Sonic hedgehog mediates a novel pathway of PDGF-BB-dependent vessel maturation.

Recruitment of mural cells (MCs), namely pericytes and smooth muscle cells (SMCs), is essential to improve the maturation of newly formed vessels. Sonic hedgehog (Shh) has been suggested to promote the formation of larger and more muscularized vessels, but the underlying mechanisms of this process have not yet been elucidated. We first identified Shh as a target of platelet-derived growth factor BB (PDGF-BB) and found that SMCs respond to Shh by upregulating extracellular signal-regulated kinase 1/2 and Akt phosphorylation. We next showed that PDGF-BB-induced SMC migration was reduced after inhibition of Shh or its signaling pathway. Moreover, we found that PDGF-BB-induced SMC migration involves Shh-mediated motility. In vivo, in the mouse model of corneal angiogenesis, Shh is expressed by MCs of newly formed blood vessels. PDGF-BB inhibition reduced Shh expression, demonstrating that Shh is a target of PDGF-BB, confirming in vitro experiments. Finally, we found that in vivo inhibition of either PDGF-BB or Shh signaling reduces NG2(+) MC recruitment into neovessels and subsequently reduces neovessel life span. Our findings demonstrate, for the first time, that Shh is involved in PDGF-BB-induced SMC migration and recruitment of MCs into neovessels and elucidate the molecular signaling pathway involved in this process.

Early activation of internal medial smooth muscle cells in the rabbit aorta after mechanical injury: relationship with intimal thickening and pharmacological applications.

1. Smooth muscle cells (SMC) participate in both inflammatory and dedifferentiation processes during atherosclerosis, as well as during mechanical injury following angioplasty. In the latter, we studied medial SMC differentiation and inflammation processes implicated early after de-endothelialization in relation to mechanical stresses. We hypothesized that activation of a subpopulation of SMC within the media plays a crucial role in the early phase of neointimal formation. 2. For this purpose, we used a rabbit model of balloon injury to study activation and differentiation of medial SMC in the early time after denudation and just before neointima thickening. Inflammation was evaluated by the expression of vascular cell adhesion molecule (VCAM)-1, integrin alpha4beta1 and nuclear factor (NF)-kB. Myosin isoforms and 2P1A2 antigen, a membrane protein expressed by rabbit dedifferentiated SMC, were used as markers of differentiation. 3. On day 2 after de-endothelialization, VCAM-1, alpha4beta1 and NF-kB were coexpressed by a well-defined subpopulation of SMC of the internal part of the media, in the vicinity of the blood stream. At the same time, the majority of SMC throughout the media expressed non-muscle myosin heavy chain-B (nm-MHC-B) and 2P1A2 antigen. On day 7, when intimal thickening appeared, SMC of the media were no longer activated, whereas some intimal SMC expressed the activation markers. Thus, after de-endothelialization, early dedifferentiation occurs in most of the medial SMC, whereas activation concerned only a subpopulation of SMC located in the internal media. Using the T-type voltage-operated calcium channel blocker mibefradil (0.1-1 micromol/L) in SMC culture, we showed that this agent exhibited an antiproliferative effect in a dose-dependent manner only on undifferentiated cells. 4. In conclusion, the results suggest that the activated SMC represent cells that are potentially able to migrate and participate in the intimal thickening process. Thus, the medial SMC inflammatory process, without any contribution of inflammatory cells, may represent a major mechanism underlying the development of intimal thickening following mechanical stress. In humans, inhibition of T-type calcium channels may be a tool to prevent the early proliferation step leading to neointimal formation.

Involvement of FrzA/sFRP-1 and the Wnt/frizzled pathway in ischemic preconditioning.

Phosphorylation and subsequent inactivation of glycogen synthase kinase (GSK)-3beta via the Akt/PI3-Kinase pathway during ischemic preconditioning (PC) has been shown to be cardioprotective. As FrzA/sFRP-1, a secreted antagonist of the Wnt/Frizzled pathway, is expressed in the heart and is able to decrease the phosphorylation of GSK-3beta in vitro on vascular cells, we examined its effect during PC using transgenic mouse overexpressing FrzA in cardiomyocytes (alpha-MHC promoter) under a conditional transgene expression approach (tet-off system). Overexpression of FrzA inhibited the increase in GSK-3beta phosphorylation as well as protein kinase C (PKC) epsilon activation in transgenic mice after PC as compared with littermates. Phospho-Akt (P-Akt), phospho-JNK, or the cytoplasmic beta-catenin levels were not modified, phospho-p38 (P-p38) was slightly increased in transgenic mice after PC as compared with littermates. FrzA transgenic mice displayed a larger infarct size and a greater worsening of cardiac function compared with littermates. All these differences were reversed by the addition of doxycycline. This study demonstrates for the first time that disruption of a beta-catenin independent Wnt/Frizzled pathway induces the activation of GSK-3beta and reverses the benefit of preconditioning.

Disruption of the elastin gene in adult Williams syndrome is accompanied by a paradoxical reduction in arterial stiffness.

Although the aetiology of Williams syndrome (WS) is related to elastin gene disruption, its pathogenesis remains unknown, particularly that of vascular lesions. The aim of the present study was to compare the elastic properties of three WS patients with age- and gender-matched normotensive and hypertensive controls. Common carotid arteries of WS patients had a higher distensibility, a thicker intima-media and a lower elastic modulus. Electron microscopy studies of one WS renal artery showed major abnormalities of the elastic fibres, which displayed a reticular structure and a thickening of the internal elastic lamina, whereas the ultrastructure of elastic fibres was normal in a control subadventitial muscular fibrodysplasia. In this WS arterial stenosis, we studied the expression patterns of several major smooth muscle (SM) phenotypic markers using immunofluorescence and used a normal renal artery as a control. In WS, SM-alpha-actin- and myosin-heavy-chain-positive cells contained low amounts of heavy caldesmon, and laminin-beta1 chain was expressed into the basement membranes, indicating a less differentiated phenotype. In conclusion, in WS patients, the carotid artery wall was abnormally distensible and thick, and major ultrastructural abnormalities of elastic fibres were observed in association with smooth muscle cell de-differentiation. These results indicate that the haplo-insufficiency of the elastin gene in WS patients leads to abnormal elastic fibre assembly within the media. Arterial wall hypertrophy found with a primary defect in elastin may represent a major factor responsible for increased distensibility. We suggest that, in WS, the increased proliferative response and the associated de-differentiation process represent two important mechanisms underlying the matrix accumulation and the development of arterial stenosis.