(1/3987) VEGF is required for growth and survival in neonatal mice.
We employed two independent approaches to inactivate the angiogenic protein VEGF in newborn mice: inducible, Cre-loxP- mediated gene targeting, or administration of mFlt(1-3)-IgG, a soluble VEGF receptor chimeric protein. Partial inhibition of VEGF achieved by inducible gene targeting resulted in increased mortality, stunted body growth and impaired organ development, most notably of the liver. Administration of mFlt(1-3)-IgG, which achieves a higher degree of VEGF inhibition, resulted in nearly complete growth arrest and lethality. Ultrastructural analysis documented alterations in endothelial and other cell types. Histological and biochemical changes consistent with liver and renal failure were observed. Endothelial cells isolated from the liver of mFlt(1-3)-IgG-treated neonates demonstrated an increased apoptotic index, indicating that VEGF is required not only for proliferation but also for survival of endothelial cells. However, such treatment resulted in less significant alterations as the animal matured, and the dependence on VEGF was eventually lost some time after the fourth postnatal week. Administration of mFlt(1-3)-IgG to juvenile mice failed to induce apoptosis in liver endothelial cells. Thus, VEGF is essential for growth and survival in early postnatal life. However, in the fully developed animal, VEGF is likely to be involved primarily in active angiogenesis processes such as corpus luteum development. (+info)
(2/3987) Microvessels from Alzheimer's disease brains kill neurons in vitro.
Understanding the pathogenesis of Alzheimer's disease is of widespread interest because it is an increasingly prevalent disorder that is progressive, fatal, and currently untreatable. The dementia of Alzheimer's disease is caused by neuronal cell death. We demonstrate for the first time that blood vessels isolated from the brains of Alzheimer's disease patients can directly kill neurons in vitro. Either direct co-culture of Alzheimer's disease microvessels with neurons or incubation of cultured neurons with conditioned medium from microvessels results in neuronal cell death. In contrast, vessels from elderly nondemented donors are significantly (P<0.001) less lethal and brain vessels from younger donors are not neurotoxic. Neuronal killing by either direct co-culture with Alzheimer's disease microvessels or conditioned medium is dose- and time-dependent. Neuronal death can occur by either apoptotic or necrotic mechanisms. The microvessel factor is neurospecific, killing primary cortical neurons, cerebellar granule neurons, and differentiated PC-12 cells, but not non-neuronal cell types or undifferentiated PC-12 cells. Appearance of the neurotoxic factor is decreased by blocking microvessel protein synthesis with cycloheximide. The neurotoxic factor is soluble and likely a protein, because its activity is heat labile and trypsin sensitive. These findings implicate a novel mechanism of vascular-mediated neuronal cell death in Alzheimer's disease. (+info)
(3/3987) Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.
Angiosarcomas apparently derive from blood vessel endothelial cells; however, occasionally their histological features suggest mixed origin from blood and lymphatic endothelia. In the absence of specific positive markers for lymphatic endothelia the precise distinction between these components has not been possible. Here we provide evidence by light and electron microscopic immunohistochemistry that podoplanin, a approximately 38-kd membrane glycoprotein of podocytes, is specifically expressed in the endothelium of lymphatic capillaries, but not in the blood vasculature. In normal skin and kidney, podoplanin colocalized with vascular endothelial growth factor receptor-3, the only other lymphatic marker presently available. Complementary immunostaining of blood vessels was obtained with established endothelial markers (CD31, CD34, factor VIII-related antigen, and Ulex europaeus I lectin) as well as podocalyxin, another podocytic protein that is also localized in endothelia of blood vessels. Podoplanin specifically immunolabeled endothelia of benign tumorous lesions of undisputed lymphatic origin (lymphangiomas, hygromas) and was detected there as a 38-kd protein by immunoblotting. As paradigms of malignant vascular tumors, poorly differentiated (G3) common angiosarcomas (n = 8), epitheloid angiosarcomas (n = 3), and intestinal Kaposi's sarcomas (n = 5) were examined for their podoplanin content in relation to conventional endothelial markers. The relative number of tumor cells expressing podoplanin was estimated and, although the number of cases in this preliminary study was limited to 16, an apparent spectrum of podoplanin expression emerged that can be divided into a low-expression group in which 0-10% of tumor cells contained podoplanin, a moderate-expression group with 30-60% and a high-expression group with 70-100%. Ten of eleven angiosarcomas and all Kaposi's sarcomas showed mixed expression of both lymphatic and blood vascular endothelial phenotypes. By double labeling, most podoplanin-positive tumor cells coexpressed endothelial markers of blood vessels, whereas few tumor cells were positive for individual markers only. From these results we conclude that (1) podoplanin is a selective marker of lymphatic endothelium; (2) G3 angiosarcomas display a quantitative spectrum of podoplanin-expressing tumor cells; (3) in most angiosarcomas, a varying subset of tumor cells coexpresses podoplanin and endothelial markers of blood vessels; and (4) all endothelial cells of Kaposi's sarcomas expressed the lymphatic marker podoplanin. (+info)
(4/3987) Microvascular function relates to insulin sensitivity and blood pressure in normal subjects.
BACKGROUND: A strong but presently unexplained inverse association between blood pressure and insulin sensitivity has been reported. Microvascular vasodilator capacity may be a common antecedent linking insulin sensitivity to blood pressure. To test this hypothesis, we studied 18 normotensive and glucose-tolerant subjects showing a wide range in insulin sensitivity as assessed with the hyperinsulinemic, euglycemic clamp technique. METHODS AND RESULTS: Blood pressure was measured by 24-hour ambulatory blood pressure monitoring. Videomicroscopy was used to measure skin capillary density and capillary recruitment after arterial occlusion. Skin blood flow responses after iontophoresis of acetylcholine and sodium nitroprusside were evaluated by laser Doppler flowmetry. Insulin sensitivity correlated with 24-hour systolic blood pressure (24-hour SBP; r=-0.50, P<0.05). Capillary recruitment and acetylcholine-mediated vasodilatation were strongly and positively related to insulin sensitivity (r=0.84, P<0.001; r=0.78, P<0.001, respectively), and capillary recruitment was inversely related to 24-hour SBP (r=-0.53, P<0.05). Waist-to-hip ratio showed strong associations with insulin sensitivity, blood pressure, and the measures of microvascular function but did not confound the associations between these variables. Subsequent regression analysis showed that the association between insulin sensitivity and blood pressure was not independent of the estimates of microvascular function, and part of the variation in both blood pressure (R2=38%) and insulin sensitivity (R2=71%) could be explained by microvascular function. CONCLUSIONS: Insulin sensitivity and blood pressure are associated well within the physiological range. Microvascular function strongly relates to both, consistent with a central role in linking these variables. (+info)
(5/3987) Endothelin up-regulation and localization following renal ischemia and reperfusion.
BACKGROUND: Endothelin (ET), a potent vasoconstrictor, is known to play a role in ischemic acute renal failure. Although preproET-1 (ppET-1) mRNA is known to be up-regulated following ischemia/reperfusion injury, it has not been determined which component of the injury (ischemia or reperfusion) leads to initial gene up-regulation. Likewise, although ET-1 peptide expression has been localized in the normal kidney, its expression pattern in the ischemic kidney has not been determined. Therefore, the purpose of this study was twofold: (a) to determine whether ischemia alone or ischemia plus reperfusion is required for the up-regulation of ppET-1 mRNA to occur, and (b) to localize ET-1 peptide expression following ischemia in the rat kidney to clarify better the role of ET in the pathophysiology of ischemia-induced acute renal failure. METHODS: Male Lewis rats underwent clamping of the right renal vascular pedicle for either 30 minutes of ischemia (group 1), 60 minutes of ischemia (group 2), 30 minutes of ischemia followed by 30 minutes of reperfusion (group 3), or 60 minutes of ischemia followed by three hours of reperfusion (group 4). The contralateral kidney acted as a control. ppET-1 mRNA up-regulation and ET-1 peptide expression were examined using the reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. RESULTS: Reverse transcription-polymerase chain reaction yielded a control (nonischemic) value of 0.6 +/- 0.2 densitometric units (DU) of ppET-1 mRNA in the kidney. Group 1 levels (30 min of ischemia alone) were 1.8 +/- 0.4 DU, a threefold increase (P < 0.05). Group 2 levels (60 min of ischemia alone) increased almost six times above baseline, 3.5 +/- 0.2 DU (P < 0.01), whereas both group 3 and group 4 (ischemia plus reperfusion) did not experience any further significant increases in mRNA levels (1.9 +/- 0.4 DU and 2.8 +/- 0.6 DU, respectively) beyond levels in group 1 or 2 animals subjected to similar ischemic periods. ET-1 peptide expression in the ischemic kidneys was significantly increased over controls and was clearly localized to the endothelium of the peritubular capillary network of the kidney. CONCLUSIONS: Initial ET-1 gene up-regulation in the kidney occurs secondary to ischemia, but reperfusion most likely contributes to sustaining this up-regulation. The marked increase of ET-1 in the peritubular capillary network suggests that ET-induced vasoconstriction may have a pathophysiological role in ischemic acute tubular necrosis. (+info)
(6/3987) Inhibition of effects of flow on potassium permeability in single perfused frog mesenteric capillaries.
1. We have investigated the effects of various potential inhibitors on flow-dependent K+ permeability (PK) of single perfused mesenteric microvessels in pithed frogs. 2. Neither superfusion with a nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (10 or 100 micromol l-1), nor the addition of indomethacin (30 micromol l-1) to both perfusate and superfusate reduced the positive correlation between PK and flow velocity (U). 3. In the presence of agents known to raise intracellular levels of adenosine 3',5'-cyclic monophosphate (noradrenaline, 8-bromo-cAMP and a combination of forskolin and rolipram) the slope of the relation between PK and U was no longer significant, so that PK was no longer flow dependent. 4. These results confirm that the flow dependence of PK is a biological process and not an artefact of measurement and suggest a role for intracellular cAMP rather than nitric oxide or prostacyclin in the flow-dependent modulation of PK in frog mesenteric microvessels. (+info)
(7/3987) Polyol formation and NADPH-dependent reductases in dog retinal capillary pericytes and endothelial cells.
PURPOSE: Dogs fed a diet containing 30% galactose experience retinal vascular changes similar to those in human diabetic retinopathy, with selective pericyte loss as an initial lesion. In the present study the relationship among reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductases, polyol formation, and flux through the polyol pathway in cultured dog retinal capillary cells were investigated. METHODS: Pericytes and endothelial cells were cultured from retina of beagle dogs. NADPH-dependent reductases were characterized by chromatofocusing after gel filtration. Sugars in cultured cells were analyzed by gas chromatography, and flux through the polyol pathway was investigated by 19F nuclear magnetic resonance (NMR) with 3-fluoro-3-deoxy-D-glucose (3FG) as a substrate. The presence of aldose reductase and sorbitol dehydrogenase in these cells was examined by northern blot analysis. RESULTS: Two distinct peaks corresponding to aldose reductase and aldehyde reductase, the latter being dominant, were observed in pericytes by chromatofocusing. Culture in medium containing either 10 mM D-galactose or 30 mM D-glucose resulted in the accumulation of sugar alcohol in pericytes that was markedly reduced by aldose reductase inhibitors. 19F NMR spectra obtained from pericytes cultured for 5 days in medium containing 2 mM 3FG displayed the marked accumulation of 3-fluoro-deoxysorbitol but not 3-fluoro-deoxyfructose. No 3FG metabolism was observed in similarly cultured endothelial cells. With northern blot analysis, aldose reductase was detected in pericytes but not in endothelial cells. Sorbitol dehydrogenase was below the detectable limit in pericytes and endothelial cells. CONCLUSIONS: Aldose, aldehyde, and glyceraldehyde reductases are present in dog retinal capillary pericytes, with aldehyde reductase being the major reductase present. Polyol accumulation easily occurs in pericytes but not in endothelial cells. (+info)
(8/3987) VEGF deprivation-induced apoptosis is a component of programmed capillary regression.
The pupillary membrane (PM) is a transient ocular capillary network, which can serve as a model system in which to study the mechanism of capillary regression. Previous work has shown that there is a tight correlation between the cessation of blood flow in a capillary segment and the appearance of apoptotic capillary cells throughout the segment. This pattern of cell death is referred to as synchronous apoptosis (Lang, R. A., Lustig, M., Francois, F., Sellinger, M. and Plesken, H. (1994) Development 120, 3395-3404; Meeson, A., Palmer, M., Calfon, M. and Lang, R. A. (1996) Development 122, 3929-3938). In the present study, we have investigated whether the cause of synchronous apoptosis might be a segmental deficiency of either oxygen or a survival factor. Labeling with the compound EF5 in a normal PM indicated no segmental hypoxia; this argued that oxygen deprivation was unlikely to be the cause of synchronous apoptosis. When rat plasma was used as a source of survival factors in an in vitro PM explant assay, inhibition of vascular endothelial growth factor (VEGF) all but eliminated the activity of plasma in suppressing apoptosis. This argued that VEGF was an important plasma survival factor. Furthermore, inhibition of VEGF in vivo using fusion proteins of the human Flk-1/KDR receptor resulted in a significantly increased number of capillaries showing synchronous apoptosis. This provides evidence that VEGF is necessary for endothelial cell survival in this system and in addition, that VEGF deprivation mediated by flow cessation is a component of synchronous apoptosis. (+info)