Leukocyte-endothelial Adherence Research Articles

TNF-\u03b1-Inhibition Improves the Biocompatibility of Porous Polyethylene Implants In Vivo

Background: To improve the biocompatibility of porous polyethylene (PPE) implants and expand their application range for reconstructive surgery in poorly vascularized environments, implants were coated with tumor necrosis factor α (TNFα) inhibitor Etanercept. While approved for systemic application, local application of the drug is a novel experimental approach. Microvascular and mechanical integration as well as parameters of inflammation were analyzed in vivo.Methods: PPE implants were coated with Etanercept and extracellular matrix (ECM) components prior to implantation into dorsal skinfold chambers of C57BL/6 mice. Fluorescence microscopy analyses of angiogenesis and local inflammatory response were thrice performed in vivo over a period of 14 days to assess tissue integration and biocompatibility. Uncoated implants and ECM-coated implants served as controls.Results: TNFα inhibition with Etanercept led to a reduced local inflammatory response: leukocyte-endothelial cell adherence was significantly lowered compared to both control groups (n = 6/group) on days 3 and 14, where the lowest values were reached: 3573.88 leukocytes/mm-2 ± 880.16 (uncoated implants) vs. 3939.09 mm-2 ± 623.34 (Matrigel only) vs. 637.98 mm-2 + 176.85 (Matrigel and Etanercept). Implant-coating with Matrigel alone and Matrigel and Etanercept led to significantly higher vessel densities 7 and 14 days vs. 3 days after implantation and compared to uncoated implants. Mechanical implant integration as measured by dynamic breaking strength did not differ after 14 days.Conclusion: Our data show a reduced local inflammatory response to PPE implants after immunomodulatory coating with Etanercept in vivo, suggesting improved biocompatibility. Application of this tissue engineering approach is therefore warranted in models of a compromised host environment.

Reduction of Leukocyte Microvascular Adherence and Preservation of Blood-Brain Barrier Function by Superoxide-Lowering Therapies in a Piglet Model of Neonatal Asphyxia.

Background: Asphyxia is the most common cause of brain damage in newborns. Substantial evidence indicates that leukocyte recruitment in the cerebral vasculature during asphyxia contributes to this damage. We tested the hypothesis that superoxide radical () promotes an acute post-asphyxial inflammatory response and blood-brain barrier (BBB) breakdown. We investigated the effects of removing by superoxide dismutase (SOD) or C3, the cell-permeable SOD mimetic, in protecting against asphyxia-related leukocyte recruitment. We also tested the hypothesis that xanthine oxidase activity is one source of this radical.Methods: Anesthetized piglets were tracheostomized, ventilated, and equipped with closed cranial windows for the assessment of post-asphyxial rhodamine 6G-labeled leukocyte-endothelial adherence and microvascular permeability to sodium fluorescein in cortical venules. Asphyxia was induced by discontinuing ventilation. SOD and C3 were administered by cortical superfusion. The xanthine oxidase inhibitor oxypurinol was administered intravenously.Results: Leukocyte-venular adherence significantly increased during the initial 2 h of post-asphyxial reperfusion. BBB permeability was also elevated relative to non-asphyxial controls. Inhibition of production by oxypurinol, or elimination of by SOD or C3, significantly reduced rhodamine 6G-labeled leukocyte-endothelial adherence and improved BBB integrity, as measured by sodium fluorescein leak from cerebral microvessels.Conclusion: Using three different strategies to either prevent formation or enhance elimination of during the post-asphyxial period, we saw both reduced leukocyte adherence and preserved BBB function with treatment. These findings suggest that agents which lower in brain may be attractive new therapeutic interventions for the protection of the neonatal brain following asphyxia.

Acclimatization of the systemic microcirculation to alveolar hypoxia is mediated by an iNOS-dependent increase in nitric oxide availability

Rats breathing 10% O2 show a rapid and widespread systemic microvascular inflammation that results from nitric oxide (NO) depletion secondary to increased reactive O2 species (ROS) generation. The inflammation eventually resolves, and the microcirculation becomes resistant to more severe hypoxia. These experiments were directed to determine the mechanisms underlying this microvascular acclimatization process. Intravital microscopy of the mesentery showed that after 3 wk of hypoxia (barometric pressure ~380 Torr; partial pressure of inspired O2 ~68-70 Torr), rats showed no evidence of inflammation; however, treatment with the inducible NO synthase (iNOS) inhibitor L-N6-(1-iminoethyl) lysine dihydrochloride led to ROS generation, leukocyte-endothelial adherence and emigration, and increased vascular permeability. Mast cells harvested from normoxic rats underwent degranulation when exposed in vitro to monocyte chemoattractant protein-1 (MCP-1), the proximate mediator of mast cell degranulation in acute hypoxia. Mast cell degranulation by MCP-1 was prevented by the NO donor spermine-NONOate. MCP-1 did not induce degranulation of mast cells harvested from 6-day hypoxic rats; however, pretreatment with either the general NOS inhibitor L-NG-monomethyl arginine citrate or the selective iNOS inhibitor N-[3-(aminomethyl) benzyl] acetamidine restored the effect of MCP-1. iNOS was demonstrated in mast cells and alveolar macrophages of acclimatized rats. Nitrate + nitrite plasma levels decreased significantly in acute hypoxia and were restored after 6 days of acclimatization. The results support the hypothesis that the microvascular acclimatization to hypoxia results from the restoration of the ROS/NO balance mediated by iNOS expression at key sites in the inflammatory cascade.NEW & NOTEWORTHY The study shows that the systemic inflammation of acute hypoxia resolves via an inducible nitric oxide (NO) synthase-induced restoration of the reactive O2 species/NO balance in the systemic microcirculation. It is proposed that the acute systemic inflammation may represent the first step of the microvascular acclimatization process.

Preconditioning-induced CXCL12 upregulation minimizes leukocyte infiltration after stroke in ischemia-tolerant mice

Repetitive hypoxic preconditioning creates long-lasting, endogenous protection in a mouse model of stroke, characterized by reductions in leukocyte–endothelial adherence, inflammation, and infarct volumes. The constitutively expressed chemokine CXCL12 can be upregulated by hypoxia and limits leukocyte entry into brain parenchyma during central nervous system inflammatory autoimmune disease. We therefore hypothesized that the sustained tolerance to stroke induced by repetitive hypoxic preconditioning is mediated, in part, by long-term CXCL12 upregulation at the blood–brain barrier (BBB). In male Swiss Webster mice, repetitive hypoxic preconditioning elevated cortical CXCL12 protein levels, and the number of cortical CXCL12+ microvessels, for at least two weeks after the last hypoxic exposure. Repetitive hypoxic preconditioning-treated mice maintained more CXCL12-positive vessels than untreated controls following transient focal stroke, despite cortical decreases in CXCL12 mRNA and protein. Continuous administration of the CXCL12 receptor (CXCR4) antagonist AMD3100 for two weeks following repetitive hypoxic preconditioning countered the increase in CXCL12-positive microvessels, both prior to and following stroke. AMD3100 blocked the protective post-stroke reductions in leukocyte diapedesis, including macrophages and NK cells, and blocked the protective effect of repetitive hypoxic preconditioning on lesion volume, but had no effect on blood–brain barrier dysfunction. These data suggest that CXCL12 upregulation prior to stroke onset, and its actions following stroke, contribute to the endogenous, anti-inflammatory phenotype induced by repetitive hypoxic preconditioning.

Dexamethasone blocks the systemic inflammation of alveolar hypoxia at several sites in the inflammatory cascade

Alveolar hypoxia produces a rapid and widespread systemic inflammation in rats. The inflammation is initiated by the release into the circulation of monocyte chemoattractant protein-1 (MCP-1) from alveolar macrophages (AMO) activated by the low alveolar Po(2). Circulating MCP-1 induces mast cell (MC) degranulation with renin release and activation of the local renin-angiotensin system, leading to microvascular leukocyte recruitment and increased vascular permeability. We investigated the effect of dexamethasone, a synthetic anti-inflammatory glucocorticoid, on the development of the systemic inflammation of alveolar hypoxia and its site(s) of action in the inflammatory cascade. The inflammatory steps investigated were the activation of primary cultures of AMO by hypoxia, the degranulation of MCs by MCP-1 in the mesentery microcirculation of rats, and the effect of angiotensin II (ANG II) on the leukocyte/endothelial interface of the mesentery microcirculation. Dexamethasone prevented the mesentery inflammation in conscious rats breathing 10% O(2) for 4 h by acting in all key steps of the inflammatory cascade. Dexamethasone: 1) blocked the hypoxia-induced AMO activation and the release of MCP-1 and abolished the increase in plasma MCP-1 of conscious, hypoxic rats; 2) prevented the MCP-1-induced degranulation of mesentery perivascular MCs and reduced the number of peritoneal MCs, and 3) blocked the leukocyte-endothelial adherence and the extravasation of albumin induced by topical ANG II in the mesentery. The effect at each site was sufficient to prevent the AMO-initiated inflammation of hypoxia. These results may explain the effectiveness of dexamethasone in the treatment of the systemic effects of alveolar hypoxia.

Salvianolic acids: small compounds with multiple mechanisms for cardiovascular protection

Salvianolic acids are the most abundant water-soluble compounds extracted from Radix Salvia miltiorrhiza (Danshen). In China, Danshen has been wildly used to treat cardiovascular diseases for hundreds of years. Salvianolic acids, especially salvianolic acid A (Sal A) and salvianolic acid B (Sal B), have been found to have potent anti-oxidative capabilities due to their polyphenolic structure. Recently, intracellular signaling pathways regulated by salvianolic acids in vascular endothelial cells, aortic smooth muscle cells, as well as cardiomyocytes, have been investigated both in vitro and in vivo upon various cardiovascular insults. It is discovered that the cardiovascular protection of salvianolic acids is not only because salvianolic acids act as reactive oxygen species scavengers, but also due to the reduction of leukocyte-endothelial adherence, inhibition of inflammation and metalloproteinases expression from aortic smooth muscle cells, and indirect regulation of immune function. Competitive binding of salvianolic acids to target proteins to interrupt protein-protein interactions has also been found to be a mechanism of cardiovascular protection by salvianolic acids. In this article, we review a variety of studies focusing on the above mentioned mechanisms. Besides, the target proteins of salvianolic acids are also described. These results of recent advances have shed new light to the development of novel therapeutic strategies for salvianolic acids to treat cardiovascular diseases.

ERYTHROPOIETIN ENHANCES OXYGENATION IN CRITICALLY PERFUSED TISSUE THROUGH MODULATION OF NITRIC OXIDE SYNTHASE

The aim of this study was to investigate the effect of human recombinant erythropoietin (EPO) on the microcirculation and oxygenation of critically ischemic tissue and to elucidate the role of endothelial NO synthase in EPO-mediated tissue protection. Island flaps were dissected from the back skin of anesthetized male Syrian golden hamsters including a critically ischemic, hypoxic area that was perfused via a collateralized vasculature. Before ischemia, animals received an injection of epoetin beta at a dose of 5,000 U/kg body weight with (n = 7) or without (n = 7) blocking NO synthase by 30 mg/kg body weight L-NAME (Nomega-nitro-L-arginine methyl ester hydrochloride). Saline-treated animals served as control (n = 7). Ischemic tissue damage was characterized by severe hypoperfusion and inflammation, hypoxia, and accumulation of apoptotic cell nuclei after 5 h of collateralization. Erythropoietin pretreatment increased arteriolar and venular blood flow by 33% and 37%, respectively (P < 0.05), and attenuated leukocytic inflammation by approximately 75% (P < 0.05). Furthermore, partial tissue oxygen tension in the ischemic tissue increased from 8.2 to 15.8 mmHg (P < 0.05), which was paralleled by a 21% increased density of patent capillaries (P < 0.05) and a 50% reduced apoptotic cell count (P < 0.05). The improved microcirculation and oxygenation were associated with a 2.2-fold (P < 0.05) increase of endothelial NO synthase protein expression. Of interest, L-NAME completely abolished all the beneficial effects of EPO pretreatment. Our study demonstrates that, in critically ischemic and hypoxic collateralized tissue, EPO pretreatment improves tissue perfusion and oxygenation in vivo. This effect may be attributed to NO-dependent vasodilative effects and anti-inflammatory actions on the altered vascular endothelium.

The systemic inflammation of alveolar hypoxia is initiated by a circulating mediator(s) released from alveolar macrophages

Alveolar hypoxia (FIO2 = 0.1) produces widespread systemic inflammation in rats. Recent evidence suggests that the inflammation is not triggered by the low systemic PO2, but by activation of mast cells (MC) by a circulating mediator released from activated alveolar macrophages (AM). If this is correct, the following should apply: 1.Neither MC nor resident tissue macrophages should be directly activated by hypoxia and 2. MC would be activated when in contact with hypoxic AM, but not with hypoxic tissue macrophages.MethodsRat mesenteric microcirculation intravital microscopy was combined with primary cultures of AM, peritoneal macrophages (PM), and peritoneal MC.Resultsa. Cultured peritoneal MC did not degranulate with hypoxia. b. Hypoxia induced a respiratory burst in AM, but not in PM cultures. c. Immersion of isolated MC in supernatant of hypoxic AM, but not in supernatant of hypoxic PM, induced MC degranulation d. Supernatant of hypoxic AM, but not of hypoxic PM produced MC degranulation and leukocyte‐endothelial adherence in mesentery.ConclusionsA mediator(s) released from hypoxic AM activates MC and triggers the systemic inflammation. Reduced systemic tissue PO2 and activation of systemic tissue macrophages do not play a role in this phenomenon. The inflammation may contribute to the systemic effects observed in conditions with reduced alveolar PO2.Supported by NIH HL39443‐18 and AHA 0815652G

Lack of effects of acemetacin on signalling pathways for leukocyte adherence may explain its gastrointestinal safety

Acemetacin is a non-steroidal anti-inflammatory drug which is rapidly bioconverted to indomethacin, but produces significantly less gastric damage than indomethacin. This study was performed to investigate several possible mechanisms that could account for the gastrointestinal tolerability of acemetacin. The gastric and intestinal damaging effects of acemetacin and indomethacin were examined in the rat. Effects of the drugs on blood levels of leukotriene B(4) and thromboxane B(2), on leukocyte-endothelial adherence in post-capillary mesenteric venules, and on gastric expression of tumour necrosis factor-alpha (TNF-alpha) were determined. The two drugs were also compared for gastric toxicity in rats pretreated with inhibitors of COX-2 and NOS. Acemetacin induced significantly less gastric and intestinal damage than indomethacin, despite markedly suppressing COX activity. Indomethacin, but not acemetacin, significantly increased leukocyte adherence within mesenteric venules, and gastric expression of TNF-alpha. Pretreatment with L-nitro-arginine methyl ester or lumiracoxib increased the severity of indomethacin-induced gastric damage, but this was not the case with acemetacin. The increased gastric and intestinal tolerability of acemetacin may be related to the lack of induction of leukocyte-endothelial adherence. This may be attributable to the reduced ability of acemetacin to elevate leukotriene-B(4) synthesis and TNF-alpha expression, compared to indomethacin, despite the fact that acemetacin is rapidly bioconverted to indomethacin after its absorption.

NADPH oxidase mediates the mesenteric inflammation initiated by alveolar macrophages in alveolar hypoxia

Alveolar hypoxia (AH), induced by reducing inspired O2 to 10%, rapidly produces inflammation in skeletal muscle, brain, and mesentery of rats. Data from our laboratory suggest that the inflammation is initiated by an agent (s) released into the circulation by alveolar macrophages (AM). This agent stimulates mast cells (MC) and the renin‐angiotensin system (RAS) to produce inflammation. Plasma from AH rats produces inflammation in normoxic muscle; the inflammation is attenuated by MC stabilization and by blockade of the RAS. Depletion of AM in vivo attenuates the inflammation in AH rats; furthermore, supernatant of AM cultured in hypoxia activates MC and produces inflammation in normoxic muscle. The present experiments investigated the interaction: AM‐MC‐inflammation using intravital microscopy; specifically, we asked whether NADPH oxidase is involved in the RAS‐mediated inflammation. Supernatant from AM cultured in hypoxia induced leukocyte endothelial adherence (LEA) and MC degranulation (MCD) in mesentery of normoxic rats. Angiotensin II receptor blocker and NADPH oxidase inhibitor (apocynin) blocked LEA but not MCD, indicating that activation of RAS and of NADPH oxidase are downstream of MCD. The data support the hypothesis that an agent(s) released from AM in AH initiate MCD; this, in turn, stimulates the RAS which leads to inflammation via activation of NADPH oxidase. Supported by NIH HL39443‐17

Alveolar macrophages are necessary for the systemic inflammation of acute alveolar hypoxia

Alveolar hypoxia (Fi(O(2)) 0.10) rapidly produces inflammation in the microcirculation of skeletal muscle, brain, and mesentery of rats. Dissociation between tissue Po(2) values and inflammation, plus the observation that plasma from hypoxic rats activates mast cells and elicits inflammation in normoxic tissues, suggest that the response to hypoxia is initiated when mast cells are activated by an agent released from a distant site and carried by the circulation. These experiments tested the hypothesis that this agent originates in alveolar macrophages (AM). Male rats were depleted of AM by tracheal instillation of clodronate-containing liposomes. Four days after treatment, AM recovered by bronchoalveolar lavage were <10% of control. Control rats received buffer-containing liposomes. As expected, alveolar hypoxia (Fi(O(2)) 0.10) in control rats increased leukocyte-endothelial adherence, produced degranulation of perivascular mast cells, and increased fluorescent albumin extravasation in the cremaster microcirculation. None of these effects was seen when AM-depleted rats were exposed to hypoxia. Plasma obtained from control rats after 5 min of breathing 10% O(2) elicited inflammation when applied to normoxic cremasters. In contrast, normoxic cremasters did not develop inflammation after application of plasma from hypoxic AM-depleted rats. Supernatant from AM cultured in 10% O(2) produced increased leukocyte-endothelial adherence, vasoconstriction, and albumin extravasation when applied to normoxic cremasters. Normoxic AM supernatant did not produce any of these responses. The effects of hypoxic supernatant were attenuated by pretreatment of the cremaster with the mast cell stabilizer cromolyn. These data support the hypothesis that AM are the source of the agent that initiates hypoxia-induced systemic inflammation by activating mast cells.

Role of the renin-angiotensin system in the systemic microvascular inflammation of alveolar hypoxia

Alveolar hypoxia (AH) induces widespread systemic inflammation. Previous studies have shown dissociation between microvascular Po(2) and inflammation. Furthermore, plasma from AH rats (PAHR) induces mast cell (MC) activation, inflammation, and vasoconstriction in normoxic cremasters, while plasma from normoxic rats does not produce these responses. These results suggest that inflammation of AH is triggered by a blood-carried agent. This study investigated the involvement of the renin-angiotensin system (RAS) in the inflammation of AH. Both an angiotensin-converting enzyme (ACE) inhibitor and an angiotensin II (ANG II) receptor blocker (ANG II RB) inhibited the leukocyte-endothelial adherence produced by AH, as well as the inflammation produced by PAHR in normoxic rat cremasters. MC stabilization with cromolyn blocked the effects of PAHR but not those of topical ANG II on normoxic cremasters, suggesting ANG II generation via MC activation by PAHR. This was supported by the observation that ACE inhibition and ANG II RB blocked the leukocyte-endothelial adherence produced by the MC secretagogue compound 48/80. These results suggest that the intermediary agent contained in PAHR activates MC and stimulates the RAS, leading to inflammation, and imply an RAS role in AH-induced inflammation.

Adequate glutathione levels are necessary to prevent hypoxia‐induced inflammation in trained rats

Systemic hypoxia (Hx) produces microvascular inflammation in several tissues, including skeletal muscle. This inflammatory response results in an increase in leukocyte-endothelial adherence (LEA) and an increase in vascular permeability during Hx. The response to Hx is absent in exercised trained (ET) rats. Our objective was to investigate the role of glutathione (GSH), an endogenous antioxidant, on the protective effect of exercise training (5 weeks treadmill exercise). Total GSH levels were measured in ET and untrained (UT) rat liver, blood, cremaster, and biceps femoris. In addition, buthionine sulfoximine (BSO) was used to deplete endogenous GSH to determine if GSH was necessary to prevent Hx-induced inflammation. GSH levels were not elevated in ET liver (2.9±0.1 vs. 2.9±0.2μmol/g in UT, P > 0.05), blood (1.1±0.1 vs. 1.0±0.1μmol/ml in UT, P > 0.05), cremaster (0.7±0.1 vs. 0.8±0.1μmol/g in UT, P > 0.05), and biceps femoris (0.5±0.1 vs. 0.5±0.1μmol/g in UT, P > 0.05). However, BSO depletion of GSH in ET rats produced increases in LEA (2.2±0.4 during Hx vs. 18.4±2.1 leukocytes per 100μm venule during Hx with BSO, P<0.05) and vascular permeability (0.11±0.02 during Hx vs. 0.53±0.05 extra/intravascular FITC-albumin ratio during Hx with BSO, P<0.05). Therefore, exercise training does not significantly elevate GSH levels in liver, blood, cremaster, or biceps femoris, but adequate GSH levels are necessary to prevent Hx-induced inflammation from occurring in ET cremaster venules. Supported by National Heart, Lung, and Blood Institute Grants HL-39443 and HL-64195.

Leukocyte-derived matrix metalloproteinase-9 mediates blood-brain barrier breakdown and is proinflammatory after transient focal cerebral ischemia

Results of recent studies reveal vascular and neuroprotective effects of matrix metalloproteinase-9 (MMP-9) inhibition and MMP-9 gene deletion in experimental stroke. However, the cellular source of MMP-9 produced in the ischemic brain and the mechanistic basis of MMP-9-mediated brain injury require elucidation. In the present study, we used MMP-9-/- mice and chimeric knockouts lacking either MMP-9 in leukocytes or in resident brain cells to test the hypothesis that MMP-9 released from leukocytes recruited to the brain during postischemic reperfusion contributes to this injury phenotype. We also tested the hypothesis that MMP-9 promotes leukocyte recruitment to the ischemic brain and thus is proinflammatory. The extent of blood-brain barrier (BBB) breakdown, the neurological deficit, and the volume of infarction resulting from transient focal stroke were abrogated to a similar extent in MMP-9-/- mice and in chimeras lacking leukocytic MMP-9 but not in chimeras with MMP-9-containing leukocytes. Zymography and Western blot analysis from these chimeras confirmed that the elevated MMP-9 expression in the brain at 24 h of reperfusion is derived largely from leukocytes. MMP-9-/- mice exhibited a reduction in leukocyte-endothelial adherence and a reduction in the number of neutrophils plugging capillaries and infiltrating the ischemic brain during reperfusion; microvessel immunopositivity for collagen IV was also preserved in these animals. These latter results document proinflammatory actions of MMP-9 in the ischemic brain. Overall, our findings implicate leukocytes, most likely neutrophils, as a key cellular source of MMP-9, which, in turn, promotes leukocyte recruitment, causes BBB breakdown secondary to microvascular basal lamina proteolysis, and ultimately contributes to neuronal injury after transient focal stroke.

Plasma from conscious hypoxic rats stimulates leukocyte-endothelial interactions in normoxic cremaster venules

Systemic hypoxia results in rapid increases in leukocyte-endothelial adherence (LEA) and emigration, vascular permeability, and mast cell activation in several microcirculations. Observations in cremaster muscle suggest that this response is initiated by a mediator released from a distant site (Dix R, Orth T, Allen JA, Wood JG, and Gonzalez NC. J Appl Physiol 95: 2495-2502, 2003). The present experiments in rat cremaster muscle tested the hypothesis that, if a circulating mediator triggers hypoxia-induced inflammation, then plasma from hypoxic rats should elicit LEA in normoxic cremaster venules. Plasma from conscious donor rats breathing 10% O2-90% N2 for 5 min was applied topically to the cremaster of normoxic anesthetized rats. In this and all other groups described below, the donor plasma had attained normoxic PO2 when applied to the cremaster. LEA (leukocytes/100-microm venule) increased from 2.7 +/- 0.8 to 12.3 +/- 2.4, and venular shear rate and arteriolar diameter decreased to 79 +/- 9% (P < 0.05, n = 6) and 77 +/- 5% of control (P < 0.05, n = 5), respectively, 10 min after application of plasma from hypoxic donors. The decrease in venular shear rate was exclusively due to a reduction of venular blood flow, secondary to the upstream arteriolar vasoconstriction. Plasma from normoxic donors had no effects. Plasma from blood equilibrated in vitro for 5 min with 5% CO2-95% N2 did not alter LEA or shear rate of normoxic cremasters, suggesting that the putative mediator does not originate in blood cells. The effects of plasma from hypoxic rats persisted when the donors were pretreated with the mast cell stabilizer cromolyn, which prevents hypoxia-induced LEA. This suggests that the effects of hypoxic plasma are not due to inflammatory mediators released by adherent leukocytes in the donor rat. There was a positive correlation between LEA and mast cell degranulation observed histologically. These results support the idea that systemic hypoxia produces the release of a substance transported by the circulation that initiates the microvascular inflammation.

Exercise training prevents the inflammatory response to hypoxia in cremaster venules

Systemic hypoxia produces microvascular inflammation in several tissues, including skeletal muscle. Exercise training (ET) has been shown to reduce the inflammatory component of several diseases. Alternatively, ET could influence hypoxia-induced inflammation by improving tissue oxygenation or increasing mechanical antiadhesive forces at the leukocyte-endothelial interface. The effect of 5 wk of treadmill ET on hypoxia-induced microvascular inflammation was studied in the cremaster microcirculation of rats using intravital microscopy. In untrained rats, hypoxia (arterial Po(2) = 32.3 +/- 2.1 Torr) increased leukocyte-endothelial adherence from 2.3 +/- 0.4 to 10.2 +/- 0.3 leukocytes per 100 microm of venule (P < 0.05) and was accompanied by extravasation of FITC-labeled albumin after 4 h of hypoxia (extra-/intravascular fluorescence intensity ratio = 0.50 +/- 0.07). These responses were attenuated in ET (leukocyte adherence was 1.5 +/- 0.4 during normoxia and 1.8 +/- 0.7 leukocytes per 100 mum venule after 10 min of hypoxia; extra-/intravascular fluorescence intensity ratio = 0.11 +/- 0.02; P < 0.05 vs. untrained) despite similar reductions of arterial (32.4 +/- 1.8 Torr) and microvascular Po(2) (measured with an oxyphor-quenching method) in both groups. Shear rate decreased during hypoxia to similar extents in ET and untrained rats. In addition, circulating blood leukocyte count was similar in ET and untrained rats. The effects of ET on hypoxia-induced leukocyte-endothelial adherence remained up to 4 wk after discontinuing training. Thus ET attenuated hypoxia-induced inflammation despite similar effects of hypoxia on tissue Po(2), venular shear rate, and circulating leukocyte count.

Activation of mast cells by systemic hypoxia, but not by local hypoxia, mediates increased leukocyte-endothelial adherence in cremaster venules.

Systemic hypoxia, produced by lowering inspired Po2, induces a rapid inflammation in several microcirculations, including cremaster muscle. Mast cell activation is a necessary element of this response. Selective reduction of cremaster microvascular Po2 (PmO2) with normal systemic arterial Po2 (PaO2; cremaster hypoxia/systemic normoxia), however, does not elicit increased leukocyte-endothelial adherence (LEA) in cremaster venules. This could be due to a short time of leukocyte exposure to the hypoxic cremaster environment. Conversely, LEA increases when PaO2 is lowered, while cremaster PmO2 remains high (cremaster normoxia/systemic hypoxia). An alternative explanation of these results is that a mediator released from a central site during systemic hypoxia initiates the inflammatory cascade. We hypothesized that if this is the case, cremaster mast cells would be activated during cremaster normoxia/systemic hypoxia, but not during cremaster hypoxia/systemic normoxia. The microcirculation of rat cremaster muscles was visualized by using intravital microscopy. Cremaster PmO2 was measured with a phosphorescence quenching method. Cremaster hypoxia/systemic normoxia (PmO2 7 +/- 1 Torr, PaO2 87 +/- 2 Torr) did not increase LEA; however, topical application of the mast cell activator compound 48/80 under these conditions did increase LEA. The effect of compound 48/80 on LEA was blocked by topical cromolyn, a mast cell stabilizer. LEA increased during cremaster normoxia/systemic hypoxia, (PmO2 64 +/- 5 Torr, PaO2 33 +/- 2 Torr); this increase was blocked by topical cromolyn. The results suggest that mast cell stimulation occurs only when PaO2 is reduced, independent of cremaster PmO2, and support the idea of a mediator that is released during systemic hypoxia and initiates the inflammatory cascade.

Evidence that 5-lipoxygenase and acetylated cyclooxygenase 2-derived eicosanoids regulate leukocyte-endothelial adherence in response to aspirin.

(1) Unlike other nonsteroidal anti-inflammatory drugs that inhibit formation of cyclooxygenase (COX)-dependent eicosanoids, acetylation of COX-2 by aspirin switches eicosanoid biosynthesis from prostaglandin E(2) (PGE(2)) to 15-epi-lipoxin A(4) (15-epi-LXA(4) or aspirin-triggered lipoxin, ATL). ATL formation by activated leukocytes (PMN) requires the intervention of 5-lipoxygenase (5-LOX), an enzyme that is involved in leukotriene B(4) (LTB(4)) formation. (2) In the present study, we have examined the role of acetylated COX-2 and 5-LOX in modulating antiadhesive effects of aspirin on adhesion of PMN to endotoxin (LPS)-primed human umbilical endothelial cells (HUVEC). (3) Treating PMN/HUVEC cocultures with aspirin resulted in a concentration-dependent inhibition of cell-to-cell adhesion induced by LPS. Treating HUVEC with selective COX-2 inhibitors, celecoxib and rofecoxib, caused an approximately 70% reversion of antiadhesive effect of aspirin. In contrast, inhibition of neutrophil's 5-LOX pathway with 1 micro M ZD2138, a selective 5-LOX inhibitor, 1 micro M BAY-X-1005, a FLAP inhibitor, or 100 micro M licofelone, a dual COX/5-LOX inhibitor, did not affect antiadhesive properties of aspirin. (4) Exposure to celecoxib (100 micro M) or rofecoxib (10 micro M) completely suppressed ATL formation caused by aspirin without affecting LTB(4) levels. ZD2138, licofelone and BAY-X-1005 inhibited ATL formation as well as LTB(4) generation. (5) Treatment with LXA(4) reduced PMN adhesion to HUVEC and counteracted the proadhesive effect of celecoxib. In contrast, exposure to Boc-1, an LXA(4) antagonist, counteracts the antiadhesive activities of aspirin. Exposure to U75302, an LTB(4) receptor antagonist, enhances the antiadesive effect of aspirin. (6) Reversal of antiadhesive activities of aspirin by celecoxib was associated with increased expression of LFA-1 on PMN and E-selectin on HUVEC. Addition of LXA(4), ZD2138 and U75302 inhibited these changes. (7) The present results support the notion that inhibition of ATL formation is mechanistically linked to the reversal of the antiadhesive activity of aspirin caused by selective COX-1 inhibitors and suggests that the LTB(4)/ATL balance modulates pro- and antiadhesive activity of nonsteroidal anti-inflammatory drugs at the leukocyte-endothelial cell interface.

Dissociation between skeletal muscle microvascular Po2and hypoxia-induced microvascular inflammation

Systemic hypoxia (SHx) produces microvascular inflammation in mesenteric, cremasteric, and pial microcirculations. In anesthetized rats, SHx lowers arterial blood pressure (MABP), which may alter microvascular blood flow and microvascular Po(2) (Pm(O(2))) and influence SHx-induced leukocyte-endothelial adherence (LEA). These experiments attempted to determine the individual contributions of the decreases in Pm(O(2)), venular blood flow and shear rate, and MABP to the hypoxia-induced increase in LEA. Cremaster microcirculation of anesthetized rats was visualized by intravital microscopy. Pm(O(2)) was measured by a phosphorescence-quenching method. SHx [inspired Po(2) of 70 Torr for 10 min, MABP of 65 +/- 3 mmHg, arterial Po(2) (Pa(O(2))) of 33 +/- 1 Torr] and cremaster ischemia (MABP of 111 +/- 7 mmHg, Pa(O(2)) of 86 +/- 3 Torr) produced similar Pm(O(2)): 7 +/- 2 and 6 +/- 2 Torr, respectively. However, LEA increased only in SHx (1.9 +/- 0.9 vs. 11.2 +/- 1.1 leukocytes/100 microm, control vs. SHx, P < 0.05). Phentolamine-induced hypotension (MABP of 55 +/- 4 mmHg) in normoxia lowered Pm(O(2)) to 26 +/- 6 Torr but did not increase LEA. Cremaster equilibration with 95% N(2)-5% CO(2) during air breathing (Pa(O(2)) of 80 +/- 1 Torr) lowered Pm(O(2)) to 6 +/- 1 Torr but did not increase LEA. On the other hand, when cremaster Pm(O(2)) was maintained at 60-70 Torr during SHx (Pa(O(2)) of 35 +/- 1 Torr), LEA increased from 2.1 +/- 1.1 to 11.1 +/- 1.5 leukocytes/100 microm (P < 0.05). The results show a dissociation between Pm(O(2)) and LEA and support the idea that SHx results in the release of a mediator responsible for the inflammatory response.

Mesenteric microvascular inflammatory responses to systemic hypoxia are mediated by PAF and LTB4.

Systemic hypoxia produces a rapid microvascular inflammatory response characterized by increased reactive oxygen species (ROS) levels, leukocyte-endothelial adherence and emigration, and increased vascular permeability. The lipid inflammatory mediator leukotriene B(4) (LTB(4)) is involved in the early hypoxia-induced responses (ROS generation and leukocyte adherence). Whether other lipid inflammatory mediators participate in this phenomenon is not known. The objective of these experiments was to study the role of platelet-activating factor (PAF) in the microvascular inflammatory response to hypoxia and its potential interactions with LTB(4) in this response. Intravital microscopy was used to examine mesenteric venules of anesthetized rats. We found that WEB-2086, a PAF receptor antagonist, completely prevented the increase in ROS levels and leukocyte adherence during a brief reduction in inspired Po(2) to anesthetized rats; administration of either WEB-2086 or the LTB(4) antagonist LTB(4)-DMA attenuated leukocyte emigration and the increase in vascular permeability to the same extent during prolonged systemic hypoxia in conscious rats. Furthermore, no additive effect was observed in either response when both antagonists were administered simultaneously. This study demonstrates a role for PAF in the rapid microvascular inflammatory response to hypoxia, as well as contributions of PAF and LTB(4) to the slowly developing responses observed during sustained hypoxia. The incomplete blockade of the hypoxia-induced increases in vascular permeability and leukocyte emigration by combined administration of both antagonists indicates that factors in addition to LTB(4) and PAF participate in these phenomena.