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B Razani,
J A Engelman,
X B Wang,
W Schubert,
X L Zhang,
C B Marks, F Macaluso,
R G Russell,
M Li,
R G Pestell,
D Di Vizio,
H Hou,
B Kneitz,
G Lagaud,
G J Christ,
W Edelmann,
M P Lisanti
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ABSTRACT: Caveolin-1 is the principal structural protein of caveolae membranes in fibroblasts and endothelia. Recently, we have shown that the human CAV-1 gene is localized to a suspected tumor suppressor locus, and mutations in Cav-1 have been implicated in human cancer. Here, we created a caveolin-1 null (CAV-1 -/-) mouse model, using standard homologous recombination techniques, to assess the role of caveolin-1 in caveolae biogenesis, endocytosis, cell proliferation, and endothelial nitric-oxide synthase (eNOS) signaling. Surprisingly, Cav-1 null mice are viable. We show that these mice lack caveolin-1 protein expression and plasmalemmal caveolae. In addition, analysis of cultured fibroblasts from Cav-1 null embryos reveals the following: (i) a loss of caveolin-2 protein expression; (ii) defects in the endocytosis of a known caveolar ligand, i.e. fluorescein isothiocyanate-albumin; and (iii) a hyperproliferative phenotype. Importantly, these phenotypic changes are reversed by recombinant expression of the caveolin-1 cDNA. Furthermore, examination of the lung parenchyma (an endothelial-rich tissue) shows hypercellularity with thickened alveolar septa and an increase in the number of vascular endothelial growth factor receptor (Flk-1)-positive endothelial cells. As predicted, endothelial cells from Cav-1 null mice lack caveolae membranes. Finally, we examined eNOS signaling by measuring the physiological response of aortic rings to various stimuli. Our results indicate that eNOS activity is up-regulated in Cav-1 null animals, and this activity can be blunted by using a specific NOS inhibitor, nitro-l-arginine methyl ester. These findings are in accordance with previous in vitro studies showing that caveolin-1 is an endogenous inhibitor of eNOS. Thus, caveolin-1 expression is required to stabilize the caveolin-2 protein product, to mediate the caveolar endocytosis of specific ligands, to negatively regulate the proliferation of certain cell types, and to provide tonic inhibition of eNOS activity in endothelial cells.
Journal of Biological Chemistry 11/2001; 276(41):38121-38. · 4.77 Impact Factor
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ABSTRACT: M. tuberculosis accesses the terminal lung and is phagocytosed by alveolar macrophages. Utilizing a mouse intratracheal challenge model, we demonstrate that M. tuberculosis rapidly enters through M cells as well. From there, bacilli are deposited within associated intraepithelial leukocytes and subsequently conveyed to the draining lymph nodes early after infection. Osteopetrotic (Csfm(op)/Csfm(op)) mice, null mutants for macrophage colony-stimulating factor, possess diminished numbers of circulating monocytes and tissue macrophages. Csfm(op)/Csfm(op) mice were highly susceptible to challenge with M. tuberculosis. In contrast to controls, tubercle bacilli were not conveyed to draining lymph nodes early after infection but were instead retained within the mucosa. These results indicate that M cells represent an alternate portal of entry for M. tuberculosis, which may contribute to the rapid development of protective lung immune responses.
Immunity 07/1999; 10(6):641-50. · 21.64 Impact Factor
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ABSTRACT: Antidiuretic hormone (arginine vasopressin) induces a cyclic process of docking, fusion, and endocytosis of water channel-containing vesicles in the collecting duct. There is now evidence that docking and endocytosis are mediated by an array of proteins associated with vesicles and target membranes. In recent studies, we have shown that cellubrevin, a member of the vesicle-associated membrane protein family, as well as other docking proteins, are expressed in the rat inner medullary collecting duct. We now show by immunogold electron microscopy that cellubrevin is present on vesicles containing water channels, that it is associated with both coated and uncoated vesicles, and that it is present on the apical membrane. Cellubrevin, therefore, is in a position to mediate one or more steps in arginine vasopressin-induced water channel cycling.
The American journal of physiology 10/1995; 269(3 Pt 1):C797-801.
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ABSTRACT: The delivery of water channels to the apical membrane in response to antidiuretic hormone (ADH) requires the targeting of channel-containing vesicles to specific sites in the membrane, followed by fusion and exocytosis. A complex array of proteins is now believed to mediate targeting and fusion in eukaryotic cells. They include N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAP), and cellubrevin, a vesicle-associated protein present in the nerve terminal. We asked whether these proteins are in epithelial cells of rat inner medullary collecting duct (IMCD) and amphibian bladder. Immunoblots on both tissues showed the presence of NSF and alpha-SNAP. Cellubrevin was present in immunoblots of the IMCD, but not the bladder. Immunogold electron microscopy showed NSF, alpha-SNAP, and cellubrevin in rat IMCD cells, with vesicular labeling. In the bladder, NSF was seen on vesicles and aggrephores. We conclude that components of the vesicle-targeting and fusion systems are present in kidney and amphibian bladder and may mediate a wide variety of fusion events, including those initiated by ADH.
The American journal of physiology 04/1995; 268(3 Pt 1):C792-7.
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ABSTRACT: The tetrameric lectin from Glycine max (soybean) (SBA) has been shown to cross-link and precipitate with N-linked multiantennary complex type oligosaccharides containing nonreducing terminal Gal residues (Bhattacharyya, L., Haraldsson, M., & Brewer, C. F. (1988) Biochemistry 27, 1034-1041). In the present study, negative stain electron micrographs of the precipitates of SBA with a series of naturally occurring and synthetic multiantennary carbohydrates with terminal Gal or GalNAc residues show the presence of highly ordered cross-linked lattices for many of the complexes. The precipitates of SBA with a "bisected" and "nonbisected" N-linked biantennary complex type oligosaccharide containing Gal residues at the nonreducing termini show similar two-dimensional patterns. However, the pattern observed for the precipitates of a tetraantennary complex type oligosaccharide with SBA is distinct from those of the two biantennary carbohydrates. Furthermore, the precipitates formed between the lectin and a synthetic O-linked biantennary ("cluster") glycoside with terminal GalNAc residues show a pattern that is different from those above. Four biantennary pentasaccharide analogs of the blood group I antigen containing beta-LacNAc moieties at the 2.3-, 2.4-, 2.6-, and 3.6-positions of the core Gal also showed ordered patterns in their precipitates with SBA. X-ray crystallographic data and mixed quantitative precipitation profiles of binary mixtures of the four analogs demonstrate that each analog possesses a unique cross-linked lattice with the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry 05/1994; 33(18):5614-22. · 3.42 Impact Factor
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ABSTRACT: We studied host endothelial growth and calcification of bovine pericardial valve prostheses treated with: (A) 0.625% glutaraldehyde + 4% formaldehyde, (B) 99.5% glycerol or (C) 99.5% glycerol + 4% formaldehyde. Twenty-three stentless chordally supported bileaflet pericardial mitral valves with treatments A (n = 6), B (n = 6) or C (n = 11) were implanted in juvenile sheep for 125-273 days. After sacrifice, the anterior cusp from the annulus to papillary muscle of each valve was examined by scanning electron microscopy for the presence of endothelial cells, and the intrinsic calcification of each valve was determined by measuring calcium (micrograms/mg dry weight) from another 1 cm2 piece of grossly normal cusp. Sixty pieces of 1 cm2 pericardium with treatment A, B or C (n = 20 in each group) were implanted in 30 rats for 70 days. Calcium analysis and histology study of the implants were performed. In sheep, within a similar range of implantation periods, the endothelial growth rate of the valves was the highest in group B, 100% (6/6); group C was 45.5% (5/11) and A 16.7% (1/6). There were no significant differences in calcium among groups A, B and C. In rat implants, the calcium of group B was much lower than that of A or C (B = 6.92 +/- 4.46 vs A = 144.52 +/- 27.66 or C = 240.54 +/- 13.47, P < 0.05) although its histology showed more severe degeneration and inflammatory changes. Pericardial mitral valves treated with glycerol show satisfactory biocompatibility with regard to host endothelial growth and prevention of calcification; however, these tissues show evidence of rapid degeneration.(ABSTRACT TRUNCATED AT 250 WORDS)
European Journal of Cardio-Thoracic Surgery 01/1993; 7(11):591-6. · 2.55 Impact Factor
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ABSTRACT: Neither homografts nor bioprostheses have previously been seen to acquire a host endothelium. We previously reported a direct relation between aldehyde tanning and bioprosthesis calcification and the absence of calcification in the absence of aldehyde.
Bovine pericardium was 1) treated with 0.625% glutaraldehyde and stored in 4% formaldehyde, 2) treated with 99.5% glycerol, and 3) treated with 99.5% glycerol and stored in formaldehyde (0.25-4%). The treated pericardium was used to construct stentless mitral valve prostheses (of a single pattern) that were implanted in weanling sheep. After the animals were killed, a strip of anterior cusp from annulus to papillary muscle was processed and examined by scanning electron microscopy for the presence of host endothelial growth. Avoidance of aldehyde allowed host endothelial growth in all cases (six of six), and pure aldehyde treatment inhibited growth in five of six animals. Exposure to aldehyde after glycerol treatment interfered with endothelialization significantly; after longer periods of implantation, however, endothelial growth occurred almost invariably in this group (12 of 13 implanted longer than 200 days). For this group, there was a statistically significant difference for duration of implantation between the valves that grew endothelium and those that did not (218.4 +/- 61.9 versus 128.5 +/- 65.4 days).
Aldehyde treatment inhibits endothelial growth. With glycerol treatment, growth is uniformly present. Limited exposure to aldehydes after glycerol treatment inhibits endothelial growth, but this effect was ameliorated by prolonged implantation. The possibility of host endothelium-covered, noncalcifying bioprostheses is now real.
Circulation 12/1992; 86(5 Suppl):II75-9. · 14.74 Impact Factor
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ABSTRACT: Studies with the confocal microscope have shown that arginine vasopressin (AVP) depolymerizes F-actin in the apical region of the toad bladder granular cell. However, the resolution of the fluorescence microscope is not great enough to reveal the exact pattern of depolymerization or the relative extent to which microvillar and subapical membrane actin pools contribute to overall depolymerization. We have developed an electron microscopic immunogold method that shows a significant decrease in immunogold labeling of actin in the region just below the apical membrane, with the decrease most pronounced in regions adjacent to the microvilli. There was no significant change of immunogold labeling within the microvilli themselves. Our studies show a reorganization of the actin cytoskeleton in the region of the granular cell, where water channel-carrying vesicles are positioned and fuse in response to AVP.
The American journal of physiology 11/1992; 263(4 Pt 1):C908-12.
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ABSTRACT: Scanning electron microscopy of bovine pericardium (BP) shows anisotropic collagen fiber orientation (CFO). We studied the effect of CFO on the breaking strength of longitudinal (L) and transverse (T) strips cut from six pieces of fresh (Fr), glutaraldehyde (Glu)-, or formaldehyde (For)-fixed BP loaded at constant rate until rupture. Maximum tensile stress (Stmax) and strain (Snmax) were measured. The Stmax of L strips were larger than T ones in all groups, and Snmax was the same. Similar results were also observed in 10 pieces of Glu fixed normal canine mitral valves (MV). Maximum tensile stress is obtained when the load is parallel to CFO in both canine MV and BP.
ASAIO transactions / American Society for Artificial Internal Organs 37(3):M349-51.
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ABSTRACT: We studied host endothelial growth and calcification of bovine pericardial valve prostheses treated with: (A) 0.625% glutaraldehyde + 4% formaldehyde, (B) 99.5% glycerol or (C) 99.5% glycerol + 4% formaldehyde. Twenty-three stentless chordally supported bileaflet pericardial mitral valves with treatments A (n = 6), B (n = 6) or C (n = 11) were implanted in juvenile sheep for 125–273 days. After sacrifice, the anterior cusp from the annulus to papillary muscle of each valve was examined by scanning electron microscopy for the presence of endothelial cells, and the intrinsic calcification of each valve was determined by measuring calcium (μg/mg dry weight) from another 1 cm2 piece of grossly normal cusp. Sixty pieces of 1 cm2 pericardium with treatment A, B or C (n = 20 in each group) were implanted in 30 rats for 70 days. Calcium analysis and histology study of the implants were performed. In sheep, within a similar range of implantation periods, the endothelial growth rate of the valves was the highest in group B, 100% (); group C was 45.5% () and A 16.7% (). There were no significant differences in calcium among groups A, B and C. In rat implants, the calcium of group B was musch lower than that of A or C (B = 6.92 ± 4.46 vs A = 144.52 ± 27.66 or C = 240.54 ± 13.47, P < 0.05) although its histology showed more severe degeneration and inflammatory changes. Pericardial mitral valves treated with glycerol show satisfactory biocompatibility with regard to host endothelial growth and prevention of calcification; however, these tissues show evidence of rapid degeneration. Exposure to aldehyde reduces the host endothelial growth on valves and causes calcification. The calcium content in grossly normal areas of the valves does not increase in the groups with less endothelial growth, although those valves tend to have more calcification, grossly, in some areas. This implies that calcium deposition is not evenly distributed in the whole valve; host endothelial growth and calcification are independent of each other in certain areas of valves.
European Journal of Cardio-Thoracic Surgery.
