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At least 775 records · Page 43Linked to original sources

PubMed1993

Identification of GTP binding proteins in brain microvessels and their role in phosphoinositide turnover.

The identity of GTP binding proteins in cerebral microvessels was investigated by immunoblotting. Data indicate the presence of a characteristic pattern of Gi, Gs and Go. The most remarkable finding is the presence of Go protein in cerebral microvessels that consist predominantly of capillary segments free of neuronal contaminants. The pattern of pertussis toxin-catalyzed ADP-ribosylation of a 40 kDa polypeptide is characteristic of the Gi/Go type alpha-subunits. In addition, we have observed that GTP tau S, a non-hydrolyzable guanine nucleotide, exerts a dual regulatory effect on phosphoinositide metabolism depending on the concentration, thus 10(-7) M concentration inhibits the 32P incorporation into PIP2 and PA by a pertussis toxin-sensitive mechanism; on the contrary, the stimulatory effect of higher concentration than 10(-6) M of GTP tau S is pertussis toxin-insensitive.

Adenosine Triphosphate↗

PubMed1993

Specific uptake of succinylated proteins via a scavenger receptor-mediated mechanism in cultured brain microvessel endothelial cells.

Cellular uptake of succinylated catalase (Suc-CAT; Mw 227 kDa), bovine serum albumin (Suc-BSA; Mw 70 kDa), superoxide dismutase (Suc-SOD; Mw 34 kDa) and soybean trypsin inhibitor (Suc-STI; Mw 21 kDa) was studied using primary cultures of bovine brain microvessel endothelial cells (BMECs) developed as an in vitro model of the blood-brain barrier. Large succinylated proteins (Suc-CAT, Suc-BSA) were taken up by BMECs whereas significant uptake was not observed for native proteins and small succinylated proteins (Suc-SOD, Suc-STI). Uptake of Suc-BSA was significantly inhibited at 4 degrees C and in the presence of endocytosis inhibitors. Large succinylated proteins, maleylated BSA and dextran sulfate also showed competitive inhibition against Suc-BSA uptake while small succinylated proteins and carboxymethyl dextran did not show any effect. These results indicate that microvessel endothelial cells obtained from the brain endocytose succinylated proteins via a scavenger receptor-mediated mechanism for polyanions, and in addition, the importance of molecular weight or total numbers of anionic charges per one molecule of proteins is suggested. Usefulness of direct succinylation of proteins for their delivery to the brain capillary endothelium is thus demonstrated.

Animals↗

PubMed1994

Nitric oxide synthase activity is elevated in brain microvessels in Alzheimer's disease.

The cerebral microcirculation undergoes specific biochemical changes in Alzheimer's disease. In this study, we have compared the nitric oxide synthase (NOS) activity of brain microvessels isolated from Alzheimer and control brains. L-[3H]-citrulline, the stable co-product generated with nitric oxide (NO) from L-[3H]-arginine, was measured as an indicator of NOS activity. The results indicated a significant increase in NOS activity in microvessels isolated from Alzheimer brains. In addition, using antibodies to both the endothelial and inducible NOS isoforms, we demonstrated a significant increase in enzyme level in Alzheimer-derived vessels. Elevated vascular production of NO, a potentially neurotoxic mediator in the CNS, may contribute to the susceptibility of neurons to injury and cell death in Alzheimer's disease.

Aged↗

PubMed1998

Expression of multidrug resistance-associated protein (MRP) in brain microvessel endothelial cells.

Multidrug resistance-associated protein (MRP) is a recently identified drug efflux transport system that actively transports organic acids and selected glucuronide or glutathione conjugates out of the cell. The current study presents, for the first time, both functional and biochemical data demonstrating the presence of MRP in the brain microvessel endothelial cells that form the blood-brain barrier (BBB). Using known MRP inhibitors, such as indomethacin and probenecid, fluorescein accumulation in primary cultured bovine brain microvessel endothelial cell (BBMEC) monolayers was significantly enhanced compared to control. The specificity of the MRP inhibitors on cellular fluorescein accumulation was confirmed using both MRP positive (Panc-1) and MRP negative (KBv) cell lines. Furthermore, western blot analysis using a specific antibody for MRP (MRPm6) and RT-PCR studies using a complementary sequence probe for human MRP demonstrate the expression of MRP in BBMEC. Previous studies have demonstrated the significance of the P-glycoprotein drug efflux transporter in the BBB. Given its function as a drug efflux transport system, it is anticipated that MRP in the BBB will also have an important role in limiting the exposure of the brain to many endogenous and exogenous compounds, including both toxic and therapeutic agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Expression of ephrinB2 identifies a stable genetic difference between arterial and venous vascular smooth muscle as well as endothelial cells, and marks subsets of microvessels at sites of adult neovascularization.

The transmembrane ligand ephrinB2 and its receptor tyrosine kinase EphB4 are molecular markers of embryonic arterial and venous endothelial cells, respectively, and are essential for angiogenesis. Here we show that expression of ephrinB2 persists in adult arteries where it extends into some of the smallest diameter microvessels, challenging the classical view that capillaries have neither arterial nor venous identity. EphrinB2 also identifies arterial microvessels in several settings of adult neovascularization, including tumor angiogenesis, contravening the dogma that tumor vessels arise exclusively from postcapillary venules. Unexpectedly, expression of ephrinB2 also defines a stable genetic difference between arterial and venous vascular smooth muscle cells. These observations argue for revisions of classical concepts of capillary identity and the topography of neovascularization. They also imply that ephrinB2 may be functionally important in neovascularization and in arterial smooth muscle, as well as in embryonic angiogenesis.

Animals↗

PubMed1995

Chronic lead treatment accelerates photochemically induced platelet aggregation in cerebral microvessels of mice, in vivo.

Effects of two chronic treatment levels with lead on platelet aggregation in cerebral (pial) microcirculation of the mouse were investigated. Exposure to lead was made by subcutaneous injections for 7 days of lead acetate dissolved in 5% glucose solution, vehicle. Two doses of lead were used, a low dose of 0.1 mg/kg and a high dose of 1.0 mg/kg. Adult male mice were divided into three groups, 10 each; one group was injected with vehicle (control), another was injected with the low dose, and the third was injected with the high dose. Additional mice were used for the determination of hematological parameters and for the lead level in serum of the three groups. On the eighth day, platelet aggregation in pial microvessels of these groups of mice was carried out in vivo. Animals were anesthetized (urethane, 1-2 mg/g, ip), the trachea was intubated, and a craniotomy was performed. Platelet aggregation in pial microvessels was induced photochemically, by activation of circulating sodium fluorescein (0.1 mg/25 g, iv) with an intense mercury light. The time required for the first platelet aggregate to appear in pial arterioles was significantly shorter in the lead-treated mice than in control. This effect was in a dose-dependent manner; 113 +/- 44 sec for low dose and 71 +/- 18 sec for high dose vs 155 +/- 25 sec for control, P < 0.02 and P < 0.001, respectively. Between the two lead-treated groups, the high dose significantly (P < 0.05) shortened the time to first aggregate. These data evidenced an increased susceptibility to cerebrovascular thrombosis as a result of exposure to lead.

Animals↗

PubMed1993

Three-dimensional analysis of contrast-filled microvessel diameters.

Organ blood flow is controlled, in part, by changes in diameter of resistance vessels. In thick tissue, vessels can be imaged with a microscope using contrast-enhancing methods (e.g., fluorescence) and image analysis techniques can be used for quantitative diameter estimations. However, a change in the position of a vessel with respect to the plane of focus can be misinterpreted as a diameter change. In order to address this problem, a 3D image in a light microscope is obtained by serial optical sectioning, and a 3D deconvolution procedure (Avinash et al., 1991, "Fourteenth Association for Research in Otolaryngology Midwinter Meeting, St. Petersberg, FL," Abstract 156) is used to deblur 3D image data. Deblurred sections are computationally projected onto a 2D plane to give an extended-focus image, from which diameter estimates of microvessels are made using a quantitative, 2D diameter-tracking algorithm (Miles, 1987, "Semiautomatic Quantitative Image Analysis of Dynamic in Vivo Cochlear Microvessel Diameters." Ph.D. dissertation, Univ. Michigan; Miles and Nuttall, 1992, IEEE Trans. Biomed. Eng.). Justification for 3D preprocessing before diameter analysis is provided by absolute and relative error analyses using computer-generated synthetic vessels. The 3D diameter analysis technique is validated using a capillary tube of known diameter, filled with fluorescent solution. Demonstration of its applicability is shown in diameter measurements from the vessels of guinea pig cochlea. Our approach, using extended-focus images, minimizes overestimation of microvascular diameters and underestimation of relative diameter changes. Therefore, unambiguous diameter measurements are possible with extended-focus images.

Algorithms↗

PubMed1993

Effect of diameter variability along a microvessel segment on pressure drop.

In most microcirculatory studies, the diameter of a blood vessel is characterized by a single, average value, obtained from a projected two-dimensional microscopic view of the vessel. Such diameter values are often used to calculate microvascular hemodynamic variables (e.g., pressure drop along vessels). The validity, and some possible consequences, of the assumption that vessel diameter is constant along vessels were examined for (a) single capillaries of the hamster cremaster muscle and (b) a network model of blood flow in a rat mesenteric network. Vessel diameter was measured in cremaster microvessels of anesthetized golden hamsters (Nembutal, 70 mg/kg ip) at regular intervals along the lengths of individual vessels. The standard deviation of the diameter measurements (ranging from 0.2 to 4.0 microns) in each vessel increased with the average diameter (ranging from 4 to 27 microns, 132 vessels, 12 animals). The coefficient of variation (CV = SD/Mean) was close to 0.1 for vessels larger than 15 microns and up to 0.4 for smaller vessels. In individual isolated vessels, the ratio of calculated pressure drop using the actual diameter measurements (delta P) to pressure drop assuming an average diameter (delta Po) was between 1.05 and 3.0 (at constant volume flow rate); this ratio correlated significantly with the CV for that vessel. Using an iterative mathematical model of network blood flow in a mesenteric network, we investigated the effects on single-vessel delta P/delta P0 of using a single average estimate of diameter for each microvessel segment, compared to the inclusion of variations in each vessel diameter expected from the single-vessel data. The results from the model indicated that single vessel delta P/delta Po values obtained with expected diameter variations in each vessel varied between 0.01 and 100. This implies that the axial variations in vessel diameter that exist in vivo may affect the calculations of vessel pressure drop both in single vessels and in microvascular networks.

Animals↗

PubMed1993

Human lung microvessel endothelial cells: isolation, culture, and characterization.

The pulmonary vasculature is of great physiological/pathological significance. We have isolated and cultured microvessel endothelial cells (HuLEC) from lung tissue obtained from lung transplant recipients by modification of published methods. Pure cultures of HuLEC were isolated by mechanical disaggregation of the tissue prior to sequential dispase and trypsin digestion to obtain microvessel fragments. Magnetic beads (Dynabeads) coated with Ulex europaeus agglutinin-1 were then used to enhance the purity of cultures at the first passage. HuLEC formed contact-inhibited "cobblestone" monolayers on gelatin and fibronectin substrates and capillary-like "tubes" on Matrigel and accumulated acetylated low-density lipoprotein. Immunofluorescent characterization of these cells revealed the presence of von Willebrand Factor, angiotensin-converting enzyme, and thrombomodulin and the expression of antigens for the endothelial cell-specific monoclonal antibodies EN4, PAL-E, and H4-7/33. The endothelial origin of these cells was confirmed by the demonstration of the cell adhesion molecules, platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), and E-selectin (endothelial leukocyte adhesion molecule-1/ELAM-1) upon stimulation with TNF alpha. These cells should provide a useful tool for studying various aspects of pathology and biology of the pulmonary microvasculature in vitro.

Animals↗

PubMed1994

A theoretical model for gas transport and acid/base regulation by blood flowing in microvessels.

An investigation was made of the coupling between O2 and CO2 transport by blood flowing in microvessels. The blood was treated as two continuous coexisting phases: a red blood cell (RBC) phase and a plasma phase. The microvessel was divided into two regions: the central, RBC-rich and the outer, cell-free region. The radial distribution of RBCs and transport of various species due to bulk convection and radial diffusion were taken into account. Chemical and transport processes which were included in the model are (1) interactions of hemoglobin with O2 and CO2, (2) the Bohr and Haldane effects (the inter-dependence of O2/CO2 transport), (3) CO2 hydration-dehydration reactions, (4) buffering actions of hemoglobin and plasma proteins, and (5) anion exchange across the red cell membrane. The governing equations of the model subjected to the imposed inlet and boundary conditions were solved numerically to provide the concentration distributions of various species in blood that are important in the simultaneous gas exchange and pH regulation process. Predictions of the new model of simultaneous O2/CO2 transport by flowing blood were shown to be in excellent agreement with prior workers' experimental results from large artificial membrane tubes. A previous mathematical model which treats blood as a homogeneous continuum and uses a local chemical equilibrium approximation to describe the gas transport was shown to satisfactorily predict the amount of O2 transport for blood oxygenation accompanied by CO2 elimination. However, the previous model significantly underpredicts O2 transfer for blood deoxygenation accompanied by CO2 uptake. Furthermore, the previous model disagrees substantially with the CO2 transport results under both oxygenation and deoxygenation conditions.

Acid-Base Equilibrium↗

PubMed1995

Relative blood velocity measurement in individual microvessels using the self-mixing effect in a fiber-coupled helium-neon laser.

A system has been developed for the measurement of relative blood velocity in micro-vessels by using the self-mixing effect of a laser. A helium-neon laser was coupled to a single-mode optical fiber and the pulled fiber tip (approximately 30 microns diameter) was positioned on a single microvessel. The backscattered Doppler-shifted laser light from moving red blood cells enters the laser cavity and modulates the laser output by influencing internal laser parameters. The signal of the laser output intensity change was produced using a fiber-coupled photodiode and processed by a signal processor. This processor yields an output signal proportional to the first moment of the power spectral density, i.e., the mean frequency of the Doppler shift, corresponding to the blood flow velocity on an arbitrary instrument scale. Results of the in vitro experiment demonstrated that the current method can detect moving particles in fluid and moving red blood cells in a small plastic tube. Data from the in vivo study showed that this system is capable of measuring relative blood velocity in arterioles and venules and can easily follow the cardiac cycle up to 360 beats/min. Primary data suggest that, in addition to high sensitivity, good spatial and temporal resolution, and convenience of use, the self-mixing technique may have an even greater capacity for analysis of blood flow in microvessels than explored in this study, since information on the absolute velocity and velocity distribution of red blood cells is available in self-mixing signal. Further study on its hematocrit dependence and particle bias effect is needed.

Animals↗

PubMed1996

Correlation of intratumoral microvessel density and p53 protein overexpression in human colorectal adenocarcinoma.

A malignant cell population needs the development of microvessels in order to grow and metastasize. Recently, a role for the p53 gene in the regulation of this angiogenic process has been suggested. Wild-type p53 is involved in the secretion of Trombospondin-1 (TSP-1), an angiogenesis inhibitor. Mutations of the p53 gene cause a downregulation of TSP-1 mRNA in cell lines. Mutant p53 also upregulates the expression of vascular endothelial cell growth factor, a potent angiogenic factor. Together with the reported association of p53 protein overexpression and microvessel density (MVD) in head-and-neck squamous-cell carcinoma, these in vitro findings led us to investigate whether this association would also apply in colorectal adenocarcinomas. Structural changes of the p53 gene are the most frequent observed mutations in colorectal carcinoma and are suspected to be involved in the carcinogenesis at a relatively early stage. Parallel tissue sections from primary colorectal adenocarcinomas were immunostained for CD31, an endothelial cell marker, and with DO7, recognizing both mutant and wild-type p53 protein overexpression. The presence of p53 protein overexpression was found to be significantly associated with high MVD in the vascular hot spots. Our results are in accordance with the in vitro studies on the involvement of p53 in angiogenesis. Mutant p53 might stimulate tumor angiogenesis both indirectly, by augmenting the tumor cell proliferation, and directly, by upregulating angiogenic factors and downregulating angiogenic inhibitors.

Adenocarcinoma↗

PubMed1996

Lung microvessel injury from peritoneal abscesses and gram-negative bacteremia.

To analyze the effect of an extrathoracic focus of infection on lung vessel wall structure, we produced peritoneal abscesses in the rat, over a period of 3 to 7 weeks, by implanting capsules containing live gram-negative bacteria (3.0 x 10(7) Escherichia coli and 5.0 x 10(7) Bactercides fragilis) with an adjutant. We document here by arteriography, morphometric analysis, and high resolution microscopy, microvessel cell injury, and wall remodeling. In the sepsis-injured lung, both dilated and thick-walled microvessels are present. The walls of dilated vessels are disrupted by extensive endothelial and precursor smooth muscle cell injury. In thick-walled vessels these cells are hypertrophied, and the precursor smooth muscle cells express filaments, demonstrating a shift toward a contractile phenotype. Infiltrating monocytic cells focally consolidate alveolar regions. In the residual nonconsolidated regions, alveolar-capillary membrane cells are attenuated and the capillaries dilated. Vessel changes after 7 weeks are similar to those after 3 weeks but alveolar-capillary membrane injury is more extensive. These structural changes, including the development of precursor smooth muscle cells, may contribute to the known increase in reactivity of these lung vessels to challenge by vasoactive agents.

Abdominal Abscess↗

PubMed1997

Permselectivity of angiogenic microvessels following alteration of the endothelial fiber matrix by oligosaccharides.

The endothelial glycocalyx, which is composed of integral and peripheral glycoconjugates, forms a fibrous matrix that confers macromolecular sieving properties on the microvascular wall. Changes in pore size within the matrix may regulate macromolecular access to the paracellular and/or vesicular transendothelial pathways. We tested the hypothesis that modifications of the endothelial glycocalyx might play a role in the ontogeny of endothelial permselectivity in proliferating microvessels of the chick chorioallantoic membrane (CAM). Accordingly, we evaluated the effects of Dolichos biflorus agglutinin (DBA) or Arachis hypogaea agglutinin (PNA) lectin binding, and N'N'diacetylchitobiose or hydroxyethyl starch polysaccharide (HES) incorporation on CAM endothelial restriction of FITC-dextrans 40 or 150 at Days 4.5 and 5.0 of development. Extravasation of FITC-dextrans was determined by recording their perivascular interstitial intensities. Following DBA, PNA, and N'N'diacetylchitobiose administration, interstitial accumulation of the tracers near first-order pre- and postcapillaries, and surrounding the capillaries, was similar to that of controls at both Days 4.5 and 5.0. At Day 4.5, pretreatment with HES significantly decreased extravasation of FITC-dextran 40. Thus, retention of HES molecules within the glycocalyx might tighten the matrix, and reduce access of dextran 40 to transendothelial pathways across the angiogenic microvessels.

Animals↗

PubMed1998

Evaluation of enhanced high-resolution laser Doppler imaging in an in vitro tube model with the aim of assessing blood flow in separate microvessels.

An enhanced high-resolution laser Doppler imaging (EHR-LDI) technique intended for visualization of separate microvessels was evaluated by use of in vitro flow models. In EHR-LDI, a laser beam focused to a half-power diameter less than 40 microm successively scans the tissue under study in steps of 25 microm. Spatial blood flow variations within microvascular structures of 1.5 x 1.5 mm are rendered by 64 x 64 measurement sites. Individual microvessel diameters could be estimated and an average difference of 11 microm compared to microscopic measurements was obtained. For the flow algorithm used, the LDI output signal was found to scale linearly with average velocity (0-3.5 mm/s) when a plastic tube of inner diameter 175 microm was perfused with human blood (correlation coefficient 0.99). The LDI output signal was further found insensitive to hematocrit variations in the range 16-44%. Due to the limited laser light penetration in blood, a reduction in the LDI output signal was observed as the inner tube diameters were successively changed from 280 to 1400 microm.

Animals↗

PubMed1999

Expression of membrane-bound and soluble cell adhesion molecules by human brain microvessel endothelial cells.

Expression of membrane-bound (mb) and soluble (s) forms of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor-alpha (TNF-alpha) has been measured by enzyme-linked immunosorbent assay in cultured human brain microvessel endothelial cells. Both the mb and the s forms of VCAM-1 and ICAM-1 were upregulated by TNF-alpha; however, the stimulation of the s forms was delayed in time. When piracetam, a neuroprotective drug, was added to the tissue culture medium simultaneously with TNF-alpha, the expression of mbVCAM-1 and ICAM-1 was lowered. Differential upregulation of mb and s forms of adhesion molecules and a novel effect of piracetam have been demonstrated in human brain microvessel endothelial cell cultures.

Brain↗

PubMed2000

Quantitative assessment of angiogenesis and tumor vessel architecture by computer-assisted digital image analysis: effects of VEGF-toxin conjugate on tumor microvessel density.

Tumor growth is angiogenesis dependent. As a consequence, strategies aimed at disrupting this mechanism are heavily investigated. Several angiogenesis assays are used to directly compare the efficacy of anti-angiogenic compounds. However, objective assessment of new vascular growth has been difficult to achieve. The aim of this study was to test and develop a computer-assisted image analysis method that would give an unbiased quantification of the microvessel density. Human tumors were grown in athymic mice and tumor biopsies were taken after a weeklong treatment with VEGF-toxin conjugate. Frozen tumor sections were prepared and stained with PE-conjugated anti-CD-31 antibodies and vessels were imaged with a fluorescence microscope. Vessel density was analyzed by quantifying PE-positive pixels per recorded field. In addition, images were further processed to investigate morphological differences by an automated binarization and skeletonization protocol. This procedure allowed the computer-assisted estimation of important angiogenic parameters such as total vessel number, length, and branch points. Based on these indices, differences in the angiogenic response between control tumors and those treated with VEGF-toxin conjugate were readily detected (P < 0.007 for all parameters). More importantly, computer-generated measurements correlated well with manual microvessel counts and showed significantly less variation. Our results suggest that computer-assisted image analysis represents a rapid, objective, and alternative method for the quantitative assessment of tumor angiogenesis and vessel architecture.

Adenocarcinoma↗

PubMed1984

Angiotensin receptors and the control of Na+ and K+ transport in cultured aortic smooth muscle and brain microvessel cells.

We examined the effect of angiotensin on Na+ and K+ transport by cultures of aortic smooth muscle and brain microvessel cells. Angiotensin II (AII) and angiotensin III (AIII) stimulated net Na+ uptake, which was assayed in the presence of ouabain to block Na+ efflux via the Na+-K+ pump. The combination of saturating concentrations of AII and AIII produced no greater stimulation of net Na+ uptake than either angiotensin by itself. AII also stimulated ouabain-sensitive 86Rb+ uptake by cultured aortic smooth muscle and brain microvessel cells. AII was not as effective as monensin, a Na+ ionophore, in stimulating the Na+-K+ pump. In the presence of monensin, AII had no effect on ouabain-sensitive 86Rb+ uptake. These data are consistent with previous observations suggesting that AII stimulates the Na+-K+ pump by supplying it with more of its rate-limiting substrate, Na+. 125I-AII bound tightly to cultured aortic muscle cells. Approximately 5 nM unlabelled AII half-maximally inhibited 125I-AII binding. Incubation of intact cultures with 1.0 microM AII produced a time-dependent decrease in specific 125I-AII binding. This slow loss of AII binding was prevented by methylamine, a known inhibitor of receptor clustering and internalization. We conclude (1) that angiotensin increases the Na+ permeability of smooth muscle cells in culture, thereby stimulating the Na+-K+ pump; (2) that AII binds to a specific receptor on the cell surface; and (3) that the AII-receptor complex may be internalized by a methylamine-inhibited pathway.

Angiotensin II↗