SHP2 mediates gp130-dependent cardiomyocyte hypertrophy via negative regulation of skeletal alpha-actin gene.

Morphological and biochemical phenotypes of cardiomyocyte hypertrophy are determined by neurohumoral factors. Stimulation of G protein-coupled receptor (GPCR) results in uniform cell enlargement in all directions with an increase in skeletal alpha-actin (alpha-SKA) gene expression, while stimulation of gp130 receptor by interleukin-6 (IL-6)-related cytokines induces longitudinal elongation with no increase in alpha-SKA gene expression. Thus, alpha-SKA is a discriminating marker for hypertrophic phenotypes; however, regulatory mechanisms of alpha-SKA gene expression remain unknown. Here, we clarified the role of SH2-containing protein tyrosine phosphatase 2 (SHP2) in alpha-SKA gene expression. In neonatal rat cardiomyocytes, endothelin-1 (ET-1), a GPCR agonist, but not leukemia inhibitory factor (LIF), an IL-6-related cytokine, induced RhoA activation and promotes alpha-SKA gene expression via RhoA. In contrast, LIF, but not ET-1, induced activation of SHP2 in cardiomyocytes, suggesting that SHP2 might negatively regulate alpha-SKA gene expression downstream of gp130. Therefore, we examined the effect of adenovirus-mediated overexpression of wild-type SHP2 (SHP2(WT)), dominant-negative SHP2 (SHP2(C/S)), or beta-galactosidase (beta-gal), on alpha-SKA gene expression. LIF did not upregulate alpha-SKA mRNA in cardiomyocytes overexpressing either beta-gal or SHP2(WT). In cardiomyocytes overexpressing SHP2(C/S), LIF induced upregulation of alpha-SKA mRNA, which was abrogated by concomitant overexpression of either C3-toxin or dominant-negative RhoA. RhoA was activated after LIF stimulation in the cardiomyocytes overexpressing SHP2(C/S), but not in myocytes overexpressing beta-gal. Furthermore, SHP2 mediates LIF-induced longitudinal elongation of cardiomyocytes via ERK5 activation. Collectively, these findings indicate that SHP2 negatively regulates alpha-SKA expression via RhoA inactivation and suggest that SHP2 implicates ERK5 in cardiomyocyte elongation downstream of gp130.

Gab family proteins are essential for postnatal maintenance of cardiac function via neuregulin-1/ErbB signaling.

Grb2-associated binder (Gab) family of scaffolding adaptor proteins coordinate signaling cascades downstream of growth factor and cytokine receptors. In the heart, among EGF family members, neuregulin-1beta (NRG-1beta, a paracrine factor produced from endothelium) induced remarkable tyrosine phosphorylation of Gab1 and Gab2 via erythroblastic leukemia viral oncogene (ErbB) receptors. We examined the role of Gab family proteins in NRG-1beta/ErbB-mediated signal in the heart by creating cardiomyocyte-specific Gab1/Gab2 double knockout mice (DKO mice). Although DKO mice were viable, they exhibited marked ventricular dilatation and reduced contractility with aging. DKO mice showed high mortality after birth because of heart failure. In addition, we noticed remarkable endocardial fibroelastosis and increase of abnormally dilated vessels in the ventricles of DKO mice. NRG-1beta induced activation of both ERK and AKT in the hearts of control mice but not in those of DKO mice. Using DNA microarray analysis, we found that stimulation with NRG-1beta upregulated expression of an endothelium-stabilizing factor, angiopoietin 1, in the hearts of control mice but not in those of DKO mice, which accounted for the pathological abnormalities in the DKO hearts. Taken together, our observations indicated that in the NRG-1beta/ErbB signaling, Gab1 and Gab2 of the myocardium are essential for both maintenance of myocardial function and stabilization of cardiac capillary and endocardial endothelium in the postnatal heart.

AMP-activated protein kinase protects cardiomyocytes against hypoxic injury through attenuation of endoplasmic reticulum stress.

Oxygen deprivation leads to the accumulation of misfolded proteins in the endoplasmic reticulum (ER), causing ER stress. Under conditions of ER stress, inhibition of protein synthesis and up-regulation of ER chaperone expression reduce the misfolded proteins in the ER. AMP-activated protein kinase (AMPK) is a key regulatory enzyme involved in energy homeostasis during hypoxia. It has been shown that AMPK activation is associated with inhibition of protein synthesis via phosphorylation of elongation factor 2 (eEF2) in cardiomyocytes. We therefore examined whether AMPK attenuates hypoxia-induced ER stress in neonatal rat cardiomyocytes. We found that hypoxia induced ER stress, as assessed by the expression of CHOP and BiP and cleavage of caspase 12. Knockdown of CHOP or caspase 12 through small interfering RNA (siRNA) resulted in decreased expression of cleaved poly(ADP-ribose) polymerase following exposure to hypoxia. We also found that hypoxia-induced CHOP expression and cleavage of caspase 12 were significantly inhibited by pretreatment with 5-aminoimidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), a pharmacological activator of AMPK. In parallel, adenovirus expressing dominant-negative AMPK significantly attenuated the cardioprotective effects of AICAR. Knockdown of eEF2 phosphorylation using eEF2 kinase siRNA abolished these cardioprotective effects of AICAR. Taken together, these findings demonstrate that activation of AMPK contributes to protection of the heart against hypoxic injury through attenuation of ER stress and that attenuation of protein synthesis via eEF2 inactivation may be the mechanism of cardioprotection by AMPK.

Haplotype of thrombomodulin gene associated with plasma thrombomodulin level and deep vein thrombosis in the Japanese population.

INTRODUCTION: Thrombomodulin (TM) is an essential cofactor in protein C activation by thrombin. Here, we evaluated the contribution of genetic variations in the TM gene to soluble TM (sTM) level and deep vein thrombosis (DVT) in Japanese. PATIENTS AND METHODS: We sequenced the TM putative promoter, exon, and 3'-untranslated region in DVT patients (n=118). Among 17 genetic variations we identified, two missense mutations (R385K, D468Y) and three common single nucleotide polymorphisms (-202G>A, 2487A>T, 2729A>C) were genotyped in a general population of 2247 subjects (1032 men and 1215 women) whose sTM levels were measured. We then compared the frequency of these mutations in DVT patients with that in the age, body mass index-adjusted population-based controls. RESULTS: We identified one neutral mutation (H381) and three missense mutations (R385K; n=2, A455V; n=53 heterozygous, n=14 homozygous, D468Y; n=2) of TM in the DVT patients. Age-adjusted mean values of sTM were lower in C-allele carriers of 2729A>C than in noncarriers in the Japanese general population (women: 16.7+/-0.3 U/ml vs. 17.9+/-0.2 U/ml, p<0.01, men: 19.4+/-0.3 U/ml vs. 20.4+/-0.3 U/ml, p=0.03). Additionally, the CC genotype of this mutation was more common in the male DVT patients than in the male individuals of the general population (odds ratio=2.76, 95% confidence interval=1.14-6.67; p=0.02). This mutation was in linkage disequilibrium (r-square>0.9) with A455V mutation. CONCLUSIONS: TM mutations, especially those with a haplotype consisting of 2729A>C and A455V missense mutation, affect sTM levels, and may be associated with DVT in Japanese.

Smad1 protects cardiomyocytes from ischemia-reperfusion injury.

BACKGROUND: We previously reported that bone morphogenetic protein 2 (BMP2) protected against apoptosis of serum-deprived cardiomyocytes via induction of Bcl-xL through the Smad1 pathway. To investigate whether Smad1 signaling promotes cell survival in the adult heart, we subjected transgenic mice with cardiac-specific overexpression of smad1 gene (Smad1TG) to ischemia-reperfusion (I/R) injury. METHODS AND RESULTS: The effects of BMP2 or adenovirus-mediated transfection of smad1 on cardiomyocyte survival in hypoxia-reoxygenation were examined using rat neonatal cardiomyocytes. BMP2 and Smad1 each significantly promoted survival and diminished apoptotic death of cardiomyocytes during hypoxia-reoxygenation. Interestingly, Smad1 was found to be activated during I/R in normal mouse heart. To examine physiological and pathological roles of Smad1 in I/R, we generated Smad1TG using the alpha-myosin heavy chain gene promoter. Phosphorylation of Smad1 was found in all smad1 transgene-positive mouse hearts. To examine whether Smad1 prevents injury of cardiomyocytes in vivo, we subjected Smad1TG and age-matched wild-type mice (WT) to I/R injury induced by 1 hour of ligation of the left coronary artery and 1 hour of reperfusion. TUNEL and DNA ladder analyses showed that Smad1TG had significantly smaller myocardial infarctions and fewer apoptotic deaths of cardiomyocytes than did WT. Interestingly, increased expression of Bcl-xL and beta-catenin was more remarkable whereas caspase3 was less activated in Smad1TG heart than in that of WT. CONCLUSIONS: These findings suggest that the Smad1 signaling pathway plays a role in cardioprotection against I/R injury.

Activation of gp130 transduces hypertrophic signal through interaction of scaffolding/docking protein Gab1 with tyrosine phosphatase SHP2 in cardiomyocytes.

Grb2-associated binder-1 (Gab1) is a scaffolding/docking protein and contains a Pleckstrin homology domain and potential binding sites for Src homology (SH) 2 and SH3 domains. Gab1 is tyrosine phosphorylated and associates with protein tyrosine phosphatase SHP2 and p85 phosphatidylinositol 3-kinase on stimulation with various cytokines and growth factors, including interleukin-6. We previously demonstrated that interleukin-6-related cytokine, leukemia inhibitory factor (LIF), induced cardiac hypertrophy through gp130. In this study, we report the role of Gab1 in gp130-mediated cardiac hypertrophy. Stimulation with LIF induced tyrosine phosphorylation of Gab1, and phosphorylated Gab1 interacted with SHP2 and p85 in cultured cardiomyocytes. We constructed three kinds of adenovirus vectors, those carrying wild-type Gab1 (AdGab1WT), mutated Gab1 lacking SHP2 binding site (AdGab1F627/659), and beta-galactosidase (Adbeta-gal). Compared with cardiomyocytes infected with Adbeta-gal, longitudinal elongation of cardiomyocytes induced by LIF was enhanced in cardiomyocytes infected with AdGab1WT but inhibited in cardiomyocytes infected with AdGab1F627/659. Upregulation of BNP mRNA expression by LIF was evoked in cardiomyocytes infected with Adbeta-gal and AdGab1WT but not in cardiomyocytes infected with AdGab1F627/659. In contrast, Gab1 repressed skeletal alpha-actin mRNA expression through interaction with SHP2. Furthermore, activation of extracellular signal-regulated kinase 5 (ERK5) was enhanced in cardiomyocytes infected with AdGab1WT compared with cardiomyocytes infected with Adbeta-gal but repressed in cardiomyocytes infected with AdGab1F627/659. Coinfection of AdGab1WT with adenovirus vector carrying dominant-negative ERK5 abrogated longitudinal elongation of cardiomyocytes induced by LIF. Taken together, these findings indicate that Gab1-SHP2 interaction plays a crucial role in gp130-dependent longitudinal elongation of cardiomyoctes through activation of ERK5.

Bcl-xl reduces doxorubicin-induced myocardial damage but fails to control cardiac gene downregulation.

OBJECTIVE: We recently reported that doxorubicin (Dox), an effective anti-cancer drug, induces apoptosis in cardiac myocytes in association with reduction of Bcl-xl expression. In the present study, we further examined whether overexpression of Bcl-xl ameliorates Dox-induced cardiac myocyte damage. METHODS AND RESULTS: Overexpression of the Bcl-xl gene by adenovirus vector resulted in an 11-fold increase in Bcl-xl protein in neonatal rat cardiac myocytes (BCL) compared to that in cells with beta-galactosidase gene transfection (CTL). Although Dox treatment generated similar amounts of reactive oxygen species (ROS) in BCL and CTL, cell viability was maintained and the number of apoptotic cardiac myocytes was significantly decreased in BCL. Cytochrome c release and enhanced caspase-3 activity after Dox treatment were significantly suppressed and Bax expression level was decreased in BCL. Cardiac-specific gene expression is known to be inhibited by Dox. The expression of cardiac alpha-actin and sarcoplasmic reticulum Ca(2+)-ATPase 2a mRNA was equally inhibited in BCL and CTL after Dox treatment. CONCLUSIONS: Overexpression of Bcl-xl in cardiac myocytes failed to regulate Dox-induced ROS generation and cardiac-specific gene downregulation but inhibited apoptosis accompanied by reduction of Bax protein.

Aldosterone augments endothelin-1-induced cardiac myocyte hypertrophy with the reinforcement of the JNK pathway.

Aldosterone is thought to regulate cardiac work independently of sodium retention, though the mechanisms remain to be known. In the present study, we have demonstrated that aldosterone reinforces endothelin-mediated cardiac hypertrophy with the increase in cell surface area and upregulation of the transcripts characteristic of hypertrophy. We have also shown that aldosterone augments c-Jun N-terminal kinase activation induced by endothelin-1. Taken together, it is suggested that aldosterone modulates cardiac hypertrophy, at least partially, synergistically with extracellular signals that have been shown to be involved in cardiac remodeling.

Solitary right ventricle metastasis by renal cell carcinoma.

A 57-year-old man with a history of renal cell carcinoma presented with presyncope. He underwent nephrectomy years earlier followed by HLA-matched allogeneic peripheral-blood stem-cell transplantation. Echocardiographic investigation revealed a solitary right ventricle mass without contiguous vena caval or right atrial involvement. The mass was pathologically confirmed to be metastatic carcinoma in the right ventricular cavity. This case highlights the need to consider an underlying neoplastic syndrome in patients presenting isolated right ventricle mass by echocardiography.

Specific cardiomyopathy caused by multisystemic lipid storage in Jordans' anomaly.

Multisystemic lipid storage disease is a rare disorder of lipid metabolism. We report one case of a Japanese man with systemic lipid storage in skeletal muscle and heart as well as in leukocytes (Jordans' anomaly). Positron emission tomography (PET) using 18F-fluoro-2-deoxyglucose (FDG) clearly revealed an abnormal increase of uptake during fasting in the left ventricle, suggesting changes in the energy metabolism in the heart.

Interaction of scaffolding adaptor protein Gab1 with tyrosine phosphatase SHP2 negatively regulates IGF-I-dependent myogenic differentiation via the ERK1/2 signaling pathway.

Grb2-associated binder 1 (Gab1) coordinates various receptor tyrosine kinase signaling pathways. Although skeletal muscle differentiation is regulated by some growth factors, it remains elusive whether Gab1 coordinates myogenic signals. Here, we examined the molecular mechanism of insulin-like growth factor-I (IGF-I)-mediated myogenic differentiation, focusing on Gab1 and its downstream signaling. Gab1 underwent tyrosine phosphorylation and subsequent complex formation with protein-tyrosine phosphatase SHP2 upon IGF-I stimulation in C2C12 myoblasts. On the other hand, Gab1 constitutively associated with phosphatidylinositol 3-kinase regulatory subunit p85. To delineate the role of Gab1 in IGF-I-dependent signaling, we examined the effect of adenovirus-mediated forced expression of wild-type Gab1 (Gab1(WT)), mutated Gab1 that is unable to bind SHP2 (Gab1(DeltaSHP2)), or mutated Gab1 that is unable to bind p85 (Gab1(Deltap85)), on the differentiation of C2C12 myoblasts. IGF-I-induced myogenic differentiation was enhanced in myoblasts overexpressing Gab1(DeltaSHP2), but inhibited in those overexpressing either Gab1(WT) or Gab1(Deltap85). Conversely, IGF-I-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation was significantly repressed in myoblasts overexpressing Gab1(DeltaSHP2) but enhanced in those overexpressing either Gab1(WT) or Gab1(Deltap85). Furthermore, small interference RNA-mediated Gab1 knockdown enhanced myogenic differentiation. Overexpression of catalytic-inactive SHP2 modulated IGF-I-induced myogenic differentiation and ERK1/2 activation similarly to that of Gab1(DeltaSHP2), suggesting that Gab1-SHP2 complex inhibits IGF-I-dependent myogenesis through ERK1/2. Consistently, the blockade of ERK1/2 pathway reversed the inhibitory effect of Gab1(WT) overexpression on myogenic differentiation, and constitutive activation of the ERK1/2 pathway suppressed the enhanced myogenic differentiation by overexpression of Gab1(DeltaSHP2). Collectively, these data suggest that the Gab1-SHP2-ERK1/2 signaling pathway comprises an inhibitory axis for IGF-I-dependent myogenic differentiation.

Effect of bosentan on plasma endothelin-1 concentration in patients with pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a progressive disease with high mortality. An orally active dual endothelin (ET) receptor antagonist, bosentan, has been reported to improve exercise capacity and survival in patients with PAH. Plasma ET-1 concentration is known to be increased in PAH patients; however, the effect of bosentan on ET-1 concentration has not yet been investigated. METHODS AND RESULTS: The concentration of ET-1 after bosentan administration was examined in 7 PAH patients, including 2 primary and 5 secondary cases. They were clinically assessed by pulmonary artery pressure (PAP), 6-min walk distance (6MWD) and plasma brain natriuretic peptide (BNP) concentration. Baseline ET-1 concentration was significantly higher in patients with PAH than in normal individuals (2.19+/-0.71 pg/ml vs 1.45+/-0.10 pg/ml, p<0.05) and was significantly correlated with 6MWD and BNP. A single dose of 62.5 mg bosentan in patients with PAH significantly increased plasma ET-1 concentration to 2.04 times the basal concentration (p<0.01) with a peak at 8.1 h. The peak to base ratio of ET-1 after bosentan administration correlated negatively with severity of PAH as assessed by PAP. CONCLUSIONS: The present study is the first study to show that bosentan administration increases plasma ET-1 in patients with PAH. The response of plasma ET-1 to bosentan administration might be useful for determining the severity of PAH.

Carbacyclin induces carnitine palmitoyltransferase-1 in cardiomyocytes via peroxisome proliferator-activated receptor (PPAR) delta independent of the IP receptor signaling pathway.

Prostacyclin (PGI2) and its analogues exert cardioprotective effects via the rhodopsin type membrane PGI2 receptor, IP. Peroxisome proliferator-activated receptor (PPAR) delta is a nuclear receptor abundantly expressed in cardiomyocytes and plays a pivotal role in maintaining constitutive mitochondrial fatty acid beta-oxidation (FAO). Recently, a novel signaling pathway of PGI2 via PPARdelta has been demonstrated in non-cardiac tissues. We therefore examined whether carbacyclin (cPGI2), a PGI2 analogue, up-regulates transcriptional expression of carnitine palmitoyltransferase-1 (CPT-1), the rate-limiting enzyme in mitochondrial FAO, via PPARdelta in cardiomyocytes. Intraperitoneal injection of cPGI2 increased CPT-1 mRNA expression in murine hearts. Transcriptional activity was evaluated by PPAR responsive element (PPRE)-luciferase reporter gene assay in cultured neonatal rat cardiomyocytes. CPT-1 mRNA expression and PPRE promoter activity were significantly increased by cPGI2 in a concentration-dependent manner, where PPRE has been mapped to the promoter region of the CPT-1 gene. Moreover, the elevation of CPT-1 mRNA expression and PPRE promoter activity by cPGI2 was not abolished by H-89, a potent protein kinase A inhibitor, but was significantly inhibited in cardiomyocytes over-expressing a dominant-negative type of PPARdelta. Furthermore, electrophoretic mobility shift assays demonstrated that binding of PPARdelta to PPRE in the CPT-1 gene promoter is enhanced in response to cPGI2 stimulation. In addition, down-regulation of CPT-1 mRNA expression in cardiomyocytes subjected to hypoxia was attenuated by cPGI2. These results indicate that cPGI2 induces CPT-1 mRNA expression through PPARdelta, independent of the IP receptor signaling pathway, suggesting a possibility that cPGI2 modulates cardiac energy metabolism by activating FAO via PPARdelta.

Cross-talk between bone morphogenetic protein 2 and leukemia inhibitory factor through ERK 1/2 and Smad1 in protection against doxorubicin-induced injury of cardiomyocytes.

The survival of cardiomyocytes is regulated by growth factors and cytokines such as bone morphogenetic protein (BMP) 2 and leukemia inhibitory factor (LIF). BMP2 and LIF induce distinct signal transduction pathways that each activate a different transcription factor [Smad1 and signal transducing activating transcriptional factor (Stat) 3, respectively] and common signal pathway [mitogen-activated protein kinase (MAPK)]. We previously demonstrated that BMP2 and LIF protect cardiomyocytes via Smad1 and STAT3 signaling pathways, respectively. On the other hand, these signals are known to act in synergy via synergistic integration of signaling pathways. Here, we examined interaction between BMP2 and LIF in primary cultured neonatal rat cardiomyocytes. LIF sustained phosphorylation/activation of Smad1 by BMP2. The role of extracellular signal-regulated kinase (ERK) 1/2 cascade activated by LIF was highlighted by the use of a MAPK/ERK kinase (MEK) 1/2 inhibitor, U0126, or overexpression of dominant-negative form of MEK1 that abolished sustained phosphorylation of Smad1 and cell survival effect induced by co-stimulation of LIF with BMP2, while BMP2 alone did not activate ERK1/2. Conversely, overexpression of the constitutive-active form of MEK1 increased BMP2-induced phosphoration of Smad1 without additional LIF. Moreover, BMP2 and LIF synergistically induced bcl-xL mRNA in doxorubicin (DOX)-injured cardiomyocytes. These findings suggest that the ERK1/2 pathway downstream of LIF is involved in sustained phosphorylation/activation of Smad1 by BMP2 and provide a possible mechanism for cooperation between intracellular signals activated by LIF and BMP2 in protection against DOX-induced injury of cardiomyocytes.

Sildenafil as adjunct therapy to high-dose epoprostenol in a patient with pulmonary veno-occlusive disease.

Pulmonary veno-occlusive disease is refractory to medical treatment and is generally associated with a poor prognosis. Treatment with vasodilators, such as prostacyclin, of patients with PVOD is controversial because of concerns regarding hemodynamic deterioration. Although a preferential pulmonary vasodilatory effect of a specific phosphodiesterase-5 inhibitor, sildenafil, has recently been reported in patients with primary pulmonary hypertension, little information is available regarding the effect of sildenafil on patients with pulmonary veno-occlusive disease. In the present case, remarkable improvement of hemodynamics and of clinical course was produced by adjunctive use of oral sildenafil in association with intravenous high-dose epoprostenol. These findings suggest that sildenafil may be a therapeutic option in the medical treatment of pulmonary veno-occlusive disease.

A case of hypereosinophilic syndrome presenting mid-ventricular obstruction.

A 59-year-old man with hypereosinophilic syndrome (HES) who had been maintained with low-dose prednisolone for 5 years developed the characteristic features of hypertrophic cardiomyopathy. Left ventricular endomyocardial biopsy revealed no eosinophilic infiltration but extensive myocardial fibrosis. Cardiac involvement in HES presents as endocardial fibrosis, resulting in a clinical presentation of restrictive cardiomyopathy. HES heart disease can also present dilated cardiomyopathy, but myocardial hypertrophy has only rarely been noted in conjunction with HES. This report concerns a patient with HES who had clinical and hemodynamic evidence of asymmetric septal hypertrophy with mid-ventricular obstruction.

Circulating interleukin-6 family cytokines and their receptors in patients with congestive heart failure.

gp130 is a common signal-transducing receptor subunit for the interleukin (IL)-6 cytokine family. Studies in genetically engineered animal models have demonstrated a critical role for the gp130-dependent cardiomyocyte survival pathway in the transition to heart failure. In the present study, we examined plasma levels of the IL-6 family of cytokines and the soluble form of their receptors in patients with congestive heart failure (CHF). Circulating levels of the IL-6 family of cytokines, soluble IL-6 receptor (sIL-6R), and soluble gp130 (sgp130) were examined in 48 patients with various degrees of CHF, including dilated cardiomyopathy (DCM), ischemic cardiomyopathy (ICM), and valvular cardiomyopathy (VCM). Circulating levels of IL-6, leukemia inhibitory factor (LIF), and sgp130 significantly increased in association with the severity of CHF. No significant difference was observed in the circulating levels of sIL-6R and IL-11 among these patients. Interestingly, DCM patients showed higher circulating sgp130 levels than patients with ICM or VCM. Our findings suggest that gp130 expression in the heart is likely to be dynamic, and that the IL-6 family of cytokines and their common receptor gp130 participates in the pathogenesis of CHF, especially in DCM.

Cardiac-specific activation of signal transducer and activator of transcription 3 promotes vascular formation in the heart.

Signal transducer and activator of transcription 3 (STAT3) functions in cell proliferation, differentiation, and cell survival. Previously, we have demonstrated that the activation of STAT3 is required for glycoprotein 130-mediated induction of VEGF in cardiac myocytes, but the functional importance of STAT3 as an angiogenic mediator remains to be determined. To address this issue, we first generated the adenoviral vector expressing constitutively active STAT3 (caSTAT3). Adenoviral gene transfer of caSTAT3 induced an increase in the expression of VEGF in cultured cardiomyocytes. The conditioned medium from caSTAT3-transfected cardiomyocyte culture promoted endothelial tubule formation, which was inhibited by anti-VEGF antibody. Next, we generated the transgenic (TG) mice with cardiac-specific overexpression of caSTAT3 and demonstrated that caSTAT3 TG mice showed evidence of VEGF induction in the hearts. The caSTAT3 TG hearts also demonstrated increased capillary density accompanied by an increase in the expression of VE-cadherin, an endothelial-specific component. These data indicate that caSTAT3 TG hearts exhibit an enriched vascular structure compared with non-transgenic hearts. The study presented here provides the first evidence that activation of STAT3 controls vessel growth in vivo and suggests that STAT3 contributes to cardiac adaptation by regulating vascular function under the conditions of stress.

Novel insertional mutation in the bone morphogenetic protein receptor type II associated with sporadic primary pulmonary hypertension.

Primary pulmonary hypertension (PPH), which results from occlusion of small pulmonary arteries, is a devastating condition. Mutations of the bone morphogenetic protein receptor type II gene (BMPR2), a component of the transforming growth factor- beta (TGF-beta) family, which plays a key role in cell growth, have recently been identified as causing familial and sporadic PPH. The first case of BMPR2 mutation found in Japan is reported here in a 19-year-old woman with a clinical diagnosis of PPH and no identifiable family history of pulmonary hypertension. Direct sequencing of the entire coding region and intron/exon boundaries of BMPR2 revealed a frameshift mutation predicted to alter the cell signaling response to specific ligands. A molecular classification of PPH, based upon the presence or absence of BMPR2 mutations, might have important implications for patient management and screening of relatives.