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Fetal hemoglobin (HbF) induction is considered to be a promising therapeutic strategy to ameliorate the clinical severity of β-hemoglobin disorders, and has gained a significant amount of attention in recent times. Despite the enormous efforts towards the pharmacological intervention of HbF reactivation, progress has been stymied due to limited understanding of γ-globin gene regulation. In this study, we intended to investigate the implications of lncRNA-associated competing endogenous RNA (ceRNA) interactions in HbF regulation. Probe repurposing strategies for extraction of lncRNA signatures and subsequent in silico analysis on publicly available datasets (GSE13284, GSE71935 and GSE7874) enabled us to identify 46 differentially expressed lncRNAs (DElncRNAs). Further, an optimum set of 11 lncRNAs that could distinguish between high HbF and normal conditions were predicted from these DElncRNAs using supervised machine learning and a stepwise selection model. The candidate lncRNAs were then linked with differentially expressed miRNAs and mRNAs to identify lncRNA-miRNA-mRNA ceRNA networks. The network revealed that 2 lncRNAs (UCA1 and ZEB1-AS1) and 4 miRNAs (hsa-miR-19b-3p,hsa-miR-3646,hsa-miR-937 and hsa-miR-548j) sequentially mediate cross-talk among different signaling pathways which provide novel insights into the lncRNA-mediated regulatory mechanisms, and thus lay the foundation of future studies to identify lncRNA-mediated therapeutic targets for HbF reactivation.

Beta-hemoglobinopathies exhibit a heterogeneous clinical picture with varying degrees of clinical severity. Pertaining to the limited treatment options available, where blood transfusion still remains the commonest mode of treatment, pharmacological induction of fetal hemoglobin (HbF) has been a lucrative therapeutic intervention. Till now more than 70 different HbF inducers have been identified. The practical usage of many pharmacological drugs has been limited due to safety concerns. Natural compounds, like Resveratrol, Ripamycin and Bergaptene, with limited cytotoxicity and high efficacy have started capturing the attention of researchers. In this review, we have summarized pharmacological drugs and bioactive compounds isolated from natural sources that have been shown to increase HbF significantly. It primarily discusses recently identified synthetic and natural compounds, their mechanism of action, and their suitable screening platforms, including high throughput drug screening technology and biosensors. It also delves into the topic of combinatorial therapy and drug repurposing for HbF induction. Overall, we aim to provide insights into where we stand in HbF induction strategies for treating β-hemoglobinopathies.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel pharmacological target for hypercholesterolemia and associated cardiovascular diseases owing to its function to mediate the degradation of low-density lipoprotein receptor (LDLR). Findings over the past two decades have identified novel binding partners and cellular functions of PCSK9. Notably, PCSK9 is aberrantly expressed in a broad spectrum of cancers and apparently contributes to disease prognosis, indicating that PCSK9 could be a valuable cancer biomarker. Experimental studies demonstrate the contribution of PCSK9 in various aspects of cancer, including cell proliferation, apoptosis, invasion, metastasis, anti-tumor immunity and radioresistance, strengthening the idea that PCSK9 could be a promising therapeutic target. Here, we comprehensively review the involvement of PCSK9 in cancer, summarizing its aberrant expression, association with disease prognosis, biological functions and underlying mechanisms in various malignancies. Besides, we highlight the potential of PCSK9 as a future therapeutic target in personalized cancer medicine.

In the last three decades, researchers have utilized genome engineering to alter the DNA sequence in the living cells of a plethora of organisms, ranging from plants, fishes, mice, to even humans. This has been conventionally achieved by using methodologies such as single nucleotide insertion/deletion in coding sequences, exon(s) deletion, mutations in the promoter region, introducing stop codon for protein truncation, and addition of foreign DNA for functional elucidation of genes. However, recent years have witnessed the advent of novel techniques that use programmable site-specific nucleases like CRISPR/Cas9, TALENs, ZFNs, Cre/loxP system, and gene trapping. These have revolutionized the field of experimental transgenesis as well as contributed to the existing knowledge base of classical genetics and gene mapping. Yet there are certain experimental/technological barriers that we have been unable to cross while creating genetically modified organisms. Firstly, while interfering with coding strands, we inadvertently change introns, antisense strands, and other non-coding elements of the gene and genome that play integral roles in the determination of cellular phenotype. These unintended modifications become critical because introns and other non-coding elements, although traditionally regarded as “junk DNA,” have been found to play a major regulatory role in genetic pathways of several crucial cellular processes, development, homeostasis, and diseases. Secondly, post-insertion of transgene, non-coding RNAs are generated by host organism against the inserted foreign DNA or from the inserted transgene/construct against the host genes. The potential contribution of these non-coding RNAs to the resulting phenotype has not been considered. We aim to draw attention to these inherent flaws in the transgenic technology being employed to generate mutant mice and other model organisms. By overlooking these aspects of the whole gene and genetic makeup, perhaps our current understanding of gene function remains incomplete. Thus, it becomes important that, while using genetic engineering techniques to generate a mutant organism for a particular gene, we should carefully consider all the possible elements that may play a potential role in the resulting phenotype. This perspective highlights the commonly used mouse strains and the most probable associated complexities that have not been considered previously, resulting in possible limitations in the currently utilized transgenic technology. This work also warrants the use of already established mouse lines in further research.

Transcription factors as multifaceted modulators of gene expression that play a central role in cell proliferation, differentiation, lineage commitment, and disease progression. They interact among themselves and create complex spatiotemporal gene regulatory networks that modulate hematopoiesis, cardiogenesis, and conditional differentiation of hematopoietic stem cells into cells of cardiovascular lineage. Additionally, bone marrow-derived stem cells potentially contribute to the cardiovascular cell population and have shown potential as a therapeutic approach to treat cardiovascular diseases. However, the underlying regulatory mechanisms are currently debatable. This review focuses on some key transcription factors and associated epigenetic modifications that modulate the maintenance and differentiation of hematopoietic stem cells and cardiac progenitor cells. In addition to this, we aim to summarize different potential clinical therapeutic approaches in cardiac regeneration therapy and recent discoveries in stem cell-based transplantation.

Objective Alterations in cardiac energy and substrate metabolism play a critical role in the development and clinical course of heart failure. We hypothesized that the cardioprotective role of the breast cancer 1, early onset (BRCA1) gene might be mediated in part by alterations in cardiac bioenergetics. Methods We generated cardiomyocyte-specific BRCA1 homozygous and heterozygous knockout mice using the Cre-loxP technology and evaluated the key molecules and pathways involved in glucose metabolism, fatty acid metabolism, and mitochondrial bioenergetics. Results Cardiomyocyte-specific BRCA1-deficient mice showed reduced cardiac expression of glucose and fatty acid transporters, reduced acetyl-coenzyme A carboxylase 2 and malonyl-coenzyme A decarboxylase (key enzymes that control malonyl coenzyme A, which in turn controls fatty acid oxidation), and reduced carnitine palmitoyltransferase I, a rate-limiting enzyme for mitochondrial fatty acid uptake. Peroxisome proliferator-activated receptor α and γ and carnitine palmitoyltransferase I levels were also downregulated in these hearts. Rates of glucose and fatty acid oxidation were reduced in the hearts of heterozygous cardiomyocyte-restricted BRCA1-deficient mice, resulting in a decrease in the rate of adenosine triphosphate production. This decrease in metabolism and adenosine triphosphate production occurred despite an increase in 5′-adenosine monophosphate-activated protein kinase and AKT activation in the heart. Conclusions Cardiomyocyte-specific loss of BRCA1 alters critical pathways of fatty acid and glucose metabolism, leading to an energy starved heart. BRCA1-based cell or gene therapy might serve as a novel target to improve cardiac bioenergetics in patients with heart failure.

Aims Foxo3 is a transcription factor involved in cell metabolism, survival, and inflammatory disease. However, mechanistic insight in Foxo3 effects is still limited. Here, we investigated the role of Foxo3 on natural killer (NK) cell responses and its effects in viral myocarditis. Methods and results Effects of Foxo3 on viral load and immune responses were investigated in a model of coxsackie virus B3 myocarditis in wild-type (WT) and Foxo3 deficient mice. Reduced immune cell infiltration, viral titres, and pro-inflammatory cytokines in cardiac tissue were observed in Foxo3(-/-) mice 7 days post-infection (p.i.). Viral titres were also attenuated in hearts of Foxo3(-/-) mice at Day 3 while interferon-gamma (IFN gamma) and NKp46 expression were up-regulated suggesting early viral control by enhanced NK cell activity. CD69 expression of NK cells, frequencies of CD11b(+)CD27(+) effector NK cells and cytotoxicity of Foxo3(-/-) mice was enhanced compared to WT littermates. Moreover, microRNA-155 expression, essential in NK cell activation, was elevated in Foxo3(-/-) NK cells while its inhibition led to diminished IFN gamma production. Healthy humans carrying the longevity-associated FOXO3 single nucleotide polymorphism (SNP) rs12212067 exhibited reduced IFN gamma and cytotoxic degranulation of NK cells. Viral inflammatory cardiomyopathy (viral CMI) patients with this SNP showed a poorer outcome due to less efficient virus control. Conclusion Our results implicate Foxo3 in regulating NK cell function and suggest Foxo3 playing an important role in the antiviral innate immunity. Thus, enhanced FOXO3 activity such as in the polymorphism rs12212067 may be protective in chronic inflammation such as cancer and cardiovascular disease but disadvantageous to control acute viral infection.

Sepsis-related complications and mortality remain a major clinical problem. Increased cell death and unresolved cellular repair have been implicated as key upstream mediators of sepsis-induced organ dysfunction and death. We hypothesised that gene therapy with BRCA1, a critical regulator of DNA damage repair and cell survival, would attenuate the sequelae of sepsis and peritonitis in mice subjected to caecal ligation and perforation (CLP) and thioglycollate stimulation. C57Bl/6J mice underwent sham or CLP surgery 3 days following treatment with either human BRCA1 adenovirus (AdBRCA1) or the adeno-CMV-null vector (Adnull). The 24-h post-CLP mortality was 2.8% vs 17.9% (P

Altered macrophage kinetics is a pivotal mechanism of visceral obesity-induced inflammation and cardiometabolic risk. Because monocytes can differentiate into either proatherogenic M1 macrophages or anti-inflammatory M2 macrophages, approaches that limit M1 while promoting M2 differentiation represent a unique therapeutic strategy. We hypothesized that adiponectin may prime human monocytes toward the M2 phenotype. Adiponectin promoted the alternative activation of human monocytes into anti-inflammatory M2 macrophages as opposed to the classically activated M1 phenotype. Adiponectin-treated cells displayed increased M2 markers, including the mannose receptor (MR) and alternative macrophage activation-associated CC chemokine-1. Incubation of M1 macrophages with adiponectin-treated M2-derived culture supernatant resulted in a pronounced inhibition of tumor necrosis factor-α and monocyte chemotactic protein-1 secretion. Activation of human monocytes into M2 macrophages by adiponectin was mediated, in addition to AMP-activated protein kinase and peroxisome proliferator-activated receptor (PPAR)-γ, via PPAR-α. Furthermore, macrophages isolated from adiponectin knockout mice demonstrated diminished levels of M2 markers such as MR, which were restored with adiponectin treatment. We report a novel immunoregulatory mechanism through which adiponectin primes human monocyte differentiation into anti-inflammatory M2 macrophages. Conditions associated with low adiponectin levels, such as visceral obesity and insulin resistance, may promote atherosclerosis, in part through aberrant macrophage kinetics.

Background— In patients with myocardial infarction, high serum levels of interleukin-6 cytokines predict a poor outcome. The common receptor of interleukin-6 cytokines, glycoprotein-130 (gp130), signals via janus kinase/signal transducer and activator of transcription (STAT), cytoplasmic protein tyrosine phosphatase/extracellular signal-regulated kinase, and phosphoinositide-3-kinase/Akt pathways, and the regulation of these pathways depends at least in part on the gp130 tyrosine-757 residue. By analyzing cardiomyocyte-specific gp130 Y757F mutant mice, we investigated the effect of disturbed gp130 signaling after myocardial infarction. Methods and Results— The cardiomyocyte-restricted α-myosin heavy chain-Cre-recombinase-loxP system was used to generate mice with gp130 Y757F mutant cardiomyocytes ( αMHC-Cre tg/− ;gp130 fl/Y757F [Y 757 F]); all other cells carried at least 1 functional gp130 gene, ensuring normal gp130 signaling. Y 757 F mice displayed normal cardiac function and morphology at 3 months of age comparable to their nonmutant littermates. In response to myocardial infarction, Y 757 F mice displayed higher mortality associated with increased left ventricular rupture rate, sustained cardiac inflammation, and heart failure. These adverse effects were associated with prolonged and enhanced STAT3 activation and increased expression of interleukin-6 and of the complement-activating mannose-binding lectin C. Pharmacological inhibition of the complement system by cobra venom factor attenuated inflammation, prevented left ventricular rupture, and improved cardiac function in Y 757 F mice. Stronger effects were observed with a genetic reduction of STAT3 (STAT3 flox/+ ) restricted to cardiomyocytes in Y 757 F mice, which prevented extensive upregulation of interleukin-6, complement activation, and sustained inflammation and lowered left ventricular rupture rate, heart failure, and mortality in subacute myocardial infarction. Conclusion— Impaired downregulation of gp130-mediated STAT3 activation in subacute infarction promotes cardiac inflammation, adverse remodeling, and heart failure, suggesting a potential causative role of high interleukin-6 serum levels after myocardial infarction.

DNA turnover through damage and repair is normal homeostatic process in viable cells. DNA damage induces sequential responses to either repair the damage or activate a programmed cell death process. Defects in this critical response to DNA damage underpin a wide array of human pathologies that include cancer predisposition, immune dysfunction, radiosensitivity, and neurodegeneration. Recent experimental and clinical studies have suggested that oxidative stress leading to catastrophic DNA damage is enhanced in failing hearts as well as other cardiovascular diseases. Accordingly, targeting the genotoxic effects of oxidative stress may prove to be a novel avenue in the development of rational therapies for cardiovascular diseases. A platform introduction to DNA repair/response are integrated, and insights into the future directions of this exciting field are offered.

Fibrillin-1 is a multifunctional extracellular protein encoded by the FBN1 gene. FBN1 is 237 kb in size and is located on chromosome 15q21. FBN1 mutations are known to cause Marfan syndrome and other fibrillinopathies. FBN1 is composed of 65 exons and 3 additional alternatively spliced exons at the 5' end. The absence of the peptide sequence from the extreme N-terminus of the fibrillin-1 protein and the presence of in-frame and alternatively spliced exons at the 5' end of the FBN1 gene create some ambiguity about the translation start site and indicate a functional role of these alternatively spliced exons. We demonstrate here the conservation of 5'-upstream region of the FBN1 gene among humans and non-human primates.

Overexpression and activation of protein kinase C-ε (PKCε) results in myocardial hypertrophy. However, these observations do not establish that PKCε is required for the development of myocardial hypertrophy. Thus, we subjected PKCε-knockout (KO) mice to a hypertrophic stimulus by transverse aortic constriction (TAC). KO mice show normal cardiac morphology and function. TAC caused similar cardiac hypertrophy in KO and wild-type (WT) mice. However, KO mice developed more interstitial fibrosis and showed enhanced expression of collagen Iα1 and collagen III after TAC associated with diastolic dysfunction, as assessed by tissue Doppler echocardiography (Ea/Aa after TAC: WT 2.1±0.3 versus KO 1.0±0.2; P

The sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) is key to Ca(2+) homeostasis and is redox-regulated by reversible glutathione (GSH) adducts on the cysteine (C) 674 thiol that stimulate Ca(2+) uptake activity and endothelial cell angiogenic responses in vitro. We found that mouse hind limb muscle ischemia induced S-glutathione adducts on SERCA in both whole muscle tissue and endothelial cells. To determine the role of S-glutathiolation, we used a SERCA 2 C674S heterozygote knock-in (SKI) mouse lacking half the key thiol. Following hind limb ischemia, SKI animals had decreased SERCA S-glutathione adducts and impaired blood flow recovery. We studied SKI microvascular endothelial cells in which total SERCA 2 expression was unchanged. Cultured SKI microvascular endothelial cells showed impaired migration and network formation compared with wild type (WT). Ca(2+) studies showed decreased nitric oxide (·NO)-induced (45)Ca(2+) uptake into the endoplasmic reticulum (ER) of SKI cells, while Fura-2 studies revealed lower Ca(2+) stores and decreased vascular endothelial growth factor (VEGF)- and ·NO-induced Ca(2+) influx. Adenoviral overexpression of calreticulin, an ER Ca(2+) binding protein, increased ionomycin-releasable stores, VEGF-induced Ca(2+) influx and endothelial cell migration. Taken together, these data indicate that the redox-sensitive Cys-674 thiol on SERCA 2 is required for normal endothelial cell Ca(2+) homeostasis and ischemia-induced angiogenic responses, revealing a novel redox control of angiogenesis via Ca(2+) stores.

Objective BRCA1 , a tumor suppressor gene implicated in breast and ovarian cancers, exerts multiple effects on DNA repair and affords resistance against cellular stress responses. We hypothesized that BRCA1 limits endothelial cell apoptosis and dysfunction, and via this mechanism attenuates atherosclerosis. Methods Loss and gain of function were achieved in cultured endothelial cells by silencing and overexpressing BRCA1, respectively. In vivo loss and gain of function were performed by generating endothelial cell-specific knockout ( EC-BRCA1 −/− ) mice and administering a BRCA1 adenovirus. Well-established cell and animal models of angiogenesis and atherosclerosis were used. Results BRCA1 is basally expressed in endothelial cells. BRCA1 overexpression protected and BRCA1 silencing exaggerated inflammation- and doxorubicin-induced endothelial cell apoptosis. Key indices of endothelial function were modulated in a manner consistent with an effect of BRCA1 to limit endothelial cell apoptosis and improve endothelial function. BRCA1 overexpression strongly attenuated the production of reactive oxygen species and upregulated endothelial nitric oxide synthase, phosphorylated endothelial nitric oxide synthase, phosphorylated Akt, and vascular endothelial growth factor-a expression. BRCA1 overexpression also improved capillary density and promoted blood flow restoration in mice subjected to hind-limb ischemia. BRCA1-overexpressing ApoE −/− mice fed a Western diet developed significantly less aortic plaque lesions, exhibited reduced macrophage infiltration, and generated less reactive oxygen species. Lung sections and aortic segments from EC-BRCA1 −/− mice demonstrated greater inflammation-associated apoptosis and impaired endothelial function, respectively. BRCA1 expression was attenuated in the plaque region of human atherosclerotic carotid artery samples compared with the adjacent plaque-free area. Conclusions These data collectively highlight a previously unrecognized role of BRCA1 as a gatekeeper of inflammation-induced endothelial cell function and a target to limit atherosclerosis. Translational studies evaluating endothelial function and atherosclerosis in individuals with BRCA1 mutations are suggested.

Background— MicroRNA are essential posttranscriptional modulators of gene expression implicated in various chronic diseases. Because microRNA-145 is highly expressed in vascular smooth muscle cells (VSMC) and regulates VSMC fate and plasticity, we hypothesized that it may be a novel regulator of atherosclerosis and plaque stability. Methods and Results— Apolipoprotein E knockout mice ( ApoE −/− ) mice were treated with either a microRNA-145 lentivirus under the control of the smooth muscle cell (SMC)-specific promoter SM22 α or a SM22 α control lentivirus before commencing the Western diet for 12 weeks. The SMC-targeted microRNA-145 treatment markedly reduced plaque size in aortic sinuses, ascending aortas, and brachiocephalic arteries. It also significantly increased fibrous cap area, reduced necrotic core area, and increased plaque collagen content. Cellular plaque composition analyses revealed significantly less macrophages in ApoE −/− mice treated with the SMC-specific microRNA-145. These mice also demonstrated marked increases in calponin levels and α-smooth muscle actin–positive SMC areas in their atherosclerotic lesions. Furthermore, lentiviral delivery of microRNA-145 resulted in reduced KLF4 and elevated myocardin expression in aortas from ApoE −/− mice, consistent with an effect of microRNA-145 to promote a contractile phenotype in VSMC. Conclusions— VSMC-specific overexpression of microRNA-145 is a novel in vivo therapeutic target to limit atherosclerotic plaque morphology and cellular composition, shifting the balance toward plaque stability vs plaque rupture.

The tumor suppressor breast cancer susceptibility gene 2 (BRCA2) plays an important role in the repair of DNA damage, and loss of BRCA2 predisposes carriers to breast and ovarian cancers. Doxorubicin (DOX) remains the cornerstone of chemotherapy in such individuals. However, it is often associated with cardiac failure, which once manifests carries a poor prognosis. Because BRCA2 regulates genome-wide stability and facilitates DNA damage repair, we hypothesized that loss of BRCA2 may increase susceptibility to DOX-induced cardiac failure. To this aim, we generated cardiomyocyte-specific BRCA2 knock-out (CM-BRCA2(-/-)) mice using the Cre-loxP technology and evaluated their basal and post-DOX treatment phenotypes. Although CM-BRCA2(-/-) mice exhibited no basal cardiac phenotype, DOX treatment resulted in markedly greater cardiac dysfunction and mortality in CM-BRCA2(-/-) mice compared with control mice. Apoptosis in left ventricular (LV) sections from CM-BRCA2(-/-) mice compared with that in corresponding sections from wild-type (WT) littermate controls was also significantly enhanced after DOX treatment. Microscopic examination of LV sections from DOX-treated CM-BRCA2(-/-) mice revealed a greater number of DNA double-stranded breaks and the absence of RAD51 focus formation, an essential marker of double-stranded break repair. The levels of p53 and the p53-related proapoptotic proteins p53-up-regulated modulator of apoptosis (PUMA) and Bax were significantly increased in samples from CM-BRCA2(-/-) mice. This corresponded with increased Bax to Bcl-2 ratios and elevated cytochrome c release in the LV sections of DOX-treated CM-BRCA2(-/-) mice. Taken together, these data suggest a critical and previously unrecognized role of BRCA2 as a gatekeeper of DOX-induced cardiomyocyte apoptosis and susceptibility to overt cardiac failure. Pharmacogenomic studies evaluating cardiac function in BRCA2 mutation carriers treated with doxorubicin are encouraged.

P53 protein levels are elevated by trastuzumab and the biologically similar rat ERBB2/HER2/NEU antibody; and that this coincides with enhanced apoptosis, increased cleaved caspase-3 levels and diminished cardiac function. We also demonstrate that MDM2 may be a regulatory target of anti-ERBB2 thereby implicating the MDM2/p53 axis as a potential molecular component for the undesirable cardiac outcomes noted with trastuzumab. Finally, we show that these MDM2/p53-mediated events are independent of both the ERK1/2 and Akt systems. In conclusion, our findings suggest that the adverse cardiac events observed with trastuzumab may stem from its negative regulation of MDM2 events which impairs p53 degradation resultantly promoting apoptosis leading to cardiac dysfunction. These observations may have important therapeutic implications since they suggest that anticancer agents that inhibit MDM2 and its downstream actions may curb tumor progression at the expense of increasing cardiac stress.

Background— Adropin is a recently identified protein that has been implicated in the maintenance of energy homeostasis and insulin resistance. Because vascular function and insulin sensitivity are closely related, we hypothesized that adropin may also exert direct effects on the endothelium. Methods and Results— In vitro cell culture models were partnered with an in vivo murine injury model to determine the potential vascular effects of adropin. Adropin was expressed in human umbilical vein and coronary artery endothelial cells (ECs). Adropin-treated endothelial cells exhibited greater proliferation, migration and capillary-like tube formation and less permeability and tumor necrosis factor-α–induced apoptosis. In keeping with a vascular protective effect, adropin stimulated Akt Ser 473 and endothelial nitric oxide (NO) synthase Ser 1177 phosphorylation. The former was abrogated in the presence of the phosphatidylinositol 3-kinase inhibitor LY294002, whereas the latter was attenuated by LY294002 and by mitogen-activated protein kinase kinase 1 inhibition with PD98059. Together, these findings suggest that adropin regulates NO bioavailability and events via the phosphatidylinositol 3-kinase-Akt and extracellular signal regulated kinase 1/2 signaling pathways. Adropin markedly upregulated vascular endothelial growth factor receptor-2 (VEGFR2) transcript and protein levels, and in VEGFR2-silenced endothelial cells, adropin failed to induce phosphorylation of endothelial NO synthase, Akt, and extracellular signal regulated kinase 1/2, supporting VEGFR2 as an upstream target of adropin-mediated endothelial NO synthase activation. Last, adropin improved murine limb perfusion and elevated capillary density following induction of hindlimb ischemia. Conclusions— We report a potential endothelial protective role of adropin that is likely mediated via upregulation of endothelial NO synthase expression through the VEGFR2-phosphatidylinositol 3-kinase-Akt and VEGFR2-extracellular signal regulated kinase 1/2 pathways. Adropin represents a novel target to limit diseases characterized by endothelial dysfunction in addition to its favorable metabolic profile.

Improving endothelial nitric oxide synthase (eNOS) bioactivity and endothelial function is important to limit native, vein graft, and transplant atherosclerosis. Visfatin, a NAD biosynthetic enzyme, regulates the activity of the cellular survival factor, Sirt1. We hypothesized that visfatin may improve eNOS expression, endothelial function, and postnatal angiogenesis. In human umbilical vein (HUVEC) and coronary artery endothelial cells, we evaluated the effects of recombinant human visfatin on eNOS protein and transcript expression and mRNA stability, in the presence and absence of visfatin RNA silencing. We also assessed visfatin-induced protein kinase B (Akt) activation and its association with src-tyrosine kinases, phosphorylation of Ser1177within eNOS in the presence and absence of phosphatidylinositol 3-kinase (PI 3-kinase) inhibition with LY-294002, and evaluated the contributory role of extracellular signal-regulated kinase 1/2. Finally, we determined the impact of visfatin on HUVEC migration, proliferation, inflammation-induced permeability, and in vivo angiogenesis. Visfatin (100 ng/ml) upregulated and stabilized eNOS mRNA and increased the production of nitric oxide and cGMP. Visfatin-treated HUVEC demonstrated greater proliferation, migration, and capillary-like tube formation but less tumor necrosis factor-α-induced permeability; these effects were decreased in visfatin gene-silenced cells. Visfatin increased total Akt and Ser473-phospho-Akt expression with concomitant rises in eNOS phosphorylation at Ser1177; these effects were blocked by LY-2940002. Studies with PP2 showed that the nonreceptor tyrosine kinase, src, is an upstream stimulator of the PI 3-kinase-Akt pathway. Visfatin also activated mitogen-activated protein (MAP) kinase through PI 3-kinase, and mitogen/extracellular signal-regulated kinase inhibition attenuated visfatin-elicited Akt and eNOS phosphorylation. Visfatin-filled Matrigel implants showed an elevated number of infiltrating vessels, and visfatin treatment produced significant recovery of limb perfusion following hindlimb ischemia. These results indicate a novel effect of visfatin to stimulate eNOS expression and function in endothelial cells, via a common upstream, src-mediated signaling cascade, which leads to activation of Akt and MAP kinases. Visfatin represents a translational target to limit endothelial dysfunction, native, vein graft and transplant atherosclerosis, and improve postnatal angiogenesis.

INTRODUCTION: Preservation of structure and function of the myocardium is critically dependent upon improving the survival of existing cardiomyocytes (CM), through strategies that limit CM apoptosis and DNA damage. BRCA1 is a tumor suppressor gene which functions to promote DNA repair, and protect cells against oxidative and genotoxic stress. We hypothesized that BRCA1 is a novel cellular target to limit CM apoptosis, and prevent aberrant cardiac remodeling. METHODS AND RESULTS: Experimental MI in mice caused a profound 16-fold upregulation in BRCA1 expression, which peaked at 72 hours (pIn vitro gain-of-function experiments demonstrated that Ad-BRCA1 overexpression protected neonatal rat CM against doxorubicin- and H 2 O 2 -induced apoptosis, as assessed by FACS (p2 O 2 . Co-immunoprecipitation studies demonstrated a distinct physical interaction of BRCA1 with p53. Inhibition of p53, with pifithrin-alpha, blocked doxorubicin-induced CM apoptosis in a manner similar to BRCA1, but BRCA1-overexpressing CM, when treated with doxorubicin did not show further reduction with pifithrin-alpha, indicating an essential requirement of BRCA1 to modulate p53. In vivo gain-of-function studies demonstrated that systemic Ad-BRCA1 delivery completely prevented doxorubicin-induced cardiac dysfunction in mice (echocardiography, pIn vivo loss-of-function studies were performed in CM -specific BRCA1-KO mice (developed using Cre-lox P technology), which demonstrated marked cardiac dysfunction and mortality in response to doxorubicin administration (p< 0.01 vs. WT + Dox). CONCLUSIONS: We report for the first time an essential role of BRCA1 to limit CM apoptosis, and improve cardiac function in response to genotoxic and oxidative stress. Heart specific deletion of BRCA1 promotes severe systolic dysfunction, and limits survival. In addition to the immediate implications for cardiovascular repair, these data may have ramifications for individuals with BRCA1 mutations or cancer syndromes, particularly in the setting of adjuvant chemotherapy.

INTRODUCTION: Endothelial cell apoptosis, in response to inflammatory, ischemic and hypoxic stressors, has been identified as an essential target for therapies aimed at improving graft patency. BRCA1 is a tumor suppressor gene which functions primarily to promote DNA repair, and afford protection against various genotoxic stimuli, including immunosuppressant and chemotherapeutic regimens often employed post-transplantation. We hypothesized that BRCA1 is a novel target to limit inflammation-induced endothelial cell apoptosis and to restore endothelial function. METHODS AND RESULTS: In the first series of studies, we used a gain-of-function approach by adenoviral BRCA1 overexpression in human umbilical vein endothelial cells (HUVECs). Ad-BRCA1-expressing HUVECs exhibited profound protection against TNFα (20 ng/ml)-induced apoptosis, as assessed by flow cytometry, DNA laddering, COMET assay and cleaved procaspase 3 (P < 0.01). This response was mediated by complete prevention of TNFα-induced increases in endothelial ROS production (P < 0.001, vs. Ad-BRCA1). Furthermore, Ad-BRCA1 overexpression prevented TNFα-induced reduction in key indices of endothelial function, namely capillary-like tube formation (P < 0.01) and cell migration (P < 0.01). The protective effects of BRCA1 were not mediated by changes in total or phosphorylated p53 (P = 0.1) but were associated with a significant 6-fold upregulation in eNOS (P < 0.05). A loss-of-function strategy with BRCA1 gene silencing reversed the effects on endothelial apoptosis, ROS production and eNOS phosporylation. CONCLUSIONS: BRCA1 protects endothelial cells against inflammation-induced apoptosis, through a mechanism that involves up-regulation of eNOS and reduced ROS production, and is a novel candidate gene to limit aberrant vascular remodeling. These data also suggest that patients with BRCA1 mutations or cancer syndromes may be at an exaggerated risk of native and transplant atherosclerosis and graft dysfunction, particularly in the setting of DNA damaging immunosuppressants.

Marfan syndrome (MFS; OMIM#154700) is a connective tissue disorder characterized by manifestations in the ocular, skeletal and cardiovascular systems. MFS is caused by mutation in the fibrillin-1 gene (FBN1; OMIM#134797) and more than 550 mutations have been identified so far. FBN1 is approximately 230 kb in size and contains three evolutionarily conserved alternatively spliced exons B, A and C at the 5'end. In a first systematic attempt to associate sequence variations in the FBN1 5' alternatively spliced exons with MFS, we investigated 41 individuals fulfilling the diagnostic criteria of Ghent nosology or with features of MFS including at least one major criterion or involvement of two organ systems but not fulfilling a strict interpretation of the Ghent nosology, and known to be negative for mutations in the FBN1 exons 1-65 as well as the TGFBR2 and TGFBR1 coding regions. We identified five novel and one previously reported variants in the six unrelated probands and provide preliminary evidence for their role in pathogenesis.

and echocardiographic follow-up. RESULTS: In-hospital mortality was 12%. One patient died in the operating room from atrioventricular groove rupture and 2 died from congestive heart failure postoperatively. During a mean follow-up of 51 31 months, 8 patients died. Of these, 3 were a result of cardiovascular causes. Four patients underwent reoperation because of failure of MV repair (2), endocarditis (1) or hemolysis (1). All survivors were NYHA 2, with none to trivial mitral regurgitation in all MV repairs (12). At 5 years, actuarial survival was 56 21%, freedom from cardiac death was 70 19% and freedom from MV reoperation was 82 16%. CONCLUSIONS: After aggressive decalcification of the posterior MV annulus both MV repair and replacement are feasible with acceptable procedure-related mortality. It is clear however, that decalcification increases complexity and risk. While an aggressive approach is advisable in young patients and those with infective endocarditis, the choice of a more conservative approach may be appropriate in fragile and elderly patients.

Background Current limitations to the experimentation on patients with peripheral arterial disease push the development of different preclinical strategies. We investigated both duration of ischemia and blood flow recovery in mouse models of partial femoral artery ligation. Methods Male BALB/c mice were used. The ligation over needle method involved placing a suture needle over the femoral artery, ligating over it and then removing the needle. The transfixation method involved transfixing the approximate center of the femoral artery and then tying the suture. Laser Doppler Perfusion Imaging was used to assess perfusion every 3rd day until 42 days after the procedure. Results Ligation over needle method: Immediately post procedure, mean perfusion was -71.87% ± 4.43. Then mean difference in perfusion remained below the base line reading on days 3, 6, 9, and 12. From day 15 on wards mean perfusion progressively improved remaining near base line. Transfixation Method: Immediately post procedure mean perfusion was -70.82% ± 4.73. Mean perfusion improved following the procedure on days 3 and 6; a plateau followed this on days 9, 12 and 15. From day 15 onwards perfusion progressively improved remaining well below base line until crossing it on day 36. Conclusion The currently described models do not pose major improvements over previously described methods.

ObjectiveBone morphogenetic protein-2 (BMP-2) is a major regulator of aortic valve calcification. MicroRNAs (miRNAs) are essential post-transcriptional modulators of gene expression and miRNA-141 is a known repressor of BMP-2–mediated osteogenesis. We hypothesized that miRNA-141 is a key regulator of aortic valve calcification.MethodsPorcine valvular interstitial cells were isolated, transfected with miRNA-141 or control, and stimulated with transforming growth factor-β. The BMP-2, extracellular signal-regulated kinase 1/2, and runt-related transcription factor 2 levels were determined by immunoblotting and reverse transcriptase polymerase chain reaction. To determine the role of miRNA-141 in bicuspid aortic valve disease, human bicuspid (n = 19) and tricuspid (n = 17) aortic valve leaflets obtained intraoperatively were submitted for GenoExplorer human microRNA array, immunoblotting, and histologic and immunohistochemical analyses.ResultsStimulation of porcine aortic valvular interstitial cells with transforming growth factor-β induced morphologic alterations consistent with myofibroblastic transformation, BMP-2 signaling, and calcification. Transfection with miRNA-141 restored transforming growth factor-β–induced valvular interstitial cell activation, BMP-2 signaling, and alkaline phosphatase activity (3.55 ± 0.18 vs 4.01 ± 0.21, P