Search

Renal reactivity to angiotensin II (ANG II) is mediated by superoxide signaling produced by NADPH oxidase (NOX)A1/NOX1. Acute vasoconstriction of renal arteries by ANG was blunted in Noxa1−/− compared with wild-type mice. NOXA1/NOX1/O2•− signaling was also observed in ANG II stimulation of vascular smooth muscle cells and isolated mesenteric resistance arteries, indicating that it contributes to ANG II-induced hypertension. A NOXA1/NOX1 assembly inhibitor (C25) has been characterized that inhibits superoxide production and ameliorates the effects of ANG II.

Rapid over-activation of β-adrenergic receptors (β-AR) following acute stress initiates cardiac inflammation and injury by activating interleukin-18 (IL-18), however, the process of inflammation cascades has not been fully illustrated. The present study aimed to determine the mechanisms of cardiac inflammatory amplification following acute sympathetic activation. With bioinformatics analysis, galectin-3 was identified as a potential key downstream effector of β-AR and IL-18 activation. The serum level of galectin-3 was positively correlated with norepinephrine or IL-18 in patients with chest pain. In the heart of mice treated with β-AR agonist isoproterenol (ISO, 5 mg kg

Activity of the epithelial Na+ channel (ENaC) in the distal nephron fine-tunes renal Na+ excretion. Angiotensin II (ANG II) has been reported to enhance ENaC activity. Emerging evidence suggests that NADPH oxidase (NOX) signaling plays an important role in the stimulation of ENaC by ANG II in principal cells. The present findings indicate that NOX activator 1/NOX1 signaling mediates ANG II stimulation of ENaC in renal principal cells.

Introduction: Low aerobic exercise capacity is an independent risk factor for cardiovascular disease (CVD) and a predictor of premature death. In combination with aging, low aerobic capacity lowers the threshold for CVD. Aim: Since low aerobic capacity and aging have been linked to mitochondrial oxidative stress and dysfunction, we investigated whether aged Low-Capacity Runner (LCR) rats (27 months) had vascular dysfunction compared to High-Capacity Runner (HCR) rats. Methods and Results: A significant decrease in aortic eNOS levels and vasodilation as well as an increase in aortic collagen and stiffness were observed in aged LCR rats compared to age and sex-matched HCR rats. There was a correlation between age-related vascular dysfunction and increased levels of ROS and DNA damage in aortas of LCR rats. Moreover, mitochondrial oxygen consumption, membrane potential, ATP levels, and mitophagy were lower in VSMCs of aged LCR rats. VSMCs from older LCR rats showed AIM2 inflammasome activation. VSMCs of young (4 months old) LCR rats treated with purified mitochondrial damage-associated molecular patterns (DAMP) recapitulated an inflammasome activation phenotype similar to that seen in aged rat VSMCs. Rapamycin, a potent immunosuppressant, induced mitophagy, stimulated electron transport chain activity, reduced inflammasome activity, mitochondrial ROS and DAMP levels in VSMCs from aged LCR rats. MitoTEMPO, a mitochondrial ROS scavenger, was similarly effective on VSMCs from aged rats. Conclusion: The findings suggest that impaired mitophagy and inflammasome activation in the vasculature under conditions of low aerobic exercise capacity during aging results in arterial dysfunction and aortic stiffness. In older adults with reduced aerobic capacity, mitochondrial antioxidants, mitophagy induction, and inflammasome inhibition may be effective therapeutic strategies for enhancing vascular health.

AIMS: NADPH oxidase (NOX)-derived reactive oxygen species (ROS) are implicated in the pathophysiology of hypertension in chronic kidney disease patients. Genetic deletion of NOX activator 1 (Noxa1) subunit of NOX1 decreases ROS under pathophysiological conditions. Here, we investigated the role of NOXA1-dependent NOX1 activity in the pathogenesis of angiotensin II (Ang II)-induced hypertension (AIH) and possible involvement of abnormal renal function. RESULTS: NOXA1 is present in epithelial cells of Henle's thick ascending limb and distal nephron. Telemetry showed lower basal systolic blood pressure (BP) in Noxa1(−/−) versus wild-type mice. Ang II infusion for 1 and 14 days increased NOXA1/NOX1 expression and ROS in kidney of male but not female wild-type mice. Mean BP increased 30 mmHg in wild-type males, with smaller increases in Noxa1-deficient males and wild-type or Noxa1(−/−) females. In response to an acute salt load, Na(+) excretion was similar in wild-type and Noxa1(−/−) mice before and 14 days after Ang II infusion. However, Na(+) excretion was delayed after 1–2 days of Ang II in male wild-type versus Noxa1(−/−) mice. Ang II increased epithelial Na(+) channel (ENaC) levels and activation in the collecting duct principal epithelial cells of wild-type but not Noxa1(−/−) mice. Aldosterone induced ROS levels and Noxa1 and Scnn1a expression and ENaC activity in a mouse renal epithelial cell line, responses abolished by Noxa1 small-interfering RNA. INNOVATION AND CONCLUSION: Ang II activation of renal NOXA1/NOX1-dependent ROS enhances tubular ENaC expression and Na(+) reabsorption, leading to increased BP. Attenuation of AIH in females is attributed to weaker NOXA1/NOX1-dependent ROS signaling and efficient natriuresis. Antioxid. Redox Signal. 36, 550–566.

Loss-of-function (LOF) variants in SCN1B, encoding the voltage-gated sodium channel β1/β1B subunits, are linked to neurological and cardiovascular diseases. Scn1b-null mice have spontaneous seizures and ventricular arrhythmias and die by approximately 21 days after birth. β1/β1B Subunits play critical roles in regulating the excitability of ventricular cardiomyocytes and maintaining ventricular rhythmicity. However, whether they also regulate atrial excitability is unknown. We used neonatal Scn1b-null mice to model the effects of SCN1B LOF on atrial physiology in pediatric patients. Scn1b deletion resulted in altered expression of genes associated with atrial dysfunction. Scn1b-null hearts had a significant accumulation of atrial collagen, increased susceptibility to pacing induced atrial fibrillation (AF), sinoatrial node (SAN) dysfunction, and increased numbers of cholinergic neurons in ganglia that innervate the SAN. Atropine reduced the incidence of AF in null animals. Action potential duration was prolonged in null atrial myocytes, with increased late sodium current density and reduced L-type calcium current density. Scn1b LOF results in altered atrial structure and AF, demonstrating the critical role played by Scn1b in atrial physiology during early postnatal mouse development. Our results suggest that SCN1B LOF variants may significantly impact the developing pediatric heart.

Reactive oxygen species (ROS) and telomere dysfunction are both associated with aging and the development of age-related diseases. Although there is evidence for a direct relationship between ROS and telomere dysfunction as well as an independent association of oxidative stress and telomere attrition with age-related disorders, there has not been sufficient exploration of how the interaction between oxidative stress and telomere function may contribute to the pathophysiology of cardiovascular diseases (CVD). To better understand the complex relationships between oxidative stress, telomerase biology and pathophysiology, we examined the telomere biology of aortic smooth muscle cells (ASMCs) isolated from mutant mouse models of oxidative stress. We discovered that telomere lengths were significantly shorter in ASMCs isolated from superoxide dismutase 2 heterozygous (Sod2

Acute sympathetic stress in adults results in catecholamine overload and often leads to development of stress cardiomyopathy complicated by long-lasting diastolic dysfunction. We previously reported that activation of β-adrenergic signaling in the heart increases reactive oxygen species-mediated inflammasome activation and IL-18 release, inducing myocardial inflammation, fibrosis, and dysfunction. Here we tested the hypothesis that NOX4 NADPH oxidase is a primary mediator of myocardial inflammation and diastolic dysfunction in the setting of acute stress. Acute stress in wild-type and mice with cardiomyocyte (CM)-specific Nox4 deletion ( Nox4 CM-/- ) was induced with a single dose of β-adrenergic receptor agonist isoproterenol (ISO; 5 mg/kg) injection. After 3 days, ISO-treated wild-type mice had higher myocardial H 2 O 2 , inflammasome activation and IL-1β, IL-18, IL-6, CCL2, and TNF-α levels and enhanced myocardial interstitial macrophage infiltration compared with vehicle-treated or Nox4 CM-/- mice (p+ ) and myofibroblasts (α-actin-2 + ) and significantly higher interstitial collagen deposition compared with Nox4 CM-/- mice 7 days post-ISO. Assessment of left ventricular function showed significantly increased mitral valve E, decreased deceleration time, decreased tissue Doppler E’ and increased E/E’ without changes in ejection fraction and end systolic or diastolic volumes, suggesting restrictive pattern of diastolic dysfunction with increased filling pressure in wild-type compared with control or Nox4 CM-/- mice. ISO treatment of ventricular CMs isolated from wild-type mice significantly increased levels of H 2 O 2 and expression of NLRP3 and cleaved caspase-1 and IL-18 as compared with Nox4 -deficient or wild-type CMs pretreated with NOX4/NOX1 inhibitor GKT137831. These data suggest that NOX4 NADPH oxidase is a critical mediator of CM sympathetic overstimulation inducing inflammasome activation and cytokine release, myocardial inflammation, and fibrosis, resulting in impaired diastolic function. Inhibiting myocardial NOX4 during acute sympathetic stress may be beneficial to preserve cardiac function.

We recently reported that increased NADPH oxidase 4 (NOX4) expression and activity during aging results in enhanced cellular and mitochondrial oxidative stress, vascular inflammation, dysfunction, and atherosclerosis. The goal of the present study was to elucidate the molecular mechanism(s) for these effects and determine the importance of NOX4 modulation of proinflammatory gene expression in mouse vascular smooth muscle cells (VSMCs). A novel peptide-mediated siRNA transfection approach was used to inhibit Nox4 expression with minimal cellular toxicity. Using melittin-derived peptide p5RHH, we achieved significantly higher transfection efficiency (92% vs. 85% with Lipofectamine) and decreased toxicity (p < 0.001 vs. Lipofectamine in MTT and p < 0.0001 vs. Lipofectamine in LDH assays) in VSMCs. TGFβ1 significantly upregulates Nox4 mRNA (p < 0.01) and protein (p < 0.01) expression in VSMCs. p5RHH-mediated Nox4 siRNA transfection greatly attenuated TGFβ1-induced upregulation of Nox4 mRNA (p < 0.01) and protein (p < 0.0001) levels and decreased hydrogen peroxide production (p < 0.0001). Expression of pro-inflammatory genes Ccl2, Ccl5, Il6, and Vcam1 was significantly upregulated in VSMCs in several settings cells isolated from aged vs. young wild-type mice, in atherosclerotic arteries of Apoe−/− mice, and atherosclerotic human carotid arteries and correlated with NOX4 expression. p5RHH-mediated Nox4 siRNA transfection significantly attenuated the expression of these pro-inflammatory genes in TGFβ1-treated mouse VSMCs, with the highest degree of inhibition in the expression of Il6. p5RHH peptide-mediated knockdown of TGFβ-activated kinase 1 (TAK1, also known as Map3k7), Jun, and Rela, but not Nfkb2, downregulated TGFβ1-induced Nox4 expression in VSMCs. Together, these data demonstrate that increased expression and activation of NOX4, which might result from increased TGFβ1 levels seen during aging, induces a proinflammatory phenotype in VSMCs, enhancing atherosclerosis.

Aging is characterized by increased aortic stiffness, an early, independent predictor and cause of cardiovascular disease. Oxidative stress from excess reactive oxygen species (ROS) production increases with age. Mitochondria and NADPH oxidases (NOXs) are two major sources of ROS in cardiovascular system. We showed previously that increased mitochondrial ROS levels over a lifetime induce aortic stiffening in a mouse oxidative stress model. Also, NADPH oxidase 4 (NOX4) expression and ROS levels increase with age in aortas, aortic vascular smooth muscle cells (VSMCs) and mitochondria, and are correlated with age-associated aortic stiffness in hypercholesterolemic mice. The present study investigated whether young mice (4 months-old) with increased mitochondrial NOX4 levels recapitulate vascular aging and age-associated aortic stiffness. We generated transgenic mice with low (Nox4TG605; 2.1-fold higher) and high (Nox4TG618; 4.9-fold higher) mitochondrial NOX4 expression. Young Nox4TG618 mice showed significant increase in aortic stiffness and decrease in phenylephrine-induced aortic contraction, but not Nox4TG605 mice. Increased mitochondrial oxidative stress increased intrinsic VSMC stiffness, induced aortic extracellular matrix remodeling and fibrosis, a leftward shift in stress-strain curves, decreased volume compliance and focal adhesion turnover in Nox4TG618 mice. Nox4TG618 VSMCs phenocopied other features of vascular aging such as increased DNA damage, increased premature and replicative senescence and apoptosis, increased proinflammatory protein expression and decreased respiration. Aortic stiffening in young Nox4TG618 mice was significantly blunted with mitochondrial-targeted catalase overexpression. This demonstration of the role of mitochondrial oxidative stress in aortic stiffness will galvanize search for new mitochondrial-targeted therapeutics for treatment of age-associated vascular dysfunction., Graphical abstract Image 1, Highlights • Aortic stiffness in aging is associated with increased mitochondrial NOX4 levels. • Young mitochondrial Nox4 overexpressing transgenic mice phenocopy aortic stiffness. • Nox4 transgenic mice show increased VSMC stiffness, aortic remodeling and fibrosis. • Nox4 transgenic mouse VSMC show DNA damage, senescence, apoptosis and inflammation. • High mitochondrial catalase levels blunt aortic stiffness in Nox4 transgenic mice.

Aims: Oxidative stress is implicated in cardiomyocyte cell death and cardiac remodeling in the failing heart. The role of NADPH oxidase 4 (NOX4) in cardiac adaptation to pressure overload is controversial, but its function in myocardial ischemic stress has not been thoroughly elucidated. This study examined the function of NOX4 in the pathogenesis of ischemic heart failure, utilizing mouse models, cell culture, and human heart samples. Results: Nox4(−/−) mice showed a protective phenotype in response to permanent left anterior descending coronary artery ligation with smaller infarction area, lower cardiomyocyte cross-sectional area, higher capillary density, and less cell death versus wild-type (WT) mice. Nox4(−/−) mice had lower activity of soluble epoxide hydrolase (sEH), a potent regulator of inflammation. Nox4(−/−) mice also showed a 50% reduction in the number of infiltrating CD68(+) macrophages in the peri-infarct zone versus WT mice. Adenoviral overexpression of NOX4 in cardiomyoblast cells increased sEH expression and activity and CCL4 and CCL5 levels; inhibition of sEH activity in NOX4 overexpressing cells attenuated the cytokine levels. Human hearts with ischemic cardiomyopathy showed adverse cardiac remodeling, increased NOX4 and sEH protein expression and CCL4 and CCL5 levels compared with control nonfailing hearts. Innovation and Conclusion: These data from the Nox4(−/−) mouse model and human heart tissues show for the first time that oxidative stress from increased NOX4 expression has a functional role in ischemic heart failure. One mechanism by which NOX4 contributes to ischemic heart failure is by increasing inflammatory cytokine production via enhanced sEH activity.

Increased reactive oxygen species (ROS) production and inflammation are key factors in the pathogenesis of atherosclerosis. We previously reported that NOX activator 1 (NOXA1) is the critical functional homolog of p67phox for NADPH oxidase activation in mouse vascular smooth muscle cells (VSMC). Here we investigated the effects of systemic and SMC-specific deletion of Noxa1 on VSMC phenotype during atherogenesis in mice. Neointimal hyperplasia following endovascular injury was lower in Noxa1-deficient mice versus the wild-type following endovascular injury. Noxa1 deletion in Apoe-/- or Ldlr-/- mice fed a Western diet showed 50% reduction in vascular ROS and 30% reduction in aortic atherosclerotic lesion area and aortic sinus lesion volume (P, Graphical abstract fx1, Highlights • NOXA1 is a VSMC-specific regulator of NADPH oxidase 1 activity and downstream cell signaling. • NOX1 NADPH oxidase-dependent ROS generation is required for VSMC proliferation and migration after endovascular injury. • NOXA1-dependent NOX1 activation of KLF4 in atherosclerotic lesions induces SMC phenotypic switch to macrophage-like cells. • Atherosclerotic lesion macrophage-like cells promote plaque inflammation, matrix remodeling and increase volume expansion.

Introduction: Increased vascular reactive oxygen species (ROS) levels and chronic inflammation are hallmarks of vascular aging. We previously reported that expression of NOX4 NADPH oxidase increases with age in mouse and human arteries and correlates with inflammatory cytokine secretion and atherosclerotic lesion progression. Hypothesis: To determine whether NOX4 promotes atherosclerosis in aging, we assessed vascular inflammation and atherosclerosis burden in Nox4 -/- / Apoe -/- and Apoe -/- mice. Results: Aortic atherosclerotic lesion area was not different between 5-month old (young) Nox4 -/- / Apoe -/- and Apoe -/- mice fed a Western diet for 3 months. Lesion area increased significantly in 16-month old (aged) compared with young mice but was 51% lower in Nox 4 -/- / Apoe -/- versus Apoe -/- mice (n=12, P Nox4 -/- / Apoe -/- versus Apoe -/- mice. Flow cytometry analysis of atherosclerotic aortas showed significantly higher number of CD11b + macrophages in the lesions of aged Apoe -/- versus Nox4 -/- /Apoe -/- mice. However, the number of M1 pro-inflammatory macrophages (CD38 + ) was not different, whereas percentage of M2 macrophages (Egr2 + ) was significantly higher in aged Nox4 -/- Apoe -/- versus Apoe -/- mice (46% vs. 33%, respectively). In addition, Nox4 deletion increased the ratio of M2/M1 macrophages in the atherosclerotic aortas from aged Apoe -/- mice (0.56 and 0.43 in Nox 4 -/- / Apoe -/- and Apoe -/- , respectively). Macrophages isolated from Nox4 -/- mice showed lower number of M1 cells after IFNγ+LPS treatment and higher number of M2 cells after IL4 treatment versus macrophages from the wild-type. Furthermore, wild-type macrophages showed significantly higher secreted IL1β levels after IFNγ+LPS treatment versus macrophages from Nox4 -/- mice. Conclusions: These data suggest that increased vascular NOX4 levels in aged Apoe -/- mice promote a proinflammatory environment, enhancing plaque macrophage accumulation and skewing polarization to M1 phenotype resulting in inflammasome activation and atherosclerotic lesion expansion.

Studies have linked exposure to ultrafine particulate matter (PM) and adverse cardiovascular events. Particulate matter-induced oxidative stress is believed to be a key mechanism underlying observed adverse vascular effects. Advanced age is one factor known to decrease anti-oxidant defenses and confer susceptibility to the detrimental vascular effects seen following PM exposure. The present study was designed to investigate the vasomotor responses following ultrafine PM exposure in wild type (WT) and superoxide dismutase 2 deficient (SOD2+/−) mice which possess decreased anti-oxidant defense. Thoracic aortic rings isolated from young and aged WT and SOD2+/− mice were exposed to ultrafine PM in a tissue bath system. Aortic rings were then constricted with increasing concentrations of phenylephrine, followed by relaxation with rising amounts of nitroglycerin (NTG). Data demonstrated that ultrafine PM decreased the relaxation response in both young WT and young SOD2+/− mouse aortas, and relaxation was significantly reduced in young SOD2+/− compared to WT mice. Ultrafine PM significantly diminished the NTG-induced relaxation response in aged compared to young mouse aortas. After ultrafine PM exposure, the relaxation response did not differ markedly between aged WT and aged SOD2+/− mice. Data demonstrated that the greater vascular effect in aortic rings in aged mice ex vivo after ultrafine PM exposure may be attributed to ultrafine PM-induced oxidative stress and loss of anti-oxidant defenses in aged vascular tissue. Consistent with this conclusion is the attenuation of NTG-induced relaxation response in young SOD2+/− mice.

Background Atherosclerosis progression during aging culminates in the development of vulnerable plaques, which may increase the risk of cardiovascular events. Increased generation and/or decreased scavenging of reactive oxygen species in the vascular wall are major contributors to atherogenesis. We previously showed that superoxide dismutase 2 deficiency increased vascular oxidative stress and reduced aortic compliance in aged wild‐type mice and that young Apoe −/− / Sod2 +/− had increased mitochondrial DNA damage and atherosclerosis versus young Apoe −/− mice. Here we investigated the effects of superoxide dismutase 2 deficiency on atherosclerosis progression and plaque morphology in middle‐aged Apoe −/− mice. Methods and Results Compared with Apoe −/− , middle‐aged Apoe −/− / Sod2 +/− mice had increased vascular wall reactive oxygen species ( P P Apoe −/− / Sod2 +/− mice had an increased necrotic core with higher inflammatory cell infiltration, a thinned fibrous cap with depleted smooth muscle content, and intraplaque hemorrhage. In addition, the plaque shoulder area had higher levels of calpain‐2, caspase‐3, and matrix metalloproteinase‐2 in intimal smooth muscle cells and depleted fibrous cap collagen. Targeting mitochondrial reactive oxygen species with Mito TEMPO attenuated features of atherosclerotic plaque vulnerability in middle‐aged Apoe −/− / Sod2 +/− mice by lowering expression of calpain‐2, caspase‐3, and matrix metalloproteinase‐2 and decreasing smooth muscle cell apoptosis and matrix degradation. Conclusions Enhanced mitochondrial oxidative stress under hyperlipidemic conditions in aging induces plaque instability, in part by increasing smooth muscle cell apoptosis, necrotic core expansion, and matrix degradation. Targeting mitochondrial reactive oxygen species or its effectors may be a viable therapeutic strategy to prevent aging‐associated and oxidative stress–related atherosclerosis complications.

Many advances have been made in the diagnosis and management of heart failure (HF) in recent years. Cardiac biomarkers are an essential tool for clinicians: point of care B-Type Natriuretic Peptide (BNP) and its N-terminal counterpart (NT-proBNP) levels help distinguish cardiac from non-cardiac causes of dyspnea and are also useful in the prognosis and monitoring of the efficacy of therapy. One of the major limitations of HF biomarkers is in obese patients where the relationship between BNP and NT-proBNP levels and myocardial stiffness is complex. Recent data suggest an inverse relationship between BNP and NT-proBNP levels and body mass index. Given the ever-increasing prevalence of obesity world-wide, it is important to understand the benefits and limitations of HF biomarkers in this population. This review will explore the biology, physiology, and pathophysiology of these peptides and the cardiac endocrine paradox in HF. We also examine the clinical evidence, mechanisms, and plausible biological explanations for the discord between BNP levels and HF in obese patients.

Objective— Age-related aortic stiffness is an independent risk factor for cardiovascular diseases. Although oxidative stress is implicated in aortic stiffness, the underlying molecular mechanisms remain unelucidated. Here, we examined the source of oxidative stress in aging and its effect on smooth muscle cell (SMC) function and aortic compliance using mutant mouse models. Methods and Results— Pulse wave velocity, determined using Doppler, increased with age in superoxide dismutase 2 (SOD2) +/− but not in wild-type, p47phox −/− and SOD1 +/− mice. Echocardiography showed impaired cardiac function in these mice. Increased collagen I expression, impaired elastic lamellae integrity, and increased medial SMC apoptosis were observed in the aortic wall of aged SOD2 +/− versus wild-type (16-month-old) mice. Aortic SMCs from aged SOD2 +/− mice showed increased collagen I and decreased elastin expression, increased matrix metalloproteinase-2 expression and activity, and increased sensitivity to staurosporine-induced apoptosis versus aged wild-type and young (4-month-old) SOD2 +/− mice. Smooth muscle α-actin levels were increased with age in SOD2 +/− versus wild-type SMCs. Aged SOD2 +/− SMCs had attenuated insulin-like growth factor-1-induced Akt and Forkhead box O3a phosphorylation and prolonged tumor necrosis factor-α–induced Jun N-terminal kinase 1 activation. Aged SOD2 +/− SMCs had increased mitochondrial superoxide but decreased hydrogen peroxide levels. Finally, dominant-negative Forkhead box O3a overexpression attenuated staurosporine-induced apoptosis in aged SOD2 +/− SMCs. Conclusion— Mitochondrial oxidative stress over a lifetime causes aortic stiffening, in part by inducing vascular wall remodeling, intrinsic changes in SMC stiffness, and aortic SMC apoptosis.

The intracellular signaling events by which NADPH oxidase-generated reactive oxygen species (ROS) modulate vascular smooth muscle cell (VSMC) function and atherogenesis are yet to be entirely elucidated. We previously demonstrated that NADPH oxidase deficiency decreased atherosclerosis in apoE(-/-) mice and identified adhesion protein CD44 as an important ROS-sensitive gene expressed in VSMC and atherosclerotic lesions. Here, we examined the molecular mechanisms by which NADPH oxidase-generated ROS regulate the expression of CD44 and its principal ligand, hyaluronan (HA), and how CD44-HA interaction affects VSMC proliferation and migration and inflammatory gene expression in apoE(-/-) mice aortas. Thrombin-induced CD44 expression is mediated by transcription factor AP-1 in a NADPH oxidase-dependent manner. NADPH oxidase-mediated ROS generation enhanced thrombin-induced HA synthesis, and hyaluronan synthase 2 expression in VSMC. Hyaluronidase, which generates low molecular weight HA (LMW-HA), is induced in VSMC in a NADPH oxidase-dependent manner and LMW-HA stimulated ROS generation and cell proliferation in wild-type but not p47(phox-/-) VSMC, effects that were enhanced by thrombin pretreatment. Haptotactic VSMC migration toward HA was increased by thrombin in a CD44-dependent manner. HA expression in atherosclerotic lesions and plasma-soluble CD44 and HA levels were higher in apoE(-/-) compared with apoE(-/-)/p47(phox-/-) mice. HA-regulated pro-inflammatory gene expression was higher in apoE(-/-) than apoE(-/-)/p47(phox-/-) mouse aortas. GKT136901, a specific inhibitor of Nox1- and Nox4-containing NADPH oxidase activity, attenuated ROS generation and atherosclerosis and decreased CD44 and HA expression in atherosclerotic lesions. Together, these data suggest that increased CD44 and HA expression and CD44-HA-dependent gene regulation may play a role in atherosclerosis stimulated by NADPH oxidase activation.

Background— Despite a concerted effort by many laboratories, the critical subunits that participate in vascular smooth muscle cell (VSMC) NADPH oxidase function have yet to be elucidated. Given the potential therapeutic importance of cell-specific inhibition of NADPH oxidase, we investigated the role of Nox activator 1 (NoxA1), a homolog of p67phox, in VSMC NADPH oxidase function and atherosclerosis. Methods and Results— The presence of NoxA1 in mouse aortic VSMCs was confirmed by reverse-transcription polymerase chain reaction and sequencing. NoxA1/p47phox interaction after thrombin treatment was observed by immunoprecipitation/Western analysis of lysates from p47phox −/− VSMCs transfected with adenoviral HA-NoxA1 and Myc-p47phox. Infection with adenoviral NoxA1 significantly enhanced thrombin-induced reactive oxygen species generation in wild-type but not in p47phox −/− and Nox1 −/− VSMCs. Thrombin-induced reactive oxygen species production and VSMC proliferation were significantly reduced after downregulation of NoxA1 with shRNA. Infection with NoxA1 shRNA but not scrambled shRNA significantly decreased thrombin-induced activation of the redox-sensitive protein kinases (Janus kinase 2, Akt, and p38 mitogen-activated protein kinase) in VSMCs. Adenovirus-mediated overexpression of NoxA1 in guidewire-injured mouse carotid arteries significantly increased superoxide production in medial VSMCs and enhanced neointimal hyperplasia. NoxA1 expression was significantly increased in aortas and atherosclerotic lesions of ApoE −/− mice compared with age-matched wild-type mice. Furthermore, in contrast to p67phox, immunoreactive NoxA1 is present in intimal and medial SMCs of human early carotid atherosclerotic lesions. Conclusions— NoxA1 is the functional homolog of p67phox in VSMCs that regulates redox signaling and VSMC phenotype. These findings support the potential for modulation of NoxA1 expression as a viable approach for the treatment of vascular diseases.

Increased production of reactive oxygen species in mitochondria, accumulation of mitochondrial DNA damage, and progressive respiratory chain dysfunction are associated with atherosclerosis or cardiomyopathy in human investigations and animal models of oxidative stress. Moreover, major precursors of atherosclerosis—hypercholesterolemia, hyperglycemia, hypertriglyceridemia, and even the process of aging—all induce mitochondrial dysfunction. Chronic overproduction of mitochondrial reactive oxygen species leads to destruction of pancreatic β-cells, increased oxidation of low-density lipoprotein and dysfunction of endothelial cells—factors that promote atherosclerosis. An additional mechanism by which impaired mitochondrial integrity predisposes to clinical manifestations of vascular diseases relates to vascular cell growth. Mitochondrial function is required for normal vascular cell growth and function. Mitochondrial dysfunction can result in apoptosis, favoring plaque rupture. Subclinical episodes of plaque rupture accelerate the progression of hemodynamically significant atherosclerotic lesions. Flow-limiting plaque rupture can result in myocardial infarction, stroke, and ischemic/reperfusion damage. Much of what is known on reactive oxygen species generation and modulation comes from studies in cultured cells and animal models. In this review, we have focused on linking this large body of literature to the clinical syndromes that predispose humans to atherosclerosis and its complications.

NADPH oxidases (NOX) are a major source of reactive oxygen species in the cardiovascular system, contributing to the pathogenesis of disease. We recently reported that increased NOX4 expression in aging results in enhanced cellular and mitochondrial oxidative stress, vascular inflammation, dysfunction, and atherosclerosis. However, the role of NOX4 in the activation of inflammatory molecular pathways in vascular smooth muscle cells (VSMC) under pathophysiological conditions has not been elucidated. In this study, we investigated whether inhibition of NOX4 expression modulates proinflammatory gene expression in mouse VSMC by suppressing its expression with a novel peptide-mediated siRNA transfection approach. Using melittin derived peptide p5RHH, we achieved significantly higher transfection efficiency (92% vs 85% with Lipofectamine) and decreased toxicity (p

New medical-education models in which research plays a modest role could engender a two-tiered educational system, cause a reduction in the physician-scientist pipeline, and diminish the translation of biomedical advances.

Aims: Increased oxidative stress and vascular inflammation are implicated in increased cardiovascular disease (CVD) incidence with age. We and others demonstrated that NOX1/2 NADPH oxidase inhibition, by genetic deletion of p47phox, in Apoe−/− mice decreases vascular reactive oxygen species (ROS) generation and atherosclerosis in young age. The present study examined whether NOX1/2 NADPH oxidases are also pivotal to aging-associated CVD. Results: Both aged (16 months) Apoe−/− and Apoe−/−/p47phox−/− mice had increased atherosclerotic lesion area, aortic stiffness, and systolic dysfunction compared with young (4 months) cohorts. Cellular and mitochondrial ROS (mtROS) levels were significantly higher in aortic wall and vascular smooth muscle cells (VSMCs) from aged wild-type and p47phox−/− mice. VSMCs from aged mice had increased mitochondrial protein oxidation and dysfunction and increased vascular cell adhesion molecule 1 expression, which was abrogated with (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) treatment. NOX4 expression was increased in the vasculature and mitochondria of aged mice and its suppression with shRNA in VSMCs from aged mice decreased mtROS levels and improved function. Increased mtROS levels were associated with enhanced mitochondrial NOX4 expression in aortic VSMCs from aged subjects, and NOX4 expression levels in arterial wall correlated with age and atherosclerotic severity. Aged Apoe−/− mice treated with MitoTEMPO and 2-(2-chlorophenyl)-4-methyl-5-(pyridin-2-ylmethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione had decreased vascular ROS levels and atherosclerosis and preserved vascular and cardiac function. Innovation and Conclusion: These data suggest that NOX4, but not NOX1/2, and mitochondrial oxidative stress are mediators of CVD in aging under hyperlipidemic conditions. Regulating NOX4 activity/expression and using mitochondrial antioxidants are potential approaches to reducing aging-associated CVD. Antioxid. Redox Signal. 23, 1389–1409.

Objective— In mice that are heterozygous for mitochondrial superoxide dismutase (SOD2 +/− ) with apoE deficiency (apoE −/− ), mitochondrial DNA damage increases formation of atherosclerotic lesions. The purpose of this study was to determine whether SOD2 provides protection against increased vascular superoxide and endothelial dysfunction in apoE-deficient mice. Methods and Results— Four groups of mice [apoE −/− /SOD2 +/− ( apoe / sod2 ), apoE −/− /SOD2 +/+ ( apoe / SOD2 ), apoE +/+ /SOD2 +/− ( apoE / sod2 ), and apoE +/+ /SOD2 +/+ ( apoE / SOD2 )] were fed normal chow diet, and studied at 15 to 17 months of age. Serum cholesterol levels were similar in apoe / sod2 and apoe / SOD2 mice, and also were similar in apoE / sod2 and apoE / SOD2 mice. Intimal area was increased in aorta, but not carotid artery, of apoe / sod2 and apoe / SOD2 mice. In carotid artery, superoxide was increased (67±5.2 relative fluorescence intensity/vessel area [RI] in apoe/sod2 mice, 31±3.1 RI in apoE / SOD2 mice, P apoe / sod2 mice versus apoe / SOD2, apoE/sod2, apoE/SOD2 mice. Tiron improved relaxation to acetylcholine. In aorta, superoxide levels were increased and relaxation to acetylcholine was impaired in apoe/sod2 and apoe/SOD2 mice, but responses were similar in apoe / sod2 and apoe / SOD2 mice. Conclusion— SOD2 protects against oxidative stress and endothelial dysfunction in carotid artery of apoE-deficient mice.

Atherosclerosis, the primary cause of coronary artery disease (CAD), is a multifactorial disease, the molecular etiology of which involves interaction of many genes and environmental factors. Reactive oxygen species are integral to many cellular and biomolecular processes that are active in the transition of incipient fatty streaks into acute coronary syndromes. Animal models of atherosclerosis and correlative data from human studies support the oxidative stress hypothesis of atherosclerosis. However, the association of genetic polymorphisms that underlie enhanced oxidative stress with CAD is controversial. In this review, we discuss polymorphisms in genes that are main sources of reactive oxygen species generation (NAD[P]H oxidase, endothelial nitric oxide synthase, and myeloperoxidase) in mitochondria and the antioxidant enzymes paraoxonase, glutathione reductase, and heme oxygenase. The contribution of defined genetic variants involved in oxidative homeostasis to human atherosclerosis susceptibility is modest because regulation of oxidative stress is multifactorial. However, the contribution of genetic haplotypes in concert with environmental factors is likely significant. A more rigorous characterization of genetic and oxidative phenotypes together with characterization of novel gene polymorphisms may help in early therapeutic intervention for CAD.

Summary. Atherosclerosis is a multifactorial disease for which the molecular etiology of many of the risk factors is still unknown. As no single genetic marker or test accurately predicts cardiovascular death, phenotyping for markers of inflammation may identify the individuals at risk for vascular diseases. Reactive oxygen species (ROS) are key mediators of signaling pathways that underlie vascular inflammation in atherogenesis, starting from the initiation of fatty streak development through lesion progression to ultimate plaque rupture. Various animal models of atherosclerosis support the notion that ROS released from NAD(P)H oxidases, xanthine oxidase, lipoxygenases, and enhanced ROS production from dysfunctional mitochondrial respiratory chain indeed have a causatory role in atherosclerosis and other vascular diseases. Human investigations also support the oxidative stress hypothesis of atherogenesis. This is further supported by the observed impairment of vascular function and enhanced atherogenesis in animal models that have deficiencies in antioxidant enzymes. The importance of oxidative stress in atherosclerosis is further emphasized because of its role as a unifying mechanism across many vascular diseases. The main contraindicator for the role oxidative stress plays in atherosclerosis is the lack of effectiveness of antioxidants in reducing primary endpoints of cardiovascular death and morbidity. However, this lack of effectiveness by itself does not negate the existence or causatory role of oxidative stress in vascular disease. Lack of proven markers of oxidative stress, which could help to identify a subset of population that can benefit from antioxidant supplementation, and the complexity and subcellular localization of redox reactions, are among the factors responsible for the mixed outcomes in the use of antioxidants for the prevention of cardiovascular diseases. To better understand the role of oxidative stress in vascular diseases, future studies should be aimed at using advances in mouse and human genetics to define oxidative stress phenotypes and link phenotype with genotype.

Growing evidence indicates that chronic and acute overproduction of reactive oxygen species (ROS) under pathophysiologic conditions is integral in the development of cardiovascular diseases (CVD). These ROS can be released from nicotinamide adenine dinucleotide (phosphate) oxidase, xanthine oxidase, lipoxygenase, mitochondria, or the uncoupling of nitric oxide synthase in vascular cells. ROS mediate various signaling pathways that underlie vascular inflammation in atherogenesis: from the initiation of fatty streak development through lesion progress to ultimate plaque rupture. Various animal models of oxidative stress support the notion that ROS have a causal role in atherosclerosis and other cardiovascular diseases. Human investigations also support the oxidative stress hypothesis of atherosclerosis. Oxidative stress is the unifying mechanism for many CVD risk factors, which additionally supports its central role in CVD. Despite the demonstrated role of antioxidants in cellular and animal studies, the ineffectiveness of antioxidants in reducing cardiovascular death and morbidity in clinical trials has led many investigators to question the importance of oxidative stress in human atherosclerosis. Others have argued that the prime factor for the mixed outcomes from using antioxidants to prevent CVD may be the lack of specific and sensitive biomarkers by which to assess the oxidative stress phenotypes underlying CVD. A better understanding of the complexity of cellular redox reactions, development of a new class of antioxidants targeted to specific subcellular locales, and the phenotype-genotype linkage analysis for oxidative stress will likely be avenues for future research in this area as we move toward the broader use of pharmacological and regenerative therapies in the treatment and prevention of CVD.

Human protease-activated receptor-1 (hPar1) plays a role in malignant and physiological invasion processes. We have identified a functional androgen response element (ARE) located in the hPar1 promoter upstream of the transcription start site at -1791 to -1777. Dihydrotestosterone treatment of the prostate cancer cell line LNCaP increased endogenous hPar1 mRNA levels, consistent with the threefold increase in promoter activity of hPar1-luciferase reporter construct. Specific binding of the hPar1-derived ARE to LNCaP nuclear extracts was demonstrated by electrophoretic mobility shift assay. This binding was abrogated by antiandrogen receptor (anti-AR) antibodies or excess cold oligonucleotide but not by a mutated oligonucleotide. Moreover, using chromatin immunoprecipitation assays, we confirm the in vivo interaction between the AR and ARE domain of the hPar1 promoter. In parallel, we show that hormone ablation therapy markedly reduces the otherwise high hPar1 expression levels in prostate cancer biopsy specimens. We suggest that the hPar1 gene is regulated transcriptionally by androgens, representing one of several target genes effectively reduced during hormone ablation therapy. A major limitation of hormonal deprivation is that it causes only a temporary remission, and the cancer eventually reappears in a more malignant, androgen-independent form. hPar1 is also overexpressed in CL1 cells, an aggressively metastasizing, hormone-independent subclone of LNCaP, and in PC3 prostate adenocarcinoma lacking AR in a mechanism yet to be fully elucidated. These data may imply that hPar1 expression correlates with prostate cancer progression in androgen-dependent and -independent phases and therefore, provides an instrumental, therapeutic target for treatment in prostate cancer.

No reflow occurs when there is inadequate myocardial perfusion of a given segment of the coronary circulation without evidence of epicardial vessel obstruction. It is a rare but clinically significant condition associated with myocardial infarction and coronary interventions. Diagnosis is usually based on clinical signs of myocardial ischemia (symptoms and/or ECG changes) combined with coronary angiography. Management can be difficult and primarily consists of intracoronary administration of vasodilators. One interesting etiology is thromboembolism and this has become the focus for new potential treatments, including distal embolic protection devices.

The development of coronary artery bypass graft (CABG) for the treatment of coronary heart disease has resulted in reduced morbidity and mortality compared with medical therapy. Even with the rapid development of improved percutaneous interventions, CABG remains an important approach for treating patients with advanced coronary heart disease. However, recent studies and commentary reporting an alarmingly high incidence of subtle, cognitive decline following CABG have generated questions about whether these adverse outcomes could be lessened. Even after considerable study, there is no consensus as to the cause of brain and lung injury after CABG and cardiopulmonary bypass, nor an agreed upon, mechanistic approach to study its prevention. The potential causes of these adverse outcomes and a simple approach are described, involving the use of the cannulae, biventricular pulsatile flow pump, and a blood substitute to optimize the perfusion of brain and alveolar cells, minimize systemic microembolization, and limit post-CABG cognitive decline.

We previously reported that aortic smooth muscle cells (SMC) from aged mice have an age-related decline in proliferative capacity compared with those derived from young mice. Here we investigated matrix metalloproteinase-9 (MMP-9) regulation in both young and aged SMC. Zymography, immunoblot, and northern blot analysis showed that MMP-9 expression is significantly reduced in response to tumor necrosis factor-α stimulation with increasing in vitro age. Mutational analysis, gel shift assays and supershift assays demonstrated that the lower MMP-9 expression in aged SMC is associated with lower activities of NF-κB and AP-1. Since mitogen-activated protein kinase ERK1/2 induce MMP-9 expression, we examined whether U0126, an ERK1/2 inhibitor, influenced MMP-9 expression in aged SMC. Treatment with U0126 successfully inhibited MMP-9 expression in both young and aged SMC. Finally, to analyze the causal relationship between replicative senescence and MMP-9 expression, we stably overexpressed the MMP-9 gene in aged SMC and we showed no alteration of the proliferative capacity of the transduced cells. Taken together, these results suggest that down-regulation of MMP-9 expression in SMC may play a role in vascular remodeling during in vitro aging.

Fibrin targeting of the thrombin inhibitor hirudin via chemical coupling is effective in vitro and in vivo. However, since chemical coupling has limitations, a recombinant approach was taken to improve the fibrin-targeting ability of hirudin. Additionally, to activate hirudin selectively at the target area and thereby limit side effects in an in vivo setting, the authors aimed to construct an inactive precursor molecule that is converted into an active thrombin inhibitor only upon cleavage by factor Xa. Using PCR, the coding region for hirudin was fused to parts of the genomic DNA of the IgG heavy chain that was cloned from the antifibrin antibody-producing hybridoma cell line 59D8. Additionally, a factor Xa recognition site was introduced between the antibody and the hirudin sequence. The fusion construct was then transfected into a heavy-chain loss variant of the hybridoma cell line 59D8. After selection of stable hybridoma clones, the expressed fusion protein was evaluated for its molecular size (57 kd) and its binding ability to the fibrin-specific peptide Bbeta 15-22. The cleavage of the fusion protein by factor Xa was demonstrated by HPLC. The recombinant anticoagulant revealed antithrombin activity only after cleavage by factor Xa. Thus, the newly designed hirudin fusion protein revealed the anticipated functions in vitro. Further experiments are needed to prove whether this precursor anticoagulant allows a highly clot-specific and efficient thrombin inhibition in vivo.

Background — Coronary atherosclerotic disease remains the leading cause of death in the Western world. Although the exact sequence of events in this process is controversial, reactive oxygen and nitrogen species (RS) likely play an important role in vascular cell dysfunction and atherogenesis. Oxidative damage to the mitochondrial genome with resultant mitochondrial dysfunction is an important consequence of increased intracellular RS. Methods and Results — We examined the contribution of mitochondrial oxidant generation and DNA damage to the progression of atherosclerotic lesions in human arterial specimens and atherosclerosis-prone mice. Mitochondrial DNA damage not only correlated with the extent of atherosclerosis in human specimens and aortas from apolipoprotein E −/− mice but also preceded atherogenesis in young apolipoprotein E −/− mice. Apolipoprotein E −/− mice deficient in manganese superoxide dismutase, a mitochondrial antioxidant enzyme, exhibited early increases in mitochondrial DNA damage and a phenotype of accelerated atherogenesis at arterial branch points. Conclusions — Mitochondrial DNA damage may result from RS production in vascular tissues and may in turn be an early event in the initiation of atherosclerotic lesions.

Activation of the human protease-activated receptor-1 (PAR-1) by thrombin leads to myriad functions essential for maintaining vascular integrity. Upregulation of PAR-1 expression is considered important in atherosclerosis, angiogenesis and tumor metastasis. In vitro analysis of the human PAR-1 promoter function revealed a positive regulatory element between −4.2 and −3.2 kb of the transcription start site. This element was examined in transgenic mice containing either 4.1 or 2.9 kb of the 5′ flanking sequence driving a LacZ reporter gene. Only the 4.1 kb PAR-1 transgene was expressed in vivo and only during embryonic development. The transgene expression was observed only in developing arteries and not in veins. Further examination of this putative regulatory sequence identified a novel noncoding RNA (ncR-uPAR:noncoding RNA upstream of the PAR-1) gene at −3.4 kb. The ncR-uPAR upregulated PAR-1-core promoter-driven luciferase activity and mRNA expression in vitro in a Pol II-dependent manner. This noncoding RNA appears to act in trans, albeit locally at the adjacent PAR-1 promoter. These data suggest that an untranslated RNA plays a role in PAR-1 gene expression during embryonic growth.

ZusammenfassungDie Atherosklerose und ihre wichtigsten Komplikationen – Myokardinfarkt und Schlaganfall – sind die Hauptursachen für Tod und Behinderung in den USA und weltweit. Eine dramatische Zunahme bei Adipositas und Diabetes mellitus wird wahrscheinlich auch in Zukunft zu einer hohen Prävalenz kardiovaskulärer Erkrankungen (CVD) und deren Auswirkungen auf das Gesundheitswesen führen. Große Fortschritte gibt es bei der Entwicklung neuer Therapien zur Senkung der Inzidenz von Atherosklerose und CVD, besonders bei der Behandlung der Hypercholesterinämie und Hypertonie. Der gemeinsame mechanistische Nenner bei vielen Risikofaktoren für CVD ist oxidativer Stress. Erst seit kurzem verfügen wir über Methoden, um die Schnittstelle zwischen oxidativem Stress und CVD im Tiermodell zu untersuchen. Die wichtigste Quelle für reaktive Sauerstoffspezies (und damit für oxidativen Stress) in vaskulären Zellen sind die Formen der Nicotin - amidadenindinukleotidphosphat-Oxidase (NADPH-Oxidase). Die jüngsten Studien belegen eindeutig, dass 1. NADPH-Oxidasen im Tiermodell von grundlegender Bedeutung für Atherosklerose und Hypertonie sind und 2. der vaskuläre oxidative Stress, angesichts der gewebespezifischen Expression wichtiger Bestandteile der NADPH-Oxidase, ein Ziel bei der Prävention der CVD sein könnte.

The prevalence of atherosclerosis and incidence of cardiovascular events increase with advancing age. Increased generation and/or decreased scavenging of reactive oxygen species (ROS) in the vascular wall are major determinants of vascular disease progression. We previously showed that SOD2 deficiency in aging increases vascular oxidative stress and reduces aortic compliance, while young ApoE-/-/SOD2+/- mice show increased mitochondrial DNA damage and accelerated atherogenesis. Here we investigated the effects of aging and SOD2 deficiency on atherosclerosis and vascular wall morphology in ApoE-/- mice. Compared with the control ApoE-/-, aged ApoE-/-/SOD2+/- mice had increased total vascular wall (P < 0.001) and mitochondrial ROS levels (P < 0.05) and 70% higher atherosclerotic lesion area (P < 0.001). Pulse wave velocity (P < 0.001) and aortic wall collagen content (P < 0.05) were significantly increased, while atherosclerotic plaque fibrous cap thickness (P < 0.01) and vascular smooth muscle cell (VSMC)-positive area (P < 0.01) were decreased in aged ApoE-/-/SOD2+/- mice. Immunofluorescence analysis of atherosclerotic lesions revealed higher levels of calpain 2, a cysteine protease implicated in apoptosis and cleaved caspase-3 localized in smooth muscle-positive areas of the plaque in aged ApoE-/-/SOD2+/- mice. VSMC isolated from aged SOD2+/- had higher levels of mitochondrial oxidative stress and dysfunction compared with VSMC from wild-type mice, as determined by increased superoxide generation and mitochondrial protein carbonylation and lower complex I and III activity. The calpain 2 protein levels were significantly higher in the SOD2+/- VSMCs compared with the wild-type cells and its expression and activity increased after TNF-α treatment, causing apoptosis. TNF-α increased MMP2 activity in SOD2+/- VSMC in Calpain 2-dependent manner. In conclusion, enhanced mitochondrial oxidative stress in aging increases aortic stiffening, atherosclerotic burden and plaque instability in advanced atherosclerosis, in part, via the activation of calpain 2 and MMP2. Targeting mitochondrial oxidative stress and calpain 2 activity may be a viable strategy to prevent aging-associated atherosclerosis complications.

Cyclic strain regulates many vascular smooth muscle cell (VSMC) functions through changing gene expression. This study investigated the effects of cyclic strain on protease-activated receptor-1 (PAR-1) expression in VSMCs and the possible signaling pathways involved, on the basis of the hypothesis that cyclic strain would enhance PAR-1 expression, reflecting increased thrombin activity. Uniaxial cyclic strain (1 Hz, 20%) of cells cultured on elastic membranes induced a 2-fold increase in both PAR-1 mRNA and protein levels. Functional activity of PAR-1, as assessed by cell proliferation in response to thrombin, was also increased by cyclic strain. In addition, treatment of cells with antioxidants or an NADPH oxidase inhibitor blocked strain-induced PAR-1 expression. Preincubation of cells with protein kinase inhibitors (staurosporine or Ro 31-8220) enhanced strain-increased PAR-1 expression, whereas inhibitors of NO synthase, tyrosine kinase, and mitogen-activated protein kinases had no effect. Cyclic strain in the presence of basic fibroblast growth factor induced PAR-1 mRNA levels beyond the effect of cyclic strain alone, whereas no additive effect was observed between cyclic strain and platelet-derived growth factor-AB. Our findings that cyclic strain upregulates PAR-1 mRNA expression but that shear stress downregulates this gene in VSMCs provide an opportunity to elucidate signaling differences by which VSMCs respond to different mechanical forces.

OBJECTIVES This randomized trial compared a strategy of predischarge coronary angiography (CA) with exercise treadmill testing (ETT) in low-risk patients in the chest pain unit (CPU) to reduce repeat emergency department (ED) visits and to identify additional coronary artery disease (CAD). BACKGROUND Patients with chest pain and normal electrocardiograms (ECGs) have a low likelihood of CAD and a favorable prognosis, but they often seek repeat evaluations in EDs. Remaining uncertainty regarding their symptoms and diagnosis may cause much of this recidivism. METHODS A total of 248 patients with no ischemic ECG changes triaged to a CPU were randomized to CA (n = 123) or ETT (n = 125). All patients had a probability of myocardial infarction ≤7% according to the Goldman algorithm, no biochemical evidence of infarction, the ability to exercise and no previous documented CAD. Patients were followed up for ≥1 year and surveyed regarding their chest pain self-perception and utility of the index evaluation. RESULTS Coronary angiography showed disease (≥50% stenosis) in 19% and ETT was positive in 7% of the patients (p = 0.01). During follow-up (374 ± 61 days), patients with a negative CA had fewer returns to the ED (10% vs. 30%, p = 0.0008) and hospital admissions (3% vs. 16%, p = 0.003), compared with patients with a negative/nondiagnostic ETT. The latter group was more likely to consider their pain as cardiac-related (15% vs. 7%), to be unsure about its etiology (38% vs. 26%) and to judge their evaluation as not useful (39% vs. 15%) (p < 0.01 for all comparisons). CONCLUSIONS In low-risk patients in the CPU, a strategy of CA detects more CAD than ETT, reduces long-term ED and hospital utilization and yields better patient satisfaction and understanding of their condition.

The cellular mechanisms that contribute to the acceleration of atherosclerosis in aging populations are poorly understood, although it is hypothesized that changes in the proliferative capacity of vascular smooth muscle cells is contributory. We addressed the relationship among aging, generation of reactive oxygen species (ROS), and proliferation in primary culture smooth muscle cells (SMC) derived from the aortas of young (4 mo old) and aged (16 mo old) mice to understand the phenotypic modulation of these cells as aging occurs. SMC from aged mice had decreased proliferative capacity in response to α-thrombin stimulation, yet generated higher levels of ROS and had constitutively increased mitogen-activated protein kinase activity, in comparison with cells from younger mice. These effects may be explained by dysregulation of cell cycle-associated proteins such as cyclin D1 and p27Kip1 in SMC from aged mice. Increased ROS generation was associated with decreased endogenous antioxidant activity, increased lipid peroxidation, and mitochondrial DNA damage. Accrual of oxidant-induced damage and decreased proliferative capacity in SMC may explain, in part, the age-associated transition to plaque instability in humans with atherosclerosis.