Identification of RSK and TTK as Modulators of Blood Vessel Morphogenesis Using an Embryonic Stem Cell-Based Vascular Differentiation Assay.

Blood vessels are formed through vasculogenesis, followed by remodeling of the endothelial network through angiogenesis. Many events that occur during embryonic vascular development are recapitulated during adult neoangiogenesis, which is critical to tumor growth and metastasis. Current antiangiogenic tumor therapies, based largely on targeting the vascular endothelial growth factor pathway, show limited clinical benefits, thus necessitating the discovery of alternative targets. Here we report the development of a robust embryonic stem cell-based vascular differentiation assay amenable to small-molecule screens to identify novel modulators of angiogenesis. In this context, RSK and TTK were identified as angiogenic modulators. Inhibition of these pathways inhibited angiogenesis in embryoid bodies and human umbilical vein endothelial cells. Furthermore, inhibition of RSK and TTK reduced tumor growth, vascular density, and improved survival in an in vivo Lewis lung carcinoma mouse model. Our study suggests that RSK and TTK are potential targets for antiangiogenic therapy, and provides an assay system for further pathway screens.

Deficiency in TIMP-3 increases cardiac rupture and mortality post-myocardial infarction via EGFR signaling: beneficial effects of cetuximab.

Cardiac rupture is a fatal complication of myocardial infarction (MI); however, its underlying molecular mechanisms are not fully understood. This study investigated the role of tissue inhibitor of metalloproteinase-3 (TIMP-3)/matrix metalloproteinase (MMP)/epidermal growth factor (EGF)/transforming growth factor (TGF)-ß1 pathway in infarct healing and effects of cetuximab on cardiac rupture after MI. Induction of MI was achieved by left coronary artery ligation in wild-type (WT) and TIMP-3(-/-) mice. TIMP-3 deficiency resulted in a fourfold increase in cardiac rupture and 50% decrease in survival after MI. Hydroxyproline content, collagen synthesis and myofibroblast cell number in the infarct region, and the force required to induce rupture of the infarct scar were significantly decreased, while MMP activity was increased in TIMP-3(-/-) mice. EGF proteins were increased by threefold in TIMP-3(-/-) mice following MI, while TGF-ß1 mRNA levels were decreased by 68%. Cell proliferation of cultured adult cardiac myofibroblasts was significantly decreased in TIMP-3(-/-) compared to WT myofibroblasts. EGF treatment significantly decreased collagen synthesis and TGF-ß1 expression. Conversely, TGF-ß1 treatment increased collagen synthesis in cardiac myofibroblasts. Treatment with cetuximab significantly decreased the incidence of cardiac rupture and improved survival post-MI in TIMP-3(-/-) mice. We conclude that deficiency in TIMP-3 increases cardiac rupture post-MI via EGF/epidermal growth factor receptor (EGFR) signaling which downregulates TGF-ß1 expression and collagen synthesis. Inhibition of EGFR by cetuximab protects against cardiac rupture and improves survival post-MI.

Neuronal nitric oxide synthase protects against myocardial infarction-induced ventricular arrhythmia and mortality in mice.

BACKGROUND: Neuronal nitric oxide synthase (nNOS) is expressed in cardiomyocytes and plays a role in regulating cardiac function and Ca2+ homeostasis. However, the role of nNOS in cardiac electrophysiology after myocardial infarction (MI) is unclear. We hypothesized that nNOS deficiency increases ventricular arrhythmia and mortality after MI. METHODS AND RESULTS: MI was induced in wild-type (WT) or nNOS(-/-) mice by ligation of the left coronary artery. Thirty-day mortality was significantly higher in nNOS(-/-) compared with WT mice. Additionally, nNOS(-/-) mice had impaired cardiac function 2 days after MI. Telemetric ECG monitoring showed that compared with WT, nNOS(-/-) mice had significantly more ventricular arrhythmias and were more likely to develop ventricular fibrillation after MI. Treatment with the L-type Ca2+ channel blocker verapamil reduced the incidence of arrhythmia and ventricular fibrillation in nNOS(-/-) mice after MI. To assess the role of nNOS in Ca2+ handling, patch-clamp and Ca2+ fluorescence techniques were used. Ca2+ transients and L-type Ca2+ currents were higher in nNOS(-/-) compared with WT cardiomyocytes. Additionally, nNOS(-/-) cardiomyocytes exhibited significantly higher systolic and diastolic Ca2+ over a range of pacing frequencies. Treatment with the NO donor S-nitroso N-acetyl-penicillamine decreased Ca2+ transients and L-type Ca2+ current in both nNOS(-/-) and WT cardiomyocytes. Furthermore, S-nitrosylation of Ca2+ handling proteins was significantly decreased in nNOS(-/-) myocardium after MI. CONCLUSIONS: Deficiency in nNOS increases ventricular arrhythmia and mortality after MI in mice. The antiarrhythmic effect of nNOS involves inhibition of L-type Ca2+ channel activity and regulation of Ca2+ handling proteins via S-nitrosylation.

Cardiomyocyte-specific overexpression of human stem cell factor improves cardiac function and survival after myocardial infarction in mice.

BACKGROUND: Soluble stem cell factor (SCF) has been shown to mobilize bone marrow stem cells and improve cardiac repair after myocardial infarction (MI). However, the effect of membrane-associated SCF on cardiac remodeling after MI is not known. The present study investigated the effects of cardiomyocyte-specific overexpression of the membrane-associated isoform of human SCF (hSCF) on cardiac function after MI. METHODS AND RESULTS: A novel mouse model with tetracycline-inducible and cardiac-specific overexpression of membrane-associated hSCF was generated. MI was induced by left coronary artery ligation. Thirty-day mortality after MI was decreased in hSCF/tetracycline transactivator (tTA) compared with wild-type mice. In vivo cardiac function was significantly improved in hSCF/tTA mice at 5 and 30 days after MI compared with wild-type mice. Endothelial progenitor cell recruitment and capillary density were increased and myocardial apoptosis was decreased in the peri-infarct area of hSCF/tTA mice. Myocyte size was decreased in hSCF/tTA mice 30 days after MI compared with WT mice. Furthermore, hSCF overexpression promoted de novo angiogenesis as assessed by matrigel implantation into the left ventricular myocardium. CONCLUSIONS: Cardiomyocyte-specific overexpression of hSCF improves myocardial function and survival after MI. These beneficial effects of hSCF may result from increases in endothelial progenitor cell recruitment and neovascularization and decreases in myocardial apoptosis and cardiac remodeling.

Erythropoietin protects the heart from ventricular arrhythmia during ischemia and reperfusion via neuronal nitric-oxide synthase.

Erythropoietin (EPO) is a potent cardioprotective agent in models of myocardial ischemia and reperfusion (I/R). It has been suggested recently that EPO may also reduce ventricular arrhythmia after I/R. The present study investigated the role of neuronal nitric oxide synthase (nNOS) on the antiarrhythmic effects of EPO. EPO treatment increased nNOS expression in isolated neonatal mouse ventricular myocytes. Cotreatment with the phosphatidylinositol 3 (PI3)-kinase inhibitor, LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], or treatment of cardiomyocytes infected with a dominant negative adenovirus targeted to Akt1 (ADV-dnAkt1) blocked the effects of EPO on nNOS expression, suggesting that EPO regulates nNOS expression via PI3-kinase and Akt. To examine the in vivo antiarrhythmic effects of EPO, wild-type (WT) and nNOS(-/-) mice were anesthetized and, after a baseline measurement, subjected to myocardial I/R to provoke ventricular arrhythmias. Pretreatment with EPO 24 h before ischemia increased nNOS expression and significantly reduced the number of premature ventricular contractions (PVCs) and the incidence of ventricular tachycardia (VT) in WT mice. In contrast, treatment with EPO had no effect on PVCs or the incidence of VT in nNOS(-/-) mice. Furthermore, EPO treatment after ischemia significantly reduced the threshold dose of cesium chloride (CsCl) to induce VT. We conclude that EPO via nNOS protects the heart from spontaneous and CsCl-induced ventricular arrhythmia during myocardial I/R.

Tissue inhibitor of metalloproteinase-3 inhibits neonatal mouse cardiomyocyte proliferation via EGFR/JNK/SP-1 signaling.

We have recently demonstrated that tissue inhibitor of metalloproteinase-3 (TIMP-3) decreases neonatal cardiomyocyte proliferation (Hammoud L, Xiang F, Lu X, Brunner F, Leco K, Feng Q. Cardiovasc Res 75: 359-368, 2007). The aim of the present study was to delineate a pathway through which TIMP-3 exerts its antiproliferative effect. Experiments were conducted on neonatal cardiomyocyte cultures and heart tissues isolated from wild-type (WT) and TIMP-3(-/-) mice. Deficiency in TIMP-3 decreased p27 expression and increased cardiomyocyte proliferation in cardiomyocytes and neonatal hearts. A TIMP-3/epidermal growth factor (EGF) receptor (EGFR)/c-Jun NH(2)-terminal kinase (JNK)/SP-1/p27 pathway was investigated. JNK phosphorylation and EGFR protein levels were increased in TIMP-3(-/-) cardiomyocytes and heart tissues. Treatment with recombinant TIMP-3 decreased JNK phosphorylation and EGFR expression/phosphorylation. Inhibition of JNK activity using SP-600125 decreased SP-1 phosphorylation, increased p27 expression, and decreased cardiomyocyte proliferation. Furthermore, treatment with the EGFR specific inhibitor PD-168393 or the EGF-neutralizing antibody decreased cardiomyocyte proliferation as well as phosphorylation of JNK and SP-1 in both WT and TIMP-3(-/-) cardiomyocytes. We conclude that TIMP-3 inhibits neonatal mouse cardiomyocyte proliferation by upregulating p27 expression. The effects of TIMP-3 are mediated via inhibition of EGFR expression/phosphorylation, and decreases in JNK and SP-1 signaling.

Role of heme oxygenase-1 in the cardioprotective effects of erythropoietin during myocardial ischemia and reperfusion.

We have recently demonstrated that erythropoietin (EPO) protects cardiomyocytes from apoptosis during myocardial ischemia-reperfusion (I/R). The objective of the present study was to investigate the role of heme oxygenase (HO)-1 in the antiapoptotic effects of EPO. Primary cultures of neonatal mouse cardiomyocytes were subjected to anoxia-reoxygenation (A/R). Pretreatment with EPO significantly reduced apoptosis in A/R-treated cells. This reduction in apoptosis was preceded by an increase in the mRNA and protein expression of HO-1. Selective inhibition of HO-1 using chromium mesoporphyrin (CrMP) significantly diminished the ability of EPO to inhibit apoptosis. Cotreatment of EPO with SB-202190, an inhibitor of p38 activation, blocked the EPO-mediated HO-1 expression and antiapoptotic effects, suggesting a p38-dependent mechanism. The in vivo significance of p38 and HO-1 as mediators of EPO's cardioprotection was investigated in mice subjected to myocardial I/R. Pretreatment with EPO decreased infarct size as well as I/R-induced apoptosis in wild-type mice. However, these effects were significantly diminished in HO-1(-/-) mice. Furthermore, EPO given during ischemia reduced infarct size in mice subjected to I/R, and this effect was blocked by CrMP treatment in wild-type mice. Moreover, inhibition of p38 diminished the cardioprotective effects of EPO. We conclude that upregulation of HO-1 expression via p38 signaling contributes to EPO-mediated cardioprotection during myocardial I/R.

Endothelial nitric oxide synthase promotes neonatal cardiomyocyte proliferation by inhibiting tissue inhibitor of metalloproteinase-3 expression.

OBJECTIVE: We have recently demonstrated that endothelial nitric oxide synthase (eNOS) promotes cardiomyocyte proliferation. However, mechanisms by which eNOS regulates cardiomyocyte proliferation are not fully understood. The goal of the present study was to investigate the role of tissue inhibitor of metalloproteinase-3 (TIMP-3) in eNOS-mediated cardiomyocyte proliferation. METHODS AND RESULTS: Experiments were conducted in cultured neonatal mouse cardiomyocytes. TIMP-3 expression was significantly decreased in wild-type (WT) cardiomyocytes treated with an adenoviral eNOS (Ad-eNOS). Furthermore, TIMP-3 levels were significantly decreased in cardiomyocytes derived from eNOS transgenic mice. Conversely, TIMP-3 transcript levels were significantly elevated in eNOS(-/-) cardiomyocytes. The inhibitory effect of NO on TIMP-3 expression was dependent on S-nitrosylation of c-jun, a subunit of AP-1. Cell proliferation was increased in TIMP-3(-/-) cardiomyocytes while recombinant TIMP-3 decreased proliferation in both TIMP-3(-/-) and WT cardiomyocytes. Furthermore, the decline in proliferation observed in eNOS(-/-) cardiomyocytes was abrogated when TIMP-3 expression was blocked by an anti-TIMP-3 antibody. In vivo cardiomyocyte proliferation was assessed by Ki67 immunostaining on postnatal day 1 hearts. Ki67-positive cardiomyocytes were decreased in eNOS(-/-), but increased in eNOS-Tg and TIMP-3(-/-) hearts compared to WT controls. CONCLUSIONS: Our study suggests that eNOS promotes neonatal cardiomyocyte proliferation by inhibiting TIMP-3 expression.

Cloning and developmental characterization of Xenopus laevis membrane type-3 matrix metalloproteinase (MT3-MMP).

Proper extracellular matrix (ECM) remodeling, mediated by matrix metalloproteinases (MMPs), is crucial for the development and survival of multicellular organisms. Full-length Xenopus laevis membrane type-3 matrix metallo proteinase (MT3-MMP) was amplified by PCR and cloned from a stage 28 Xenopus head cDNA library. A comparison of the derived Xenopus MT3-MMP protein sequence to that of other vertebrates revealed 86% identity with human and mouse and 85% identity with chicken. The expression profile of MT3-MMP was examined during Xenopus embryogenesis: MT3-MMP transcripts were first detected at the later stages of development and were localized to dorsal and anterior structures. During metamorphosis and in the adult frog, MT3-MMP expression was restricted to specific tissues and organs. Treatment of Xenopus embryos with lithium chloride (LiCl), ultraviolet irradiation (UV), or retinoic acid (RA) revealed that MT3-MMP levels increased with LiCl-dorsalizing treatments and decreased with UV-ventralizing and RA-anterior neural truncating treatments. Overexpression of MT3-MMP through RNA injections led to dose-dependent developmental abnormalities and death. Moreover, MT3-MMP overexpression resulted in neural and head structure abnormalities, as well as truncated axes. Taken together, these results indicate that MT3-MMP expression in Xenopus is spatially and temporally restricted. Furthermore, deregulation of MT3-MMP during early embryogenesis has detrimental effects on development.