Marker Of Cell Injury Research Articles

MicroRNA‐17‐3p inhibits excessive post‐hypoxic autophagy and attenuates H9C2 cardiomyocytes reoxygenation injury via PTEN‐Akt‐mTOR signaling

BackgroundThe microRNA (miR)‐17~92 is one of the best‐characterized polycistronic miRNA clusters, which encodes six individual miRNAs and contributes to various cellular and biological processes. In particular, miR‐17‐3p (a passenger miRNA of miR‐17) has been shown to indirectly inhibit Phosphatase and tensin homolog (PTEN) and attenuate myocardial ischemia/reperfusion (I/R) injury, but the underneath mechanism is unclear. Inhibition of PTEN can upregulate Akt/mTOR signaling and therefore suppresses the excessive autophagy, which may be a mechanism that underlies myocardial I/R injury. We, thus, hypothesized that miR‐17‐3p may protect against hypoxia/reoxygenation (H/R) induced cell injury by inhibiting post‐hypoxic excessive autophagy in H9C2 cardiomyocytes via PTEN‐Akt‐mTOR signaling pathway.Methods and resultsIn rat H9C2 cardiomyocytes, H/R (6 hours hypoxia followed by 6 hours reoxygenation) significantly enhanced the expression of miR‐17‐3p (P <0.05 vs. Control), which was concomitant by increased lactic acid dehydrogenase (LDH) leakage (cell injury marker, P<0.05 vs. Control) and the levels of p62 and the ratio of LC3II/I (autophagy markers, P<0.05 vs. Control), suggesting that miR‐17‐3p may be involved in the pathogenesis of H/R induced autophagic cell death. To explore the potential role of miR‐17‐3p in H/R‐induced cell injury, H9C2 cells were transfected with the miR‐17‐3p agomir or its negative control. The overexpression of miR‐17‐3p can significantly attenuate H/R‐induced cell injury (reduced LDH level, P <0.05 vs. H/R) and inhibit H/R‐induced excessive autophagy (decreased levels of p62 and LC3II/I, P <0.05 vs. H/R). Given the important role of PTEN‐Akt‐mTOR signaling in autophagy, the effects of miR‐17‐3p on the key events in the PTEN‐Akt‐mTOR signaling cascade during H/R were examined. As anticipated, miR‐17‐3p overexpression significantly down‐regulated PTEN expression and up‐regulated the levels of phosphorylated Akt1 (Thr308) and mTOR (P<0.05 vs. H/R), indicating that the miR‐17‐3p may protect against excessive post‐hypoxic autophagic cell death in H9C2 cardiomyocytes via PTEN‐Akt‐mTOR signaling pathway.ConclusionUpregulating PTEN‐Akt‐mTOR axis and the subsequent inhibition of excessive autophagy may represent the major mechanism whereby miR‐17‐3p attenuates H/R injury in H9C2 cardiomyocytes.Support or Funding InformationStudied supported by RGC/GRF grants (17158616M, 17117217 M)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression of Endothelial Cell Injury Marker Cd146 Correlates with Disease Severity and Predicts the Renal Outcomes in Patients with Diabetic Nephropathy

Background/Aims: Glomerular endothelial cell injury plays a crucial role in the development of diabetic nephropathy (DN). CD146, an endothelial marker, was shown to increase in chronic kidney disease (CKD), but its role in DN remains unknown. We aim to assess whether CD146 could be used to evaluate disease severity and predict renal outcomes in DN at early stages. Methods: 159 non-dialysis type 2-DN patients from 2008 to 2015 were enrolled to measure the plasma concentration of soluble CD146 (sCD146). 94 type 2 diabetes mellitus patients without DN and 100 healthy subjects were used as controls. The patients with CKD stage 1-3 were referred as early stage patients. Another independent cohort of 48 patients with biopsy-proved DN was used for the immunohistochemistry study of CD146. Renal outcomes were defined as doubling of serum creatinine, initiation of renal replacement therapy or death. Results: We found that plasma level of sCD146 was upregulated and associated with renal function in DN patients. sCD146 was proved to be a more optimal marker than urine albumin creatinine ratio to evaluate disease severity in these DN patients. The kidney expression of CD146 was co-localized with endothelial marker CD31 and increased in DN. CD146 staining in kidney was correlated with the severity of pathological changes in DN patients. Survival analysis suggested that both plasma and biopsy expression of CD146 were correlated with renal outcomes. Conclusions: CD146 is associated with kidney injury and could be a good marker to predict renal outcomes in patients with early stages of DN.

Plasmatic Villin 1 Is a Novel In Vivo Marker of Proximal Tubular Cell Injury During Renal Ischemia-Reperfusion.

Early detection of acute kidney injury (AKI), a common condition with a high mortality risk, can be facilitated by specific and reliable biomarkers. Villin 1, a protein typically found in the brush borders of proximal tubular cells, has been detected in urine of patients with AKI, but its possible release in plasma remains unexplored. We measured the presence of villin 1 by immunohistochemistry on kidney sections and by Western blotting in plasma samples from rats subjected to renal ischemia-reperfusion injury, pigs subjected to renal transplantation and liver transplantation patients that developed AKI. Moreover, rats were treated with necrostatin-1, an inhibitor of programmed necrosis (necroptosis), which occurs in renal tubular cells during AKI. Villin 1 levels were compared with other renal injury markers (creatinine, aspartate transaminase, and heart-type fatty acid binding protein). During AKI, plasmatic villin 1 levels corresponded with the severity of kidney injury and dysfunction. Its detection in plasma was associated with a redistribution in the kidney tissue. Unlike the levels of other markers, plasmatic villin 1 decreased already after a short (3 hours) treatment with necrostatin-1 during renal ischemia-reperfusion injury. The presence of plasmatic villin 1 was confirmed in patients who experienced AKI after liver transplantation. Villin 1 is released in plasma during AKI and shows potential as an early marker for proximal tubular injury/necrosis and warrants further investigation.

α1A-Subtype adrenergic agonist therapy for the failing right ventricle.

Failure of the right ventricle (RV) is a serious disease with a poor prognosis and limited treatment options. Signaling by α1-adrenergic receptors (α1-ARs), in particular the α1A-subtype, mediate cardioprotective effects in multiple heart failure models. Recent studies have shown that chronic treatment with the α1A-subtype agonist A61603 improves function and survival in a model of left ventricular failure. The goal of the present study was to determine if chronic A61603 treatment is beneficial in a RV failure model. We used tracheal instillation of the fibrogenic antibiotic bleomycin in mice to induce pulmonary fibrosis, pulmonary hypertension, and RV failure within 2 wk. Some mice were chronically treated with a low dose of A61603 (10 ng·kg-1·day-1). In the bleomycin model of RV failure, chronic A61603 treatment was associated with improved RV fractional shortening and greater in vitro force development by RV muscle preparations. Cell injury markers were reduced with A61603 treatment (serum cardiac troponin I, RV fibrosis, and expression of matrix metalloproteinase-2). RV oxidative stress was reduced (using the probes dihydroethidium and 4-hydroxynonenal). Consistent with lowered RV oxidative stress, A61603 was associated with an increased level of the cellular antioxidant superoxide dismutase 1 and a lower level of the prooxidant NAD(P)H oxidase isoform NOX4. In summary, in the bleomycin model of RV failure, chronic A61603 treatment reduced RV oxidative stress, RV myocyte necrosis, and RV fibrosis and increased both RV function and in vitro force development. These findings suggest that in the context of pulmonary fibrosis, the α1A-subtype is a potential therapeutic target to treat the failing RV.NEW & NOTEWORTHY Right ventricular (RV) failure is a serious disease with a poor prognosis and no effective treatments. In the mouse bleomycin model of RV failure, we tested the efficacy of a treatment using the α1A-adrenergic receptor subtype agonist A61603. Chronic A61603 treatment improved RV contraction and reduced multiple indexes of RV injury, suggesting that the α1A-subtype is a therapeutic target to treat RV failure.

Retinoic acid improves nephrotoxic serum–induced glomerulonephritis through activation of podocyte retinoic acid receptor α

Proliferation of glomerular epithelial cells, including podocytes, is a key histologic feature of crescentic glomerulonephritis. We previously found that retinoic acid (RA) inhibits proliferation and induces differentiation of podocytes by activating RA receptor-α (RARα) in a murine model of HIV-associated nephropathy. Here, we examined whether RA would similarly protect podocytes against nephrotoxic serum-induced crescentic glomerulonephritis and whether this effect was mediated by podocyte RARα. RA treatment markedly improved renal function and reduced the number of crescentic lesions in nephritic wild-type mice, while this protection was largely lost in mice with podocyte-specific ablation of Rara (Pod-Rara knockout). At a cellular level, RA significantly restored the expression of podocyte differentiation markers in nephritic wild-type mice, but not in nephritic Pod-Rara knockout mice. Furthermore, RA suppressed the expression of cell injury, proliferation, and parietal epithelial cell markers in nephritic wild-type mice, all of which were significantly dampened in nephritic Pod-Rara knockout mice. Interestingly, RA treatment led to the coexpression of podocyte and parietal epithelial cell markers in a small subset of glomerular cells in nephritic mice, suggesting that RA may induce transdifferentiation of parietal epithelial cells toward a podocyte phenotype. Invitro, RA directly inhibited the proliferation of parietal epithelial cells and enhanced the expression of podocyte markers. Invivo lineage tracing of labeled parietal epithelial cells confirmed that RA increased the number of parietal epithelial cells expressing podocyte markers in nephritic glomeruli. Thus, RA attenuates crescentic glomerulonephritis primarily through RARα-mediated protection of podocytes and in part through the inhibition of parietal epithelial cell proliferation and induction of their transdifferentiation into podocytes.

Graft-derived macrophage migration inhibitory factor correlates with hepatocellular injury in patients undergoing liver transplantation.

Human liver grafts release MIF in proportion to hepatocellular injury. Greater MIF concentrations in effluent and recipient's serum are associated with EAD after liver transplantation.

Response of the human myocardium to ischemic injury and preconditioning: The role of cardiac and comorbid conditions, medical treatment, and basal redox status.

BackgroundThe diseased human myocardium is highly susceptible to ischemia/reoxygenation (I/R)-induced injury but its response to protective interventions such as ischemic preconditioning (IPreC) is unclear. Cardiac and other pre-existing clinical conditions as well as previous or ongoing medical treatment may influence the myocardial response to I/R injury and protection. This study investigated the effect of both on myocardial susceptibility to I/R-induced injury and the protective effects of IPreC.Methods and resultsAtrial myocardium from cardiac surgery patients (n = 300) was assigned to one of three groups: aerobic control, I/R alone, and IPreC. Lactate dehydrogenase leakage, as a marker of cell injury, and cell viability were measured. The basal redox status was determined in samples from 90 patients.The response to I/R varied widely. Myocardium from patients with aortic valve disease was the most susceptible to injury whereas myocardium from dyslipidemia patients was the least susceptible. Tissue from females was better protected than tissue from males. Myocardium from patients with mitral valve disease was the least responsive to IPreC. The basal redox status was altered in the myocardium from patients with mitral and aortic valve disease.ConclusionsThe response of the myocardium to I/R and IPreC is highly variable and influenced by the underlying cardiac pathology, dyslipidemia, sex, and the basal redox status. These results should be taken into account in the design of future clinical studies on the prevention of I/R injury and protection.

Circulating Extracellular DNA: Cause or Consequence of Thrombosis?

Thrombosis leads to ischemic organ damage in cardiovascular and thromboembolic diseases. Neutrophils promote thrombosis in vitro and in vivo by releasing neutrophil extracellular traps (NETs). NETs are composed of DNA filaments coated with histones and neutrophil enzymes such as myeloperoxidase (MPO). Circulating extracellular DNA (ceDNA) is widely used as a surrogate marker to monitor NET formation in thrombosis. This narrative review summarizes the association of ceDNA with human thrombosis. Levels of ceDNA indicate the extent and outcome of several cardiovascular and thromboembolic diseases, including myocardial infarction, stroke, and venous thromboembolism. ceDNA correlates with markers of coagulation and platelet consumption, thus supporting the hypothesis that ceDNA may be a surrogate marker of thrombus formation. In addition, ceDNA levels correlate with markers of cell injury and size of ischemic lesions, suggesting that ceDNA does not derive from NETs but is probably released from damaged organs. Few studies identified NET-specific biomarkers such as DNA-MPO complexes in the blood of patients with thrombosis. In conclusion, it remains to be established whether ceDNA in patients derives from NETs and is a cause or consequence of thrombosis.

Hepatoprotective effect of blocking N-methyl-d-aspartate receptors in male albino rats exposed to acute and repeated restraint stress.

Stress affects many organs in addition to the brain, including the liver. We assessed the effects on the liver of blocking N-methyl-d-aspartate (NMDA) glutamate receptors with memantine in acute and repeated restraint stress. Forty-two male albino rats were divided into 7 groups; control, acute restraint stress (ARS), ARS + memantine, repeated restraint stress, repeated restraint + memantine, and positive control groups. We measured serum iron, zinc, alanine transferase and aspartame transferase, hepatic malondialdehyde, tumor necrosis factor-α (TNF-α), glutathione peroxidase, superoxide dismutase, metallothionein content, zinc transporter ZRT/IRT-like protein 14 mRNA expression, and hepcidin expression. We conducted a histopathological evaluation via histological staining and immunostaining for glial fibrillary acidic protein and synaptophysin expression, both of which are markers of hepatic stellate cell (HSC) activation. Both ARS and repeated stress increased markers of hepatic cell injury, oxidative stress, and HSC activation. Blocking NMDA with memantine provided a hepatoprotective effect in acute and repeated restraint stress and decreased hepatic cell injury, oxidative stress, and HSC activation.

Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema.

Endothelial injury is implicated in the pathogenesis of COPD and emphysema; however the role of endothelial progenitor cells (EPCs), a marker of endothelial cell repair, and circulating endothelial cells (CECs), a marker of endothelial cell injury, in COPD and its subphenotypes is unresolved. We hypothesized that endothelial progenitor cell populations would be decreased in COPD and emphysema and that circulating endothelial cells would be increased. Associations with other subphenotypes were examined. The Multi-Ethnic Study of Atherosclerosis COPD Study recruited smokers with COPD and controls age 50–79 years without clinical cardiovascular disease. Endothelial progenitor cell populations (CD34+KDR+ and CD34+KDR+CD133+ cells) and circulating endothelial cells (CD45dimCD31+CD146+CD133-) were measured by flow cytometry. COPD was defined by standard spirometric criteria. Emphysema was assessed qualitatively and quantitatively on CT. Full pulmonary function testing and expiratory CTs were measured in a subset. Among 257 participants, both endothelial progenitor cell populations, and particularly CD34+KDR+ endothelial progenitor cells, were reduced in COPD. The CD34+KDR+CD133+ endothelial progenitor cells were associated inversely with emphysema extent. Both endothelial progenitor cell populations were associated inversely with extent of panlobular emphysema and positively with diffusing capacity. Circulating endothelial cells were not significantly altered in COPD but were inversely associated with pulmonary microvascular blood flow on MRI. There was no consistent association of endothelial progenitor cells or circulating endothelial cells with measures of gas trapping. These data provide evidence that endothelial repair is impaired in COPD and suggest that this pathological process is specific to emphysema.

Ethyl pyruvate treatment ameliorates pancreatic damage: evidence from a rat model of acute necrotizing pancreatitis.

IntroductionEthyl pyruvate (EP), a natural flavoring and fragrance agent, has been shown to exert anti-inflammatory and antioxidant actions. We tested the potential beneficial effects of EP in a rat model of acute necrotizing pancreatitis (ANP), a serious condition with a significant inflammatory explosion and oxidative stress.Material and methodsFifty-two adult male Sprague-Dawley rats were divided into four groups: sham + saline, sham + EP, ANP + saline, and ANP + EP. The ANP was induced by glycodeoxycholic acid and cerulein. Animals were sacrificed at 48 h and biochemical, hematological, and histological markers of ANP and inflammation were assessed. The extent of mortality, systemic cardiorespiratory variables, pancreatic microcirculation, renal/hepatic functions, acinar cell injury and enzyme markers for pancreas and lung tissues were investigated.ResultsThe EP-treated ANP group presented significantly lower mortality than the untreated ANP group (44% (7/16) vs. 19% (3/16), respectively, p < 0.05). Administration of EP resulted in significantly lower levels of IL-6 (ANP + saline: 5470 ±280 vs. ANP + EP: 2250 ±180 pg/ml, p < 0.05). Compared with the ANP group, the ANP + EP group had a lower pancreatic necrosis score (1.45 ±0.2 vs. 0.96 ±0.2, p < 0.05). Moreover, intraperitoneal EP administration had a positive effect on most indices of pancreatitis (amylase and alanine transaminase levels) and lung damage (except lung malondialdehyde levels) as they decreased towards baseline values.ConclusionsThe results from this experimental study indicate that EP, a nontoxic chemical approved by the Food and Drug Administration as a food additive, provides positive effects on the course of pancreatitis, suggesting potential usefulness in management of ANP.

Elevated Levels of Circulating Nucleosomes Are Not Associated with Venous Thrombosis or Neutrophil Activation in Patients with Multiple Myeloma

Background: Patients with multiple myeloma (MM) are at a 9-fold increased risk of venous thromboembolism (VTE). Mechanisms underlying the increased VTE risk in MM have not yet been clarified. Recently, extracellular DNA, e.g. nucleosomes, has been shown to play a central role in coagulation activation and propagation in vivo and in animal models. Increased nucleosome levels were demonstrated in patients with VTE, supporting their role in VTE development. The prothrombotic activity of nucleosomes may be of importance in systemic inflammatory states, in which activated immune cells can release nucleosomes, such as neutrophils by forming neutrophil extracellular traps (NETs). However, during inflammation and upon chemotherapy, parenchymal cells and tumor cells, respectively, can also release extracellular DNA in the form of nucleosomes, which could promote coagulation. As MM is regarded as a systemic inflammatory disease, we hypothesized to find elevated levels of nucleosomes in MM patients, with an association between the highest levels of nucleosomes and development of VTE. Therefore, we assessed levels of nucleosomes and neutrophil activation, and their association with VTE, in a cohort of patients with newly diagnosed MM.Methods: We assessed levels of circulating nucleosomes and systemic neutrophil activation, by presence of elastase-α1-antitrypsin (EA) complexes, in 131 patients with newly diagnosed MM using plasma samples. Interleukin (IL)-6 and IL-8 levels were also assessed to estimate the inflammatory state. We used a case-cohort design to investigate the association between nucleosomes, neutrophil activation and VTE, by calculating odds ratios (ORs) with corresponding 95% confidence intervals (CIs) using nucleosomes and EA complexes as categorical variables, based on the 80th percentile for MM patients without VTE, with ≤80th percentile being the reference category. To compare levels of nucleosomes and neutrophil activation complexes in MM patients with patients with other malignancies, a comparison was made with a cohort of cases with objectified deep-vein thrombosis (DVT) of the leg and screened controls without DVT, stratified by malignancy status.Results: 19 of 131 MM patients (14.5%) developed VTE during MM treatment. Levels of nucleosomes (median 39.5 U/mL; interquartile range [IQR] 7.9-156.8, vs. median 25.6 U/mL; IQR 10.9-76.4; p=0.61) and neutrophil activation, as evidenced by presence of EA complexes (median 30.1 U/mL; IQR 22.6-39.3, vs. median 31.6 U/mL; IQR 24.3-46.0; p=0.45), did not differ between MM patients with and without VTE. No association was found between high levels (>80th percentile of non-VTE MM patients) of nucleosomes or neutrophil activation, and VTE (OR 0.5; 95% CI 0.2-1.6, and OR 2.1; 95% CI 0.5-9.7, respectively) in MM patients. IL-6 and IL-8 levels were low in all MM patients, irrespective of VTE. Nucleosome levels were similarly high in MM patients compared with DVT patients with other malignancies, and significantly higher compared with DVT patient without malignancies, and with DVT controls, irrespective of malignancy status (Fig. 1A). In contrast, levels of neutrophil activation were significantly lower in MM patients in all comparisons (Fig. 1B).Conclusions: Elevated levels of nucleosomes were found in MM patients, but without correlating high levels of neutrophil activation. Nucleosome levels in MM patients with and without VTE were similar, and there was no association between high levels of circulating nucleosomes or neutrophil activation, and VTE in MM patients. Our results suggest that elevated levels of nucleosomes in MM patients are independent of neutrophil activation or inflammatory status, but rather a marker of plasma cell injury or death. This could also explain absence of a clear association with VTE. Future studies are necessary to confirm the source and the clinical relevance of high levels of nucleosomes in MM patients. [Display omitted] DisclosuresLeebeek:Baxter: Research Funding; UniQure: Consultancy; Netherlands Hemophilia Foundation: Research Funding; CSL Behring: Research Funding.

Evaluation of an in vitro screening model to assess phosgene inhalation injury

Therapeutic development against exposure to toxic gases is hindered by the lack of appropriate models to evaluate candidate compounds prior to animal efficacy studies. In this study, an in vitro, air-liquid interface exposure model has been tested to examine its potential application for screening treatments for phosgene (carbonyl chloride)-induced pulmonary injury. Epithelial cultures on Transwell® inserts, combined with a Vitrocell® exposure apparatus, provided a physiologically relevant exposure environment. Differentiated human bronchial epithelial (16HBE) cultures were exposed for 8 min to phosgene ranging from 0 to 64 ppm and assessed for changes in transepithelial electrical resistance (TEER, epithelial barrier integrity), cellular viability (XTT) and post-exposure (PE) cellular metabolic energy status. Exposure to phosgene concentrations ≥8 ppm caused dose-dependent and significant decreases in TEER and XTT which did not recover within 24-h PE. In addition, at 64 ppm the rate of oxidative glutamine metabolism was significantly inhibited at 6 and 24 h after exposure. Glycolytic activities (glucose utilization and lactate production) were also inhibited, but to a lesser extent. Decreased glycolytic function can translate to insufficient energy sources to counteract barrier function failure. Consistent and sensitive markers of phosgene exposure were TEER, cell viability and decreased metabolism. As such, we have assessed an appropriate in vitro model of phosgene inhalation that produced quantifiable alterations in markers of lung cell metabolism and injury in human airway epithelial cells. Data indicate the suitability of this model for testing classes of anti-edemagenic compounds such as corticosteroids or phosphodiesterase inhibitors for evaluating phosgene therapeutics.

A serine protease inhibitor attenuates aldosterone-induced kidney injuries via the suppression of plasmin activity

Emerging evidence has suggested that aldosterone has direct deleterious effects on the kidney independently of its hemodynamic effects. However, the detailed mechanisms of these direct effects remain to be elucidated. We have previously reported that camostat mesilate (CM), a synthetic serine protease inhibitor, attenuated kidney injuries in Dahl salt-sensitive rats, remnant kidney rats, and unilateral ureteral obstruction rats, suggesting that some serine proteases would be involved in the pathogenesis of kidney injuries. The current study was conducted to investigate the roles of serine proteases and the beneficial effects of CM in aldosterone-related kidney injuries. We observed a serine protease that was activated by aldosterone/salt in rat kidney lysate, and identified it as plasmin with liquid chromatography-tandem mass spectrometry. Plasmin increased pro-fibrotic and inflammatory gene expressions in rat renal fibroblast cells. CM inhibited the protease activity of plasmin and suppressed cell injury markers induced by plasmin in the fibroblast cells. Furthermore, CM ameliorated glomerulosclerosis and interstitial fibrosis in the kidney of aldosterone/salt-treated rats. Our findings indicate that plasmin has important roles in kidney injuries that are induced by aldosterone/salt, and that serine protease inhibitor could provide a new strategy for the treatment of aldosterone-associated kidney diseases in humans.

Abstract P340: Molecular Mechanisms of Fetuin-a-induced Vascular Dysfunction and Inflammation: Role of Oxidative Stress and Toll-like Receptor 4

Fetuin-A (FetA) is an endogenous agonist to toll-like receptor 4 (TLR4) and regulates insulin resistance and inflammation. FetA has been associated with endothelial dysfunction during metabolic diseases. Exact mechanisms whereby FetA influences vascular function in pathological conditions remain unknown, but we demonstrated that FetA regulates vascular function by Nox1 and TLR4 activation. Here we hypothesized that FetA, through changes in cell metabolism and activation of TLR4-Nox1 axis induces ROS formation and inflammation in hypertension. Normotensive (WKY) and hypertensive (SHRSP) vascular cells, as well as human microvascular endothelial cells, were stimulated with FetA (50 ng/mL). ROS production was measured by lucigenin and Amplex red, while gene expression was assessed by qPCR. FetA increased ROS production (131±49.2%), decreased H 2 O 2 intracellular levels (63±14%) and increased gene levels of IL6 (2 fold), IL1β (1 fold), RANTES (1 fold) and MMP2/9 (2 fold) in rat endothelial cells ( vs. veh, p&lt;0.05); all effects were blocked by TLR4 inhibitor (CLI095) and Nox1 inhibitor (ML171). FetA increased JNK (184±19%), but not p38 MAPK, activation in endothelial cells. In VSMCs, FetA-induced TLR4-dependent ROS generation was similar in WKY (136±9%) and SHRSP (144±14%) (p&lt;0.05 vs veh). However, while IL6 gene expression was increased by FetA in WKY (4 fold) and SHRSP (0.5 fold), IL-1β gene levels were only increased by FetA in SHRSP (1 fold) derived VSMCs (p&lt;0.05). CLI095 inhibited FetA effects on IL6 expression; however, TLR4 inhibition did not block FetA effects on IL-1β gene levels. In human endothelial cells, FetA increased ROS levels, was inhibited by CLI095 and a glucose-6-phosphate dehydrogenase (G6PD) inhibitor (6-aminonicotinamide), suggesting that FetA effects may be related to control of cell metabolism. In conclusion, FetA seems to regulate ROS and pro-inflammatory responses by TLR4, Nox1 and G6PD in endothelial cells. In VSMCs, FetA effects on oxidative stress and markers of cell injury are partially dependent on TLR4 activation and may involve other molecular partners.

Inhibition Of COX‐2/PGE2/EP4 Signaling Protects Against Post‐hypoxic Apoptosis In H9C2 Cardiomyocytes

Myocardial infarction is a condition associated with significant mortality. Reperfusion therapy restores blood flow, it may also induce lethal tissue injury, known as ischemia‐reperfusion injury (IRI) in clinical practice. Thus, exploring ways to control or attenuate IRI is of clinical interest for improving post‐ischemic recovery. Cyclooxyenase‐2 (COX‐2) – which is induced during ischemia ‐ is highly expressed at the site of recent acute myocardial infarction (Heart. 2004;90:440–443). Furthermore, the presence of COX‐2 is positively correlated with progressively increased apoptosis which played a central role in the pathophysiology of IRI. However, whether there is causal link or underlying signaling cascade between ischemia‐driven COX‐2 expression and apoptosis in IRI is unclear. Thus, the present study aimed to investigate the molecular basis of potential cause‐effect link between COX‐2 expression and enhanced apoptosis during ischemia using rat origin cardiomyocytes (H9C2) subjected to hypoxia/reoxygenation (H/R, 6 hours hypoxia followed by 12 hours reoxygenation). The results showed that H/R significantly increased the protein expression of COX‐2 (P&lt;0.05 vs. Control), which was prevented by 2 hours pretreatment with Helenalin (NFkB inhibitor, at 10μM, P&lt;0.05 vs. H/R) but not by pretreatment with SC‐205346A [Hypoxia inducible factor alpha (HIF‐1α) inhibitor, at 20μM]. This suggests that NFkB but not HIF‐1α mediated the expression of COX‐2 during H/R in H9C2 cardiomyocytes. Further, under the experimental setting, H/R induced H9C2 cells damage [evidenced by increased lactic acid dehydrogenase (LDH) leakage (marker of cell injury, P&lt;0.05 vs. Control)] was concomitant with increased cleaved caspase‐3 expression (marker of cell apoptosis, P&lt;0.05 vs. Control) but no changes in the protein expressions of the anti‐apoptotic Bcl‐2 and pro‐apoptotic Bax, suggesting that H/R induced caspase‐3 dependent cell apoptosis which was independent of the intrinsic mitochondrial pathway. Pretreat the cells with NS398 (COX‐2 selective inhibitor, at 10μM, 1 hour) or L161,982 [prostaglandin E receptor subtype 4 (EP4) selective antagonist, at 100nM, 1 hour] suppressed the H/R induced increases of LDH leakage and cleaved caspase‐3 expression (P&lt;0.05 vs. H/R), indicating that both COX‐2 and EP4 mediated cell apoptosis during H/R. Given that prostaglandin E2 (PGE2) is a major metabolite of COX‐2 and an endogenous ligand of EP4 receptor, H9C2 cardiomyocytes were exposed to exogenous PGE2 (from 5nM to 50μM, 18 hours) in a concentration dependent manner. As anticipated, PGE2, at a high concentration (50μM), significantly increased cellular injury as evidenced by enhanced LDH leakage (P&lt;0.05 vs. Control). It is concluded that H/R may induce cardiomyocytes apoptosis via NFkB/COX‐2/PGE2/EP4 signaling, which may serve as a novel target for anti‐ischemic therapy.Support or Funding InformationThis study was supported by General Research Fund (17124614, and 17123915) from the Research Grants Council of Hong Kong and by National Natural Science Foundation of China (NSFC 81270899).

Keratins are novel markers of renal epithelial cell injury

Keratins, the intermediate filaments of the epithelial cell cytoskeleton, are up-regulated and post-translationally modified in stress situations. Renal tubular epithelial cell stress is a common finding in progressive kidney diseases, but little is known about keratin expression and phosphorylation. Here, we comprehensively describe keratin expression in healthy and diseased kidneys. In healthy mice, the major renal keratins, K7, K8, K18, and K19, were expressed in the collecting ducts and K8, K18 in the glomerular parietal epithelial cells. Tubular expression of all 4 keratins increased by 20- to 40-fold in 5 different models of renal tubular injury as assessed by immunohistochemistry, Western blot, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The up-regulation became significant early after disease induction, increased with disease progression, was found de novo in distal tubules and was accompanied by altered subcellular localization. Phosphorylation of K8 and K18 increased under stress. In humans, injured tubules also exhibited increased keratin expression. Urinary K18 was only detected in mice and patients with tubular cell injury. Keratins labeled glomerular parietal epithelial cells forming crescents in patients and animals. Thus, all 4 major renal keratins are significantly, early, and progressively up-regulated upon tubular injury regardless of the underlying disease and may be novel sensitive markers of renal tubular cell stress.

Evaluation of Significance of Liver Enzymes as Screening Tests for the Early Detection of Clinically Asymptomatic Non Alcoholic Fatty Liver Disease in Type 2 Diabetes Mellitus Patients

Background: Type 2 diabetes mellitus has been linked with abnormal liver function tests. Increased activities of liver enzymes such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), ?-glutamyl transferase (GGT) are markers of liver cell injury. Increased activity of these markers is associated with metabolic syndrome, insulin resistance and type 2 diabetes mellitus. This study was aimed to evaluate the significance of liver enzymes such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), ?-glutamyl transferase (GGT) in type 2 diabetes mellitus patients. Materials & Methods: Total of 108 cases of type 2 diabetes mellitus patients, comprising of 68 males (62.9%) and 40 females (37%) were included in the study. 110 healthy subjects were includes as controls. Age of the subjects ranged between 27 to 75 years. Serum sample was used for the estimation of study parameters such as FBS, PPBS, AST, ALT, ALP, GGT, Total bilirubin, Direct bilirubin, Total proteins and Albumin. Results: In the present study, mean values of AST, ALT, ALP and GGT were statistically significantly increased in type 2 diabetes mellitus patients when compared to controls (P value 0.001). Conclusion: In this study, we found an association between the increased levels of liver enzymes such as AST, ALT, ALP and GGT in type 2 diabetes mellitus patients. Increased levels of ALT and AST are the surrogate markers for associated non alcoholic fatty liver disease in type 2 diabetes mellitus patients. Hence, testing for AST, ALT, ALP, and GGT should be carried out to screen for underlying non alcoholic fatty liver disease (NAFLD) in type 2 diabetes mellitus patients.

Abstract 18190: Associations Between Endothelial Injury, Severity of Post-cardiac Arrest Syndrome and Mortality After Out-of-hospital Cardiac Arrest

Background: Post-cardiac arrest syndrome (PCAS) is characterized by systemic inflammation and endothelial dysfunction, which may exacerbate organ failure and may be related to adverse outcome. We sought to investigate the associations between trombomodulin (TM), a marker of endothelial cell injury, and severity of PCAS and the possible prognostic importance in comatose survivors of out-of-hospital cardiac arrest treated with targeted temperature management (TTM). Methods: We included 163 patients, randomly assigned to TTM at 33°C or 36°C for 24 h in the TTM-trial at a single center. Serum TM was analyzed at 0, 24, 48 and 72 hours after cardiac arrest and stratified in median value of TM for outcome analysis at each time point to a high or low TM group. Severity of PCAS was assessed daily by the Sequential Organ Failure Assessment score. Survival status was recorded at 180 days. Results: Corresponding values of TM and severity of PCAS correlated at each time point, baseline (r=0.31, p=0.0001), 24 h (r=0.52, p&lt;0.0001), 48 h (r=0.50, p&lt;0.0001) and 72 h (r=0.36, p&lt;0.0001). Levels of TM changed significantly within the first 3 days (p&lt;0.0001) with no significant difference between temperature groups, p interaction =0.32. At 24 h, the 180-day mortality rate in patients with high TM levels (&gt; 6.9 ng·ml -1 = median) was 42% compared to 18% in patients with low TM levels, p=0.002 (Figure), corresponding to an unadjusted hazard ratio of 2.6 (95%CI: 1.4-4.9), p=0.003. When adjusting for pre-hospital prognostic factors the hazard ratio for high TM levels at 24 h was 1.5 (95%CI: 0.7-3.1), p=0.31, with no significant interaction between level of TM and TTM-group, p=0.74. Analysis of TM at 48 and 72 hours showed similar results. Conclusions: Levels of TM are significantly elevated and associated with severity of PCAS with no effect of target temperature at 33°C or 36°C. High TM levels were associated with increased mortality, but the association was explained by confounding factors.

Label-Free and Sensitive Detection of Thrombomodulin, a Marker of Endothelial Cell Injury, Using Quartz Crystal Microbalance.

Thrombomodulin (TM), an integral glycoprotein on the surface of endothelial cells, can be released during endothelial cell injury and the levels of serum TM are regarded as an important parameter of activity in vasculitides in vivo. Quantitative detection of TM and investigation on the release of soluble thrombomodulin (sTM) by the injured HUVEC-C cells using quartz crystal microbalance (QCM) were achieved in this work. Anti-antibody (AAb) and bovine serum albumin (BSA) were bound on gold nanoparticles (GNPs) to construct BSA-GNPs-AAb nanocomposites and they were characterized by transmission electron microscope, UV-vis, and infrared spectrophotometry, respectively. The capture of the nanocomposites on the TM antibody modified electrode, which was tested by scanning electron microscope, could result in a great decrease of the resonant frequency (f0). This binding was effectively inhibited by the beforehand immobilized TM proteins on the electrode surface due to the strong steric hindrance effect. It led to the decrease of the frequency changing extent. The relative frequency-shift was found to be proportional to the logarithm of the TM concentration from 10 to 5000 ng mL(-1) with a detection limit of 2 ng mL(-1). By analyzing the growth medium used for cell incubation, the release of sTM by the injured HUVEC-C cells in the presence of H2O2 was confirmed. The sTM amount in the growth medium was increased with the enhancement of contact time of the cells with H2O2, proving that sTM may serve as a specific marker of endothelial cell injury.