Venular Microvessels Research Articles

Epac1-/- mice have elevated baseline permeability and do not respond to histamine as measured with dynamic contrast-enhanced magnetic resonance imaging with contrast agents of different molecular weights.

AimEpac1−/− mice, but not Epac2−/− mice have elevated baseline permeability to albumin. This study extends the investigations of how Epac‐dependent pathways modulate transvascular exchange in response to the classical inflammatory agent histamine. It also evaluates the limitations of models of blood‐to‐tissue exchange in transgenic mice in DCE‐MRI measurements.MethodsWe measured DCE‐MRI signal intensity in masseter muscle of wt and Epac1−/− mice with established approaches from capillary physiology to determine how changes in blood flow and vascular permeability contribute to overall changes of microvascular flux. We used two tracers, the high molecular weight tracer (Gadomer‐17, MW 17 kDa, apparent MW 30‐35 kDa) is expected to be primarily limited by diffusion and therefore less dependent on changes in blood flow and the low molecular weight tracer (Dotarem (MW 0.56 kDa) whose transvascular exchange is determined by both blood flow and permeability. Paired experiments in each animal combined with analytical methods provided an internally consistent description of microvascular transport.ResultsEpac1−/− mice had elevated baseline permeability relative to wt control mice for Dotarem and Gadomer‐17. In contrast to wt mice, Epac1−/− mice failed to increase transvascular permeability in response to histamine. Dotarem underestimated blood flow and vascular volume and Gadomer‐17 has limited sensitivity in extravascular accumulation.ConclusionThe study suggests that the normal barrier loosening effect of histamine in venular microvessels do not function when the normal barrier tightening effect of Epac1 is already compromised. The study also demonstrated that the numerical analysis of DCE‐MRI data with tracers of different molecular weight has significant limitations.

Platelets Guide Leukocytes to Their Sites of Extravasation.

Effective immune responses require the directed migration of leukocytes from the vasculature to the site of injury or infection. How immune cells “find” their site of extravasation remains largely obscure. Here, we identified a previously unrecognized role of platelets as pathfinders guiding leukocytes to their exit points in the microvasculature: upon onset of inflammation, circulating platelets were found to immediately adhere at distinct sites in venular microvessels enabling these cellular blood components to capture neutrophils and, in turn, inflammatory monocytes via CD40-CD40L-dependent interactions. In this cellular crosstalk, ligation of PSGL-1 by P-selectin leads to ERK1/2 MAPK-dependent conformational changes of leukocyte integrins, which promote the successive extravasation of neutrophils and monocytes to the perivascular tissue. Conversely, blockade of this cellular partnership resulted in misguided, inefficient leukocyte responses. Our experimental data uncover a platelet-directed, spatiotemporally organized, multicellular crosstalk that is essential for effective trafficking of leukocytes to the site of inflammation.

Platelets Direct Leukocytes to their Sites of Extravasation

BackgroundLeukocyte recruitment from the microvasculature to the site of injury or infection is a key event in the inflammatory response. Whereas the principles of this highly complex process have been elucidated in the past decades, it remained poorly understood how these immune cells can ‘find’ their site of extravasation.ObjectiveThe objective of this study was to identify the exit points of myeloid leukocytes in the inflamed microvasculature and to characterize the mechanisms underlying the recruitment of these immune cells to these sites.Materials and methodsInteractions of platelets, neutrophils, inflammatory monocytes, and endothelial cells were analyzed by multi‐color in vivo microscopy in the cremaster muscle of male CX3CR‐1GFP/+ mice (exhibiting fluorescence‐labeled monocytes) upon stimulation with CCL2. Expression profiles of adhesion and signaling molecules in the microvasculature were assessed ex vivo by confocal microscopy in cremasteric tissue whole mounts. Different in vitro assays were used to further characterize the underlying mechanisms.ResultsUpon onset of inflammation, circulating platelets immediately adhered at distinct sites in venular microvessels enabling these cellular blood components to capture neutrophils and, in turn, inflammatory monocytes via CD40‐CD40L‐dependent interactions. In this cellular crosstalk, ligation of leukocyte PSGL‐1 by P‐selectin initiates conformational changes in surface‐expressed leukocyte integrins via ERK1/2 MAPK which subsequently promote the successive extravasation of neutrophils and inflammatory monocytes to the perivascular tissue. Conversely, blockade of this cellular partnership resulted in misguided, inefficient leukocyte responses.ConclusionHere, we report a previously unrecognized role of platelets as pathfinders navigating neutrophils and inflammatory monocytes to their exit points in the inflamed microvasculature. This platelet‐directed guidance enhances the efficacy of the leukocyte transmigration process and is essential for effective leukocytes responses.Support or Funding InformationSupported by the CRC914 (DFG)

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery.

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

Albumin modulates S1P delivery from red blood cells in perfused microvessels: mechanism of the protein effect

Removal of plasma proteins from perfusates increases vascular permeability. The common interpretation of the action of albumin is that it forms part of the permeability barrier by electrostatic binding to the endothelial glycocalyx. We tested the alternate hypothesis that removal of perfusate albumin in rat venular microvessels decreased the availability of sphingosine-1-phosphate (S1P), which is normally carried in plasma bound to albumin and lipoproteins and is required to maintain stable baseline endothelial barriers (Am J Physiol Heart Circ Physiol 303: H825-H834, 2012). Red blood cells (RBCs) are a primary source of S1P in the normal circulation. We compared apparent albumin permeability coefficients [solute permeability (Ps)] measured using perfusates containing albumin (10 mg/ml, control) and conditioned by 20-min exposure to rat RBCs with Ps when test perfusates were in RBC-conditioned protein-free Ringer solution. The control perfusate S1P concentration (439 ± 46 nM) was near the normal plasma value at 37 °C and established a stable baseline Ps (0.9 ± 0.4 × 10(-6) cm/s). Ringer solution perfusate contained 52 ± 8 nM S1P and increased Ps more than 10-fold (16.1 ± 3.9 × 10(-6) cm/s). Consistent with albumin-dependent transport of S1P from RBCs, S1P concentrations in RBC-conditioned solutions decreased as albumin concentration, hematocrit, and temperature decreased. Protein-free Ringer solution perfusates that used liposomes instead of RBCs as flow markers failed to maintain normal permeability, reproducing the "albumin effect" in these mammalian microvessels. We conclude that the albumin effect depends on the action of albumin to facilitate the release and transport of S1P from RBCs that normally provide a significant amount of S1P to the endothelium.

Sphingosine‐1‐phosphate and the “albumin effect” on rat venular microvessels

Sphingosine‐1‐phosphate (S1P) is carried in plasma bound to albumin (40%) and high density lipoprotein (60%). Because constant S1P is required to maintain a stable baseline endothelial barrier in individually perfused microvessels (Am J Physiol 303: H825, 2012), we tested the hypothesis that removal of albumin from a perfusate in rat venular microvessels would decrease available S1P and increase permeability. We measured the apparent solute permeability coefficient (Ps) of the vessel wall to fluorescently labeled albumin. Unlike the Landis‐Michel method to measure hydraulic conductivity, Ps measurement does not require the use of RBCs, a principal source of S1P, as flow markers. Perfusates used to measure Ps were initially conditioned by 20 min exposure to rat RBCs to provide a baseline level of S1P. Control perfusate was Ringer with fatty acid free albumin (10 mg/ml); test was Ringer alone. The control solution had a stable S1P concentration (0.34 ± 0.03 μM) close to normal plasma value and established a stable baseline Ps of 0.8 ± 0.2 × 10−6 cm/sec. The test solution with no albumin contained only 0.03 ± 0.01 μM S1P and increased permeability more than 10‐fold (n=4). We conclude that part of the well‐known “albumin effect” depends on the transport of S1P by albumin from RBCs to the endothelium. NIH HL28607.

Microvascular permeability to water is independent of shear stress, but dependent on flow direction

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 (Lp), was stable [mean 1.0 × 10(-7) cm/(s × cmH2O)] and independent of shear stress (3-14 dyn/cm(2)) for up to 3 h. Vessels perfused opposite to the direction of normal blood flow exhibited a delayed Lp increase [ΔLp was 7.6 × 10(-7) cm/(s × cmH2O)], 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 Lp 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 Lp 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.

Erythrocyte-derived sphingosine-1-phosphate stabilizes basal hydraulic conductivity and solute permeability in rat microvessels.

Exogenous sphingosine-1-phosphate (S1P), a lipid mediator in blood, attenuates acute microvascular permeability increases via receptor S1P1 to stabilize the endothelium. To evaluate the contribution of erythrocytes as an endogenous source of S1P to the regulation of basal permeability, we measured permeability coefficients in intact individually perfused venular microvessels of rat mesentery. This strategy also enabled the contributions of other endogenous S1P sources to be evaluated. Apparent permeability coefficients (P(S)) to albumin and α-lactalbumin and the hydraulic conductivity of mesenteric microvessels were measured in the presence or absence of rat erythrocytes or rat erythrocyte-conditioned perfusate. Rat erythrocytes added to the perfusate were the principal source of S1P in these microvessels. Basal P(S) to albumin was stable and typical of blood-perfused microvessels (mean 0.5 × 10(-6) cm/s) when erythrocytes or erythrocyte-conditioned perfusates were present. When they were absent, P(S) to albumin or α-lactalbumin increased up to 40-fold (over 10 min). When exogenous S1P was added to perfusates, permeability returned to levels comparable with those seen in the presence of erythrocytes. Addition of SEW 2871, an agonist specific for S1P1, in the absence of red blood cells reduced P(S)(BSA) (40-fold reduction) toward basal. The specific S1P1 receptor antagonist (W-146) reversed the stabilizing action of erythrocytes and increased permeability (27-fold increase) in a manner similar to that seen in the absence of erythrocytes. Erythrocytes are a primary source of S1P that maintains normal venular microvessel permeability. Absence of erythrocytes or conditioned perfusate in in vivo and in vitro models of endothelial barriers elevates basal permeability.

Sphingosine‐1‐phosphate from erythrocytes modulates water and macromolecule permeability in rat microvessels

Exogenous sphingosine‐1‐phosphate (S1P) attenuates acute increases in vascular permeability acting through receptor S1P1 to stabilize the endothelial barrier. Recent evidence shows that S1P is synthesized, stored in and released from erythrocytes. This study directly evaluates the contribution of erythrocyte derived S1P to maintain basal permeability to serum albumin in intact individually perfused venular microvessels of rat mesentery. Permeability coefficients (Ps) to albumin, α‐lactalbumin and the hydraulic conductivity (Lp) of mesenteric venules were measured in the presence or absence of perfusates containing rat erythrocytes (RBCs) or RBC conditioned media. Under these conditions RBCs added to the perfusate were the principal source of S1P in the microvessel. Albumin Ps values were stable and within the range expected of blood perfused microvessels (mean 0.5 × 10−6 cm/sec) when RBCs or RBC conditioned media were present. When absent, Ps to albumin or α‐lactalbumin was increased up to 10 fold. The S1P1 receptor antagonist (W146) reversed the stabilizing action of RBCs and Ps increased to the same extent as in the absence of RBCs. W146 also increased Lp in the presence of RBCs used as flow markers. We conclude that erythrocytes are the primary source of S1P that maintains normal venular microvessel permeability. Disease states which reduce RBC derived S1P may increase basal permeability. Supp NIH HL28607

Microvascular injury: Mechanisms and modulation

A common end point of many signaling pathways to increase permeability in cultured endothelial cell monolayers is increased phosphorylation of myosin light chains, a step assumed to be necessary for activation of contractile mechanisms in endothelial cells. However, the signaling pathways which modulate permeability in cultured endothelial cells, differ significantly from those that regulate permeability in intact venular microvessels. One reason for this difference may be that endothelial cells in culture express a phenotype with exaggerated contractile machinery. If this phenotype arises as the result of the process of harvesting and culturing endothelial cells, it is possible that some endothelial cells in culture are more characteristic of microvessel endothelium that has been subjected to injury (e.g. from oxidant stress, leukocyte attachment, or repeated expsoure to inflammatory conditions) than the endothelium of uninjured microvessels. It follows that agents that attenuate increased endothelial barrier permeability in cultured endothelial monolayers (e.g. regulators of the Rho-Rho kinase pathway, including the statins) may be useful candidates for the treatment of chronic dysfunction of the endothelial barrier.

Sphingosine-1-phosphate modulation of basal permeability and acute inflammatory responses in rat venular microvessels

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.

Vascular permeability modulation at the cell, microvessel, or whole organ level: towards closing gaps in our knowledge

Multiple processes modulate net blood-to-tissue exchange in a microvascular unit in normal and pathophysiological conditions. These include mechanisms that control the number and type of microvessels perfused, the balance of adhesion and contractile forces that determine the conductance of the spaces between endothelial cells to water and solutes, the pressure and chemical potential gradients determining the driving forces through these conductive pathways, and the organization of barriers to macromolecules in the endothelial glycocalyx. Powerful methods are available to investigate these mechanisms at the levels of cultured endothelial monolayers, isolated microvessels, and the microvascular units within intact organs. Here we focus on current problems that limit the integration of our knowledge of mechanisms investigated in detail at the cellular level into a more complete understanding of modulation of blood-to-tissue exchange in whole organs when the endothelial barrier is exposed to acute and more long-term inflammatory conditions. First, we review updated methods, applicable in mouse models of vascular permeability regulation, to investigate both acute and long-term changes in permeability. Methods to distinguish tracer accumulation due to change in perfusion from real increases in extravascular accumulation are emphasized. The second part of the review compares normal and increased permeability in individually perfused venular microvessels and endothelial cell monolayers. The heterogeneity of endothelial cell phenotypes in the baseline state and after exposure to injury and inflammatory conditions is emphasized. Lastly, we review new approaches to investigation of the glycocalyx barrier properties in cultured endothelial monolayers and in whole-body investigations.

Shear stress Lp response enhanced by chondroitin sulfate and hyaluronic acid is Rho kinase dependent

Vascular endothelial cells (EC) orient their long axes parallel to the fluid flow direction both in vivo and in culture. The surface glycocalyx of cultured EC transduces the fluid flow shear stress that mediates nitric oxide production and EC cytoskeletal rearrangement (Thi et al., PNAS 2004 101(47): 16483). Previously we showed that for intact microvessels perfusion against the normal flow direction stimulated increased hydraulic permeability (Lp) correlated with shear stress and induced rearrangement of cytoskeletal structures using Ringer's solution containing bovine serum albumin (BSA; 10 mg/ml) as perfusate. As a further test we added chondroitin sulfate (C; 0.2 mg/ml) and hyaluronic acid (H; 0.2 mg/ml) to the perfusate (CH; to maintain glycocalyx structure; Potter & Damiano, Circ Res 2008; 102:770) and we measured Lp in rat mesentery venular microvessels perfused for 2 hours against the normal flow direction (Reverse). Reverse perfused CH vessels had a mean Lp change (ΔLp) of 18 ± 9 (sem; ×10−7 cm/(s×cmH2O)) while for Reverse perfused BSA vessels ΔLp was 7.1 ± 3.5. Forward perfusion did not change Lp in either group. For Reverse perfusion using CH and the Rho kinase inhibitor Y27632 (30 μM) ΔLp was 6.2 ± 3.5. The data suggest that CH helps maintain an intact glycocalyx which is more sensitive to altered shear stress than that of BSA perfused vessels and that the ΔLp response is Rho kinase dependent. Supp NIH HL44485.

Retrograde shear stress modulates rat mesentery microvessel permeability and endothelial adhesion structures

Vascular endothelial cells (EC) orient their long axis parallel to the fluid flow direction both in vivo and in culture. The EC surface glycocalyx transduces the fluid flow shear stress that mediates nitric oxide production (Florian et al. Circ Res 2003) and EC cytoskeletal rearrangement (Thi et al. PNAS 2004). To test that in vivo shear stress regulates EC adhesion structures and permeability we compared final hydraulic permeability (Lp) measured in rat mesentery venular microvessels perfused orthograde with Lp measured in vessels perfused retrograde, both for about 3 hours. Shear stress ranged from 0.2 to 17 dyne/cm2 in the retrograde group and from 3.2 to 36 in the orthograde group. For retrograde vessels Lp (range 0.4 to 30 x 10−7 (cm/s*cmH2O)) was significantly correlated (p < 0.05) to mean shear stress, with a linear regression slope 1.5 x 10−7 (cm/s*cmH2O)/(dyne/cm2) and Lp intercept ‐0.6 x 10−7 (cm/s*cmH2O). For orthograde vessels the Lp (2.0 ± 0.6 x 10−7 (cm/s*cmH2O)) was not correlated with mean shear stress. We also examined the continuity of the adhesion protein VE cadherin (VE) and that of the tight junction protein occludin (Oc). Significantly more discontinuities were observed for both VE and Oc in the retrograde group than in the orthograde group. In vivo reversal of flow direction stimulates increased Lp correlated with shear stress and induces rearrangement of cytoskeletal structures. HL44485

Three-dimensional localization and quantification of PAF-induced gap formation in intact venular microvessels

Combining single-vessel perfusion technique with confocal microscopy, this study presents a new approach that allows three-dimensional visualization and quantification of endothelial gaps under experimental conditions identical to those used to measure permeability coefficients, endothelial calcium concentration, and nitric oxide production in individually perfused intact microvessels. This approach provides an efficient means for defining the transport pathways and cellular mechanisms of increased microvascular permeability during inflammation. Platelet-activating factor (PAF) was used to increase the permeability of individually perfused rat mesenteric venules. Fluorescent microspheres (FMs, 100 nm) were used as leakage markers, and confocal images were acquired at successive focal planes through the perfused microvessel. Perfusion of FMs under control conditions produced a thin, uniform layer of FMs in the vessel lumen, but in PAF-stimulated microvessels significant amounts of FMs accumulated at endothelial junctions. Reconstructed confocal images three-dimensionally delineated the temporal and spatial development of endothelial gaps in PAF-stimulated microvessels. The FM accumulation, quantified as the total fluorescence intensity per square micrometer of vessel wall, was 8.4 +/- 1.8 times the control value within 10 min of PAF perfusion and declined to 5.0 +/- 0.6 and 1.4 +/- 0.2 times the control value when FMs were applied 30 and 60 min after PAF perfusion. The changes in the magnitude of FM accumulation closely correlated with the time course of PAF-induced increases in hydraulic conductivity (L(p)), indicating that the opening and closing of endothelial gaps contributed to the transient increase in L(p) in PAF-stimulated microvessels. Electron microscopic evaluations confirmed PAF-induced gap formation and FM accumulation at endothelial clefts.

Vasopressin V2 receptor (V2R)‐mediated potentiation of platelet activating factor (PAF)‐induced increase in rat mesentery venular microvessel permeability

We tested the hypothesis that V2R activation potentiates the increase in microvessel permeability induced by PAF, an inflammatory mediator involved in sepsis. Individual venular microvessels in rat mesentery were perfused with 10mg/ml albumin in Ringer to measure hydraulic conductivity (Lp) by the modified Landis technique. The vessels were exposed to PAF (10nM), which induced an Lp increase peaking in 5–10min followed by return toward baseline values. The same vessels were then exposed to the selective V2R agonist desmopressin (dDAVP) for 40min followed by a second exposure to PAF in the presence of dDAVP. Peak PAF‐induced Lp significantly increased at dDAVP concentrations of 30pM (2.1 fold ±0.4 SEM, n=8) and 100pM (2.8 fold ±0.9 SEM, n=5) relative to peak Lp with PAF alone. In contrast, 10pM dDAVP did not increase PAF‐induced peak Lp. In control experiments, a second exposure to PAF alone did not cause an increase in peak Lp relative to the first PAF exposure. Also, dDAVP alone had no effect on baseline Lp. In further experiments, the V2R antagonist AdAc‐D‐Tyr(Et)‐Phe‐Val‐Asn‐Abu‐Pro‐Arg‐Arg‐NH2 (86nM) abolished potentiation by dDAVP (30pM) of PAF‐induced peak Lp (n=6). These results show that V2R activation potentiates PAF‐induced increase in vascular permeability, suggesting that V2R activation may exacerbate vascular leak syndrome in conditions such as sepsis. Supported by Ferring Research Institute Inc.

Transient regulation of transport by pericytes in venular microvessels via trapped microdomains

A phenomenon that has defied explanation for two decades is the time scale for transient reabsorption in the classic experiments of Michel and Phillips on individually perfused frog mesentery microvessels. One finds that transient reabsorption lasts <2 min before a new steady state of low filtration is established when the lumen pressure is abruptly dropped from a high to a low value. Our experiments in frog and rat venular microvessels under a variety of conditions revealed the same time trend for new steady states to be established as in Michel and Phillips' experiments. In contrast, one theoretically predicts herein that the time required for the tissue albumin concentration to increase to values for a new steady state to be achieved through reabsorption is in the order of several hours. In this paper we propose a new hypothesis and theoretical model for this rapid regulation, namely that pericytes covering the interendothelial cleft exits create small trapped microdomains outside the cleft exits which regulate this transient behavior. Our electron microscopy studies on rat mesenteric venular microvessels reveal an average pericyte coverage of approximately 85%. The theoretical model based on this ultrastructural study predicts an equilibration time on the order of 1 min when the lumen pressure is abruptly lowered. The basic concept of a trapped microdomain can also be extended to microdomains in the interstitial space surrounding skeletal muscle capillaries.

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.

Leukocyte-platelet aggregate adhesion and vascular permeability in intact microvessels: role of activated endothelial cells

Leukocyte-platelet aggregation and aggregate adhesion have been indicated as biomarkers of the severity of tissue injury during inflammation or ischemic reperfusion. The objective of this study is to investigate the mechanisms of the aggregate adhesion and quantitatively evaluate its relationship with microvessel permeability. A combined autologous blood perfusion with single microvessel perfusion technique was employed in rat mesenteric venular microvessels. The aggregate adhesion was induced by systemic application of TNF-alpha plus local application of platelet-activating factor (PAF). Changes in permeability were determined by measurements of hydraulic conductivity (Lp) before and after aggregate adhesion in the same individually perfused microvessels. The compositions of the adherent aggregates were identified with fluorescent labeling and confocal imaging. In contrast to leukocyte adhesion as single cells resulting in no increase in microvessel permeability, aggregate adhesion induced prolonged increases in microvessel Lp (6.1 +/- 0.9 times the control, n = 9) indicated by the initial Lp measurements after 3 h of blood perfusion, which is distinct from the transient Lp increase caused by PAF-induced endothelial activation in the absence of blood. Isoproteronol (Iso) attenuated aggregate adhesion-mediated Lp increases if applied after autologous blood perfusion and prevented the aggregate adhesion if the initial endothelial activation is inhibited by applying Iso before PAF administration but showed less effect on single leukocyte adhesion. This study demonstrated that leukocyte-platelet aggregate adhesion via a mechanism different from that of single leukocyte adhesion caused a prolonged increase in microvessel permeability. Our results also indicate that the initial activation of endothelial cells by PAF plays a crucial role in the initiation of leukocyte-platelet aggregate adhesion.

FMLP-stimulated release of reactive oxygen species from adherent leukocytes increases microvessel permeability

Our previous study (Am J Physiol Heart Circ Physiol 288: H1331-H1338, 2005) demonstrated that TNF-alpha induced significant leukocyte adhesion without causing increases in microvessel permeability, and that formyl-Met-Leu-Phe-OH (fMLP)-stimulated neutrophils in the absence of adhesion increased microvessel permeability via released reactive oxygen species (ROS). The objective of our present study is to investigate the mechanisms that regulate neutrophil respiratory burst and the roles of fMLP-stimulated ROS release from adherent leukocytes in microvessel permeability. A technique that combines single-microvessel perfusion with autologous blood perfusion was employed in venular microvessels of rat mesenteries. Leukocyte adhesion was induced by systemic application of TNF-alpha. Microvessel permeability was assessed by measuring hydraulic conductivity (L(p)). The 2-h autologous blood perfusion after TNF-alpha application increased leukocyte adhesion from 1.2 +/- 0.2 to 13.3 +/- 1.6 per 100 microm of vessel length without causing increases in L(p). When fMLP (10 microM) was applied to either perfusate (n = 5) or superfusate (n = 8) in the presence of adherent leukocytes, L(p) transiently increased to 4.9 +/- 0.9 and 4.4 +/- 0.3 times the control value, respectively. Application of superoxide dismutase or an iron chelator, deferoxamine mesylate, after fMLP application prevented or attenuated the L(p) increase. Chemiluminescence measurements in isolated neutrophils demonstrated that TNF-alpha alone did not induce ROS release but that preexposure of neutrophils to TNF-alpha in vivo or in vitro potentiated fMLP-stimulated ROS release. These results suggest a priming role of TNF-alpha in fMLP-stimulated neutrophil respiratory burst and indicate that the released ROS play a key role in leukocyte-mediated permeability increases during acute inflammation.