Masahisa Shimpo

Identification of Serum Soluble ST2 Receptor as a Novel Heart Failure Biomarker

Background—Using genomic technology, we previously identified an interleukin-1 receptor family member, ST2, as a gene markedly induced by mechanical strain in cardiac myocytes. The soluble receptor form of ST2 is secreted and detectable in human serum. This study tested the hypothesis that soluble ST2 levels in the serum of patients with severe chronic heart failure are increased in patients with neurohormonal activation. Methods and Results—Serum samples, clinical variables, and neurohormone levels from the PRAISE-2 heart failure trial (NYHA functional class III-IV; end point, mortality or transplantation) were analyzed. ST2 serum measurements were performed with ELISA on samples from 161 patients obtained at trial enrollment and from 139 of the same patients obtained 2 weeks after trial enrollment. Baseline ST2 levels were correlated with baseline B-type natriuretic peptide (BNP) levels (r =0.36, P <0.0001), baseline proatrial natriuretic peptide (ProANP) levels (r =0.36, P <0.0001), and baseline norepinephrine levels (r =0.39, P <0.0001). The change in ST2 was significant as a univariate predictor of subsequent mortality or transplantation (P =0.048), as was baseline BNP (P <0.0001) and baseline ProANP (P <0.0001). In multivariate models including BNP and ProANP, the change in ST2 remained significant as a predictor of mortality or transplantation independent of BNP and ProANP. Conclusions—Serum soluble ST2 is a novel biomarker for neurohormonal activation in patients with heart failure. In patients with severe chronic NYHA class III to IV heart failure, the change in ST2 levels is an independent predictor of subsequent mortality or transplantation.

Expression and Regulation of ST2, an Interleukin-1 Receptor Family Member, in Cardiomyocytes and Myocardial Infarction

Background—We identified an interleukin-1 receptor family member, ST2, as a gene markedly induced by mechanical strain in cardiac myocytes and hypothesized that ST2 participates in the acute myocardial response to stress and injury. Methods and Results—ST2 mRNA was induced in cardiac myocytes by mechanical strain (4.7±0.9-fold) and interleukin-1&bgr; (2.0±0.2-fold). Promoter analysis revealed that the proximal and not the distal promoter of ST2 is responsible for transcriptional activation in cardiac myocytes by strain and interleukin-1&bgr;. In mice subjected to coronary artery ligation, serum ST2 was transiently increased compared with unoperated controls (20.8±4.4 versus 0.8±0.8 ng/mL, P <0.05). Soluble ST2 levels were increased in the serum of human patients (N=69) 1 day after myocardial infarction and correlated positively with creatine kinase (r =0.41, P <0.001) and negatively with ejection fraction (P =0.02). Conclusions—These data identify ST2 release in response to myocardial infarction and suggest a role for this innate immune receptor in myocardial injury.

Serum Levels of the Interleukin-1 Receptor Family Member ST2 Predict Mortality and Clinical Outcome in Acute Myocardial Infarction

Background—Mechanically overloaded cardiomyocytes secrete a soluble interleukin-1 receptor family member called ST2. Serum levels of ST2 are associated with prognosis in nonischemic heart failure, but the predictive value of ST2 in patients with acute myocardial infarction is unknown. Methods and Results—ST2 levels were measured in serum from 810 patients with acute myocardial infarction in the Thrombolysis In Myocardial Infarction (TIMI) 14 (362 patients) and Enoxaparin and TNK-tPA With or Without GPIIb/IIIa Inhibitor as Reperfusion Strategy in STEMI (ENTIRE)-TIMI 23 (448 patients) clinical trials. Baseline levels of ST2 were significantly higher in those patients who died (0.379 versus 0.233 ng/mL, P = 0.0001) or developed new congestive heart failure (0.287 versus 0.233 ng/mL, P = 0.009) by 30 days. In an analysis of outcomes at 30 days by ST2 quartiles, both death (P = 0.001) and the combined death/heart failure end point (P = 0.001) showed a significant graded association with levels of ST2; furthermore, in-hospital death (P = 0.003) and death/heart failure (P = 0.004) were also significantly associated with higher ST2 levels. In a logistic regression analysis that controlled for important clinical factors, increasing levels of ST2 remained associated with death at 30 days (P = 0.047). ST2 levels rose during the first day after infarction and were maximal at 12 hours; ST2 levels at 12 hours were also independently associated with death at 30 days (P < 0.001). Conclusions—Serum levels of the interleukin-1 receptor family member ST2 predict mortality and heart failure in patients with acute myocardial infarction. These data suggest that ST2 may be a useful biomarker and that this novel inflammatory receptor may play a role in cardiac pathophysiology.

Fluvastatin Inhibits Matrix Metalloproteinase-1 Expression in Human Vascular Endothelial Cells

Matrix metalloproteinase-1 (MMP-1), also called interstitial collagenase, may play an important role in the pathogenesis of atherosclerosis and atherosclerotic plaque rupture. We investigated the effects of fluvastatin on MMP-1 expression in human vascular endothelial cells (ECs). The addition of fluvastatin decreased the basal MMP-1 levels in the culture media of ECs in a time-dependent (0 to 48 hours) and dose-dependent (10−8 to 10−5 mol/L) manner. On the other hand, fluvastatin did not affect tissue inhibitor of metalloproteinase-1 levels. Collagenolytic activity in conditioned media of ECs was also dose-dependently reduced by fluvastatin. The effect of fluvastatin on MMP-1 expression was completely reversed in the presence of mevalonate or geranylgeranyl-pyrophosphate, but not in the presence of squalene. Inhibition of Rho by C3 exoenzyme also significantly decreased MMP-1 expression in ECs. Our findings revealed that fluvastatin decreases MMP-1 expression in human vascular ECs through inhibition of Rho.

Endothelin-1 Inhibits Nitric Oxide Synthesis in Vascular Smooth Muscle Cells

We investigated the effects of endothelin-1 on nitric oxide synthesis in vascular smooth muscle cells. We measured the production of nitrite, a stable metabolite of nitric oxide, and the expression of inducible nitric oxide synthase mRNA and protein in cultured rat vascular smooth muscle cells. Incubation of the cultures with interleukin-1 beta (10 ng/mL) for 24 hours caused a significant increase in nitrite production. Endothelin-1 significantly decreased the interleukin-1 beta-induced nitrite production by vascular smooth muscle cells in a dose-dependent manner (10(-11) to 10(-8) mol/L). Incubation with interleukin-1 beta for 24 hours induced expression of inducible nitric oxide synthase mRNA and protein in vascular smooth muscle cells, whereas endothelin-1 showed a suppressive effect on their expressions. Addition of the endothelin type A receptor antagonist BQ-485, but not the endothelin type B receptor antagonist BQ-788, dose-dependently inhibited the effect of endothelin-1. After protein kinase C activity was functionally depleted by treatment of cells with phorbol 12-myristate 13-acetate for 24 hours, the effect of endothelin-1 was abolished. These results indicate that endothelin-1 acts on endothelin type A receptors and inhibits nitric oxide synthesis in interleukin-1 beta-stimulated vascular smooth muscle cells at least partially through a protein kinase C-dependent pathway.

Macrophage migration inhibitory factor as a redox-sensitive cytokine in cardiac myocytes

OBJECTIVE Macrophage migration inhibitory factor (MIF), which plays a pivotal role in the control of inflammatory responses, was first characterized as a T-cell cytokine, but later was also found as a pituitary peptide released in response to infection and stress. However, MIFs role and expression in the myocardium has never been reported. The goal of this study is to examine MIF in the myocardium. METHODS AND RESULTS MIF protein and mRNA levels were assayed using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR), respectively. Increased MIF concentrations were detected in the sera of patients with acute myocardial infarction (AMI). In cultured rat cardiac myocytes, significant amounts of MIF were produced in response to hypoxia and hydrogen peroxide (H(2)O(2)), but not to angiotensin II, endothelin-1, interleukin-1beta (IL-1beta) or tumor necrosis factor alpha (TNFalpha). H(2)O(2)-induced MIF production increased in a time- and dose-dependent manner and was completely abolished in the presence of catalase. H(2)O(2) also induced MIF mRNA expression. The H(2)O(2)-induced MIF production was completely inhibited by the protein kinase C (PKC) inhibitor GF109203X, partially inhibited by the tyrosine kinase inhibitor herbimycin A, and uninhibited by calcium chelation or phorbol ester-sensitive PKC down-regulation. This suggests that H(2)O(2)-induced MIF production is mediated by an atypical PKC isoform. DNA microarray analysis revealed that 52 genes were preferentially expressed in response to MIF. Of these, the MIF-induced expression of both glutathione S-transferase (GST) and lipopolysaccharide-induced CXC chemokine (LIX) mRNAs was confirmed using RT-PCR analysis. CONCLUSION The present results suggest that MIF is expressed by the myocardium in response to redox stress and may play a role in the pathogenesis of myocardial ischemia.

Serotonin Increases Interleukin-6 Synthesis in Human Vascular Smooth Muscle Cells

BackgroundInterleukin-6 (IL-6) is a key molecule in chronic inflammation and has been implicated in the progression of atherosclerosis. Serotonin (5-hydroxytryptamine; 5-HT) causes vascular contraction and proliferation, but its role in atherogenesis has not been clarified. We investigated the effects of 5-HT on IL-6 synthesis in human vascular smooth muscle cells (VSMCs). Methods and ResultsIL-6 levels in the culture medium of VSMCs were determined by ELISA. IL-6 mRNA accumulation was determined by use of a Quantikine mRNA colorimetric quantification kit. NF-&kgr;B activation was tested by gel retardation assay. 5-HT induced IL-6 production by VSMCs in a time- and dose-dependent manner, with increased IL-6 mRNA accumulation and nuclear factor-&kgr;B activation. The effect of 5-HT on IL-6 production was significantly inhibited by the 5-HT2 receptor antagonist ketanserin and the selective 5-HT2A receptor antagonist sarpogrelate. Conversely, the 5-HT2 receptor agonist &agr;-methyl-5-HT increased IL-6 production. The protein kinase C (PKC) inhibitor calphostin C, but not the protein kinase A inhibitor KT5720, suppressed 5-HT–induced IL-6 production. The effect of 5-HT was also abolished in PKC-depleted VSMCs after pretreatment with phorbol 12-myristate 13-acetate for 24 hours. Conclusions5-HT acts on 5-HT2A receptors and increases IL-6 synthesis in human VSMCs at least partially through a PKC-dependent pathway. These results suggested that 5-HT may contribute to inflammatory activation of the vessels during atherogenesis.

Adeno-associated virus vector-mediated interleukin-10 gene transfer inhibits atherosclerosis in apolipoprotein E-deficient mice

Inflammation is a major contributor to atherosclerosis by its effects on arterial wall biology and lipoprotein metabolism. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that may modulate the atherosclerotic disease process. We investigated the effects of adeno-associated virus (AAV) vector-mediated gene transfer of IL-10 on atherogenesis in apolipoprotein E (ApoE)-deficient mice. A murine myoblast cell line, C2C12, transduced with AAV encoding murine IL-10 (AAV2-mIL10) secreted substantial amounts of IL-10 into conditioned medium. The production of monocyte chemoattractant protein-1 (MCP-1) by the murine macrophage cell line, J774, was significantly inhibited by conditioned medium from AAV2-mIL10-transduced C2C12 cells. ApoE-deficient mice were injected with AAV5-mIL10 into their anterior tibial muscle at 8 weeks of age. The expression of MCP-1 in the vascular wall of the ascending aorta and serum MCP-1 concentration were decreased in AAV5-mIL10-transduced mice compared with AAV5-LacZ-transduced mice. Oil red-O staining of the ascending aorta revealed that IL-10 gene transfer resulted in a 31% reduction in plaque surface area. Serum cholesterol concentrations were also significantly reduced in AAV5-mIL10-transduced mice. To understand the cholesterol-lowering mechanism of IL-10, we measured the cellular cholesterol level in HepG2 cells, resulting in its significant decrease by the addition of IL-10 in a dose-dependent manner. Furthermore, IL-10 suppressed HMG-CoA reductase expression in the HepG2 cells. These observations suggest that intramuscular injection of AAV5-mIL10 into ApoE-deficient mice inhibits atherogenesis through anti-inflammatory and cholesterol-lowering effects.

AAV-mediated VEGF gene transfer into skeletal muscle stimulates angiogenesis and improves blood flow in a rat hindlimb ischemia model

OBJECTIVES Clinical trials on therapeutic angiogenesis using vascular endothelial growth factor (VEGF) are ongoing, however the benefits of these therapies are still controversial. To establish a more efficient gene transfer method for ischemic diseases, we investigated the therapeutic potential of adeno-associated virus (AAV)-mediated VEGF gene transfer. METHODS We produced VEGF(165)-expressing AAV vectors (AAV-VEGF). HEK-293 cells were transduced with AAV-VEGF in vitro and VEGF expression and secretion were examined. We used a rat ischemic hindlimb model and AAV-VEGF was administered intramuscularly into the ischemic limb. Gene expression was evaluated by RT-PCR and ELISA. Six weeks after gene transfer, we measured the blood flow of limb vessels and the skin temperature of limbs. Histochemical examination was performed to illustrate capillary growth. RESULTS Western blotting and ELISA revealed VEGF protein expression and secretion from AAV-VEGF-transduced HEK-293 cells. VEGF mRNA and protein expression was consistently observed in the injected muscle at least 10 weeks after the injection, while no VEGF mRNA could be detected at remote organs. The mean blood flow in AAV-VEGF-transduced ischemic limbs was significantly higher than in AAV-LacZ-transduced limbs. Capillary density was significantly higher in AAV-VEGF-injected tissues than in AAV-LacZ-injected tissues. CONCLUSIONS This study demonstrates that (1) AAV-mediated VEGF gene transfer into rat skeletal muscles is efficient and stable without ectopic expression, and (2) AAV-mediated VEGF gene transfer stimulates angiogenesis and thereby improves blood flow in a rat hindlimb ischemia model. These findings suggest that AAV-mediated VEGF gene transfer may be useful for treatment of ischemic diseases.

Monocyte-endothelial cell interaction induces expression of adhesion molecules on human umbilical cord endothelial cells

OBJECTIVE The adhesive interaction of monocytes and endothelial cells has been implicated as a regulatory signal in the cell activation that is involved in the pathogenesis of atherosclerosis. We investigated the effect of monocyte-endothelial cell interaction on the expression of adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), in human umbilical cord vein endothelial cells (HUVECs). METHODS ICAM-1 and VCAM-1 protein and mRNA expression were determined by cellular ELISA and Northern blot analysis, respectively. RESULTS The addition of unstimulated human monocytes, as well as interleukin-1 beta (IL-1 beta: 25 U/ml) and tumor necrosis factor-alpha (TNF: 100 U/ml), to HUVECs rapidly induced the expression of ICAM-1 and VCAM-1 protein and mRNA in HUVECs, whereas the addition of polymorphonuclear leukocytes (PMNs) had no significant effect on their expression. The induction of ICAM-1 and VCAM-1 by the co-culture of HUVECs and monocytes was significantly, but partially, inhibited by the combination of anti-IL-1 alpha, anti-IL-1 beta and anti-TNF Abs. Actinomycin D and genistein, but not calphostin C, also significantly inhibited the co-culture-induced adhesion molecule expression. CONCLUSIONS These results suggest that the monocyte-endothelial cell interaction induces the expression of ICAM-1 and VCAM-1 in endothelial cells partially through the production of IL-1 and TNF. These findings also suggest that the monocyte-endothelial interaction further augments their interaction through the up-regulation of endothelial adhesion molecules, as a positive feedback mechanism.