5'-AMP-activated protein kinase activation prevents postischemic leukocyte-endothelial cell adhesive interactions

Preconditioning (PC) with nitric oxide (NO) donors or agents that increase endothelial NO synthase (eNOS) activity 24 h before ischemia-reperfusion (I/R) prevents postischemic leukocyte rolling (LR) and stationary leukocyte adhesion (LA). Since 5'-AMP-activated protein kinase (AMPK) phosphorylates eNOS at Ser1177, resulting in activation, we postulated that AMPK activation may trigger the development of a preconditioned anti-inflammatory phenotype similar to that induced by NO donors. Wild-type (WT) C57BL/6J and eNOS(-/-) mice were treated with the AMPK agonist 5-aminoimidazole-4-carboxamide 1-beta-d-furanoside (AICAR) 30 min (early AICAR PC) or 24 h (late AICAR PC) before I/R; LR and LA were quantified in single postcapillary venules in the jejunum using intravital microscopy. I/R induced comparable marked increases in LR and LA in WT and eNOS(-/-) mice relative to sham-operated (no ischemia) animals. Late AICAR PC prevented postischemic LR and LA, whereas early AICAR PC prevented LA in WT mice. Late AICAR PC was ineffective in preventing I/R-induced LR but not LA in the eNOS(-/-) mice, and the same pattern was seen in WT animals treated with the NOS inhibitor N(omega)-nitro-l-arginine. Early AICAR PC remained effective in preventing LA in eNOS(-/-) mice. Our results indicate that both early and late PC with an AMPK agonist produces an anti-inflammatory phenotype in postcapillary venules. Since the protection afforded by late AICAR PC on postischemic LR was prevented by NOS inhibition in WT mice and absent in eNOS-deficient mice, it appears that eNOS triggers this protective effect. In stark contrast, antecedent AMPK activation prevented I/R-induced LA by an eNOS-independent mechanism.     

Human recombinant erythropoietin protects the striated muscle microcirculation of the dorsal skinfold from postischemic injury in mice

Erythropoietin (EPO) has been proposed as a novel cytoprotectant in ischemia-reperfusion (I/R) injury of the brain, heart, and kidney. However, whether EPO exerts its protection by prevention of postischemic microcirculatory deterioration is unknown. We have investigated the effect of EPO on I/R-induced microcirculatory dysfunctions. We used the mouse dorsal skinfold chamber preparation to study nutritive microcirculation and leukocyte-endothelial cell interaction in striated muscle of the dorsal skinfold by in vivo fluorescence microscopy before 3 h of ischemia and during 5 days of reperfusion. Animals were pretreated with EPO (5,000 U/kg body wt) 1 or 24 h before ischemia. Vehicle-treated I/R-injured animals served as controls. Additional animals underwent sham operation only or were pretreated with EPO but not subjected to I/R. I/R significantly (P < 0.05) reduced functional capillary density, increased microvascular permeability, and enhanced venular leukocyte-endothelial cell interaction during early reperfusion. These findings were associated with pronounced (P < 0.05) arteriolar constriction and diminution of blood flow during late reperfusion. Pretreatment with EPO induced EPO receptor and endothelial nitric oxide synthase expression at 6 h of reperfusion (P < 0.05). In parallel, EPO significantly (P < 0.05) reduced capillary perfusion failure and microvascular hyperpermeability during early reperfusion and arteriolar constriction and flow during late reperfusion. EPO pretreatment substantially (P < 0.05) diminished I/R-induced leukocytic inflammation by reducing the number of rolling and firmly adhering leukocytes in postcapillary venules. EPO applied 1 h before ischemia induced angiogenic budding and sprouting at 1 and 3 days of reperfusion and formation of new capillary networks at 5 days of reperfusion. Thus our study demonstrates for the first time that EPO effectively attenuates I/R injury by preserving nutritive perfusion, reducing leukocytic inflammation, and inducing new vessel formation.     

Antecedent hydrogen sulfide elicits an anti-inflammatory phenotype in postischemic murine small intestine: role of heme oxygenase-1

We recently demonstrated that preconditioning with an exogenous hydrogen sulfide donor (NaHS-PC) 24 h before ischemia and reperfusion (I/R) causes postcapillary venules to shift to an anti-inflammatory phenotype in C57BL/6J wild-type (WT) mice such that these vessels fail to support increases in postischemic leukocyte rolling (LR) and leukocyte adhesion (LA). The objective of the present study was to determine whether heme oxygenase-1 (HO-1) is a mediator of these anti-inflammatory effects noted during I/R in mice preconditioned with NaHS. Intravital fluorescence microscopy was used to visualize LR and LA in single postcapillary venules of the murine small intestine. I/R induced marked increases in LR and LA, effects that were prevented by NaHS-PC. Treatment with the HO inhibitor tin protoporphyrin IX, but not the inactive protoporphyrin CuPPIX, just before reperfusion prevented the anti-inflammatory effects of antecedent NaHS. The anti-inflammatory effects of NaHS-PC were mimicked by preconditioning with hemin, an agent that induces HO-1 expression. We then evaluated the effect of NaHS as a preconditioning stimulus in mice that were genetically deficient in HO-1 (HO-1(-/-) on an H129 background with appropriate WT strain controls). NaHS-PC was ineffective in HO-1(-/-) mice. Our work indicates that HO-1 serves as an effector of the anti-inflammatory effects of NaHS-PC during I/R 24 h later.     

Bradykinin-induced proinflammatory signaling mechanisms

Intravital microscopic techniques were used to examine the mechanisms underlying bradykinin-induced leukocyte/endothelial cell adhesive interactions (LECA) and venular protein leakage (VPL) in single postcapillary venules of the rat mesentery. The effects of bradykinin superfusion to increase LECA and VPL were prevented by coincident topical application of either a bradykinin-B(2) receptor antagonist, a cell-permeant superoxide dismutase (SOD) mimetic or antioxidant, or inhibitors of cytochrome P-450 epoxygenase (CYPE) or protein kinase C (PKC) but not by concomitant treatment with either SOD, a mast cell stabilizer, or inhibitors of nitric oxide synthase, cyclooxygenase, xanthine oxidase, NADPH oxidase, or platelet-activating factor. Immunoneutralizing P-selectin or intercellular adhesion molecule-1 (ICAM-1) completely prevented bradykinin-induced leukocyte adhesion and emigration but did not affect VPL. On the other hand, stabilization of F-actin with phalloidin prevented bradykinin-induced leukocyte emigration and VPL but did not alter leukocyte adhesion. These data indicate that bradykinin induces LECA in rat mesenteric venules via a B(2)-receptor-initiated, CYPE-, oxidant- and PKC-mediated, P-selectin- and ICAM-1-dependent mechanism. Bradykinin also produced VPL, an effect that was initiated by stimulation of B(2) receptors and involved CYPE and PKC activation, oxidant generation, and cytoskeletal reorganization but was independent of leukocyte adherence and emigration.     

Time-dependent platelet-vessel wall interactions induced by intestinal ischemia-reperfusion

Platelets roll and adhere in venules exposed to ischemia-reperfusion (I/R). This platelet-endothelial adhesion may influence leukocyte trafficking because platelet depletion decreases I/R-induced leukocyte emigration. The objectives of this study were 1) to assess the time course of platelet adhesion in the small bowel after I/R and 2) to determine the roles of endothelial and/or platelet P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) in this adhesion. The adhesion of fluorescently labeled platelets was monitored by intravital microscopy in postcapillary venules exposed to 45 min of ischemia and up to 8 h of reperfusion. Peak platelet adhesion was observed at 4 h of reperfusion. To assess the contributions of platelet and endothelial cell P-selectin, platelets from P-selectin-deficient and wild-type mice were infused into wild-type and P-selectin-deficient mice, respectively. Platelets deficient in P-selectin exhibited low levels of adhesion comparable to that in sham-treated animals. In the absence of endothelial P-selectin, platelet adhesion was reduced by 65%. Treatment with a blocking antibody against PSGL-1 reduced adhesion by 57%. These results indicate that I/R induces a time-dependent platelet-endothelial adhesion response in postcapillary venules via a mechanism that involves PSGL-1 and both platelet and endothelial P-selectin, with platelet P-selectin playing a greater role.     

Leukocyte dependence of platelet adhesion in postcapillary venules

Reperfusion of ischemic tissues results in development of a proinflammatory, prothrombogenic phenotype, culminating in the recruitment of leukocytes and platelets within postcapillary venules. Recent studies have indicated an interdependence of platelet and leukocyte adhesion, suggesting that heterotypic blood cell interactions may account for postischemic platelet recruitment. The objectives of this study were to 1) determine whether ischemia-reperfusion (I/R)-induced platelet recruitment is leukocyte dependent and 2) quantify the contributions of leukocytes and endothelial cells in this platelet recruitment. Intravital microscopy was used to monitor the recruitment of fluorescently labeled platelets in postcapillary venules of the small intestine after 45-min ischemia and 4-h reperfusion. To assess the leukocyte dependence of platelet adhesion, platelets from wild-type mice were infused into mice deficient in neutrophils and/or lymphocytes and mice deficient in key leukocyte adhesion molecules (CD18 and ICAM-1). These antileukocyte strategies resulted in significantly reduced platelet recruitment. Simultaneous visualization of platelets and leukocytes enabled quantification of leukocyte-dependent and endothelium-dependent platelet adhesion. It was observed that in wild-type animals 74% of I/R-induced platelet adhesion was a result of platelet-leukocyte interactions. Although the majority of adherent platelets were associated with leukocytes, <50% of adherent leukocytes were platelet bearing, suggesting that not all adherent leukocytes support platelet adhesion. These results are consistent with leukocytes playing a major role in supporting I/R-induced platelet adhesion.     

Oxygen radical-dependent expression of CXC chemokines regulate ischemia/reperfusion-induced leukocyte adhesion in the mouse colon

Activation and accumulation of leukocytes constitute a rate-limiting step in ischemia/reperfusion (I/R)-induced tissue injury. The signalling mechanisms, however, that regulate leukocyte rolling and adhesion in the colonic microcirculation are not known. The objective of the study was to define the role of CXC chemokines (MIP-2 and KC) in I/R-induced leukocyte-endothelial cell interactions in the mouse colon. In C57/B16 mice, colonic ischemia was induced by clamping the superior mesenteric artery for 30 min and leukocyte rolling and stationary adhesion were examined in venules after 120 and 240 min of reperfusion. I/R provoked a clear-cut increase in leukocyte rolling and adhesion in colonic venules. Both MIP-2 and KC were upregulated at the gene and protein level in the reperfused colon. Immunoneutralization of MIP-2 and KC by monoclonal antibodies reduced reperfusion-induced firm adhesion of leukocytes by 73% and 75%, respectively. Interestingly, combined inhibition of MIP-2 and KC additionally decreased leukocyte rolling by 79%, but did not further reduce the number of firmly adherent leukocytes. To study the role of oxygen free radicals (OFRs) in the regulation of CXC chemokine expression, additional animals were pretreated with the xanthine-oxidase inhibitor allopurinol. In fact, allopurinol treatment reduced the colonic levels of MIP-2 and KC by 62% and 64%, respectively. This study elucidates important interactions between OFRs and chemokines in the I/R-induced leukocyte response in the mouse colon. Moreover, our data demonstrate that CXC chemokines play a fundamental role in colonic I/R and that functional interference with CXC chemokines may protect against pathological inflammation in the colon.     

Ethanol modulates gut ischemia/reperfusion-induced liver injury in rats

Whereas both ethanol and gut ischemia/reperfusion (I/R) are known to alter hepatic microvascular function, little is known about the influence of ethanol consumption on the hepatic microvascular responses to I/R. The objective of this study was to determine whether acute ethanol administration exacerbates the hepatic microvascular dysfunction induced by gut I/R. Rats were exposed to gut ischemia for 30 min followed by reperfusion. Intravital videomicroscopy was used to monitor leukocyte recruitment and the number of nonperfused sinusoids (NPS). Plasma alanine aminotransferase (ALT), tumor necrosis factor-alpha (TNF-alpha), and endotoxin concentrations were monitored. In separate experiments, ethanol was administered 15 min or 24 h before gut ischemia. In control rats, gut I/R increased the number of stationary leukocytes and NPS. It also elevated the plasma ALT, TNF-alpha, and endotoxin with a corresponding increase in intestinal mucosal permeability. Low-dose ethanol consumption 15 min before gut ischemia blunted the gut I/R-induced leukostasis and elevations in plasma TNF-alpha and ALT. However, high-dose ethanol consumption aggravated the gut I/R-induced increases in leukostasis and increases in plasma endotoxin and ALT. When ethanol was administered 24 h before, high-dose ethanol aggravated the gut I/R-induced hepatocellular injury, but low-dose ethanol did not have any effects on it. These results suggest that low-dose ethanol consumption shortly before gut ischemia attenuates the hepatic inflammatory responses, microvascular dysfunction, and hepatocellular injury elicited by gut I/R, whereas high-dose ethanol consumption appears to significantly aggravate these gut I/R-induced responses.     

Hypercholesterolemia promotes inflammation and microvascular dysfunction: role of nitric oxide and superoxide

Relatively brief periods (days) of hypercholesterolemia can exert profound effects on endothelium-dependent functions of the microcirculation, including dilation of arterioles, fluid filtration across capillaries, and regulation of leukocyte recruitment in postcapillary venules. Hypercholesterolemia appears to convert the normal anti-inflammatory phenotype of the microcirculation to a proinflammatory phenotype. This phenotypic change appears to result from a decline in nitric oxide (NO) bioavailability that results from a reduction in NO biosynthesis, inactivation of NO by superoxide (O(2)(*)(-)), or both. A consequence of the hypercholesterolemia-induced microvascular responses is an enhanced vulnerability of the microcirculation to the deleterious effects of ischemia and other inflammatory conditions. Hence, therapeutic strategies that are directed towards preventing the early microcirculatory dysfunction and inflammation caused by hypercholesterolemia may prove effective in reducing the high mortality associated with ischemic tissue diseases. Agents that act to maintain the normal balance between NO and reactive oxygen species (ROS) in vascular endothelial cells may prove particularly useful in this regard.     

Hypothermia-induced cardioprotection using extended ischemia and early reperfusion cooling

Optimal timing of therapeutic hypothermia for cardiac ischemia is unknown. Our prior work suggests that ischemia with rapid reperfusion (I/R) in cardiomyocytes can be more damaging than prolonged ischemia alone. Also, these cardiomyocytes demonstrate protein kinase C (PKC) activation and nitric oxide (NO) signaling that confer protection against I/R injury. Thus we hypothesized that hypothermia will protect most using extended ischemia and early reperfusion cooling and is mediated via PKC and NO synthase (NOS). Chick cardiomyocytes were exposed to an established model of 1-h ischemia/3-h reperfusion, and the same field of initially contracting cells was monitored for viability and NO generation. Normothermic I/R resulted in 49.7 +/- 3.4% cell death. Hypothermia induction to 25 degrees C was most protective (14.3 +/- 0.6% death, P < 0.001 vs. I/R control) when instituted during extended ischemia and early reperfusion, compared with induction after reperfusion (22.4 +/- 2.9% death). Protection was completely lost if onset of cooling was delayed by 15 min of reperfusion (45.0 +/- 8.2% death). Extended ischemia/early reperfusion cooling was associated with increased and sustained NO generation at reperfusion and decreased caspase-3 activation. The NOS inhibitor N(omega)-nitro-L-arginine methyl ester (200 microM) reversed these changes and abrogated hypothermia protection. In addition, the PKCepsilon inhibitor myr-PKCepsilon v1-2 (5 microM) also reversed NO production and hypothermia protection. In conclusion, therapeutic hypothermia initiated during extended ischemia/early reperfusion optimally protects cardiomyocytes from I/R injury. Such protection appears to be mediated by increased NO generation via activation of protein kinase Cepsilon; nitric oxide synthase.     

Preconditioning with ethanol prevents postischemic leukocyte-endothelial cell adhesive interactions

Long-term ethanol consumption at low to moderate levels exerts cardioprotective effects in the setting of ischemia and reperfusion (I/R). The aims of this study were to determine whether 1) a single orally administered dose of ethanol [ethanol preconditioning (EtOH-PC)] would induce a biphasic temporal pattern of protection (early and late phases) against the inflammatory responses to I/R and 2) adenosine and nitric oxide (NO) act as initiators of the late phase of protection. Ethanol was administered as a bolus to C57BL/6 mice at a dose that achieved a peak plasma concentration of ~45 mg/dl 30 min after gavage and returned to control levels within 60 min of alcohol ingestion. The superior mesenteric artery was occluded for 45 min followed by 60 min of reperfusion beginning 10 min or 1, 2, 3, 4, or 24 h after ethanol ingestion, and the numbers of fluorescently labeled rolling and firmly adherent (stationary) leukocytes in single postcapillary venules of the small intestine were quantified using intravital microscopic approaches. I/R induced marked increases in leukocyte rolling and adhesion, effects that were attenuated by EtOH-PC 2-3 h before I/R (early phase), absent when assessed after 10 min, 1 h, and 4 h of ethanol ingestion, with an even more powerful late phase of protection reemerging when I/R was induced 24 h later. The anti-inflammatory effects of late EtOH-PC were abolished by treatment with adenosine deaminase, an adenosine A(2) (but not A(1)) receptor antagonist, or a NO synthase (NOS) inhibitor during the period of EtOH-PC. Preconditioning with an adenosine A(2) (but not an A(1)) receptor agonist in lieu of ethanol 24 h before I/R mimicked the protective actions of late phase EtOH-PC. Like EtOH-PC, the effect of preconditioning with an adenosine A(2) receptor agonist was abrogated by coincident NOS inhibition. These findings suggest that EtOH-PC induces a biphasic temporal pattern of protection against the proinflammatory effects of I/R. In addition, our observations are consistent with the hypothesis that the late phase of EtOH-PC is triggered by NO formed secondary to adenosine A(2) receptor-dependent activation of NOS during the period of ethanol exposure.     

C5aR-mediated myocardial ischemia/reperfusion injury

The complement system activation can mediate myocardial ischemia and reperfusion (I/R). Inhibition of C5a activity reveals attenuation of I/R-induced myocardial infarct size. However, the contribution of C5a receptor (C5aR) to I/R injury remains to be unknown. Here, we reported that both mRNA and protein for the C5aR were constitutively expressed on cardiomyocytes and upregulated as a function of time after I/R-induced myocardial cell injury in mice. Blockade of C5aR markedly decreased microvascular permeability in ischemic myocardial area and leukocyte adherence to coronary artery endothelium. Importantly, the blocking of C5aR with an anti-C5aR antibody was associated with inhibition in activation of protein kinase C delta (PKC-delta) and induction of PKC-mediated mitogen-activated protein kinase phosphatases-1 (MKP-1) leading to the increased activity of p42/p44 mitogen-activated protein (MAP) kinase cascade. These data provide evidence that C5aR-mediated myocardial cell injury is an important pathogenic factor, and that C5aR blockade may be useful therapeutic targets for the prevention of myocardial I/R injury.     

Pathophysiological role of platelets in acute experimental pancreatitis: influence of endothelin A receptor blockade

The potential pathophysiological role of platelet-endothelium interactions was investigated during ischemia/reperfusion (I/R), and the effect of a selective endothelin(A) receptor antagonist (ET(A)-RA) was evaluated in an acute pancreatitis model. Acute pancreatitis was induced by warm ischemia (60 min) in Wistar rats, and its effects with and without antagonist treatment were investigated. Equivalent sham-operated animals were also studied. Microcirculatory changes were assessed by in vivo microscopy, and serum levels for lipase/amylase and histological specimens were investigated. Capillary constriction to 83.7 +/- 6.7% of sham-operated diameters was observed after 60 min of ischemia. A capillary density of 56.8 +/- 9.3% of the sham-operated group (396.3 +/- 15.8 mm(-1)) was measured after reperfusion. Stagnant leukocytes (329.5 +/- 30.4%) and platelets (337.5 +/- 32.3%) increased in postcapillary venules (P < 0.05). Administration of the ET(A)-RA significantly reduced I/R injury. Capillary diameters were maintained (101.4 +/- 4.5%), and capillary density was improved to 73.3 +/- 7.6% of sham-operated animals (P < 0.05). Stagnant leukocytes (152.3 +/- 10.6%) and platelets (207.1 +/- 19.8%) in sinusoids and postcapillary venules were reduced (P < 0.05). The extent of acute pancreatitis was reduced in the therapy group as indicated by serum lipase/amylase values and histological tissue damage (P < 0.05). Thus, ET(A)-RA therapy was effective in reducing I/R-induced pancreatitis in this experimental model.     

Role of ICAM-1 in chronic ethanol consumption-enhanced liver injury after gut ischemia-reperfusion in rats

Intercellular adhesion molecule-1 (ICAM-1) has been implicated in the hepatic microvascular dysfunction elicited by gut ischemia-reperfusion (I/R). Although the effects of chronic ethanol (EtOH) consumption on the liver are well known, it remains unclear whether this condition renders the hepatic microcirculation more vulnerable to the deleterious effects of gut and/or hepatic I/R. The objectives of this study were to determine whether chronic EtOH consumption alters the severity of gut I/R-induced hepatic microvascular dysfunction and hepatocellular injury and to determine whether ICAM-1 contributes to this response. Male Wistar rats, pair fed for 6 wk a liquid diet containing EtOH or an isocaloric control diet, were exposed to gut I/R. Intravital video microscopy was used to monitor leukocyte recruitment in the hepatic microcirculation, the number of nonperfused sinusoids (NPS), and plasma concentrations of endotoxin and tumor necrosis factor-alpha. Plasma alanine aminotransferase (ALT) levels were measured 6 h after the onset of reperfusion. In control rats, gut I/R elicited increases in the number of stationary leukocytes, NPS, and plasma endotoxin, tumor necrosis factor-alpha, and ALT. In EtOH-fed rats, the gut I/R-induced increases in NPS and leukostasis were blunted in the midzonal region, while exaggerated leukostasis was noted in the pericentral region and terminal hepatic venules. Chronic EtOH consumption also enhanced the gut I/R-induced increase in plasma endotoxin and ALT. The exaggerated responses to gut I/R normally seen in EtOH-fed rats were largely prevented by pretreatment with a blocking anti-ICAM-1 monoclonal antibody. In conclusion, these results suggest that chronic EtOH consumption enhances gut I/R-induced hepatic microvascular dysfunction and hepatocellular injury in the pericentral region and terminal hepatic venules via an enhanced hepatic expression of ICAM-1.     

Protective effects of chlorogenic acid against ischemia/reperfusion injury in rat liver: molecular evidence of its antioxidant and anti-inflammatory properties

Hepatic ischemia and reperfusion injury (I/R) is accompanied by excessive reactive oxygen species and resultant sterile inflammation. Chlorogenic acid (CGA), one of the most abundant polyphenols in the human diet, has been shown to exert potent anti-inflammatory, antibacterial and antioxidant activities. Thus, the purpose of the present study was to investigate protective effects of CGA and its molecular mechanisms against hepatic I/R injury. Rats were subjected to 60 min of partial hepatic ischemia followed by 5 h of reperfusion. CGA (2.5, 5 and 10 mg/kg, ip) was administered twice: 10 min prior to ischemia and 10 min before reperfusion. CGA treatment resulted in marked improvement of hepatic function and histology, and suppressed oxidative stress, as indicated by hepatic lipid peroxidation and glutathione level. Levels of serum tumor necrosis factor-α, inducible nitric oxide synthase and cyclooxygenase-2 protein and mRNA expressions were up-regulated after I/R; these effects were attenuated by CGA. Immunoblot results showed that CGA reduced I/R-induced toll-like receptor 4 overexpression, nuclear translocation of nuclear factor kappa B and interferon regulatory factor-1, high-mobility group box-1 release into extracellular milieu, and enhanced heme oxygenase-1 expression and nuclear translocation of nuclear factor erythroid 2-related factor 2. Our results suggest that CGA alleviates I/R-induced liver injury and that this protection is likely due to inhibition of inflammatory response and enhancement of antioxidant defense systems. Therefore, CGA might have potential as an agent for use in clinical treatment of hepatic I/R injury.     

Immune complexes alter cerebral microvessel permeability: roles of complement and leukocyte adhesion

Immune complexes (ICs) are potent inflammatory mediators in peripheral tissues. However, very few studies have examined the ability of ICs to induce inflammatory responses in the brain. Therefore, using preformed ICs or the reverse passive Arthus (RPA) model to localize ICs to the pial microvasculature of mice, we aimed to investigate the ability of ICs to induce an inflammatory response in the cerebral (pial) microvasculature. Application of preformed ICs immediately increased pial microvascular permeability, with a minimal change in leukocyte adhesion in pial postcapillary venules. In contrast, initiation of the RPA response in the pial microvasculature induced changes in cerebral microvascular permeability and increased leukocyte adhesion in pial postcapillary venules. The RPA response induced deposition of C3 in perivascular regions adjacent to sites of IC formation. Depletion of C3 abrogated RPA-induced microvascular permeability and leukocyte adhesion, indicating that the complement pathway was critical for this response. Inhibition of leukocyte adhesion via CD18 blockade also reduced IC-induced microvascular permeability. However, this did not require intercellular adhesion molecule-1, inasmuch as blockade of intercellular adhesion molecule-1 did not alter RPA-induced microvascular permeability and adhesion. These findings demonstrate that ICs are capable of rapidly inducing inflammatory responses in the cerebral microvasculature, with the complement pathway and leukocyte recruitment playing critical roles in microvascular dysfunction.     

Neutrophil elastase contributes to the development of ischemia-reperfusion-induced liver injury by decreasing endothelial production of prostacyclin in rats

We previously reported that nitric oxide (NO) derived from endothelial NO synthase (NOS) increased endothelial prostacyclin (PGI(2)) production in rats subjected to hepatic ischemia-reperfusion (I/R). The present study was undertaken to determine whether neutrophil elastase (NE) decreases endothelial production of PGI(2), thereby contributing to the development of I/R-induced liver injury by decreasing hepatic tissue blood flow in rats. Hepatic tissue levels of 6-keto-PGF(1alpha), a stable metabolite of PGI(2), were transiently increased and peaked at 1 h after reperfusion, followed by a gradual decrease until 3 h after reperfusion. Sivelestat sodium hydrochloride and L-658,758, two NE inhibitors, reduced I/R-induced liver injury. These substances inhibited the decreases in hepatic tissue levels of 6-keto-PGF(1alpha) at 2 and 3 h after reperfusion but did not affect the levels at 1 h after reperfusion. These NE inhibitors significantly increased hepatic tissue blood flow from 1 to 3 h after reperfusion. Both hepatic I/R-induced increases in the accumulation of neutrophils and the microvascular permeability were inhibited by these two NE inhibitors. Protective effects induced by the two NE inhibitors were completely reversed by pretreatment with nitro-l-arginine methyl ester, an inhibitor of NOS, or indomethacin. Administration of iloprost, a stable derivative of PGI(2), produced effects similar to those induced by NE inhibitors. These observations strongly suggest that NE might play a critical role in the development of I/R-induced liver injury by decreasing endothelial production of NO and PGI(2), leading to a decrease in hepatic tissue blood flow resulting from inhibition of vasodilation and induction of activated neutrophil-induced microvascular injury.     

Inflammatory responses to ischemia,and reperfusion in skeletal muscle

Skeletal muscle ischemia and reperfusion is now recognized as one form of acute inflammation in which activated leukocytes play a key role. Although restoration of flow is essential in alleviating ischemic injury, reperfusion initiates a complex series of reactions which lead to neutrophil accumulation, microvascular barrier disruption, and edema formation. A large body of evidence exists which suggests that leukocyte adhesion to and emigration across postcapillary venules plays a crucial role in the genesis of reperfusion injury in skeletal muscle. Reactive oxygen species generated by xanthine oxidase and other enzymes promote the formation of proinflammatory stimuli, modify the expression of adhesion molecules on the surface of leukocytes and endothelial cells, and reduce the bioavailability of the potent antiadhesive agent nitric oxide. As a consequence of these events, leukocytes begin to form loose adhesive interactions with postcapillary venular endothelium (leukocyte rolling). If the proinflammatory stimulus is sufficient, leukocytes may become firmly adherent (stationary adhesion) to the venular endothelium. Those leukocytes which become firmly adherent may then diapedese into the perivascular space. The emigrated leukocytes induce parenchymal cell injury via a directed release of oxidants and hydrolytic enzymes. In addition, the emigrating leukocytes also exacerbate ischemic injury by disrupting the microvascular barrier during their egress across the vasculature. As a consequence of this increase in microvascular permeability, transcapillary fluid filtration is enhanced and edema results. The resultant increase in interstitial tissue pressure physically compresses the capillaries, thereby preventing microvascular perfusion and thus promoting the development of the no-reflow phenomenon. The purpose of this review is to summarize the available information regarding these mechanisms of skeletal muscle ischemia/reperfusion injury.     

A Korean herbal medicine, Panax notoginseng, prevents liver fibrosis and hepatic microvascular dysfunction in rats

We assessed the prevention of hepatic fibrogenesis by water-extract of Panax notoginseng Buck F.H. Chen. (Arialiaceae) root (PNS) in Long-Evans rats with cinnamon coat color (LEC rats). LEC rats were divided into three groups A, fed on a basal diet (BD); B, fed on BD plus 1% PNS; and C), fed on BD plus 0.005% lycopene as a control. All rats were sacrificed at 26 weeks of age. The percentage of the total area involved by fibrosis was 1.46 +/- 0.47 in group A, 0.83 +/- 0.10 in B (P=0.0030, B vs A) and 0.91 +/- 0.45 in C (P=0.0035, C vs. A). The percentage of the total area that was stained for alpha-SMA was 0.56 +/- 0.34 in group A, 0.15 +/- 0.02 in B (P=0.0016, B vs. A and 0.11 +/- 0.01 in C (P=0.0025, C vs. A. In group B, malondialdehyde (MDA) in the liver was lower than in group C (P=0.007). In group C, the concentration of iron in the liver was lower than in group A (P=0.0053). Thus, PNS suppressed fibrogenesis through reduced generation of lipid peroxides. The mechanisms of this preventive effect of fibrogenesis with PNS were suggested to inhibit the stellate cell activity. Second objective of this study was to determine whether PNS affects hepatic microvascular dysfunction elicited by gut ischemia and reperfusion (I/R), since gut I/R causes hepatic microvascular dysfunction, and to investigate the role of nitric oxide (NO). Male Wistar rats were exposed to 30 min of gut ischemia followed by 60 min of reperfusion. Intravital microscopy was used to monitor the number of non-perfused sinusoids (NPS). In another set of experiments, PNS (1 g/kg per day intragastrically) was administered to rats for 7 days. In some experiments, dexamethasone (ST) (2 mg/kg per day intravenously) was administered. In control rats, gut I/R elicited increases in the number of NPS, and plasma TNF-alpha and ALT activities, and these changes were mitigated by the pretreatment with PNS. Pretreatment with an NO synthase inhibitor diminished the protective effects of PNS on the increase in NPS and plasma TNF-alpha levels, but not its effect on the increase in plasma ALT activities. Pretreatment with PNS increased plasma nitrite/nitrate levels. The responses caused by gut I/R were attenuated by the pretreatment with ST. Pretreatment with an NO synthase inhibitor did not affect the effect of ST. These results suggest that PNS attenuates the gut I/R-induced hepatic microvascular dysfunction and inflammatory responses such as TNF-alpha production in the early phase via enhancement of NO production, and sequential hepatocellular damage via its anti-inflammatory effect.     

Angiotensin II mediates postischemic leukocyte-endothelial interactions: role of calcitonin gene-related peptide

Vascular inflammation and enhanced production of angiotensin II (ANG II) are involved in the pathogenesis of hypertension and diabetes, disease states that predispose the afflicted individuals to ischemic disorders. In light of these observations, we postulated that ANG II may play a role in promoting leukocyte rolling (LR) and adhesion (LA) in postcapillary venules after exposure of the small intestine to ischemia-reperfusion (I/R). Using an intravital microscopic approach in C57BL/6J mice, we showed that ANG II type I (AT(1)) or type II (AT(2)) receptor antagonism (with valsartan or PD-123319, respectively), inhibition of angiotensin-converting enzyme (ACE) with captopril, or calcitonin gene-related peptide (CGRP) receptor blockade (CGRP8-37) prevented postischemic LR but did not influence I/R-induced LA. However, both postischemic LR and LA were largely abolished by concomitant AT(1) and AT(2) receptor blockade or chymase inhibition (with Y-40079). Additionally, exogenously administered ANG II increased LR and LA, effects that were attenuated by pretreatment with a CGRP receptor antagonist or an NADPH oxidase inhibitor (apocynin). Our work suggests that ANG II, formed by the enzymatic activity of ACE and chymase, plays an important role in inducing postischemic LR and LA, effects that involve the engagement of both AT(1) and AT(2) receptors and may be mediated by CGRP and NADPH oxidase.