How Senescent Vascular Cells Lose Their <i>Clock</i> Age-Dependent Impairment of Circadian Rhythmicity in Smooth Muscle Cells

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Combinatorial Effects of Double Cardiomyopathy Mutant Alleles in Rodent Myocytes: A Predictive Cellular Model of Myofilament Dysregulation in Disease

Inherited cardiomyopathy (CM) represents a diverse group of cardiac muscle diseases that present with a broad spectrum of symptoms ranging from benign to highly malignant. Contributing to this genetic complexity and clinical heterogeneity is the emergence of a cohort of patients that are double or compound heterozygotes who have inherited two different CM mutant alleles in the same or different sarcomeric gene. These patients typically have early disease onset with worse clinical outcomes. Little experimental attention has been directed towards elucidating the physiologic basis of double CM mutations at the cellular-molecular level. Here, dual gene transfer to isolated adult rat cardiac myocytes was used to determine the primary effects of co-expressing two different CM-linked mutant proteins on intact cardiac myocyte contractile physiology. Dual expression of two CM mutants, that alone moderately increase myofilament activation, tropomyosin mutant A63V and cardiac troponin mutant R146G, were shown to additively slow myocyte relaxation beyond either mutant studied in isolation. These results were qualitatively similar to a combination of moderate and strong activating CM mutant alleles αTmA63V and cTnI R193H, which approached a functional threshold. Interestingly, a combination of a CM myofilament deactivating mutant, troponin C G159D, together with an activating mutant, cTnIR193H, produced a hybrid phenotype that blunted the strong activating phenotype of cTnIR193H alone. This is evidence of neutralizing effects of activating/deactivating mutant alleles in combination. Taken together, this combinatorial mutant allele functional analysis lends molecular insight into disease severity and forms the foundation for a predictive model to deconstruct the myriad of possible CM double mutations in presenting patients

Oxidative Stress Induces Protein Phosphatase 2A-dependent Dephosphorylation of the Pocket Proteins pRb, p107, and p130

Oxidative stress induces cell death and growth arrest. In this study, the regulation and the functional role of the retinoblastoma family proteins pRb, p107, and p130 in the cellular response to oxidative stress were investigated. Treatment of endothelial cells with H2O2 induced rapid hypophosphorylation of the retinoblastoma family proteins. This event did not require p53 or p21Waf1/Cip1/Sdi1 and was not associated with cyclin/cyclin-dependent kinase down-modulation. Four lines of evidence indicate that H2O2-induced hypophosphorylation of pRb, p107, and p130 was because of the activity of protein phosphatase 2A (PP2A). First, cell treatment with two phosphatase inhibitors, okadaic acid and calyculin A, prevented the hypophosphorylation of the retinoblastoma family proteins, at concentrations that specifically inhibit PP2A. Second, SV40 small t, which binds and inhibits PP2A, when overexpressed prevented H2O2-induced dephosphorylation of the retinoblastoma family proteins, whereas a SV40 small t mutant unable to bind PP2A was totally inert. Third, PP2A core enzyme physically interacted with pRb and p107, both in H2O2-treated and untreated cells. Fourth, a PP2A phosphatase activity was co-immunoprecipitated with pRb, and the activity of pRb-associated PP2A was positively modulated by cell treatment with H2O2. Because DNA damaging agents inhibit DNA synthesis in a pRb-dependent manner, it was determined whether the PP2A-mediated dephosphorylation of the retinoblastoma family proteins played a role in this S-phase response. Indeed, it was found that inhibition of PP2A by SV40 small t over-expression prevented DNA synthesis inhibition induced by H2O2

Regenerative Therapy in Peripheral Artery Disease

Patients with peripheral artery disease (PAD) and critical limb ischemia are the main candidates for limb amputations and have a poor life expectancy. Frequently, these patients are not eligible for either surgical or percutaneous interventions aimed at mechanical revascularization. Therefore, new strategies need to be identified to offer these patients a viable therapeutic option. Gene and cell therapy hold great promise for the treatment of peripheral vascular diseases because, in animal models, local delivery of growth factors and endothelial progenitor cells result in new blood vessel formation and regeneration of ischemic tissues. In this article, are reviewed phase I and phase II gene, and cell therapy clinical trials in patients with PAD

Intracellular targets of RGDS peptide in melanoma cells

<p>Abstract</p> <p>Background</p> <p>RGD-motif acts as a specific integrins-ligand and regulates a variety of cell-functions via extracellular action affecting cell-adhesion properties. However, increasing evidence identifies additional RGDS-functions at intracellular level. Previous reports show RGDS-internalization in endothelial cells, cardiomyocytes and lymphocytes, indicating intracellular targets such as caspase-8 and caspase-9, and suggest RGDS specific activity at cytoplasmic level. Given the role RGDS-peptides play in controlling proliferation and apoptosis in several cell types, investigating intracellular targets of RGDS in melanoma cells may un-reveal novel molecular targets and key pathways, potentially useful for a more effective approach to melanoma treatment.</p> <p>Results</p> <p>In the present study we show for the first time that RGDS-peptide is internalized in melanoma cells in a time-dependent way and exerts strong anti-proliferative and pro-apoptotic effects independently from its extracellular anti-adhesive action. RGES control-peptide did not show biological effects, as expected; nevertheless it is internalized, although with slower kinetics. Survivin, a known cell-cycle and survival-regulator is highly expressed in melanoma cells. Co-immunoprecipitation assays in cell lysates and overlay assays with the purified proteins showed that RGDS interacts with survivin, as well as with procaspase-3, -8 and -9. RGDS-peptide binding to survivin was found to be specific, at high affinity (Kd 27.5 μM) and located at the survivin C-terminus. RGDS-survivin interaction appeared to play a key role, since RGDS lost its anti-mitogenic effect in survivin-deprived cells with a specific siRNA.</p> <p>Conclusions</p> <p>RGDS inhibits melanoma growth with an adhesion-independent mechanism; it is internalized in melanoma cells and specifically interacts with survivin. The present data may indicate a novel role of RGDS-containing peptides physiologically released from the extracellular matrix and may suggest a possible novel anti-proliferation strategy in melanoma.</p

Enhanced Healing of Diabetic Wounds by Topical Administration of Adipose Tissue-Derived Stromal Cells Overexpressing Stromal-Derived Factor-1: Biodistribution and Engraftment Analysis by Bioluminescent Imaging

Chronic ulcers represent a major health problem in diabetic patients resulting in pain and discomfort. Conventional therapy does not guarantee adequate wound repair. In diabetes, impaired healing is partly due to poor endothelial progenitor cells mobilisation and homing, with altered levels of the chemokine stromal-derived factor-1 (SDF-1) at the wound site. Adipose tissue-associated stromal cells (AT-SCs) can provide an accessible source of progenitor cells secreting proangiogenic factors and differentiating into endothelial-like cells. We demonstrated that topical administration of AT-SCs genetically modified ex vivo to overexpress SDF-1, promotes wound healing into diabetic mice. In particular, by in vivo bioluminescent imaging analysis, we monitored biodistribution and survival after transplantation of luciferase-expressing cells. In conclusion, this study indicates the therapeutic potential of AT-SCs administration in wound healing, through cell differentiation, enhanced cellular recruitment at the wound site, and paracrine effects associated with local growth-factors production

Role of miR-200c in myogenic differentiation impairment via p66Shc: implication in skeletal muscle regeneration of dystrophic mdx mice

Duchenne muscular dystrophy (DMD) is a genetic disease associated with mutations of Dystrophin gene that regulate myofiber integrity and muscle degeneration, characterized by oxidative stress increase. We previously published that reactive oxygen species (ROS) induce miR-200c that is responsible for apoptosis and senescence. Moreover, we demonstrated that miR-200c increases ROS production and phosphorylates p66Shc in Ser-36. p66Shc plays an important role in muscle differentiation; we previously showed that p66Shc(-/-) muscle satellite cells display lower oxidative stress levels and higher proliferation rate and differentiated faster than wild-type (wt) cells. Moreover, myogenic conversion, induced by MyoD overexpression, is more efficient in p66Shc(-/-) fibroblasts compared to wt cells. Herein, we report that miR-200c overexpression in cultured myoblasts impairs skeletal muscle differentiation. Further, its overexpression in differentiated myotubes decreases differentiation indexes. Moreover, anti-miR-200c treatment ameliorates myogenic differentiation. In keeping, we found that miR-200c and p66Shc Ser-36 phosphorylation increase in mdx muscles. In conclusion, miR-200c inhibits muscle differentiation, whereas its inhibition ameliorates differentiation and its expression levels are increased in mdx mice and in differentiated human myoblasts of DMD. Therefore, miR-200c might be responsible for muscle wasting and myotube loss, most probably via a p66Shc-dependent mechanism in a pathological disease such as DMD

Different Effects of High and Low Shear Stress on Platelet-Derived Growth Factor Isoform Release by Endothelial Cells

In the present study, we analyzed the effect of conditioned media (CM) from bovine aortic endothelial cells exposed to laminar shear stress (SS) of 5 dyne/cm 2 (SS5) or 15 dyne/cm 2 (SS15) for 16 hours on smooth muscle cell (SMC) migration. In response to CM from bovine aortic endothelial cells exposed to SS5 (CMSS5) and SS15 (CMSS15), migration was 45±5.5 and 30±1.5 cells per field, respectively ( P <0.05). Similar results were obtained with SS of 2 versus 20 dyne/cm 2 and also when SS of 5 and 15 dyne/cm 2 lasted 24 hours. Platelet-derived growth factor (PDGF)-AA levels in CMSS5 and CMSS15 were 9±7 and 18±5 ng/10 6 cells for 16 hours, respectively ( P <0.05); PDGF-BB levels in CMSS5 and CMSS15 were 38±10 and 53±10 ng/10 6 cells for 16 hours, respectively ( P CMSS5. In response to CMSS15, a neutralizing antibody against PDGF-AA enhanced SMC migration to a level comparable to that of CMSS5; in contrast, antibodies against PDGF-BB abolished SMC migration. Transfection of SMCs with a dominant-negative PDGFRα or PDGFRβ increased or inhibited, respectively, SMC migration in response to CMSS15. Overexpression of wild-type PDGFRα inhibited SMC migration in response to CMSS5, CMSS15, or recombinant PDGF-BB ( P <0.001). These results suggest that the ability of high SS to inhibit arterial wall thickening in vivo may be related to enhanced activation of PDGFRα in SMCs by PDGF isoforms secreted by the endothelium