Frizzled 4 regulates arterial network organization through noncanonical Wnt/planar cell polarity signaling.

RATIONALE: A growing body of evidence supports the hypothesis that the Wnt/planar cell polarity (PCP) pathway regulates endothelial cell proliferation and angiogenesis, but the components that mediate this regulation remain elusive. OBJECTIVE: We investigated the involvement of one of the receptors, Frizzled4 (Fzd4), in this process because its role has been implicated in retinal vascular development. METHODS AND RESULTS: We found that loss of fzd4 function in mice results in a striking reduction and impairment of the distal small artery network in the heart and kidney. We report that loss of fzd4 decreases vascular cell proliferation and migration and decreases the ability of the endothelial cells to form tubes. We show that fzd4 deletion induces defects in the expression level of stable acetylated tubulin and in Golgi organization during migration. Deletion of fzd4 favors Wnt noncanonical AP1-dependent signaling, indicating that Fzd4 plays a pivotal role favoring PCP signaling. Our data further demonstrate that Fzd4 is predominantly localized on the top of the plasma membrane, where it preferentially induces Dvl3 relocalization to promote its activation and α-tubulin recruitment during migration. In a pathological mouse angiogenic model, deletion of fzd4 impairs the angiogenic response and leads to the formation of a disorganized arterial network. CONCLUSIONS: These results suggest that Fzd4 is a major receptor involved in arterial formation and organization through a Wnt/PCP pathway.

Thrombocytopenia resulting from mutations in filamin A can be expressed as an isolated syndrome.

Filaminopathies A caused by mutations in the X-linked FLNA gene are responsible for a wide spectrum of rare diseases including 2 main phenotypes, the X-linked dominant form of periventricular nodular heterotopia (FLNA-PVNH) and the otopalatodigital syndrome spectrum of disorders. In platelets, filamin A (FLNa) tethers the principal receptors ensuring the platelet-vessel wall interaction, glycoprotein Ibα and integrin αIIbß3, to the underlying cytoskeleton. Hemorrhage, coagulopathy, and thrombocytopenia are mentioned in several reports on patients with FLNA-PVNH. Abnormal platelet morphology in 2 patients with FLNA-PVNH prompted us to examine a third patient with similar platelet morphology previously diagnosed with immunologic thrombocytopenic purpura. Her enlarged platelets showed signs of FLNa degradation in Western blotting, and a heterozygous missense mutation in FLNA was detected. An irregular distribution of FLNa within the total platelet population was shown by confocal microscopy for all 3 patients. In vitro megakaryocyte cultures showed an abnormal differentiation, including an irregular distribution of FLNa with a frayed aspect, the presence of enlarged α-granules, and an abnormal fragmentation of the cytoplasm. Mutations in FLNA may represent an unrecognized cause of macrothrombocytopenia with an altered platelet production and a modified platelet-vessel wall interaction.

Secreted frizzled-related protein-1 improves postinfarction scar formation through a modulation of inflammatory response.

OBJECTIVE: The inflammatory response after myocardial infarction plays a crucial role in the healing process. Lately, there is accumulating evidence that the Wnt/Frizzled pathway may play a distinct role in inflammation. We have shown that secreted frizzled-related protein-1 (sFRP-1) overexpression reduced postinfarction scar size, and we noticed a decrease in neutrophil infiltration in the ischemic tissue. We aimed to further elucidate the role of sFRP-1 in the postischemic inflammatory process. METHODS AND RESULTS: We found that in vitro, sFRP-1 was able to block leukocyte activation and cytokine production. We transplanted bone marrow cells (BMCs) from transgenic mice overexpressing sFRP-1 into wild-type recipient mice and compared myocardial healing with that of mice transplanted with wild-type BMCs. These results were compared with those obtained in transgenic mice overexpressing sFRP-1 specifically in endothelial cells or in cardiomyocytes to better understand the spatiotemporal mechanism of the sFRP-1 effect. Our findings indicate that when overexpressed in the BMCs, but not in endothelial cells or cardiomyocytes, sFRP-1 was able to reduce neutrophil infiltration after ischemia, by switching the balance of pro- and antiinflammatory cytokine expression, leading to a reduction in scar formation and better cardiac hemodynamic parameters. CONCLUSION: sFRP-1 impaired the loop of cytokine amplification and decreased neutrophil activation and recruitment into the scar, without altering the neutrophil properties. These data support the notion that sFRP-1 may be a novel antiinflammatory factor protecting the heart from damage after myocardial infarction.

Mapping 3-dimensional neovessel organization steps using micro-computed tomography in a murine model of hindlimb ischemia-brief report.

OBJECTIVE: Studying the mechanisms of neovascularization and evaluating the effects of proangiogenic strategies require accurate analysis of the neovascular network. We sought to evaluate the contribution of the microcomputed tomography (mCT) providing high-resolution 3-dimensional (3D) structural data, to a better comprehension of the well-studied mouse hindlimb postischemic neovascularization. METHODS AND RESULTS: We showed a predominant arteriogenesis process in the thigh and a predominant angiogenesis-related process in the tibiofibular region, in response to ischemia during the first 15 days. After 15 days, mCT quantitative analysis reveals a remodeling of arterial neovessels and a regression depending on the restoration of the blood flow. We provided also new mCT data on the rapid and potent angiogenic effects of mesenchymal stem cell therapy on vessel formation and organization. We discussed the contribution of this technique compared with or in addition to data generated by the more conventional approaches. CONCLUSIONS: This study demonstrated that optimized mCT is a robust method for providing new insights into the 3D understanding of postischemic vessel formation.

Secreted frizzled-related protein-1 enhances mesenchymal stem cell function in angiogenesis and contributes to neovessel maturation.

Mesenchymal stem cell (MSC) transplantation offers a great angiogenic opportunity in vascular regenerative medicine. The canonical Wnt/beta-catenin signaling pathway has been demonstrated to play an essential role in stem cell fate. Recently, genetic studies have implicated the Wnt/Frizzled (Fz) molecular pathway, namely Wnt7B and Fz4, in blood growth regulation. Here, we investigated whether MSC could be required in shaping a functional vasculature and whether secreted Frizzled-related protein-1 (sFRP1), a modulator of the Wnt/Fz pathway, could modify MSC capacities, endowing MSC to increase vessel maturation. In the engraftment model, we show that murine bone marrow-derived MSC induced a beneficial vascular effect through a direct cellular contribution to vascular cells. MSC quickly organized into primitive immature vessel tubes connected to host circulation; this organization preceded host endothelial cell (EC) and smooth muscle cell (SMC) recruitment to later form mature neovessel. MSC sustained neovessel organization and maturation. We report here that sFRP1 forced expression enhanced MSC surrounding neovessel, which was correlated with an increase in vessel maturation and functionality. In vitro, sFRP1 strongly increased platelet-derived growth factor-BB (PDGF-BB) expression in MSC and enhanced beta-catenin-dependent cell-cell contacts between MSC themselves and EC or SMC. In vivo, sFRP1 increased their functional integration around neovessels and vessel maturation through a glycogen synthase kinase 3 beta (GSK3beta)-dependent pathway. sFRP1-overexpressing MSC compared with control MSC were well elongated and in a closer contact with the vascular wall, conditions required to achieve an organized mature vessel wall. We propose that genetically modifying MSC to overexpress sFRP1 may be potentially effective in promoting therapeutic angiogenesis/arteriogenesis processes. Disclosure of potential conflicts of interest is found at the end of this article.

Association between semicarbazide-sensitive amine oxidase, a regulator of the glucose transporter, and elastic lamellae thinning during experimental cerebral aneurysm development: laboratory investigation.

OBJECT: Amine oxidases play a key role in the polymerization and cross-linking of the collagens and elastic lamellae of the arterial wall. The loss of elastic lamellae integrity is one of the first steps in the genesis of a cerebral aneurysm. The authors investigated the relation between semicarbazide-sensitive amine oxidase (SSAO) and the organization of the cerebral arterial wall during aneurysm development. METHODS: Intracranial aneurysms were induced in rats via unilateral carotid artery ligation and renovascular hypertension. This modified Hashimoto model was used to create elevated blood pressure associated with shear stress in cerebral arteries. The authors immunohistologically investigated some markers of the extracellular matrix (Types I, III, and IV collagen and elastin), vascular smooth muscle cell differentiation (smooth muscle myosin heavy chain [sm-MHC], alpha-smooth muscle actin, and desmin), and amine oxidases (SSAO and lysyl oxidase [LOX]) in the cerebral arterial wall in control and treated rats 1, 2, 3, 4, and 6 months after the surgical procedure. RESULTS: The authors found severe disorganization and thinning of the elastic lamellae and a dramatic reduction in SSAO activity and immunostaining during cerebral aneurysm development. In contrast, LOX markers were slightly increased. Elastic lamellae thinning was highly correlated with decreases in SSAO (r = 0.76, p < 0.0001). There was also a correlation between sm-MHC and SSAO levels. CONCLUSIONS: The data suggested that cerebral hemodynamic modifications induce decreases in SSAO activity resulting in cell dedifferentiation and inducing dysregulation of glucose transport.

Epicardial deposition of endothelial progenitor and mesenchymal stem cells in a coated muscle patch after myocardial infarction in a murine model.

OBJECTIVES: To assess, using an in vivo engraftment strategy combining bone marrow cell (BMC) transplantation and tissue cardiomyoplasty, the functional outcome of distinct vascular progenitor cell therapy (endothelial progenitor (EPC) and mesenchymal stem (MSC) cells) at distance of myocardium infarction (MI). The study was also designed to test whether scaffold mixing progenitors with unfractionated BMC could improve progenitor recruitment in the damaged myocardium. METHODS: To track engrafted progenitor cells in vivo, cultured murine MSC and EPC were transduced with eGFP lentiviruses. Thirty days after cryogenical induction of MI, C57BL/6J mice were randomized to receive muscle patch placement coated or not (control group), labeled EPC or MSC mixed to the ration of 1:10, or not with unfractionated BMC. Two weeks after transplantation, cardiac function was recorded and heart sections were examined to detect GFP-labeled progenitor cells and analyze cell differentiation. RESULTS: This study showed that either type of mono cell therapy improved angiogenesis and cell survival in the scar but only MSC exhibited the capacity to invade the scar. We found no evidence of myocardial or vascular regeneration from progenitor cells. Engraftment of the progenitors/unfractionated BMC mix increased repopulation and thickness of the scar. CONCLUSION: Combined therapy with unfractionated BMC and expanded MSC appeared thus promising for scar repopulation.

Semicarbazide-sensitive amine oxidase in annulo-aortic ectasia disease: relation to elastic lamellae-associated proteins.

Lysyl oxidases (Lox), which are members of the amine oxidase family, are involved in the maturation of elastic lamellae and collagen fibers. Modifications of amine oxidases in idiopathic annulo-aortic ectasia disease (IAAED) have never been investigated. Our aim was to examine the expression of several proteins that might interfere with elastic fiber organization in control (n=10) and IAAED (n=18) aortic tissues obtained at surgery. Expression of amine oxidases and semicarbazide-sensitive amine oxidase (SSAO), and cellular phenotypic markers were examined by immunohistopathology and confocal microscopy. The expression of these proteins was assessed in relation to clinical and histomorphological features of the arterial wall. In control aorta, SSAO staining was expressed along elastic lamellae, whereas in aneurysmal areas of IAAED, SSAO was markedly decreased, in association with severe disorganization of elastic lamellae. Smooth muscle myosin heavy chain was also decreased in IAAED compared with controls, indicating smooth muscle cell dedifferentiation. Multiple regression analysis showed that elastic lamellar thickness (ELT) was correlated positively with the SSAO:elastin ratio and negatively with the Lox:elastin ratio, and that the clinical features of IAAED (aneurysm, thoracic aorta diameter, and aortic insufficiency) were positively correlated with ELT but not with SSAO. The relationship between SSAO expression and ELT suggests that this amine oxidase may be involved in elastic fiber organization. However, in advanced IAAED, the deficit in SSAO expression could be secondary to the decrease and fragmentation of elastic fibers and/or to vascular smooth muscle cell dedifferentiation.

Regulation of endothelial cell cytoskeletal reorganization by a secreted frizzled-related protein-1 and frizzled 4- and frizzled 7-dependent pathway: role in neovessel formation.

Consistent with findings of Wnt pathway members involved in vascular cells, a role for Wnt/Frizzled signaling has recently emerged in vascular cell development. Among the few Wnt family members implicated in vessel formation in adult, Wnt7b and Frizzled 4 have been shown as involved in vessel formation in the lung and in the retina, respectively. Our previous work has shown a role for secreted Frizzled-related protein-1 (sFRP-1), a proposed Wnt signaling inhibitor, in neovascularization after an ischemic event and demonstrated its role as a potent angiogenic factor. However the mechanisms involved have not been investigated. Here, we show that sFRP-1 treatment increases endothelial cell spreading on extracellular matrix as revealed by actin stress fiber reorganization in an integrin-dependent manner. We demonstrate that sFRP-1 can interact with Wnt receptors Frizzled 4 and 7 on endothelial cells to transduce downstream to cellular machineries requiring Rac-1 activity in cooperation with GSK-3beta. sFRP-1 overexpression in endothelium specifically reversed the inactivation of GSK-3 beta and increased neovascularization in ischemia-induced angiogenesis in mouse hindlimb. This study illustrates a regulated pathway by sFRP-1 involving GSK-3beta and Rac-1 in endothelial cell cytoskeletal reorganization and in neovessel formation.

Modifications of arterial phenotype in response to amine oxidase inhibition by semicarbazide.

Semicarbazide-sensitive amine oxidase (SSAO)-deficient mice present no alteration in elastin cross-linking processes and carotid mechanical properties. In contrast, previous studies have shown that SSAO inhibitors induced marked anomalies in arterial structure and function. The aim of the present study was to examine the effect of semicarbazide (SCZ), an efficient SSAO inhibitor, on the arterial phenotype of the carotid artery in relation to modulation of SSAO and lysyl oxidase activities in growing rats. We first show that after 6 weeks of SCZ treatment (100 mg/kg per day), SSAO activity was reduced by 90%, whereas lysyl oxidase activity was only partially inhibited (<60%) in carotid artery, compared with controls. There was significant growth inhibition and no difference in mean arterial pressure but an increase in pulse pressure with a smaller arterial diameter in SCZ-treated rats. SCZ decreased aortic insoluble elastin without a change in total collagen. In addition, extracellular proteins other than insoluble elastin and collagen were increased in SCZ-treated rats. All of the elastic lamellae presented globular masses along their periphery, and focal disorganization was observed in the ascending aorta. Carotid artery mechanical strength was lower in SCZ-treated rats, and the elastic modulus-wall stress curve was shifted leftward compared with controls, indicating increased stiffness. Thus, SCZ modifies arterial geometry and mechanical properties, alters elastic fiber structure, and reduces the content of cross-linked elastin. Because these abnormalities are essentially absent in SSAO-deficient mice, our results suggest that lysyl oxidase inhibition is responsible for the major part of the vascular phenotype of SCZ-treated rats.

Activation of the Ca(2+)/calcineurin/NFAT2 pathway controls smooth muscle cell differentiation.

Cellular mechanisms controlling smooth muscle cells (SMCs) phenotypic modulation are largely unknown. Intracellular Ca2+ movements are essential to ensure SMC functions; one of the roles of Ca2+ is to regulate calcineurin, which in turn induces nuclear localization of the nuclear factor of activated T-cell (NFAT). In order to investigate, during phenotypic differentiation of SMCs, the effect of calcineurin inhibition on NFAT2 nuclear translocation, we used a culture model of SMC differentiation in serum-free conditions. We show that the treatment of cultured SMC with the calcineurin inhibitor cyclosporine A induced their dedifferentiation while preventing their differentiation. These findings suggest that nuclear translocation of NFAT2 is dependent of calcineurin activity during the in vitro SMC differentiation kinetic and that the nuclear presence of NFAT2 is critical in the acquisition and maintenance of SMC differentiation.