Epac/Rap1 pathway regulates microvascular hyperpermeability induced by PAF in rat mesentery.

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.

Oncotic pressures opposing filtration across non-fenestrated rat microvessels.

We hypothesized that ultrafiltrate crossing the luminal endothelial glycocalyx through infrequent discontinuities (gaps) in the tight junction (TJ) strand of endothelial clefts reduces albumin diffusive flux from tissue into the 'protected region' of the cleft on the luminal side of the TJ. Thus, the effective oncotic pressure difference (sigma black triangle down pi) opposing filtration is greater than that measured between lumen and interstitial fluid. To test this we measured sigma black triangle down pi across rat mesenteric microvessels perfused with albumin (50 mg ml(-1)) with and without interstitial albumin at the same concentration within a few micrometres of the endothelium as demonstrated by confocal microscopy. We found sigma black triangle down pi was near 70% of luminal oncotic pressure when the tissue concentration equalled that in the lumen. We determined size and frequency of TJ strand gaps in endothelial clefts using serial section electron microscopy. We found nine gaps in the reconstructed clefts having mean spacing of 3.59 microm and mean length of 315 nm. The mean depth of the TJ strand near gaps was 67 nm and the mean cleft path length from lumen to interstitium was 411 nm. With these parameters our three-dimensional hydrodynamic model confirmed that fluid velocity was high at gaps in the TJ strand so that even at relatively low hydraulic pressures the albumin concentration on the tissue side of the glycocalyx was significantly lower than in the interstitium. The results conform to the hypothesis that colloid osmotic forces opposing filtration across non-fenestrated continuous capillaries are developed across the endothelial glycocalyx and that the oncotic pressure of interstitial fluid does not directly determine fluid balance across microvascular endothelium.

PAF- and bradykinin-induced hyperpermeability of rat venules is independent of actin-myosin contraction.

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 (Lp) was measured to monitor hyperpermeability response to the platelet-activating factor (PAF) 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine or bradykinin. Perfusion with PAF (10 nM) induced a robust transient high Lp [24.3 +/- 1.7 x 10-7 cm/(s.cmH2O)] that peaked in 8.9 +/- 0.5 min and then returned toward control Lp [1.6 +/- 0.1 x 10-7 cm/(s.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 microM 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7) failed to block the PAF Lp 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 +/- 0.7 to 3.2 +/- 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 endothelial-endothelial cell adhesion structures in the absence of contraction by the actin-myosin network.

Quantitative laser scanning confocal microscopy on single capillaries: permeability measurement.

OBJECTIVE: We tested the hypothesis that there is significant solute loss to the superfusate during microvessel solute permeability measurement in frog mesentery which leads to underestimation of permeability coefficient. This is the first application of laser-scanning confocal microscopy to measure microvessel permeability. METHODS: Microvessel permeability to sodium fluorescein (0.5 mg/ml, MW 376) was measured in the presence of bovine serum albumin (10 mg/ml) in Ringer's solution. Every 11-12s an optical section transverse to the vessel axis (x-z section) was collected. The images were corrected for fluorescence attenuation in the z direction and nonuniformity of light collection (shading) in the x direction by comparison to a fluorescent standard. Extravascular fluorescence, which increased linearly with time, and the step increase in fluorescence intensity of the vessel lumen were used to calculate an apparent permeability. Measurements were made first with the tissue superfused with Ringer and second after changing the superfusate to mineral oil in order to retain solute potentially lost to Ringer superfusate. RESULTS: The permeability to fluorescein measured with Ringer superfusate (5.0 +/- SD 2.6 x 10(-5) cm/s; n = 3) was not different from that measured with mineral oil covering the upper mesothelial surface (4.5 +/- 1.9). CONCLUSIONS: Therefore, the loss of sodium fluorescein through the upper mesothelium does not significantly alter permeability under these conditions. These techniques enable detailed analysis of solute concentration gradients surrounding microvessels.

Low density lipoprotein transport across a microvascular endothelial barrier after permeability is increased.

We investigated the pathways for low density lipoprotein (LDL) transport across an endothelial barrier in individually perfused microvessels before and after an increase in permeability. The divalent cation ionophore A23187 (5 microM) was used to increase microvessel permeability. LDL permeability coefficients (PsLDL) were measured using quantitative fluorescence microscopy. In the control state, PsLDL measured after 10-23 minutes of accumulation of fluorescent-labeled LDL outside the microvessel wall was 4.8 x 10(-8) cm/sec. The transvascular vesicular exchange of approximately 50 vesicles/sec would account for the measured flux. The flux of LDL across the microvessel wall increased as much as 170-fold at the peak of the permeability increase (2-4 minutes after ionophore infusion). Permeability returned toward control values 10 minutes after ionophore infusion but remained elevated for as long as ionophore was present in the perfusate. The effective PsLDL was similar in magnitude to the Ps for fluorescent-labeled dextran (MW 20,000) when permeability was increased. To investigate the nature of pathways for LDL in the high-permeability state, PsLDL was measured at a series of microvessel pressures. LDL transport increased as microvessel pressure increased, demonstrating coupling of LDL flux to transvascular water flow. Solvent drag accounted for more than 95% of the increased flux of LDL in the period 2-10 minutes after permeability increased. Our results conform to the hypothesis that porous pathways between adjacent endothelial cells contribute to LDL transport across an endothelial barrier when permeability is increased.

Modulation of microvessel wall charge by plasma glycoprotein orosomucoid.

Haraldsson and Rippe suggested that the circulating glycoprotein orosomucoid (alpha 1-acid glycoprotein) contributes to the net charge on microvessel walls (Acta Physiol. Scand. 129: 127-135, 1987). We tested their hypothesis in individually perfused microvessels of frog mesentery by measuring solute permeability coefficients of two globular proteins (alpha-lactalbumin and ribonuclease) having approximately the same size (Stokes radius, 2 nm) but different charge (-11 and +3, respectively). In vessels perfused with orosomucoid (0.1 and 1 mg/ml) in a Ringer-albumin perfusate, the solute permeability coefficient of alpha-lactalbumin decreased to one-half [0.47 +/- 0.25 (SD)] the value in the absence of orosomucoid, and the solute permeability coefficient of ribonuclease was close to six times as large as alpha-lactalbumin permeability. Both results may be accounted for if orosomucoid increases the net negative charge on microvessel walls in frog mesentery from 11.2 to 28 meq/l. A similar change in microvessel charge would be more than sufficient to account for the decrease in albumin clearance in the presence of orosomucoid reported by Haraldsson and Rippe in rat muscle microvessels.