Biomedical subjects

J M Carbajal

Publications and source records attributed to J M Carbajal.

4 recordsLinked to original sources

PubMed2000

ROCK mediates thrombin's endothelial barrier dysfunction.

Thrombin-induced endothelial monolayer hyperpermeability is thought to result from increased F-actin stress fiber-related contractile tension, a process regulated by the small GTP-binding protein Rho. We tested whether this process was dependent on the Rho-associated protein kinase, ROCK, using a specific ROCK inhibitor, Y-27632. The effects of Y-27632 on thrombin-induced myosin light chain phosphorylation (MLCP) and tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) and paxillin were measured by Western blotting. F-actin organization and content were analyzed by digital imaging, and endothelial monolayer permeability was measured in bovine pulmonary artery endothelial cell (EC) monolayers using a size-selective permeability assay. Y-27632 enhanced EC monolayer barrier function due to a decline in small-pore number that was associated with increased EC surface area, reduced F-actin content, and reorganization of F-actin to beta-catenin-containing cell-cell adherens junctions. Although Y-27632 prevented thrombin-induced MLCP, stress fiber formation, and the increased phosphotyrosine content of paxillin and p125(FAK), it attenuated but did not prevent the thrombin-induced formation of large paracellular holes. These data indicate that thrombin-induced stress fiber formation is ROCK dependent. In contrast, thrombin-induced paracellular hole formation occurs in a ROCK-independent manner, whereas thrombin-induced monolayer hyperpermeability appears to be partially ROCK dependent.

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PubMed1999

RhoA inactivation enhances endothelial barrier function.

The modulation of endothelial barrier function is thought to be a function of contractile tension mediated by the cell cytoskeleton, which consists of actomyosin stress fibers (SF) linked to focal adhesions (FA). We tested this hypothesis by dissociating SF/FA with Clostridium botulinum exoenzyme C3 transferase (C3), an inhibitor of the small GTP-binding protein RhoA. Bovine pulmonary artery endothelial cell (EC) monolayers given C3, C3 + thrombin, thrombin, or no treatment were examined using a size-selective permeability assay and quantitative digital imaging measurements of SF/FA. C3 treatment disassembled SF/FA, stimulated diffuse myosin II immunostaining, and reduced the phosphotyrosine (PY) content of paxillin and 130- to 140-kDa proteins that included p125(FAK). C3-treated monolayers displayed a 60-85% decline in F-actin content and a 170-300% increase in EC surface area with enhanced endothelial barrier function. This activity correlated with reorganization of F-actin and PY protein(s) to beta-catenin-containing cell-cell junctions. Because C3 prevented the thrombin-induced formation of myosin ribbons, SF/FA, and the increased PY content of proteins, these characteristics were Rho dependent. Our data show that C3 inhibition of Rho proteins leads to cAMP-like characteristics of reduced SF/FA and enhanced endothelial barrier function.

ADP Ribose Transferases↗

PubMed1999

VEGF stimulates tyrosine phosphorylation of beta-catenin and small-pore endothelial barrier dysfunction.

The purpose of this study was to test the hypothesis that tyrosine phosphorylation signaling events and protein kinase C (PKC) activation mediate vascular endothelial growth factor-A(165) (VEGF)-induced endothelial cell (EC) proliferation and barrier dysfunction in bovine pulmonary artery EC monolayers. A size-selective permeability assay showed that VEGF stimulated a delayed, prolonged (6-45 h), concentration-dependent (50-200 ng/ml, approximately 1-4 nM) increase in the number of predominantly small-"pore" transport pathways (<60 A) across EC monolayers. The tyrosine kinase inhibitor herbimycin A (HA) and the selective PKC inhibitor bisindolylmaleimide (BIM) prevented this phenomenon. After 6-24 h, VEGF-treated monolayers displayed an HA- and BIM-sensitive reorganization of beta-catenin adherens junctions with fingerlike projections and the loss of beta-catenin at sites of small paracellular hole formation. HA and BIM prevented the VEGF-induced increase in EC growth. HA blocked the VEGF-induced rapid and prolonged (10 min-45 h) increases in the phosphotyrosine (PY) contents of VEGF receptor 2, phospholipase C-gamma1, paxillin, and beta-catenin as well as approximately 140- and 128- to 117-kDa proteins, whereas BIM inhibited only the tyrosine phosphorylation of beta-catenin. These data suggest that VEGF initiates increased EC growth and chronic, small-pore endothelial barrier dysfunction by PY signaling through beta-catenin that depends on PKC.

Actomyosin↗

PubMed1998

H2O2 and genistein differentially modulate protein tyrosine phosphorylation, endothelial morphology, and monolayer barrier function.

The effects of hydrogen peroxide (H2O2) and the protein tyrosine kinase (PTK) inhibitor, genistein, to modulate protein tyrosine phosphorylation (PTP) and endothelial barrier function were examined in bovine pulmonary artery endothelial cell (EC) monolayers. H2O2 stimulated a concentration (100-800 microM) and time-dependent increase in the phosphotyrosine (PY) content of multiple (56-72, 93-97, 113-142, and 161-183 kDa) EC proteins. A size-selected solute permeability assay of EC monolayer barrier function showed that (200 microM) H2O2 elevated EC monolayer permeability to large solutes. This effect was associated with paracellular hole formation and a loss of beta-catenin immunostaining at these sites. In contrast, genistein (100 microM, 1 h) reduced basal PY protein content and reorganized F-actin to beta-catenin containing cell-cell junctions, enhancing endothelial monolayer barrier function. In addition, genistein prevented the H2O2-induced increases in tyrosine phosphorylation, monolayer permeability, and paracellular hole formation. These data suggest that H2O2 and genistein differentially regulate PTP, endothelial morphology, and monolayer barrier function.

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