Rat Mesenteric Postcapillary Venules Research Articles

Autotaxin inhibition attenuates endothelial permeability after ischemia-reperfusion injury.

Autotaxin (ATX-secretory lysophospholipase D) is the primary lysophosphatidic acid (LPA) producing enzyme. LPA promotes endothelial hyper-permeability and microvascular dysfunction following cellular stress. We sought to assess whether ATX inhibition would attenuate endothelial monolayer permeability after anoxia-reoxygenation (A-R) in vitro and attenuate the increase in hydraulic permeability observed after ischemia-reperfusion injury (IRI) in vivo. A permeability assay assessed bovine endothelial monolayer permeability during anoxia-reoxygenation with/without administration of pipedimic acid, a specific inhibitor of ATX, administered either pre-anoxia or post-anoxia. Hydraulic permeability (Lp) of rat mesenteric post-capillary venules was evaluated after IRI, with and without ATX inhibition. Lastly, Lp was evaluated after the administration of ATX alone. Anoxia-reoxygenation increased monolayer permeability 4-fold (p < 0.01). Monolayer permeability was reduced to baseline similarly in both the pre-anoxia and post-anoxia ATX inhibition groups (each p < 0.01, respectively). Lp was attenuated by 24% with ATX inhibition (p < 0.01). ATX increased Lp from baseline in a dose dependent manner (p < 0.05). Autotaxin inhibition attenuated increases in endothelial monolayer permeability during A-R in vitro and hydraulic permeability during IRI in vivo. Targeting ATX may be especially beneficial by limiting its downstream mediators that contribute to mechanisms associated with endothelial permeability. ATX inhibitors may therefore have potential for pharmacotherapy during IRI.

The Role of NADPH Oxidase Isoform 1 (NOX1) in L‐NAME‐Induced Leukocyte‐Endothelial Interactions in Rat Mesenteric Postcapillary Venules

Vascular endothelial dysfunction and subsequent inflammation underlie many pathological diseases such as cardiovascular disease, diabetes, and hypertension. Both events are principally initiated by reduced endothelial‐derived nitric oxide (NO) bioavailability and/or activation of vascular/leukocyte NADPH oxidase. NADPH oxidase has 7 isoforms (NOX1‐5 and Duox1‐2). We currently have limited knowledge regarding the roles of individual NOX isoforms in diseases, particularly NOX1, which is a major NADPH oxidase isoform in the vasculature but not in leukocytes. In this study, we evaluated the roles of NOX1 in vascular endothelial dysfunction‐induced inflammation by recording leukocyte rolling, adherence, and transmigration in a postcapillary venule of rat mesenteric tissue in real time via intravital microscopy. We superfused the mesenteric tissue with Krebs’ buffer, and Krebs’ buffer with a non‐selective NO synthase inhibitor, NG‐nitro‐L‐arginine‐methyl‐ester (L‐NAME, 50 μM) with/without a selective NOX1 inhibitor, ML171. We found that at 2 hrs of superfusion, leukocyte rolling (21±8 cells/min), adherence (3±2 cells/100 μm), and transmigration (2±1 cells/200 μm2) was minimal in Krebs’ control (n=8). By contrast, L‐NAME (n=6) significantly increased leukocyte rolling (74±12), adhesion (19±6), and transmigration (18±4) (all P&lt;0.05 compared to Krebs’ control). Whereas, ML171 (1 μM, n=7) prevented the L‐NAME‐induced effect on leukocyte rolling (25±5), adhesion (5±2), and transmigration (5±1). These findings suggest that NOX‐1 activation participates in L‐NAME induced inflammatory responses; whereas inhibition of NOX1 can mitigate inflammation following vascular endothelial dysfunction.Support or Funding InformationThis study was supported by Division of Research, the Center for Chronic Disorders of Aging, and Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.

The effects of modulating eNOS activity and coupling on leukocyte‐endothelial interactions in rat mesenteric postcapillary venules

Leukocyte‐endothelial interactions associated with vascular injury are attenuated by endothelial‐derived nitric oxide (NO). Endothelial NO synthase (eNOS) in the presence of tetrahydrobiopterin (BH4) produces NO from L‐arginine and is termed eNOS coupling. However, when the ratio of dihydrobiopterin (BH2) to BH4 is increased, eNOS becomes uncoupled and produces superoxide instead of NO. Protein kinase C epsilon (PKC ε) positively regulates eNOS activity. This study examined modulating eNOS activity and coupling by superfusing BH2 (100 μM) by itself, combined with PKC ε activator (10μM) or PKC ε inhibitor (10μM), or combined with BH4 (100μM) and PKC ε activator in rat mesenteric venules. Intravital microscopy was used to evaluate leukocyte endothelial interactions within a 2 hr period. We found that BH2 (100μM n=3, P&lt;0.05) or combined with PKC ε activator (n=5) significantly increased leukocyte rolling, adherence, and transmigration, compared to Krebs’ buffer controls (n=4, P&lt;0.05). By contrast, the combination of PKC ε activator and BH4 significantly attenuated (n=5, P&lt;0.05) BH2 induced leukocyte rolling/adherence. The BH2 induced response on all three leukocyte endothelial interactions was significantly attenuated by PKC ε inhibitor (n=4 P&lt;0.05). The data suggest that promoting eNOS coupling or inhibiting eNOS uncoupling may be important mechanisms mediating inflammation induced vascular injury. .

Inhibition of VE-Cadherin Proteasomal Degradation Attenuates Microvascular Hyperpermeability

VE-cadherin, an integral component of the adherens junction complex, is processed through the endosome-lysosome pathway and proteasome system for degradation. Our objective was to determine if inhibition of this pathway would protect against microvascular hyperpermeability. To induce VE-cadherin degradation, we utilized a mutant VE-cadherin protein that lacks the extracellular domain (rVE-cad CPD). Intravital microscopy was employed to study the changes in microvascular permeability in rat mesenteric postcapillary venules. Rat lung microvascular endothelial cell (RLMEC) monolayers were utilized in parallel studies. The adherens junction integrity was determined using VE-cadherin and β-catenin immunofluorescence. TOPflash/FOPflash transfection and luciferase reporter assay were performed to study β-catenin-mediated transcriptional activation. rVE-cad CPD (2.5 μg/mL of blood volume) increased hyperpermeability significantly (p < 0.05). The VE-cadherin siRNA as well as rVE-cad CPD induced significant increase in monolayer hyperpermeability (p < 0.05). Transfection of rVE-cad CPD disrupted adherens junctions evidenced by discontinuity in β-catenin and VE-cadherin immunofluorescence (p < 0.05). Proteasome inhibitor MG132 attenuated rVE-cad CPD induced monolayer hyperpermeability and adherens junction damage. VE-cadherin disruption in animals results in hyperpermeability. Parallel studies in RLMEC demonstrated similar results. In addition, inhibition of proteasomal degradation attenuated microvascular hyperpermeability. These findings have significance in understanding the role of VE-cadherin in regulating vascular hyperpermeability.

Curcumin inhibits reactive oxygen species formation and vascular hyperpermeability following haemorrhagic shock

1. Oxidative stress induced by reactive oxygen species (ROS) is a key mediator of haemorrhagic shock (HS)-induced vascular hyperpermeability. In the present study, curcumin, a natural anti-oxidant obtained from turmeric (Curcuma longa), was tested against HS-induced hyperpermeability and associated ROS formation in rat mesenteric post-capillary venules in vivo and in rat lung microvascular endothelial cells (RLMEC) in vitro. 2. In rats, HS was induced by withdrawing blood to reduce mean arterial pressure to 40 mmHg for 60 min, followed by resuscitation for 60 min. To investigate vascular permeability, rats were given fluorescein isothiocyanate (FITC)-albumin (50 mg/kg, i.v.). The FITC-albumin flux was measured in mesenteric post-capillary venules by determining optical intensity intra- and extravascularly under intravital microscopy. Mitochondrial ROS formation was determined using dihydrorhodamine 123 in vivo. Parallel studies were conducted in vitro using serum collected after HS. The serum was tested on rat lung microvascular endothelial cell RLMEC monolayers. 3. In rats, HS induced a significant increase in vascular hyperpermeability and ROS formation in vivo (P < 0.05). Treatment with curcumin (20 micromol/L) attenuated both these effects (P < 0.05). In RLMEC in vitro, HS serum induced monolayer permeability and ROS formation. Curcumin (10 micromol/L) attenuated HS serum-induced monolayer hyperpermeability and ROS formation. Curcumin (2-100 micromol/L) scavenged 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and 1,1-diphenyl-2-picrylhydrazyl radicals in vitro, indicating its potential as a free radical scavenger. 4. The present study demonstrates that curcumin is an inhibitor of vascular hyperpermeability following HS, with its protective effects mediated through its anti-oxidant properties.

ENDOTHELIN 1 AND PROSTACYCLIN ATTENUATE INCREASES IN HYDRAULIC PERMEABILITY CAUSED BY PLATELET-ACTIVATING FACTOR IN RATS

We have previously documented that endothelin 1 (ET-1) and prostacyclin (PGI2) decrease basal state hydraulic permeability (Lp). The aim of this study was to investigate the ability of ET-1 and PGI2 to modulate transendothelial fluid flux during situations in which Lp was artificially elevated with platelet-activating factor (PAF). We hypothesized that ET-1 and PGI2 administration before PAF exposure would prevent the increase in Lp secondary to PAF. In addition, in a potentially more clinically relevant situation, we also hypothesized that ET-1 and PGI2 administration after PAF exposure would attenuate the increase in Lp secondary to PAF. Microvascular Lp was measured in rat mesenteric postcapillary venules. Exposure to 10 nM PAF increased Lp 4-fold (P < 0.001). If the administration of 80 pM ET-1 or 10 microM PGI2 was completed before PAF exposure, no PAF-associated increase in Lp was observed (P < 0.001). The administration of ET-1 or PGI2 after PAF exposure attenuated the peak increase in Lp caused by PAF alone by 55% and 57%, respectively (P < 0.001). We conclude that ET-1 and PGI2 administration before PAF exposure prevents PAF-induced elevations in Lp, and in a more clinically relevant situation, ET-1 and PGI2 administered after PAF exposure attenuate the PAF-induced increase in Lp. Endothelin 1 and PGI2 receptors may provide important therapeutic targets for decreasing the microvascular fluid leak-associated morbidity resulting from shock and sepsis.

Ghrelin Decreases Microvascular Leak During Inflammation

Obesity is a risk factor for poor outcomes after trauma, and circulating levels of ghrelin are decreased in obese patients. We hypothesized that ghrelin modifies microvascular permeability. The purposes of this study were to determine (1) the effect of ghrelin on microvascular permeability, (2) the effect of ghrelin on microvascular permeability during lipopolysaccharide (LPS)-induced inflammation, (3) the involvement of the growth hormone secretagogue receptor (GHS-R1a) cell receptor, and (4) the involvement of nuclear factor kappa B (NF-kappaB). Hydraulic permeability (Lp), a measure of transendothelial fluid leak, was measured in rat mesenteric postcapillary venules. Lp was measured during continuous administration of (1) ghrelin (3 micromol/L), (2) ghrelin and systemic LPS (10 mg/kg), (3) the GHS-R1a receptor antagonist, (D-Arg1 D-Phe5 D-Trp7,9 Leu11)-substance P (9 micromol/L) plus ghrelin and LPS, and (4) an NF-kappaB inhibitor, parthenolide (10 micromol/L) plus ghrelin and LPS. Ghrelin alone had no effect (p > 0.7). Compared with LPS alone, ghrelin plus LPS decreased Lp (Lp: ghrelin + LPS = 1.60 +/- 0.16 vs. LPS = 2.27 +/- 0.14, p < 0.006). The GHS-R1a ghrelin receptor antagonist blunted the effect of ghrelin by 86% during LPS-induced inflammation (Lp: ghrelin + LPS = 1.60 +/- 0.16 vs. ghrelin antagonist + ghrelin + LPS = 2.17 +/- 0.27, p < 0.018). NF-kappaB inhibition did not influence the initial increased microvascular leak effect of ghrelin (p > 0.8). Although ghrelin has no effect on basal microvascular permeability, it has a biphasic effect with an overall decrease in microvascular permeability during LPS-induced inflammation through the GHS-R1a receptor, independent of NF-kappaB. Ghrelin is a key mediator of inflammation and may contribute to the increased morbidity and mortality in obese trauma patients.

Glucagon-like peptide-1 protects mesenteric endothelium from injury during inflammation

Glucagon-like peptide-1 (GLP-1) is a proglucagon-derived hormone with cellular protective actions. We hypothesized that GLP-1 would protect the endothelium from injury during inflammation. Our aims were to determine the: (1) effect of GLP-1 on basal microvascular permeability, (2) effect of GLP-1 on increased microvascular permeability induced by lipopolysaccaride (LPS), (3) involvement of the GLP-1 receptor in GLP-1 activity, and (4) involvement of the cAMP/PKA pathway in GLP-1 activity. Microvascular permeability ( L p) of rat mesenteric post-capillary venules was measured in vivo. First, the effect of GLP-1 on basal L p was measured. Second, after systemic LPS injection, L p was measured after subsequent perfusion with GLP-1. Thirdly, L p was measured after LPS injection and perfusion with GLP-1 + GLP-1 receptor antagonist. Lastly, L p was measured after LPS injection and perfusion with GLP-1 + inhibitors of the cAMP/PKA pathway. Results are presented as mean area under the curve (AUC) ± SEM. GLP-1 had no effect on L p (AUC: baseline = 27 ± 1.4, GLP-1 = 1 ± 0.4, p = 0.08). LPS increased L p two-fold (AUC: LPS = 54 ± 1.7, p < 0.0001). GLP-1 reduced the LPS increase in L p by 75% (AUC: LPS + GLP-1 = 34 ± 1.5, p < 0.0001). GLP-1 antagonism reduced the effects of GLP-1 by 60% (AUC: LPS + GLP-1 + antagonist = 46 ± 2.0, p < 0.001). The cAMP synthesis inhibitor reduced the effects of GLP-1 by 60% (AUC: LPS + GLP-1 + cAMP inhibitor = 46 ± 1.5, p < 0.0001). The PKA inhibitor reduced the effects of GLP-1 by 100% (AUC: LPS + GLP-1 + PKA inhibitor = 56 ± 1.5, p < 0.0001). GLP-1 attenuates the increase in microvascular permeability induced by LPS. GLP-1 may protect the endothelium during inflammation, thus decreasing third-space fluid loss.

Lipopolysaccharide-induced endothelial barrier breakdown is cyclic adenosine monophosphate dependent in vivo and in vitro*

To determine whether cyclic adenosine monophosphate (cAMP) is critically involved in lipopolysaccharide (LPS)-induced breakdown of endothelial barrier functions in vivo and in vitro. Experimental laboratory research. Research laboratory. Wistar rats and cultured human microvascular endothelial cells. Permeability measurements in single postcapillary venules in vivo and permeability measurements and cell biology techniques in vitro. We demonstrate that within 120 minutes LPS increased endothelial permeability in rat mesenteric postcapillary venules in vivo and caused a barrier breakdown in human dermal microvascular endothelial cells in vitro. This was associated with the formation of large intercellular gaps and fragmentation of vascular endothelial cadherin immunostaining. Furthermore, claudin 5 immunostaining at cell borders was drastically reduced after LPS treatment. Interestingly, activity of the small GTPase Rho A, which has previously been suggested to mediate the LPS-induced endothelial barrier breakdown, was not increased after 2 hours. However, activity of Rac 1, which is known to be important for maintenance of endothelial barrier functions, was significantly reduced to 64 +/- 8% after 2 hours. All LPS-induced changes of endothelial cells were blocked by a forskolin-mediated or rolipram-mediated increase of cAMP. Consistently, enzyme-linked immunosorbent assay-based measurements demonstrated that LPS significantly decreased intracellular cAMP. In summary, our data demonstrate that LPS disrupts endothelial barrier properties by decreasing intracellular cAMP. This mechanism may involve inactivation of Rac 1 rather than activation of Rho A.

The attenuation of leukocyte‐endothelial interactions by a unique broad‐spectrum protein kinase C inhibitor (Gö 6983) in rat mesenteric postcapillary venules

Protein kinase C (PKC) is a very important enzyme that regulates endothelial and leukocyte function. However, the role of broad‐spectrum PKC inhibition involving the atypical zeta isoform in regulating leukocyte‐endothelial interactions has not been clarified in vivo in real‐time. The microcirculation of postcapillary venules in rat mesentery was observed by intravital microscopy. We found that superfusion of NG‐nitro‐L‐arginine methyl ester (L‐NAME, a nitric oxide synthase (NOS) inhibitor, 50 µM, n=10) significantly increased leukocyte rolling, adherence and transmigration within a 2 hour period compared to superfusion of Krebs' buffer (n=8) (P&lt;0.01). By contrast, in the presence of Gö 6983 (25 nM, n=5/ 100 nM, n=5/ 200 nM, n=6), a unique broad‐spectrum inhibitor of PKC that inhibits isoforms from all three biochemical classes, these leukocyte‐endothelial interactions were dose‐dependently attenuated compared to L‐NAME alone (P&lt;0.05). Moreover, Gö 6983 dose‐dependently decreased superoxide release from neutrophils compared to control (P&lt;0.01). In summary, inhibition of endothelial NOS activity (i.e., superfusion of L‐NAME) enhances leukocyte‐endothelial interactions. By contrast, inhibition of PKC can decrease these increased interactions, which may be related to decreasing superoxide release from neutrophils. This study was supported by Center for Chronic Disorders of Aging at PCOM.

THE EFFECT OF HYPOXIA, REOXYGENATION, ISCHEMIA, AND REPERFUSION ON HYDRAULIC PERMEABILITY IN RAT MESENTERIC VENULES

Little is known regarding the effects of I/R on hydraulic permeability (Lp). We sought to compare the individual influences of hypoxia, ischemia, reoxygenation, and reperfusion on Lp. We hypothesized that (1) hypoxia increases Lp; (2) reoxygenation further increases Lp; (3) ischemia results in greater increases in Lp compared with hypoxia; (4) reperfusion causes additional increases in Lp compared with hypoxia, ischemia, and reoxygenation; and (5) xanthine oxidase (XO) and white blood cell adherence play important roles in hypoxia, ischemia, and reperfusion. Hydraulic permeability was measured by an in vivo microcannulation technique during hypoxia, reoxygenation, ischemia, and reperfusion in rat mesenteric postcapillary venules. Additional rats were fed a Tungsten-enriched diet to inhibit XO activity, and the studies were repeated. White blood cell adherence was also documented. Hypoxia and ischemia both increased Lp 2-fold from baseline levels (P < 0.001). Reoxygenation did not alter Lp compared with 15 min of hypoxia alone (P > 0.07). Reperfusion after hypoxia increased Lp 6-fold (P < 0.001). Reperfusion after ischemia also increased Lp 6-fold (P < 0.001). Inhibition of XO had no effect on the increase in Lp after both hypoxia and ischemia. However, inhibition of XO attenuated the 6-fold increase in Lp observed during reperfusion after both hypoxia and ischemia by approximately 50% (P < 0.001). White blood cell adherence increased during reperfusion but not hypoxia or ischemia. The complexity of I/R injury makes it a difficult clinical scenario to model for research. We have demonstrated in an in vivo model that hypoxia and ischemia increase Lp similarly, and that reperfusion has a profound deleterious effect on Lp. These changes in Lp seem to be XO and white blood cell dependent.

Ischemia-reperfusion injury in rats affects hydraulic conductivity in two phases that are temporally and mechanistically separate

Ischemia-reperfusion (IR) injury is a major insult to postcapillary venules. We hypothesized that IR increases postcapillary venular hydraulic conductivity and that IR-mediated changes in hydraulic conductivity result from temporally and mechanistically separate processes. A microcannulation technique was used to determine hydraulic conductivity (Lp) in rat mesenteric postcapillary venules serially throughout ischemia (45 min) and reperfusion (5 h) induced by superior mesenteric artery occlusion and release. Mesenteric IR resulted in a biphasic increase in Lp. White blood cell (WBC) adhesion slowly increased with maximal adhesion corresponding to the second peak (P < 0.005). After IR, tissue was harvested for RT-PCR analysis of ICAM-1, E-selectin, and P-selectin mRNA. Intercellular adhesion molecule-1 (ICAM-1) mRNA in the gut showed the most significant upregulation. Quantitative real-time PCR revealed that ICAM-1 mRNA was upregulated 60-fold in the gut. An ICAM-1 antibody was therefore used to determine the effect of WBC adhesion on Lp during IR. ICAM-1 inhibition attenuated Lp during the first peak and completely blocked the second peak (P < 0.005). When rats were fed a tungsten diet to inhibit xanthine oxidase and then underwent IR, Lp was dramatically attenuated during the first peak and mildly decreased the second peak (P < 0.005). Inhibition of xanthine oxidase by oxypurinol decreased Lp during IR by over 60% (P < 0.002). Tempol, a superoxide dismutase mimetic, decreased Lp during IR by over 30% (P < 0.01). We conclude that IR induces a biphasic increase in postcapillary hydraulic conductivity. Reactive oxygen species impact both the first transient peak and the sustained second peak. However, the second peak is also dependent on WBC-endothelial cell adhesion. These serial measurements of postcapillary hydraulic conductivity may lead the way for optimal timing of pharmaceutical therapies in IR injury.

Estradiol inhibits vascular hyperpermeability following hemorrhagic shock

Recent studies from our laboratory showed the involvement of mitochondria mediated ‘intrinsic’ apoptotic signaling in vascular hyperpermeability following hemorrhagic shock (HS). We hypothesized that 17ß‐estradiol, which shows anti‐apoptotic properties, would attenuate HS‐induced vascular hyperpermeability. The objective of this study was to determine if 17ß‐Estradiol would inhibit hyperpermeability in rat mesenteric post capillary venules in vivo and protect mitochondrial transmembrane potential (MTP), following HS. In rats, HS was induced by withdrawing blood to reduce the MAP to 40 mmHg for 60 minutes followed by resuscitation to 90 mmHg. To study vascular permeability, the rats were injected intravenously with FITC‐albumin (50 mg/kg) and the FITC‐albumin flux was measured by determining the changes in integrated optical intensity obtained intra and extra vascularly using intravital microscopy. Changes in MTP were determined using the fluorescent dye JC‐1. HS induced hyperpermeability (p&lt;0.05). 17ß‐Estradiol (2 mg/kg body weight) attenuated HS‐induced hyperpermeability (p&lt;0.05). 17ß‐Estradiol pre‐treatment prevented HS‐induced decrease in MTP (p&lt;0.05). These findings demonstrate that 17ß‐estradiol maintains vascular endothelial barrier functions following HS. Its protective effects may be mediated through the inhibition of ‘intrinsic’ apoptotic signaling.

Roflumilast inhibits leukocyte‐endothelial cell interactions, expression of adhesion molecules and microvascular permeability

The present study addressed the effects of the investigational PDE4 inhibitor roflumilast on leukocyte-endothelial cell interactions and endothelial permeability in vivo and in vitro. In vivo, intravital video-microscopy was used to determine effects of roflumilast p.o. on leukocyte-endothelial cell interactions and microvascular permeability in rat mesenteric venules. In vitro, the effects of roflumilast N-oxide, the active metabolite of roflumilast in humans, and other PDE4 inhibitors on neutrophil adhesion to tumour necrosis factor alpha (TNFalpha)-activated human umbilical vein endothelial cells (HUVEC), E-selectin expression and thrombin-induced endothelial permeability was evaluated. Flow cytometry was used to determine the effect of roflumilast on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced CD11b upregulation on human neutrophils. In vivo, roflumilast, given 1 h before lipopolysaccharide (LPS), dose-dependently reduced leukocyte-endothelial cell interactions in rat mesenteric postcapillary venules. It also diminished histamine-induced microvascular permeability. Immunohistochemical analyses revealed that roflumilast prevented LPS-induced endothelial P- and E-selectin expression. In vitro, roflumilast N-oxide concentration-dependently suppressed neutrophil adhesion to TNFalpha-activated HUVEC and CD11b expression on fMLP-stimulated neutrophils. It also reduced TNFalpha-induced E-selectin expression on HUVEC, when PDE3 activity was blocked. HUVEC permeability elicited by thrombin was concentration-dependently suppressed by roflumilast N-oxide. While roflumilast N-oxide was as potent as roflumilast at inhibiting stimulated endothelial cell and neutrophil functions, both compounds were significantly more potent than the structurally unrelated PDE4 inhibitors, rolipram or cilomilast. These findings further support earlier observations on the inhibition of inflammatory cell influx and protein extravasation by roflumilast in vivo.

Osteoprotegerin increases leukocyte adhesion to endothelial cells both in vitro and in vivo

AbstractRecombinant osteoprotegerin (OPG) promoted the adhesion of both primary polymorphonuclear neutrophils (PMNs) and leukemic HL60 cells to endothelial cells. Leukocyte/endothelial cell adhesion was promoted by short (peak at 1 hour) preincubation of either endothelial cells or PMNs with OPG, and the peak of proadhesive activity was observed in the same range of OPG concentrations detected in the sera of patients affected by cardiovascular diseases. Although the cognate high-affinity ligands for OPG, membrane receptor activator of nuclear factor-κB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were detected at significant levels on both PMNs and HL60 cells, they were not expressed on the surface of endothelial cells. However, preincubation of OPG with heparin abrogated its proadhesive activity, whereas pretreatment of endothelial cells with chondroitinase plus heparinases significantly decreased the proadhesive activity of OPG. Taken together, these findings suggest the involvement of both the ligand binding and the N-terminal heparin-binding domains of OPG in mediating its pro-adhesive activity. The relevance of these in vitro findings was underscored by in vivo experiments, in which the topical administration of recombinant OPG increased leukocyte rolling and adhesion to rat mesenteric postcapillary venules. Our data suggest that a pathological increase of OPG serum levels might play an important role in promoting leukocyte/endothelial cell adhesion.

P268: Urocortins govern microvascular permeabilty during inflammation

Introduction: Urocortins (Ucns) are known to regulate vascular tone and more recently have been implicated in mediating intestinal inflammation. Our hypothesis is that Ucn1 promotes microvascular permeability and this effect is exacerbated during systemic inflammation. The purposes of this study are: 1) to determine the effect of Ucn1 on microvascular permeability, and 2) to determine the effect of Ucn1 on microvascular permeability during inflammation. Methods: First, basal microvascular permeability (Lp) of rat mesenteric post-capillary venules was measured using an in vivo micro-cannulation technique for 50 minutes (control 1). Second, the effect of Ucn1 on microvascular permeability (Lp-Ucn1) was obtained by continuous perfusion of 10-7M Ucn1; Lp-Ucn1 was recorded at 10 min intervals for 50 min. Third, a bolus of lipopolysaccharide (LPS, 10mg/kg) into the rat femoral vein was used to induce inflammation. The effect of LPS on Lp was measured for 50 min (control 2). Fourth, to examine if Ucn1 exacerbated the effects of LPS-induced changes in Lp, 10mg/kg LPS and 10-7M Ucn1 were continuously perfused in post capillary venules and changes in Lp were measured at 10 min intervals for 50 min. Results: Ucn1 alone increased Lp 2-fold over baseline with a peak effect at 30 min (Lp baseline = 0.96 ± 0.03, Lp Ucn1 = 2.02 ± 0.20) (p = 0.005). LPS-induced inflammation also increased Lp 2-fold over baseline (Lp baseline = 0.92 ± 0.04, Lp-LPS= 1.93 ± 0.14) (p = 0.005). Surprisingly, Ucn1 perfusion increased Lp 3-fold (Lp = 6.42 ± 0.23) within 2 minutes of administration and exhibited a maximum effect of a 5-fold increase in Lp at 20 minutes (Lp = 9.87 ± 0.14) (p = 0.001). Conclusion: Ucn1 increases microvascular fluid leak. Ucn1 acts synergistically with LPS to increase microvascular fluid losses during inflammation. Thus, Ucn1 acts as a key inflammatory mediator in endotoxic shock in the gut.

The influence of angiotensin II and cyclic nucleotide second messenger signal transduction on ischemia-reperfusion-induced elevations in microvascular hydraulic permeability

Introduction: Intravascular volume loss from ischemia-reperfusion injury is a major clinical concern. We hypothesize that angiotensin II influences the ischemia-reperfusion-associated microvascular fluid leak through cyclic nucleotide second messenger signal transduction mechanisms. The purposes are to determine hydraulic permeability after ischemia-reperfusion of venules treated with 1) angiotensin II, 2) a cAMP synthesis inhibitor, 3) a cGMP inhibitor, and 4) simultaneous administration of angiotensin II and either a cAMP synthesis inhibitor or a cGMP synthesis inhibitor. Methods: Rat mesenteric postcapillary venules were micro-cannulated to measure hydraulic permeability (Lp). Ischemia-reperfusion was achieved by placing animals in a 5% oxygen environment and preventing venular flow followed by allowing blood flow to resume. Lp was measured after ischemia-reperfusion and treatment with 1) angiotensin II (20nM), 2) cAMP synthesis inhibitor (DDA,10uM), 3) cGMP synthesis inhibitor (LY83583, 10uM), and 4) angiotensin II plus either cAMP inhibition or cGMP inhibition, (n=6 in each group). Results: Compared to the 7-fold increase in Lp due to ischemia-reperfusion alone: 1) angiotensin II attenuated the 7-fold increase by 50% (p<0.001), 2) cAMP inhibition attenuated the 7-fold increase by 45% (p<0.001), 3) cGMP inhibition completed blocked any elevation in Lp due to ischemia-reperfusion (p<0.001), and 4) angiotensin II + cAMP inhibition was not statistically different from angiotensin II alone (p=0.16) while angiotensin II + cGMP inhibition attenuated the 7-fold increase in Lp due to ischemia-reperfusion by 65% (p<0.001). Conclusion: Treatment with angiotensin II attenuated increases in hydraulic permeability due to ischemia-reperfusion by 50%. Inhibition of cGMP synthesis completely blocked any increase in permeability due to ischemia-reperfusion while cAMP inhibition appears to play a lesser role. This emphasizes the major impact that cyclic nucleotide second messengers play in ischemia-reperfusion. A better understanding of mediators that reduce intravascular fluid loss from IR-induced microvascular dysfunction may help clinicians treat uncontrolled fluid extravasation that occurs during shock and sepsis.

L‐NAME Induces Direct Arteriolar Leukocyte Adhesion, Which Is Mainly Mediated by Angiotensin‐II

Acute inhibition (1 h) of nitric oxide synthase (NOS) with L-NAME causes leukocyte recruitment in the rat mesenteric postcapillary venules that is angiotensin-II (Ang-II) dependent. Since 4-h exposure to Ang-II provokes arteriolar leukocyte adhesion, this study was designed to investigate whether subacute (4-h) NOS inhibition also causes this effect. Rats were intraperitoneally injected with saline, L-NAME, or 1H-[1,2,4]-oxidazolol-[4,3-a]-quinoxalin-1-one (ODQ). Leukocyte accumulation in the mesenteric microcirculation was examined 4 h later via intravital microscopy. Some groups were pretreated with losartan, an AT(1) Ang-II receptor antagonist. At 4-h, L-NAME caused a significant increase in arteriolar leukocyte adhesion and leukocyte-endothelial cell interactions in postcapillary venules. Mononuclear cells were the predominant leukocytes attached to the arteriolar endothelium. Administration of losartan inhibited L-NAME-induced arteriolar leukocyte adhesion by 90%. L-NAME provoked increased expression of P-selectin, E-selectin, ICAM-1, and VCAM-1 in arterial endothelium, which was attenuated by losartan pretreatment. Inhibition of guanylyl cyclase with ODQ mimicked the effects exerted by L-NAME and losartan also reduced these effects. NOS inhibition for 4-h results in the attachment of leukocytes to the arterial endothelium, a critical event in disease states such as hypertension and atherosclerosis, which could be prevented by the administration of AT(1)Ang-II receptor antagonists.

Effect of fibrinogen on leukocyte margination and adhesion in postcapillary venules.

To quantitatively evaluate the role of fibrinogen (Fb) as a determinant of leukocyte (WBC) margination in postcapillary venules in light of its ability to induce red blood cell (RBC) aggregation with reductions in shear rate (gamma) and increase adhesiveness of WBCs to endothelium (EC). Red cell aggregation (RCA), WBC margination (flux at the EC), rolling velocity, and adhesion to the EC were measured in rat mesenteric postcapillary venules upon reducing gamma, prior to and following systemic infusion of Fb. Proximal occlusion of feeding microvessels with a blunted probe facilitated reductions in gamma from 600 to 50 s(-1). An index of aggregation (G) was derived from light-scattering properties of RBCs, where G was proportional to the number of RBCs per aggregate. WBC margination was measured as the percentage of total luminal WBC flux that rolled on the EC, F*(WBC). For normal levels of Fb (0.07 g%), reductions in gamma resulted in a 4-fold rise in F*(WBC) and no change in G as gamma was reduced to 50 s(-1). Infusion of Fb to achieve a plasma concentration to 0.7 g% caused a modest 20% increase in G and a 2.5-fold increase in F*(WBC) at gamma = 50 s(-1). WBC-EC adhesion appeared to increase significantly, but much less than with infusion of high molecular weight dextran (Dx). With Dx, G increased 3-fold, with reductions in gamma, but F*(WBC) increased only half the amount incurred with Fb at low shear. The greater margination in the presence of Fb results from RBC rouleaux that promote radial migration of WBCs. In contrast, clumps of RBCs resulting from high molecular weight Dx entrain WBCs within plasma gaps along the vessel centerline. In the presence of Fb, margination of WBCs increases dramatically at low shear due to rouleaux formation, which enhances radial migration of WBCs. This effect is much greater than with Dx because disruption of the much weaker Fb induced rouleaux precludes reductions in H(MICRO), whereas clumping aggregates induced by Dx form plasma gaps. Thus, modest levels of RCA caused by increased Fb may greatly enhance margination and with an enhancement of adhesiveness synergistically promote firm WBC-EC adhesion in the low flow state.

Cytolytically inactive terminal complement complex causes transendothelial migration of polymorphonuclear leukocytes in vitro and in vivo

Intravital microscopy was used to monitor leukocyte traffic across rat mesenteric postcapillary venules induced by the inactive terminal complement (C) complex (iTCC) topically applied to ileal mesentery. Leukocytes started rolling within 15 minutes from the administration of iTCC, and by 1 hour they adhered almost completely to the endothelium emigrating from the vessels in the next 3 hours. C5a caused a similar, though less marked, effect, whereas boiled iTCC was inactive, excluding the contribution of contaminating lipopolysaccharide. The complex stimulated the migration of polymorphonuclear neutrophils (PMNs) across endothelial cells (ECs) in a transwell system after a 4-hour incubation of ECs with iTCC added to the lower chamber of the transwell, whereas a 30-minute incubation was sufficient for C5a and interleukin (IL)-8 to induce the passage of PMNs. C5a was not responsible for the effect of iTCC because this complex had no chemotactic activity and contained too small an amount of C5a to account for the transendothelial migration of PMNs. Similarly, the effect of iTCC was not mediated by IL-8 released by stimulated ECs because anti-IL-8 failed to inhibit the migration of PMNs induced by the complex. Unlike tumor necrosis factor-alpha, iTCC did not cause the redistribution of platelet-endothelial cell adhesion molecule-1 (PECAM-1), and PMN mobilization was partially blocked by anti-PECAM-1 antibodies.