Yingjin Wang

S100A8 and S100A9 Mediate Endotoxin-Induced Cardiomyocyte Dysfunction via the Receptor for Advanced Glycation End Products

Cardiovascular dysfunction as a result of sepsis is the leading cause of death in the critically ill. Cardiomyocytes respond to infectious pathogens with a Toll-like receptor–initiated proinflammatory response in conjunction with a decrease in contractility, although the downstream events linking Toll-like receptor activation and reduced cardiac contractility remain to be elucidated. Using microarray analysis of cardiac tissue exposed to systemic lipopolysaccharide (LPS), we discovered that 2 small calcium-regulating proteins (S100A8 and S100A9) are highly upregulated. HL-1 cardiomyocytes, isolated primary cardiomyocytes, and live mice were exposed to LPS, whereas beating HL-1 cells had S100A8 and S100A9 overexpressed and their calcium flux quantified. Using in vivo microbubble technology, we delivered S100A8 and S100A9 to normal mouse hearts; using the same technology, we inhibited S100A9 production in mouse hearts and subsequently exposed them to LPS. Coimmunoprecipitation of S100A8 and S100A9 identified interaction with RAGE (the receptor for advanced glycation end products), the cardiac function and postreceptor signaling of which were investigated. HL-1 cardiomyocytes, isolated primary cardiomyocytes, and whole hearts exposed to LPS have large increases in S100A8 and S100A9. Cardiac overexpression of S100A8 and S100A9 led to a RAGE-dependent decrease in calcium flux and, in the intact mouse, to a decreased cardiac ejection fraction, whereas knockdown of S100A9 attenuated LPS-induced cardiac dysfunction. Cardiomyocytes exposed to LPS express S100A8 and S100A9, leading to a RAGE-mediated decrease in cardiomyocyte contractility. This finding provides a novel mechanistic link between circulating pathogen-associated molecular products and subsequent cardiac dysfunction.

Low-dose vasopressin infusion results in increased mortality and cardiac dysfunction following ischemia-reperfusion injury in mice

IntroductionArginine vasopressin is a vasoactive drug commonly used in distributive shock states including mixed shock with a cardiac component. However, the direct effect of arginine vasopressin on the function of the ischemia/reperfusion injured heart has not been clearly elucidated.MethodsWe measured left ventricular ejection fraction using trans-thoracic echocardiography in C57B6 mice, both in normal controls and following ischemia/reperfusion injury induced by a one hour ligation of the left anterior descending coronary artery. Mice were treated with one of normal saline, dobutamine (8.33 μg/kg/min), or arginine vasopressin (0.00057 Units/kg/min, equivalent to 0.04 Units/min in a 70 kg human) delivered by an intraperitoneal micro-osmotic pump. Arterial blood pressure was measured using a micromanometer catheter. In addition, mortality was recorded and cardiac tissues processed for RNA and protein.ResultsBaseline left ventricular ejection fraction was 65.6% (60 to 72). In normal control mice, there was no difference in left ventricular ejection fraction according to infusion group. Following ischemia/reperfusion injury, AVP treatment significantly reduced day 1 left ventricular ejection fraction 46.2% (34.4 to 52.0), both in comparison with baseline and day 1 saline treated controls 56.9% (42.4 to 60.2). There were no significant differences in preload (left ventricular end diastolic volume), afterload (blood pressure) or heart rate to account for the effect of AVP on left ventricular ejection fraction. The seven-day mortality rate was highest in the arginine vasopressin group. Following ischemia/reperfusion injury, we found no change in cardiac V1 Receptor expression but a 40% decrease in Oxytocin Receptor expression.ConclusionsArginine vasopressin infusion significantly depressed the myocardial function in an ischemia/reperfusion model and increased mortality in comparison with both saline and dobutamine treated animals. The use of vasopressin may be contraindicated in non-vasodilatory shock states associated with significant cardiac injury.

Lipopolysaccharide Is Cleared from the Circulation by Hepatocytes via the Low Density Lipoprotein Receptor

Sepsis is the leading cause of death in critically ill patients. While decreased Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) function improves clinical outcomes in murine and human sepsis, the mechanisms involved have not been fully elucidated. We tested the hypothesis that lipopolysaccharide (LPS), the major Gram-negative bacteria endotoxin, is cleared from the circulation by hepatocyte Low Density Lipoprotein Receptors (LDLR)—receptors downregulated by PCSK9. We directly visualized LPS uptake and found that LPS is rapidly taken up by hepatocytes into the cell periphery. Over the course of 4 hours LPS is transported towards the cell center. We next found that clearance of injected LPS from the blood was reduced substantially in Ldlr knockout (Ldlr-/-) mice compared to wild type controls and, simultaneously, hepatic uptake of LPS was also reduced in Ldlr-/- mice. Specifically examining the role of hepatocytes, we further found that primary hepatocytes isolated from Ldlr-/- mice had greatly decreased LPS uptake. In the HepG2 immortalized human hepatocyte cell line, LDLR silencing similarly resulted in decreased LPS uptake. PCSK9 treatment reduces LDLR density on hepatocytes and, therefore, was another independent strategy to test our hypothesis. Incubation with PCSK9 reduced LPS uptake by hepatocytes. Taken together, these findings demonstrate that hepatocytes clear LPS from the circulation via the LDLR and PCSK9 regulates LPS clearance from the circulation during sepsis by downregulation of hepatic LDLR.

Vasopressin decreases sepsis-induced pulmonary inflammation through the V2R☆

UNLABELLED The early use of vasopressors in sepsis has been associated with a decrease in immune activation independent of hemodynamic effects, although the mechanism behind this remains unclear. We hypothesize that low dose vasopressin will reduce the pulmonary inflammation associated with sepsis. Our aims were to (1) determine whether vasopressin reduces lipopolysaccharide (LPS)-induced pulmonary inflammation and (2) determine which vasopressin receptor is responsible for pulmonary immune modulation. Mice were treated with intraperitoneal LPS to induce both systemic and pulmonary inflammation. Vasopressin or saline was infused via peritoneal pump and interleukin 6 (IL-6) in lung and serum was measured at 6h. NF-kappaB activation as was determined in the lung through immunoblotting total and phospho-IkappaB. Hemodynamic data was also obtained at the 6h mark. In a separate series of experiments mice received both LPS and vasopressin infusion following pretreatment with vasopressin receptor antagonists to V1R, V2R and OTR. Low dose LPS dramatically raises both serum IL-6 and pulmonary levels of IL-6 and phospho-IkappaB despite no significant changes in mean arterial pressure at 6h. Compared to saline, vasopressin infusion significantly decreases both the pulmonary IL-6 levels and phospho-IkappaB in LPS treated mice without raising arterial pressure. Pretreatment with V2R antagonist results in complete attenuation of vasopressins immunosuppressive effects, with restoration of pulmonary IL-6 and phospho-IkappaB levels to those seen with LPS alone. CONCLUSIONS Vasopressin exerts a local anti-inflammatory effect on the lung through the V2R in a model of sepsis.

Albumin resuscitation improves ventricular contractility and myocardial tissue oxygenation in rat endotoxemia.

Objective:Fluid resuscitation to improve delivery of oxygen to vital organs is a principal clinical intervention for septic patients. We previously reported that albumin resuscitation in rat endotoxemia improved contractility in isolated cardiomyocytes, but whether this effect occurs in vivo is unknown. We hypothesized that albumin resuscitation would improve decreased ventricular contractility and myocardial tissue oxygenation in vivo. Design:Randomized, controlled, prospective animal study. Setting:University animal laboratory. Subjects:Male Sprague-Dawley rats (250–350 g). Interventions:Rats were randomized into three groups: control with no lipopolysaccharide (n = 8), lipopolysaccharide (10 mg/kg) without albumin resuscitation (n = 8), and lipopolysaccharide with albumin resuscitation (n = 6). Five hours after lipopolysaccharide injection, animals were resuscitated with 10 mL/kg 5% rat albumin in 0.9% saline. Six hours after 10 mL/kg lipopolysaccharide, a pressure-volume conductance catheter (MIKRO-Tip 2.0-Fr, Millar Instruments, Houston, TX) was inserted into the left ventricle to quantify maximum elastance as an index of contractility. Myocardial tissue Po2 was measured using a fiberoptic oxygen probe. Measurements and Main Results:Maximum elastance decreased after lipopolysaccharide relative to control (47%, from 5.9 ± 0.8 to 3.1 ± 0.4 mm Hg/&mgr;L, p < .05). Albumin resuscitation prevented the lipopolysaccharide-induced decrease in maximum elastance (7.0 ± 1.2 mm Hg/&mgr;L, p < .05 vs. lipopolysaccharide). Myocardial tissue Po2 was reduced in endotoxemia compared with control (53%, from 10.1 ± 0.9 to 4.7 ± 0.6 mm Hg, p < .05), and albumin resuscitation improved the lipopolysaccharide-induced tissue hypoxia toward the control value (9.0 ± 1.4 mm Hg, p < .05). Conclusions:Albumin resuscitation improved decreased ventricular contractility and myocardial oxygenation in endotoxemic rats. This result suggests that albumin resuscitation may improve ventricular dysfunction by improving myocardial hypoxia.

Notch-Dependent Regulation of the Ischemic Vasodilatory Response—Brief Report

Objective—We have recently described that Notch activates nitric oxide (NO) signaling in the embryonic endocardium. Both Notch signaling and NO signaling have been shown to be important during adult arteriogenesis. Notch has been shown to be required for remodeling of the collateral vessels, whereas NO is required for the initial vasodilatory response to ischemia. Whether Notch also has an impact on the vasodilatory phase of arteriogenesis after ischemia is not known. We tested the hypothesis that endothelial cell-Notch function is required for NO induction and vasodilation, in response to ischemia in the adult vasculature. Methods and Results—We observed a significant decrease in NO levels in the dorsal aorta using a mouse model where Notch was inhibited in endothelial cell in a Tet-inducible fashion. In a femoral artery ligation model, inhibition of endothelial cell-Notch reduced reperfusion and NO generation, as quantified by laser Doppler perfusion imaging and by phosphoendothelial NO synthase, nitrotyrosine, and phosphovasodilator-stimulated phosphoprotein staining, respectively. Conclusion—Endothelial Notch activation is required for NO production and reactive vasodilation in a femoral artery ligation model.

Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft involves anastomosis of donor inferior vena cava (IVC) to recipient IVC, and donor ascending aorta (Ao) to recipient abdominal Ao. The right upper and middle lung lobes are preserved and function as conduits for blood flow from right heart to left heart. There are several advantages to using this technique, and it lends itself to a broad range of applications. Because the graft is transplanted in a configuration that allows for dyamic volume-loading, cardiac function may be directly assessed in vivo. The use of pressure-volume conductance catheters permits characterization of load-dependent and load-independent hemodynamic parameters. The graft may be converted to a loaded configuration by applying a clamp to the recipients infra-hepatic IVC. We describe modified surgical techniques for both donor and recipient operations, and an ideal myocardial protection strategy. Depending on the experimental aim, this model may be adapted for use in both acute and chronic studies of graft function, immunologic status, and variable ventricular loading conditions. The conducting airways to the transplanted lung are preserved, and allow for acute lung re-ventilation. This facilitates analysis of the effects of the mixed venous and arterial blood providing coronary perfusion to the graft. A limitation of this model is its technical complexity. There is a significant learning curve for new operators, who should ideally be mentored in the technique. A surgical training background is advantageous for those wishing to apply this model. Despite its complexity, we aim to present the model in a clear and easily applicable format. Because of the physiologic similarity of this model to orthotopic transplantation, and its broad range of study applications, the effort invested in learning the technique is likely to be worthwhile.