endothelial cells express ANP (and BNP) specific ANPA receptors. CNP may play an important role in the regulation of water and electrolyte movements across the blood brain barrier.

Animals↗

Potassium activation of the Na,K-pump in isolated brain microvessels and synaptosomes.

Brain capillary endothelial cells play an important role in ion homeostasis of the brain through the transendothelial transport of Na and K. Since little is known about the regulation of ion transport in these cells, we determined the effect of extracellular potassium concentration ([K]o) on the kinetics of the Na,K-pump in isolated cerebral microvessels using both K uptake and Na efflux as measures of pump activity. In addition, we studied K activation of K uptake into synaptosomes under similar conditions to compare this neuronal system to the capillary. When microvessels were preloaded with 22Na by 30 min incubation in K-free buffer, efflux of 22Na into buffer with varying [K]o was dependent on [K]o and inhibited by 7 mM ouabain. This activation of Na efflux was half maximal at 4.2 mM [K]. Ouabain-sensitive K uptake was also half maximally stimulated by a similar [K] in both Na loaded and non-loaded microvessels. In contrast, K uptake into synaptosomes was half maximal at 0.47 mM K. These results demonstrate that both active Na efflux and K uptake into microvessels in vitro are dependent on [K]o in the physiological range. In contrast, synaptosomal K uptake is near maximal at 3 mM K. This suggests that increases in brain [K]o may stimulate ion transport across the cerebral capillary, but will have little effect on Na,K-pump activity in neurons.

Animals↗

Atrial natriuretic peptide receptors in cerebral microvessels and choroid plexus of spontaneously hypertensive rats.

Binding sites of atrial natriuretic peptide in the cerebral microvessels and choroid plexus of spontaneously hypertensive rats (SHR) and Wistar Kyoto control rats (WKY) were measured. In the microvessels, the number of ANP binding sites was lower in SHR than WKY, but there was no difference in affinity of binding sites for the ligand between SHR and WKY. In the choroid plexus, the number of ANP binding sites was lower and the affinity for the ligand was higher in SHR than in WKY. These results suggest that the physiological function regulated by the ANP receptors in the cerebral microvessels and choroid plexus were altered in hypertension and that ANP receptors in the cerebral microvessels and choroid plexus were differentially regulated.

Animals↗

Identification of proton-translocating adenosine triphosphatases in rat cerebral microvessels.

To determine if proton translocating adenosine triphosphatases (ATPases) can be localized at the blood-brain barrier, isolated rat cerebral microvessels and cerebral synaptosomal preparations were assayed for ATPase activity in the presence of various inhibitors. N-ethylmaleimide-sensitivity could be consistently found in both cerebral microvessel and synaptosomal preparations. There was no vanadate sensitive component in the presence of ouabain, oligomycin and EGTA. Immunoblotting of cerebral microvessels and synaptosomes with a monoclonal antibody (E11) against the 31 kDa subunit of the vacuolar type H-ATPase pump identified a discrete 31 kDa band. Diffuse immunocytochemical staining of cerebral cortical tissue, predominantly in choroid plexus, could be found with E11 but not with HK alpha N2, an H,K-ATPase specific antibody, nor with a non-specific mouse monoclonal antibody (MOPC-21). Immunoblotting with HK alpha N2 showed an immunoreactive 76 kDa band, not present with the preimmune serum or the antibody preabsorbed with the immunizing synthetic peptide. It is concluded that the vascular type H-ATPase and not the gastric H,K-ATPase is present in cerebral tissue including cerebral microvessels and choroid plexus. Non-specific immunoreactivity may account for the 76 kDa band observed in the immunoblots using the HK alpha N2 antibody although presence of a degradation product of H,K-ATPase can not be ruled out. The functional role of the vacuolar H-ATPase in the blood-brain barrier remains to be determined.

Animals↗