No abstract

Our previous experiments indicated that GTPases, other than RhoA, are important for the maintenance of endothelial barrier integrity in both intact microvessels of rats and mice and cultured mouse myocardial endothelial (MyEnd) cell monolayers. In the present study, we inhibited the endothelial GTPase Rac by Clostridium sordellii lethal toxin (LT) and investigated the relation between the degree of inhibition of Rac by glucosylation and increased endothelial barrier permeability. In rat venular microvessels, LT (200 ng/ml) increased hydraulic conductivity from a control value of 2.5 +/- 0.6 to 100.8 +/- 18.7 x 10-7 cm x s(-1) x cm H2O(-1) after 80 min. In cultured MyEnd cells exposed to LT (200 ng/ml), up to 60% of cellular Rac was glucosylated after 90 min, resulting in depolymerization of F-actin and interruptions of junctional distribution of vascular endothelial cadherin (VE-cadherin) and beta-catenin as well as the formation of intercellular gaps. To understand the mechanism by which inhibition of Rac caused disassembly of adherens junctions, we used laser tweezers to quantify VE-cadherin-mediated adhesion. LT and cytochalasin D, an actin depolymerizing agent, both reduced adhesion of VE-cadherin-coated microbeads to the endothelial cell surface, whereas the inhibitor of Rho kinase Y-27632 did not. Stabilization of actin filaments by jasplakinolide completely blocked the effect of cytochalasin D but not of LT on bead adhesion. We conclude that Rac regulates endothelial barrier properties in vivo and in vitro by 1) modulation of actin filament polymerization and 2) acting directly on the tether between VE-cadherin and the cytoskeleton.

Development of an optimal systemic drug delivery strategy to the brain will require noninvasive or minimally invasive methods to quantify the permeability of the cerebral microvessel wall or blood-brain barrier (BBB) to various therapeutic agents and to measure their transport in the brain tissue. To address this problem, we used laser-scanning multiphoton microscopy to determine BBB permeability to solutes (P) and effective solute diffusion coefficients (Deff) in rat brain tissue 100-250 μm below the pia mater. The cerebral microcirculation was observed through a section of frontoparietal bone thinned with a microgrinder. Sodium fluorescein, fluorescein isothiocyanate (FITC)-dextrans, or Alexa Fluor 488-immunoglobulin G (IgG) in 1% bovine serum albumin (BSA) mammalian Ringer's solution was injected into the cerebral circulation via the ipsilateral carotid artery by a syringe pump at a constant rate of ∼3 ml/min. P and Deff were determined from the rate of tissue solute accumulation and the radial concentration gradient around individual microvessels in the brain tissue. The mean apparent permeability P values for sodium fluorescein (molecular weight (MW) 376 Da), dextran-4k, -20k, -40k, -70k, and IgG (MW ∼160 kDa) were 14.6, 6.2, 1.8, 1.4, 1.3, and 0.54 × 10-7 cm/s, respectively. These P values were not significantly different from those of rat pial microvessels for the same-sized solutes (Yuan et al., 2009, "Non-Invasive Measurement of Solute Permeability in Cerebral Microvessels of the Rat," Microvasc. Res., 77(2), pp. 166-73), except for the small solute sodium fluorescein, suggesting that pial microvessels can be a good model for studying BBB transport of relatively large solutes. The mean Deff values were 33.2, 4.4, 1.3, 0.89, 0.59, and 0.47 × 10-7 cm2/s, respectively, for sodium fluorescein, dextran-4k, -20k, -40k, -70k, and IgG. The corresponding mean ratio of Deff to the free diffusion coefficient Dfree, Deff/Dfree, were 0.46, 0.19, 0.12, 0.12, 0.11, and 0.11 for these solutes. While there is a significant difference in Deff/Dfree between small (e.g., sodium fluorescein) and larger solutes, there is no significant difference in Deff/Dfree between solutes with molecular weights from 20,000 to 160,000 Da, suggesting that the relative resistance of the brain tissue to macromolecular solutes is similar over a wide size range. The quantitative transport parameters measured from this study can be used to develop better strategies for brain drug delivery.

We reported previously that increasing cAMP levels in endothelial cells attenuated ATP-induced increases in hydraulic conductivity (L(p)), and that the activation of cGMP-dependent pathways was a necessary step to increase L(p) in response to inflammatory mediators. The aim of the present study was to evaluate the role of basal levels of cAMP in microvessel permeability under resting conditions and to evaluate the cross talk between cAMP- and cGMP-dependent signaling mechanisms in regulation of microvessel permeability under stimulated conditions, using individually perfused microvessels from frog and rat mesenteries. We found that reducing cAMP levels by inhibition of adenylate cyclase or inhibiting cAMP-dependent protein kinase through the use of H-89 increased basal L(p) in both frog and rat mesenteric venular microvessels. We also found that 8-bromocAMP (8-BrcAMP, 0.2 and 2 mM) was sufficient to attenuate or abolish the increases in L(p) due to exposure of frog mesenteric venular microvessels to 8-BrcGMP (2 mM) and ATP (10 microM). Similarly, in rat mesenteric venular microvessels, application of 8-BrcAMP (2 mM) abolished the increases in L(p) due to exposure to 8-BrcGMP alone (2 mM) or with the combination of bradykinin (1 nM). In addition, application of erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of cGMP-stimulated phosphodiesterase, significantly attenuated both 8-BrcGMP- and bradykinin-induced increases in L(p). These results demonstrate that basal levels of cAMP are critical to maintaining normal permeability under resting conditions, and that increased levels of cAMP are capable of overcoming the activation of cGMP-dependent pathways, therefore preventing increases in microvessel permeability. The balance between endothelial concentrations of these two opposing cyclic nucleotides controls microvessel permeability, and cAMP levels play a dominant role.

Due to its unique location, the endothelial surface glycocalyx (ESG) at the luminal side of the microvessel wall may serve as a mechano-sensor and transducer of blood flow and thus regulate endothelial functions. To examine this role of the ESG, we used fluorescence microscopy to measure nitric oxide (NO) production in post-capillary venules and arterioles of rat mesentery under reduced (low) and normal (high) flow conditions, with and without enzyme pretreatment to remove heparan sulfate (HS) of the ESG and in the presence of an endothelial nitric oxide synthase (eNOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA). Rats (SD, 250–300g) were anesthetized. The mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. An individual post-capillary venule or arteriole was cannulated and loaded for 45 min with 5 μM 4, 5-Diaminofluorescein diacetate, a membrane permeable fluorescent indictor for NO, then the NO production was measured for ~10 min under a low flow (~300 μm/s) and for ~60 min under a high flow (~1000 μm/s). In the 15 min after switching to the high flow, DAF-2-NO fluorescence intensity increased to 1.27-fold of its baseline, DAF-2-NO continuously increased under the high flow, to 1.53-fold of its baseline in 60 min. Inhibition of eNOS by 1 mM L-NMMA attenuated the flow-induced NO production to 1.13-fold in 15 min and 1.30-fold of its baseline in 60 min, respectively. In contrast, no significant increase in NO production was observed after switching to the high flow for 60 min when 1 h pretreatment with 50 mU/mL heparanase III to degrade the ESG was applied. Similar NO production was observed in arterioles under low and high flows and under eNOS inhibition. Our results suggest that ESG participates in endothelial cell mechanosensing and transduction through its heparan sulfate to activate eNOS.

. PAF-and bradykinin-induced hyperpermeability of rat venules is independent of actin-myosin contraction. Am J Physiol Heart Circ Physiol 285: H406-H417, 2003. First published March 20, 2003 10.1152/ajpheart. 00021.2003.-We tested the hypothesis that acutely induced hyperpermeability is dependent on actin-myosin contractility by using individually perfused mesentery venules of pentobarbital-anesthetized rats. Venule hydraulic conductivity (L p) was measured to monitor hyperpermeability response to the platelet-activating factor (PAF) 1-O-hexadecyl-2-acetylsn-glycero-3-phosphocholine or bradykinin. Perfusion withϪ7 cm/(s ⅐ cmH2O)] that peaked in 8.9 Ϯ 0.5 min and then returned toward control L p [1.6 Ϯ 0.1 ϫ 10 Ϫ7 cm/(s ⅐ cmH2O)]. Reconstruction of venular segments with the use of transmission electron microscopy of serial sections confirmed that PAF induces paracellular inflammatory gaps. Specific inhibition of myosin light chain kinase (MLCK) with 1-10 M 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7) failed to block the PAF L p response or change the time-to-peak Lp. ML-7 reduced baseline Lp 50% at 40 min of pretreatment. ML-7 also increased the rate of recovery from PAF hyperpermeability measured as the decrease of half-time of recovery from 4.8 Ϯ 0.7 to 3.2 Ϯ 0.3 min. Inhibition of myosin ATPase with 5-20 mM 2,3-butanedione 2-monoxime also failed to alter the hyperpermeability response to PAF. Similar results were found using ML-7 to modulate responses. These experiments indicate that an actin-myosin contractile mechanism modulated by MLCK does not contribute significantly to the robust initial increase in permeability of rat venular microvessels exposed to two common inflammatory mediators. The results are consistent with paracellular gap formation by local release of endothelialendothelial cell adhesion structures in the absence of contraction by the actin-myosin network.bradykinin; platelet-activating factor; inflammatory gaps; vascular endothelium; ML-7; myosin light chain kinase

To investigate the effect of tumor cell adhesion on microvascular permeability (P) in intact microvessels, we measured the adhesion rate of human mammary carcinoma MDA-MB-231, the hydraulic conductivity (L(p)), the P, and reflection coefficient (σ) to albumin of the microvessels at the initial tumor cell adhesion and after ∼45 min cell perfusion in the postcapillary venules of rat mesentery in vivo. Rats (Sprague-Dawley, 250-300 g) were anesthetized with pentobarbital sodium given subcutaneously. A midline incision was made in the abdominal wall, and the mesentery was gently taken out and arranged on the surface of a glass coverslip for the measurement. An individual postcapillary venule was perfused with cells at a rate of ∼1 mm/s, which is the mean blood flow velocity in this type of microvessels. At the initial tumor cell adhesion, which was defined as one adherent cell in ∼100- to 145-μm vessel segment, L(p) was 1.5-fold and P was 2.3-fold of their controls, and σ decreased from 0.92 to 0.64; after ∼45-min perfusion, the adhesion increased to ∼5 adherent cells in ∼100- to 145-μm vessel segment, while L(p) increased to 2.8-fold, P to 5.7-fold of their controls, and σ decreased from 0.92 to 0.42. Combining these measured data with the predictions from a mathematical model for the interendothelial transport suggests that tumor cell adhesion to the microvessel wall degrades the endothelial surface glycocalyx (ESG) layer. This suggestion was confirmed by immunostaining of heparan sulfate of the ESG on the microvessel wall. Preserving of the ESG by a plasma glycoprotein orosomucoid decreased the P to albumin and reduced the tumor cell adhesion.

The objective of this study was to investigate whether leukocyte adhesion and/or emigration are critical steps in increased microvessel permeability during acute inflammation. To conduct this study, we combined autologous blood perfusion with a single microvessel perfusion technique, which allows microvessel permeability to be measured precisely after the endothelium has interacted with blood-borne stimuli. Experiments were carried out in intact venular microvessels in rat mesenteries. Firm attachment of leukocytes to endothelial cells was induced by intravenous injection of TNF-alpha (3.5 microg/kg) and resuming autoperfusion in a precannulated microvessel. Leukocyte emigration was facilitated by superfusion of formyl-Met-Leu-Phe-OH. Microvessel permeability was measured as hydraulic conductivity (L(p)) or the solute permeability coefficient to tetramethylrhodamine isothiocyanate-labeled alpha-lactalbumin before and after leukocyte adhesion and emigration in individually perfused microvessels. We found that perfusion of a microvessel with TNF-alpha did not affect basal microvessel permeability, but intravenous injection of TNF-alpha caused significant leukocyte adhesion. However, the significant leukocyte adhesion and emigration did not cause corresponding increases in either L(p) or solute permeability. Thus our results suggest that leukocyte adhesion and emigration do not necessarily increase microvessel permeability and the mechanisms that regulate the adhesion process act independently from mechanisms that regulate permeability. In addition, silver staining of endothelial boundaries demonstrated that leukocytes preferentially adhere at the junctions of endothelial cells. The appearance of the silver lines indicates that the TNF-alpha-induced firm adhesion of leukocyte to microvessel walls did not involve apparent changes in the junctional structure of endothelial cells, which is consistent with the results of permeability measurements.