Raffaella Soleti

Microparticles harboring Sonic Hedgehog promote angiogenesis through the upregulation of adhesion proteins and proangiogenic factors

Microparticles (MPs) are small fragments generated from the plasma membrane after cell stimulation or apoptosis. We have recently shown that MPs harboring the morphogen Sonic Hedgehog (MPs(Shh+)) correct endothelial injury by release of nitric oxide from endothelial cells [Agouni, Mostefai, Porro, Carusio, Favre, Richard, Henrion, Martínez and Andriantsitohaina (2007) FASEB J., 21, 2735-2741]. Here, we show that MPs(Shh+) induce the formation of capillary-like structures in an in vitro model using human endothelial cells, although they inhibited cell migration. Besides, MPs(Shh+) regulate cell proliferation. Both cell adhesion and expression of proteins involved in this process such as Rho A and phosphorylation of focal-activated kinase were increased by MPs(Shh+), via a Rho-associated coiled-coil-containing protein kinase inhibitor-sensitive pathway. We demonstrate that MPs(Shh+) increase messenger RNA and protein levels of proangiogenic factors as measured by quantitative reverse transcription-polymerase chain reaction and western blot. In spite of vascular endothelial growth factor expression, conditioned media from endothelial cells treated avec MPs(Shh+) reduces angiogenesis. Interestingly, the effects induced by MPs(Shh+) on the formation of capillary-like structures, expression of adhesion molecules and proangiogenic factors were reversed after silencing of the Shh receptor, using small interfering RNA or when Sonic Hedgehog (Shh) signaling was pharmacologically inhibited with cyclopamine. Taken together, we show that Shh carried by MPs(Shh+) regulate angiogenesis probably through both a direct and an indirect mechanisms, and we propose that MPs harboring Shh may contribute to the generation of a vascular network in pathologies associated with tumor growth.

Microparticles carrying Sonic hedgehog favor neovascularization through the activation of nitric oxide pathway in mice.

Background Microparticles (MPs) are vesicles released from plasma membrane upon cell activation and during apoptosis. Human T lymphocytes undergoing activation and apoptosis generate MPs bearing morphogen Shh (MPsShh+) that are able to regulate in vitro angiogenesis. Methodology/Principal Findings Here, we investigated the ability of MPsShh+ to modulate neovascularization in a model of mouse hind limb ischemia. Mice were treated in vivo for 21 days with vehicle, MPsShh+, MPsShh+ plus cyclopamine or cyclopamine alone, an inhibitor of Shh signalling. Laser doppler analysis revealed that the recovery of the blood flow was 1.4 fold higher in MPsShh+-treated mice than in controls, and this was associated with an activation of Shh pathway in muscles and an increase in NO production in both aorta and muscles. MPsShh+-mediated effects on flow recovery and NO production were completely prevented when Shh signalling was inhibited by cyclopamine. In aorta, MPsShh+ increased activation of eNOS/Akt pathway, and VEGF expression, being inhibited by cyclopamine. By contrast, in muscles, MPsShh+ enhanced eNOS expression and phosphorylation and decreased caveolin-1 expression, but cyclopamine prevented only the effects of MPsShh+ on eNOS pathway. Quantitative RT-PCR revealed that MPsShh+ treatment increased FGF5, FGF2, VEGF A and C mRNA levels and decreased those of α5-integrin, FLT-4, HGF, IGF-1, KDR, MCP-1, MT1-MMP, MMP-2, TGFβ1, TGFβ2, TSP-1 and VCAM-1, in ischemic muscles. Conclusions/Significance These findings suggest that MPsShh+ may contribute to reparative neovascularization after ischemic injury by regulating NO pathway and genes involved in angiogenesis.

Systems biology of antioxidants.

Understanding the role of oxidative injury will allow for therapy with agents that scavenge ROS (reactive oxygen species) and antioxidants in the management of several diseases related to free radical damage. The majority of free radicals are generated by mitochondria as a consequence of the mitochondrial cycle, whereas free radical accumulation is limited by the action of a variety of antioxidant processes that reside in every cell. In the present review, we provide an overview of the mitochondrial generation of ROS and discuss the role of ROS in the regulation of endothelial and adipocyte function. Moreover, we also discuss recent findings on the role of ROS in sepsis, cerebral ataxia and stroke. These results provide avenues for the therapeutic potential of antioxidants in a variety of diseases.

Modulation of mitochondrial capacity and angiogenesis by red wine polyphenols via estrogen receptor, NADPH oxidase and nitric oxide synthase pathways.

Red wine polyphenolic compounds (RWPC) are reported to exert vasculoprotective properties on endothelial cells, involving nitric oxide (NO) release via a redox-sensitive pathway. This NO release involves the activation of the estrogen receptor-alpha (ERα). Paradoxical effects of a RWPC treatment occur in a rat model of post-ischemic neovascularization, where a low-dose is pro-angiogenic while a higher dose is anti-angiogenic. NO and ERα are key regulators of mitochondrial capacity, and angiogenesis is a highly energetic process associated with mitochondrial biogenesis. However, whether RWPC induces changes in mitochondrial capacity has never been addressed. We investigated the effects of RWPC at low (10(-4)g/l, LCP) and high concentration (10(-2)g/l, HCP) in human endothelial cells. Mitochondrial respiration, expression of mitochondrial biogenesis factors and mitochondrial DNA content were assessed using oxygraphy and quantitative PCR respectively. In vitro capillary formation using ECM gel(®) was also performed. Treatment with LCP increased mitochondrial respiration, with a maximal effect achieved at 48h. LCP also increased expression of several mitochondrial biogenesis factors and mitochondrial DNA content. In contrast, HCP did not affect these parameters. Furthermore, LCP modulated both mitochondrial capacity and angiogenesis through mechanisms sensitive to ER, NADPH oxidase and NO-synthase inhibitors. Finally, the inhibition of mitochondrial protein synthesis abolished the pro-angiogenic capacity of LCP. These results suggest a possible association between the modulation of mitochondrial capacity by LCP and its pro-angiogenic activity. These data provide evidence for a role of mitochondria in the regulation of angiogenesis by RWPC.

Microparticles from apoptotic monocytes enhance nitrosative stress in human endothelial cells.

Microparticles are membrane vesicles with procoagulant and proinflammatory properties released during cell activation or apoptosis. Microparticles from monocytes have been implicated in atherosclerosis and vascular inflammation, but their direct effects on endothelial cells are not completely elucidated. The present study was designed to dissect the signaling pathways of monocytic microparticles in endothelial cells with respect to both NO pathway and reactive oxygen species. Microparticles were produced by treatment of human monocytic cell line THP‐1 with the apoptotic agent VP‐16. Human endothelial cells were treated with monocytic microparticles and then, we studied their effects on nitrosative and oxidative stresses. Incubation of human endothelial cells with microparticles enhanced the production of NO without affecting superoxide anions generation. Microparticles did not affect endothelial NO synthase expression and its phosphorylation. Interestingly, microparticles decreased caveolin‐1 expression and increased its phosphorylation. Inhibition of PI‐3‐kinase or MEK1/2 reversed the effects of microparticles on caveolin‐1 expression but not its phosphorylation. Moreover, microparticles increased nitration of several proteins, reflecting peroxynitrite production, which was prevented by blockade of PI‐3‐kinase pathway. In summary, monocyte microparticles active multiple pathways related to nitrosative stress in endothelial cells including both PI‐3‐kinase and ERK1/2 in the regulation of caveolin‐1 expression. These data underscore the pleiotropic effect of microparticles on endothelial cells and suggest that they probably play a critical role on vascular function.

Activation of Sonic hedgehog signaling in ventricular cardiomyocytes exerts cardioprotection against ischemia reperfusion injuries

Sonic hedgehog (SHH) is a conserved protein involved in embryonic tissue patterning and development. SHH signaling has been reported as a cardio-protective pathway via muscle repair–associated angiogenesis. The goal of this study was to investigate the role of SHH signaling pathway in the adult myocardium in physiological situation and after ischemia-reperfusion. We show in a rat model of ischemia-reperfusion that stimulation of SHH pathway, either by a recombinant peptide or shed membranes microparticles harboring SHH ligand, prior to reperfusion reduces both infarct size and subsequent arrhythmias by preventing ventricular repolarization abnormalities. We further demonstrate in healthy animals a reduction of QTc interval mediated by NO/cGMP pathway leading to the shortening of ventricular cardiomyocytes action potential duration due to the activation of an inward rectifying potassium current sharing pharmacological and electrophysiological properties with ATP-dependent potassium current. Besides its effect on both angiogenesis and endothelial dysfunction we demonstrate here a novel cardio-protective effect of SHH acting directly on the cardiomyocytes. This emphasizes the pleotropic effect of SHH pathway as a potential cardiac therapeutic target.

Internalization and induction of antioxidant messages by microvesicles contribute to the antiapoptotic effects on human endothelial cells

Microvesicles are plasma membrane-derived fragments released from various cell types during activation and/or apoptosis and posses the ability to deliver biological information between cells. Microvesicles generated from T lymphocytes undergoing activation and apoptosis bear the morphogen Sonic Hedgehog, and exert a beneficial potential effect on the cardiovascular system through their dual capacity to increase nitric oxide and reduce reactive oxygen species production. This study investigated the effect of microvesicles on the apoptosis of human umbilical vein endothelial cells triggered by actinomycin D. Microvesicles prevented apoptosis induced by actinomycin D by modulating reactive oxygen species production: during the early phase of apoptosis, microvesicles might act directly as reactive oxygen species scavengers, owing to their ability to carry active antioxidant enzymes, catalase, and isoforms of the superoxide dismutase. Furthermore, their effects were associated with the ability to increase the expression of manganese-superoxide dismutase in endothelial cells, through the internalization process. Interestingly, microvesicles bearing Sonic Hedgehog induced cytoprotection in endothelial cells through the activation of the Sonic Hedgehog pathway. These findings provide additional evidence that microvesicles from T lymphocytes exert their vasculoprotective effects by promoting internalization and induction of antioxidant messages to the endothelial monolayer.

Sonic Hedgehog on Microparticles and Neovascularization

Neovascularization represents a pivotal process consisting in the development of vascular network during embryogenesis and adult life. Postnatally, it arises mainly through angiogenesis, which has physiological and pathological roles in health and disease. Blood vessel formation results as tightly regulated multistep process which needs coordination and precise regulation of the balance of proangiogenic and antiangiogenic factors. Sonic Hedgehog (SHH), a morphogen belonging to Hedgehog (HH) family proteins, is implicated in a remarkably wide variety of process, including vessel development. Recent evidence demonstrate that, in addition to the classic factors, microvesicles (MVs), both microparticles (MPs) and exosomes, small vesicles released distinct cellular compartments, are involved in modulation of neovascularization. MPs generated from T lymphocytes undergoing both activation and apoptosis harbor at their surface SHH and play a crucial role in modulation of neovascularization. They are able to modulate the different steps implicated in angiogenesis process in vitro and to enhance postischemic neovascularization in vivo. As the consequence, we suggest that the MPs carrying SHH contribute to generation of a vascular network and may represent a new therapeutic approach to treat pathologies associated with failed angiogenesis.

Apoptotic process in cystic fibrosis cells

Cystic fibrosis (CF) is a recessively inherited disease caused by genetic lesions in CF transmembrane conductance regulator (CFTR) gene. CF is characterized by exaggerated inflammation, progressive tissue damage, and chronic bacterial colonization, mainly in the respiratory tract. The mechanisms underlying these pathological changes are increasingly well understood. However, apoptotic dysfunction in CF disease is still debated since studies report controversial results. Nonetheless, it is clear that apoptosis participates to onset of pathology and concerns various types of cells with variable susceptibility. Apoptosis is a physiological process necessary for the preservation of homeostasis of epithelial organization and function for clearance of inflammatory cells. Increased susceptibility to apoptosis in epithelial cells and failed apoptosis in neutrophils would contribute to the self-perpetuating inflammatory cycle in CF. Also, retention of mutated CFTR in the endoplasmic reticulum participates to inflammation which may trigger apoptosis. Independently of the sensibility to apoptosis of CF cells, it has been shown that clearance of apoptotic cells, due in part to decrease in efferocytosis, is flawed and that accumulation of such cells may contribute to ongoing inflammation in CF patients. Despite great advance in understanding CF pathophysiology, there is still no cure for the disease. The most recent therapeutic strategies are directed to target CFTR protein using cell and gene therapy as well as pharmacotherapy.

Delphinidin inhibits VEGF induced-mitochondrial biogenesis and Akt activation in endothelial cells

Delphinidin, an anthocyanin present in red wine, has been reported to exert vasculoprotective properties on endothelial cells, including vasorelaxing and anti-apoptotic effects. Moreover, delphinidin treatment in a rat model of post-ischemic neovascularization has been described to exert anti-angiogenic property. Angiogenesis is an energetic process and VEGF-induced angiogenesis is associated with mitochondrial biogenesis. However, whether delphinidin induces changes in mitochondrial biogenesis has never been addressed. Effects of delphinidin were investigated in human endothelial cells at a concentration described to be anti-angiogenic in vitro (10(-2)g/l). mRNA expression of mitochondrial biogenesis factors, mitochondrial respiration, DNA content and enzyme activities were assessed after 48 h of stimulation. Delphinidin increased mRNA expression of several mitochondrial biogenesis factors, including NRF1, ERRα, Tfam, Tfb2m and PolG but did not affect neither mitochondrial respiration, DNA content nor enzyme activities. In presence of delphinidin, VEGF failed to increase mitochondrial respiration, DNA content, complex IV activity and Akt activation in endothelial cells. These results suggest a possible association between inhibition of VEGF-induced mitochondrial biogenesis through Akt pathway by delphinidin and its anti-angiogenic effect, providing a novel mechanism sustaining the beneficial effect of delphinidin against pathologies associated with excessive angiogenesis such as cancers.