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 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 (290Ϯ103 vessels/mm 2 versus 104Ϯ43 in controls at day 15; PϽ0.001). Vessels were more muscularized, and mean lumen area was 3-fold higher in Tg animals. Conclusions-Overexpression

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 orders. 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.

Undifferentiated cells have been identified in the prenatal blastocyst, inner cell mass, and gonadal ridges of rodents and primates, including humans. After isolation these cells express molecular and immunological markers for embryonic cells, capabilities for extended self‐renewal, and telomerase activity. When allowed to differentiate, embryonic stem cells express phenotypic markers for tissues of ectodermal, mesodermal, and endodermal origin. When implanted in vivo, undifferentiated noninduced embryonic stem cells formed teratomas. In this report we describe a cell clone isolated from postnatal rat skeletal muscle and derived by repetitive single‐cell clonogenic analysis. In the undifferentiated state it consists of very small cells having a high ratio of nucleus to cytoplasm. The clone expresses molecular and immunological markers for embryonic stem cells. It exhibits telomerase activity, which is consistent with its extended capability for self‐renewal. When induced to differentiate, it expressed phenotypic markers for tissues of ectodermal, mesodermal, and endodermal origin. The clone was designated as a postnatal pluripotent epiblastic‐like stem cell (PPELSC). The undifferentiated clone was transfected with a genomic marker and assayed for alterations in stem cell characteristics. No alterations were noted. The labeled clone, when implanted into heart after injury, incorporated into myocardial tissues undergoing repair. The labeled clone was subjected to directed lineage induction in vitro, resulting in the formation of islet‐like structures (ILSs) that secreted insulin in response to a glucose challenge. This study suggests that embryonic‐like stem cells are retained within postnatal mammals and have the potential for use in gene therapy and tissue engineering. Anat Rec Part A 277A:178–203, 2004. © 2004 Wiley‐Liss, Inc.

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.

Profilin is an actin monomer binding protein that provides ATP-actin for incorporation into actin filaments. In contrast to higher eukaryotic cells with their large filamentous actin structures, apicomplexan parasites typically contain only short and highly dynamic microfilaments. In apicomplexans, profilin appears to be the main monomer-sequestering protein. Compared to classical profilins, apicomplexan profilins contain an additional arm-like β-hairpin motif, which we show here to be critically involved in actin binding. Through comparative analysis using two profilin mutants, we reveal this motif to be implicated in gliding motility of Plasmodium berghei sporozoites, the rapidly migrating forms of a rodent malaria parasite transmitted by mosquitoes. Force measurements on migrating sporozoites and molecular dynamics simulations indicate that the interaction between actin and profilin fine-tunes gliding motility. Our data suggest that evolutionary pressure to achieve efficient high-speed gliding has resulted in a unique profilin-actin interface in these parasites.

Migration of malaria parasites is powered by a myosin motor that moves actin filaments, which in turn link to adhesive proteins spanning the plasma membrane. The retrograde flow of these adhesins appears to be coupled to forward locomotion. However, the contact dynamics between the parasite and the substrate as well as the generation of forces are complex and their relation to retrograde flow is unclear. Using optical tweezers we found retrograde flow rates up to 15 μm/s contrasting with parasite average speeds of 1-2 μm/s. We found that a surface protein, TLP, functions in reducing retrograde flow for the buildup of adhesive force and that actin dynamics appear optimized for the generation of force but not for maximizing the speed of retrograde flow. These data uncover that TLP acts by modulating actin dynamics or actin filament organization and couples retrograde flow to force production in malaria parasites.

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 ␣-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. Key Words: blood vessels Ⅲ imaging Ⅲ ischemia Ⅲ transgenic mice Ⅲ vascular biology D uring development, blood vessel formation ensures tissue growth and organ function in the entire organism. The essential role of Wnt/Frizzled signaling in the development of the vascular network was established when it was demonstrated that deletion of distinct Wnt genes caused embryonic lethality with severe phenotypes. A growing body of evidence supports the hypothesis that the Wnt/planar cell polarity (PCP) pathway regulates endothelial cell proliferation and angiogenesis, 1-3 but the components that mediate this regulation remain elusive. Embryo-specific deletion of Wnt7b/7a, which bypassed early lethality because of Wnt7b effect on placenta formation, demonstrated a role of Wnt7a/7b ligands in blood-brain barrier formation through Wnt canonical signaling. 4 These models also indicated that Fzd4 is a prominent receptor involved in vascular formation. Fzd4 has been linked to genetic diseases altering retinal vascular development in Norrie disease, familial exudative vitreoretinopathy, 5,6 and osteoporosis-pseudoglioma. 7 In mice, Fzd4 controls retinal vascular growth and organization, 8 and blood-brain barrier formation in the cerebellum. 9 Moreover, Fzd4 is linked to sterility. 10 We have previously demonstrated that the action of sFRP1, a secreted regulator of the Wnt pathway, is mediated in part by Fzd4 in endothelial cells. 2 The sFRP1 stimulates angiogenesis in vivo and in vitro 11 via a noncanonical Wnt-dependent mechanism and activates downstream signaling factors such as GSK3␤ and Rac1. There is growing evidence of a link between noncanonical Wnt/PCP signaling and angi...

Abstract-Phosphorylation and subsequent inactivation of glycogen synthase kinase (GSK)-3␤ 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␤ in vitro on vascular cells, we examined its effect during PC using transgenic mouse overexpressing FrzA in cardiomyocytes (␣-MHC promoter) under a conditional transgene expression approach (tet-off system). Overexpression of FrzA inhibited the increase in GSK-3␤ 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 ␤-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 ␤-catenin independent Wnt/Frizzled pathway induces the activation of GSK-3␤ and reverses the benefit of preconditioning. Key Words: animal models of human disease Ⅲ cell signaling/signal transduction Ⅲ genetically-altered mice Ⅲ ischemia Ⅲ heart B rief episodes of ischemia/reperfusion, termed ischemic preconditioning (PC), protect the myocardium from the damage induced by subsequent and more prolonged ischemia. 1 PC has proved crucial in the protection of surviving cells, resulting in a reduction of infarct size, arrhythmias, and postischemic contractile dysfunction in all species tested so far. There is also evidence that it might be operative in man. 2,3 The different signaling pathways involved in PC result in protein kinase C (PKC) activation and in phosphatidylinositol-3-kinase (PI3-kinase) activation and translocation. 2,4 Downstream targets of PKC include the mitogen-activated protein kinase pathway, the activation of the mitochondrial ATPsensitive potassium channel, 5 and the modulation of energy and substrate metabolism. 6 Downstream targets of PI3-kinase in PC include the phosphorylation of the PKB/Akt pathway and the phosphorylation of glycogen synthase kinase-3␤ (GSK-3␤). 4 Phosphorylation at serine-9 residue (Ser9) and subsequent inactivation of GSK-3␤ has recently been demonstrated to play an important role in the protection and enhanced cell survival afforded by PC in the heart. 7 Multiple pathways other than the PI3-kinase-PKB/Akt-dependent pathway regulate the activity of GSK-3␤, eg, the Wnt signaling pathway. Wnt can inactivate GSK-3␤ through 2 distinct pathways. 8,9 In the Wnt canonical pathway, phosphorylation of GSK-3␤ (at a residue different from Ser9) results in increased ␤-catenin levels and its translocation to the nucleus. 10 Additionally, PKC could be involved in Wnt-induced GSK-3␤ inhibition by a phosphorylation at Ser9. [11][1...