Na + /H + exchanger isoform 1-induced osteopontin expression facilitates cardiac hypertrophy through p90 ribosomal S6 kinase

Cardiovascular diseases are the leading cause of death worldwide. One in three cases of heart failure is due to dilated cardiomyopathy. The Na+/H+ exchanger isoform 1 (NHE1), a multifunctional protein and the key pH regulator in the heart, has been demonstrated to be increased in this condition. We have previously demonstrated that elevated NHE1 activity induced cardiac hypertrophy in vivo. Furthermore, the overexpression of active NHE1 elicited modulation of gene expression in cardiomyocytes including an upregulation of myocardial osteopontin (OPN) expression. To determine the role of OPN in inducing NHE1-mediated cardiomyocyte hypertrophy, double transgenic mice expressing active NHE1 and OPN knockout were generated and assessed by echocardiography and the cardiac phenotype. Our studies showed that hearts expressing active NHE1 exhibited cardiac remodeling indicated by increased systolic and diastolic left ventricular internal diameter and increased ventricular volume. Moreover, these hearts demonstrated impaired function with decreased fractional shortening and ejection fraction. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNA was upregulated, and there was an increase in heart cell cross-sectional area confirming the cardiac hypertrophic effect. Moreover, NHE1 transgenic mice also showed increased collagen deposition, upregulation of CD44 and phosphorylation of p90 ribosomal s6 kinase (RSK), effects that were regressed in OPN knockout mice. In conclusion, we developed an interesting comparative model of active NHE1 transgenic mouse lines which express a dilated hypertrophic phenotype expressing CD44 and phosphorylated RSK, effects which were regressed in absence of OPN.Scopu

Vitamin D deficiency causes inward hypertrophic remodeling and alters vascular reactivity of rat cerebral arterioles

BACKGROUND AND PURPOSE: Vitamin D deficiency (VDD) is a global health problem, which can lead to several pathophysiological consequences including cardiovascular diseases. Its impact on the cerebrovascular system is not well understood. The goal of the present work was to examine the effects of VDD on the morphological, biomechanical and functional properties of cerebral arterioles. METHODS: Four-week-old male Wistar rats (n = 11 per group) were either fed with vitamin D deficient diet or received conventional rat chow with per os vitamin D supplementation. Cardiovascular parameters and hormone levels (testosterone, androstenedione, progesterone and 25-hydroxyvitamin D) were measured during the study. After 8 weeks of treatment anterior cerebral artery segments were prepared and their morphological, biomechanical and functional properties were examined using pressure microangiometry. Resorcin-fuchsin and smooth muscle actin staining were used to detect elastic fiber density and smooth muscle cell counts in the vessel wall, respectively. Sections were immunostained for eNOS and COX-2 as well. RESULTS: VDD markedly increased the wall thickness, the wall-to-lumen ratio and the wall cross-sectional area of arterioles as well as the number of smooth muscle cells in the tunica media. As a consequence, tangential wall stress was significantly lower in the VDD group. In addition, VDD increased the myogenic as well as the uridine 5'-triphosphate-induced tone and impaired bradykinin-induced relaxation. Decreased eNOS and increased COX-2 expression were also observed in the endothelium of VDD animals. CONCLUSIONS: VDD causes inward hypertrophic remodeling due to vascular smooth muscle cell proliferation and enhances the vessel tone probably because of increased vasoconstrictor prostanoid levels in young adult rats. In addition, the decreased eNOS expression results in endothelial dysfunction. These morphological and functional alterations can potentially compromise the cerebral circulation and lead to cerebrovascular disorders in VDD

P2 receptors in atherosclerosis and postangioplasty restenosis

Atherosclerosis is an immunoinflammatory process that involves complex interactions between the vessel wall and blood components and is thought to be initiated by endothelial dysfunction [Ross (Nature 362:801–09, 1993); Fuster et al. (N Engl J Med 326:242–50, 1992); Davies and Woolf (Br Heart J 69:S3–S11, 1993)]. Extracellular nucleotides that are released from a variety of arterial and blood cells [Di Virgilio and Solini (Br J Pharmacol 135:831–42, 2002)] can bind to P2 receptors and modulate proliferation and migration of smooth muscle cells (SMC), which are known to be involved in intimal hyperplasia that accompanies atherosclerosis and postangioplasty restenosis [Lafont et al. (Circ Res 76:996–002, 1995)]. In addition, P2 receptors mediate many other functions including platelet aggregation, leukocyte adherence, and arterial vasomotricity. A direct pathological role of P2 receptors is reinforced by recent evidence showing that upregulation and activation of P2Y2 receptors in rabbit arteries mediates intimal hyperplasia [Seye et al. (Circulation 106:2720–726, 2002)]. In addition, upregulation of functional P2Y receptors also has been demonstrated in the basilar artery of the rat double-hemorrhage model [Carpenter et al. (Stroke 32:516–22, 2001)] and in coronary artery of diabetic dyslipidemic pigs [Hill et al. (J Vasc Res 38:432–43, 2001)]. It has been proposed that upregulation of P2Y receptors may be a potential diagnostic indicator for the early stages of atherosclerosis [Elmaleh et al. (Proc Natl Acad Sci U S A 95:691–95, 1998)]. Therefore, particular effort must be made to understand the consequences of nucleotide release from cells in the cardiovascular system and the subsequent effects of P2 nucleotide receptor activation in blood vessels, which may reveal novel therapeutic strategies for atherosclerosis and restenosis after angioplasty

Probable insensitivity of mollicutes to rifampin and characterization of spiroplasmal DNA-dependent RNA polymerase.

The effect of rifampin on five mollicutes (Spiroplasma citri, Spiroplasma melliferum, Spiroplasma apis, Acholeplasma laidlawii, and Mycoplasma mycoides) was compared with that on Escherichia coli. We found that, in contrast to wild-type E. coli, mollicutes were insensitive to rifampin. DNA-dependent RNA polymerases from S. melliferum and S. apis were purified to the stage where the enzymes were dependent on the addition of exogenous templates for activity. The enzymes were then tested for their sensitivity to rifampin. Spiroplasmal enzymes were at least 1,000 times less sensitive to rifampin than the corresponding E. coli enzyme. This result provides a molecular basis for the resistance of mollicutes to rifampin. The RNA polymerase of S. melliferum was further purified and its subunit composition was investigated. The RNA polymerase has one small and two large subunits. The structure of S. melliferum RNA polymerase therefore resembles that of the eubacterial enzymes in spite of its insensitivity to rifampin

Osteopontin: A Promising Therapeutic Target in Cardiac Fibrosis

Osteopontin (OPN) is recognized for its significant roles in both physiological and pathological processes. Initially, OPN was recognized as a cytokine with pro-inflammatory actions. More recently, OPN has emerged as a matricellular protein of the extracellular matrix (ECM). OPN is also known to be a substrate for proteolytic cleavage by several proteases that form an integral part of the ECM. In the adult heart under physiological conditions, basal levels of OPN are expressed. Increased expression of OPN has been correlated with the progression of cardiac remodeling and fibrosis to heart failure and the severity of the condition. The intricate process by which OPN mediates its effects include the coordination of intracellular signals necessary for the differentiation of fibroblasts into myofibroblasts, promoting angiogenesis, wound healing, and tissue regeneration. In this review, we discuss the role of OPN in contributing to the development of cardiac fibrosis and its suitability as a therapeutic target.Scopu

C006 Nadph-oxidases and uncoupled endothelial NO-synthase in pulmonary arterial hypertension induced by chronic hypoxia

Nitric oxide (NO) production by endothelial NO-synthase (eNOS) is critically dependent on the cofactor, tetrahydrobiopterin (BH4). Depletion in BH4 consecutive to an increase of reactive oxygen species (ROS) production by NADPH-oxidases and/or eNOS over-expression, favour eNOS uncoupling. This study investigates the potential role of NADPH-oxidases and uncoupled eNOS in pulmonary arterial hypertension induced by chronic hypoxia.Male C57BL/6 and eNOS knockout (eNOS-/-) mice were exposed or not to hypobaric hypoxia (0.5atm) for 21 days. Fulton index (right ventricular / left ventricular + septum weight ratio) was determined. Lungs were used for measurement of BH4 (by HPLC), for expression of eNOS (by western-blotting) and of the NADPH-oxidases subunits Nox1, Nox2 and Nox4 (by RT-PCR). Pulmonary arteries were also mounted in a wire myograph for evaluation of vasomotor responses.Chronic hypoxia induced a marked up-regulation of Nox1, Nox2 and Nox4 mRNAs in lungs, and an increase of ROS levels in pulmonary arteries. BH4 levels, as well as eNOS expression, were enhanced in lungs from hypoxic WT mice (1.25 and 4 fold increase compared to normoxic WT mice, respectively). In pulmonary arteries from hypoxic WT mice, the contractile response to phenylephrine was about 1.8 greater than in those from normoxic WT mice. The use of ROS scavengers (PEG-SOD or catalase) and NOS inhibitor (L-NAME) revealed the involvement of ROS in hypoxia-induced hyper-reactivity to phenylephrine, and a loss of NO-dependent relaxation. Chronic treatment of hypoxic WT mice with the BH4 precursor sepiapterin preserved the vasorelaxant effect of NO. This treatment and the deletion of eNOS gene abolished the inhibitory effect of catalase on phenylephrine-induced contraction, and also attenuated hypoxia-induced right ventricular hypertrophy.These data show that chronic hypoxia induced an up-regulation of Nox isoforms and eNOS in lungs. They suggest that uncoupled eNOS participates to right ventricular hypertrophy and to alterations of vasomotor responses in pulmonary arteries in hypoxia-induced pulmonary hypertension. The weak increase in BH4 and the large over-expression of eNOS suggest the existence of compensatory mechanisms on BH4 synthesis, which may moderate eNOS dysfunction.Grants: Fondation de France, ANR, GRRC (E.D PhD grant

H020 Chronic expression of osteopontin in cardiomyocytes led to dilated cardiomyopathy

Osteopontin (OPN) is not expressed in healthy heart while, its expression is dramatically increased in cardiomyocytes and inflammatory cells during cardiomyopathies and heart failure. However its role in the development of heart diseases is not known.To understand whether OPN is involved in cardiomyopathy, we created a transgenic mouse (MHC-OPN) expressing recombinant OPN (rOPN) specifically in cardiomyocytes using aMHC promoter-directed OPN expression and tTA technology. In these mice, rOPN expression could be regulated by doxycyclin oral administration.After birth, MHC-OPN young mice were phenotypically indistinguishable from their littermate controls, but most of them died early between the 8 th and 15 th weeks after birth with a half life of 12 weeks. However, less than 10 % MHC-OPN mice survived and were still alive 30 weeks after birth. Inhibition of recombinant OPN expression by doxycyclin at the beginning of T cell infiltration (5 weeks after birth) or when DCM was initiated (11 weeks after birth) reduced myocarditis and thus avoided the early death.Electrocardiography demonstrated atrio-ventricular and intra ventricular defects. Moreover, echocardiography showed left ventricular dilation without hypertrophy and a systolic dysfunction, as indicated by reduced left ventricular fractional shortening (control mice: 29,8±2,0 %, n=13 ; and MHC-OPN mice: 13,7±4,0 %, n=5 ; T test p<0.05). In vitro histology confirmed that mice died because of a dilated cardiomyopathy associated with a strong fibrosis.By immunohistology, we demonstrated that OPN expression in cardiomyocytes induced an important cell infiltration including some macrophages and a large number of fibroblasts and activated CD4+ and CD8+ T cells.All together these experiments suggested that chronic OPN expression is required for DCM development inducing Tcell activation and thus a chronic myocarditis resulting in the dilated cardiomyopathy