Praphulla C. Shukla
St. Michael's Hospital
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Featured researches published by Praphulla C. Shukla.
Circulation | 2012
Fina Lovren; Yi Pan; Adrian Quan; Krishna K. Singh; Praphulla C. Shukla; Nandini Gupta; Brent M. Steer; Alistair J. Ingram; Milan Gupta; Mohammed Al-Omran; Hwee Teoh; Philip A. Marsden; Subodh Verma
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&agr; or a SM22&agr; 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 &agr;-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.
American Journal of Physiology-heart and Circulatory Physiology | 2010
Fina Lovren; Yi Pan; Adrian Quan; Paul E. Szmitko; Krishna Singh; Praphulla C. Shukla; Milan Gupta; Lawrence Chan; Mohammed Al-Omran; Hwee Teoh; Subodh Verma
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-alpha 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)-gamma, via PPAR-alpha. 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.
American Journal of Physiology-heart and Circulatory Physiology | 2008
Fina Lovren; Yi Pan; Adrian Quan; Hwee Teoh; Guilin Wang; Praphulla C. Shukla; Kevin S. Levitt; Gavin Y. Oudit; Mohammed Al-Omran; Duncan J. Stewart; Arthur S. Slutsky; Mark D. Peterson; Peter H. Backx; Josef M. Penninger; Subodh Verma
The endothelium plays a central role in the maintenance of vascular homeostasis. One of the main effectors of endothelial dysfunction is ANG II, and pharmacological approaches to limit ANG II bioactivity remain the cornerstone of cardiovascular therapeutics. Angiotensin converting enzyme-2 (ACE2) has been identified as a critical negative modulator of ANG II bioactivity, counterbalancing the effects of ACE in determining net tissue ANG II levels; however, the role of ACE2 in the vasculature remains unknown. In the present study, we hypothesized that ACE2 is a novel target to limit endothelial dysfunction and atherosclerosis. To this aim, we performed in vitro gain and loss of function experiments in endothelial cells and evaluated in vivo angiogenesis and atherosclerosis in apolipoprotein E-knockout mice treated with AdACE2. ACE2-deficient mice exhibited impaired endothelium-dependent relaxation. Overexpression of ACE2 in human endothelial cells stimulated endothelial cell migration and tube formation, and limited monocyte and cellular adhesion molecule expression; effects that were reversed in ACE2 gene silenced and endothelial cells isolated from ACE2-deficient animals. ACE2 attenuated ANG II-induced reactive oxygen species production in part through decreasing the expression of p22phox. The effects of ACE2 on endothelial activation were attenuated by pharmacological blockade of ANG-(1-7) with A779. ACE2 promoted capillary formation and neovessel maturation in vivo and reduced atherosclerosis in apolipoprotein E-knockout mice These data indicate that ACE2, in an ANG-(1-7)-dependent fashion, functions to improve endothelial homeostasis via a mechanism that may involve attenuation of NADPHox-induced reactive oxygen species production. ACE2-based treatment approaches may be a novel approach to limit aberrant vascular responses and atherothrombosis.
Circulation | 2010
Fina Lovren; Yi Pan; Adrian Quan; Krishna Singh; Praphulla C. Shukla; Milan Gupta; Mohammed Al-Omran; Hwee Teoh; Subodh Verma
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-&agr;-induced apoptosis. In keeping with a vascular protective effect, adropin stimulated Akt Ser473 and endothelial nitric oxide (NO) synthase Ser1177 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.
Circulation Research | 2005
Gunnar Klein; Arnd Schaefer; Denise Hilfiker-Kleiner; Dagmar Oppermann; Praphulla C. Shukla; Anja Quint; Eva Podewski; Andres Hilfiker; Frank Schröder; Michael Leitges; Helmut Drexler
Overexpression and activation of protein kinase C-&egr; (PKC&egr;) results in myocardial hypertrophy. However, these observations do not establish that PKC&egr; is required for the development of myocardial hypertrophy. Thus, we subjected PKC&egr;-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 I&agr;1 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<0.05). To explore underlying mechanisms, we analyzed the left ventricular (LV) expression pattern of additional PKC isoforms (ie, PKC&agr;, PKC&bgr;, and PKC&dgr;). After TAC, expression and activation of PKC&dgr; protein was increased in KO LVs. Moreover, KO LVs displayed enhanced activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), whereas p42/p44–MAPK activation was attenuated. Under stretch, cultured KO fibroblasts showed a 2-fold increased collagen I&agr;1 (col I&agr;1) expression, which was prevented by PKC&dgr; inhibitor rottlerin or by p38 MAPK inhibitor SB 203580. In conclusion, PKC&egr; is not required for the development of a pressure overload–induced myocardial hypertrophy. Lack of PKC&egr; results in upregulation of PKC&dgr; and promotes activation of p38 MAPK and JNK, which appears to compensate for cardiac hypertrophy, but in turn, is associated with increased collagen deposition and impaired diastolic function.
Circulation | 2010
Denise Hilfiker-Kleiner; Praphulla C. Shukla; Gunnar Klein; Arnd Schaefer; Britta Stapel; Melanie Hoch; Werner Müller; Michaela Scherr; Gregor Theilmeier; Matthias Ernst; Andres Hilfiker; Helmut Drexler
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 gp130Y757F mutant mice, we investigated the effect of disturbed gp130 signaling after myocardial infarction. Methods and Results— The cardiomyocyte-restricted &agr;-myosin heavy chain-Cre-recombinase-loxP system was used to generate mice with gp130Y757F mutant cardiomyocytes (&agr;MHC-Cretg/−;gp130fl/Y757F [Y757F]); all other cells carried at least 1 functional gp130 gene, ensuring normal gp130 signaling. Y757F mice displayed normal cardiac function and morphology at 3 months of age comparable to their nonmutant littermates. In response to myocardial infarction, Y757F 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 Y757F mice. Stronger effects were observed with a genetic reduction of STAT3 (STAT3flox/+) restricted to cardiomyocytes in Y757F 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.
American Journal of Physiology-endocrinology and Metabolism | 2009
Fina Lovren; Yi Pan; Praphulla C. Shukla; Adrian Quan; Hwee Teoh; Paul E. Szmitko; Mark D. Peterson; Milan Gupta; Mohammed Al-Omran; Subodh Verma
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 Ser(1177) within 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-alpha-induced permeability; these effects were decreased in visfatin gene-silenced cells. Visfatin increased total Akt and Ser(473)-phospho-Akt expression with concomitant rises in eNOS phosphorylation at Ser(1177); 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.
Nature Communications | 2011
Praphulla C. Shukla; Krishna K. Singh; Adrian Quan; Mohammed Al-Omran; Hwee Teoh; Fina Lovren; Liu Cao; Ilsa I. Rovira; Yi Pan; Christine Brezden-Masley; Bobby Yanagawa; Aanika Gupta; Chu-Xia Deng; John G. Coles; Howard Leong-Poi; William L. Stanford; Thomas G. Parker; Michael D. Schneider; Toren Finkel; Subodh Verma
The tumour suppressor BRCA1 is mutated in familial breast and ovarian cancer but its role in protecting other tissues from DNA damage has not been explored. Here we show a new role for BRCA1 as a gatekeeper of cardiac function and survival. In mice, loss of BRCA1 in cardiomyocytes results in adverse cardiac remodelling, poor ventricular function and higher mortality in response to ischaemic or genotoxic stress. Mechanistically, loss of cardiomyocyte BRCA1 results in impaired DNA double-strand break repair and activated p53-mediated pro-apoptotic signalling culminating in increased cardiomyocyte apoptosis, whereas deletion of the p53 gene rescues BRCA1-deficient mice from cardiac failure. In human adult and fetal cardiac tissues, ischaemia induces double-strand breaks and upregulates BRCA1 expression. These data reveal BRCA1 as a novel and essential adaptive response molecule shielding cardiomyocytes from DNA damage, apoptosis and heart dysfunction. BRCA1 mutation carriers, in addition to risk of breast and ovarian cancer, may be at a previously unrecognized risk of cardiac failure.
The Journal of Thoracic and Cardiovascular Surgery | 2012
Bobby Yanagawa; Fina Lovren; Yi Pan; Vinay Garg; Adrian Quan; Gilbert Tang; Krishna K. Singh; Praphulla C. Shukla; Nikhil P. Kalra; Mark D. Peterson; Subodh Verma
OBJECTIVE Bone 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. METHODS Porcine 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. RESULTS Stimulation 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 < .05), suggesting upstream regulation by miRNA-141. miRNA microarray demonstrated differential expression of 35 of 1583 miRNA sequences in the bicuspid versus tricuspid aortic valve leaflets, with a 14.5-fold decrease in miRNA-141 in the bicuspid versus tricuspid leaflets (P < .05). This was associated with significantly increased BMP-2 protein expression in bicuspid aortic valve compared with the tricuspid aortic valve leaflets (P < .001). CONCLUSIONS We report a completely novel role of miRNA-141 as a regulator of BMP-2-dependent aortic valvular calcification and demonstrate marked attenuation of miRNA-141 expression in patients with bicuspid aortic valve-associated aortic stenosis. Therapeutic targeting of miRNA-141 could serve as a novel strategy to limit progressive calcification in aortic stenosis.
Journal of Biological Chemistry | 2012
Krishna Singh; Praphulla C. Shukla; Adrian Quan; Jean-Francois Desjardins; Fina Lovren; Yi Pan; Vinay Garg; Sumandeep Gosal; Ankit Garg; Paul E. Szmitko; Michael D. Schneider; Thomas G. Parker; William Stanford; Howard Leong-Poi; Hwee Teoh; Mohammed Al-Omran; Subodh Verma
Background: BRCA2 is widely implicated in breast and ovarian cancers, but the role of BRCA2 in the heart is unknown. Results: Loss of BRCA2 in the heart resulted in increased doxorubicin-induced DNA damage, apoptosis, and cardiac dysfunction. Conclusion: BRCA2 is a novel regulator of cardiomyocyte genomic integrity, survival, and function. Significance: BRCA2 mutation carriers may be at a heightened risk of anthracycline-induced cardiac failure. 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.