Catherine Moreau

Reduction of Infarct Size and Prevention of Cardiac Rupture in Transgenic Mice Overexpressing FrzA

Background—FrzA/sFRP-1, a secreted, frizzled-related protein and antagonist for the wnt/frizzled pathway, is expressed in the heart and vessels during mouse embryogenesis and adulthood. FrzA is involved in cell cycle control of vascular cells and angiogenesis. We assessed the hypothesis that FrzA could control the healing process after myocardial infarction (MI). Methods and Results—We demonstrated an upregulation of sFRP-1 and distinct wnt and fz member expression after MI. We established transgenic (Tg) mice that overexpress FrzA under a cytomegalovirus promoter and developed a model of MI by coronary artery ligation. FrzA reduced cardiac rupture after MI in Tg (6.5% versus 26.4% in controls; n=165, P <0.01). MI was smaller in Tg at each time point (18±10.8% of left ventricular circumference versus 30±14.2% in controls at day 30; P <0.001). Similar results were found in cryolesion-induced MI. Cardiac function was improved in Tg mice (3800±370 mm Hg/s dP/dtmax versus 2800±840 in controls; −2800±440 dP/dtmin versus −1800±211 in controls at day 15; P <0.001). Early leukocyte infiltration had decreased in Tg mice during the first week. Apoptotic index was decreased by 50% in Tg mice at day 7. Matrix metalloproteinase-2 and −9 activity was reduced in Tg mice at day 4, and collagen deposition in the scar was increased in Tg mice. Capillary density in the scar was higher in Tg mice (290±103 vessels/mm2 versus 104±43 in controls at day 15; P <0.001). Vessels were more muscularized, and mean lumen area was 3-fold higher in Tg animals. Conclusions—Overexpression of FrzA, through direct or indirect interaction with different phases of infarct healing, reduced infarct size and improved cardiac function.

Regulation of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in human vascular smooth muscle cells.

Vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin are inducible proteins involved in cell-cell adhesion. Immunohistochemical studies have indicated that human atherosclerotic plaques contain smooth muscle cells (SMCs) that express ICAM-1 and VCAM-1. Recently, we demonstrated that SMCs in culture express a functionally active cytokine-inducible ICAM-1. SMCs and mononuclear cells participate in the local accumulation of cytokines and related growth factors in atherosclerotic lesions. Therefore, we determined the effects of different cytokines and growth factors on mRNA content and cell surface expression of VCAM-1, ICAM-1, and E-selectin in cultured human aortic SMCs by Northern blotting, quantitative polymerase chain reaction amplification, and immunofluorescence flow cytometry. Under basal conditions of cultivation, both VCAM-1 mRNA and membrane expression of VCAM-1 were low and were induced very little by interleukin-1 beta (100 U/mL). Platelet-derived growth factor or transforming growth factor-beta decreased VCAM-1 mRNA basal expression. Treatment of SMCs with tumor necrosis factor-alpha (TNF-alpha) led to an increase in both VCAM-1 mRNA and cell surface expression for VCAM-1 in a dose- and time-dependent manner. Interferon-gamma induced a weak increase in VCAM-1 mRNA expression, with no synergistic effect on the stimulation by TNF-alpha. Various differences were noted between the expression of ICAM-1 and VCAM-1 genes, because interleukin-1 beta induced substantial amounts of ICAM-1 but not VCAM-1. The addition of interferon-gamma delays the time at which peak expression of ICAM-1 in response to TNF-alpha stimulation occurs. Under our conditions, we did not detect any expression of E-selectin by SMCs. These results suggest that cytokines regulate VCAM-1 and ICAM-1 expression on arterial SMCs and could play an important role in the pathophysiology of inflammatory and immune processes in atherosclerosis.

Hypoxia Preconditioned Mesenchymal Stem Cells Improve Vascular and Skeletal Muscle Fiber Regeneration After Ischemia Through a Wnt4-dependent Pathway

Mesenchymal stem cells (MSC) are multipotent postnatal stem cells, involved in the treatment of ischemic vascular diseases. We investigate the ability of MSC, exposed to short-term hypoxic conditions, to participate in vascular and tissue regeneration in an in vivo model of hindlimb ischemia. Transplantation of hypoxic preconditioned murine MSC (HypMSC) enhanced skeletal muscle regeneration at day 7, improved blood flow and vascular formation compared to injected nonpreconditioned MSC (NormMSC). These observed effects were correlated with an increase in HypMSC engraftment and a putative role in necrotic skeletal muscle fiber clearance. Moreover, HypMSC transplantation resulted in a large increase in Wnt4 (wingless-related MMTV integration site 4) expression and we demonstrate its functional significance on MSC proliferation and migration, endothelial cell (EC) migration, as well as myoblast differentiation. Furthermore, suppression of Wnt4 expression in HypMSC, abrogated the hypoxia-induced vascular regenerative properties of these cells in the mouse hindlimb ischemia model. Our data suggest that hypoxic preconditioning plays a critical role in the functional capabilities of MSC, shifting MSC location in situ to enhance ischemic tissue recovery, facilitating vascular cell mobilization, and skeletal muscle fiber regeneration via a paracrine Wnt-dependent mechanism.Mesenchymal stem cells (MSC) are multipotent postnatal stem cells, involved in the treatment of ischemic vascular diseases. We investigate the ability of MSC, exposed to short-term hypoxic conditions, to participate in vascular and tissue regeneration in an in vivo model of hindlimb ischemia. Transplantation of hypoxic preconditioned murine MSC (HypMSC) enhanced skeletal muscle regeneration at day 7, improved blood flow and vascular formation compared to injected nonpreconditioned MSC (NormMSC). These observed effects were correlated with an increase in HypMSC engraftment and a putative role in necrotic skeletal muscle fiber clearance. Moreover, HypMSC transplantation resulted in a large increase in Wnt4 (wingless-related MMTV integration site 4) expression and we demonstrate its functional significance on MSC proliferation and migration, endothelial cell (EC) migration, as well as myoblast differentiation. Furthermore, suppression of Wnt4 expression in HypMSC, abrogated the hypoxia-induced vascular regenerative properties of these cells in the mouse hindlimb ischemia model. Our data suggest that hypoxic preconditioning plays a critical role in the functional capabilities of MSC, shifting MSC location in situ to enhance ischemic tissue recovery, facilitating vascular cell mobilization, and skeletal muscle fiber regeneration via a paracrine Wnt-dependent mechanism.

FrzA, a Secreted Frizzled Related Protein, Induced Angiogenic Response

Background—The secreted frizzled related proteins (sFRP) are soluble proteins thought to interfere with the Wnt signaling. Our group previously demonstrated that one of these members, sFRP-1/FrzA, is strongly expressed during early phases of the vascularization process in embryonic vasculature and in the endothelium of arteries and capillaries in adults and modulated vascular cell proliferation. Methods and Results—Analysis of the expression of sFRP-1 during cyclic ovarian angiogenesis revealed that sFRP-1 is expressed during the formation of neovessels and becomes undetectable when the vasculature is fully maturated. We then studied the role of FrzA in several distinct angiogenic models. FrzA induced angiogenesis in a chick chorioallantoic membrane model. Moreover, gene transfer of AdFrzA in grafted mesenchymal and glioma cells increased vessel density and tumor growth. FrzA induced formation of vessels, which were enlarged, longer, and appeared to be more mature compared with vessels formed under control treatments. In vitro, FrzA increased migration and tube formation of endothelial cells and seemed to protect them from apoptosis. FrzA-angiogenic effect in vitro was independent of vascular endothelial growth factor, fibroblast growth factor-2, or angiopiotin-1 induction and Akt activation. In contrast, FrzA decreased glycogen synthase kinase-3 phosphorylation. Conclusions—These results showed that FrzA has proangiogenic effects and suggest that Wnt signaling may be involved in normal differentiation as well as in the pathological development of vasculature.

Tumor necrosis factor-alpha stimulates ICAM-1 expression in human vascular smooth muscle cells.

Human atherosclerotic plaques contain numerous smooth muscle cells (SMCs) that express intercellular adhesion molecule-1 (ICAM-1). Expression of ICAM-1 in different cells is known to be regulated by tumor necrosis factor-alpha (TNF-alpha), which has recently been found to be present in the intimal thickening of human arteries. Therefore, we studied the effect of TNF-alpha on ICAM-1 mRNA content and surface expression in cultured human aortic SMCs by using the methods of Northern blotting and immunofluorescence flow cytometry. Under basal conditions of cultivation, ICAM-1 mRNA was not revealed in SMCs. However, treatment of the cells with recombinant human TNF-alpha induced substantial levels of ICAM-1 mRNA. The content of ICAM-1 on the surface of SMCs also increased in a dose- and time-dependent manner after incubation with TNF-alpha. Twenty-four hours of treatment with 10 ng/mL TNF-alpha led to an approximately 10-fold increase in ICAM-1 surface expression in the SMCs. Under the same conditions, pretreatment of SMCs with TNF-alpha resulted in a twofold increase of their adhesiveness for monocytes. In the presence of anti-ICAM-1 monoclonal antibody 10F3, monocyte adhesion to TNF-alpha-pretreated SMCs was significantly inhibited, suggesting that the observed monocyte-SMC interaction involved the ICAM-1 expressed on SMC surfaces as a result of TNF-alpha stimulation. These results led us to propose that TNF-alpha may act a regulator of functional ICAM-1 expression on the SMC surface and thus can increase the possibility of interactions between mononuclear cells and SMCs in atherosclerotic plaques.

Vitronectin Expression and Interaction With Receptors in Smooth Muscle Cells From Human Atheromatous Plaque

Vitronectin (VN) is a plasma glycoprotein that promotes cell attachment and induces migration of human smooth muscle cells (SMCs) in culture. VN has been observed to accumulate in human atherosclerotic plaques, although its origin and role in atherosclerosis are not yet established. In the present experiments, synthesis of VN by intimal cells and its colocalization with receptors, alphavbeta3 and alphavbeta5, were studied by in situ hybridization and immunohistochemistry on 15 human atherosclerotic plaques from carotid arteries obtained after surgery. Strong VN protein and mRNA expression was observed in the intima and in the media. In the intima, VN mRNA expression was colocalized with SMCs, indicating that these cells produce VN, which may account for its accumulation in atherosclerotic plaques. In SMCs in culture, immunoprecipitation after metabolic labeling demonstrated that human SMCs do synthesize vitronectin. Confocal microscopic examination showed that VN colocalized with its receptors, alphavbeta3 and alphavbeta5, in the atherosclerotic intima. However, the distribution of the VN receptors on SMCs in culture in contact with VN was different. These observations suggest that VN plays various parts in atherogenesis via different SMC membrane receptors.

Adult reserve stem cells and their potential for tissue engineering

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories, have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher order. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve, precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including, tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50–70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.

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/β‐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 β‐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 (GSK3β)‐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.

The Integrin Very Late Antigen-4 Is Expressed in Human Smooth Muscle Cell Involvement of α4 and Vascular Cell Adhesion Molecule-1 During Smooth Muscle Cell Differentiation

Vascular cell adhesion molecule-1 (VCAM-1) and its counterreceptor, the integrin very late antigen-4 (VLA-4), have recently been identified in smooth muscle cells during intimal thickening in humans and in newly forming vessels during ontogeny in mice, respectively. We examined the coexpression of VCAM-1 and the alpha 4 integrin subunit in human smooth muscle cells. The expression of VCAM-1 and alpha 4 subunit were studied during development of the aorta. In the 10-week-old human fetal aorta, VCAM-1 and alpha 4 were strongly expressed in smooth muscle cells. Their expression was dramatically reduced within the 24th week of gestation and disappeared in the adult aortic media. However, smooth muscle cells from intimal atherosclerotic thickening of adult aorta reexpressed both VCAM-1 and alpha 4. In a culture model mimicking smooth muscle differentiation, VCAM-1 mRNA and protein and alpha 4 integrin protein were coexpressed with smooth muscle-specific variants of cytoskeletal and contractile proteins, smooth muscle myosin heavy chain, caldesmon heavy chain, and desmin. Treatment with antibodies against VCAM-1 or alpha 4 integrin subunit interfered with the mRNA induction of smooth muscle-specific markers of differentiation. These results in vitro, associated with the transitory expression of VCAM-1 and VLA-4 during vascular ontogeny and the atherosclerosis process, point to a possible role of VCAM-1 and VLA-4 in the induction of smooth muscle differentiation.

Involvement of FrzA/sFRP-1 and the Wnt/Frizzled Pathway in Ischemic Preconditioning

Phosphorylation and subsequent inactivation of glycogen synthase kinase (GSK)-3&bgr; 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-3&bgr; in vitro on vascular cells, we examined its effect during PC using transgenic mouse overexpressing FrzA in cardiomyocytes (&agr;-MHC promoter) under a conditional transgene expression approach (tet-off system). Overexpression of FrzA inhibited the increase in GSK-3&bgr; 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 &bgr;-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 &bgr;-catenin independent Wnt/Frizzled pathway induces the activation of GSK-3&bgr; and reverses the benefit of preconditioning.