Kathy K Griendling

Publications of Kathy K Griendling

• Biochemistry, physiology, and pathophysiology of NADPH oxidases in the cardiovascular system.

  Authors: Bernard Lassègue, Alejandra San Martín, Kathy K Griendling

  Circulation research. 05/2012; 110(10):1364-90.

  atherosclerosis, hypertension, cardiac hypertrophy and remodeling, angiogenesis and collateral formation, stroke, and heart failure. In this review, we discuss in detail the biochemistry of Noxatherosclerosis, hypertension, cardiac hypertrophy and remodeling, angiogenesis and collateral formation, stroke, and heart failure. In this review, we discuss in detail the biochemistry of Nox enzymes expressed in the cardiovascular system (Nox1, 2, 4, and 5), their roles in cardiovascular cell biology, and their contributions to disease development.

• Effects of the antioxidant drug tempol on renal oxygenation in mice with reduced renal mass.

  Authors: En Yin Lai, Zaiming Luo, Maristela L Onozato, Earl H Rudolph, Glenn Solis, Pedro A Jose, Anton Wellstein, Shakil Aslam, Mark T Quinn, Kathy K Griendling, Thu Huy Le, Ping Li, Fredrik Palm, William J Welch, Christopher S Wilcox

  American journal of physiology. Renal physiology. 04/2012;

  We tested the hypothesis that reactive oxygen species (ROS) contributed to renal hypoxia in C57BL/6 mice with 5/6 surgical reduction of renal mass (RRM). ROS can activate the mitochondrial uncouplingWe tested the hypothesis that reactive oxygen species (ROS) contributed to renal hypoxia in C57BL/6 mice with 5/6 surgical reduction of renal mass (RRM). ROS can activate the mitochondrial uncoupling protein 2 (UCP-2) and increase O(2) usage. However, UCP-2 can be inactivated by glutathionylation. Mice were fed normal (NS) or high salt (HS) diets and HS mice received the antioxidant drug tempol or vehicle for 3 months. Since salt intake did not affect the tubular Na(+) transport per O(2) consumed (T(Na)/Q(O2)), further studies were confined to HS mice. RRM mice had increased excretion of 8-isoprostane F(2α) and H(2)O(2), renal expression of UCP-2 and renal O(2) extraction, and reduced T(Na)/Q(O2) (Sham: 20±2 vs RRM: 10±1 μmol×μmol(-1); P<0.05) and cortical PO(2) (sham: 43±2, RRM: 29±2 mmHg; P<0.02). Tempol normalized all these parameters while further increasing compensatory renal growth and glomerular volume. RRM mice had preserved blood pressure, glomeruli, and patchy tubulointerstitial fibrosis. The patterns of protein expression in the renal cortex suggested that RRM kidneys had increased ROS from upregulated p22(phox), NOX-2 and -4 and that ROS-dependent increases in UCP-2 led to hypoxia that activated transforming growth factor beta whereas Nrf-2, glutathione peroxidase-1 and glutathione-s-transferase- mu-1 were upregulated independent of ROS. We conclude that RRM activated distinct processes: a ROS-dependent activation of UCP-2 leading to inefficient renal O(2) usage and cortical hypoxia that was offset by Nrf-2-dependent glutathionylation. Thus, hypoxia in RRM may be the outcome of NADPH oxidase-initiated ROS generation leading to mitochondrial uncoupling counteracted by defense pathways coordinated by Nrf-2.

• Differential roles of NADPH oxidases in vascular physiology and pathophysiology.

  Authors: Angelica M Amanso, Kathy K Griendling

  Frontiers in bioscience (Scholar edition). 01/2012; 4:1044-64.

  Reactive oxygen species (ROS) are produced by all vascular cells and regulate the major physiological functions of the vasculature. Production and removal of ROS are tightly controlled and occur inReactive oxygen species (ROS) are produced by all vascular cells and regulate the major physiological functions of the vasculature. Production and removal of ROS are tightly controlled and occur in discrete subcellular locations, allowing for specific, compartmentalized signaling. Among the many sources of ROS in the vessel wall, NADPH oxidases are implicated in physiological functions such as control of vasomotor tone, regulation of extracellular matrix and phenotypic modulation of vascular smooth muscle cells. They are involved in the response to injury, whether as an oxygen sensor during hypoxia, as a regulator of protein processing, as an angiogenic stimulus, or as a mechanism of wound healing. These enzymes have also been linked to processes leading to disease development, including migration, proliferation, hypertrophy, apoptosis and autophagy. As a result, NADPH oxidases participate in atherogenesis, systemic and pulmonary hypertension and diabetic vascular disease. The role of ROS in each of these processes and diseases is complex, and a more full understanding of the sources, targets, cell-specific responses and counterbalancing mechanisms is critical for the rational development of future therapeutics.

• Vascular smooth muscle insulin resistance, but not hypertrophic signaling, is independent of angiotensin II-induced IRS-1 phosphorylation by JNK.

  Authors: Hirofumi Hitomi, Puja K Mehta, Yoshihiro Taniyama, Bernard Lassègue, Bonnie Seidel-Rogol, Alejandra San Martin, Kathy K Griendling

  American journal of physiology. Cell physiology. 09/2011; 301(6):C1415-22.

  Angiotensin II (ANG II) has been implicated in the pathogenesis of diabetic micro- and macrovascular disease. In vascular smooth muscle cells (VSMCs), ANG II phosphorylates and degrades insulinAngiotensin II (ANG II) has been implicated in the pathogenesis of diabetic micro- and macrovascular disease. In vascular smooth muscle cells (VSMCs), ANG II phosphorylates and degrades insulin receptor substrate-1 (IRS-1). While the pathway responsible for IRS-1 degradation in this system is unknown, c-Jun NH(2)-terminal kinase (JNK) has been linked with serine phosphorylation of IRS-1 and insulin resistance. We investigated the role of JNK in ANG II-induced IRS-1 phosphorylation, degradation, Akt activation, glucose uptake, and hypertrophic signaling, focusing on three IRS-1 phosphorylation sites: Ser302, Ser307, and Ser632. Maximal IRS-1 phosphorylation on Ser632 occurred at 5 min, on Ser307 at 30 min, and on Ser302 at 60 min. The JNK inhibitor SP600125 reduced ANG II-induced IRS-1 Ser307 phosphorylation (by 80%), IRS-1 Ser302 phosphorylation (by 70%), and IRS-1 Ser632 phosphorylation (by 50%). However, JNK inhibition had no effect on ANG II-mediated IRS-1 degradation, nor did it reverse the ANG II-induced decrease in Akt phosphorylation or glucose uptake. Transfection of VSMCs with mutants S307A, S302A, or S632A of IRS-1 did not block ANG II-mediated IRS-1 degradation. In contrast, JNK inhibition attenuated insulin-induced upregulation of collagen and smooth muscle α-actin in ANG II-pretreated cells. We conclude that phosphorylation of Ser307, Ser302, and Ser632 of IRS-1 is not involved in ANG II-mediated IRS-1 degradation, and that JNK alone does not mediate ANG II-stimulated IRS-1 degradation, but rather is responsible for the hypertrophic effects of insulin on smooth muscle.

• Platelet-derived growth factor (PDGF) regulates Slingshot phosphatase activity via Nox1-dependent auto-dephosphorylation of serine 834 in vascular smooth muscle cells.

  Authors: Mithunan Maheswaranathan, Hope K A Gole, Isabel Fernandez, Bernard Lassègue, Kathy K Griendling, Alejandra San Martín

  The Journal of biological chemistry. 08/2011; 286(41):35430-7.

  Migration of vascular smooth muscle cells (VSMCs) contributes to vascular pathology. PDGF induces VSMC migration by a Nox1-based NADPH oxidase mediated mechanism. We have previously shown thatMigration of vascular smooth muscle cells (VSMCs) contributes to vascular pathology. PDGF induces VSMC migration by a Nox1-based NADPH oxidase mediated mechanism. We have previously shown that PDGF-induced migration in VSMCs requires Slingshot-1L (SSH1L) phosphatase activity. In the present work, the mechanism of SSH1L activation by PDGF is further investigated. We identified a 14-3-3 consensus binding motif encompassing Ser-834 in SSH1L that is constitutively phosphorylated. PDGF induces SSH1L auto-dephosphorylation at Ser-834 in wild type (wt), but not in Nox1(-/y) cells. A SSH1L-S834A phospho-deficient mutant has significantly lower binding capacity for 14-3-3 when compared with the phospho-mimetic SSH1L-S834D mutant, and acts as a constitutively active phosphatase, lacking of PDGF-mediated regulation. Given that Nox1 produces reactive oxygen species, we evaluated their participation in this SSH1L activation mechanism. We found that H(2)O(2) activates SSH1L and this is accompanied by SSH1L/14-3-3 complex disruption and 14-3-3 oxidation in wt, but not in Nox1(-/y) cells. Together, these data demonstrate that PDGF activates SSH1L in VSMC by a mechanism that involves Nox1-mediated oxidation of 14-3-3 and Ser-834 SSH1L auto-dephosphorylation.

• Mechanical stretch augments insulin-induced vascular smooth muscle cell proliferation by insulin-like growth factor-1 receptor.

  Authors: Gang Liu, Hirofumi Hitomi, Naohisa Hosomi, Bai Lei, Daisuke Nakano, Kazushi Deguchi, Hirohito Mori, Tsutomu Masaki, Hong Ma, Kathy K Griendling, Akira Nishiyama

  Experimental cell research. 08/2011; 317(17):2420-8.

  Insulin resistance and hypertension have been implicated in the pathogenesis of cardiovascular disease; however, little is known about the roles of insulin and mechanical force in vascular smoothInsulin resistance and hypertension have been implicated in the pathogenesis of cardiovascular disease; however, little is known about the roles of insulin and mechanical force in vascular smooth muscle cell (VSMC) remodeling. We investigated the contribution of mechanical stretch to insulin-induced VSMC proliferation. Thymidine incorporation was stimulated by insulin in stretched VSMCs, but not in un-stretched VSMCs. Insulin increased 2-deoxy-glucose incorporation in both stretched and un-stretched VSMCs. Mechanical stretch augmented insulin-induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation. Inhibitors of epidermal growth factor (EGF) receptor tyrosine kinase and Src attenuated insulin-induced ERK and Akt phosphorylation, as well as thymidine incorporation, whereas 2-deoxy-glucose incorporation was not affected by these inhibitors. Moreover, stretch augmented insulin-like growth factor (IGF)-1 receptor expression, although it did not alter the expression of insulin receptor and insulin receptor substrate-1. Insulin-induced ERK and Akt activation, and thymidine incorporation were inhibited by siRNA for the IGF-1 receptor. Mechanical stretch augments insulin-induced VSMC proliferation via upregulation of IGF-1 receptor, and downstream Src/EGF receptor-mediated ERK and Akt activation. Similar to in vitro experiment, IGF-1 receptor expression was also augmented in hypertensive rats. These results provide a basis for clarifying the molecular mechanisms of vascular remodeling in hypertensive patients with hyperinsulinemia.

• NADPH oxidase 4 mediates TGF-β-induced smooth muscle α-actin via p38MAPK and serum response factor.

  Authors: Abel Martin-Garrido, David I Brown, Alicia N Lyle, Anna Dikalova, Bonnie Seidel-Rogol, Bernard Lassègue, Alejandra San Martín, Kathy K Griendling

  Free radical biology & medicine. 11/2010; 50(2):354-62.

  In contrast to other cell types, vascular smooth muscle cells modify their phenotype in response to external signals. NADPH oxidase 4 (Nox4) is critical for maintenance of smooth muscle geneIn contrast to other cell types, vascular smooth muscle cells modify their phenotype in response to external signals. NADPH oxidase 4 (Nox4) is critical for maintenance of smooth muscle gene expression; however, the underlying mechanisms are incompletely characterized. Using smooth muscle α-actin (SMA) as a prototypical smooth muscle gene and transforming growth factor-β (TGF-β) as a differentiating agent, we examined Nox4-dependent signaling. TGF-β increases Nox4 expression and activity in human aortic smooth muscle cells (HASMC). Transfection of HASMC with siRNA against Nox4 (siNox4) abolishes TGF-β-induced SMA expression and stress fiber formation. siNox4 also significantly inhibits TGF-β-stimulated p38MAPK phosphorylation, as well as that of its substrate, mitogen-activated protein kinase-activated protein kinase-2. Moreover, the p38MAPK inhibitor SB-203580 nearly completely blocks the SMA increase induced by TGF-β. Inhibition of either p38MAPK or NADPH oxidase-derived reactive oxygen species impairs the TGF-β-induced phosphorylation of Ser103 on serum response factor (SRF) and reduces its transcriptional activity. Binding of SRF to myocardin-related transcription factor (MRTF) is also necessary, because downregulation of MRTF by siRNA abolishes TGF-β-induced SMA expression. Taken together, these data suggest that Nox4 regulates SMA expression via activation of a p38MAPK/SRF/MRTF pathway in response to TGF-β.

• Reactive oxygen species, NADPH oxidases, and hypertension.

  Authors: Srinivasa Raju Datla, Kathy K Griendling

  Hypertension. 09/2010; 56(3):325-30.

• Upregulation of Nox1 in vascular smooth muscle leads to impaired endothelium-dependent relaxation via eNOS uncoupling.

  Authors: Anna E Dikalova, María Carolina Góngora, David G Harrison, J David Lambeth, Sergey Dikalov, Kathy K Griendling

  American journal of physiology. Heart and circulatory physiology. 09/2010; 299(3):H673-9.

  Recent work has made it clear that oxidant systems interact. To investigate potential cross talk between NADPH oxidase (Nox) 1 upregulation in vascular smooth muscle and endothelial function,Recent work has made it clear that oxidant systems interact. To investigate potential cross talk between NADPH oxidase (Nox) 1 upregulation in vascular smooth muscle and endothelial function, transgenic mice overexpressing Nox1 in smooth muscle cells (Tg(SMCnox1)) were subjected to angiotensin II (ANG II)-induced hypertension. As expected, NADPH-dependent superoxide generation was increased in aortas from Nox1-overexpressing mice. Infusion of ANG II (0.7 mg x kg(-1) x day(-1)) for 2 wk potentiated NADPH-dependent superoxide generation and hydrogen peroxide production compared with similarly treated negative littermate controls. Endothelium-dependent relaxation was impaired in transgenic mice, and bioavailable nitric oxide was markedly decreased. To test the hypothesis that eNOS uncoupling might contribute to endothelial dysfunction, the diet was supplemented with tetrahydrobiopterin (BH(4)). BH(4) decreased aortic superoxide production, partially restored bioavailable nitric oxide in aortas of ANG II-treated Tg(SMCnox1) mice, and significantly improved endothelium-dependent relaxation in these mice. Western blot analysis revealed less dimeric eNOS in Tg(SMCnox1) mice compared with the wild-type mice; however, total eNOS was equivalent. Pretreatment of mouse aortas with the eNOS inhibitor N(G)-nitro-L-arginine methyl ester decreased ANG II-induced superoxide production in Tg(SMCnox1) mice compared with wild-type mice, indicating that uncoupled eNOS is also a significant source of increased superoxide in transgenic mice. Thus overexpression of Nox1 in vascular smooth muscle leading to enhanced production of reactive oxygen species in response to ANG II causes eNOS uncoupling and a decrease in nitric oxide bioavailability, resulting in impaired vasorelaxation.

• NADPH Oxidases: Functions and Pathologies in the Vasculature.

  Authors: Bernard Lassègue, Kathy K Griendling

  Arteriosclerosis, thrombosis, and vascular biology. 11/2009;

  Reactive oxygen species are ubiquitous signaling molecules in biological systems. Four members of the NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species in theReactive oxygen species are ubiquitous signaling molecules in biological systems. Four members of the NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species in the vasculature: Nox1, Nox2, Nox4, and Nox5. Signaling cascades triggered by stresses, hormones, vasoactive agents, and cytokines control the expression and activity of these enzymes and of their regulatory subunits, among which p22phox, p47phox, Noxa1, and p67phox are present in blood vessels. Vascular Nox enzymes are also regulated by Rac, ClC-3, Poldip2, and protein disulfide isomerase. Multiple Nox subtypes, simultaneously present in different subcellular compartments, produce specific amounts of superoxide, some of which is rapidly converted to hydrogen peroxide. The identity and location of these reactive oxygen species, and of the enzymes that degrade them, determine their downstream signaling pathways. Nox enzymes participate in a broad array of cellular functions, including differentiation, fibrosis, growth, proliferation, apoptosis, cytoskeletal regulation, migration, and contraction. They are involved in vascular pathologies such as hypertension, restenosis, inflammation, atherosclerosis, and diabetes. As our understanding of the regulation of these oxidases progresses, so will our ability to alter their functions and associated pathologies.

• Poldip2, a Novel Regulator of Nox4 and Cytoskeletal Integrity in Vascular Smooth Muscle Cells.

  Authors: Alicia N Lyle, Nita N Deshpande, Yoshihiro Taniyama, Bonnie Seidel-Rogol, Lily Pounkova, Pingfeng Du, Christopher Papaharalambus, Bernard Lassegue, Kathy K Griendling

  Circulation research. 08/2009;

  Rationale: NADPH oxidases (Noxs) regulate vascular physiology and contribute to the pathogenesis of vascular disease. In vascular smooth muscle cells (VSMCs), the interactions of individual NoxRationale: NADPH oxidases (Noxs) regulate vascular physiology and contribute to the pathogenesis of vascular disease. In vascular smooth muscle cells (VSMCs), the interactions of individual Nox homologs with regulatory proteins are poorly defined. Objective: The objective of this study was to identify novel NADPH oxidase regulatory proteins. Methods and Results: Using a yeast 2-hybrid screen, we identified a novel binding partner, Poldip2, and demonstrated that it associates with p22phox, NADPH oxidase (Nox)1, and Nox4 and colocalizes with p22phox at sites of Nox4 localization. Poldip2 increases Nox4 enzymatic activity by 3-fold and positively regulates basal reactive oxygen species production in VSMCs (O2(.-): 86.3+/-15.6% increase; H2O2: 40.7+/-4.5% increase). Overexpression of Poldip2 activates Rho (180.2+/-24.8% increase), strengthens focal adhesions, and increases stress fiber formation. These phenotypic changes are blocked by dominant negative Rho. In contrast, depletion of either Poldip2 or Nox4 results in a loss of these structures, which is rescued by adding back active Rho. Cell migration, which requires dynamic cytoskeletal remodeling, is impaired by either excess (70.1+/-14.7% decrease) or insufficient Poldip2 (63.5+/-5.9% decrease). Conclusions: These results suggest that Poldip2 associates with p22phox to activate Nox4, leading to regulation of focal adhesion turnover and VSMC migration, thus linking reactive oxygen species production and cytoskeletal remodeling. Poldip2 may be a novel therapeutic target for vascular pathologies with a significant VSMC migratory component, such as restenosis and atherosclerosis.

• Insulin-Like Growth Factor-1 Receptor Expression Masks the Antiinflammatory and Glucose Uptake Capacity of Insulin in Vascular Smooth Muscle Cells.

  Authors: Niels Engberding, Alejandra San Martín, Abel Martin Garrido, Mitsuhisa Koga, Lily Pounkova, Erin Lyons, Bernard Lassègue, Kathy K Griendling

  Arteriosclerosis, thrombosis, and vascular biology. 02/2009;

  OBJECTIVE: Insulin resistance of vascular smooth muscle cells (VSMCs) has been linked to accelerated atherosclerosis in diabetes; however, the effects of insulin on VSMCs remain controversial. MostOBJECTIVE: Insulin resistance of vascular smooth muscle cells (VSMCs) has been linked to accelerated atherosclerosis in diabetes; however, the effects of insulin on VSMCs remain controversial. Most VSMC insulin receptors are sequestered into insulin-insensitive hybrids with insulin-like growth factor-1 receptors (IGF1R). Thus we hypothesized that regulation of IGF1R expression may impact cellular insulin sensitivity. METHODS AND RESULTS: IGF1R expression was increased in aortas from diabetic mice. IGF1R overexpression in VSMCs impaired insulin-induced Akt phosphorylation. Conversely, IGF1R downregulation by siRNA allowed assembly of insulin holoreceptors, enhanced insulin-induced phosphorylation of its receptor, Akt, Erk1/2, and further augmented insulin-induced glucose uptake. IGF1R downregulation uncovered an insulin-induced reduction in activation of NF-kappaB and inhibition of MCP-1 upregulation in response to TNF-alpha. CONCLUSIONS: Downregulation of IGF1R increases the fraction of insulin receptors organized in holoreceptors, which leads to enhanced insulin signaling and unmasks potential antiinflammatory properties of insulin in VSMCs. Therefore, IGF1R, which is susceptible to feedback regulation by its own ligand, may represent a novel target for interventions designed to treat insulin resistance in the vasculature.

• Mechanisms of Vascular Smooth Muscle NADPH Oxidase 1 (Nox1) Contribution to Injury-Induced Neointimal Formation.

  Authors: Moo Yeol Lee, Alejandra San Martin, Puja K Mehta, Anna E Dikalova, Abel Martin Garrido, Erin Lyons, Karl-Heinz Krause, Botond Banfi, J David Lambeth, Bernard Lassègue, Kathy K Griendling

  Arteriosclerosis, thrombosis, and vascular biology. 02/2009;

  OBJECTIVE: Vascular NADPH oxidases (Noxes) have been implicated in cardiovascular diseases; however, the importance of individual Nox homologues remains unclear. Here, the role of the vascular smoothOBJECTIVE: Vascular NADPH oxidases (Noxes) have been implicated in cardiovascular diseases; however, the importance of individual Nox homologues remains unclear. Here, the role of the vascular smooth muscle cell (VSMC) Nox1 in neointima formation was studied using genetically modified animal models. METHODS AND RESULTS: Wire injury-induced neointima formation in the femoral artery, along with proliferation and apoptosis, was reduced in Nox1(y/-) mice, but there was little difference in Tg(SMCnox1) mice compared with wild-type (WT) mice. Proliferation and migration were reduced in cultured Nox1(y/-) VSMCs and increased in Tg(SMCnox1) cells. Tg(SMCnox1) cells exhibited increased fibronectin secretion, but neither collagen I production nor cell adhesion was affected by alteration of Nox1. Using antibody microarray and Western blotting analysis, increased cofilin phosphorylation and mDia1 expression and decreased PAK1 expression were detected in Nox1(y/-) cells. Overexpression of S3A, a constitutively active cofilin mutant, partially recovered reduced migration of Nox1(y/-) cells, suggesting that reduction in cofilin activity contributes to impaired migration of Nox1(y/-) VSMCs. CONCLUSIONS: These results indicate that Nox1 plays a critical role in neointima formation by mediating VSMC migration, proliferation, and extracellular matrix production, and that cofilin is a major effector of Nox1-mediated migration. Inhibition of Nox1 may be an efficient strategy to suppress neointimal formation.

• NADPH oxidases and angiotensin II receptor signaling.

  Authors: Abel Martin Garrido, Kathy K Griendling

  Molecular and cellular endocrinology. 12/2008;

  Over the last decade many studies have demonstrated the importance of reactive oxygen species (ROS) production by NADPH oxidases in angiotensin II (Ang II) signaling, as well as a role for ROS in theOver the last decade many studies have demonstrated the importance of reactive oxygen species (ROS) production by NADPH oxidases in angiotensin II (Ang II) signaling, as well as a role for ROS in the development of different diseases in which Ang II is a central component. In this review, we summarize the mechanism of activation of NADPH oxidases by Ang II and describe the molecular targets of ROS in Ang II signaling in the vasculature, kidney and brain. We also discuss the effects of genetic manipulation of NADPH oxidase function on the physiology and pathophysiology of the renin-angiotensin system.

• Distinct roles of Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production.

  Authors: Sergey I Dikalov, Anna E Dikalova, Alfiya T Bikineyeva, Harald H H W Schmidt, David G Harrison, Kathy K Griendling

  Free radical biology & medicine. 09/2008;

  NADPH oxidases are major sources of superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)) in vascular cells. Production of these reactive oxygen species (ROS) is essential for cell proliferation andNADPH oxidases are major sources of superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)) in vascular cells. Production of these reactive oxygen species (ROS) is essential for cell proliferation and differentiation, while ROS overproduction has been implicated in hypertension and atherosclerosis. It is known that the heme-containing catalytic subunits Nox1 and Nox4 are responsible for oxygen reduction in vascular smooth muscle cells from large arteries. However, the exact mechanism of ROS production by NADPH oxidases is not completely understood. We hypothesized that Nox1 and Nox4 play distinct roles in basal and angiotensin II (AngII)-stimulated production of O(2)(-) and H(2)O(2). Nox1 and Nox4 expression in rat aortic smooth muscle cells (RASMCs) was selectively reduced by treatment with siNox4 or antisense Nox1 adenovirus. Production of O(2)(-) and H(2)O(2) in intact RASMCs was analyzed by dihydroethidium and Amplex Red assay. Activity of NADPH oxidases was measured by NADPH-dependent O(2)(-) and H(2)O(2) production using electron spin resonance (ESR) and 1-hydroxy-3-carboxypyrrolidine (CPH) in the membrane fraction in the absence of cytosolic superoxide dismutase. It was found that production of O(2)(-) by quiescent RASMC NADPH oxidases was five times less than H(2)O(2) production. Stimulation of cells with AngII led to a 2-fold increase of O(2)(-) production by NADPH oxidases, with a small 15 to 30% increase in H(2)O(2) formation. Depletion of Nox4 in RASMCs led to diminished basal H(2)O(2) production, but did not affect O(2)(-) or H(2)O(2) production stimulated by AngII. In contrast, depletion of Nox1 in RASMCs inhibited production of O(2)(-) and AngII-stimulated H(2)O(2) in the membrane fraction and intact cells. Our data suggest that Nox4 produces mainly H(2)O(2), while Nox1 generates mostly O(2)(-) that is later converted to H(2)O(2.) Therefore, Nox4 is responsible for basal H(2)O(2) production, while O(2)(-) production in nonstimulated and AngII-stimulated cells depends on Nox1. The difference in the products generated by Nox1 and Nox4 may help to explain the distinct roles of these NADPH oxidases in cell signaling. These findings also provide important insight into the origin of H(2)O(2) in vascular cells, and may partially account for the limited pharmacological effect of antioxidant treatments with O(2)(-) scavengers that do not affect H(2)O(2).

• Nox5 mediates PDGF-induced proliferation in human aortic smooth muscle cells.

  Authors: Desmond B Jay, Christopher A Papaharalambus, Bonnie Seidel-Rogol, Anna E Dikalova, Bernard Lassègue, Kathy K Griendling

  Free radical biology & medicine. 09/2008; 45(3):329-35.

  The proliferation of vascular smooth muscle cells is important in the pathogenesis of many vascular diseases. Reactive oxygen species (ROS) produced by NADPH oxidases in smooth muscle cells have beenThe proliferation of vascular smooth muscle cells is important in the pathogenesis of many vascular diseases. Reactive oxygen species (ROS) produced by NADPH oxidases in smooth muscle cells have been shown to participate in signaling cascades regulating proliferation induced by platelet-derived growth factor (PDGF), a powerful smooth muscle mitogen. We sought to determine the role of Nox5 in the regulation of PDGF-stimulated human aortic smooth muscle cell (HASMC) proliferation. Cultured HASMC were found to express four isoforms of Nox5. When HASMC stimulated with PDGF were pretreated with N-acetyl cysteine (NAC), proliferation was significantly reduced. Proliferation induced by PDGF was also heavily dependent on JAK/STAT activation, as the JAK inhibitor, AG490, was able to completely abolish PDGF-stimulated HASMC growth. Specific knockdown of Nox5 with a siRNA strategy reduced PDGF-induced HASMC ROS production and proliferation. Additionally, siRNA to Nox5 inhibited PDGF-stimulated JAK2 and STAT3 phosphorylation. ROS produced by Nox5 play an important role in PDGF-induced JAK/STAT activation and HASMC proliferation.

• Redox signaling, vascular function, and hypertension.

  Authors: Moo Yeol Lee, Kathy K Griendling

  Antioxidants & redox signaling. 07/2008; 10(6):1045-59.

  Accumulating evidence supports the importance of redox signaling in the pathogenesis and progression of hypertension. Redox signaling is implicated in many different physiological and pathologicalAccumulating evidence supports the importance of redox signaling in the pathogenesis and progression of hypertension. Redox signaling is implicated in many different physiological and pathological processes in the vasculature. High blood pressure is in part determined by elevated total peripheral vascular resistance, which is ascribed to dysregulation of vasomotor function and structural remodeling of blood vessels. Aberrant redox signaling, usually induced by excessive production of reactive oxygen species (ROS) and/or by decreases in antioxidant activity, can induce alteration of vascular function. ROS increase vascular tone by influencing the regulatory role of endothelium and by direct effects on the contractility of vascular smooth muscle. ROS contribute to vascular remodeling by influencing phenotype modulation of vascular smooth muscle cells, aberrant growth and death of vascular cells, cell migration, and extracellular matrix (ECM) reorganization. Thus, there are diverse roles of the vascular redox system in hypertension, suggesting that the complexity of redox signaling in distinct spatial spectrums should be considered for a better understanding of hypertension.

• Dual regulation of cofilin activity by LIM kinase and Slingshot-1L phosphatase controls platelet-derived growth factor-induced migration of human aortic smooth muscle cells.

  Authors: Alejandra San Martín, Moo Yeol Lee, Holly C Williams, Kensaku Mizuno, Bernard Lassègue, Kathy K Griendling

  Circulation research. 02/2008; 102(4):432-8.

  Platelet-derived growth factor (PDGF) plays a central role in vascular healing, atherosclerosis, and restenosis, partly by stimulating vascular smooth muscle cell (VSMC) migration. Migration requiresPlatelet-derived growth factor (PDGF) plays a central role in vascular healing, atherosclerosis, and restenosis, partly by stimulating vascular smooth muscle cell (VSMC) migration. Migration requires rapid turnover of actin filaments, which is partially controlled by cofilin. Although cofilin is negatively regulated by Ser3 phosphorylation, the upstream signaling pathways have not been defined, nor has its role in VSMC migration been studied. We hypothesized that PDGF-induced migration of VSMCs involves cofilin activation and that this is regulated by the serine kinase LIM kinase (LIMK) and the novel phosphatase Slingshot (SSH)1L. In human VSMCs, stimulation with PDGF increased G-actin incorporation into the actin cytoskeleton. PDGF transiently activated the cofilin kinase, LIMK, with a peak at 5 minutes. However, cofilin was dephosphorylated between 5 and 45 minutes, with a maximum of 43+/-5% dephosphorylation at 30 minutes, suggesting that PDGF also activates a cofilin phosphatase. We found that VSMCs express SSH1L, which is induced and activated (564+/-73 versus 1021+/-141 picomoles of PO(4); P=0.015) by PDGF. Of importance, small interfering RNA directed against SSH1L blocked cofilin dephosphorylation and decreased migration (528+/-33 versus 318+/-25 cells/field; P<0.01). Taken together, our results suggest that PDGF participates in actin dynamics by dual regulation of cofilin activity via LIMK and SSH1L.

• Differential effects of AT1 receptor and Ca2+ channel blockade on atherosclerosis, inflammatory gene expression, and production of reactive oxygen species.

  Authors: Derek E Doran, Daiana Weiss, Yong Zhang, Kathy K Griendling, W Robert Taylor

  Atherosclerosis. 12/2007; 195(1):39-47.

  Angiotensin II receptor blockade has been shown to inhibit atherosclerosis in several different animal models. We sought to determine if this effect was the result of blood pressure reduction per seAngiotensin II receptor blockade has been shown to inhibit atherosclerosis in several different animal models. We sought to determine if this effect was the result of blood pressure reduction per se or a result of the anti-inflammatory effects of receptor blockade. ApoE-deficient mice were fed a high fat diet and treated with either an angiotensin II receptor antagonist, candesartan (0.5 mg/kg/day, s.c.) or a calcium channel blocker, amlodipine (7.5 mg/kg/day, mixed with food). Atherosclerotic lesion area, aortic inflammatory gene expression as well as aortic H2O2 and superoxide production were assayed. We found that candesartan but not amlodipine treatment dramatically attenuated the development of atherosclerosis despite a similar reduction in blood pressure. Similarly, candesartan treatment inhibited aortic expression of inflammatory genes and production of reactive oxygen species, effects not seen with amlodipine. These data demonstrate that angiotensin II receptor blockade inhibits atherosclerosis by reducing vascular oxidative stress and inflammatory gene production independent of blood pressure reduction.

• NADPH oxidase inhibitors: new antihypertensive agents?

  Authors: Holly C Williams, Kathy K Griendling

  Journal of cardiovascular pharmacology. 08/2007; 50(1):9-16.

  NADPH oxidases have recently been shown to contribute to the pathogenesis of hypertension. The development of specific inhibitors of these enzymes has focused attention on their potential therapeuticNADPH oxidases have recently been shown to contribute to the pathogenesis of hypertension. The development of specific inhibitors of these enzymes has focused attention on their potential therapeutic use in hypertensive disease. Two of the most specific inhibitors, gp91ds-tat and apocynin, have been shown to decrease blood pressure in animal models of hypertension. Other inhibitors, including diphenylene iodonium, aminoethyl benzenesulfono fluoride, S17834, PR39, protein kinase C inhibitors, and VAS2870, have shown promise in vitro, but their in vivo specificity, pharmacokinetics, and effectiveness in hypertension remains to be determined. Of importance, the currently available antihypertensive agents angiotensin-converting enzyme inhibitors and angiotensin receptor blockers also effectively inhibit NADPH oxidase activation. Similarly, the cholesterol-lowering agents, statins, have been shown to attenuate NADPH oxidase activation. Although, antioxidants act to scavenge the reactive oxygen species produced by these enzymes, their effectiveness is limited. Targeting NADPH homologues may have a distinct advantage over current therapies because it would specifically prevent the pathophysiological formation of reactive oxygen species that contributes to hypertension.