Microparticles harboring sonic hedgehog: Role in angiogenesis regulation

Sonic Hedgehog (Shh) is a morphogen involved in embryonic development of nervous system. Also, it has been shown that recombinant Shh can modulate angiogenesis under ischemic conditions. However, angiogenic effects of endogenous Shh have not been completely elucidated. Using small membrane-derived vesicles expressing Shh (MPsShh+), we have shown that, although MPsShh+ decrease endothelial cell proliferation and migration, they are able to favour angiogenesis through the increase of both endothelial cell adhesion and expression of pro-angiogenenic factors. Activation of proteins implicated in cell adhesion, such as Rho A, as well as upregulation of pro-angiogenic factors were sensitive to inhibition of Shh pathway. Although whole composition of MPsShh+ needs to be characterized to understand potential effects of MPsShh+, these results highlight a new role of MPsShh+ in vascular pathophysiology and may have significant implication for therapy in pathologies associated with altered angiogenesis in order to re-address angiogenic switch

Engineered microparticles bearing the morphogen Sonic Hedgehog protect endothelial cells against actinomycin D-induced apoptosis

It has been reported that microparticles generated from T lymphocytes undergoing activation and apoptosis, bear the morphogen Sonic Hedgehog (MPsShh+), and possessed the dual ability to increase NO and reduce ROS productions. Here, we investigated whether MPsShh+ protected human umbical vein endothelial cells (HUVECs) against actinomycin D (ActD)-induced apoptosis. MPsShh+ were obtained by activation of human lymphocyte with phytohemagglutinin and then, by stimulation with phorbol-12 myristate-13 acetate and ActD. HUVECs were grown for 24 h in absence or presence of pro-apoptotic agent, ActD (1 μg/mL), and/or 10 μg protein/mL of MPsShh+. Apoptosis evaluation was based on flow cytometry, TUNEL labelling and cytochrome c release. We showed that MPsShh+ treatment significantly prevented HUVECs apoptosis evoked by ActD. Also, caspases inhibitor z-vad.fmk (50 μM ) reduced cell death either in presence or in absence of MPsShh+, indicating the implication of caspases in ActD-induced apoptosis. To investigate the implication of Shh pathway in this effect, its agonist SAG and its antagonist CUR61414 were tested. SAG reduced apoptosis in a dose-dependent manner; by itself, CUR61414 had no effect on its own but abolished the antiapoptotic effect of MPsShh, revealing a contribution of Shh pathway. In contrast, MPsShh+ were still able to reduce apoptosis in the presence of NO synthase inhibitor, L-NA (100 μM ), or when the PI3-kinase and ERK were inhibited with LY294002 (10 μM ) and U0126 (10 μM ) respectively, showing that these pathways were not associated with protection against apoptosis. Besides, we explored changes in ROS production at different times, by electronic paramagnetic resonance. ROS levels were increased in ActD-treated cells at 2 h and 10 h. This elevation was prevented by MPsShh+ only at 2 h. When sources of ROS, xanthine oxidase, NAD(P)H oxidase and respiratory chain complex I, were inhibited using allopurinol (50 μM ), apocynin (100 μM ) and rotenone (5 μM), respectively, we found that only rotenone reduced ActD-induced apoptosis. Also, the superoxide dismutase (SOD) mimetic, MnTMPyP (100 μM ), reduced ActD-evoked cell death and the protective effect of MPsShh+. These results indicate that, under these experimental conditions, MPsShh+ may act in the early phase of apoptosis at mitochondrial level and behave as a SOD mimetic. These findings provide additional mechanisms by which MPsShh+ exert their vasculoprotective effects, preserving integrity of endothelial monolayer. Supported by ANR-07-PHYSIO-010-01

The role of Smoothened and Hh signaling in neovascularization

New vessel formation plays a key role not only in physiological processes such as embryonic development and wound repair but also during several pathological situations. In this respect, favoring neovascularization represents a promising therapeutic approach that would allow inducing tissue repair. Among the candidate proteins able to modulate neovascularization, evidence show that the administration of recombinant hedgehog (Hh) protein, gene, or cell therapy based on Hh transfer or using extracellular vesicles as vectors enhance new vessel formation. Here, we summarized the role of Hh pathway on angiogenesis and its therapeutic potential during myocardial infarction and diabetes

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

Lymphocytic microparticles confer endothelial protection through activation of antioxidant defenses

Oxidative stress plays an important role in the initial occurrence of cardiovascular diseases. Previous studies demonstrated that microparticles (MPs) released from activated and apoptotic lymphocytes improve endothelial function due to their ability to decrease reactive oxygen species (ROS). Here, we evaluated the capacity of these MPs to modulate ROS production during actinomicyn D (ActD)-induced apoptosis in human umbilical vein endothelial cells (HUVECs). MPs were generated from T lymphocytes undergoing activation with phytohemagglutinin and then, by stimulation with phorbol-12 myristate-13 acetate and ActD. HUVECs were grown for 24h in absence or presence of ActD (1g/ml), and/or 10Pg protein/ml of MPs. Once evaluated that MPs are able to prevent ActD-evoked apoptosis, we investigated the time course of modifcations in ROS production (i.e. 1, 2, 4, 6, 8, 10 and 24h) by electron paramagnetic resonance. ActD enhances ROS production after 2h and 10h of treatment that was prevented by MPs after 2h but not at 10h, indicating that they act during the early phase of apoptosis. Interestingly at 24h, superoxide dismutase (SOD) mimetic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP) decreases cell death and inhibition of apoptosis induced by MPs, suggesting that MPs may behave as an SOD mimetic. Although these MPs carry functionally active extracellular SOD and catalase, as demonstrated by western blot analysis, treatment of HUVECs with these MPs does not modify expression of these enzymes. Under the same experimental conditions, MPs increase the expressions of other antioxidant enzymes such as Cu/Zn SOD and Mn SOD in HUVECs that may explain the protective effects induced by MPs. Thus, the present study illustrates new mechanisms by which MPs from activated/apoptotic lymphocytes may confer endothelial protection under pathological conditions in which oxidative stress is enhanced by a subtle modulation of antioxidant defense mechanism

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

Sonic Hedgehog Pathway as a Target for Therapy in Angiogenesis-Related Diseases

Hedgehog (Hh) proteins belong to a class of morphogens involved in many biological processes during embryonic development; they are relatively silent during normal adult life although they may be recruited postnatally in response to tissue injury. Three secreted proteins have been identified: Sonic hedgehog (Shh), Desert hedgehog and Indian hedgehog. The interaction of Hh ligand with its receptor Patched-1 triggers the activation of smoothened and initiates transduction events that lead to the regulation of transcriptional factors belonging to the Gli family. Hh pathway orchestrates both coronary development and adult coronary neovascularisation by controlling the expression of multiple proangiogenic genes and anti-apoptotic cytokines. Shh pathway enhances the recruitment of endothelial progenitor cells in addition to the mechanisms described for other Hh and concurs to its myocardial protection. In cerebral ischemia, Hh mimicking molecules has been reported to limit damages caused by vessel occlusion. Besides, Shh carried by microparticles corrects endothelial injury through nitric oxide release. Anomalous activations of Hh pathway are implicated in various types of tumours including medulloblastoma, carcinoma of esophagus, stomach, pancreas and colon. Hh can influence angiogenesis in both positive and negative manner and they may have implication for therapeutic strategies to treat either ischemic or cancer diseases

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