Cyclase-associated proteins are highly conserved proteins that have a role in the regulation of actin dynamics. Higher eukaryotes have two isoforms, CAP1 and CAP2. To study the in vivo function of CAP2, we generated mice in which the CAP2 gene was inactivated by a gene-trap approach. Mutant mice showed a decrease in body weight and had a decreased survival rate. Further, they developed a severe cardiac defect marked by dilated cardiomyopathy (DCM) associated with drastic reduction in basal heart rate and prolongations in atrial and ventricular conduction times. Moreover, CAP2-deficient myofibrils exhibited reduced cooperativity of calciumregulated force development. At the microscopic level, we observed disarrayed sarcomeres with development of fibrosis. We analyzed CAP2's role in actin assembly and found that it sequesters G-actin and efficiently fragments filaments. This activity resides completely in its WASP homology domain. Thus CAP2 is an essential component of the myocardial sarcomere and is essential for physiological functioning of the cardiac system, and a deficiency leads to DCM and various cardiac defects.

The coordination of cell proliferation and cell fate determination is critical during development but the mechanisms through which this is accomplished are unclear. We present evidence that the Snail-related transcription factor CES-1 of Caenorhabditis elegans coordinates these processes in a specific cell lineage. CES-1 can cause loss of cell polarity in the NSM neuroblast. By repressing the transcription of the BH3-only gene egl-1, CES-1 can also suppress apoptosis in the daughters of the NSM neuroblasts. We now demonstrate that CES-1 also affects cell cycle progression in this lineage. Specifically, we found that CES-1 can repress the transcription of the cdc-25.2 gene, which encodes a Cdc25-like phosphatase, thereby enhancing the block in NSM neuroblast division caused by the partial loss of cya-1, which encodes Cyclin A. Our results indicate that CDC-25.2 and CYA-1 control specific cell divisions and that the over-expression of the ces-1 gene leads to incorrect regulation of this functional ‘module’. Finally, we provide evidence that dnj-11 MIDA1 not only regulate CES-1 activity in the context of cell polarity and apoptosis but also in the context of cell cycle progression. In mammals, the over-expression of Snail-related genes has been implicated in tumorigenesis. Our findings support the notion that the oncogenic potential of Snail-related transcription factors lies in their capability to, simultaneously, affect cell cycle progression, cell polarity and apoptosis and, hence, the coordination of cell proliferation and cell fate determination.

IntroductionCell therapy using adipose-derived stromal cells (ADSC) is an intensively developing approach to promote angiogenesis and regeneration. Administration technique is crucial and among others minimal constructs - cell sheets (CS) have certain advantages. Delivery of CS allows transplantation of cells along with matrix proteins to facilitate engraftment. Cells’ therapeutic potential can be also increased by expression of proangiogenic factors by viral transduction. In this work we report on therapeutic efficacy of CS from mouse ADSC transduced to express human vascular endothelial growth factor 165 a/a isoform (VEGF165), which showed potency to restore perfusion and protect tissue in a model of limb ischemia.MethodsMouse ADSC (mADSC) isolated from C57 male mice were expanded for CS formation (106cells per CS). Constructs were transduced to express human VEGF165 by baculoviral (BV) system. CS were transplanted subcutaneously to mice with surgically induced limb ischemia and followed by laser Doppler perfusion measurements. At endpoint animals were sacrificed and skeletal muscle was evaluated for necrosis and vessel density; CS with underlying muscle was stained for apoptosis, proliferation, monocytes and blood vessels.ResultsUsing BV system and sodium butyrate treatment we expressed human VEGF165 in mADSC (production of VEGF165 reached ≈ 25-27 ng/ml/105 cells) and optimized conditions to ensure cells’ viability after transduction. Implantation of mock-transduced CS resulted in significant improvement of limb perfusion, increased capillary density and necrosis reduction at 2 weeks post-surgery compared to untreated animals. Additional improvement of blood flow and angiogenesis was observed after transplantation of VEGF165-expressing CS indicating enhanced therapeutic potential of genetically modified constructs. Moreover, we found delivery of mADSC as CS to be superior to equivalent dose of suspended cells in terms of perfusion and angiogenesis. Histology analysis of extracted CS detected limited proliferation and approximately 10 % prevalence of apoptosis in transplanted mADSC. Significant vascularization of CS and infiltration by monocytes were found in both – BV-transduced and control CS indicating graft and host interaction after transplantation.ConclusionsDelivery of ADSC by subcutaneous transplantation of CS is effective for stimulation of angiogenesis and tissue protection in limb ischemia with a potential for efficacy improvement by BV transduction to express VEGF165.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0199-6) contains supplementary material, which is available to authorized users.

Since development of plasmid gene therapy for therapeutic angiogenesis by J. Isner this approach was an attractive option for ischemic diseases affecting large cohorts of patients. However, first placebo-controlled clinical trials showed its limited efficacy questioning further advance to practice. Thus, combined methods using delivery of several angiogenic factors got into spotlight as a way to improve outcomes. This study provides experimental proof of concept for a combined approach using simultaneous delivery of VEGF165 and HGF genes to alleviate consequences of myocardial infarction (MI). However, recent studies suggested that angiogenic growth factors have pleiotropic effects that may contribute to outcome so we expanded focus of our work to investigate potential mechanisms underlying action of VEGF165, HGF and their combination in MI. Briefly, Wistar rats underwent coronary artery ligation followed by injection of plasmid bearing VEGF165 or HGF or mixture of these. Histological assessment showed decreased size of post-MI fibrosis in both—VEGF165- or HGF-treated animals yet most prominent reduction of collagen deposition was observed in VEGF165+HGF group. Combined delivery group rats were the only to show significant increase of left ventricle (LV) wall thickness. We also found dilatation index improved in HGF or VEGF165+HGF treated animals. These effects were partially supported by our findings of c-kit+ cardiac stem cell number increase in all treated animals compared to negative control. Sporadic Ki-67+ mature cardiomyocytes were found in peri-infarct area throughout study groups with comparable effects of VEGF165, HGF and their combination. Assessment of vascular density in peri-infarct area showed efficacy of both–VEGF165 and HGF while combination of growth factors showed maximum increase of CD31+ capillary density. To our surprise arteriogenic response was limited in HGF-treated animals while VEGF165 showed potent positive influence on a-SMA+ blood vessel density. The latter hinted to evaluate infiltration of monocytes as they are known to modulate arteriogenic response in myocardium. We found that monocyte infiltration was driven by VEGF165 and reduced by HGF resulting in alleviation of VEGF-stimulated monocyte taxis after combined delivery of these 2 factors. Changes of monocyte infiltration were concordant with a-SMA+ arteriole density so we tested influence of VEGF165 or HGF on endothelial cells (EC) that mediate angiogenesis and inflammatory response. In a series of in vitro experiments we found that VEGF165 and HGF regulate production of inflammatory chemokines by human EC. In particular MCP-1 levels changed after treatment by recombinant VEGF, HGF or their combination and were concordant with NF-κB activation and monocyte infiltration in corresponding groups in vivo. We also found that both–VEGF165 and HGF upregulated IL-8 production by EC while their combination showed additive type of response reaching peak values. These changes were HIF-2 dependent and siRNA-mediated knockdown of HIF-2α abol...

The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire-actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire-actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott-Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire-actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire-actin module. In addition, we find that preformed, isolated Spire-actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs-even a single WH2 repeat-sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem.cytoskeleton | nucleation T he first step in the assembly of actin filaments is nucleation (1, 2). Three major classes of nucleating proteins have been identified until today: the Arp2/3 complex together with newly identified nucleation-promoting factors such as WASH, WHAMM and JMY (3-7), formins (8, 9), and a third class which comprises the proteins commonly named tandem-monomer-binding nucleators (10). This last group of nucleators contains 17-27 amino acid long actin-binding motifs called the WH2 repeats-the name derived from the Wiskott-Aldrich syndrome protein homology domain 2 (11, 12). The group consists of Spire (13), , Leiomodin from muscle cells (15), JMY (6), and the recently discovered adenomatous polyposis coli (APC) protein (16). These proteins share the common ability, mediated by WH2 domains, to gather actin monomers into a nucleation complex, but the arrangement of nuclei might vary significantly among these nucleators. Spire contains four consecutive WH2 domains and is the most important representative member of the group. The molecular mechanism of actin nucleation is well described for the Arp2/3 complex (17) and formins (18), whereas several different mechanisms have been proposed for Spire (19)(20)(21)(22), Leiomodin (15), JMY (6), and the APC protein (16). The N-terminal domain of Spire (SpireNT, residues 1-520 in Drosophila melanogaster Spire; see Fig. S1) has the potential to form a string of four actin monomers through the interaction with four WH2 repeats (13,19,20). WH2 motifs are known to be intrinsically disordered, adopting an α-helical structure only upon binding to actin (23). Alignments of WH2 domains indicate that the most conserved regions are the LKK motif and an α-helix at the N terminus, which is shown to be the principal structural actin-binding element that binds to actin in its hydrophobic pocket between actin's subdomains 1 and 3 (20,24,25). The rest of the WH2, including the LKK motif, exten...

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NICD and activated expression of Notch target genes (Hes, Hey). Activation of Notch signaling upregulated expression of endothelial cell transcription factors Gata 4 and Vezf1 in CSCs but γ-secretase inhibitor prevented Notch signaling activation and endothelial cell commitment of CSCs. CONCLUSIONS: These results revealed that Notch signaling activation promote CSCs commitment toward vascular cells. These findings suggest that modulation of Notch signaling can be promising therapeutic strategy for the treatment of myocardial ischemic damage.

Cell sheet (CS) approach is extensively developed in recent years for a number of applications in regenerative medicine including lesion treatment, tissue-engineered constructs and organoplastics. In