Alejandra San Martin

Publications of Alejandra San Martin

• 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.

• Early endosomal antigen 1 (EEA1) is an obligate scaffold for angiotensin II-induced, PKC-alpha-dependent Akt activation in endosomes.

  Authors: Rafal Robert Nazarewicz, Gloria Salazar, Nikolay Patrushev, Alejandra San Martin, Lula Hilenski, Shiqin Xiong, R Wayne Alexander

  The Journal of biological chemistry. 01/2011; 286(4):2886-95.

  Akt/protein kinase B (PKB) activation/phosphorylation by angiotensin II (Ang II) is a critical signaling event in hypertrophy of vascular smooth muscle cells (VSMCs). Conventional wisdom asserts thatAkt/protein kinase B (PKB) activation/phosphorylation by angiotensin II (Ang II) is a critical signaling event in hypertrophy of vascular smooth muscle cells (VSMCs). Conventional wisdom asserts that Akt activation occurs mainly in plasma membrane domains. Recent evidence that Akt activation may take place within intracellular compartments challenges this dogma. The spatial identity and mechanistic features of these putative signaling domains have not been defined. Using cell fractionation and fluorescence methods, we demonstrate that the early endosomal antigen-1 (EEA1)-positive endosomes are a major site of Ang II-induced Akt activation. Akt moves to and is activated in EEA1 endosomes. The expression of EEA1 is required for phosphorylation of Akt at both Thr-308 and Ser-473 as well as for phosphorylation of its downstream targets mTOR and S6 kinase, but not for Erk1/2 activation. Both Akt and phosphorylated Akt (p-Akt) interact with EEA1. We also found that PKC-α is required for organizing Ang II-induced, EEA1-dependent Akt phosphorylation in VSMC early endosomes. EEA1 expression enables PKC-α phosphorylation, which in turn regulates Akt upstream signaling kinases, PDK1 and p38 MAPK. Our results indicate that PKC-α is a necessary regulator of EEA1-dependent Akt signaling in early endosomes. Finally, EEA1 down-regulation or expression of a dominant negative mutant of PKC-α blunts Ang II-induced leucine incorporation in VSMCs. Thus, EEA1 serves a novel function as an obligate scaffold for Ang II-induced Akt activation in early endosomes.

• PGC-1{alpha} serine570 phosphorylation and GCN5-mediated acetylation by Angiotensin II drive catalase downregulation and vascular hypertrophy.

  Authors: Shiqin Xiong, Gloria Salazar, Alejandra San Martin, Mushtaq Ahmad, Nikolay Patrushev, Lula Hilenski, Rafal Robert Nazarewicz, Minhui Ma, Masuko Ushio-Fukai, R Wayne Alexander

  The Journal of biological chemistry. 11/2009;

  Angiotensin II (Ang II) is a pleuripotential hormone that is important in the pathophysiology of multiple conditions including ageing, cardiovascular and renal diseases and insulin resistance.Angiotensin II (Ang II) is a pleuripotential hormone that is important in the pathophysiology of multiple conditions including ageing, cardiovascular and renal diseases and insulin resistance. Reactive oxygen species (ROS) are important mediators of Ang II-induced signaling generally and have a well defined role in vascular hypertrophy, which is inhibited by overexpression of catalase, inferring a specific role of H(2)O(2). Molecular mechanisms are understood incompletely. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) is a key regulator of energy metabolism and ROS-scavenging enzymes including catalase. We show that Ang II stimulates Akt-dependent PGC-1alpha serine570 phosphorylation, which is required for the binding of the histone acetyltransferase general control nonderepressible 5 (GCN5) to PGC-1alpha and for its lysine acetylation. These sequential posttranslational modifications suppress PGC-1alpha activity and prevent its binding to the catalase promoter, thus decreasing catalase expression. We demonstrate that overexpression of the phosphorylation defective mutant PGC-1alpha (S570A) prevents Ang II-induced increases in H(2)O(2) levels and hypertrophy ([(3)H] leucine incorporation). Knock-down of PGC-1alpha by siRNA promotes basal and Ang II-stimulated ROS and hypertrophy, which is reversed by PEG-catalase. Thus, endogenous PGC-1alpha is a negative regulator of vascular hypertrophy by upregulating catalase expression and thus reducing ROS levels. We provide novel mechanistic insights by which Ang II may mediate its ROS-dependent pathophysiologic effects on multiple cardiometabolic diseases.

• Redox control of vascular smooth muscle migration.

  Authors: Alejandra San Martin, Kathy Griendling

  Antioxidants & redox signaling. 10/2009;

  Vascular smooth muscle cell migration is important during vascular development and contributes to lesion formation in the adult vasculature. The mechanisms regulating migration of this cell type areVascular smooth muscle cell migration is important during vascular development and contributes to lesion formation in the adult vasculature. The mechanisms regulating migration of this cell type are therefore of great interest. Recent work has shown that reactive oxygen species (ROS) derived from NADPH oxidases are important mediators of pro-migratory signaling pathways. ROS regulate the intracellular signals responsible for lamellipodia formation, actin cytoskeleton remodeling, focal adhesion turnover and contraction of the cell body. In addition, they contribute to matrix remodeling, a critical step to initiate and support vascular smooth muscle cell motility. Despite these recent advances in our understanding of the redox mechanisms that contribute to migration, additional work is needed to fully evaluate the potential of ROS-sensitive molecular signals as therapeutic targets to prevent inappropriate smooth muscle cell migration.

• 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.

• Nox1-based NADPH oxidase-derived superoxide is required for VSMC activation by advanced glycation end-products.

  Authors: Alejandra San Martin, Rocio Foncea, Francisco R Laurindo, Roberto Ebensperger, Kathy K Griendling, Federico Leighton

  Free radical biology & medicine. 06/2007; 42(11):1671-9.

  Vascular diseases are important clinical complications of diabetes. Advanced glycation end-products (AGE) are mediators of vascular dysfunction, but their effects on vascular smooth muscle cellVascular diseases are important clinical complications of diabetes. Advanced glycation end-products (AGE) are mediators of vascular dysfunction, but their effects on vascular smooth muscle cell (VSMC) ROS production are unclear. We studied the source and downstream targets of AGE-mediated ROS and reactive nitrogen species production in these cells. Significant increases in superoxide production in AGE-treated VSMC were measured using lucigenin (7650+/-433 vs 4485+/-424 LU/10(6) cells, p<0.001) or coelenterazine (277,907+/-71,295 vs 120,456+/-4140 LU/10(6) cells, p<0.05) and confirmed by ESR spectroscopy. These signals were blocked by the flavin-containing oxidase inhibitor diphenylene iodonium (DPI). AGE-stimulated NF-kappaB activity was abolished by DPI and the superoxide scavenger MnTBAP. AGE differentially regulated VSMC NADPH oxidase catalytic subunits, stimulating the transcription of Nox1 (201+/-12.7%, p<0.0001), while having no effect on Nox4. AGE also increased 3-nitrotyrosine formation, which was inhibited by MnTBAP, DPI, or the NOS inhibitor L-NAME. Regarding the source of NO, AGE stimulated inducible nitric oxide synthase mRNA (1 vs 9.7+/-3.0, p=0.046), which was abolished by a NF-kappaB inhibitor, SOD, catalase, or siRNA against Nox1. This study establishes that AGE activate iNOS in VSMC through a ROS-sensitive, NF-kappaB-dependent mechanism involving ROS generation by a Nox1-based oxidase.

• Nox1 overexpression potentiates angiotensin II-induced hypertension and vascular smooth muscle hypertrophy in transgenic mice.

  Authors: Anna Dikalova, Roza Clempus, Bernard Lassègue, Guangjie Cheng, James McCoy, Sergey Dikalov, Alejandra San Martin, Alicia Lyle, David S Weber, Daiana Weiss, W Robert Taylor, Harald H H W Schmidt, Gary K Owens, J David Lambeth, Kathy K Griendling

  Circulation. 11/2005; 112(17):2668-76.

  BACKGROUND: Reactive oxygen species (ROS) have been implicated in the development of cardiovascular pathologies. NAD(P)H oxidases (Noxes) are major sources of reactive oxygen species in the vesselBACKGROUND: Reactive oxygen species (ROS) have been implicated in the development of cardiovascular pathologies. NAD(P)H oxidases (Noxes) are major sources of reactive oxygen species in the vessel wall, but the importance of individual Nox homologues in specific layers of the vascular wall is unclear. Nox1 upregulation has been implicated in cardiovascular pathologies such as hypertension and restenosis. METHODS AND RESULTS: To investigate the pathological role of Nox1 upregulation in vascular smooth muscle, transgenic mice overexpressing Nox1 in smooth muscle cells (TgSMCnox1) were created, and the impact of Nox1 upregulation on the medial hypertrophic response during angiotensin II (Ang II)-induced hypertension was studied. These mice have increased expression of Nox1 protein in the vasculature, which is accompanied by increased superoxide production. Infusion of Ang II (0.7 mg/kg per day) into these mice for 2 weeks led to a potentiation of superoxide production compared with similarly treated negative littermate controls. Systolic blood pressure and aortic hypertrophy were also markedly greater in TgSMCnox1 mice than in their littermate controls. To confirm that this potentiation of vascular hypertrophy and hypertension was due to increased ROS formation, additional groups of mice were coinfused with the antioxidant Tempol. Tempol decreased the level of Ang II-induced aortic superoxide production and partially reversed the hypertrophic and hypertensive responses in these animals. CONCLUSIONS: These data indicate that smooth muscle-specific Nox1 overexpression augments the oxidative, pressor, and hypertrophic responses to Ang II, supporting the concept that medial Nox1 participates in the development of cardiovascular pathologies.