Endothelial Cell-Derived von Willebrand Factor Is the Major Determinant That Mediates von Willebrand Factor-Dependent Acute Ischemic Stroke by Promoting Postischemic Thrombo-Inflammation.

OBJECTIVE: von Willebrand factor (VWF), which is synthesized in endothelial cells and megakaryocytes, is known to worsen stroke outcome. In vitro studies suggest that platelet-derived VWF (Plt-VWF) is biochemically different from the endothelial cell-derived VWF (EC-VWF). However, little is known about relative contribution of different pools of VWF in stroke. APPROACH AND RESULTS: Using bone marrow transplantation, we generated chimeric Plt-VWF mice, Plt-VWF mice that lack ADAMTS13 in platelets and plasma (Plt-VWF/Adamts13(-/-)), and EC-VWF mice to determine relative contribution of different pools of VWF in stroke. In brain ischemia/reperfusion injury model, we found that infarct size and postischemic intracerebral thrombo-inflammation (fibrin(ogen) deposition, neutrophil infiltration, interleukin-1ß, and tumor necrosis factor-α levels) within lesions were comparable between EC-VWF and wild-type mice. Infarct size and postischemic thrombo-inflammation were comparable between Plt-VWF and Plt-VWF/Adamts13(-/-) mice, but decreased compared with EC-VWF and wild-type mice (P<0.05) and increased compared with Vwf(-/-) mice (P<0.05). Susceptibility to FeCl3 injury-induced carotid artery thrombosis was comparable between wild-type and EC-VWF mice, whereas Plt-VWF and Plt-VWF/Adamts13(-/-) mice exhibited defective thrombosis. Although most of the injured vessels did not occlude, slope over time showed that thrombus growth rate was increased in both Plt-VWF and Plt-VWF/Adamts13(-/-) mice compared with Vwf(-/-) mice (P<0.05), but decreased compared with wild-type or EC-VWF mice. CONCLUSIONS: Plt-VWF, either in presence or absence of ADAMTS13, partially contributes to VWF-dependent injury and postischemic thrombo-inflammation after stroke. EC-VWF is the major determinant that mediates VWF-dependent ischemic stroke by promoting postischemic thrombo-inflammation.

TLR4-HMGB1-, MyD88- and TRIF-dependent signaling in mouse intestinal ischemia/reperfusion injury.

AIM: To characterize high-mobility group protein 1-toll-like receptor 4 (HMGB1-TLR4) and downstream signaling pathways in intestinal ischemia/reperfusion (I/R) injury. METHODS: Forty specific-pathogen-free male C57BL/6 mice were randomly divided into five groups (n = 8 per group): sham, control, anti-HMGB1, anti-myeloid differentiation gene 88 (MyD88), and anti-translocating-chain-associating membrane protein (TRIF) antibody groups. Vehicle with the control IgG antibody, anti-HMGB1, anti-MyD88, or anti-TRIF antibodies (all 1 mg/kg, 0.025%) were injected via the caudal vein 30 min prior to ischemia. After anesthetization, the abdominal wall was opened and the superior mesenteric artery was exposed, followed by 60 min mesenteric ischemia and then 60 min reperfusion. For the sham group, the abdominal wall was opened for 120 min without I/R. Levels of serum nuclear factor (NF)-κB p65, interleukin (IL)-6, and tumor necrosis factor (TNF)-α were measured, along with myeloperoxidase activity in the lung and liver. In addition,morphologic changes that occurred in the lung and intestinal tissues were evaluated. Levels of mRNA transcripts encoding HMGB1 and NF-κB were measured by real-time quantitative PCR, and levels of HMGB1 and NF-κB protein were measured by Western blot. Results were analyzed using one-way analysis of variance. RESULTS: Blocking HMGB1, MyD88, and TRIF expression by injecting anti-HMGB1, anti-MyD88, or anti-TRIF antibodies prior to ischemia reduced the levels of inflammatory cytokines in serum; NF-κB p65: 104.64 ± 11.89, 228.53 ± 24.85, 145.00 ± 33.63, 191.12 ± 13.22, and 183.73 ± 10.81 (P < 0.05); IL-6: 50.02 ± 6.33, 104.91 ± 31.18, 62.28 ± 6.73, 85.90 ± 17.37, and 78.14 ± 7.38 (P < 0.05); TNF-α, 43.79 ± 4.18, 70.81 ± 6.97, 52.76 ± 5.71, 63.19 ± 5.47, and 59.70 ± 4.63 (P < 0.05) for the sham, control, anti-HMGB1, anti-MyD88, and anti-TRIF groups, respectively (all in pg/mL).Antibodies also alleviated tissue injury in the lung and small intestine compared with the control group in the mouse intestinal I/R model. The administration of anti-HMGB1, anti-MyD88, and anti-TRIF antibodies markedly reduced damage caused by I/R, for which anti-HMGB1 antibody had the most obvious effect. CONCLUSION: HMGB1 and its downstream signaling pathway play important roles in the mouse intestinal I/R injury, and the effect of the TRIF-dependent pathway is slightly greater.

Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators.

Nucleotide-based drug candidates such as antisense oligonucleotides, aptamers, immunoreceptor-activating nucleotides, or (anti)microRNAs hold great therapeutic promise for many human diseases. Phosphorothioate (PS) backbone modification of nucleotide-based drugs is common practice to protect these promising drug candidates from rapid degradation by plasma and intracellular nucleases. Effects of the changes in physicochemical properties associated with PS modification on platelets have not been elucidated so far. Here we report the unexpected binding of PS-modified oligonucleotides to platelets eliciting strong platelet activation, signaling, reactive oxygen species generation, adhesion, spreading, aggregation, and thrombus formation in vitro and in vivo. Mechanistically, the platelet-specific receptor glycoprotein VI (GPVI) mediates these platelet-activating effects. Notably, platelets from GPVI function-deficient patients do not exhibit binding of PS-modified oligonucleotides, and platelet activation is fully abolished. Our data demonstrate a novel, unexpected, PS backbone-dependent, platelet-activating effect of nucleotide-based drug candidates mediated by GPVI. This unforeseen effect should be considered in the ongoing development programs for the broad range of upcoming and promising DNA/RNA therapeutics.

Role of calreticulin mutations in the aetiological diagnosis of splanchnic vein thrombosis.

BACKGROUND & AIMS: Myeloproliferative neoplasms are the most common aetiological cause of splanchnic vein thrombosis (SVT). In these patients, the JAK2V617F mutation has facilitated the diagnosis of an underlying myeloproliferative neoplasm (MPN). Recently, somatic mutations of the calreticulin (CALR) gene have been identified in MPN patients lacking the JAK2 mutation. The aim of the present study was to ascertain whether CALR mutations could also play a role in the diagnosis of masked MPN in SVT. METHODS: We included 209 patients with SVT (140 with PVT and 69 with Budd-Chiari syndrome) who had a complete aetiological diagnostic work-out. They were investigated for CALR mutations. RESULTS: CALR mutations were found in 4 of the 209 patients (1.9%). They represented 5.4% of patients with an underlying MPN of whom all had already been diagnosed with a MPN using conventional criteria including bone marrow biopsy findings. CONCLUSIONS: In the screening of underlying MPNs in patients with SVT, given its high frequency in these disorders, the JAK2 mutation must be evaluated first and, if negative, CALR mutations should also be investigated. This approach would increase the diagnostic yield of masked MPNs by reducing the need for additional studies.

Intraperitoneal metabolic consequences of supraceliac aortic balloon occlusion in an experimental animal study using microdialysis.

BACKGROUND: To investigate the effects of supraceliac aortic balloon occlusion (ABO) and superior mesenteric artery (SMA) occlusion on abdominal visceral metabolism in an animal model using intraperitoneal microdialysis (IPM) and laser Doppler flowmetry. METHODS: A total of 9 pigs were subjected to ABO and 7 animals were subjected to SMA occlusion for 1 hour followed by 3 hours of reperfusion. Seven animals served as controls. Hemodynamic data, arterial blood samples, urinary output, and intestinal mucosal blood flow (IBF) were followed hourly. Intraperitoneal (i.p) glucose, glycerol, lactate, and pyruvate concentrations and lactate-to-pyruvate (l/p) ratio were measured using IPM. RESULTS: Compared with the baseline, ABO reduced IBF by 76% and decreased urinary output. SMA occlusion reduced IBF by 75% without affecting urinary output. ABO increased the i.p l/p ratio from 18 at baseline, peaking at 46 in early reperfusion. SMA occlusion and reperfusion tended to increase the i.p l/p ratio, peaking at 36 in early reperfusion. ABO increased the i.p glycerol concentration from 87 µM at baseline to 579 µM after 3 hours of reperfusion. SMA occlusion and reperfusion increased the i.p glycerol concentration but to a lesser degree. CONCLUSIONS: Supraceliac ABO caused severe hemodynamic, renal, and systemic metabolic disturbances compared with SMA occlusion, most likely because of the more extensive ischemia-reperfusion injury. The intra-abdominal metabolism, measured by microdialysis, was affected by both ABO and SMA occlusion but the most severe disturbances were caused by ABO. The i.p l/p ratios and the glycerol concentrations increased during ischemia and reperfusion and may serve as markers of these events and indicate anaerobic metabolism and cell damages respectively.

MiR-874 promotes intestinal barrier dysfunction through targeting AQP3 following intestinal ischemic injury.

Intestinal ischemic injury is a significant clinical problem arising from diseases or as a complication of abdominal surgery. Our previous study showed aquaporin 3 is involved in intestinal barrier impairment. Here, we revealed that intestinal ischemia induced a time-dependent increase of miR-874 expression and a time-dependent decrease of AQP3 expression, and the level of miR-874 expression was inversely related to AQP3 protein expression. In addition, miR-874 promoted the paracellular permeability in vitro through targeting 3'UTR of AQP3. Two of the tight junction proteins, Occludin and Claudin-1, were found to be involved in miR-874-induced intestinal barrier dysfunction.

Effects of exogenous vasoactive intestinal peptide on mesenteric lymph pathway during early intestinal ischemia-reperfusion injury in rats.

Mesenteric lymph pathway serves as the primary route by which gut injury leads to systemic inflammation and distant organ injury. The inflammation of the intestinal tract is partially mediated by vasoactive intestinal peptide (VIP). Therefore, the aim of this study was to test whether exogenous VIP affects mesenteric lymph pathway during early intestinal ischemia-reperfusion (IIR) injury. Rats were randomized into control, control+VIP, IIR and IIR+VIP groups. The observation of mesenteric lymph flow was carried out by cannulation of mesenteric lymphatics. The distribution of in vivo lymphocyte trafficking was performed by (51)Cr labeled lymphocytes and was measured by γ-counter. Endotoxin concentration was assayed using the limulus test kit and TNF-α level was detected by ELISA. When IIR injury treated with VIP, the volumes of lymph flow increased by 80%, which caused the number of lymphocytes exiting in mesenteric lymphatic increased by 50% while the proportion of (51)Cr-lymphocytes in Peyer's patches, intestinal effector tissues, mesenteric nodes, large intestine, stomach decreased by 58%, 51%, 58%, 63%, 64% respectively at the 6th h after reperfusion following intestinal ischemia. Meanwhile, endotoxin and TNF-α levels in intestinal lymph decreased by 51% and 83%. These results suggest that exogenous VIP ameliorates IIR induced splanchnic organ damage via inhibition of toxic mediators reaching systemic circulation and reinforcement of the effective immune responses in gut-associated lymphoid tissues (GALT).

Pentoxifylline protects the small intestine after severe ischemia and reperfusion.

OBJECTIVES: Pentoxifylline, a methylxanthine derivative with significant hemorheologic properties, is used for claudication in patients with peripheral vascular disease, and experimentally for ischemic injury to organs because of its antioxidant and antiinflammatory effects. We used a rat model of severe small intestinal ischemia and reperfusion to determine the ability of pentoxifylline in improving survival, molecular response, and pathological protection. MATERIALS AND METHODS: We used 6 groups of male Wistar rats (n=25 each). The superior mesenteric artery was occluded for 120 minutes. Laboratory and tissue studies were done on 5 animals, 1 hour after reperfusion, and animal survival was assessed at 7 days. There were 2 control groups that received normal saline, either before ischemia or during reperfusion. The 4 treated groups received pentoxifylline 1 or 10 mg/kg at the same times mentioned above. Laboratory studies included measuring serum lactic acid dehydrogenase, tumor necrosis factor-α, interleukin-1ß, and interleukin-6.Intestinal tissue malondialdehyde and myeloperoxidase in small intestine tissue also were measured. Histology and laser vascular blood flow at baseline and reperfusion were obtained, and survival was determined 7 days after ischemia. RESULTS: A significant survival benefit in the animals treated with 10 mg/kg of pentoxifylline at reperfusion was noted. This coincided with a reduction in biochemical markers of cell damage - specifically, serum lactic acid dehydrogenase, and tissue malondialdehyde, ischemia, and reperfusion. Additionally, we saw decreased levels of tumor necrosis factor-α, interleukin-1ß, and interleukin-6. Improved postreperfusion blood flow shown by laser Doppler technology also was seen in the treated groups. Histologically, we observed less neutrophil infiltration in the intestine of ischemic-treated rats. Also seen in the control animals were increased necrotic lesions in the microvilli with a higher presence of lysozyme in the Paneth cells. Survival was significantly better at 7 days (70% vs 40%) when we compared the pentoxifylline group treated at reperfusion (10 mg/kg) to the ischemic controls. CONCLUSIONS: Pentoxifylline had a significant protective effect on severely ischemic bowel when administered during reperfusion at a dosage of 10 mg/kg. Better survival, improved histology, and molecular response should urge consideration of the consideration of applying these findings in some general surgery and transplant conditions.

Challenges in diagnosing mesenteric ischemia.

Early identification of acute mesenteric ischemia (AMI) is challenging. The wide variability in clinical presentation challenges providers to make an early accurate diagnosis. Despite major diagnostic and treatment advances over the past decades, mortality remains high. Arterial embolus and superior mesenteric artery thrombosis are common causes of AMI. Non-occlusive causes are less common, but vasculitis may be important, especially in younger people. Because of the unclear clinical presentation and non-specific laboratory findings, low clinical suspicion may lead to loss of valuable time. During this diagnostic delay, progression of ischemia to transmural bowel infarction with peritonitis and septicemia may further worsen patient outcomes. Several diagnostic modalities are used to assess possible AMI. Multi-detector row computed tomographic angiography is the current gold standard. Although computed tomographic angiography leads to an accurate diagnosis in many cases, early detection is a persistent problem. Because early diagnosis is vital to commence treatment, new diagnostic strategies are needed. A non-invasive simple biochemical test would be ideal to increase clinical suspicion of AMI and would improve patient selection for radiographic evaluation. Thus, AMI could be diagnosed earlier with follow-up computed tomographic angiography or high spatial magnetic resonance imaging. Experimental in vitro and in vivo studies show promise for alpha glutathione S transferase and intestinal fatty acid binding protein as markers for AMI. Future research must confirm the clinical utility of these biochemical markers in the diagnosis of mesenteric ischemia.

Ischemic postconditioning attenuate reperfusion injury of small intestine: impact of mitochondrial permeability transition.

BACKGROUND: Ischemic postconditioning (IPoC) modulates the reperfusion maneuver to mitigate ischemia-reperfusion (I/R) injury. This study aims to investigate the effects and protective mechanism of IPoC on intestinal I/R injury. METHODS: Intestinal I/R was induced by occluding the superior mesenteric artery for 30 min followed by reperfusion for 60 min on male Wistar rats. IPoC was elicited by three cycles of 30-sec reperfusion and reocclusion of superior mesenteric artery at the initiation of reperfusion. Carboxyatractyloside (CATR), a mitochondrial permeability transition pore (mPTP) opener, and N-methyl-4-isoleucine cyclosporine (NIM811), an mPTP inhibitor, were administered separately in selected groups. The serum and intestinal sections were collected for analysis. RESULTS: IPoC and the administration of NIM811 significantly diminished the expression of intestinal-type fatty acid-binding protein and lactate dehydrogenase (3427±236.8 U/L for I/R, 1190.5±36.7 U/L for IPoC, 1399.3±295.6 U/L for I/R+NIM811, and 2002±370.9 IU/L for IPoC+CATR) in portal blood, the release of cytosolic cytochrome c, and the cleaved caspase 9 expression in intestinal mucosa after intestinal I/R injury (P<0.05). Histopathologically, IPoC and NIM811 mitigated mucosal damage after I/R as well (Chiu's score, 3.8±0.4 for I/R, 0.2±0.2 for IPoC, 0.4±0.2 for I/R+NIM811, and 4.2±0.2 for IPoC+CATR; apoptotic index, 59.5%±4.6% for I/R, 15.7%±15.7% for I/R+IPoC, 3.5%±3.5% for I/R+NIM811, and 67.1%±9.3% in IPoC+CATR). CATR negated the protection conferred by IPoC. CONCLUSIONS: IPoC and NIM811 attenuate intestinal I/R injury. The addition of CATR negated the effects of IPoC, indicating that the protective mechanism of IPoC was associated with the modulation of mPTP opening.

Diagnostic pitfalls at admission in patients with acute superior mesenteric artery occlusion.

BACKGROUND: Acute superior mesenteric artery (SMA) occlusion leads to acute intestinal ischemia and is associated with high mortality. Early diagnosis is often missed, and confounding factors leading to diagnostic delays need to be highlighted. OBJECTIVES: To identify potential diagnostic laboratory pitfalls at admission in patients with acute SMA occlusion. METHODS: Fifty-five patients with acute SMA occlusion were identified from the in-hospital register during a 4-year period, 2005-2009. RESULTS: The median age was 76 years; 78% were women. The occlusion was embolic in 53% and thrombotic in 47% of patients. At admission, troponin I was above the clinical decision level (> 0.06 µg/L) for acute ischemic myocardial injury in 9/19 (47%) patients with embolic occlusion. Elevated pancreas amylase and normal plasma lactate were found in 12/45 and 13/27, respectively. A troponin I (TnI) above the clinical decision level was associated with a high frequency of referrals from the general surgeon to a specialist in internal medicine (p = 0.011) or a cardiologist (p = 0.024). The diagnosis was established after computed tomography angiography in 98% of the patients. The overall in-hospital mortality rate was 33%. Attempting intestinal revascularization (n = 43; p < 0.001), with a 95% frequency rate of completion control of the vascular procedure, was associated with a higher survival rate, whereas referral to the cardiologist was associated with a higher mortality rate (p = 0.018). CONCLUSION: Elevated TnI was common in acute SMA occlusion, and referral to the cardiologist was found to be associated with adverse outcome. Elevated pancreas amylase and normal plasma lactate values are also potential pitfalls at admission in patients with acute SMA occlusion.

Effects of peripherally and centrally applied ghrelin in the pathogenesis of ischemia-reperfusion induced injury of the small intestine.

Ghrelin is an important hormone involved in the control of the human appetite center. Recently, protective properties of this hormone have been recognized in various models of impairment of the gastric mucosa, including stress, ischemia and reperfusion (I/R). Ghrelin is predominantly secreted by the gastric mucosa of stomach, but there are other sources of ghrelin, for example in the hypothalamus and various parts of the central nervous system (CNS) that should be taken into consideration. This hormone exerts biological effects via the activation of growth hormone secretagogue receptor (GHSR), the presence of which was confirmed in different parts of the gastrointestinal (GI) tract and midbrain structures. Although substantial evidence of the divergent biological effects of ghrelin and the mechanism of its action has been emphasized, the precise mechanisms of ghrelin which affords GI protection is still unclear. Particularly, there is a sparse amount of evidence concerning its action on the GI system. The major aim of the present study was to evaluate the importance of peripherally and centrally administered ghrelin at different times of the ischemia and reperfusion (I/R period in the modulation of resistance of the intestinal mucosa to the injury induced by ischemia and subsequent reperfusion. Secondly, we wanted to evaluate the possible mechanism of the action of ghrelin with a particular focus on its influence on the intestinal blood flow. Male Wistar rats were divided into 4 series (A-D) of the experimental groups (n=7). In series A the importance of peripherally administered ghrelin at different time of I/R period was studied. In series B the importance of centrally administered ghrelin at different time of I/R period was evaluated. In series C and D, the mechanisms of peripherally and centrally administered hormone were examined, respectively. Two models of the I/R period were selected: short lasting (30/60 min) and long lasting (60/120 min). The following drugs were used: ghrelin (50 µg/kg i.p. or 1 nmol in 10 µl i.c.v.), 6 hydroxy dopamine (50 mg/kg i.p.), nadolol (0.5 mg/kg i.p.), calcitonin gene related peptide fragment (CGRP(8-37), 100 µg /kg i.p.), capsaicin (5-10 mg/100 ml solution s.c.). The mesenteric blood flow (MBF-ml/min), the intestinal microcirculatory blood flow (LDBF-PU), the arterio-venous oxygen difference (AVO(2)-ml/O(2)/100 ml blood), and the intestinal oxygen uptake (VO(2)) in ml O(2)/min were measured. Mucosal impairment was assessed planimetrically with the use of a digital photo analyzer (LA) and histologically with the use of the six-point Park/Chiu scale. Peripheral administration of ghrelin evoked marked increase of MBF and LDBF by 42% and 48%, respectively, with significant reduction of LA by 38%. When ghrelin was administered at the beginning of the reperfusion period during the short I/R period or prior to the long lasting I/R period, the vascular reactions and protective effects were reduced, but not completely abolished. The central administration of ghrelin before the short I/R period significantly increased the MBF and LDBF by about 32% and 35%, respectively, as well as LA reduction by about 20% in comparison to the control group. However, when ghrelin was administered prior to the long I/R period or after the onset of completed ischemia, neither vascular nor protective effects were noticed. Sensory denervation and the blockade of the CGRP1 receptors totally blocked the protective and hyperemic effects of the peripherally administered ghrelin. Selective blockade of the adrenergic system or blunting of the vagal nerves (vagotomy) significantly but not totally eliminated the effects of centrally applied ghrelin, which were abolished when both adrenergic and parasympathetic pathways were ablated. These results indicate that ghrelin applied centrally or peripherally markedly increases resistance of the intestinal tissue during the I/R period induced mucosal and hyperemic impairment evoked by I/R. Ghrelin is an important mediator of the increase in the intestinal microcirculation and elevation of the intestinal metabolism, which seems to be, at least in part, responsible for the observed protection of the intestine subjected to I/R. Impairment of this microvasculature response due to I/R seems to be responsible for a markedly observed weaker effect of ghrelin when this hormone was administered after the ischemic period. The lack of a protective effect observed after central administration of this peptide against a long lasting I/R period is probably due to damage of neural pathways caused by I/R. Finally, the peripheral activity of ghrelin in the intestine is mediated by the sensory neurons with a prominent role of CGRP released from their endings. However, this peripheral action of ghrelin depends upon the proper functioning of both the sympathetic and parasympathetic system.

Analysis of exhaled volatile compounds following acute superior mesenteric artery occlusion in a pilot rat study.

BACKGROUND: Prompt diagnosis and treatment of acute mesenteric ischemia (AMI) requires a high index of suspicion for timely management. Poor clinical outcomes and delays in surgical treatment are demonstrated even in modern clinical series. Recognition of exhaled volatile organic compounds (VOCs) specific to AMI may facilitate early detection and diagnosis and improve patient outcomes. METHODS: Adult Wistar rats (n = 5) were intubated and anesthetized, and control tracheostomy breath samples were collected using Tedlar gas sample bags. Intestinal ischemia was induced by placing an occlusive clip across the superior mesenteric artery, and breath samples were collected after 1 hour of intestinal ischemia and after 15 minutes of intestinal reperfusion. Gas chromatography was used to identify and measure levels of VOCs obtained, and measured retention indices were compared with known values in the Kovats retention index database. RESULTS: Multiple retention indices (n = 41) were noted on gas chromatography, representing a variety of VOCs detected. Z,Z-farnesol (C15H26O), an isoprenoid, was the only compound detected that was undetectable during the control phase (median = 0 cts/sec) but which significantly elevated during the ischemic (median = 34 cts/sec, range = 25-37) and reperfusion (median = 148 cts/sec, range = 42-246) phases. Three other isoprenoid compounds (E,E-alpha-farnesene, germacrene A, and Z,Z-4,6,8-megastigmatriene) were also detected in all five animals, but their levels did not differ significantly between control, ischemic, and reperfusion phases. CONCLUSIONS: This pilot study demonstrates the feasibility of analyzing exhaled VOCs using a novel rat model for AMI. These findings may be useful for the development and identification of similar assays for the rapid diagnosis of AMI.

Acute venous occlusion enhances matrix metalloprotease activity: Implications on endothelial dysfunction.

Venous hypertension is associated with microvascular inflammation, restructuring, and apoptosis, but the cellular and molecular mechanisms underlying these events remain uncertain. In the present study, we tested the hypothesis that elevated venous pressure and reduction of shear stress induce elevated enzymatic activity. This activity in turn may affect endothelial surface receptors and promote their dysfunction. Using a rodent model for venous hypertension using acute venular occlusion, microzymographic techniques for enzyme detection, and immunohistochemistry for receptor labeling, we found increased activity of the matrix metalloproteases (MMPs) -1, -8, and -9 and tissue inhibitors of metalloproteases (TIMPs) -1 and -2 in both high- and low-pressure regions. In this short time frame, we also observed that elevated venule pressure led to two different fates for the vascular endothelial growth factor receptor-2 (VEGFR2); in higher-pressure upstream regions, some animals exhibited higher VEGFR2 expression, while others displayed lower levels upstream compared to their downstream counterparts with lower pressure. VEGFR2 expression was, on average, more pronounced upon application of MMP inhibitor, suggesting possible cleavage of the receptor by activated enzymes in this model. We conclude that venous pressure elevation increases enzymatic activity which may contribute to inflammation and endothelial dysfunction associated with this disease by influencing critical surface receptors.

Antiallergic cromones inhibit neutrophil recruitment onto vascular endothelium via annexin-A1 mobilization.

OBJECTIVE: To determine whether the inhibitory action of the antiallergic cromone "mast cell stabilizing" drugs on polymorphonuclear leukocyte (PMN) trafficking is mediated through an annexin-A1 (Anx-A1) dependent mechanism. METHODS AND RESULTS: Intravital microscopy was used to monitor the actions of cromones in the inflamed microcirculation. Reperfusion injury provoked a dramatic increase in adherent and emigrated leukocytes in the mesenteric vascular bed, associated with augmented tissue levels of myeloperoxidase. Nedocromil, 2 to 20 mg/kg, significantly (P<0.05) inhibited cell adhesion and emigration, as well as myeloperoxidase release, in wild-type but not Anx-A1(-/-) mice. Short pretreatment of human PMNs with nedocromil, 10 nmol/L, inhibited cell adhesion (P<0.05) in the flow chamber assay, and this effect was reversed by specific anti-AnxA1 or a combination of antiformyl peptide receptors 1 and 2, but not irrelevant control, antibodies. Western blotting experiments revealed that cromones stimulate protein kinase C-dependent phosphorylation and release Anx-A1 in human PMNs. CONCLUSIONS: We propose a novel mechanism to explain the antiinflammatory actions of cromones on PMN trafficking, an effect that has long puzzled investigators.

Antioxidants reverse age-related collateral growth impairment.

Aging is a major risk factor for the development of cardiovascular diseases, including arterial occlusive disease. Oxidant stress increases with age, and may be a significant factor contributing to vascular dysfunction and disease. We have shown that aging and hypertension impair collateral growth, the natural compensatory response to arterial occlusive disease, and that antioxidants restore collateral growth in young hypertensive rats. The aim of this study was to test the hypothesis that oxidant stress mediates collateral growth impairment in nondiseased, aged rats. Ileal arteries were induced to become collaterals via ligation of adjacent arteries. Growth was assessed at 7 days by repeated in vivo measurements and comparison to same-animal control arteries. Collateral diameter enlargement did not occur in aged rats, but luminal expansion was stimulated by pretreatment with tempol. Co-administration of L-NAME with tempol prevented tempol-mediated collateral development. Expression of p22(phox) mRNA was increased in aged versus young rat arteries, suggesting NAD(P)H oxidase as a source of reactive oxygen species. Treatment with apocynin increased collateral growth capacity, whether administered prior to, or 7 days following, arterial ligation. The results suggest that antioxidant treatment may be useful in promoting collateral growth to compensate for age-related arterial occlusive disease.

Carbon monoxide liberated from CO-releasing molecule (CORM-2) attenuates ischemia/reperfusion (I/R)-induced inflammation in the small intestine.

CORM-released CO has been shown to be beneficial in resolution of acute inflammation. The acute phase of intestinal ischemia-reperfusion (I/R) injury is characterized by oxidative stress-related inflammation and leukocyte recruitment. In this study, we assessed the effects and potential mechanisms of CORM-2-released CO in modulation of inflammatory response in the small intestine following I/R-challenge. To this end mice (C57Bl/6) small intestine were challenged with ischemia by occluding superior mesenteric artery (SMA) for 45 min. CORM-2 (8 mg/kg; i.v.) was administered immediately before SMA occlusion. Sham operated mice were injected with vehicle (0.25% DMSO). Inflammatory response in the small intestine (jejunum) was assessed 4 h following reperfusion by measuring tissue levels of TNF-alpha protein (ELISA), adhesion molecules E-selectin and ICAM-1 (Western blot), NF-kappaB activation (EMSA), along with PMN tissue accumulation (MPO assay) and leukocyte rolling/adhesion in the microcirculation of jejunum (intravital microscopy). The obtained results indicate that tissue levels of TNF-alpha, E-selectin and ICAM-1 protein expression, activation of NF-kappaB, and subsequent accumulation of PMN were elevated in I/R-challenged jejunum. The above changes were significantly attenuated in CORM-2-treated mice. Taken together these findings indicate that CORM-2-released CO confers anti-inflammatory effects by interfering with NF-kappaB activation and subsequent up-regulation of vascular pro-adhesive phenotype in I/R-challenged small intestine.

Low-density lipoproteins oxidized after intestinal ischemia/reperfusion in rats.

BACKGROUND: Intestinal ischemia/reperfusion (I/R) is a complex phenomenon causing destruction of both local and remote tissues, as well as multiple-organ failure. We investigated the role of lipid peroxidation in damage to intestinal, liver, and lung tissues in this pathology. MATERIALS AND METHODS: The superior mesenteric artery was blocked for 30 min followed by 24 h of reperfusion. Tissues were removed and the presence of oxidized LDL, the activities of the superoxide dismutase enzyme, malondialdehyde levels, and inducible nitric oxide synthase expression were each evaluated in the intestinal, liver, and lung tissues. RESULTS: While there was no staining in the control group tissues, ischemia/reperfusion resulted in positive oxidized LDL staining in all of the I/R test group tissue samples. Inducible nitric oxide synthase expression was significantly increased in the ischemia/reperfusion group tissues. Compared with those of the control group rats, the ischemia/reperfusion group tissues showed significantly higher malondialdehyde levels and lower superoxide dismutase activities. CONCLUSIONS: This study demonstrated for the first time that oxidized LDL accumulated in the terminal ileum, liver, and lung tissues after intestinal ischemia/reperfusion. This occurrence (or the presence of oxidized LDL) may be an indicator of ongoing oxidative stress and enhanced lipid peroxidation. Augmentation of inducible nitric oxide synthase expression may play a role in progression of inflammation and LDL oxidation. These data support the hypothesis that cellular oxidative stress is a critical step in reperfusion-mediated injury in both the intestine and end organs, and that antioxidant strategies may provide organ protection in patients with reperfusion injury, at least through affecting interaction with free radicals, nitric oxide, and oxidized LDL.

[Hepato-renal syndrome and problem of protein-energy metabolism correction during urgent surgery of the abdominal organs].

Early connecting of hardware detoxification methods allow more efficient use of amino acid preparations for action. The use of efferent technology provides a full correction of protein and energy metabolism in the immediate surgical patients with complicated diseases of the abdominal cavity. This will reduce bed-day in patient and surgical mortality.

Immediate but not delayed postconditioning during reperfusion attenuates acute lung injury induced by intestinal ischemia/reperfusion in rats: comparison with ischemic preconditioning.

BACKGROUND: A previous study has shown that brief period of repetitive superior mesenteric artery (SMA) occlusion and reperfusion applied at the onset of reperfusion, ischemic postconditioning (IPo), attenuates intestinal injury after intestinal ischemia/reperfusion (II/R). This study tested the hypothesis that IPo would attenuate II/R-induced acute lung injury, which is comparable to ischemic preconditioning (IPC) and the brief period of postconditioning applied at the onset of reperfusion is critical to pulmonary protection by IPo. METHODS: Rat II/R injury was produced by clamping SMA for 60 min followed by 60 min of reperfusion. The rats were randomly allocated into one of five groups based upon the intervention (n = 8): sham operation (Sham): sham surgical preparation including isolation of the SMA without occlusion was performed; Injury: there was no intervention either before or after SMA occlusion; ischemia preconditioning (IPC): the SMA was occluded for 10 min followed by 10 min of reperfusion before prolonged occlusion; ischemia postconditioning (IPo): three cycles of 30 sec reperfusion-30 sec reocclusion were imposed immediately upon reperfusion (3 min total intervention); delayed postconditioning: clamping was completely released for full reperfusion for 3 min (the duration of the IPo algorithm), after which three cycles of 30 sec occlusion and reperfusion were applied. RESULTS: Histologic results showed severe damage in rat lungs in the injury group evidenced by increased lung wet/dry weight ratio and pulmonary permeability index, which was accompanied by increases in the levels of plasma TNFalpha and IL-6, the pulmonary malondialdehyde (MDA), and the pulmonary myeloperoxidase (MPO) activity and a decrease in superoxide dismutase (SOD) activity. IPo, not delayed IPo, could significantly attenuate lung injury and improve the above variables, which was comparable to IPC. CONCLUSIONS: IPo at onset of reperfusion reduces acute lung injury induced by II/R, which may be mediated, in part, by inhibiting oxidant generation, neutrophils filtration, and proinflammatory mediators releases. The early period of reperfusion in the rat model is critical to pulmonary protection by IPo. IPo may improve outcome in clinical conditions associated with II/R.