Oxidative Stress and Hypertension.

A link between oxidative stress and hypertension has been firmly established in multiple animal models of hypertension but remains elusive in humans. While initial studies focused on inactivation of nitric oxide by superoxide, our understanding of relevant reactive oxygen species (superoxide, hydrogen peroxide, and peroxynitrite) and how they modify complex signaling pathways to promote hypertension has expanded significantly. In this review, we summarize recent advances in delineating the primary and secondary sources of reactive oxygen species (nicotinamide adenine dinucleotide phosphate oxidases, uncoupled endothelial nitric oxide synthase, endoplasmic reticulum, and mitochondria), the posttranslational oxidative modifications they induce on protein targets important for redox signaling, their interplay with endogenous antioxidant systems, and the role of inflammasome activation and endoplasmic reticular stress in the development of hypertension. We highlight how oxidative stress in different organ systems contributes to hypertension, describe new animal models that have clarified the importance of specific proteins, and discuss clinical studies that shed light on how these processes and pathways are altered in human hypertension. Finally, we focus on the promise of redox proteomics and systems biology to help us fully understand the relationship between ROS and hypertension and their potential for designing and evaluating novel antihypertensive therapies.

NADPH Oxidase 1 Mediates Acute Blood Pressure Response to Angiotensin II by Contributing to Calcium Influx in Vascular Smooth Muscle Cells.

BACKGROUND: Reactive oxygen species (ROS) and calcium ions (Ca2+) are among the major effectors of Ang II (angiotensin II) in vascular smooth muscle cells. ROS are related to Ca2+ signaling or contraction induced by Ang II, but little is known about their detailed functions. Here, NOX (NADPH oxidase), a major ROS source responsive to Ang II, was investigated regarding its contribution to Ca2+ signaling. METHODS: Vascular smooth muscle cells were primary cultured from rat aorta. Ca2+ and ROS were monitored mainly using fura-2 and HyPer family probes' respectively. Signals activating NOX were examined with relevant pharmacological inhibitors and genetic manipulation techniques. RESULTS: Ang II-induced ROS generation was found to be biphasic: the first phase of ROS production, which was mainly mediated by NOX1, was small and transient, preceding a rise in Ca2+, and the second phase of ROS generation, mediated by NOX1 and NOX4, was slow but sizeable, continuing over tens of minutes. NOX1-derived superoxide in the first phase is required for Ca2+ influx through nonselective cation channels. AT1R (Ang II type 1 receptor)-Gßγ-PI3Kγ (phosphoinositide 3-kinase γ) signaling pathway was responsible for the rapid activation of NOX1 in the first phase, while in the second phase, NOX1 was further activated by a separate AT1R-Gαq/11-PLC (phospholipase C)-PKCß (protein kinase C ß) signaling axis. Consistent with these observations, aortas from NOX1-knockout mice exhibited reduced contractility in response to Ang II, and thus the acute pressor response to Ang II was also attenuated in NOX1-knockout mice. CONCLUSIONS: NOX1 mediates Ca2+ signal generation and thereby contributes to vascular contraction and blood pressure elevation by Ang II.

ß1- and ß2-integrins: central players in regulating vascular permeability and leukocyte recruitment during acute inflammation.

The integrin family, an indispensable part of cell-cell and cell-matrix interactions, consists of a group of heterodimeric adhesion receptors formed by α- and ß-integrin subunits. Their wide expression and unique bidirectional signaling pathways allow them to play roles in a variety of biological activities including blood clot formation, cell attachment, and migration. Evidence suggests that integrins are essential regulators of the initiation of acute inflammation, especially two key aspects of this process i.e., vascular permeability and leukocyte recruitment. This mini-review discusses the importance of integrins at the onset of the acute inflammatory response and outlines research advances regarding the function of integrins and their modulators at different stages of this process. Insights into the fine-tuning of integrin signaling during acute inflammation may inspire the design of new drugs for inflammatory diseases.

Poldip2 is an oxygen-sensitive protein that controls PDH and αKGDH lipoylation and activation to support metabolic adaptation in hypoxia and cancer.

Although the addition of the prosthetic group lipoate is essential to the activity of critical mitochondrial catabolic enzymes, its regulation is unknown. Here, we show that lipoylation of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (αKDH) complexes is a dynamically regulated process that is inhibited under hypoxia and in cancer cells to restrain mitochondrial respiration. Mechanistically, we found that the polymerase-δ interacting protein 2 (Poldip2), a nuclear-encoded mitochondrial protein of unknown function, controls the lipoylation of the pyruvate and α-KDH dihydrolipoamide acetyltransferase subunits by a mechanism that involves regulation of the caseinolytic peptidase (Clp)-protease complex and degradation of the lipoate-activating enzyme Ac-CoA synthetase medium-chain family member 1 (ACSM1). ACSM1 is required for the utilization of lipoic acid derived from a salvage pathway, an unacknowledged lipoylation mechanism. In Poldip2-deficient cells, reduced lipoylation represses mitochondrial function and induces the stabilization of hypoxia-inducible factor 1α (HIF-1α) by loss of substrate inhibition of prolyl-4-hydroxylases (PHDs). HIF-1α-mediated retrograde signaling results in a metabolic reprogramming that resembles hypoxic and cancer cell adaptation. Indeed, we observe that Poldip2 expression is down-regulated by hypoxia in a variety of cell types and basally repressed in triple-negative cancer cells, leading to inhibition of lipoylation of the pyruvate and α-KDH complexes and mitochondrial dysfunction. Increasing mitochondrial lipoylation by forced expression of Poldip2 increases respiration and reduces the growth rate of cancer cells. Our work unveils a regulatory mechanism of catabolic enzymes required for metabolic plasticity and highlights the role of Poldip2 as key during hypoxia and cancer cell metabolic adaptation.

Reactive Oxygen Species in Metabolic and Inflammatory Signaling.

Reactive oxygen species (ROS) are well known for their role in mediating both physiological and pathophysiological signal transduction. Enzymes and subcellular compartments that typically produce ROS are associated with metabolic regulation, and diseases associated with metabolic dysfunction may be influenced by changes in redox balance. In this review, we summarize the current literature surrounding ROS and their role in metabolic and inflammatory regulation, focusing on ROS signal transduction and its relationship to disease progression. In particular, we examine ROS production in compartments such as the cytoplasm, mitochondria, peroxisome, and endoplasmic reticulum and discuss how ROS influence metabolic processes such as proteasome function, autophagy, and general inflammatory signaling. We also summarize and highlight the role of ROS in the regulation metabolic/inflammatory diseases including atherosclerosis, diabetes mellitus, and stroke. In order to develop therapies that target oxidative signaling, it is vital to understand the balance ROS signaling plays in both physiology and pathophysiology, and how manipulation of this balance and the identity of the ROS may influence cellular and tissue homeostasis. An increased understanding of specific sources of ROS production and an appreciation for how ROS influence cellular metabolism may help guide us in the effort to treat cardiovascular diseases.

Polymerase-δ-interacting protein 2 activates the RhoGEF epithelial cell transforming sequence 2 in vascular smooth muscle cells.

Polymerase-δ-interacting protein 2 (Poldip2) controls a wide variety of cellular functions and vascular pathologies. To mediate these effects, Poldip2 interacts with numerous proteins and generates reactive oxygen species via the enzyme NADPH oxidase 4 (Nox4). We have previously shown that Poldip2 can activate the Rho family GTPase RhoA, another signaling node within the cell. In this study, we aimed to better understand how Poldip2 activates Rho family GTPases and the functions of the involved proteins in vascular smooth muscle cells (VSMCs). RhoA is activated by guanine nucleotide exchange factors. Using nucleotide-free RhoA (isolated from bacteria) to pulldown active RhoGEFs, we found that the RhoGEF epithelial cell transforming sequence 2 (Ect2) is activated by Poldip2. Ect2 is a critical RhoGEF for Poldip2-mediated RhoA activation, because siRNA against Ect2 prevented Poldip2-mediated RhoA activity (measured by rhotekin pulldowns). Surprisingly, we were unable to detect a direct interaction between Poldip2 and Ect2, as they did not coimmunoprecipitate. Nox4 is not required for Poldip2-driven Ect2 activation, as Poldip2 overexpression induced Ect2 activation in Nox4 knockout VSMCs similar to wild-type cells. However, antioxidant treatment blocked Poldip2-induced Ect2 activation. This indicates a novel reactive oxygen species-driven mechanism by which Poldip2 regulates Rho family GTPases. Finally, we examined the function of these proteins in VSMCs, using siRNA against Poldip2 or Ect2 and determined that Poldip2 and Ect2 are both essential for vascular smooth muscle cell cytokinesis and proliferation.

Poldip2 knockdown inhibits vascular smooth muscle proliferation and neointima formation by regulating the expression of PCNA and p21.

Polymerase delta-interacting protein 2 (Poldip2) is a multi-functional protein with numerous roles in the vasculature, including the regulation of cell apoptosis and migration, as well as extracellular matrix deposition; however, its role in VSMC proliferation and neointimal formation is unknown. In this study, we investigated the role of Poldip2 in intraluminal wire-injury induced neointima formation and proliferation of vascular smooth muscle cells in vitro and in vivo. Poldip2 expression was observed in the intima and media of human atherosclerotic arteries, where it colocalized with proliferating cell nuclear antigen (PCNA). Wire injury of femoral arteries of Poldip2+/+ mice induced robust neointimal formation after 2 weeks, which was impaired in Poldip2+/‒ mice. PCNA expression was significantly reduced and expression of the cell cycle inhibitor p21 was significantly increased in wire-injured arteries of Poldip2+/‒ animals compared to wild-type controls. No difference was observed in apoptosis. Downregulation of Poldip2 in rat aortic smooth muscle cells significantly reduced serum-induced proliferation and PCNA expression, but upregulated p21 expression. Downregulation of p21 using siRNA reversed the inhibition of proliferation induced by knockdown of Poldip2. These results indicate that Poldip2 plays a critical role in the proliferation of VSMCs.

Poldip2 mediates blood-brain barrier disruption in a model of sepsis-associated encephalopathy.

BACKGROUND: Sepsis-associated encephalopathy (SAE), a diffuse cerebral dysfunction in the absence of direct CNS infection, is associated with increased rates of mortality and morbidity in patients with sepsis. Increased cytokine production and disruption of the blood-brain barrier (BBB) are implicated in the pathogenesis of SAE. The induction of pro-inflammatory mediators is driven, in part, by activation of NF-κΒ. Lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, potently activates NF-κΒ and its downstream targets, including cyclooxygenase-2 (Cox-2). Cox-2 catalyzes prostaglandin synthesis and in the brain prostaglandin, E2 is capable of inducing endothelial permeability. Depletion of polymerase δ-interacting protein 2 (Poldip2) has previously been reported to attenuate BBB disruption, possibly via regulation of NF-κΒ, in response to ischemic stroke. Here we investigated Poldip2 as a novel regulator of NF-κΒ/cyclooxygenase-2 signaling in an LPS model of SAE. METHODS: Intraperitoneal injections of LPS (18 mg/kg) were used to induce BBB disruption in Poldip2+/+ and Poldip2+/- mice. Changes in cerebral vascular permeability and the effect of meloxicam, a selective Cox-2 inhibitor, were assessed by Evans blue dye extravasation. Cerebral cortices of Poldip2+/+ and Poldip2+/- mice were further evaluated by immunoblotting and ELISA. To investigate the role of endothelial Poldip2, immunofluorescence microscopy and immunoblotting were performed to study the effect of siPoldip2 on LPS-mediated NF-κΒ subunit p65 translocation and Cox-2 induction in rat brain microvascular endothelial cells. Finally, FITC-dextran transwell assay was used to assess the effect of siPoldip2 on LPS-induced endothelial permeability. RESULTS: Heterozygous deletion of Poldip2 conferred protection against LPS-induced BBB permeability. Alterations in Poldip2+/+ BBB integrity were preceded by induction of Poldip2, p65, and Cox-2, which was not observed in Poldip2+/- mice. Consistent with these findings, prostaglandin E2 levels were significantly elevated in Poldip2+/+ cerebral cortices compared to Poldip2+/- cortices. Treatment with meloxicam attenuated LPS-induced BBB permeability in Poldip2+/+ mice, while having no significant effect in Poldip2+/- mice. Moreover, silencing of Poldip2 in vitro blocked LPS-induced p65 nuclear translocation, Cox-2 expression, and endothelial permeability. CONCLUSIONS: These data suggest Poldip2 mediates LPS-induced BBB disruption by regulating NF-κΒ subunit p65 activation and Cox-2 and prostaglandin E2 induction. Consequently, targeted inhibition of Poldip2 may provide clinical benefit in the prevention of sepsis-induced BBB disruption.

Poldip2 deficiency protects against lung edema and vascular inflammation in a model of acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) in a deadly disease that can be brought on by endotoxins such as lipopolysaccharide (LPS). ARDS is characterized by vascular permeability, a severe inflammatory response, lung leukocyte infiltration, and resultant lung edema. Polymerase δ-interacting protein 2 (Poldip2) is a novel regulator of blood-brain barrier permeability; however, its role in regulating lung permeability and vascular inflammation is unknown. Here, the role of Poldip2 in regulating vascular permeability and inflammation in a mouse model of ARDS was assessed. Heterozygous deletion of Poldip2 was found to reduce LPS-induced mortality within 20 h, lung inflammatory signaling, and leukocyte infiltration. Moreover, reduced Poldip2-suppressed LP-induced vascular cell adhesion molecule (VCAM)-1 induction, leukocyte recruitment, and mitochondrial reactive oxygen species (ROS) production in vitro These data indicate that Poldip2 is an important regulator of the debilitating consequences of ARDS, potentially through the regulation of mitochondrial ROS-induced inflammatory signaling. Consequently, inhibition of Poldip2 may be a viable option for therapeutic discovery moving forward.

Fundamental Cardiovascular Research: Returns on Societal Investment: A Scientific Statement From the American Heart Association.

Recent decades have witnessed robust successes in conquering the acutely lethal manifestations of heart and vascular diseases. Many patients who previously would have died now survive. Lifesaving successes like these provide a tremendous and easily recognized benefit to individuals and society. Although cardiovascular mortality has declined, the devastating impact of chronic heart disease and comorbidities on quality of life and healthcare resources continues unabated. Future strides, extending those made in recent decades, will require continued research into mechanisms underlying disease prevention, pathogenesis, progression, and therapeutic intervention. However, severe financial constraints currently jeopardize these efforts. To chart a path for the future, this report analyzes the challenges and opportunities we face in continuing the battle against cardiovascular disease and highlights the return on societal investment afforded by fundamental cardiovascular research.

NOX4 (NADPH Oxidase 4) and Poldip2 (Polymerase δ-Interacting Protein 2) Induce Filamentous Actin Oxidation and Promote Its Interaction With Vinculin During Integrin-Mediated Cell Adhesion.

Objective- Actin cytoskeleton assembly and organization, as a result of focal adhesion (FA) formation during cell adhesion, are dependent on reactive oxygen species and the cellular redox environment. Poldip2 (polymerase δ-interacting protein 2), a novel regulator of NOX4 (NADPH oxidase 4), plays a significant role in reactive oxygen species production and cytoskeletal remodeling. Thus, we hypothesized that endogenous reactive oxygen species derived from Poldip2/NOX4 contribute to redox regulation of actin and cytoskeleton assembly during integrin-mediated cell adhesion. Approach and Results- Using vascular smooth muscle cells, we verified that hydrogen peroxide (H2O2) levels increase during integrin-mediated cell attachment as a result of activation of NOX4. Filamentous actin (F-actin) was oxidized by sulfenylation during cell attachment, with a peak at 3 hours (0.80±0.04 versus 0.08±0.13 arbitrary units at time zero), which was enhanced by overexpression of Poldip2. Depletion of Poldip2 or NOX4 using siRNA, or scavenging of endogenous H2O2 with catalase, inhibited F-actin oxidation by 78±26%, 99±1%, and 98±1%, respectively. To determine the consequence of F-actin oxidation, we examined the binding of F-actin to vinculin, a protein involved in FA complexes that regulates FA maturation. Vinculin binding during cell adhesion as well as migration capacity were inhibited after transfection with actin containing 2 oxidation-resistant point mutations (C272A and C374A). Silencing of Poldip2 or NOX4 also impaired actin-vinculin interaction, which disturbed maturation of FAs and inhibited cell migration. Conclusions- These results suggest that integrin engagement during cell attachment activates Poldip2/Nox4 to oxidize actin, which modulates FA assembly.

Polymerase delta-interacting protein 2 deficiency protects against blood-brain barrier permeability in the ischemic brain.

BACKGROUND: Polymerase δ-interacting protein 2 (Poldip2) is a multifunctional protein that regulates vascular extracellular matrix composition and matrix metalloproteinase (MMP) activity. The blood-brain barrier (BBB) is a dynamic system assembled by endothelial cells, basal lamina, and perivascular astrocytes, raising the possibility that Poldip2 may be involved in maintaining its structure. We investigated the role of Poldip2 in the late BBB permeability induced by cerebral ischemia. METHODS: Transient middle cerebral artery occlusion (tMCAO) was induced in Poldip2+/+ and Poldip2+/- mice. The volume of the ischemic lesion was measured in triphenyltetrazolium chloride-stained sections. BBB breakdown was evaluated by Evans blue dye extravasation. Poldip2 protein expression was evaluated by western blotting. RT-PCR, zymography, and ELISAs were used to measure mRNA levels, activity, and protein levels of cytokines and MMPs. Cultured astrocytes were transfected with Poldip2 siRNA, and mRNA levels of cytokines were evaluated as well as IκBα protein degradation. RESULTS: Cerebral ischemia induced the expression of Poldip2. Compared to Poldip2+/+ mice, Poldip2+/- animals exhibited decreased Evans blue dye extravasation and improved survival 24 h following stroke. Poldip2 expression was upregulated in astrocytes exposed to oxygen and glucose deprivation (OGD) and siRNA-mediated downregulation of Poldip2 abrogated OGD-induced IL-6 and TNF-α expression. In addition, siRNA against Poldip2 inhibited TNF-α-induced IκBα degradation. TNF-α, IL-6, MCP-1, VEGF, and MMP expression induced by cerebral ischemia was abrogated in Poldip2+/- mice. The protective effect of Poldip2 depletion on the increased permeability of the BBB was partially reversed by systemic administration of TNF-α. CONCLUSIONS: Poldip2 is upregulated following ischemic stroke and mediates the breakdown of the BBB by increasing cerebral cytokine production and MMP activation. Therefore, Poldip2 appears to be a promising novel target for the development of therapeutic strategies to prevent the development of cerebral edema in the ischemic brain.

Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From the American Heart Association.

Reactive oxygen species and reactive nitrogen species are biological molecules that play important roles in cardiovascular physiology and contribute to disease initiation, progression, and severity. Because of their ephemeral nature and rapid reactivity, these species are difficult to measure directly with high accuracy and precision. In this statement, we review current methods for measuring these species and the secondary products they generate and suggest approaches for measuring redox status, oxidative stress, and the production of individual reactive oxygen and nitrogen species. We discuss the strengths and limitations of different methods and the relative specificity and suitability of these methods for measuring the concentrations of reactive oxygen and reactive nitrogen species in cells, tissues, and biological fluids. We provide specific guidelines, through expert opinion, for choosing reliable and reproducible assays for different experimental and clinical situations. These guidelines are intended to help investigators and clinical researchers avoid experimental error and ensure high-quality measurements of these important biological species.

Design, synthesis, and biological evaluation of inhibitors of the NADPH oxidase, Nox4.

NADPH oxidases (Nox enzymes) are critical mediators of both physiologic and pathophysiologic processes. Nox enzymes catalyze NADPH-dependent generation of reactive oxygen species (ROS), including superoxide and hydrogen peroxide. Until recently, Nox4 was proposed to be involved exclusively in normal physiologic functions. Compelling evidence, however, suggests that Nox4 plays a critical role in fibrosis, as well as a host of pathologies and diseases. These considerations led to a search for novel, small molecule inhibitors of this important enzyme. Ultimately, a series of novel tertiary sulfonylureas (23-25) was designed using pharmacophore modeling, synthesized, and evaluated for inhibition of Nox4-dependent signaling.

Superoxide and hydrogen peroxide counterregulate myogenic contractions in renal afferent arterioles from a mouse model of chronic kidney disease.

Myogenic contractions protect kidneys from barotrauma but are impaired in chronic kidney disease (CKD). Since myogenic contractions are enhanced by superoxide but impaired by hydrogen peroxide, we tested the hypothesis that they are counterregulated by superoxide and H2O2 from NOX2/p47phox and/or NOX4/POLDIP2 in CKD. Myogenic contraction in isolated perfused afferent arterioles from mice with surgical 5/6 nephrectomy or sham operations fed a 6% sodium chloride diet was measured directly while superoxide and H2O2 were measured by fluorescence microscopy. Compared to sham-operated animals, an increase in perfusion pressure of arterioles from CKD mice doubled superoxide (21 versus 11%), increased H2O2 seven-fold (29 versus 4%), and reduced myogenic contractions profoundly (-1 versus -14%). Myogenic contractions were impaired further by PEG-superoxide dismutase or in arterioles from p47phox-/- (versus wild type) mice but became supra-normal by PEG-catalase or in mice with transgenic expression of catalase in vascular smooth muscle cells (-11 versus -1%). Single arterioles from mice with CKD expressed over 40% more mRNA and protein for NOX4 and POLDIP2. Myogenic responses in arterioles from POLDIP2 +/- (versus wild type) mice with CKD had over an 85% reduction in H2O2, but preserved superoxide and a normal myogenic response. Tempol administration to CKD mice for 3 months decreased afferent arteriolar superoxide and H2O2 and maintained myogenic contractions. Thus, afferent arteriolar superoxide generated by NOX2/p47phox opposes H2O2 generated by NOX4/POLDIP2 whose upregulation in afferent arterioles from mice with CKD accounts for impaired myogenic contractions.

Regulation of signal transduction by reactive oxygen species in the cardiovascular system.

Oxidative stress has long been implicated in cardiovascular disease, but more recently, the role of reactive oxygen species (ROS) in normal physiological signaling has been elucidated. Signaling pathways modulated by ROS are complex and compartmentalized, and we are only beginning to identify the molecular modifications of specific targets. Here, we review the current literature on ROS signaling in the cardiovascular system, focusing on the role of ROS in normal physiology and how dysregulation of signaling circuits contributes to cardiovascular diseases, including atherosclerosis, ischemia-reperfusion injury, cardiomyopathy, and heart failure. In particular, we consider how ROS modulate signaling pathways related to phenotypic modulation, migration and adhesion, contractility, proliferation and hypertrophy, angiogenesis, endoplasmic reticulum stress, apoptosis, and senescence. Understanding the specific targets of ROS may guide the development of the next generation of ROS-modifying therapies to reduce morbidity and mortality associated with oxidative stress.

Polymerase δ-interacting Protein 2: A Multifunctional Protein.

Polymerase δ-interacting protein 2 (Poldip2) is a multifunctional protein originally described as a binding partner of the p50 subunit of DNA polymerase δ and proliferating cell nuclear antigen. In addition to its role in DNA replication and damage repair, Poldip2 has been implicated in mitochondrial function, extracellular matrix regulation, cell cycle progression, focal adhesion turnover, and cell migration. However, Poldip2 functions are incompletely understood. In this review, we discuss recent literature on Poldip2 tissue distribution, subcellular localization, and function. We also address the putative function of Poldip2 in cardiovascular disease, neurodegenerative conditions and in renal pathophysiology.