Haim Bitterman

Regulation of Endothelial Matrix Metalloproteinase-2 by Hypoxia/Reoxygenation

Among the consequences resulting from the exposure of endothelial cells (ECs) to ischemia/reperfusion is angiogenesis, involving degradation of vascular basement membrane and extracellular matrix. Matrix metalloproteinase (MMP)-2, a member of the MMP family, partakes in this process. MMP-2, secreted as a proenzyme, undergoes activation through interaction with membrane type (MT)1-MMP and the endogenous tissue inhibitor of MMPs (TIMP)-2. Although hypoxia and reoxygenation (H/R) are major constituents of ischemia/reperfusion processes, their direct effects on endothelial MMP-2 have been scarcely investigated. This study examined the in vitro effects of H/R on human macrovascular ECs (EAhy 926). The level of MMP-2 mRNA (Northern blot) and protein (zymography, ELISA) and the mRNA of its activator (MT1-MMP) and inhibitor (TIMP-2) were analyzed. Short (6-hour) hypoxia inhibited the mRNA expression of MMP-2, MT1-MMP, and TIMP-2, culminating in reduced latent and active MMP-2 protein. Prolonged (24-hour) hypoxia further suppressed MT1-MMP and TIMP-2 mRNA, whereas it enhanced MMP-2 mRNA and enzyme secretion (after 48-hour hypoxia). Reoxygenation did not influence the inhibited TIMP-2 but upregulated MMP-2 and MT1-MMP mRNA expression, leading to enhanced secretion of active MMP-2 protein. These results demonstrate H/R-mediated modulation of EC MMP-2 at both transcriptional and posttranscriptional levels. Prolonged hypoxia of ECs appears to enhance MMP-2 production and secretion, whereas reoxygenation further increases its level. These H/R-mediated effects on MMPs have the potential of enabling EC migration and possible angiogenesis.

Nicotine and angiogenesis: a new paradigm for tobacco‐related diseases

The pathophysiology of tobacco‐related diseases is complex and multifactorial. Among the approximately 4,000 compounds in tobacco smoke are carcinogens such as nitro‐samines, irritants such as a variety of phenolic compounds, volatiles such as carbon monoxide, and of course nicotine. Nicotine itself has quite complex actions, mediated in part by nicotinic cholinergic receptors that may have extraneuronal, as well as neuronal distribution. This review discusses the mechanisms by which nicotine contributes to tobacco‐related disease, with a focus on the surprising new finding that nicotine is a potent angiogenic agent. Nicotine hijacks an endogenous nicotinic cholinergic pathway present in endothelial cells that is involved in physiological, as well as pathological angiogenesis.

Bench-to-bedside review: Oxygen as a drug

Oxygen is one of the most commonly used therapeutic agents. Injudicious use of oxygen at high partial pressures (hyperoxia) for unproven indications, its known toxic potential, and the acknowledged roles of reactive oxygen species in tissue injury led to skepticism regarding its use. A large body of data indicates that hyperoxia exerts an extensive profile of physiologic and pharmacologic effects that improve tissue oxygenation, exert anti-inflammatory and antibacterial effects, and augment tissue repair mechanisms. These data set the rationale for the use of hyperoxia in a list of clinical conditions characterized by tissue hypoxia, infection, and consequential impaired tissue repair. Data on regional hemodynamic effects of hyperoxia and recent compelling evidence on its anti-inflammatory actions incited a surge of interest in the potential therapeutic effects of hyperoxia in myocardial revascularization and protection, in traumatic and nontraumatic ischemicanoxic brain insults, and in prevention of surgical site infections and in alleviation of septic and nonseptic local and systemic inflammatory responses. Although the margin of safety between effective and potentially toxic doses of oxygen is relatively narrow, the ability to carefully control its dose, meticulous adherence to currently accepted therapeutic protocols, and individually tailored treatment regimens make it a cost-effective safe drug.

Hypoxia Inactivates Inducible Nitric Oxide Synthase in Mouse Macrophages by Disrupting Its Interaction with α-Actinin 4

Nitric oxide, produced in macrophages by the high output isoform inducible NO synthase (iNOS), is associated with cytotoxic effects and modulation of Th1 inflammatory/immune responses. Ischemia and reperfusion lead to generation of high NO levels that contribute to irreversible tissue damage. Ischemia and reperfusion, as well as their in vitro simulation by hypoxia and reoxygenation, induce the expression of iNOS in macrophages. However, the molecular regulation of iNOS expression and activity in hypoxia and reoxygenation has hardly been studied. We show in this study that IFN-γ induced iNOS protein expression (by 50-fold from control, p < 0.01) and nitrite accumulation (71.6 ± 14 μM, p < 0.01 relative to control), and that hypoxia inhibited NO production (7.6 ± 1.7 μM, p < 0.01) without altering iNOS protein expression. Only prolonged reoxygenation restored NO production, thus ruling out the possibility that lack of oxygen, as a substrate, was the cause of hypoxia-induced iNOS inactivation. Hypoxia did not change the ratio between iNOS monomers and dimers, which are essential for iNOS activity, but the dimers were unable to produce NO, despite the exogenous addition of all cofactors and oxygen. Using immunoprecipitation, mass spectroscopy, and confocal microscopy, we demonstrated in normoxia, but not in hypoxia, an interaction between iNOS and α-actinin 4, an adapter protein that anchors enzymes to the actin cytoskeleton. Furthermore, hypoxia caused displacement of iNOS from the submembranal zones. We suggest that the intracellular localization and interactions of iNOS with the cytoskeleton are crucial for its activity, and that hypoxia inactivates iNOS by disrupting these interactions.

Hypoxia reduces the output of matrix metalloproteinase-9 (MMP-9) in monocytes by inhibiting its secretion and elevating membranal association

Cellular hypoxia, characterizing tumors, ischemia, and inflammation induce recruitment of monocytes/macrophages, immobilize them at the hypoxic site, and alter their function. To migrate across the extracellular matrix and as part of their inflammatory functions, monocytes and macrophages secrete proteases, including matrix metalloproteinase‐9 (MMP‐9), whose expression is induced by proinflammatory cytokines [e.g., tumor necrosis factor α (TNF‐α)]. We show that hypoxia (<0.3% O2 for 48 h) reduced the output of TNF‐α‐induced proMMP‐9 by threefold (P<0.01) in the U937 monocytic cell line and in primary human monocytes. TNF‐α induced MMP‐9 transcription by threefold, but no significant difference was observed in MMP‐9 mRNA steady‐state between normoxia and hypoxia, which inhibited the trafficking of proMMP‐9 via secretory vesicles and increased the intracellular accumulation of proMMP‐9 in the cells by 47% and 62% compared with normoxia (P<0.05), as evaluated by zymography of cellular extracts and confocal microscopy, respectively. Secretion of proMMP‐9 was reduced by the addition of cytochalazin B or nocodazole, which inhibits the polymerization of actin and tubulin fibers, or by the addition of the Rho kinase inhibitor Y27632, suggesting the involvement of the cytoskeleton and the Rho GTPases in the process of enzyme secretion. Furthermore, attachment of proMMP‐9 to the cell membrane increased after hypoxia via its interactions with surface molecules such as CD44. In addition, the reduced migration of monocytes in hypoxia was shown to be mediated, at least partially, by secreted MMP‐9. Thus, hypoxia post‐translationally reduced the secreted amounts of proMMP‐9 by using two mutually nonexclusive mechanisms: mostly, inhibition of cellular trafficking and to a lesser extent, attachment to the membrane.

Acute release of cytokines is proportional to tissue injury induced by surgical trauma and shock in rats.

Cytokines are important mediators of the inflammatory reaction and microvascular injury after trauma and tissue ischemia. The plasma activity of a cytokine reflects the net effect of positive and negative signals. We examined the sequential serum activity of IL-1, IL-2, IL-6, and TNF in a severe model of splanchnic artery occlusion (SAO) shock induced in rats by total occlusion of the superior mesenteric and the celiac arteries for 40 min. A control group with negligible surgical intervention and two sham-shock groups, one with minor operation and another with major surgery employed in SAO rats, both without vascular occlusion, were also studied. No IL-1 activity was detected throughout the 190-min experimental protocol in any of the groups. Low activity of IL-2 was measured only in SAO rats (∼1 U/ml at the peak). We found graded increases in serum TNF and IL-6 activities which were proportional to the surgical trauma and were highest in SAO rats (IL-6 up to 30 U/ml,P<0.01 from both sham groups; TNF, 2500 pg/ml 30 min after reperfusion,P<0.01 from both sham groups). These data further support the role played by cytokines in the early mediation of surgical trauma and shock.

Exposure to hyperbaric oxygen induces tumour necrosis factor‐alpha (TNF‐α) secretion from rat macrophages

We investigated the secretion of TNF‐α by monocytes and macrophages derived from the peripheral blood, spleen, and lungs after a single exposure to a therapeutic profile of hyperbaric oxygen (HBO). Rats were exposed for 90 min to either 100% oxygen at 0 28 MPa (2–8 atmospheres absolute) or air. Immediately after exposure, mononuclear cells were isolated from blood, spleen, and lungs and cultured for 18h. The secretion of TNF‐α from the cultured monocytes/macrophages was determined with and without stimulation with lipopolysaccharide (LPS). Exposure to hyperbaric oxygen induced a significant increase in the spontaneous ex vivo secretion of TNF‐α (without LPS) by mononuclear cells from the blood, spleen, and lung (P < 0 05 from air controls). Stimulation with LPS after exposure to HBO induced a significant increase in TNF‐α secretion by lung and spleen macrophages compared with air controls (P<005). However, absolute TNF‐α levels were not significantly higher than those achieved ‘spontaneously’ in macrophages exposed to HBO without LPS. Stimulation with LPS induced a marked increase in secretion of TNF‐α from blood monocytes after exposure to air, but not after exposure to HBO. These results provide evidence in support of a role played by TNF‐α in mediating HBO effects on different tissues and their immune responses.

Hypoxia reduces CD80 expression on monocytes but enhances their LPS‐stimulated TNF‐α secretion

Monocytes/macrophages in ischemic tissues are involved in inflammation and suppression of adaptive immunity via secretion of proinflammatory cytokines and reduced ability to trigger T cells, respectively. We subjected human mononuclear cells and mouse macrophages to hypoxia and reoxygenation, the main constituents of ischemia and reperfusion, and added lipopolysaccharide (LPS) to simulate bacterial translocation, which frequently accompanies ischemia. We monitored the secretion of tumor necrosis factor α (TNF‐α) and the surface expression of human leukocyte antigen‐DR and the costimulatory molecules CD80 and CD86 on monocytes/macrophages. Hypoxia selectively reduced the surface expression of CD80 (P<0.01), and synergistically with LPS, it enhanced TNF‐α secretion (P<0.003). Reoxygenation reversed both phenomena. In the mouse macrophage cell line RAW 264.7, hypoxia reduced the surface expression of CD80 and increased its concentrations in the supernatants (P<0.01). Down‐regulation of the mRNA coding for the membrane‐anchored CD80 was observed, suggesting that hypoxia triggers alternative splicing to generate soluble CD80. Cumulatively, these results suggest that hypoxia simultaneously affects monocytes/macrophages to enhance inflammation and reduce their ability to initiate adaptive‐immunity responses associated with ischemic injury.

Predicting 30-day readmissions with preadmission electronic health record data.

Background:Readmission prevention should begin as early as possible during the index admission. Early identification may help target patients for within-hospital readmission prevention interventions. Objectives:To develop and validate a 30-day readmission prediction model using data from electronic health records available before the index admission. Research Design:Retrospective cohort study of admissions between January 1 and March 31, 2010. Subjects:Adult enrollees of Clalit Health Services, an integrated delivery system, admitted to an internal medicine ward in any hospital in Israel. Measures:All-cause 30-day emergency readmissions. A prediction score based on before admission electronic health record and administrative data (the Preadmission Readmission Detection Model—PREADM) was developed using a preprocessing variable selection step with decision trees and neural network algorithms. Admissions with a recent prior hospitalization were excluded and automatically flagged as “high-risk.” Selected variables were entered into multivariable logistic regression, with a derivation (two-thirds) and a validation cohort (one-third). Results:The derivation dataset comprised 17,334 admissions, of which 2913 (16.8%) resulted in a 30-day readmission. The PREADM includes 11 variables: chronic conditions, prior health services use, body mass index, and geographical location. The c-statistic was 0.70 in the derivation set and of 0.69 in the validation set. Adding length of stay did not change the discriminatory power of the model. Conclusions:The PREADM is designed for use by health plans for early high-risk case identification, presenting discriminatory power better than or similar to that of previously reported models, most of which include data available only upon discharge.

Assessment of disease-severity scoring systems for patients with sepsis in general internal medicine departments

IntroductionDue to the increasing burden on hospital systems, most elderly patients with non-surgical sepsis are admitted to general internal medicine departments. Disease-severity scoring systems are used for stratification of patients for utilization management, performance assessment, and clinical research. Some widely used scoring systems for septic patients are inappropriate when rating non-surgical patients in a non-intensive care unit (ICU) environment mainly because their calculations require types of data that are frequently unavailable. This study aimed to assess the fitness of four scoring systems for septic patients hospitalized in general internal medicine departments: modified early warning score (MEWS), simple clinical score (SCS), mortality in emergency department sepsis (MEDS) score, and rapid emergency medicine score (REMS).MethodsWe prospectively collected computerized data of septic patients admitted to general internal medicine departments in our community-based university hospital. We followed 28-day in-hospital mortality, overall in-hospital mortality, and 30- and 60-day mortality. Using a logistic regression procedure we calculated the area under ROC curve (AUC) for every scoring system.ResultsBetween February 1st, 2008 and April 30th, 2009 we gathered data of 1,072 patients meeting sepsis criteria on admission to general internal medicine departments. The 28-day mortality was 19.4%. The AUC for the MEWS was 0.65-0.70, for the SCS 0.76-0.79, for the MEDS 0.73-0.75, and for the REMS, 0.74-0.79. Using Hosmer-Lemeshow statistics, a lack of fit was found for the MEDS model. All scoring systems performed better than calculations based on sepsis severity.ConclusionsThe SCS and REMS are the most appropriate clinical scores to predict the mortality of patients with sepsis in general internal medicine departments.