Adam Khader

Blocking cold-inducible RNA-binding protein protects liver from ischemia-reperfusion injury.

ABSTRACT Cold-inducible RNA-binding protein (CIRP) is a nuclear protein that has been recently identified as a novel inflammatory mediator in hemorrhagic shock and sepsis. We hypothesized that CIRP acts as a potent inflammatory mediator in hepatic ischemia-reperfusion (I/R), and thus blocking CIRP protects against I/R-induced liver injury. Male C57BL/6 mice were subjected to 70% hepatic ischemia by microvascular clamping of the hilum of the left and median liver lobes for 60 min, followed by reperfusion. Anti-CIRP antibody (1 mg/kg body weight) or vehicle (normal saline) in 0.2 mL was injected via the internal jugular vein at the beginning of the reperfusion. Blood and liver tissues were collected 24 h after I/R for various measurements, and a 10-day survival study was performed. Cold-inducible RNA-binding protein released into the circulation was significantly increased 24 h after hepatic I/R. Anti-CIRP antibody treatment markedly reduced hepatocellular damage markers and significantly improved the liver microarchitecture. Anti-CIRP also reduced the systemic and local inflammation demonstrated by attenuation in both serum and hepatic levels of interleukin 6. The expression of neutrophil-attracting chemokine as well as liver neutrophil infiltration was reduced by anti-CIRP treatment. Anti-CIRP also dramatically decreased the amount of apoptosis and nitrosative stress, evidenced by decrease in TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining and inducible nitric oxide synthase and cyclooxygenase 2 levels, respectively. Finally, the 10-day survival rate was increased from 37.5% in the vehicle group to 75% in the anti-CIRP treatment group. Thus, targeting CIRP offers potential therapeutic implications in the treatment of hepatic I/R injury.

WNT Agonist Decreases Tissue Damage and Improves Renal Function After Ischemia-Reperfusion.

ABSTRACT Renal ischemia-reperfusion (IR) injury (IRI) after shock states or transplantation causes tissue damage and delayed graft function, respectively. The Wnt/&bgr;-catenin signaling pathway plays a critical role in nephrogenesis. We therefore hypothesized that pharmacological activation of the Wnt/&bgr;-catenin signaling by the Wnt agonist, a synthetic pyrimidine, could protect kidneys from IRI. Adult male rats were subjected to bilateral clamping of the renal pedicles with microvascular clips for 60 min, followed by reperfusion. The Wnt agonist (5 mg/kg body weight) or vehicle (20% dimethyl sulfoxide in saline) was administered intravenously 1 h before ischemia. Blood and renal tissues were collected 24 h after IR for evaluation. Renal IR caused a significant reduction of &bgr;-catenin and its downstream target gene cyclin D1 by 65% and 39%, respectively, compared with the sham, whereas the Wnt agonist restored them to sham levels. The number and intensity of cells staining with the proliferation marker Ki67 in ischematized kidneys were enhanced by the Wnt agonist. The integrity of the renal histological architecture in the Wnt agonist group was better preserved than the vehicle group. The Wnt agonist significantly lowered serum levels of creatinine, aspartate aminotransferase, and lactate dehydrogenase and inhibited the production of interleukin 6 and interleukin 1&bgr; and myeloperoxidase activities. Lastly, the Wnt agonist reduced inducible nitric oxide synthase, nitrotyrosine proteins, and 4-hydroxynonenal in the kidneys by 60%, 47%, and 21%, respectively, compared with the vehicle. These results indicate that the Wnt agonist improves renal regeneration and function while attenuating inflammation and oxidative stress in the kidneys after IR. Thus, pharmacologic stimulation of the Wnt/&bgr;-catenin signaling provides a beneficial effect on the prevention of renal IRI.

Sirtuin 1 Stimulation Attenuates Ischemic Liver Injury and Enhances Mitochondrial Recovery and Autophagy.

Objectives:Hepatic ischemia-reperfusion is a major clinical problem with limited treatment options. The pathophysiology of hepatic ischemia-reperfusion is characterized by mitochondrial dysfunction and cellular energy deficits. Sirtuin 1 is an energy-sensing enzyme known to modulate mitochondrial biogenesis. We hypothesized that pharmacologic activation of sirtuin 1 is protective after hepatic ischemia-reperfusion injury. Design:Animal study. Setting:University-based experimental laboratory. Subjects:Wild-type C57BL/6 mice. Interventions:C57BL/6 mice were subjected to 60-minute partial hepatic ischemia-reperfusion and posttreated with sirtuin 1 activator, SRT1720 (20 mg/kg), or vehicle. Blood and liver were collected at 24 hours after ischemia-reperfusion for analyses of hepatic injury, adenosine triphosphate levels, mitochondrial mass, autophagy, inflammation, and oxidative stress. H4IIE hepatoma cells and rat primary hepatocytes were incubated with oxyrase to induce hypoxia followed by reoxygenation in the presence or absence of SRT1720 for assessment of mitochondrial mass, mitochondrial membrane potential, and autophagy. Measurements and Main Results:SRT1720 restored the reduction in mitochondrial mass, enhanced autophagy, and preserved adenosine triphosphate levels in the liver after ischemia-reperfusion, which was associated with a decrease in ischemia-reperfusion–induced hepatic injury, apoptosis, and necrosis. Ischemia-reperfusion–induced inflammation was also significantly reduced by SRT1720 as measured by systemic and hepatic cytokine and chemokine levels, as well as a decrease in neutrophil infiltration to the liver. Furthermore, oxidative stress was markedly attenuated in the SRT1720-treated mice compared with the vehicle. SRT1720 treatment increased adenosine triphosphate levels and survival of cultured hepatocytes after hypoxia-reoxygenation. SRT1720 not only increased the mitochondrial mass but also increased mitochondrial membrane potential per cell in cultured hepatocytes after hypoxia-reoxygenation. Moreover, SRT1720 prevented the hypoxia-reoxygenation–induced mitochondrial depolarization and resulted in an enhancement of autophagy in cultured hepatocytes after hypoxia-reoxygenation. Conclusions:Pharmacologic stimulation of sirtuin 1 attenuates liver injury after hepatic ischemia-reperfusion by restoring mitochondrial mass and membrane potential, which is associated with the enhancement of autophagy.

Novel resveratrol analogues attenuate renal ischemic injury in rats

BACKGROUND Renal ischemia-reperfusion (I/R) is a severe clinical complication with no specific treatment. Resveratrol has been shown as a promising experimental agent in renal I/R due to its effect on cellular energy metabolism, oxidative stress, and inflammation. Recently, we identified two biologically active resveratrol analogues (RSVAs), RSVA405 and RSVA314. We hypothesized that both RSAVs would attenuate I/R-induced renal injury. METHODS Adult male rats were subjected to renal I/R through bilateral renal pedicle clamping for 60 min, followed by reperfusion. RSVA405 (3 mg/kg Body Weight), RSVA314 (3 mg/kg Body Weight), or vehicle (10% dimethyl sulfoxide and 33% Solutol in phosphate buffered saline) were administered by intraperitoneal injection 1 h before ischemia. Blood and renal tissues were collected 24 h after I/R for evaluation. RESULTS Administration of RSVA405 and RSVA314 significantly reduced the serum levels of renal dysfunction and injury markers, including creatinine, blood urea nitrogen, aspartate aminotransferase, and lactate dehydrogenase, compared with vehicle. The protective effect of RSVA405 and RSVA314 was also reflected on histologic evaluation. Both RSVAs reduced the number of apoptotic cells by more than 60% as determined by transferase dUTP nick end labeling assay, compared with vehicle. The renal adenosine triphosphate levels of the vehicle group was decreased to 52.4% of control, whereas those of the RSVA405 and RSVA314 groups were restored to 72.3% and 79.6% of control, respectively. Both RSVAs significantly reduced the protein expression of inducible nitric oxide synthase and nitrotyrosine and the messenger RNA levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β. CONCLUSIONS RSVA405 and RSVA314 attenuate I/R-induced renal injury through the modulation of energy metabolism, oxidative stress, and inflammation.

SIRTUIN 1 ACTIVATOR SRT1720 PROTECTS AGAINST ORGAN INJURY INDUCED BY INTESTINAL ISCHEMIA-REPERFUSION.

ABSTRACT Intestinal ischemia-reperfusion (I/R) occurs in various clinical situations and causes local and remote organ injury, especially in the lungs, leading to significant morbidity and mortality. The maintenance of mitochondrial biogenesis is essential for cell survival and is regulated in part by sirtuin 1 (SIRT1), an energy-sensing enzyme. We hypothesized that SIRT1 activation with SRT1720 would reduce local and remote organ injury after intestinal I/R. Intestinal I/R was induced by the occlusion of the superior mesenteric artery of adult male C57BL/6 mice for 45 min, followed by reperfusion for 4 h. SRT1720 or vehicle was injected intravenously at the time of reperfusion. Blood, small intestine, and lung tissues were collected for analysis. The SRT1720 treatment of I/R mice resulted in a 57% increase in protein levels of succinate dehydrogenase, an index of mitochondrial mass, and a 120% increase in messenger RNA levels of mitochondrial transcription factor A, a marker for mitochondrial biogenesis. The microscopic architecture and apoptosis of the gut tissue was improved in the SRT1720-treated I/R mice. SRT1720 decreased intestinal messenger RNA levels of tumor necrosis factor-&agr; by 60% and inducible nitric oxide synthase to baseline after I/R. Systemic inflammation, as determined by serum interleukin-6, was reduced in treated mice. Lung injury, as measured by histological architecture and myeloperoxidase activity, and lung apoptosis were also improved after the SRT1720 treatment. SRT1720 preserved mitochondrial biogenesis and mass, leading to inhibition of inflammation and oxidative stress, thereby protecting against intestinal I/R-induced injury. Thus, the SIRT1-mediated pathway is a promising target for the treatment of intestinal I/R injury.

Stimulation of Wnt/β-catenin signaling pathway with Wnt agonist reduces organ injury after hemorrhagic shock.

BACKGROUND Hemorrhagic shock is a leading cause of morbidity and mortality in surgery and trauma patients. Despite a large number of preclinical trials conducted to develop therapeutic strategies against hemorrhagic shock, there is still an unmet need for effective therapy for hemorrhage patients. Wnt/&bgr;-catenin signaling controls developmental processes and cellular regeneration owing to its central role in cell survival and proliferation. We therefore hypothesized that the activation of Wnt signaling reduces systemic injury caused by hemorrhagic shock. METHODS Adult male Sprague-Dawley rats underwent hemorrhagic shock by controlled bleeding of the femoral artery to maintain a mean arterial pressure of 30 mm Hg for 90 minutes, followed by resuscitation with crystalloid equal to two times the shed blood volume. After resuscitation, animals were infused with Wnt agonist (5 mg/kg) or vehicle (20% dimethyl sulfoxide in saline). Blood and tissue samples were collected 6 hours after resuscitation for analysis. RESULTS Hemorrhagic shock increased serum levels of aspartate aminotransferase, lactate, and lactate dehydrogenase. Treatment with Wnt agonist significantly reduced these levels by 40%, 36%, and 77%, respectively. Wnt agonist also decreased blood urea nitrogen and creatinine by 34% and 56%, respectively. The treatment reduced lung myeloperoxidase activity and interleukin 6 messenger RNA by 55% and 68%, respectively, and significantly improved lung histology. Wnt agonist treatment increased Bcl-2 protein to sham values and decreased cleaved caspase 3 by 46%, indicating attenuation of hemorrhage-induced apoptosis in the lungs. Hemorrhage resulted in significant reductions of &bgr;-catenin protein levels in the lungs as well as down-regulation of a Wnt target gene, cyclin D1, while Wnt agonist treatment preserved these levels. CONCLUSION The administration of Wnt agonist attenuated hemorrhage-induced organ injury, inflammation, and apoptosis. This was correlated with the preservation of the Wnt signaling pathway. Thus, Wnt/&bgr;-catenin activation could be protective in hemorrhagic shock.

Deficiency in milk fat globule-epidermal growth factor-factor 8 exacerbates organ injury and mortality in neonatal sepsis

INTRODUCTION Neonatal sepsis is a systemic inflammation occurring in neonates because of a proven infection within the first 28days of birth. It is the third leading cause of morbidity and mortality in the newborns. The mechanism(s) underlying the systemic inflammation in neonatal sepsis has not been completely understood. We hypothesize that the deficiency of milk fat globule-epidermal growth factor-factor 8 (MFG-E8), a protein commonly found in human milk, could be responsible for the increased inflammatory response leading to morbidity and mortality in neonatal sepsis. METHODS Male and female newborn mice aged 5-7days were injected intraperitoneally with 0.9mg/g body weight cecal slurry (CS). At 10h after CS injection, they were euthanized, and blood, lungs and gut tissues were obtained for further analyses. Control newborn mice underwent similar procedures with the exception of the CS injection. In duplicate newborn mice after CS injection, they were returned to their respective cages with their mothers and were closely monitored for 7days and survival rate recorded. RESULTS At 10h after CS injection, serum LDH in the MFG-E8 knockout (KO) newborn mice was significantly increased by 58% and serum IL-6, IL-1β and TNF-α in the MFG-E8KO newborn mice were also significantly increased by 56%, 65%, and 105%, respectively, from wild type (WT) newborn mice. There were no significant difference between WT control and MFG-E8 control newborn mice. The lung architecture was severely damaged and a significant 162% increase in injury score was observed in the CS MFG-E8KO newborn mice. The MPO, TUNEL staining, and cytokine levels in the lungs and the intestine in CS MFG-E8KO newborn mice were significantly increased from CS WT newborn mice. Similarly, intestinal integrity was also compromised in the CS MFG-E8KO newborn mice. In a survival study, while the mortality rate within 7days was only 29% in the CS WT newborn mice, 80% of the CS MFG-E8KO newborn mice died during the same time period with the majority of mortality occurring within 48h. CONCLUSION The deficiency in MFG-E8 caused increases in inflammation, tissue injury, neutrophil infiltration and apoptosis, which led to morbidity and mortality in murine neonatal sepsis. These studies suggest that MFG-E8 has a protective role in fighting against neonatal sepsis.

Milk Fat Globule-EGF Factor VIII Reduces Inflammation and Attenuates Lung Injury in Neonatal Sepsis

RESULTS: Two hundred seven patients were enrolled, of whom 68 patients had appendicitis. Thirty-one (46%) of these patients had complicated appendicitis. Among the individual candidate biomarkers, MMP-8 had the greatest area under the curve (AUC 1⁄4 0.7; p 1⁄4 0.005). White blood cell (WBC) count had an AUC of 0.80, p < 0.0001. The CART-generated decision tree included WBC count, MMP-8, TIMP-2, MMP-10, MMP-12, and MMP-13, and had the following test characteristics for estimating the probability of complicated appendicitis (95% CI): AUC 0.97 (95% CI, 0.93-1.00); sensitivity 94% (95% CI, 79%-99%); specificity 88% (95% CI, 71%-96%); positive predictive value 89% (95% CI, 74%-96%); and negative predictive value 94% (95% CI, 77%-99%).