Olmesartan Ameliorates Organ Injury and Mortality in Rats With Peritonitis-Induced Sepsis.

INTRODUCTION: Sepsis and related complications lead to high morbidity and mortality in humans and animals. Olmesartan medoxomil (OLM), a nonpeptide angiotensin II type 1 receptor blocker, has antiinflammatory and antioxidative effects in various experimental animal models. The present study aimed to investigate whether OLM protects against sepsis in a clinically relevant model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). METHODS: Sepsis was induced by CLP in anesthetized rats. OLM was administered intraperitoneally 3 h after CLP onset. Hemodynamic, biochemical, and inflammatory parameters were analyzed. RESULTS: The administration of OLM in CLP rats significantly improved their survival rate. Moreover, OLM mitigated CLP-induced hypotension and organ injury (indicated by biochemical parameters), but not tachycardia. OLM significantly reduced the plasma levels of interleukin-6 and nitric oxide. CONCLUSIONS: OLM markedly attenuated CLP-induced hypotension and organ injury, and hence improved survival by inhibiting the inflammatory response and nitrosative stress in this clinically relevant model of sepsis.

A bioassay system of autologous human endothelial, smooth muscle cells, and leukocytes for use in drug discovery, phenotyping, and tissue engineering.

Blood vessels are comprised of endothelial and smooth muscle cells. Obtaining both types of cells from vessels of living donors is not possible without invasive surgery. To address this, we have devised a strategy whereby human endothelial and smooth muscle cells derived from blood progenitors from the same donor could be cultured with autologous leukocytes to generate a same donor "vessel in a dish" bioassay. Autologous sets of blood outgrowth endothelial cells (BOECs), smooth muscle cells (BO-SMCs), and leukocytes were obtained from four donors. Cells were treated in monoculture and cumulative coculture conditions. The endothelial specific mediator endothelin-1 along with interleukin (IL)-6, IL-8, tumor necrosis factor α, and interferon gamma-induced protein 10 were measured under control culture conditions and after stimulation with cytokines. Cocultures remained viable throughout. The profile of individual mediators released from cells was consistent with what we know of endothelial and smooth muscle cells cultured from blood vessels. For the first time, we report a proof of concept study where autologous blood outgrowth "vascular" cells and leukocytes were studied alone and in coculture. This novel bioassay has usefulness in vascular biology research, patient phenotyping, drug testing, and tissue engineering.

Antiviral Activity of Compound L3 against Dengue and Zika Viruses In Vitro and In Vivo.

Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne flaviviruses that cause severe illness after infection. Currently, there are no specific or effective treatments against DENV and ZIKV. Previous studies have shown that tyrosine kinase activities and signal transduction are involved in flavivirus replication, suggesting a potential therapeutic strategy for DENV and ZIKV. In this study, we found that compound L3 can significantly reduce viral protein expression and viral titers in HEK-293, MCF-7, HepG2, and Huh-7 cells and exhibits superior therapeutic efficacy against flaviviral infection compared to other tyrosine kinase inhibitors. In addition, compound L3 can decrease endogenous HER2 activation and inhibit the phosphorylation of the HER2 downstream signaling molecules Src and ERK1/2, the levels of which have been associated with viral protein expression in MCF-7 cells. Moreover, silencing HER2 diminished DENV-2 and ZIKV expression in MCF-7 cells, which suggests that HER2 activity is involved in flavivirus replication. Furthermore, in DENV-2-infected AG129 mice, treatment with compound L3 increased the survival rates and reduced the viremia levels. Overall, compound L3 demonstrates therapeutic efficacy both in vitro and in vivo and could be developed as a promising antiviral drug against emerging flaviviruses or for concurrent DENV and ZIKV outbreaks.

Anti-inflammatory Compound Shows Therapeutic Safety and Efficacy against Flavivirus Infection.

Flaviviruses comprise several medically important viruses, including Japanese encephalitis virus, West Nile virus, dengue virus (DENV), yellow fever virus, and Zika virus (ZIKV). A large outbreak of DENV and ZIKV occurred recently, leading to many cases of illness and death. However, despite decades of effort, we have no clinically specific therapeutic drugs against DENV and ZIKV. Previous studies showed that inflammatory responses play a critical role in dengue and Zika virus pathogenesis. Thus, in this study, we examined a series of novel anti-inflammatory compounds and found that treatment with compound 2d could dose dependently reduce viral protein expression and viral progeny production in HEK-293 and Raw264.7 cells infected with four serotypes of DENV and ZIKV. In addition, considering medication safety, compound 2d could not suppress cyclooxygenase-1 (COX-1) enzymatic activities and thus could prevent the side effect of bleeding. Moreover, compound 2d significantly inhibited COX-2 enzymatic activities and prostaglandin E2 levels, associated with viral replication, compared to results with a selective COX-2 inhibitor, celecoxib. Furthermore, administering 5 mg/kg compound 2d to DENV-2-infected AG129 mice prolonged survival and reduced viremia and serum cytokine levels. Overall, compound 2d showed therapeutic safety and efficacy in vitro and in vivo and could be further developed as a potential therapeutic agent for flavivirus infection.

Angiotensin-(1-7) attenuates organ injury and mortality in rats with polymicrobial sepsis.

BACKGROUND: Sepsis and related multiple organ dysfunction result in high morbidity and mortality. Angiotensin (Ang)-(1-7), a biologically active peptide, has various opposing effects of Ang II. Because the effect of Ang-(1-7) on sepsis is unknown, in this study we aimed to determine the impact of Ang-(1-7) on pathophysiologic changes in a clinically relevant model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). METHODS: Sepsis was induced by CLP in rats under anesthesia. Rats were randomized to one of the following five groups: (1) sham-operated group, (2) Ang-(1-7) (1 mg/kg intravenously infused for 1 h) at 3 h and 6 h after sham operation, (3) CLP, (4) Ang-(1-7) at 3 h after CLP, and (5) Ang-(1-7) at 3 h and 6 h after CLP. Rats were observed for 24 h after CLP surgery and then killed for subsequent histological examination. RESULTS: Ang-(1-7) significantly improved the survival of septic rats (83.3% vs. 36.4% at 24 h following CLP; p = 0.009). Ang-(1-7) attenuated the CLP-induced decreased arterial pressure and organ dysfunction, indicated by diminished biochemical variables and fewer histological changes. Ang-(1-7) significantly reduced the level of plasma interleukin-6 and pulmonary superoxide production (p < 0.05). Moreover, caspase-3 and cytoplasmic IκB expression in liver was significantly lower in the Ang-(1-7)-treated CLP rats (p < 0.05). CONCLUSIONS: In this clinically relevant model of sepsis, Ang-(1-7) ameliorates CLP-induced organ dysfunction and improves survival, possibly through suppressing the inflammatory response, oxidative stress, and apoptosis, suggesting that Ang-(1-7) could be a potential novel therapeutic approach to treatment of peritonitis and polymicrobial sepsis.

Loss of plasma fibrinogen contributes to platelet hyporeactivity in rats with septic shock.

INTRODUCTION: Dysregulated host response to infection causes life-threatening organ dysfunction. Excessive inflammation and abnormal blood coagulation can lead to disseminated intravascular coagulation (DIC) and multiple-organ failure in the late sepsis stages. Platelet function impairment in sepsis contributes to bleeding, secondary infection, and tissue injury. Platelet transfusion is considered in patients with sepsis with DIC and bleeding; however, its benefits are limited and of low quality. Fibrinogen plays a crucial role in platelet function, and establishing a fibrin network binds to activated integrin αIIbß3 and promotes outside-in signaling that amplifies platelet functions. However, the role of fibrinogen in sepsis-induced platelet dysfunction remains unclear. MATERIALS AND METHODS: We evaluated the effects of fibrinogen on platelet hyporeactivity during septic shock in adult male Wistar rats using lipopolysaccharide (LPS) injection and cecal ligation and puncture (CLP) surgery. Changes in the hemodynamic, biochemical, and coagulation parameters were examined. Platelet activation and aggregation were measured using whole-blood assay, 96-well plate-based aggregometry, and light-transmission aggregometry. Additionally, platelet adhesion, spreading, and fibrin clot retraction were evaluated. RESULTS: Rats with LPS- and CLP-induced sepsis displayed considerable decreases in plasma fibrinogen levels and platelet aggregation, adhesion, spreading, and clot retraction. The aggregation of platelets obtained from rats with sepsis was markedly augmented by fibrinogen supplementation. Additionally, fibrinogen administration improved platelet adhesion, spreading, and clot retraction in rats with sepsis. CONCLUSIONS: Fibrinogen supplementation could serve as a potential therapeutic intervention for alleviating platelet hyporeactivity in patients with sepsis and bleeding.

Inferring the evolutionary histories of divergences in Hylobates and Nomascus gibbons through multilocus sequence data.

BACKGROUND: Gibbons (Hylobatidae) are the most diverse group of living apes. They exist as geographically-contiguous species which diverged more rapidly than did their close relatives, the great apes (Hominidae). Of the four extant gibbon genera, the evolutionary histories of two polyspecific genera, Hylobates and Nomascus, have been the particular focus of research but the DNA sequence data used was largely derived from the maternally inherited mitochondrial DNA (mtDNA) locus. RESULTS: To investigate the evolutionary relationships and divergence processes of gibbon species, particularly those of the Hylobates genus, we produced and analyzed a total of 11.5 kb DNA of sequence at 14 biparentally inherited autosomal loci. We find that on average gibbon genera have a high average sequence diversity but a lower degree of genetic differentiation as compared to great ape genera. Our multilocus species tree features H. pileatus in a basal position and a grouping of the four Sundaic island species (H. agilis, H. klossii, H. moloch and H. muelleri). We conducted pairwise comparisons based on an isolation-with-migration (IM) model and detect signals of asymmetric gene flow between H. lar and H. moloch, between H. agilis and H. muelleri, and between N. leucogenys and N. siki. CONCLUSIONS: Our multilocus analyses provide inferences of gibbon evolutionary histories complementary to those based on single gene data. The results of IM analyses suggest that the divergence processes of gibbons may be accompanied by gene flow. Future studies using analyses of multi-population model with samples of known provenance for Hylobates and Nomascus species would expand the understanding of histories of gene flow during divergences for these two gibbon genera.

Possible biomarkers of early mortality in peritonitis-induced sepsis rats.

BACKGROUND: Sepsis induced by cecal ligation and puncture (CLP) is accompanied by circulatory failure, multiple organ dysfunction syndrome, metabolic acidosis, and electrolyte imbalance in rats. However, it remains uncertain which parameters can be used to predict the mortality of septic rats. Thus, the aim of this study was to examine which possible biomarkers were associated with mortality in the CLP-induced sepsis model. MATERIALS AND METHODS: After the carotid artery and vein were cannulated, rats were subsequently subjected to CLP or sham operation. The changes of hemodynamics, biochemical variables, blood gas, and electrolytes were monitored during the 18-h observation. RESULTS: The CLP surgery caused circulatory failure, multiple organ dysfunction syndrome, metabolic acidosis, electrolyte imbalance, and death. Compared with survivors, nonsurvivors showed significant difference in (1) blood glucose; (2) lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, creatinine, and blood urea nitrogen in serum; and (3) base excess, HCO3(-), PaCO2, potassium, and calcium in whole blood at 9 h after CLP. No significant difference in blood pressure, heart rate, pressor response to noradrenaline, rectal temperature, total protein, albumin, PaO2, and sodium was observed between nonsurvivors and survivors. However, after multifactor dimensionality reduction analysis, the union of HCO3(-) and blood glucose had the biggest testing balanced accuracy. CONCLUSIONS: These results indicate that HCO3(-) plus blood glucose serves as the best biomarker of early death in rats with CLP-induced sepsis. Thus, these parameters could guide experimental procedures for making the right interventions when utilizing CLP as a sepsis model in rats.

Stimulation of angiotensin II type 2 receptor attenuates organ injury in rats with polymicrobial sepsis.

BACKGROUND: Both inflammation and oxidative stress contribute to the pathogenesis of sepsis and its associated organ damage. Angiotensin-(1-7), acting through the Mas receptor and angiotensin II-type 2 receptors (AT2R), could attenuate organ dysfunction and improve survival in rats with sepsis. However, the role of AT2R in inflammation and oxidative stress in rats with sepsis is unclear. Therefore, this study examined the modulatory effects and molecular mechanism of AT2R stimulation in rats with polymicrobial sepsis. METHODS: Male Wistar rats underwent cecal ligation and puncture (CLP) or sham surgery followed by the administration of saline or CGP42112 (a selective, high-affinity agonist of AT2R, 50 µg/kg intravenously) at 3 hours after sham surgery or CLP. The changes in hemodynamics, biochemical variables, and plasma levels of chemokines and nitric oxide were detected during the 24-hour observation. Organ injury was evaluated by histological examination. RESULTS: We found that CLP evoked delayed hypotension, hypoglycemia, and multiple organ injuries, characterized by elevated plasma biochemical parameters and histopathological changes. These effects were attenuated by treatment with CGP42112. CGP42112 significantly attenuated plasma chemokines and nitric oxide production and reduced liver inducible nitric oxide synthase and nuclear factor kappa-B expression. More importantly, CGP42112 significantly improved the survival of rats with sepsis (50% vs. 20% at 24 h after CLP, p < 0.05). CONCLUSION: The protective effects of CGP42112 may be related to anti-inflammatory responses, suggesting that the stimulation of AT2R is a promising therapeutic candidate for the treatment of sepsis.

Antimicrobial peptide cathelicidin LL-37 preserves intestinal barrier and organ function in rats with heat stroke.

Global warming increases the incidence of heat stroke (HS) and HS causes the reduction of visceral blood flow during hyperthermia, leading to intestinal barrier disruption, microbial translocation, systemic inflammation and multiple organ failure. Cathelicidin LL-37 exhibits antimicrobial activities, helps innate immunity within the gut to maintain intestinal homeostasis, and augments intestinal wound healing and barrier function. Thus, we evaluated the effects and possible mechanisms of cathelicidin LL-37 on HS. Wistar rats were placed in a heating-chamber of 42 ̊C to induce HS. Changes in rectal temperature, hemodynamic parameters, and survival rate were measured during the experimental period. Blood samples and ilea were collected to analyze the effects of LL-37 on systemic inflammation, multiple organ dysfunction, and intestinal injury. Furthermore, LS174T and HT-29 cells were used to assess the underlying mechanisms. Our data showed cathelicidin LL-37 ameliorated the damage of intestinal cells induced by HS. Intestinal injury, systemic inflammation, and nitrosative stress (high nitric oxide level) caused by continuous hyperthermia were attenuated in HS rats treated with cathelicidin LL-37, and hence, improved multiple organ dysfunction, coagulopathy, and survival rate. These beneficial effects of cathelicidin LL-37 were attributed to the protection of intestinal goblet cells (by increasing transepithelial resistance, mucin-2 and Nrf2 expression) and the improvement of intestinal barrier function (less cyclooxygenase-2 expression and FITC-dextran translocation). Interestingly, high cathelicidin expression in the ileal samples of inflammatory bowel disease patients was associated with better clinical outcome. These results suggest that cathelicidin LL-37 could prevent heat stress-induced intestinal damage and heat-related illnesses.

A comparative analysis of Y chromosome and mtDNA phylogenies of the Hylobates gibbons.

BACKGROUND: The evolutionary relationships of closely related species have long been of interest to biologists since these species experienced different evolutionary processes in a relatively short period of time. Comparison of phylogenies inferred from DNA sequences with differing inheritance patterns, such as mitochondrial, autosomal, and X and Y chromosomal loci, can provide more comprehensive inferences of the evolutionary histories of species. Gibbons, especially the genus Hylobates, are particularly intriguing as they consist of multiple closely related species which emerged rapidly and live in close geographic proximity. Our current understanding of relationships among Hylobates species is largely based on data from the maternally-inherited mitochondrial DNAs (mtDNAs). RESULTS: To infer the paternal histories of gibbon taxa, we sequenced multiple Y chromosomal loci from 26 gibbons representing 10 species. As expected, we find levels of sequence variation some five times lower than observed for the mitochondrial genome (mtgenome). Although our Y chromosome phylogenetic tree shows relatively low resolution compared to the mtgenome tree, our results are consistent with the monophyly of gibbon genera suggested by the mtgenome tree. In a comparison of the molecular dating of divergences and on the branching patterns of phylogeny trees between mtgenome and Y chromosome data, we found: 1) the inferred divergence estimates were more recent for the Y chromosome than for the mtgenome, 2) the species H. lar and H. pileatus are monophyletic in the mtgenome phylogeny, respectively, but a H. pileatus individual falls into the H. lar Y chromosome clade. CONCLUSIONS: Based on the ~6.4 kb of Y chromosomal DNA sequence data generated for each of the 26 individuals in this study, we provide molecular inferences on gibbon and particularly on Hylobates evolution complementary to those from mtDNA data. Overall, our results illustrate the utility of comparative studies of loci with different inheritance patterns for investigating potential sex specific processes on the evolutionary histories of closely related taxa, and emphasize the need for further sampling of gibbons of known provenance.

The IgE gene in primates exhibits extraordinary evolutionary diversity.

Membrane-bound IgE (mIgE) on B lymphocytes is essential for IgE production. Earlier studies showed that the ε chain of mIgE (mε) on human B cells has a "long" isoform, with an extra "CεmX" domain of 52 amino acid (aa) residues between the CH4 domain and the membrane-anchor segment, as compared to the conventional "short" isoform. Because CεmX provides an antigenic site for targeting IgE-expressing B cells to down-regulate IgE production in patients with allergy, analysis of CεmX in various animals is of great interest. Hence, we analyzed the ε Ig gene, in particular, its membrane exon regions encoding the membrane anchor peptide segment and CεmX domain, of 26 species of the order Primates and 12 species of seven non-Primate orders using data obtained experimentally or retrieved from GenBank. Our analyses reveal the unexpected finding that the genes of three extant tarsier species do not contain the membrane exons for mIgE. Another striking finding is that early evolved Strepsirhini primates such as lemurs and lorises do not have gene segments for the long isoform, whereas New World monkeys such as marmosets and squirrel monkeys allow the transcription of only the long isoform. In Old World monkeys and apes, including humans, the ε gene allows the transcription of both isoforms. This work thus reveals the dramatic differences in the gene segment encoding the mε C terminal region among the four major primate lineages: the Strepsirhini primates, the tarsiers, New World monkeys, and Old World monkeys and apes/humans.

Improvement in heat stress-induced multiple organ dysfunction and intestinal damage through protection of intestinal goblet cells from prostaglandin E1 analogue misoprostol.

AIMS: Heat stroke is a life-threatening disorder triggered by thermoregulatory failure. Hyperthermia-induced splanchnic hypoperfusion has been reported to induce intestinal barrier dysfunction and systemic immune response that ultimately cause multiple-organ failure and death. Intestinal goblet cells contribute greatly to the formation of mucus barrier, which hinders translocation of gut microorganisms. Studies have reported that misoprostol can not only alleviate ischemic injury but also protect GI mucosal layer. Therefore, we evaluated the effects of misoprostol on intestinal goblet cells after heat stress and on multiple-organ dysfunction in heat stroke rats. MAIN METHODS: Heat stress was established in the heating chamber and followed by misoprostol treatment. Changes in hemodynamics, organ function indices, inflammation, oxidative stress, and survival rate were analyzed. Furthermore, ilea and LS174T cells were used to examine intestinal functions. KEY FINDINGS: Heat stress caused dysfunction of intestinal goblet cells and damage to ilea by increasing oxidative stress and apoptosis. Increased nitrosative stress and inflammation accompanied by hypotension, hypoperfusion, tachycardia, multiple-organ dysfunction, and death were observed in the heat stroke rat model. Treatment of LS174T cells with misoprostol not only decreased oxidative stress and apoptosis but also reduced cytotoxicity caused by heat stress. Moreover, misoprostol prevented disruption of the enteric barrier, multiple-organ injury, and death in rats with heat stroke. SIGNIFICANCE: This study indicates that misoprostol could alleviate intestinal damage and organ injury caused by heat stress and be a potential therapy for heat-related illnesses.

Angiotensin-(1-7) treatment blocks lipopolysaccharide-induced organ damage, platelet dysfunction, and IL-6 and nitric oxide production in rats.

Sepsis can lead to shock, multiple organ failure, and even death. Platelets play an active role in the pathogenesis of sepsis-induced multiple organ failure. Angiotensin (Ang)-(1-7), a biologically active peptide, counteracts various effects of Ang II and attenuates inflammatory responses, reactive oxygen species production, and apoptosis. We evaluated the effects of Ang-(1-7) on organ injury and platelet dysfunction in rats with endotoxaemia. We treated male Wistar rats with saline or lipopolysaccharide (LPS, 10 mg, intravenously) then Ang-(1-7) (1 mg/kg, intravenous infusion for 3 h beginning 30 min after LPS administration). We analysed several haemodynamic, biochemical, and inflammatory parameters, as well as platelet counts and aggregation. Ang-(1-7) improved hypotension and organ dysfunction, and attenuated plasma interleukin-6, chemokines and nitric oxide production in rats after LPS administration. The LPS-induced reduction in platelet aggregation, but not the decreased platelet count, was restored after Ang-(1-7) treatment. The protein expression of iNOS and IκB, but not phosphorylated ERK1/2 and p38, was diminished in Ang-(1-7)-treated LPS rats. The histological changes in liver and lung were significantly attenuated in Ang-(1-7)-treated LPS rats. Our results suggest that Ang-(1-7) ameliorates endotoxaemic-induced organ injury and platelet dysfunction, likely through the inhibition of the inflammatory response and nitric oxide production.

Platelet inhibition by P2Y12 antagonists is potentiated by adenosine signalling activators.

BACKGROUND AND PURPOSE: P2Y12 receptor antagonists reduce platelet aggregation and the incidence of arterial thrombosis. Adenosine signalling in platelets directly affects cyclic nucleotide tone, which we have shown to have a synergistic relationship with P2Y12 inhibition. Several studies suggest that ticagrelor inhibits erythrocyte uptake of adenosine and that this could also contribute to its antiplatelet effects. We therefore examined the effects on platelet activation of adenosine signalling activators in combination with the P2Y12 receptor antagonists ticagrelor and prasugrel. EXPERIMENTAL APPROACH: Human washed platelets, platelet-rich plasma and whole blood were used to test the interactions between ticagrelor or prasugrel and adenosine or 5'-N-ethylcarboxamidoadenosine (NECA). Platelet reactivity to thrombin, protease-activated receptor 1 (PAR-1) activation or collagen was assessed by a combination of 96-well plate aggregometry, light transmission aggregometry, whole blood aggregometry, ATP release assay and levels of cAMP. KEY RESULTS: The inhibitory effects of ticagrelor and prasugrel on platelet aggregation and ATP release were enhanced in the presence of adenosine or NECA. Isobolographic analysis indicated a powerful synergy between P2Y12 receptor inhibition and adenosine signalling activators. Increased levels of cAMP in platelets were also observed. In all cases, ticagrelor showed similar synergistic effects on platelet inhibition as prasugrel in the presence of adenosine or NECA. CONCLUSION AND IMPLICATIONS: These results indicate that P2Y12 antagonists have a synergistic relationship with adenosine signalling and that their efficacy may depend partly upon the presence of endogenous adenosine. This effect was common for prasugrel and ticagrelor despite reports of differences in their effects upon adenosine reuptake.

Low-dose hydralazine improves endotoxin-induced coagulopathy and multiple organ dysfunction via its anti-inflammatory and anti-oxidative/nitrosative properties.

Coagulopathy is the major cause of organ injury as well as a strong predictor of mortality in septic patients. Systemic inflammatory response and redox imbalance are regarded as the major causes of sepsis-induced coagulopathy. There is growing evidence that a vasodilator hydralazine has beneficial effects on heart failure, hypertension, and ischemia/reperfusion injury via its antioxidant and anti-inflammatory properties. However, the effects of hydralazine on sepsis have not been examined. Therefore, we evaluated the effects of low-dose hydralazine on coagulopathy and multiple organ dysfunction in septic rats induced by endotoxin. Sepsis-induced coagulopathy was established by intravenous injection of rats with lipopolysaccharide (LPS). The changes of blood pressure, heart rate, blood glucose, hemostatic variables, prothrombin time, organ function indices, interleukin-6 (IL-6) concentration, and nitric oxide (NO) level were assessed during the experimental period. In addition, the aortas, lungs, livers, and kidneys were dissected to analyze superoxide levels and protein expressions. LPS induced (i) coagulopathy, multiple organ dysfunction, and circulatory failure successfully, and (ii) excessive superoxide, NO, and IL-6 production, accompanied by the overexpression of iNOS and Wnt5a in animals. Treatment of LPS-induced septic rats with low-dose hydralazine not only improved coagulopathy but also ameliorated multiple organ dysfunction. These could be due to attenuation of the overproduction of superoxide, NO, and IL-6, which were attributed to reduction of the overexpression of iNOS and Wnt5a. Thus, these findings indicate that low-dose hydralazine could be a potential therapy for sepsis-induced coagulopathy and multiple organ dysfunction via its anti-inflammatory and anti-oxidative/nitrosative properties.

Combination of cyclic nucleotide modulators with P2Y12 receptor antagonists as anti-platelet therapy.

BACKGROUND: Endothelium-derived prostacyclin and nitric oxide elevate platelet cyclic nucleotide levels and maintain quiescence. We previously demonstrated that a synergistic relationship exists between cyclic nucleotides and P2Y12 receptor inhibition. A number of clinically approved drug classes can modulate cyclic nucleotide tone in platelets including activators of NO-sensitive guanylyl cyclase (GC) and phosphodiesterase (PDE) inhibitors. However, the doses required to inhibit platelets produce numerous side effects including headache. OBJECTIVE: We investigated using GC-activators in combination with P2Y12 receptor antagonists as a way to selectively amplify the anti-thrombotic effect of both drugs. METHODS: In vitro light transmission aggregation and platelet adhesion under flow were performed on washed platelets and platelet rich plasma. Aggregation in whole blood and a ferric chloride-induced arterial thrombosis model were also performed. RESULTS: The GC-activator BAY-70 potentiated the action of the P2Y12 receptor inhibitor prasugrel active metabolite in aggregation and adhesion studies and was associated with raised intra-platelet cyclic nucleotide levels. Furthermore, mice administered sub-maximal doses of the GC activator cinaciguat together with the PDE inhibitor dipyridamole and prasugrel, showed significant inhibition of ex vivo platelet aggregation and significantly reduced in vivo arterial thrombosis in response to injury without alteration in basal carotid artery blood flow. CONCLUSIONS: Using in vitro, ex vivo, and in vivo functional studies, we show that low dose GC activators synergize with P2Y12 inhibition to produce powerful anti-platelet effects without altering blood flow. Therefore, modulation of intra-platelet cyclic nucleotide levels alongside P2Y12 inhibition can provide a strong, focused anti-thrombotic regimen while minimizing vasodilator side effects.

Therapeutic effects of hypertonic saline on peritonitis-induced septic shock with multiple organ dysfunction syndrome in rats.

OBJECTIVE: Significant mortality in patients with sepsis results from the development of multiple organ dysfunction syndrome. Small-volume resuscitation with 7.5% NaCl hypertonic saline has been proposed to restore physiologic hemodynamics in hemorrhagic shock. Therefore, we hypothesized that hypertonic saline resuscitation could alleviate the development of multiple organ dysfunction syndrome in sepsis induced by cecal ligation and puncture. DESIGN: Randomized, prospective animal experiment. SETTING: Academic research laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: The animals were randomly allocated to one of four groups: 1) sham operation (0.9% NaCl, 4 mL/kg intravenously, at 3 hrs after laparotomy); 2) sham operation plus hypertonic saline (7.5% NaCl, 4 mL/kg intravenously, at 3 hrs after laparotomy); 3) cecal ligation and puncture (0.9% NaCl, 4 mL/kg intravenously, at 3 hrs after cecal ligation and puncture); and 4) cecal ligation and puncture plus hypertonic saline (7.5% NaCl, 4 mL/kg intravenously, at 3 hrs after cecal ligation and puncture). MEASUREMENTS AND MAIN RESULTS: Cecal ligation and puncture for 18 hrs was associated with circulatory failure (i.e., hypotension and vascular hyporeactivity to norepinephrine), multiple organ dysfunction syndrome (examined by biochemical variables and histologic studies), and 18-hr mortality. Hypertonic saline not only ameliorated the deterioration of hemodynamic changes but also attenuated neutrophil infiltration in the lung and the liver of septic animals. Hypertonic saline increased the survival rate at 9 and 18 hrs compared with the cecal ligation and puncture group. Moreover, hypertonic saline reduced plasma nitric oxide and interleukin-1beta and organ O2-* levels in rats that underwent cecal ligation and puncture. CONCLUSIONS: Hypertonic saline prevented circulatory failure, alleviated multiple organ dysfunction syndrome, and decreased the mortality rate in animals receiving cecal ligation and puncture. These beneficial effects of hypertonic saline may be attributed to reducing the plasma concentration of nitric oxide and interleukin-1beta as well as the organ O2-* level and decreasing lung neutrophil infiltration and liver necrosis. Our study suggests that hypertonic saline could be a potential and inexpensive therapeutic agent in the early sepsis of animals or patients.

Macrophage expression of E3 ubiquitin ligase Grail protects mice from lipopolysaccharide-induced hyperinflammation and organ injury.

Multiple organ dysfunction caused by hyperinflammation remains the major cause of mortality during sepsis. Excessive M1-macrophage activation leads to systemic inflammatory responses. Gene related to anergy in lymphocytes (Grail) is regarded as an important regulator of T cells that functions by diminishing cytokine production. However, its role in regulating macrophage activation and organ injury during sepsis remains unclear. Our aim was to examine the effects of Grail on macrophage reactivity and organ injury in endotoxemic animals. Wild-type and Grail knockout mice were injected with vehicle or Escherichia coli lipopolysaccharide and observed for 24 h. Changes in blood pressure, heart rate, blood glucose, and biochemical variables were then examined. Moreover, levels of neutrophil infiltration, MMP-9, and caspase 3 were analyzed in the lungs of animals. The expression of pro-inflammatory cytokines in J774A, RAW264.7, and primary peritoneal macrophages stimulated with LPS were also assessed in the presence or absence of Grail. Results indicated that loss of Grail expression enhances the induction of pro-inflammatory cytokines in J774A, RAW264.7, and primary peritoneal macrophages treated with LPS. Furthermore, LPS-induced macrophage hyperactivation was alleviated by ectopic Grail overexpression. In vivo studies showed that Grail deficiency exacerbates organ damage in endotoxemic animals. Levels of neutrophil infiltration, MMP-9, and caspase 3 were significantly increased in the lungs of Grail-deficient endotoxemic mice. Thus, these results suggest that Grail contributes to the attenuation of hyperinflammation caused by activated macrophages and prevents organ damage in endotoxemic mice. We suggest that Grail signaling could be a therapeutic target for endotoxemia.