[Show abstract][Hide abstract] ABSTRACT: Endothelial cells in a cultured monolayer change from a "cobblestone" configuration when grown under static conditions to a more elongated shape, aligned with the direction of flow, after exposure to sustained uniform shear stress. Sustained blood flow acts to protect regions of large arteries from injury. We tested the hypothesis that the stable permeability state of individually perfused microvessels is also characteristic of flow-conditioning. In individually perfused rat mesenteric venular microvessels, microvascular permeability, measured as hydraulic conductivity (L(p)) was stable (mean 1.0 × 10(-7) cm/(sec × cmH(2)O)) and independent of shear stress (3-14 dynes/cm(2)) for up to 3 hours. Vessels perfused opposite to the direction of normal blood flow exhibited a delayed L(p) increase (ΔLp was 7.6 × 10(-7) cm/(sec × cmH(2)O)), but the increase was independent of wall shear stress. Addition of chondroitin sulfate and hyaluronic acid to perfusates increased the shear stress range, but did not modify the asymmetry in response to flow direction. Increased L(p) in reverse-perfused vessels was associated with numerous discontinuities of VE-cadherin and occludin, while both proteins were continuous around the periphery of forward-perfused vessels. The results are not consistent with a general mechanism for graded shear dependent permeability increase, but they are consistent with the idea that a stable L(p) under normal flow contributes to prevention of edema formation and also enables physiological regulation of shear dependent small solute permeabilities (e.g., glucose). The responses during reverse-flow are consistent with reports that disturbed flows result in a less stable endothelial barrier in venular microvessels.
Preview · Article · Feb 2013 · AJP Heart and Circulatory Physiology
[Show abstract][Hide abstract] ABSTRACT: To evaluate the hypothesis that sphingosine-1-phosphate (S1P) and cAMP attenuate increased permeability of individually perfused mesenteric microvessels through a common Rac1-dependent pathway, we measured the attenuation of the peak hydraulic conductivity (L(p)) in response to the inflammatory agent bradykinin (BK) by either S1P or cAMP. We varied the extent of exposure to each agent (test) and measured the ratio L(p)(test)/L(p)(BK alone) for each vessel (anesthetized rats). S1P (1 μM) added at the same time as BK (concurrent, no pretreatment) was as effective to attenuate the response to BK (L(p) ratio: 0.14 ± 0.05; n = 5) as concurrent plus pretreatment with S1P for 30 min (L(p) ratio: 0.26 ± 0.06; n = 11). The same pretreatment with S1P, but with no concurrent S1P, caused no inhibition of the BK response (L(p) ratio 1.07 ± 0.11; n = 8). The rapid on and off action of S1P demonstrated by these results was in contrast to cAMP-dependent changes induced by rolipram and forskolin (RF), which developed more slowly, lasted longer, and resulted in partial inhibition when given either as pretreatment or concurrent with BK. In cultured endothelium, there was no Rac activation or peripheral cortactin localization at 1 min with RF, but cortactin localization and Rac activation were maximal at 1 min with S1P. When S1P was removed, Rac activation returned to control within 2 min. Because of such differing time courses, S1P and cAMP are unlikely to act through fully common effector mechanisms.
No preview · Article · Mar 2012 · AJP Heart and Circulatory Physiology
[Show abstract][Hide abstract] ABSTRACT: Although several cultured endothelial cell studies indicate that sphingosine-1-phosphate (S1P), via GTPase Rac1 activation, enhances endothelial barriers, very few in situ studies have been published. We aimed to further investigate the mechanisms whereby S1P modulates both baseline and increased permeability in intact microvessels.
We measured attenuation by S1P of platelet-activating factor (PAF)- or bradykinin (Bk)-induced hydraulic conductivity (L(p)) increase in mesenteric microvessels of anaesthetized rats. S1P alone (1-5 µM) attenuated by 70% the acute L(p) increase due to PAF or Bk. Immunofluorescence methods in the same vessels under identical experimental conditions showed that Bk or PAF stimulated the loss of peripheral endothelial cortactin and rearrangement of VE-cadherin and occludin. Our results are the first to show in intact vessels that S1P pre-treatment inhibited rearrangement of VE-cadherin and occludin induced by PAF or Bk and preserved peripheral cortactin. S1P (1-5 µM, 30 min) did not increase baseline L(p). However, 10 µM S1P (60 min) increased L(p) two-fold.
Our results conform to the hypothesis that S1P inhibits acute permeability increase in association with enhanced stabilization of peripheral endothelial adhesion proteins. These results support the idea that S1P can be useful to attenuate inflammation by enhancing endothelial adhesion through activation of Rac-dependent pathways.
Full-text · Article · Nov 2010 · Cardiovascular Research
[Show abstract][Hide abstract] ABSTRACT: Experiments in cultured endothelial cell monolayers demonstrate that increased intracellular cAMP strongly inhibits the acute permeability responses by both protein kinase A (PKA)-dependent and -independent pathways. The contribution of the PKA-independent pathways to the anti-inflammatory mechanisms of cAMP in intact mammalian microvessels has not been systematically investigated. We evaluated the role of the cAMP-dependent activation of the exchange protein activated by cAMP (Epac), a guanine nucleotide exchange factor for the small GTPase Rap1, in rat venular microvessels exposed to the platelet-activating factor (PAF). The cAMP analog 8-pCPT-2'-O-methyl-cAMP (O-Me-cAMP), which stimulates the Epac/Rap1 pathway but has no effect on PKA, significantly attenuated the PAF increase in microvessel permeability as measured by hydraulic conductivity (Lp). We also demonstrated that PAF induced a rearrangement of vascular endothelial (VE)-cadherin seen as numerous lateral spikes and frequent short breaks in the otherwise continuous peripheral immunofluorescent label. Pretreatment with O-Me-cAMP completely prevented the PAF-induced rearrangement of VE-cadherin. We conclude that the action of the Epac/Rap1 pathway to stabilize cell-cell adhesion is a significant component of the activity of cAMP to attenuate an acute increase in vascular permeability. Our results indicate that increased permeability in intact microvessels by acute inflammatory agents such as PAF is the result of the decreased effectiveness of the Epac/Rap1 pathway modulation of cell-cell adhesion.