Dan Sorescu

Publications of Dan Sorescu

• Ranolazine improves cardiac diastolic dysfunction through modulation of myofilament calcium sensitivity.

  Authors: Joshua D Lovelock, Michelle M Monasky, Euy-Myoung Jeong, Harvey A Lardin, Hong Liu, Bindiya G Patel, Domenico M Taglieri, Lianzhi Gu, Praveen Kumar, Narayan Pokhrel, Dewan Zeng, Luiz Belardinelli, Dan Sorescu, R John Solaro, Samuel C Dudley

  Circulation research. 02/2012; 110(6):841-50.

  Rationale: Previously, we demonstrated that a deoxycorticosterone acetate (DOCA)-salt hypertensive mouse model produces cardiac oxidative stress and diastolic dysfunction with preserved systolicRationale: Previously, we demonstrated that a deoxycorticosterone acetate (DOCA)-salt hypertensive mouse model produces cardiac oxidative stress and diastolic dysfunction with preserved systolic function. Oxidative stress has been shown to increase late inward sodium current (I(Na)), reducing the net cytosolic Ca(2+) efflux. Objective: Oxidative stress in the DOCA-salt model may increase late I(Na), resulting in diastolic dysfunction amenable to treatment with ranolazine. Methods and Results: Echocardiography detected evidence of diastolic dysfunction in hypertensive mice that improved after treatment with ranolazine (E/E':sham, 31.9±2.8, sham+ranolazine, 30.2±1.9, DOCA-salt, 41.8±2.6, and DOCA-salt+ranolazine, 31.9±2.6; P=0.018). The end-diastolic pressure-volume relationship slope was elevated in DOCA-salt mice, improving to sham levels with treatment (sham, 0.16±0.01 versus sham+ranolazine, 0.18±0.01 versus DOCA-salt, 0.23±0.2 versus DOCA-salt+ranolazine, 0.17±0.0 1 mm Hg/L; P<0.005). DOCA-salt myocytes demonstrated impaired relaxation, τ, improving with ranolazine (DOCA-salt, 0.18±0.02, DOCA-salt+ranolazine, 0.13±0.01, sham, 0.11±0.01, sham+ranolazine, 0.09±0.02 seconds; P=0.0004). Neither late I(Na) nor the Ca(2+) transients were different from sham myocytes. Detergent extracted fiber bundles from DOCA-salt hearts demonstrated increased myofilament response to Ca(2+) with glutathionylation of myosin binding protein C. Treatment with ranolazine ameliorated the Ca(2+) response and cross-bridge kinetics. Conclusions: Diastolic dysfunction could be reversed by ranolazine, probably resulting from a direct effect on myofilaments, indicating that cardiac oxidative stress may mediate diastolic dysfunction through altering the contractile apparatus.

• Role of Endoplasmic Reticulum Stress in Age-Related Susceptibility to Lung Fibrosis.

  Authors: Edilson Torres-González, Marta Bueno, Atsuko Tanaka, Laurie T Krug, Dong-Sheng Cheng, Vasiliy V Polosukhin, Dan Sorescu, William E Lawson, Timothy S Blackwell, Mauricio Rojas, Ana L Mora

  American journal of respiratory cell and molecular biology. 01/2012;

  The incidence of Idiopathic pulmonary fibrosis (IPF) increases with age. The mechanisms that underlie the age-dependent risk for IPF are unknown. Based on studies that suggest an association of IPFThe incidence of Idiopathic pulmonary fibrosis (IPF) increases with age. The mechanisms that underlie the age-dependent risk for IPF are unknown. Based on studies that suggest an association of IPF and gammaherpesvirus infection, we infected young (2-3 mo) and old (≥18 mo) C57BL/6 mice with the murine gammaherpesvirus 68 (MHV68). Acute MHV68 infection in aging mice resulted in severe pneumonitis and fibrosis compared with young animals. Progressive clinical deterioration and lung fibrosis in the late chronic phase of infection was observed exclusively in old mice with diminution of tidal volume. Infected aging mice showed higher expression of TGF-β during the acute phase of infection. In addition, aging infected mice showed elevation of pro-inflammatory cytokines and the fibrocyte recruitment chemokine CXCL12 in bronchoalveolar lavage. Analyses of lytic virus infection and virus reactivation indicate that old mice were able to control chronic infection and elicit anti-virus immune responses. However, old infected mice showed a significant increase in apoptotic responses determined by in situ TUNEL assay, levels of caspase-3 and expression of the pro-apoptotitc molecule BIM. Apoptosis of type II lung epithelial cells in aging lungs was accompanied by upregulation of ER stress marker BiP and splicing of XBP1. These results indicate that the aging lung is more susceptible to injury and fibrosis associated with ER stress, apoptosis of type II lung epithelial cells and activation of pro-fibrotic pathways.

• Glp-1 analog, liraglutide, ameliorates hepatic steatosis and cardiac hypertrophy in C57BL/6J mice fed a Western diet.

  Authors: Jamie E Mells, Ping P Fu, Shvetank Sharma, Darin Olson, Lihong Cheng, Jeffrey A Handy, Neeraj K Saxena, Dan Sorescu, Frank A Anania

  American journal of physiology. Gastrointestinal and liver physiology. 01/2012; 302(2):G225-35.

  The aims of this study were designed to determine whether liraglutide, a long-acting glucagon-like peptide, could reverse the adverse effects of a diet high in fat that also contained trans-fat andThe aims of this study were designed to determine whether liraglutide, a long-acting glucagon-like peptide, could reverse the adverse effects of a diet high in fat that also contained trans-fat and high-fructose corn syrup (ALIOS diet). Specifically, we examined whether treatment with liraglutide could reduce hepatic insulin resistance and steatosis as well as improve cardiac function. Male C57BL/6J mice were pair fed or fed ad libitum either standard chow or the ALIOS diet. After 8 wk the mice were further subdivided and received daily injections of either liraglutide or saline for 4 wk. Hyperinsulinemic-euglycemic clamp studies were performed after 6 wk, revealing hepatic insulin resistance. Glucose tolerance and insulin resistance tests were performed at 8 and 12 wk prior to and following liraglutide treatment. Liver pathology, cardiac measurements, blood chemistry, and RNA and protein analyses were performed. Clamp studies revealed hepatic insulin resistance after 6 wk of ALIOS diet. Liraglutide reduced visceral adiposity and liver weight (P < 0.001). As expected, liraglutide improved glucose and insulin tolerance. Liraglutide improved hypertension (P < 0.05) and reduced cardiac hypertrophy. Surprisingly, liver from liraglutide-treated mice had significantly higher levels of fatty acid binding protein, acyl-CoA oxidase II, very long-chain acyl-CoA dehydrogenase, and microsomal triglyceride transfer protein. We conclude that liraglutide reduces the harmful effects of an ALIOS diet by improving insulin sensitivity and by reducing lipid accumulation in liver through multiple mechanisms including, transport, and increase β-oxidation.

• Diastolic dysfunction is associated with cardiac fibrosis in the senescence-accelerated mouse.

  Authors: Alana L Reed, Atsuko Tanaka, Dan Sorescu, Hong Liu, Euy-Myoung Jeong, Megan Sturdy, Erik R Walp, Samuel C Dudley, Roy L Sutliff

  American journal of physiology. Heart and circulatory physiology. 07/2011; 301(3):H824-31.

  Diastolic heart failure is a major cause of mortality in the elderly population. It is often preceded by diastolic dysfunction, which is characterized by impaired active relaxation and increasedDiastolic heart failure is a major cause of mortality in the elderly population. It is often preceded by diastolic dysfunction, which is characterized by impaired active relaxation and increased stiffness. We tested the hypothesis that senescence-prone (SAMP8) mice would develop diastolic dysfunction compared with senescence-resistant controls (SAMR1). Pulsed-wave Doppler imaging of the ratio of blood flow velocity through the mitral valve during early (E) vs. late (A) diastole was reduced from 1.3 ± 0.03 in SAMR1 mice to 1.2 ± 0.03 in SAMP8 mice (P < 0.05). Tissue Doppler imaging of the early (E') and late (A') diastolic mitral annulus velocities found E' reduced from 25.7 ± 0.9 mm/s in SAMR1 to 21.1 ± 0.8 mm/s in SAMP8 mice and E'/A' similarly reduced from 1.1 ± 0.02 to 0.8 ± 0.03 in SAMR1 vs. SAMP8 mice, respectively (P < 0.05). Invasive hemodynamics revealed an increased slope of the end-diastolic pressure-volume relationship (0.5 ± 0.05 vs. 0.8 ± 0.14; P < 0.05), indicating increased left ventricular chamber stiffness. There were no differences in systolic function or mean arterial pressure; however, diastolic dysfunction was accompanied by increased fibrosis in the hearts of SAMP8 mice. In SAMR1 vs. SAMP8 mice, interstitial collagen area increased from 0.3 ± 0.04 to 0.8 ± 0.09% and perivascular collagen area increased from 1.0 ± 0.11 to 1.6 ± 0.14%. Transforming growth factor-β and connective tissue growth factor gene expression were increased in the hearts of SAMP8 mice (P < 0.05 for all data). In summary, SAMP8 mice show increased fibrosis and diastolic dysfunction similar to those seen in humans with aging and may represent a suitable model for future mechanistic studies.

• Patient perception versus medical record entry of health-related conditions among patients with heart failure.

  Authors: Adnan S Malik, Grigorios Giamouzis, Vasiliki V Georgiopoulou, Lucy V Fike, Andreas P Kalogeropoulos, Catherine R Norton, Dan Sorescu, Sidra Azim, Sonjoy R Laskar, Andrew L Smith, Sandra B Dunbar, Javed Butler

  The American journal of cardiology. 02/2011; 107(4):569-72.

  A shared understanding of medical conditions between patients and their health care providers may improve self-care and outcomes. In this study, the concordance between responses to a medical historyA shared understanding of medical conditions between patients and their health care providers may improve self-care and outcomes. In this study, the concordance between responses to a medical history self-report (MHSR) form and the corresponding provider documentation in electronic health records (EHRs) of 19 select co-morbidities and habits in 230 patients with heart failure were evaluated. Overall concordance was assessed using the κ statistic, and crude, positive, and negative agreement were determined for each condition. Concordance between MHSR and EHR varied widely for cardiovascular conditions (κ = 0.37 to 0.96), noncardiovascular conditions (κ = 0.06 to 1.00), and habits (κ = 0.26 to 0.69). Less than 80% crude agreement was seen for history of arrhythmias (72%), dyslipidemia (74%), and hypertension (79%) among cardiovascular conditions and lung disease (70%) and peripheral arterial disease (78%) for noncardiovascular conditions. Perfect agreement was observed for only 1 of the 19 conditions (human immunodeficiency virus status). Negative agreement >80% was more frequent than >80% positive agreement for a condition (15 of 19 [79%] vs 8 of 19 [42%], respectively, p = 0.02). Only 20% of patients had concordant MSHRs and EHRs for all 7 cardiovascular conditions; in 40% of patients, concordance was observed for ≤5 conditions. For noncardiovascular conditions, only 28% of MSHR-EHR pairs agreed for all 9 conditions; 37% agreed for ≤7 conditions. Cumulatively, 39% of the pairs matched for ≤15 of 19 conditions. In conclusion, there is significant variation in the perceptions of patients with heart failure compared to providers' records of co-morbidities and habits. The root causes of this variation and its impact on outcomes need further study.

• Inhibition of NF-kappaB signaling reduces virus load and gammaherpesvirus-induced pulmonary fibrosis.

  Authors: Laurie T Krug, Edilson Torres-González, Qianhong Qin, Dan Sorescu, Mauricio Rojas, Arlene Stecenko, Samuel H Speck, Ana L Mora

  The American journal of pathology. 08/2010; 177(2):608-21.

  Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disorder of unknown etiology. Several studies have demonstrated an association between pulmonary infection with a herpesvirus andIdiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disorder of unknown etiology. Several studies have demonstrated an association between pulmonary infection with a herpesvirus and IPF. Based on those observations, we have developed a mouse model in which interferon (IFN)gammaR(-/-) mice infected intranasally with murine gammaherpesvirus 68 (MHV68) develop lung fibrosis. We hypothesize that viral load was a critical factor for the development of fibrosis. Because nuclear factor (NF)-kappaB signaling is required to efficiently establish gammaherpesvirus, latency we infected IFNgammaR(-/-) mice with a MHV68 virus that expresses a mutant dominant inhibitor of the NF-kappaB signaling pathway, called IkappaBalphaM. Striking differences were observed at the onset of the chronic infection, which correlated with a decreased virus load in mice infected with MHV68-IkappaBalphaM compared with mice infected with control MHV68 (MHV68-MR). IFNgammaR(-/-) mice infected with MHV68-IkappaBalphaM lacked vasculitis and fibrosis 15 to 120 days post infection. Inhibition of NF-kappaB in MHV68-infected cells of the lungs diminished the expression of the fibrocyte recruiting chemokines monocyte chemoattractant protein 1 (MCP-1) and CXCL12, ameliorated macrophage expression of markers of alternative activation, and failed to increase expression of the integrin alphavbeta6, which is implicated in the activation of the profibrotic factor TGF-beta. Thus, the inhibition of NF-kappaB signaling in the infected lung cells of IFNgammaR(-/-) mice reduces virus persistence and ameliorates profibrotic events. Host determinants of latency might therefore represent new therapeutic targets for gammaherpesvirus-associated pulmonary fibrosis.

• NADPH oxidases 1 and 4 mediate cellular senescence induced by resveratrol in human endothelial cells.

  Authors: Yvonne D C Schilder, Elke H Heiss, Daniel Schachner, Jürgen Ziegler, Gottfried Reznicek, Dan Sorescu, Verena M Dirsch

  Free radical biology & medicine. 04/2009;

  Resveratrol is believed to be partially responsible for the French paradox-the low risk of cardiovascular disease despite a high-fat diet in the French population. Recently, resveratrol has also beenResveratrol is believed to be partially responsible for the French paradox-the low risk of cardiovascular disease despite a high-fat diet in the French population. Recently, resveratrol has also been discussed as a life-span booster in several organisms. Age-related diseases are associated on the cellular level with senescence. We, therefore, hypothesized that resveratrol is vasoprotective by counteracting endothelial cell senescence. Surprisingly, we observed that chronic treatment with resveratrol (10 muM) was prosenescent in primary human endothelial cells. Resveratrol induced elevated reactive oxygen species (ROS) levels that were associated with and causally linked to an accumulation of cells in the S phase of the cell cycle, as measured by flow cytometry. We further show that cell accumulation in S phase leads to increased ROS and finally senescence. Using an siRNA approach, we clearly identified two NADPH oxidases, Nox1 and Nox4, as major targets of resveratrol and primary sources of ROS that act upstream of the observed S-phase accumulation.

• FOXO3A regulates peroxiredoxin III expression in human cardiac fibroblasts.

  Authors: Calin B Chiribau, Lihong Cheng, Ioan C Cucoranu, Yong-Shen Yu, Roza E Clempus, Dan Sorescu

  The Journal of biological chemistry. 04/2008; 283(13):8211-7.

  Human cardiac fibroblasts are protected from oxidative stress triggered by inflammation after myocardial injury (Li, P. F., Dietz, R., and von Harsdorf, R. (1999) FEBS Lett. 448, 206-210) byHuman cardiac fibroblasts are protected from oxidative stress triggered by inflammation after myocardial injury (Li, P. F., Dietz, R., and von Harsdorf, R. (1999) FEBS Lett. 448, 206-210) by expressing potent antioxidant defenses such as superoxide dismutases, catalases, glutathione-peroxidases, and peroxiredoxins. Recently the transcription factor FOXO3A has been shown to increase resistance to oxidative stress by up-regulation of mitochondrial superoxide dismutase and peroxisomal catalase (Kops, G. J., Dansen, T. B., Polderman, P. E., Saarloos, I., Wirtz, K. W., Coffer, P. J., Huang, T. T., Bos, J. L., Medema, R. H., and Burgering, B. M. (2002) Nature 419, 316-321; Nemoto, S., and Finkel, T. (2002) Science 295, 2450-2452). We hypothesized that FOXO3A also regulates the expression of Prx III, the mitochondrial peroxiredoxin, in human cardiac fibroblasts. We found that depletion of FOXO3A leads to a dramatic reduction of Prx III mRNA and protein in serum-deprived human cardiac fibroblasts. These data suggest that endogenous FOXO3A is necessary for base-line expression of Prx III. Next, we identified two putative FOXO3A DNA binding sites in Prx III promoter at -267 and -244 nucleotides relative to the start codon. We demonstrated that both sequences are required for binding of endogenous FOXO3A to the Prx III promoter by performing electromobility shift assays and chromatin immunoprecipitation assays. Inhibition of endogenous FOXO3A by insulin growth factor 1 prevented binding of FOXO3A to Prx III promoter. In contrast, overexpression of FOXO3A increased Prx III promoter activity. Furthermore, depletion of Prx III was associated with enhanced apoptosis and oxidative stress after serum deprivation. We conclude that FOXO3A mediates Prx III expression, and this may play a critical role in the resistance to oxidative stress in cardiac fibroblasts.

• Speckle-tracking strain echocardiography for detecting cardiac dyssynchrony in a canine model of dyssynchrony and heart failure.

  Authors: Takeshi Arita, George P Sorescu, Brian T Schuler, Laura S Schmarkey, John D Merlino, Jakob Vinten-Johansen, Angel R Leon, Randolph P Martin, Dan Sorescu

  American journal of physiology. Heart and circulatory physiology. 08/2007; 293(1):H735-42.

  Multiple echocardiographic criteria have been proposed to diagnose mechanical dyssynchrony in patients with heart failure without being validated against a model of cardiac dyssynchrony with heartMultiple echocardiographic criteria have been proposed to diagnose mechanical dyssynchrony in patients with heart failure without being validated against a model of cardiac dyssynchrony with heart failure. This study examines which of these methods can detect dyssynchrony in a canine model. Adult mongrel dogs underwent His-bundle ablation and right-ventricular pacing for 4 wk at either 110 bpm to induce dyssynchrony without heart failure (D group, n = 12) or 170 bpm to induce dyssynchrony with heart failure (DHF group, n = 9). To induce heart failure with narrow QRS, atria were paced at 190 bpm for 4 wk (HF group, n = 8). Tissue Doppler imaging (TDI) and two-dimensional echocardiography were performed at baseline and at end of study. Standard deviation of time to peak systolic velocity (color-coded TDI), time to peak S wave on pulse-wave TDI, time to peak radial and circumferential strain by speckle-tracking analysis (E(rr) and E(cc), respectively), and septal-to-posterior wall motion delay on M mode were obtained. In D group, only E(rr) and E(cc) were increased by dyssynchrony. In contrast, all the echocardiographic parameters of dyssynchrony appeared significantly augmented in the DHF group. Receiver-operator curve analysis showed good sensitivity of E(rr) (90%) and E(cc) (100%) to detected dyssynchrony without heart failure and excellent sensitivity and specificity of E(rr) and E(cc) to detect dyssynchrony with heart failure. Radial strain by speckle tracking is more accurate than TDI velocity to detect cardiac dyssynchrony in a canine model of dyssynchrony with or without heart failure.

• Hemodynamic shear stresses in mouse aortas: implications for atherogenesis.

  Authors: Jin Suo, Dardo E Ferrara, Dan Sorescu, Robert E Guldberg, W Robert Taylor, Don P Giddens

  Arteriosclerosis, thrombosis, and vascular biology. 03/2007; 27(2):346-51.

  OBJECTIVE: The hemodynamic environment is a determinant of susceptibility to atherosclerosis in the vasculature. Although mouse models are commonly used in atherosclerosis studies, little is knownOBJECTIVE: The hemodynamic environment is a determinant of susceptibility to atherosclerosis in the vasculature. Although mouse models are commonly used in atherosclerosis studies, little is known about local variations in wall shear stress (WSS) in the mouse and whether the levels of WSS are comparable to those in humans. The objective of this study was to determine WSS values in the mouse aorta and to relate these to expression of gene products associated with atherosclerosis. METHODS AND RESULTS: Using micro-CT and ultrasound methodologies we developed a computational fluid dynamics model of the mouse aorta and found values of WSS to be much larger than those for humans. We also used a quantum dot-based approach to study vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression on the aortic intima and demonstrated that increased expression for these molecules occurs where WSS was relatively low for the mouse. CONCLUSIONS: Despite large differences in WSS in the two species, the spatial distributions of atherogenic molecules in the mouse aorta are similar to atherosclerotic plaque localization found in human aortas. These results suggest that relative differences in WSS or in the direction of WSS, as opposed to the absolute magnitude, may be relevant determinants of flow-mediated inflammatory responses.

• Nox4 is required for maintenance of the differentiated vascular smooth muscle cell phenotype.

  Authors: Roza E Clempus, Dan Sorescu, Anna E Dikalova, Lily Pounkova, Patricia Jo, George P Sorescu, Harald H H Schmidt, Bernard Lassègue, Kathy K Griendling

  Arteriosclerosis, thrombosis, and vascular biology. 02/2007; 27(1):42-8.

  OBJECTIVE: The mechanisms responsible for maintaining the differentiated phenotype of adult vascular smooth muscle cells (VSMCs) are incompletely understood. Reactive oxygen species (ROS) have beenOBJECTIVE: The mechanisms responsible for maintaining the differentiated phenotype of adult vascular smooth muscle cells (VSMCs) are incompletely understood. Reactive oxygen species (ROS) have been implicated in VSMC differentiation, but the responsible sources are unknown. In this study, we investigated the role of Nox1 and Nox4-derived ROS in this process. METHODS AND RESULTS: Primary VSMCs were used to study the relationship between Nox homologues and differentiation markers such as smooth muscle alpha-actin (SM alpha-actin), smooth muscle myosin heavy chain (SM-MHC), heavy caldesmon, and calponin. We found that Nox4 and differentiation marker genes were downregulated from passage 1 to passage 6 to 12, whereas Nox1 was gradually upregulated. Nox4 co-localized with SM alpha-actin-based stress fibers in differentiated VSMC, and moved into focal adhesions in de-differentiated cells. siRNA against nox4 reduced NADPH-driven superoxide production in serum-deprived VSMCs and downregulated SM-alpha actin, SM-MHC, and calponin, as well as SM-alpha actin stress fibers. Nox1 depletion did not decrease these parameters. CONCLUSIONS: Nox4-derived ROS are critical to the maintenance of the differentiated phenotype of VSMCs. These findings highlight the importance of identifying the specific source of ROS involved in particular cellular functions when designing therapeutic interventions.

• NAD(P)H oxidase 4 mediates transforming growth factor-beta1-induced differentiation of cardiac fibroblasts into myofibroblasts.

  Authors: Ioan Cucoranu, Roza Clempus, Anna Dikalova, Patrick J Phelan, Srividya Ariyan, Sergey Dikalov, Dan Sorescu

  Circulation research. 10/2005; 97(9):900-7.

  Human cardiac fibroblasts are the main source of cardiac fibrosis associated with cardiac hypertrophy and heart failure. Transforming growth factor-beta1 (TGF-beta1) irreversibly converts fibroblastsHuman cardiac fibroblasts are the main source of cardiac fibrosis associated with cardiac hypertrophy and heart failure. Transforming growth factor-beta1 (TGF-beta1) irreversibly converts fibroblasts into pathological myofibroblasts, which express smooth muscle alpha-actin (SM alpha-actin) de novo and produce extracellular matrix. We hypothesized that TGF-beta1-stimulated conversion of fibroblasts to myofibroblasts requires reactive oxygen species derived from NAD(P)H oxidases (Nox). We found that TGF-beta1 potently upregulates the contractile marker SM alpha-actin mRNA (7.5+/-0.8-fold versus control). To determine whether Nox enzymes are involved, we first performed quantitative real time polymerase chain reaction and found that Nox5 and Nox4 are abundantly expressed in cardiac fibroblasts, whereas Nox1 and Nox2 are barely detectable. On stimulation with TGF-beta1, Nox4 mRNA is dramatically upregulated by 16.2+/-0.8-fold (n=3, P<0.005), whereas Nox5 is downregulated. Small interference RNA against Nox4 downregulates Nox4 mRNA by 80+/-5%, inhibits NADPH-driven superoxide production in response to TGF-beta1 by 65+/-7%, and reduces TGF-beta1-induced expression of SM alpha-actin by 95+/-2% (n=6, P<0.05). Because activation of small mothers against decapentaplegic (Smads) 2/3 is critical for myofibroblast conversion in response to TGF-beta1, we also determined whether Nox4 affects Smad 2/3 phosphorylation. Depletion of Nox4 but not Nox5 inhibits baseline and TGF-beta1 stimulation of Smad 2/3 phosphorylation by 75+/-5% and 68+/-3%, respectively (n=7, P<0.0001). We conclude that Nox 4 mediates TGF-beta1-induced conversion of fibroblasts to myofibroblasts by regulating Smad 2/3 activation. Thus, Nox4 may play a critical role in the pathological activation of cardiac fibroblasts in cardiac fibrosis associated with human heart failure.

• Resveratrol inhibits angiotensin II- and epidermal growth factor-mediated Akt activation: role of Gab1 and Shp2.

  Authors: Ursula G B Haider, Thomas U Roos, Maria I Kontaridis, Benjamin G Neel, Dan Sorescu, Kathy K Griendling, Angelika M Vollmar, Verena M Dirsch

  Molecular pharmacology. 08/2005; 68(1):41-8.

  trans-Resveratrol (RV), a polyphenolic stilbene derivative found in grape skin and other food products, has been proposed to exert beneficial effects in cardiovascular disease. Our group has showntrans-Resveratrol (RV), a polyphenolic stilbene derivative found in grape skin and other food products, has been proposed to exert beneficial effects in cardiovascular disease. Our group has shown previously that RV inhibits angiotensin II (Ang II)-induced Akt activation and, consequently, vascular smooth muscle cell (VSMC) hypertrophy. In this work, to identify the molecular target of RV, we investigated the impact of RV on early signaling cascades in rat aortic VSMCs triggered by Ang II and epidermal growth factor (EGF). We show that RV does not influence Ang II-mediated transactivation of EGF-receptor but potently inhibits EGF-induced phosphorylation of Akt kinase, suggesting that RV acts downstream of EGF-receptor transactivation in VSMCs. Recent evidence indicates that the adapter molecule Gab1, together with the protein tyrosine phosphatase Shp2, is critically involved in regulating the strength and duration of phosphatidylinositol-3-kinase (PI3K) and Akt activation upon EGF stimulation in fibroblasts. Our results show that stimulation of VSMCs with EGF as well as Ang II leads to a rapid tyrosine phosphorylation of Gab1 and its association with the p85 subunit of PI3K. RV attenuates these processes. Experiments performed in Shp2-deficient fibroblasts revealed that RV does not inhibit EGF-stimulated Akt activation in these cells, suggesting that Shp2 is necessary for the inhibitory effect of RV on the PI3K/Akt pathway. Furthermore, RV treatment activates Shp2. We therefore propose that RV blocks Akt activation in Ang II- and EGF-stimulated VSMCs by activating Shp2, thus preventing interaction between Gab1 and PI3K that is necessary for further signal transduction.

• Resveratrol increases serine15-phosphorylated but transcriptionally impaired p53 and induces a reversible DNA replication block in serum-activated vascular smooth muscle cells.

  Authors: Ursula G B Haider, Dan Sorescu, Kathy K Griendling, Angelika M Vollmar, Verena M Dirsch

  Molecular pharmacology. 05/2003; 63(4):925-32.

  Resveratrol (RV), a polyphenolic stilbene derivative, has been proposed to exert a plethora of beneficial cardiovascular effects. Of these, in particular, inhibition of vascular smooth muscle cellResveratrol (RV), a polyphenolic stilbene derivative, has been proposed to exert a plethora of beneficial cardiovascular effects. Of these, in particular, inhibition of vascular smooth muscle cell (VSMC) proliferation shows great promise for preventing cardiovascular disease. In the present study, we show that RV leads to a reversible arrest in early S phase of the VSMC cycle, accompanied by an accumulation of hyperphosphorylated retinoblastoma protein. In contrast to studies with other cell systems, RV decreases cellular levels of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1). This is of particular interest because phosphorylated p53 protein (serine(15)) is strongly enhanced by this substance. We further found that RV only slightly inhibits phosphorylation of Erk 1/2, protein kinase B/Akt, and p70(S6) kinase upon serum stimulation. Thus, inhibition of these kinases is not likely to contribute to the cell cycle effect of RV. Importantly, the observed S phase arrest is not linked to an increase in apoptotic cell death: there was no detectable increase in apoptotic nuclei and in levels of the proapoptotic protein Bax. This is the first study elucidating the molecular pathways mediating the antiproliferative properties of RV in VSMCs.

• Resveratrol suppresses angiotensin II-induced Akt/protein kinase B and p70 S6 kinase phosphorylation and subsequent hypertrophy in rat aortic smooth muscle cells.

  Authors: Ursula G B Haider, Dan Sorescu, Kathy K Griendling, Angelika M Vollmar, Verena M Dirsch

  Molecular pharmacology. 11/2002; 62(4):772-7.

  Resveratrol (RV), a polyphenolic substance found in grape skin, is proposed to account in part for the protective effect of red wine in the cardiovascular system. Angiotensin II (Ang II)-inducedResveratrol (RV), a polyphenolic substance found in grape skin, is proposed to account in part for the protective effect of red wine in the cardiovascular system. Angiotensin II (Ang II)-induced hypertrophy of vascular smooth muscle cells (VSMCs) is a pivotal step in the development of cardiovascular disease. The aims of this study were to test the hypothesis that RV may alter Ang II-mediated hypertrophic VSMC growth and to identify the putative underlying signaling pathways. We show that RV indeed potently inhibits Ang II-induced [(3)H]leucine incorporation in a concentration-dependent manner (50 microM RV, 71% inhibition). Western blot analysis reveals that phosphorylation of Akt/protein kinase B (PKB) and to a lesser extent the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) 1/2, both essentially involved in Ang II-mediated hypertrophy, is dose dependently reduced by RV. Consistent with these results, we show that RV attenuates phosphorylation of the p70 ribosomal protein S6 kinase (p70(S6K)), a kinase downstream of the ERK 1/2 as well as the Akt pathway, that is implicated in Ang II-induced protein synthesis. Upstream of Akt/PKB RV seems to mediate its antihypertrophic effect by inhibiting phosphorylation of the phosphatidylinositol 3-kinase (PI(3)K) rather than by activating phosphatases. In summary, we demonstrate for the first time that RV inhibits Ang II-induced VSMC hypertrophy, possibly by interfering mainly with the PI(3)K/Akt and p70(S6K) but also with the ERK 1/2 signaling pathway. Thus, this study delivers important new insight in the molecular pathways that may contribute to the proposed beneficial effects of RV in cardiovascular disease.

• Mechanism of hydrogen peroxide-induced cell cycle arrest in vascular smooth muscle.

  Authors: Nita N Deshpande, Dan Sorescu, Puvi Seshiah, Masuko Ushio-Fukai, Marjorie Akers, Qiqin Yin, Kathy K Griendling

  Antioxidants & redox signaling. 11/2002; 4(5):845-54.

  Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) can positively and negatively modulate vascular smooth muscle cell (VSMC) growth. To investigate these paradoxical effects of H(2)O(2), weReactive oxygen species such as hydrogen peroxide (H(2)O(2)) can positively and negatively modulate vascular smooth muscle cell (VSMC) growth. To investigate these paradoxical effects of H(2)O(2), we examined its effect on apoptosis, cell cycle progression, and cell cycle proteins. High concentrations of H(2)O(2) (500 microM to 1 mM) induced apoptosis, whereas moderate concentrations (100 microM) caused cell cycle arrest in G1. H(2)O(2) (100 microM) blocked serum-stimulated cyclin-dependent kinase-2 (CDK2) activity, but not CDK4 activity, suggesting that cell cycle arrest occurred in part by inhibiting CDK2 activity. The serum-induced increase in cyclin A mRNA was also completely suppressed by H(2)O(2), whereas cyclin D1 mRNA was not affected. In addition, H(2)O(2) caused a dramatic increase in expression of the cell cycle inhibitor p21 mRNA (9.67 +/- 0.94-fold at 2 h) and protein (8.75 +/- 0.08-fold at 8 h), but no change in p27 protein. Finally, H(2)O(2 )transiently increased p53 protein levels (3.16 +/- 1.2-fold at 2 h). Thus, whereas high levels of H(2)O(2) induce apoptosis, moderate concentrations of H(2)O(2) coordinate a set of molecular events leading to arrest of VSMCs at the G1/S checkpoint of the cell cycle. These results provide insight into the mechanisms underlying positive and negative regulation of VSMC growth by H(2)O(2) in vascular disease.

• Superoxide production and expression of nox family proteins in human atherosclerosis.

  Authors: Dan Sorescu, Daiana Weiss, Bernard Lassègue, Roza E Clempus, Katalin Szöcs, George P Sorescu, Liisa Valppu, Mark T Quinn, J David Lambeth, J David Vega, W Robert Taylor, Kathy K Griendling

  Circulation. 04/2002; 105(12):1429-35.

  BACKGROUND: NAD(P)H oxidases are important sources of superoxide in the vasculature, the activity of which is associated with risk factors for human atherosclerosis. This study was designed toBACKGROUND: NAD(P)H oxidases are important sources of superoxide in the vasculature, the activity of which is associated with risk factors for human atherosclerosis. This study was designed to investigate the localization of superoxide production and the expression of the Nox family of NAD(P)H oxidase proteins (gp91phox, Nox1, and Nox4) in nonatherosclerotic and atherosclerotic human coronary arteries. METHODS AND RESULTS: In coronary artery segments from explanted human hearts, we examined intracellular superoxide production with dihydroethidium. In nonatherosclerotic coronary arteries, superoxide was present homogenously throughout the intima, media, and adventitia. In atherosclerotic arteries, there was an additional intense area of superoxide in the plaque shoulder, which is rich in macrophages and alpha-actin-positive cells. p22phox colocalized with gp91phox mainly in macrophages, whereas Nox4 was found only in nonphagocytic vascular cells. Expression of gp91phox and p22phox mRNA was associated with the severity of atherosclerosis. gp91phox correlated with the plaque macrophage content, whereas Nox4 correlated with the content of alpha-actin-positive cells. Nox1 expression was low both in human coronary arteries and isolated vascular cells. CONCLUSIONS: Several Nox proteins, including gp91phox and Nox4, may contribute to increased intracellular oxidative stress in human coronary atherosclerosis in a cell-specific manner and thus may be involved in the genesis and progression of human coronary atherosclerotic disease.

• Upregulation of Nox-based NAD(P)H oxidases in restenosis after carotid injury.

  Authors: Katalin Szöcs, Bernard Lassègue, Dan Sorescu, Lula L Hilenski, Liisa Valppu, Tracey L Couse, Josiah N Wilcox, Mark T Quinn, J David Lambeth, Kathy K Griendling

  Arteriosclerosis, thrombosis, and vascular biology. 02/2002; 22(1):21-7.

  Restenosis, a frequent complication of coronary angioplasty, is associated with increased superoxide (O2*(-)) production. Although the molecular identity of the responsible oxidase is unclear, anRestenosis, a frequent complication of coronary angioplasty, is associated with increased superoxide (O2*(-)) production. Although the molecular identity of the responsible oxidase is unclear, an NAD(P)H oxidase appears to be involved. In smooth muscle, p22phox and 2 homologues of gp91phox, nox1 and nox4, are expressed, whereas fibroblasts contain gp91phox. To begin investigating the possibility that these oxidase components might contribute to the increased O2*(-) that accompanies neointimal formation, we measured their expression after balloon injury of the rat carotid artery. The increase in O2*(-) production 3 to 15 days after surgery was not due to inflammatory cell infiltration but appeared to be derived from medial and neointimal smooth muscle cells and adventitial fibroblasts. Nox1 and p22phox mRNAs were increased 2.7- and 3.6-fold, respectively, at day 3 after injury and remained elevated for 15 days. gp91Phox was increased 7 to 15 days after injury, and nox4 expression was increased 2-fold, but only at day 15 after surgery. These results confirm and extend our previous in vitro data and suggest that in the vasculature, the nox-based NAD(P)H oxidases serve different functions. This dynamic regulation of oxidase components may be critical to smooth muscle phenotypic modulation in restenosis and atherosclerosis.

• Electron spin resonance characterization of the NAD(P)H oxidase in vascular smooth muscle cells

  Authors: Dan Sorescu, Mark J Somers, Bernard Lassègue, Sharon Grant, David G Harrison, Kathy K Griendling

  Free Radical Biology and Medicine.

  Endogenously produced reactive oxygen species are important for intracellular signaling mechanisms leading to vascular smooth muscle cell (VSMC) growth. It is therefore critical to define theEndogenously produced reactive oxygen species are important for intracellular signaling mechanisms leading to vascular smooth muscle cell (VSMC) growth. It is therefore critical to define the potential enzymatic sources of ROS and their regulation by agonists in VSMCs. Previous studies have investigated O2•− production using lucigenin-enhanced chemiluminescence. However, lucigenin has been recently criticized for its ability to redox cycle and its propensity to measure cellular reductase activity independent from O2•−. To perform a definitive characterization of VSMC oxidase activity, we used electron spin resonance trapping of O2•− with DEPMPO. We confirmed that the main source of O2•− from VSMC membranes is an NAD(P)H oxidase and that the O2•− formation from mitochondria, xanthine oxidase, arachidonate-derived enzymes, and nitric oxide synthases in VSMC membranes was minor. The VSMC NAD(P)H oxidase(s) are able to produce more O2•− when NADPH is used as the substrate compared to NADH (the maximal NADPH signal is 2.4- ± 0.4-fold higher than the NADH signal). The two substrates had similar EC50’s (∼10–50 μM). Stimulation with angiotensin II and platelet-derived growth factor also predominantly increased the NADPH-driven signal (101 ± 8% and 83 ± 1% increase above control, respectively), with less of an effect on NADH-dependent O2•− (17 ± 3% and 36 ± 5% increase, respectively). Moreover, incubation of the cells with diphenylene iodonium inhibited predominantly NADPH-stimulated O2•−. In conclusion, electron spin resonance characterization of VSMC oxidase activity supports a major role for an NAD(P)H oxidase in O2•− production in VSMCs, and provides new evidence concerning the substrate dependency and agonist-stimulated activity of this key enzyme.

• NAD(P)H Oxidases and Their Relevance to Atherosclerosis

  Authors: Dan Sorescu, Katalin Szöcs, Kathy K Griendling

  Trends in Cardiovascular Medicine.

  Studies performed during the last decade have identified NAD(P)H oxidases unique to nonphagocytic vascular cells. The reactive oxygen species released from these enzymes regulate fundamental cellularStudies performed during the last decade have identified NAD(P)H oxidases unique to nonphagocytic vascular cells. The reactive oxygen species released from these enzymes regulate fundamental cellular functions such as growth (hyperplastic or hypertrophic), endothelial dysfunction, migration and inflammation, which have been demonstrated to play a role in atherogenesis. Evidence from experimental animal and human studies implicate the nonphagocytic NAD(P)H oxidases in multiple aspects of atherogenesis, suggesting that these enzymes may be important determinants of the course of vascular disease.