Lihua Ao

Preconditioning of Isolated Rat Heart Is Mediated by Protein Kinase C

Catecholamines have been implicated in the phenomenon of ischemic preconditioning. We have previously demonstrated that ischemic preconditioning against postischemic mechanical dysfunction in the isolated rat heart is mediated by the alpha 1-adrenergic receptor. The purpose of this study was to delineate the signal transduction of preconditioning distal to the alpha 1-adrenergic receptor. Our results suggest that (1) transient ischemia and alpha 1-adrenergic receptor-induced preconditioning is inhibited by protein kinase C (PKC) antagonists, (2) functional protection against global ischemia/reperfusion injury can be induced by infusion of diacylglycerol, the second messenger of the alpha 1-adrenergic pathway, and (3) transient ischemia and alpha 1-adrenergic preconditioning are both characterized by similar translocation of PKC-delta to the sarcolemma of myocardium. These findings suggest that PKC is an effector of preconditioning in the isolated rat heart.

Pro-osteogenic phenotype of human aortic valve interstitial cells is associated with higher levels of Toll-like receptors 2 and 4 and enhanced expression of bone morphogenetic protein 2.

OBJECTIVES Our aim was to determine whether aortic valve interstitial cells (AVICs) and pulmonary valve interstitial cells (PVICs) differ in expression of Toll-like receptor (TLR)2 and TLR4, response to TLR agonists, and osteogenic phenotypic changes. BACKGROUND Calcific stenosis occurs frequently in aortic valves but rarely in pulmonary valves. Studies have implicated AVICs in the inflammation associated with calcification and progression to stenosis. We previously reported that human AVICs express functional TLR2 and TLR4 and that stimulation of these receptors induces pro-osteogenic factor expression. METHODS Human aortic and pulmonary valve leaflets from the same heart were collected and interstitial cells isolated. RESULTS Aortic valves express more TLR2 and TLR4, in both tissue and isolated interstitial cells, than pulmonary valves. After stimulation with TLR2 and TLR4 agonists, AVICs express higher levels of pro-inflammatory and pro-osteogenic mediators (bone morphogenetic protein [BMP]-2, runt-related transcription factor 2) and greater osteogenic phenotypic changes (alkaline phosphatase [ALP] activity, calcified nodule formation) than PVICs. Silencing TLR2 and TLR4 in AVICs reduced BMP-2 expression and ALP activity to PVIC levels. ALP activity in AVICs induced by TLR2 and TLR4 agonists was abolished by BMP antagonism with Noggin and mimicked by stimulation with recombinant BMP-2. AVICs isolated from stenotic valves had greater expression of TLR2 and TLR4 and a greater BMP-2 response than AVICs from normal valves. CONCLUSIONS Greater expression of TLR2 and TLR4 and greater pro-inflammatory and pro-osteogenic responses to TLR2 and TLR4 agonists in AVICs than PVICs are associated with osteogenic phenotypic changes. These innate immune receptors may play a critical role in aortic valve calcification and stenosis.

Norepinephrine induces cardiac heat shock protein 70 and delayed cardioprotection in the rat through α1 adrenoceptors

OBJECTIVE Previous studies have demonstrated that norepinephrine (NE) confers immediate cardioprotection against ischemia and reperfusion injuries. The present study tests the hypothesis that in vivo treatment with NE induces delayed cardioprotection against postischemic dysfunction in the rat heart which is associated with expression of c-fos and heat shock protein (HSP) 70 in the myocardium. METHODS Rats were treated with NE (3.1 mumol/kg, ip) and hearts isolated and perfused with a modified Langendorff technique 2 or 24 h after injection. Left ventricular developed pressure (LVDP) and left ventricular end-diastolic pressure (LVEDP) were recorded during 25 min normothermic global ischemia and 40 min reperfusion. Total RNA was extracted from left ventricular tissue at various time points and Northern hybridization was applied to detect c-fos and HSP70 mRNAs. Expression and distribution of c-fos and HSP72 proteins in the myocardium were examined by immunohistochemical staining. RESULTS In vivo NE treatment improved postischemic recovery of both LVDP and LVEDP in the hearts isolated at 24 h after treatment but not in those isolated at 2 h. LVDP was 68.9 +/- 4.8 mmHg in NE 24 h group at the end of reperfusion in comparison to 43.6 +/- 2.9 mmHg in saline control group (P < 0.01). Pretreatment with prazosin abolished NE-induced cardioprotection while propranolol pretreatment had no effect. Northern analysis demonstrated rapid and transient increases in c-fos and HSP70 mRNAs in the myocardium after NE treatment. Accumulation of c-fos protein was observed at 3 h and increased amount of HSP72 protein was demonstrated at 24 h in the myocardium. Pretreatment of rats with prazosin eliminated NE-induced increase in cardiac HSP70 mRNA. CONCLUSIONS The results suggest that in vivo treatment with NE upregulates the expression of c-fos and HSP70 in the myocardium and induces delayed protection against postischemic myocardial dysfunction in the isolated rat heart. Induction of both the expression of cardiac HSP70 and the delayed cardioprotection by NE appears to be mediated by alpha 1 adrenoceptors.

Bone morphogenic protein 2 induces Runx2 and osteopontin expression in human aortic valve interstitial cells: Role of Smad1 and extracellular signal-regulated kinase 1/2

OBJECTIVE Bone morphogenic protein 2 is found in calcified areas of stenotic aortic valves, and prolonged stimulation of aortic valve interstitial cells with bone morphogenic protein 2 results in increased expression of alkaline phosphatase, indicating a mechanistic role for bone morphogenic protein 2 in aortic valve calcification. The purposes of this study were to assess the effect of bone morphogenic protein 2 on the expression of the osteogenic factors Runx2 and osteopontin in human aortic valve interstitial cells and to determine the signaling mechanisms that mediate the expression of these early osteogenic factors. METHODS Interstitial cells were isolated from normal and stenotic human aortic valve leaflets, and cellular bone morphogenic protein 2 levels were analyzed by means of immunoblotting. Cultured interstitial cells from normal aortic valves were stimulated with bone morphogenic protein 2 to determine its effect on cellular Runx2 and osteopontin levels. RESULTS Interstitial cells from stenotic aortic valves express greater levels of bone morphogenic protein 2 than cells from normal valves. Stimulation of human aortic valve interstitial cells with bone morphogenic protein 2 induced marked increases in Runx2 and osteopontin levels at 48 hours. The changes in Runx2 and osteopontin levels were preceded by phosphorylation of Smad1 and extracellular signal-regulated kinase 1/2 but not p38 mitogen-activated protein kinase. Silencing Smad1 reduced Runx2 and osteopontin levels, whereas inhibition of extracellular signal-regulated kinase 1/2 reduced osteopontin expression without an influence on Runx2 expression. CONCLUSIONS Interstitial cells of stenotic human aortic valves are characterized by increased bone morphogenic protein 2 levels. A short period of exposure of human aortic valve interstitial cells to bone morphogenic protein 2 induces the expression of Runx2 and osteopontin. The extracellular signal-regulated kinase 1/2 pathway modulates bone morphogenic protein 2-induced osteopontin expression, and the Smad1 pathway plays a role in regulating the expression of both Runx2 and osteopontin induced by bone morphogenic protein 2.

Critical role of extracellular heat shock cognate protein 70 in the myocardial inflammatory response and cardiac dysfunction after global ischemia-reperfusion.

Previous studies showed that Toll-like receptor 4 (TLR4) modulates the myocardial inflammatory response to ischemia-reperfusion injury, and we recently found that cytokines link TLR4 to postischemic cardiac dysfunction. Although TLR4 can be activated in cultured cells by endogenous agents including heat shock protein 70, how it is activated during myocardial ischemia-reperfusion is unknown. In the present study, we examined 1) whether heat shock cognate protein 70 (HSC70), which is constitutively expressed in the myocardium, is released during ischemia-reperfusion; 2) whether extracellular HSC70 induces the myocardial inflammatory response and modulates cardiac function; and 3) whether HSC70 exerts these effects via TLR4. We subjected isolated mouse hearts to global ischemia-reperfusion via the Langendorff technique. Immunoblotting and immunostaining detected the release of HSC70 from the myocardium during reperfusion. Treatment with an antibody specific to HSC70 suppressed myocardial cytokine expression and improved cardiac functional recovery after ischemia-reperfusion. Recombinant HSC70 induced NF-kappaB activation and cytokine expression and depressed myocardial contractility in a TLR4-dependent manner. These effects required the substrate-binding domain of HSC70. Fluorescence resonance energy transfer analysis of isolated macrophages demonstrated that extracellular HSC70 interacts with TLR4. Therefore, this study demonstrates for the first time that 1) the myocardium releases HSC70 during ischemia-reperfusion, 2) extracellular HSC70 contributes to the postischemic myocardial inflammatory response and to cardiac dysfunction, 3) HSC70 exerts these effects through a TLR4-dependent mechanism, and 4) the substrate-binding domain of HSC70 is required to induce these effects. Thus extracellular HSC70 plays a critical role in regulating the myocardial innate immune response and cardiac function after ischemia-reperfusion.

Oxidized Low Density Lipoprotein Induces Bone Morphogenetic Protein-2 in Coronary Artery Endothelial Cells via Toll-like Receptors 2 and 4

Vascular calcification is a common complication in atherosclerosis. Bone morphogenetic protein-2 (BMP-2) plays an important role in atherosclerotic vascular calcification. The aim of this study was to determine the effect of oxidized low density lipoprotein (oxLDL) on BMP-2 protein expression in human coronary artery endothelial cells (CAECs), the roles of Toll-like receptor (TLR) 2 and TLR4 in oxLDL-induced BMP-2 expression, and the signaling pathways involved. Human CAECs were stimulated with oxLDL. The roles of TLR2 and TLR4 in oxLDL-induced BMP-2 expression were determined by pretreatment with neutralizing antibody, siRNA, and overexpression. Stimulation with oxLDL increased cellular BMP-2 protein levels in a dose-dependent manner (40–160 μg/ml). Pretreatment with neutralizing antibodies against TLR2 and TLR4 or silencing of these two receptors reduced oxLDL-induced BMP-2 expression. Overexpression of TLR2 and TLR4 enhanced the cellular BMP-2 response to oxLDL. Furthermore, oxLDL was co-localized with TLR2 and TLR4. BMP-2 expression was associated with activation of nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase (ERK)1/2. Inhibition of NF-κB and ERK1/2 reduced BMP-2 expression whereas inhibition of p38 MAPK had no effect. In conclusion, oxLDL induces BMP-2 expression through TLR2 and TLR4 in human CAECs. The NF-κB and ERK1/2 pathways are involved in the signaling mechanism. These findings underscore an important role for TLR2 and TLR4 in mediating the BMP-2 response to oxLDL in human CAECs and indicate that these two immunoreceptors contribute to the mechanisms underlying atherosclerotic vascular calcification.

Nitric oxide synthase is not involved in cardiac contractile dysfunction in a rat model of endotoxemia without shock.

Endotoxin and proinflammatory cytokines induce nitric oxide synthase (NOS), and nitric oxide (NO) plays an important role in promoting endotoxin shock. However, the role of NOS in endotoxemic cardiac contractile dysfunction is not defined. To determine whether endotoxemic cardiac contractile dysfunction involves NOS, the present study used a rat model of endotoxemia without shock and examined the effects of glucocorticoids (dexamethasone, a potent inhibitor of inducible NOS, iNOS, expression), isoform nonselective NOS inhibitor (NG-monomethyl-L-arginine, L-NMA) and iNOS selective inhibitor (S-methylisothiourea sulfate, SMT) on cardiac contractile dysfunction. A sublethal dose of endotoxin (from Salmonella typhimurium, .5 mg/kg, i.p.) was given to adult rats, and left ventricular developed pressure (LVDP) examined by Langendorff technique was attenuated in hearts isolated at 4 or 6 h (66.7 ± 3.4 and 60.3 ± 5.5 mmHg, respectively, p < .05 vs. 102 ± 2.4 mmHg in saline control) after endotoxin treatment. Pretreatment of rats with dexamethasone (4.0 mg/kg, i.v., −30 min) partially abolished endotoxin-induced contractile dysfunction at 6 h (LVDP 87.6 ± 6.8 mmHg, p < .05 vs. endotoxin alone at 6 h). However, pretreatment with L-NMA (30 mg/kg, i.v., −5 min) or SMT (5.0 mg/kg, i.v., −1 min) failed to prevent the contractile dysfunction. Moreover, infusion of L-NMA or SMT/n vitro could not restore contractile function in hearts isolated at 6 h after endotoxin treatment. In contrast, inhibition of NOS with L-NMA or SMT in vitro further attenuated coronary flow in endotoxin-treated hearts. Thus, endotoxemic cardiac contractile dysfunction in this non-shock rat model may not involve NOS, and inhibition of NOS may deteriorate coronary perfusion in endotoxemic heart.

Myocardial TLR4 is a determinant of neutrophil infiltration after global myocardial ischemia: mediating KC and MCP-1 expression induced by extracellular HSC70

Cardiac surgery with global myocardial ischemia-reperfusion (I/R) induces a myocardial inflammatory response that impairs cardiac recovery. Chemokines contribute to the overall myocardial inflammatory response through inducing leukocyte infiltration. Although Toll-like receptor 4 (TLR4) has an important role in postischemic myocardial injury, the relative roles of myocardial tissue and leukocyte TLR4 in leukocyte infiltration, as well as the role of TLR4 in myocardial chemokine expression, are unclear. Our recent study, in an isolated mouse heart model of global I/R, found that the 70-kDa heat shock cognate protein (HSC70) is released from cardiac cells and mediates the expression of cardiodepressant cytokines via a TLR4-dependent mechanism. In the present study, we tested the hypotheses that myocardial tissue TLR4 has a major role in mediating neutrophil infiltration and that myocardial TLR4 and extracellular HSC70 contribute to the mechanisms underlying cardiac chemokine response to global I/R. We subjected hearts isolated from TLR4-defective and TLR4-competent mice to global I/R and examined myocardial neutrophil infiltration and expression of keratinocyte-derived chemokine (KC) and monocyte chemoattractant protein-1 (MCP-1). TLR4-defective hearts exhibited reduced neutrophil infiltration regardless of the phenotypes of neutrophils perfused during reperfusion and expressed lower levels of KC and MCP-1. HSC70-specific antibody reduced myocardial expression of KC and MCP-1 after I/R. Furthermore, perfusion of HSC70 increased KC and MCP-1 expression in TLR4-competent hearts but not in TLR4-defective hearts, and HSC70 also induced the chemokine response in macrophages in a TLR4-dependent fashion. A recombinant HSC70 fragment lacking the substrate-binding domain was insufficient to induce chemokine expression in hearts and cells. This study demonstrates that myocardial tissue TLR4, rather than neutrophil TLR4, is the determinant of myocardial neutrophil infiltration after global I/R. TLR4 mediates myocardial chemokine expression, and the mechanisms involve extracellular HSC70. These results imply the HSC70-TLR4 interaction as a novel mechanism underlying the myocardial chemokine response to global I/R.

Cytokines Link Toll-Like Receptor 4 Signaling to Cardiac Dysfunction After Global Myocardial Ischemia

BACKGROUND Although Toll-like receptor 4 (TLR4) has been implicated in the myocardial injury caused by regional ischemia/reperfusion, its role in the myocardial inflammatory response and in contractile dysfunction after global ischemia/reperfusion is unclear. Cytokines, particularly tumor necrosis factor-alpha (TNF-alpha), contribute to the mechanism of myocardial dysfunction after global ischemia/reperfusion. We hypothesized that a TLR4-mediated cytokine cascade modulates myocardial contractile function after global ischemia/reperfusion. This study examined whether TLR4 regulates TNF-alpha and interleukin (IL)-1beta peptide production during global ischemia/reperfusion and whether TLR4 signaling influences postischemic cardiac function through TNF-alpha and IL-1beta. METHODS Isolated hearts from wild-type mice, two strains of TLR4 mutants, TNF-alpha knockouts, and IL-1beta knockouts underwent global ischemia/reperfusion. Cardiac contractile function was analyzed, and myocardial nuclear factor-kappaB activity and TNF-alpha and IL-1beta levels were measured. RESULTS In wild-type hearts, global ischemia/reperfusion induced nuclear factor-kappaB activation and the production of TNF-alpha and IL-1beta peptides. In TLR4-mutant hearts, these changes were significantly reduced and postischemic functional recovery was improved. Application of TNF-alpha and IL-1beta to TLR4-mutant hearts abrogated this improvement in postischemic functional recovery. Postischemic functional recovery also improved in TNF-alpha knockout and IL-1beta knockout hearts, as well as in wild-type hearts treated with TNF-binding protein or IL-1 receptor antagonist. CONCLUSIONS This study demonstrates that TLR4 signaling contributes to cardiac dysfunction after global ischemia/reperfusion. TLR4 signaling mediates the production of TNF-alpha and IL-1beta peptides, and these two cytokines link TLR4 signaling to postischemic cardiac dysfunction.

TNF-α and myocardial depression in endotoxemic rats: temporal discordance of an obligatory relationship

Exogenous tumor necrosis factor-alpha (TNF-alpha) induces delayed myocardial depression in vivo but promotes rapid myocardial depression in vitro. The temporal relationship between endogenous TNF-alpha and endotoxemic myocardial depression is unclear, and the role of TNF-alpha in this myocardial disorder remains controversial. Using a rat model of endotoxemia not complicated by shock, we sought to determine 1) the temporal relationship of changes in circulating and myocardial TNF-alpha with myocardial depression, 2) the influences of protein synthesis inhibition or immunosuppression on TNF-alpha production and myocardial depression, and 3) the influence of neutralization of TNF-alpha on myocardial depression. Rats were treated with lipopolysaccharide (LPS, 0.5 mg/kg ip). Circulating and myocardial TNF-alpha increased at 1 and 2 h, whereas myocardial contractility was depressed at 4 and 6 h. Pretreatment with cycloheximide or dexamethasone abolished the increase in circulating and myocardial TNF-alpha and preserved myocardial contractile function. Similarly, treatment with TNF binding protein immediately after LPS prevented myocardial depression. We conclude that endogenous TNF-alpha mediates delayed myocardial depression in endotoxemic rats and that inhibition of TNF-alpha production or neutralization of TNF-alpha preserves myocardial contractile function in endotoxemia.Exogenous tumor necrosis factor-α (TNF-α) induces delayed myocardial depression in vivo but promotes rapid myocardial depression in vitro. The temporal relationship between endogenous TNF-α and endotoxemic myocardial depression is unclear, and the role of TNF-α in this myocardial disorder remains controversial. Using a rat model of endotoxemia not complicated by shock, we sought to determine 1) the temporal relationship of changes in circulating and myocardial TNF-α with myocardial depression, 2) the influences of protein synthesis inhibition or immunosuppression on TNF-α production and myocardial depression, and 3) the influence of neutralization of TNF-α on myocardial depression. Rats were treated with lipopolysaccharide (LPS, 0.5 mg/kg ip). Circulating and myocardial TNF-α increased at 1 and 2 h, whereas myocardial contractility was depressed at 4 and 6 h. Pretreatment with cycloheximide or dexamethasone abolished the increase in circulating and myocardial TNF-α and preserved myocardial contractile function. Similarly, treatment with TNF binding protein immediately after LPS prevented myocardial depression. We conclude that endogenous TNF-α mediates delayed myocardial depression in endotoxemic rats and that inhibition of TNF-α production or neutralization of TNF-α preserves myocardial contractile function in endotoxemia.