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PubMed1986

Three-dimensional reconstruction of vesicles in endothelium of blood-brain barrier versus highly permeable microvessels.

In this study the three-dimensional organization of pinocytotic vesicles in mouse endothelia from permeable (choroid plexus, area postrema, and skeletal muscle) and blood-brain barrier (bbb) (cerebral gray and white matter) microvessels was examined. Reconstructions of 75 segments of endothelial cells from microvessels were done with very thin (less than 23 nm) serial sections and tracings. A total of 2,013 vesicles from five tissues were reconstructed for this study. Vesicles were classified as to whether they were attached to other vesicles (fused), connected to golgi or endoplasmic peticulum (tubule-connected), open to vessel lumen or ablumen (surface-connected) or isolated in the cytoplasm (free). The densities of tubule-connected vesicles and free vesicles were the same in all four types of vessels. It seems unlikely, therefore, that these vesicles are related to vascular permeability. Vesicular clusters and surface-connected vesicles were found in much higher densities in area postrema, choroid plexus, and skeletal muscle vessels than in bbb vessels. Single-vesicle transendothelial channels were found in attenuated endothelia of area postrema and choroid plexus. These results support the hypothesis that endothelial vesicles play a role in vascular permeability, possibility by transient fusion of vesicle clusters to the plasmalemma, to form transendothelial channels.

Animals↗

PubMed1990

SEM and TEM analyses of isolated human retinal microvessel basement membranes in diabetic retinopathy.

Human retinas from persons with diabetic retinopathy and age-matched controls were rendered acellular by sequential detergent treatment. The resulting network of microvascular extracellular matrix (ECM) materials, including basement membranes (BMs), was compared by TEM and, following cryofracture, by SEM. Our study demonstrates that in diabetics, retinal capillary BM complexes are generally thickened and that their ECM subcomponents, including BM leaflets and BM-like pericytic matrix (PCM), are differentially altered. Two diabetic microvessel types were identified. In type A vessels, ECM expansion is manifested by loosely arranged combinations of concentric PCM layers and collagen fibrils with thickened subendothelial (EBM) and pericyte (PBM) BM leaflets. Type B vessels show densely compact central PCM masses and poorly recognizable EBMs and PBMs. In both types, Müller cell BMs (MBMs) are relatively unaffected. High-resolution SEM shows tissue-specific features in normal EBM and MBM surfaces, but disease-related topographic changes are not evident. It is possible that the ECM arrangements identified in our study relate to different microvessel domains and that their specific morphological features may play important roles in the pathogenesis of diabetic retinopathy including capillary closure and neovascularization.

Aged↗

PubMed2004

Microvessel density, cyclo-oxygenase 2 expression, K-ras mutation and p53 overexpression in colonic cancer.

BACKGROUND: Tumour angiogenesis, cyclo-oxygenase (COX) 2 expression, K-ras mutation and p53 overexpression are commonly involved in colorectal tumorigenesis, but their interrelationship and clinicopathological effects remain inconclusive. METHODS: Clinicopathological data from 114 consecutive patients with primary stage III colorectal cancer were evaluated prospectively. Microvessel density (MVD) of the tumour was defined by counting the number of microvessels in hotspots, visualized by immunocytochemical staining of endothelial CD34. K-ras mutation was analysed by the restriction enzyme cleavage method. COX-2 expression and p53 overexpression were determined by immunocytochemistry. RESULTS: Increased MVD in hotspots was significantly associated with COX-2 expression (P < 0.001), K-ras mutation (P = 0.007) and p53 overexpression (P = 0.006). COX-2 expression was not associated with either K-ras mutation or p53 overexpression. Clinicopathologically, greater MVD and COX-2 expression were significantly associated with vascular invasion of cancer cells (MVD, P = 0.027 and COX-2 expression, P = 0.006), but p53 overexpression and K-ras mutation were not. Multivariate analysis indicated that greater MVD (P = 0.002) and p53 overexpression (P = 0.016) were significant independent predictors of tumour recurrence, whereas COX-2 expression (P = 0.634) and K-ras mutation (P = 0.356) were not. CONCLUSION: Tumour angiogenesis may be associated with tumour metastasis and is significantly influenced by K-ras mutation, p53 overexpression and COX-2 expression in patients with colonic cancer.

Adult↗

PubMed1995

Anergy induction in encephalitogenic T cells by brain microvessel endothelial cells is inhibited by interleukin-1.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) which can be induced, in susceptible strains like Lewis rats, by transfer of activated myelin basic protein (MBP)-specific CD4+ T lymphocytes. The role of cerebral endothelium in the onset of EAE, with regard to adhesion, activation and infiltration in the CNS of encephalitogenic T lymphocytes, is not fully understood. When pretreated by interferon-gamma, the immortalized Lewis rat brain microvessel endothelial (RBE4) cells expressed major histocompatibility complex class II molecules and stimulated MBP-specific proliferation and cytolytic activity of the syngeneic encephalitogenic T cell line, designated PAS. However, RBE4-stimulated PAS lymphocytes subsequently entered an unresponsive state, known as anergy. When inoculated in syngeneic animals, anergic PAS cells, although still cytotoxic, failed to induce EAE, and no cell infiltration was detectable within CNS. The addition of interleukin-1 beta (IL-1 beta) during MBP presentation by RBE4 cells prevented T cell anergy induction, and maintained T cell encephalitogenicity, although PAS cells stimulated in these conditions caused delayed and attenuated clinical signs of EAE, with only discrete inflammatory lesions in the CNS, compared with EAE induced by PAS cells fully activated by thymic cells. Altogether, our results indicate that MBP presentation by brain microvessel endothelial cells to encephalitogenic T cells induces T cell anergy and loss of pathogenicity. In addition, IL-1 beta co-stimulation of T cells prevents anergy induction in vitro and at least partially maintains encephalitogenicity in vivo.

Animals↗

PubMed1995

Microvessel density and vessel invasion in lymph-node-negative breast cancer: effect on recurrence-free survival.

Microvessel density (MVD) and blood and lymphatic vessel invasion (BLVI) were investigated with regard to their influence on the disease-free survival (DFS) in node-negative breast cancer patients. Paraffin embedded microsections of 230 patients with T1,2 N0 breast cancer were immunohistochemically stained for factor VIII-related antigen. Every cluster consisting of more than highlighted endothelial cells was considered a countable microvessel. MVD was counted in 4 fields of 0.25 mm2 each. All MVD values are given as value for the sum of 4 fields of 0.25 mm2 each, that is, I mm2. BLVI was considered positive, when at least one tumor cell could be identified in a stained lumen. Out of 230 patients, 49 experienced local or distant recurrence and had a mean MVD of 72.4/mm2, whereas 181 patients who lived without recurrent disease had a mean MVD of 45.3/mm2. BLVI was negative in 6.2% of the cases with and in 93.8% of the cases without recurrent disease. BLVI was positive in 59.4% of the cases without and 40.6% of the cases with recurrent disease. MVD and BLVI remained the only significant prognostic factors of DFS in the Cox-Model. Tumor size, histological grade, and hormonal-receptor status were not prognostically relevant in the Cox-model. 10-year-DFS was 93.3% in BLVI-negative/MVD < or = 40/mm2 patients, 88.1% when MVD was high or BLVI was positive and 48.9% in BLVI positive/MVD < or = 40/mm2 patients. Our present data indicate that MVD and BLVI identify a very-low risk group among node-negative breast cancer patients, who will not benefit from systemic adjuvant therapy. MVD and BLVI should be used as stratification criteria in clinical trails on node-negative breast cancer patients.

Adult↗

PubMed1991

Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells.

Atrial natriuretic peptide (ANP) binding and ANP-induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid-regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time- and concentration-dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase-linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5-27 (ANP 5-27) to modulate the accumulation of cGMP was found to be ANP greater than BNP much greater than ANP 5-27. Affinity cross-linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60-70 kD receptor, indicating the presence of the nonguanylate cyclase-linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5-27 and suggested that a large proportion of the ANP receptors present in blood-brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood-brain barrier endothelial cells.

Affinity Labels↗

PubMed1992

Effect of L-fucose on proliferation and myo-inositol metabolism in cultured cerebral microvessel and aortic endothelial cells.

Decreased myo-inositol metabolism possibly contributes to the development of diabetic complications including micro and macrovascular disease. Previous studies have shown that hyperglycemia may be partially responsible for this defect. We have found that L-fucose, a monosaccharide present in low concentrations in normal circulation and found to be elevated in diabetes, causes defects in cultured endothelial cells, including alterations in myo-inositol metabolism and proliferation. Murine cerebral microvessel and bovine aortic endothelial cells take up L-fucose from the medium in a time and concentration-dependent manner. Both acute and chronic exposure of these cultured endothelial cells to media containing L-fucose at concentrations that may exist in diabetic sera cause a significant decrease in the accumulation of myo-inositol and its incorporation into inositol phospholipids. There is a concomitant decrease in the intracellular levels of myo-inositol. Kinetic analysis of the effect of L-fucose on myo-inositol uptake suggests that L-fucose competitively inhibits the transport of myo-inositol, exhibiting a Ki in the range of 1.6-4.1 mM for both cell types. Endothelial cells exposed to L-fucose concentrations of 0.5-20 mM exhibit depressed rates of proliferation in a concentration-dependent fashion. Furthermore, L-fucose causes a concentration-dependent decrease in synthesis of proteoglycan by cultured cerebral microvessel endothelial cells as measured by incorporation of 35S; however, this effect is not observed in the aortic endothelia. These data suggest that L-fucose at concentrations that may exist in diabetic sera may impair myo-inositol metabolism and proliferation of the vascular endothelium.

Amino Acids↗

PubMed1994

Regulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells.

Rat brain microvessel endothelial cells were immortalized by transfection with a plasmid containing the E1A adenovirus gene. One clone, called RBE4, was further characterized. These cells display a nontransformed phenotype and express typical endothelial markers, Factor VIII-related antigen and Bandeiraea simplicifolia binding sites. When RBE4 cells were grown in the presence of bFGF and on collagen-coated dishes, confluent cultures developed sprouts that extend above the monolayer and organized into three-dimensional structures. The activity of the blood-brain barrier-associated enzyme, gamma-glutamyl transpeptidase (gamma GTP), was expressed in these structures, not in the surrounding monolayer. Similar results were obtained with the microvessel-related enzyme alkaline phosphatase (ALP). Addition of agents that elevate intracellular cAMP reduced the formation of three-dimensional structures, but every cell inside the aggregates still expressed gamma GTP and ALP activities. Such structures, associated with high levels of gamma GTP and ALP activities, were also induced by astroglial factors, including (1) plasma membranes from newborn rat primary astrocytes or rat glioma C6 cells, (2) C6 conditioned media, or (3) diffusible factors produced by primary astrocytes grown in the presence of, but not in contact with RBE4 cells. RBE4 cells thus remain sensitive to angiogenic and astroglial factors for the expression of the blood-brain barrier-related gamma GTP activity, as well as for ALP activity, and could constitute the basis of a valuable in vitro model of the blood-brain barrier.

Alkaline Phosphatase↗

PubMed1994

Identification and distribution of insulin receptors on cultured bovine brain microvessel endothelial cells: possible function in insulin processing in the blood-brain barrier.

The binding of 125I-insulin to primary cultures of bovine brain microvessel endothelial cells was examined. Insulin binding was both time and temperature dependent and inhibited by excess unlabeled insulin. Furthermore, the specific binding of insulin was polarized to the apical side of the cell monolayers. Upon binding, the labeled insulin was internalized, with approximately 70% resistant to acid wash over a 90-min period. The inhibition of insulin internalization observed with cell monolayers exposed to either phenylarsine oxide or unlabeled insulin suggests a receptor-mediated endocytic process. Furthermore, the ability of chloroquine to reduce the metabolism of insulin indicates a significant portion of the peptide is processed through a lysosomal pathway. In contrast to the fluid-phase endocytosis marker, Lucifer yellow, as much as 65% of internalized insulin undergoes apical to basolateral trancytosis in brain microvessel endothelial cells. While most of the effluxed insulin was degraded, as assessed by trichloroacetic acid precipitation, the results of the present study suggest insulin receptors within the brain microvasculature may be involved in the processing and transport of blood-borne insulin.

Animals↗

PubMed1995

L-fucose reduces collagen and noncollagen protein production in cultured cerebral microvessel endothelial cells.

L-fucose is a monosaccharide which is present in low concentrations in normal serum but is increased in diabetes, cancer, and inflammatory diseases. The contribution that abnormal L-fucose levels make to the progression of these disorders is unknown. In a previous study we showed that increased L-fucose concentration reduced proliferation and proteoglycan production by cultured cerebral microvessel endothelial cells. In the present study we show that exposing cerebral microvessel endothelial cells for 2 weeks to medium containing an increased concentration of L-fucose causes a significant decrease in collagen and to a lesser extent noncollagen protein production. The effect of L-fucose on collagen and noncollagen protein production is concentration-dependent: 1 mM L-fucose causes a significant decrease in collagen production but has no effect on noncollagen protein production; a 5 mM L-fucose concentration causes a maximum decrease in both collagen and noncollagen protein production. This defect is unrelated to the reduction in myo-inositol uptake caused by L-fucose and is not prevented by aminoguanidine. Collagen production can be improved by restoring L-fucose-conditioned cells to normal medium. Culturing cells for 2 weeks in medium containing 10 mM L-fucose resulted in a 50% decrease in collagen production, which was restored to 75% of control after cells were transferred to normal medium for 7 days. In contrast, noncollagen protein production was totally restored after 3 days in normal medium. Increasing levels of L-fucose in serum of rats also resulted in a decrease in collagen production. Collagenase digestible incorporation of L-[2,3,4,5-3H]proline into protein of the articular cartilage from rats fed a diet containing 20% L-fucose for 3 weeks was reduced by about 40% compared to rats fed a normal diet. The decrease in collagen production in L-fucose fed rats was less than the reduction that occurred in streptozotocin-induced diabetic rats. These data suggest that changes in L-fucose concentration itself may be a factor in the regulation of collagen production.

Animals↗

PubMed1991

Microfibril-microvessel connections in the rabbit eye: correlative confocal and electron microscopy.

The connections between elastic tissue and microvessels (arterioles, capillaries, and venules) in the rabbit eye were examined by light and electron microscopy. In particular, confocal scanning laser microscopy of tissue stained with orcein and examined by fluorescence using a rhodamine filter was correlated with electron microscopic observations. The goal was an analysis of the way in which elastic tissue of the uvea (i.e., choroid, ciliary body, and iris) and the optic nerve of the eye connect to the microvessels in these structures. Confocal microscopy revealed these connections advantageously and showed how they link the elastic tissue meshwork in the perivascular tissue spaces with the wall of the blood vessels. Electron microscopy showed that the connections consist of bundles of 10-12 nm diameter microfilaments that insert into vascular basement membranes. These connections may contribute to the vascular response to changes in blood pressure or intraocular pressure in the eye.

Animals↗

PubMed2006

Cytoskeletal response of microvessel endothelial cells to an applied stress force at the submicrometer scale studied by atomic force microscopy.

Cytoskeleton fibers form an intricate three-dimensional network to provide structure and function to microvessel endothelial cells. During accommodation to blood flowing, stress fiber bundles become more prominent and align with the direction of blood flow. This network either mechanically resists the applied shear stress (lateral force) or, if deformed, is dynamically remodeled back to a preferred architecture. However, the detailed response of these stress fiber bundles to applied lateral force at submicrometer scales are as yet poorly understood. In our in vitro study, the tip, topography probe in lateral force microscopy of atomic force microscopy, acted as a tool for exerting quantitative vertical and lateral force on the filaments of the cytoskeleton. Moreover, the authors developed a formula to calculate the value of lateral force exerted on every point of the filaments. The results show that cytoskeleton fibers of healthy tight junctions in rat cerebral microvessel endothelial cells formed a cross-type network, and were reinforced and elongated in the direction of scanning under lateral force of 15-42 nN. Under peroxidation (H(2)O(2) of 300 micromol/L), the cytoskeleton remodeled at intercellular junctions, and changed over the meshwork structures into a dense bundle, that redistributed the stress. Once mechanical forces were exerted on an area, the cells shrank and lost morphologic tight junctions. It would be useful in our understanding of certain pathological processes, such as cerebral ischemia/reperfusion injury, which maybe caused by biomechanical forces and which are overlooked in current disease models.

Animals↗

PubMed2003

Correlation of dynamic contrast enhancement MRI parameters with microvessel density and VEGF for assessment of angiogenesis in breast cancer.

PURPOSE: To investigate the association between parameters obtained from dynamic contrast enhanced MRI (DCE-MRI) of breast cancer using different analysis approaches, as well as their correlation with angiogenesis biomarkers (vascular endothelial growth factor and vessel density). MATERIALS AND METHODS: DCE-MRI results were obtained from 105 patients with breast cancer (108 lesions). Three analysis methods were applied: 1) whole tumor analysis, 2) regional hot-spot analysis, and 3) intratumor pixel-by-pixel analysis. Early enhancement intensities and fitted pharmacokinetic parameters were studied. Paraffin blocks of 71 surgically resected specimens were analyzed by immunohistochemical staining to measure microvessel counts (with CD31) and vascular endothelial growth factor (VEGF) expression levels. RESULTS: MRI parameters obtained from the three analysis methods showed significant correlations (P < 0.0001), but a substantial dispersion from the linear regression line was noted (r = 0.72-0.97). The entire region of interest (ROI) vs. pixel population analyses had a significantly higher association compared to the entire ROI vs. hot-spot analyses. Cancer specimens with high VEGF expression had significantly higher CD31 microvessel densities than did specimens with low VEGF levels (P < 0.005). No significant association was found between MRI parameters obtained from the three analysis strategies and IHC based measurements of angiogenesis. CONCLUSION: A consistent analysis strategy was important in the DCE-MRI study. In this series, none of these strategies yielded results for MRI based quantitation of tumor vascularity that were associated with IHC based measurements. Therefore, different analyses could not account for the lack of association.

Breast↗

PubMed2004

Effect of heat preconditioning on the uptake and permeability of R123 in brain microvessel endothelial cells during mild heat treatment.

The purpose of this study was to assess the effect of mild heat and heat preconditioning on the uptake and permeability of a P-glycoprotein (P-gp) substrate, rhodamine 123 (R123), in a cell culture model of the blood-brain barrier (BBB). An immediate goal was to determine whether prior mild heat treatment could render brain microvessel endothelial cells more resistant to future heat stress and affect BBB drug permeation by future ultrasound-induced mild heat (USMH) treatment. To address this issue, the expression level of two proteins, P-gp and heat shock protein 70 (Hsp70), and their effects on uptake of R123 and permeability of R123 and [14C]-sucrose in combination with mild heat and P-gp modulator PSC833 during and after mild heat treatment in heat-preconditioned and heat-unconditioned bovine brain microvessel endothelial cell (BBMEC) monolayers were studied. Mild heat caused a significant increase in BBB permeability of R123 and [14C]-sucrose when compared with control and PSC833. Exposure of BBMECs to heat preconditioning caused a slight but insignificant decrease in cellular uptake of R123 both during and immediately after mild heat treatment. Heat preconditioning also caused a slight but insignificant decrease in permeability of R123 and [14C]-sucrose in BBMEC monolayers during mild heat treatment. Because exposure of BBMEC monolayers to mild heat did not affect P-gp expression but slightly affected Hsp70 expression, a heat preconditioning that results in a reinforcement of the BBB other than increased expression of P-gp is suggested. However, heat preconditioning is not sufficient to override the permeation-enhancing effects of mild heat because mild heat caused a significant increase in R123 uptake and permeability of R123 and [14C]-sucrose in both heat-preconditioned and heat-unconditioned cells. Because Hsp70 is known to play a major role in cellular repair and protective mechanisms, our results would imply a relative benign nature of mild heat treatment. Because heating produced by ultrasonic waves can be controlled and localized to a small volume within the tissue, the present results also suggest that USMH could play a pivotal role in the treatment of brain tumors and other brain-related diseases.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1987

Uptake of propranolol by microvessels isolated from bovine brain.

To study the transport system of propranolol (PL), a basic drug, in the blood-brain barrier, the uptake of PL into isolated bovine brain microvessels was investigated. The uptake of PL was a concentrative one via saturable process (Km = 42.5 microM) that was decreased by hypothermia (Q10 = 2.2), but not by metabolic inhibitors (2,4-dinitrophenol, KCN, ouabain). Although basic drugs such as quinidine and imipramine decreased both the initial rate of uptake and the steady-state cell-to-medium concentration ratio (C/M) of PL, acidic drugs (phenobarbital, salicylic acid) did not affect them. These results suggest that PL is taken up by the endothelial cells of the isolated brain microvessels by facilitated diffusion which is specific for basic drugs and then binds to certain binding sites in the cells.

2,4-Dinitrophenol↗

PubMed1995

Early increase precedes a depletion of endothelin-1 but not of von Willebrand factor in cutaneous microvessels of diabetic patients. A quantitative immunohistochemical study.

Endothelin-1 (ET-1) is a vasoconstrictor peptide which is produced by endothelial cells. The subcellular distribution of ET-1 in human skin and the variation of immunostaining for ET-1 by light microscopy in skin biopsies of diabetic patients have been analysed using immunohistochemistry and image analysis quantification. Skin biopsies were collected from 17 patients with type 1 diabetes of different durations and with presence or absence of microangiopathy in the retina; skin biopsies of healthy subjects were utilized as controls. The distribution of ET-1 immunoreactivity (IR) at both light and electron microscopy was compared to that of von Willebrand factor (vWf), a general marker of total cutaneous microvessels. Immunohistochemistry revealed that in controls the distribution of immunostaining was similar for ET-1 and vWf, being localized to microvessels in all areas of the skin. However, at the electron microscopical level ET-1-IR was localized in the endothelial cytoplasm rather than in specific organelles, while vWf immunostaining was associated with Weibel-Palade bodies. ET-1-IR was observed in 4/8 (50 per cent) biopsies from healthy subjects; this increased to 81.8 per cent in biopsies of patients affected by diabetes for less than 10 years and decreased to 16.6 per cent in patients with diabetes for more than 10 years. Quantification of ET-1 staining showed a significant decrease of ET-1-IR in patients affected by diabetes for more than 10 years compared with those affected by diabetes for less than 10 years (P < 0.05). Also, the percentage of biopsies showing positive ET-1 staining was lower in patients with retinopathy than in patients without retinopathy. On the contrary, vWf-IR was observed in all skin specimens and its quantification showed no differences between diabetic patients and controls. These changes are not related to variations in the number of blood vessels, and it is suggested that they reflect a possible functional alteration of the endothelial cells during diabetes.

Adolescent↗

PubMed1995

Microvessel count predicts metastatic disease and survival in non-small cell lung cancer.

The growth of newly formed vessels, or neoangiogenesis, represents an important step in both physiological and pathological situations: in particular, tumour growth and metastasis require angiogenesis. Microvessel count (MC), which represents a measure of tumour angiogenesis, has been associated with metastatic spread in cutaneous, mammary, prostatic, head and neck, and early-stage lung cancer. In this study, the role of tumour angiogenesis as a prognostic indicator was examined in 253 primary non-small lung cancer (NSCLC) patients. Microvessels were counted by highlighting endothelial cells with anti-Factor VIII monoclonal antibody (Mab) in methacarn-fixed tumour samples. In univariat analysis, MC (P< 0.000001), sex (P=0.0036), histotype (P < 0.014), tumour status (P <0.007), and vessel invasion (P < 0.019) were significantly related to hilar and/or mediastinal nodal involvement. However, in the stepwise logistic regression analysis, MC (P<0.000003) retained the most important influence on nodal metastasis. The overall survival analysis calculated by the Kaplan-Meier method revealed that tumours with high MC ( > 25 vessels/field) were significantly associated with increased death risk (log-rank test P = 0.00067; Cox's test P = 0.00046; Gehan's Wilcoxon test P = 0.00108). In 94 patients, the development of metastatic disease during follow-up was significantly related to MC. Indeed, patients who developed metastasis during follow-up showed a higher MC, either as a dichotomous (P = 0.01) or as a continuous (P = 0.003) variable, than patients who had developed no metastasis at the time of the analysis. Moreover, in the stepwise logistic regression analysis, MC retained the most important influence on distant metastases.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1993

Quantitative reverse transcriptase-polymerase chain reaction of glucose transporter 1 mRNA levels in rat brain microvessels.

We investigated the usefulness of reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify glucose transporter 1 (GLUT1) mRNA in cerebral microvessels. The technique was validated using an in vitro-transcribed RNA fragment (riboprobe) of partial 3' noncoding sequence of rat brain GLUT1 gene. A known amount of the riboprobe was reverse-transcribed to cDNA (target DNA). PCR primers were made to amplify a 292-bp fragment of the target DNA. The 5' primer was end labeled with 32P. An oligonucleotide of 100 bp containing the same sequences as the first 30 and the last 70 bases of the 292-bp fragment of the target DNA was synthesized and used as competitive DNA. The target DNA was coamplified with increasing amounts of competitive DNA using the same two primers. The ratio of radioactivity between amplified products of the target DNA (292-bp fragment) and the competitive DNA (100-bp fragment) was determined quantitatively after separation by gel electrophoresis and radioactivity counting. This method gave an accurate estimation of the amount of the riboprobe in the reaction and a 2- to 5-fold change in the amounts could be detected. By this method, the mean amount of GLUT1 mRNA from purified rat brain microvessels was estimated to be 1.5 +/- 0.1 x 10(-6) ng/ng total RNA. This value was about 10-fold higher than that in rat cell line PC12.

Animals↗