Effects of Human α-Thrombin and 8Bromo-cAMP on Large and Microvessel Endothelial Monolayer Equivalent "Pore" Radii

Schaeffer, Richard C.; Bitrick, Michael S.

doi:10.1006/mvre.1995.1031

Cited by 11 publications

(7 citation statements)

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“…CALCIUM AND ENDOTHELIAL CELL PERMEABILITY different processes (store-operated entry, intracellular store release, and ionomycin-induced extracellular influx) is uncoupled from cellular events governing gap formation and permeability in pulmonary MVECs. This conclusion is consistent with published in vivo and in vitro evidence showing that pulmonary MVECs (bovine) differ phenotypically from PAECs (9,34,35). Earlier studies have also shown differences in pulmonary MVEC permeability from ECs derived from other sources (4).…”

Section: L815supporting

confidence: 92%

“…However, little is known about responses of microvascular ECs (MVECs) from the pulmonary circulation to these inflammatory mediators due to the difficulty and technical limitations in harvesting and maintaining MVECs in culture. Recent data on pulmonary MVECs show im-proved monolayer permeability (10) compared with previous results using conduit cell isolates (4,33,34). Moreover, comparative studies from bovine endothelium have shown that pulmonary MVECs form a more restrictive barrier to macromolecules than to pulmonary artery or vein ECs, suggesting phenotypic differences (9,35,43).…”

mentioning

confidence: 83%

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Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca²⁺and permeability

Kelly¹,

Moore²,

Babál

et al. 1998

American Journal of Physiology-Lung Cellular and Molecular Phys

121

144

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Cytosolic Ca2+ concentration ([Ca2+]i) plays an important role in control of pulmonary vascular endothelial cell (ECs) barrier function. In this study, we investigated whether thapsigargin- and ionomycin-induced changes in cytosolic Ca2+ induce permeability in rat pulmonary microvascular (RPMV) versus macrovascular (RPA) ECs. In Transwell cultures, RPMVECs formed a tighter, more restrictive barrier than RPAECs to 12,000-, 72,000-, and 150,000-molecular-weight FITC-labeled dextrans. Thapsigargin (1 μM) produced higher [Ca2+]ilevels in RPAECs than in RPMVECs and increased permeability in RPAEC but not in RPMVEC monolayers. Due to the attenuated [Ca2+]iresponse in RPMVECs, we investigated whether reduced activation of store-operated Ca2+ entry was responsible for the insensitivity to thapsigargin. Addition of the drug in media containing 100 nM extracellular Ca2+ followed by readdition media with 2 mM extracellular Ca2+increased RPMVEC [Ca2+]ito a level higher than that in RPAECs. Under these conditions, RPMVEC permeability was not increased, suggesting that [Ca2+]iin RPMVECs does not initiate barrier disruption. Also, ionomycin (1.4 μM) did not alter RPMVEC permeability, but the protein phosphatase inhibitor calyculin A (100 nM) induced permeability in RPMVECs. These data indicate that, whereas increased [Ca2+]ipromotes permeability in RPAECs, it is not sufficient in RPMVECs, which show an apparent uncoupling of [Ca2+]isignaling pathways or dominant Ca2+-independent mechanisms from controlling cellular gap formation and permeability.

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Section: L815supporting

confidence: 92%

mentioning

confidence: 83%

Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca²⁺and permeability

Kelly¹,

Moore²,

Babál

et al. 1998

American Journal of Physiology-Lung Cellular and Molecular Phys

121

144

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“…Because these CalA-dependent characteristics were in- hibited by HA, they appear to be Src dependent. In addition, HA alone selectively reduced monolayer permeability to small solutes (a e Ͻ30 Å) by reducing the number of large paracellular holes as previously observed after treatment with cAMP or the MLCK inhibitor KT-5926 (25,26). This HA-induced improvement in endothelial barrier function was associated with the loss of Src kinase activity and reduced amounts of MLCP and PY-containing substrates.…”

Section: Discussionsupporting

confidence: 63%

“…Our personal stock of low-passage (P5-P11) EC, isolated from bovine pulmonary arteries as previously described (25), were grown in DMEM containing 10% fetal calf serum (Hyclone; Ogden, UT) and used between 4 and 5 days postconfluence.…”

Section: Methodsmentioning

confidence: 99%

Endothelial contraction and monolayer hyperpermeability are regulated by Src kinase

Mucha¹,

Myers²,

Schaeffer³

2003

American Journal of Physiology-Heart and Circulatory Physiology

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In this study, we tested the role of Src-dependent tyrosine phosphorylation to modulate endothelial contraction and monolayer barrier function with the use of the myosin phosphatase inhibitor calyculin A (CalA) to directly elevate MLCP with the Src family tyrosine kinase inhibitor herbimycin A (HA) in bovine pulmonary artery endothelial cells (EC). CalA stimulated an increase in MLCP, Src kinase activity, an increase in the tyrosine phosphorylation of paxillin and focal adhesion (FA) kinase (p125 FAK ), and monolayer hyperpermeability. Microscopic examination of CalAtreated EC revealed a contractile morphology characterized by peripheral contractile bands of actomyosin filaments and stress fibers linked to phosphotyrosine-containing FAs. These CalA-dependent events were HA sensitive. HA alone stimulated an improvement in monolayer barrier formation by reducing the levels of MLCP and phosphotyrosine-containing proteins and the number of large paracellular holes. These data show that Src kinase plays an important role in regulating monolayer hyperpermeability through adjustments in tyrosine phosphorylation, MLCP, and EC contraction. nonmuscle myosin; myosin light chain phosphorylation; paxillin; immunofluorescent digital imaging; size-selective solute permeability THE VASCULAR ENDOTHELIUM provides the permeability barrier that controls the passage of fluid and solutes into the perivascular space. Inflammation of this barrier initiates a hyperpermeability state characterized by the formation of large paracellular holes between adjacent endothelial cells (EC). These events occur most frequently in the systemic postcapillary venule and pulmonary arteriole circulations. The formation of paracellular gaps is the result of reorganization of the endothelial cell-cell junctional morphology that permits the enhanced leakage of plasma proteins and fluid into the tissues, thus causing edema and organ dys-

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“…The response of BECs to edemagenic agents generally shows similar trends in vitro and in vivo, although these effects may differ quantitatively Patterson et al, 1994). In particular, cyclic AMP (cAMP) appears to play a dominant role in regulating the barrier function of blood microvessels and BECs, and higher levels of intracellular cAMP correlate with increased tightness of the barrier (Adamson et al, 1998;Casnocha et al, 1989;He et al, 2000;Patterson et al, 1994;Schaeffer and Bitrick, 1995).…”

Section: Introductionmentioning

confidence: 99%

Effect of cyclic AMP on barrier function of human lymphatic microvascular tubes

Price

Chrobak

Tien

2008

Microvascular Research

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This work examines the effect of cyclic AMP (cAMP) on the in vitro barrier function of tubes of human dermal lymphatic microvascular endothelial cells (LECs). Under baseline conditions, the barrier function of LEC tubes was weak, with diffusional permeability coefficients to bovine serum albumin and 10 kDa dextran of cm/s and cm/s (geometric mean ± 95% CI), respectively, and 1.2 ± 0.5 (mean ± 95% CI) focal leaks per mm. Exposure to low concentrations (3 μM) of a cell-permeant analog of cAMP did not alter the barrier function. Exposure to higher concentrations (80 and 400 μM) and/or the phosphodiesterase inhibitor Ro-20−1724 (20 μM) lowered permeabilities and the number of focal leaks, and increased the selectivity of the barrier. Decreased permeabilities were accompanied by an increase in continuous VE-cadherin staining at cell-cell borders. Exposure to 1 mM 2′,5′-dideoxyadenosine, an inhibitor of adenylate cyclase, did not increase permeabilities. LECs expressed the lymphatic-specific master transcription factor Prox-1, regardless of whether barrier function was weak or strong. Our results indicate that the permeability of LEC tubes in vitro responds to cAMP in a manner similar to that well-described for the permeability of blood microvessels.

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Effects of Human α-Thrombin and 8Bromo-cAMP on Large and Microvessel Endothelial Monolayer Equivalent "Pore" Radii

Cited by 11 publications

References 0 publications

Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca²⁺and permeability

Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca²⁺and permeability

Endothelial contraction and monolayer hyperpermeability are regulated by Src kinase

Effect of cyclic AMP on barrier function of human lymphatic microvascular tubes

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Effects of Human α-Thrombin and 8Bromo-cAMP on Large and Microvessel Endothelial Monolayer Equivalent "Pore" Radii

Cited by 11 publications

References 0 publications

Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+and permeability

Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+and permeability

Endothelial contraction and monolayer hyperpermeability are regulated by Src kinase

Effect of cyclic AMP on barrier function of human lymphatic microvascular tubes

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Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca²⁺and permeability

Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca²⁺and permeability