Gediminas Cepinskas

Inflammatory Response in Microvascular Endothelium in Sepsis: Role of Oxidants

Sepsis, as a severe systemic inflammatory response to bacterial infection, represents a major clinical problem. It is characterized by the excessive production of reactive oxygen species (ROS) both in the circulation and in the affected organs. The excessive generation of ROS inevitably leads to oxidative stress in the microvasculature and has been implicated as a causative event in a number of pathologies including sepsis. In this review, we focus on the role of oxidative and nitrosative stress during the early onset of sepsis. Changes in microvascular endothelial cells, the cell type that occurs in all organs, are discussed. The mechanisms underlying septic induction of oxidative and nitrosative stresses, the functional consequences of these stresses, and potential adjunct therapies for microvascular dysfunction in sepsis are identified.

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 45xa0min. CORM-2 (8xa0mg/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 4xa0h following reperfusion by measuring tissue levels of TNF-α protein (ELISA), adhesion molecules E-selectin and ICAM-1 (Western blot), NF-κB 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-α, E-selectin and ICAM-1 protein expression, activation of NF-κB, 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-κB activation and subsequent up-regulation of vascular pro-adhesive phenotype in I/R-challenged small intestine.

Neutrophils Induce Sequential Focal Changes in Endothelial Adherens Junction Components: Role of Elastase

Objective: In vitro studies have indicated that polymorphonuclear leukocytes (PMNs) traverse endothelial cell monolayers via the paracellular pathway (i.e., through endothelial cell–cell junctions. Herein, we assessed whether the adherens junctions (AJs) are disrupted during PMN transendothelial cell migration.

Cardiac Myocytes Activated by Septic Plasma Promote Neutrophil Transendothelial Migration. Role of Platelet-Activating Factor and the Chemokines LIX and KC

Abstract— Cardiac myocytes isolated from rats with peritonitis (cecal ligation and perforation; CLP) promote PMN transendothelial migration. Herein, we assessed (1) the mechanisms involved in cardiac myocyte activation during peritonitis and (2) the means by which these activated myocytes promote PMN transendothelial migration. Plasma obtained from mice subjected to CLP (septic plasma) activated isolated cardiac myocytes as evidenced by (1) increased nuclear levels of nuclear factor-κB (NF-κB) and (2) their ability to promote PMN migration across endothelial cell monolayers. Pretreatment of septic plasma with an antibody against tumor necrosis factor-&agr; (TNF-&agr;), but not interleukin-1β (IL-1β), blunted the ability of septic plasma to activate the myocytes. However, septic plasma obtained from TNF-&agr;–deficient mice could still activate the myocytes; an effect attenuated by an antibody against IL-1β. If the myocytes were pretreated with a proteasome inhibitor (MG 132) to prevent NF-κB activation, the myocyte-induced PMN transendothelial migration was compromised. The activated myocytes released platelet-activating factor (PAF), and myocyte-induced PMN migration was abrogated by a PAF receptor antagonist (WEB 2086). These myocytes also released the CXC chemokines LIX and KC; an event prevented by MG 132. Antibodies against LIX and KC abrogated the myocyte-induced PMN migration. However, LIX and KC, but not PAF, could promote PMN migration when used at concentrations produced by activated myocytes. These observations indicate that TNF-&agr; and IL-1β are, in part, responsible for the ability of septic plasma to activate cardiac myocytes. The activated myocytes promote PMN transendothelial migration, an effect attributable to LIX and KC, and possibly, PAF.

INTERACTION BETWEEN REACTIVE OXYGEN METABOLITES AND NITRIC OXIDE IN OXIDANT TOLERANCE

The excessive generation of reactive oxygen metabolites (ROM) leads to an oxidative stress in the microvasculature of a variety of tissues and has been implicated as a causative event in a number of pathologies. There are numerous reviews on this topic that have been published recently. Herein, we will focus on a beneficial effect of ROM generation that leads to the development of an adaptive response that protects tissue from a subsequent oxidative stress (oxidant tolerance). We will focus on reductionist approaches (studies in isolated cells) used by our laboratory and those of others to define the mechanisms involved in this adaptational response and potential interactions between different cells within the tissue. As our prototype organ system, we target the heart, which has received the greatest amount of attention in this area. We will summarize evidence from isolated endothelial cells and cardiac myocytes that supports (i) the role of ROM in the development of oxidant tolerance, (ii) the possibility of an interaction between cardiac myocytes and endothelial cells in this phenomenon, and (iii) the potential interactions between ROMs and nitric oxide.

Dextran sulfate sodium-induced acute colonic inflammation in angiotensin II type 1a receptor deficient mice

Abstract.Objective:Angiotensin II (Ang II) receptor blockers have been reported to contribute to cytoprotective effects in various organs. However, the role of renin-angiotensin system (RAS) in modulation of the inflammatory bowel disease (IBD) remains unclear. In this study we assessed the role of angiotensin II type 1a (AT1a) receptor on the outcome of dextran sulfate sodium (DSS)-induced acute colitis by employing AT1a receptor deficient mice.Materials and methods:The acute colitis was induced in wild type (WT) and AT1a receptor deficient mice by giving orally 3% DSS in drinking water for 7 days.Results:Induction of DSS colitis resulted in up-regulation of Ang II and AT1a receptor in the colonic mucosa of WT mice. In parallel, loss of body weight, an increase in disease activity index (DAI), and the shortening of colon were found in DSS-challenged WT mice. In addition, an increase in thiobarbituric acid (TBA)-reactive substances and myeloperoxidase (MPO) activity, along with the up-regulation of tumor necrosis factor (TNF)-α were detected in the colonic mucosa of DSS-challenged WT mice. The endpoints mentioned above were significantly ameliorated in DSS-challenged AT1a receptor deficient mice.Conclusions:RAS is involved in the pathophysiology of DSS-induced colitis and AT1a receptor may be a novel therapeutic target for the treatment of IBD.

Delayed preconditioning in cardiac myocytes with respect to development of a proinflammatory phenotype: role of SOD and NOS

OBJECTIVEnBoth superoxide dismutase (SOD) and nitric oxide synthase (NOS) have been implicated in delayed preconditioning (DP) to ischemia/reperfusion (I/R) in the heart. We used isolated cardiac myocytes to test the hypothesis that SOD and NOS may interact in the development of DP.nnnMETHODSnMouse neonatal cardiac myocytes were challenged with anoxia/reoxygenation (A/R; an in vitro counterpart to I/R) and normoxia/normoxia (N/N) served as the control. Two indices of inflammation were measured: oxidant stress (DHR oxidation) and polymorphonuclear leukocyte (PMN) transendothelial migration (cell culture inserts). The role of SOD was assessed using an antisense approach and the role of NOS was assessed using iNOS and eNOS deficient myocytes.nnnRESULTSnCardiac myocytes exposed to A/R (1) produced more oxidants (intracellular fluorescence emission from 2.0 +/- 0.1 for N/N to 3.0 +/- 0.3 for A/R; P<0.05) and (2) promoted PMN migration (% migration from 8.4 +/- 0.9 for N/N to 14.1 +/- 1.1 for A/R; P<0.05). DP occurred if the myocytes were pretreated with an A/R challenge 24 h earlier. That is, these A/R-induced responses were significantly reduced (fluorescence emission 1.9 +/- 0.1 and % migration 8.4 +/- 0.7; P<0.05 as compared to A/R with no pretreatment). Myocyte Mn-SOD, but not Cu/Zn-SOD, activity increased 24 h after the initial A/R challenge. A Mn-SOD antisense oligonucleotide prevented the development of DP. DP occurred in iNOS, but not eNOS, deficient myocytes. A/R increased mRNA for eNOS, but not iNOS, in wild-type myocytes. A/R increased Mn-SOD protein in both iNOS and eNOS deficient myocytes. However, Mn-SOD activity increased only in iNOS deficient myocytes.nnnCONCLUSIONSnCollectively, these findings suggest that Mn-SOD and eNOS may act in concert in the development of DP in cardiac myocytes.

Anti-inflammatory Effects of Carbon Monoxide-Releasing Molecule on Trinitrobenzene Sulfonic Acid-Induced Colitis in Mice

Background and AimRecent findings indicate that carbon monoxide (CO) in non-toxic doses exerts a beneficial anti-inflammatory action in various experimental models. However, the precise anti-inflammatory mechanism of CO in the intestine remains unclear. Here, we assessed the effects of a novel water-soluble CO-releasing molecule, CORM-3, on trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice.MethodsTo induce colitis, C57BL/6 male mice received an enema of TNBS. CORM-3 or its inactive compound, iCORM-3, were administered intraperitoneally, once immediately before, and twice daily after receiving an enema of TNBS. Three days after TNBS administration, the distal colon was removed, assessed for colonic damage and histological scores, polymorphonuclear leukocyte recruitment (tissue-associated myeloperoxidase, MPO activity), and TNF-α, IFN-γ and IL-17A expression (mRNA and protein levels in the colon mucosa). CD4+ T cells isolated from murine spleens were stimulated with anti-CD3/CD28, in the presence or absence of CORM-3/iCORM-3. The cell supernatants were assessed for TNF-α and IFN-γ expression, 24xa0h following stimulation.ResultsColonic damage and histological scores were significantly increased in TNBS-induced mice compared to sham-operated mice. Tissue-associated MPO activity and expression of TNF-α, IFN-γ, and IL-17A in the colonic mucosa were higher in TNBS-induced colitis mice. The above changes were attenuated in CORM-3-treated mice. Further, CORM-3 was effective in reducing TNF-α and IFN-γ production in anti-CD3/CD28-stimulated CD4+ T cells.ConclusionsThese findings indicate that CO released from CORM-3 ameliorates inflammatory responses in the colon of TNBS-challenged mice at least in part through a mechanism that involves the suppression of inflammatory cell recruitment/activation.

Inhibition of calpain reduces oxidative stress and attenuates endothelial dysfunction in diabetes

AimsThe present study was to investigate the role of calpain in reactive oxygen species (ROS) production in endothelial cells and endothelium-dependent vascular dysfunction under experimental conditions of diabetes.Methods and resultsExposure to high glucose activated calpain, induced apoptosis and reduced nitric oxide (NO) production without changing eNOS protein expression, its phosphorylation and dimers formation in primary human umbilical vein endothelial cells (HUVECs). These effects of high glucose correlated with intracellular ROS production and mitochondrial superoxide generation. Selectively scavenging mitochondrial superoxide increased NO production in high glucose-stimulated HUVECs. Inhibition of calpain using over-expression of calpastatin or pharmacological calpain inhibitor prevented high glucose-induced ROS production, mitochondrial superoxide generation and apoptosis, which were concurrent with an elevation of NO production in HUVECs. In mouse models of streptozotocin-induced type-1 diabetes and OVE26 type-1 diabetic mice, calpain activation correlated with an increase in ROS production and peroxynitrite formation in aortas. Transgenic over-expression of calpastatin reduced ROS production and peroxynitrite formation in diabetic mice. In parallel, diabetes-induced reduction of endothelium-dependent relaxation in aortic ring was reversed by over-expression of calpastatin in mouse models of diabetes. However, the protective effect of calpastatin on endothelium-dependent relaxation was abrogated by eNOS deletion in diabetic mice.ConclusionsThis study suggests that calpain may play a role in vascular endothelial cell ROS production and endothelium-dependent dysfunction in diabetes. Thus, calpain may be an important therapeutic target to overcome diabetes-induced vascular dysfunction.

Mechanisms and consequences of acquired brain injury during development

The brain of the infant and young child is a developing, dynamic, structure subject to functional remodelling under the influence of factors responsible for optimal neuronal development and synaptogenesis. It exhibits age dependent variation in metabolic rate, blood flow, and ability to tolerate oxidative stress. It is also characterized by an exuberance of neurotransmitter activity, particularly in the first few years of life. The dynamic evolution and adaptability of early brain function permits the organization of neuronal networks to be influenced by environmental stimulation, and, to reduce the functional impact of injury. However, these same processes may also exacerbate the harm sustained by the brain following an acquired brain injury (ABI). The developing neurons are susceptible to excitotoxicity, oxidative stress, and, inflammation, often leading to cellular necrosis and apoptosis. Despite being immunologically privileged via the blood brain barrier, the developing brain is susceptible to injury from systemic inflammation through alteration of normally protective cerebrovascular endothelial cell function. Finally, many of the therapeutic agents currently employed in post-ABI hospital care may also compromise ABI outcome via non-intended pharmacological effects. These agents include analgesic, sedative and anti-convulsant medications. This review emphasizes those physiological considerations in the developing brain which may impact the outcome after ABI, including, the cellular mechanisms of neuronal and cerebrovascular endothelial cell injury, ABI outcome and future therapeutic directions.