Canan G. Nebigil

Serotonin is a novel survival factor of cardiomyocytes: mitochondria as a target of 5-HT2B-receptor signaling

Identification of factors regulating cardiomyocyte survival and growth is important to understand the pathogenesis of congenital heart diseases. Little is known about the molecular mechanism of cardiac functions triggered by serotonin. The link between signaling circuitry of external stimuli and the mitochondrial apoptotic machinery is of wide interest in cardiac diseases. Using cultured cardiomyocytes and 5‐hydroxytryptamine (5‐HT)2B‐receptor knockout mice as an animal model of dilated cardiomyopathy, for the first time we show that serotonin via the Gq‐coupled 5‐HT2B‐receptor protect cardiomyocytes against serum deprivation‐induced apoptosis as manifested by DNA fragmentation, nuclear chromatin condensation, and TUNEL labeling. Serotonin prevents cytochrome c release and caspase‐9 and ‐3 activation after serum deprivation via cross‐talks between phosphatidylinositol‐3 kinase/Akt and extracellular signal‐regulated kinase (ERK) 1/2 signaling pathways. Serotonin binding to 5‐HT2B‐receptor activates ERK kinases to inhibit Bax expression induced by serum deprivation. Serotonin via phosphatidylinositol‐3 kinase/Akt can activate NF‐κB that is required for the regulation of the mitochondrial adenine nucleotide translocator (ANT‐1). Parallel to these observations, ultrastructural analysis in the 5‐HT2B‐receptor knockout mice heart revealed pronounced mitochondrial defects in addition to altered mitochondrial enzyme activities (cytochrome oxidase and succinate dehydrogenase) and ANT‐1 and Bax expressions. These findings identify 5‐HT as a novel survival factor targeting mitochondria in cardiomyocytes.

Overexpression of the Serotonin 5-HT2B Receptor in Heart Leads to Abnormal Mitochondrial Function and Cardiac Hypertrophy

Background—Identification of factors regulating myocardial structure and function is important to understand the pathogenesis of heart disease. We previously reported that 5-HT2B receptor ablation in mice leads to dilated cardiomyopathy. In this study, we investigated the pathological consequence of overexpressing 5-HT2B receptors in heart in vivo. Methods and Results—We have generated transgenic mice overexpressing the Gq-coupled 5-HT2B receptor specifically in heart. We found that overexpression of 5-HT2B receptor in heart leads to ventricular hypertrophy as the result of increased cell number and size. Increased atrial natriuretic peptide and myosin heavy chain expression demonstrated activation of the molecular program for cardiac hypertrophy. Echocardiographic analysis indicated the presence of thickened ventricular free wall without alteration of the systolic function, showing that transgenic mice have compensated hypertrophy. Electron microscopic analysis revealed structural abnormalities including mitochondrial proliferation, as also manifested by histological staining. Transgenic mouse heart displayed a specific reduction in the expression levels of the adenine nucleotide translocator associated to increase in the succinate dehydrogenase and cytochrome C oxidase mitochondrial activities. Conclusions—Our results constitute the first genetic evidence that overexpression of the 5-HT2B receptor in the heart leads to compensated hypertrophic cardiomyopathy associated with proliferation of the mitochondria. This observation suggests a role for mitochondria in the hypertrophic signaling that is regulated by serotonin. These transgenic mice provide a new genetic model for hypertrophic heart disease.

Ablation of Serotonin 5-HT2B Receptors in Mice Leads to Abnormal Cardiac Structure and Function

Background—Identification of factors regulating myocardial structure and function is important to understand the pathogenesis of heart disease. Because little is known about the molecular mechanism of cardiac functions triggered by serotonin, the link between downstream signaling circuitry of its receptors and the heart physiology is of widespread interest. None of the serotonin receptor (5-HT1A, 5-HT1B, or 5-HT2C) disruptions in mice have resulted in cardiovascular defects. In this study, we examined 5-HT2B receptor-mutant mice to assess the putative role of serotonin in heart structure and function. Methods and Results—We have generated Gq-coupled 5-HT2B receptor-null mice by homologous recombination. Surviving 5-HT2B receptor-mutant mice exhibit cardiomyopathy with a loss of ventricular mass due to a reduction in number and size of cardiomyocytes. This phenotype is intrinsic to cardiac myocytes. 5-HT2B receptor-mutant ventricles exhibit dilation and abnormal organization of contractile elements, including Z-stripe enlargement and N-cadherin downregulation. Echocardiography and ECG both confirm the presence of left ventricular dilatation and decreased systolic function in the adult 5-HT2B receptor-mutant mice. Conclusions—Mutation of 5-HT2B receptor leads to a cardiomyopathy without hypertrophy and a disruption of intercalated disks. 5-HT2B receptor is required for cytoskeleton assembly to membrane structures by its regulation of N-cadherin expression. These results constitute, for the first time, strong genetic evidence that serotonin, via the 5-HT2B receptor, regulates cardiac structure and function.

Involvement of the serotonin 5-HT2B receptor in cardiac hypertrophy linked to sympathetic stimulation: control of interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha cytokine production by ventricular fibroblasts.

Background—The serotonergic 5-HT2B receptor regulates cardiomyocyte development and growth. A putative contribution of this receptor to fibroblast-dependent cardiac function has not been identified. Methods and Results—By mimicking sympathetic stimulation with chronic isoproterenol perfusion in vivo, we found that mice developed a cardiac hypertrophy, which was prevented by exposure to the 5-HT2B receptor antagonists SB206553 or SB215505 or in 5-HT2B receptor–knockout mice. The isoproterenol-induced hypertrophy was associated with an increase in the plasma levels of interleukin-1&bgr; and tumor necrosis factor-&agr; but not interleukin-6. In contrast, the plasma isoproterenol-induced cytokine increase was not observed in either 5-HT2B receptor–mutant or wild-type mice perfused with isoproterenol+SB206553. We demonstrated that stimulation of wild-type cardiac fibroblasts by isoproterenol markedly increased the production of the interleukin-6, interleukin-1&bgr;, and tumor necrosis factor-&agr; cytokines. Strikingly, we found that this isoproterenol-induced cytokine production was abolished by SB206553 or in 5-HT2B receptor–knockout fibroblasts. Serotonin also stimulated production of the 3 cytokines in wild-type fibroblasts, which was effectively reduced in 5-HT2B receptor–knockout fibroblasts. Conclusions—Our results demonstrate for the first time that 5-HT2B receptors are essential for isoproterenol-induced cardiac hypertrophy, which involves the regulation of interleukin-6, interleukin-1&bgr;, and tumor necrosis factor-&agr; cytokine production by cardiac fibroblasts.

Prohibitin Ligands in Cell Death and Survival: Mode of Action and Therapeutic Potential

Prohibitins (PHBs) are scaffold proteins that modulate many signaling pathways controlling cell survival, metabolism, and inflammation. Several drugs that target PHBs have been identified and evaluated for various clinical applications. Preclinical and clinical studies indicate that these PHB ligands may be useful in oncology, cardiology, and neurology, as well as against obesity. This review covers the physiological role of PHBs in health and diseases and current developments concerning PHB ligands.

Prokineticin Receptor-1 Induces Neovascularization and Epicardial-Derived Progenitor Cell Differentiation

Objective—Identification of novel factors that contribute to myocardial repair and collateral vessel growth hold promise for treatment of heart diseases. We have shown that transient prokineticin receptor-1 (PKR1) gene transfer protects the heart against myocardial infarction in a mouse model. Here, we investigated the role of excessive PKR1 signaling in heart. Methods and Results—Transgenic mice overexpressing PKR1 in cardiomyocytes displayed no spontaneous abnormalities in cardiomyocytes but showed an increased number of epicardial-derived progenitor cells (EPDCs), capillary density, and coronary arterioles. Coculturing EPDCs with H9c2 cardiomyoblasts overexpressing PKR1 promotes EPDC differentiation into endothelial and smooth muscle cells, mimicking our transgenic model. Overexpressing PKR1 in H9c2 cardiomyoblasts or in transgenic hearts upregulated prokineticin-2 levels. Exogenous prokineticin-2 induces significant outgrowth from neonatal and adult epicardial explants, promoting EPDC differentiation. These prokineticin-2 effects were abolished in cardiac explants from mice with PKR1-null mutation. Reduced capillary density and prokineticin-2 levels in PKR1-null mutant hearts supports the hypothesis of an autocrine/paracrine loop between PKR1 and prokineticin-2. Conclusion—Cardiomyocyte-PKR1 signaling upregulates its own ligand prokineticin-2 that acts as a paracrine factor, triggering EPDCs proliferation/differentiation. This study provides a novel insight for possible therapeutic strategies aiming at restoring pluripotency of adult EPDCs to promote neovasculogenesis by induction of cardiomyocyte PKR1 signaling.

Synthetic Analogue of Rocaglaol Displays a Potent and Selective Cytotoxicity in Cancer Cells: Involvement of Apoptosis Inducing Factor and Caspase-12

Flavaglines constitute a family of natural anticancer compounds. We present here 3 (FL3), the first synthetic flavagline that inhibits cell proliferation and viability (IC(50) approximately 1 nM) at lower doses than did the parent compound, racemic rocaglaol. Compound 3 enhanced doxorubicin cytotoxicity in HepG2 cells and retained its potency against adriamycin-resistant cell lines without inducing cardiomyocyte toxicity. Compound 3 induced apoptosis of HL60 and Hela cells by triggering the translocation of Apoptosis Inducing Factor (AIF) and caspase-12 to the nucleus. A fluorescent conjugate of 3 accumulated in endoplasmic reticulum (ER), suggesting that flavaglines bind to their target in the ER, where it triggers a cascade of events that leads to the translocation of AIF and caspase-12 to the nucleus and probably inhibition of eIF4A. Our studies highlight structural features critical to their antineoplastic potential and suggest that these compounds would retain their activity in cells refractory to caspase activation.

The prokineticin receptor-1 (GPR73) promotes cardiomyocyte survival and angiogenesis

Prokineticins are potent angiogenic factors that bind to two G protein‐coupled receptors to initiate their biological effects. We hypothesize that prokineticin receptor‐1 (PKR1/GPR73) signaling may contribute to cardiomyocyte survival or repair in myo‐cardial infarction. Since we showed that prokineticin‐2 and PKR1 are expressed in adult mouse heart and cardiac cells, we investigated the role of prokineticin‐2 on capillary endothelial cell and cardiomyocyte function. In cultured cardiac endothelial cells, prokineti‐cin‐2 or overexpression of PKR1 induces vessel‐like formation without increasing VEGF levels. In cardiomy‐ocytes and H9c2 cells, prokineticin‐2 or overexpressing PKR1 activates Akt to protect cardiomyocytes against oxidative stress. The survival and angiogenesis promoting effects of prokineticin‐2 in cardiac cells were com‐pletely reversed by siRNA‐PKR1, indicating PKR1 involvement. We thus, further investigated whether intramyocardial gene transfer of DNA encoding PKR1 may rescue the myocardium against myocardial infarction in mouse model. Transient PKR1 gene transfer after coronary ligation reduces mortality and preserves left ventricular function by promoting neovascularization and protecting cardiomyocytes without altering VEGF levels. In human end‐stage failing heart samples, reduced PKR1 and prokineticin‐2 transcripts and pro‐tein levels implicate a more important role for proki‐neticin‐2/PKR1 signaling in heart. Our results suggest that PKR1 may represent a novel therapeutic target to limit myocardial injury following ischemic events.—Urayama K., Guilini, C., Messaddeq, N., Hu, K., Steenman, M., Kurose, H., Ert, G., Nebigil C. G. The prokineticin receptor‐1 (GPR73) promotes cardiomyo‐cyte survival and angiogenesis FASEB J. 21, 2980–2993 (2007)

Cancer wars: natural products strike back.

Natural products have historically been a mainstay source of anticancer drugs, but in the 90s they fell out of favor in pharmaceutical companies with the emergence of targeted therapies, which rely on antibodies or small synthetic molecules identified by high throughput screening. Although targeted therapies greatly improved the treatment of a few cancers, the benefit has remained disappointing for many solid tumors, which revitalized the interest in natural products. With the approval of rapamycin in 2007, 12 novel natural product derivatives have been brought to market. The present review describes the discovery and development of these new anticancer drugs and highlights the peculiarities of natural product and new trends in this exciting field of drug discovery.

Divergent roles of prokineticin receptors in the endothelial cells: angiogenesis and fenestration

Prokineticins are secreted peptides that activate two G protein-coupled receptors: PKR1 and PKR2. Prokineticins induce angiogenesis and fenestration, but the cognate receptors involved in these functions are unknown. We hypothesized a role for prokineticin receptor signaling pathways and expression profiles in determining the selective effects of prokineticins on coronary endothelial cells (H5V). Activation of the PKR1/MAPK/Akt signaling pathway stimulates proliferation, migration, and angiogenesis in H5V cells, in which PKR1 predominates over PKR2. PKR1 was colocalized with Galpha(11) and was internalized following the stimulation of these cells with prokineticin-2. Knock down of PKR1 or Galpha(11) expression in H5V cells effectively inhibited prokineticin-2-induced vessel formation and MAPK/Akt activation, indicating a role for PKR1/Galpha(11) in this process. However, in conditions in which PKR2 predominated over PKR1, these cells displayed a fenestrated endothelial cell phenotype. H5V cells overexpressing PKR2 displayed large numbers of multivesicular bodies and caveolar clusters and a disruption of the distribution of zonula occluden-1 tight junction protein. Prokineticin-2 induced the colocalization of PKR2 with Galpha(12), and activated Galpha(12), which bound to zonula occluden-1 to trigger the degradation of this protein in these cells. Prokineticin-2 induced the formation of vessel-like structures by human aortic endothelial cells expressing only PKR1, and disorganized the tight junctions in human hepatic sinusoidal endothelial cells expressing only PKR2, confirming the divergent roles of these receptors. Our findings show the functional characteristics of coronary endothelial cells depend on the expression of PKR1 and PKR2 levels and the divergent signaling pathways used by these receptors.