Mediator Of Vascular Dysfunction Research Articles

The multidirectional effects of thrombin and the possibility of their control in neurology

The review presents the main physiological functions of thrombin. The procoagulant and anticoagulant activities of the key serine protease are discussed in both physiological and pathological conditions of hemostasis. The involvement of thrombin in atherogenesis, as well as its role as a mediator of vascular dysfunction and inflammation in both the peripheral and central nervous system, is highlighted. A pronounced imbalance between the pro- and anticoagulant systems leads to an increase in thrombin formation and creates conditions for the development of thrombosis. Tests that allow direct or indirect assessment of thrombin's functional activity are presented. The potential applications of direct thrombin inhibitors and direct blockers of thrombin PAR receptors in vascular neurology are also considered.

Endothelial mitochondrial dysfunction during Cerebral Amyloid Angiopathy and Hyperhomocysteinemia

AbstractBackgroundHyperhomocysteinemia (HHcy), or high levels of homocysteine, is considered a risk factor for vascular disease. Cardiovascular (CV) risk factors, such as HHcy, may lead to exacerbation of cerebral amyloid angiopathy (CAA) due to their detrimental effects on cerebrovascular function. Cerebrovascular function is regulated, in part, by endothelial cells (EC), the most important cellular component of the blood‐brain barrier (BBB). Dysfunctional ECs may lead to deficits in vascular perfusion, clearance, and may eventually result in neuroinflammation, microhemorrhages, and neurodegeneration. EC mitochondria serve as sensors of cellular damage and mediate downstream pathways such as cell death and inflammation. In ECs, mitochondrial dysfunction may lie as the point of convergence of CV risk factors and amyloid β (Aβ)‐induced neurovascular dysfunction. Understanding the mitochondrial deficits elicited by CV risk factors, Aβ, or their combination, may open roads to new therapeutics aimed at preserving mitochondrial function. Indeed, we have previously shown that carbonic anhydrase inhibitors (CAi) reduce amyloid‐induced cell death and mitochondrial dysfunction. Here, we aim to understand the changes occurring in EC mitochondria during Aβ‐ and mixed (Aβ+HHcy)‐ vascular pathologies. In addition, we will assess the therapeutic potential of CAi in such pathologies.MethodHuman brain microvascular ECs were challenged with Aβ, Hcy, or the combination in the presence or absence of CAi. EC barrier properties and changes in mitochondrial function were assessed.ResultThe presence of Aβ or Hcy independently increased BBB permeability, and, when both present, Hcy worsened Aβ‐induced barrier permeability; these effects were prevented by CAi. Preliminary data suggests that the mitochondrial ATP production is impaired in the presence of Aβ40‐E22Q (Dutch mutant) but not in the presence of Hcy. These results may be a reflection of the observed deficits in mitochondrial oxygen consumption rate (OCR).ConclusionThe EC barrier is negatively affected by the presence of Aβ, and Hcy aggravates Aβ‐induced BBB permeability. The presence of Aβ40‐E22Q affects mitochondrial OCR and ATP production, indicative of changes in cellular metabolism and mitochondrial dysfunction. These results suggest a central role of the mitochondria as a mediator of vascular dysfunction in the presence of Aβ.

Tubulin-folding cofactor E deficiency is associated with vascular dysfunction and endoplasmatic reticulum stress of vascular smooth muscle cells

Abstract Introduction Endothelial function assessed via flow mediated dilatation (FMD) has shown to predict risk in individuals with established cardiovascular diseases, whereas its predictive value is uncertain in the setting primary prevention. Purpose The aim of the current work was to discover and evaluate novel mediators of vascular dysfunction in the general population and in conditional knock-out transgenic animal models. Methods In order to identify novel targets that were negatively correlated with FMD and investigate their contribution in vascular function, a Genome Wide Association Study (GWAS) of 5,000 participants was performed and subsequently immune cell-, endothelial- and vascular smooth muscle cell (VSMCs)-targeted conditional knockout mouse models were generated and characterized. Results GWAS analysis revealed that single nucleotide polymorphisms (SNPs) in the tubulin folding cofactor E (TBCE) gene were negatively correlated with FMD and TBCE expression in the peripheral blood mononuclear cells (PBMCs). Myelomonocytic cell-targeted TBCE deficiency did not lead to any vascular dysfunction in vivo in the LysM+Cre+/−TBCEfl/fl mice. Endothelial-targeted TBCE deficiency led to an NLR family pyrin domain containing 3 (NLRP3)-dependent activation of the inflammasome in the endothelial cells of Tie2-ERT2Cre+/−TBCEfl/fl mice. Importantly, VSMC-targeted TBCE deficiency was associated with endothelial dysfunction, increased aortic wall thickness and endoplasmatic reticulum (ER) stress-mediated VSMC hyperproliferation in vivo (SMMHC-ERT2Cre+/−TBCEfl/fl), paralleled by calnexin upregulation. Administration of the blood pressure hormone angiotensin II exacerbated the vascular dysfunction and phenotype. Administration of the ER stress modulator tauroursodeoxycholic acid to the SMMHC-ERT2Cre+/−TBCEfl/fl mice reversed vascular dysfunction, paralleled by induction of Raptor/Beclin-1-dependent autophagy both in vitro and in vivo. Conclusion TBCE and tubulin homeostasis in the vascular musculature seem to be novel markers of vascular function and represent a new druggable target for the treatment of ER-stress-mediated vascular dysfunction. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): This work was supported by grants of the German Federal Ministry for Education and Research (BMBF01EO1003 and BMBF01EO1503), the DFG Major Research Instrumentation Programme (DFG INST 371/47-1 FUGG) as well as the Boehringer Ingelheim Foundation. PW received funds from the German Research Foundation in support of his work (DFG WE4361-4-1 and WE 4361/7-1). KS, TM and PW are PIs of the DZHK.

Maternal high-fat-diet exposure is associated with elevated blood pressure and sustained increased leptin levels through epigenetic memory in offspring

Maternal metabolism dysregulation during pregnancy predisposes offspring to major diseases, including hypertension, in later life, but the mechanism involved remains to be fully elucidated. A high-fat-diet (HFD) pregnant rat model was used to investigate whether excessive intrauterine lipid exposure was associated with elevated blood pressure in offspring and increased levels of leptin, an important biomarker and mediator of vascular dysfunction and hypertension. We found that gestational hyperlipidemia predisposed offspring to blood pressure elevation and sustained increases in leptin levels with no difference in body weight in the rat model. Increased leptin expression and leptin promoter hypomethylation were found in adipose tissues of HFD-exposed offspring. The treatment of mesenchymal stem cells with free fatty acids during adipogenic differentiation resulted in increased leptin expression, accompanied by leptin promoter hypomethylation. In addition, we also followed up 121 children to evaluate the association between maternal triglyceride levels and offspring blood pressure. Consistent with the animal study results, we observed elevated serum leptin levels and blood pressure in the offspring born to women with gestational hypertriglyceridemia. Our findings provide new insights that maternal hyperlipidemia is associated with elevated blood pressure in offspring and is associated with increases in leptin levels through epigenetic memory.

Vascular dysfunction in CAA in the presence of cardiovascular risk factors: The role of the mitochondria and therapeutic approaches

AbstractBackgroundThe accumulation of amyloid‐β (Aβ) on the brain vasculature, known as Cerebral Amyloid Angiopathy (CAA), induces vascular dysfunction contributing to neurodegeneration. Cardiovascular (CV) risk factors, which are also mediators of vascular dysfunction and may increase the risk for developing Alzheimer’s Disease and dementia, may potentiate CAA‐mediated neurovascular pathology. Mitochondria are key cellular organelles that have a dual role as mediators of cell survival and death and have an important role in disease pathogenesis. However, the role of brain endothelial mitochondria in vascular amyloidosis complicated with CV risk factors is unknown. The carbonic anhydrase inhibitors (CAi) methazolamide and acetazolamide were able to reduce amyloid‐induced cell death and mitochondrial dysfunction. Our work focuses on understanding the mechanisms of vascular dysfunction induced by Aβ complicated with CV risk factors. We aim to understand the protective role of CAi in preventing the pathological changes associated to neurovascular dysfunction and neurodegeneration.MethodImmortalized human cerebral microvascular endothelial cells (ECs) were used to study the molecular changes that occur in the cerebral vasculature during amyloid challenge complicated with CV risk factors, such as hyperhomocysteinemia (HHC). We then tested CAi and their potential to prevent cell death and mitochondrial dysfunction in ECs challenged with Aβ in the presence or absence of CV risk factors such as HHC.ResultAβ and HHC increased EC barrier permeability and that the presence of HHC worsened amyloid‐induced barrier permeability. Aβ‐induced EC apoptosis was reduced by treatment with CAi. We further demonstrate that both Aβ and HHC decrease mitochondrial basal respiration. The presence of Aβ induces cytochrome‐c release, mitochondrial membrane potential loss, and ROS production which were reduced by treatment with CAi.ConclusionHHC and Aβ induce brain microvascular EC dysfunction and increase permeability of the EC barrier. Aβ induces cell death and mitochondrial dysfunction, which are both prevented by CAi. We also demonstrate that CV risk factors, specifically HHC, induce mitochondrial dysfunction on brain ECs which could further contribute to neurovascular dysfunction and neurodegeneration observed in CAA. Our studies suggest that CAi could serve as a novel therapeutic strategy towards vascular dysfunction during CAA complicated with CV risk factors.

Thrombin, a Mediator of Coagulation, Inflammation, and Neurotoxicity at the Neurovascular Interface: Implications for Alzheimer's Disease.

The societal burden of Alzheimer’s disease (AD) is staggering, with current estimates suggesting that 50 million people world-wide have AD. Identification of new therapeutic targets is a critical barrier to the development of disease-modifying therapies. A large body of data implicates vascular pathology and cardiovascular risk factors in the development of AD, indicating that there are likely shared pathological mediators. Inflammation plays a role in both cardiovascular disease and AD, and recent evidence has implicated elements of the coagulation system in the regulation of inflammation. In particular, the multifunctional serine protease thrombin has been found to act as a mediator of vascular dysfunction and inflammation in both the periphery and the central nervous system. In the periphery, thrombin contributes to the development of cardiovascular disease, including atherosclerosis and diabetes, by inducing endothelial dysfunction and related inflammation. In the brain, thrombin has been found to act on endothelial cells of the blood brain barrier, microglia, astrocytes, and neurons in a manner that promotes vascular dysfunction, inflammation, and neurodegeneration. Thrombin is elevated in the AD brain, and thrombin signaling has been linked to both tau and amyloid beta, pathological hallmarks of the disease. In AD mouse models, inhibiting thrombin preserves cognition and endothelial function and reduces neuroinflammation. Evidence linking atrial fibrillation with AD and dementia indicates that anticoagulant therapy may reduce the risk of dementia, with targeting thrombin shown to be particularly effective. It is time for “outside-the-box” thinking about how vascular risk factors, such as atherosclerosis and diabetes, as well as the coagulation and inflammatory pathways interact to promote increased AD risk. In this review, we present evidence that thrombin is a convergence point for AD risk factors and as such that thrombin-based therapeutics could target multiple points of AD pathology, including neurodegeneration, vascular activation, and neuroinflammation. The urgent need for disease-modifying drugs in AD demands new thinking about disease pathogenesis and an exploration of novel drug targets, we propose that thrombin inhibition is an innovative tactic in the therapeutic battle against this devastating disease.

Are Reactive Oxygen Species Important Mediators of Vascular Dysfunction?

Reactive Oxygen Species (ROS) are reactive derivatives of oxygen metabolism. The ROS generation can be mediated by distinctive enzymatic systems including NADPH oxidases. The components of this enzyme are expressed in endothelial and vascular smooth muscle cells, adventitial fibroblasts, and infiltrating monocytes/macrophages. Oxidative stress is a molecular dysregulation in ROS generation/elimination, which plays a key role in the development of vascular dysfunction in distinctive conditions including hypertension. It is characterized by vascular inflammation, a loss of NO bioavailability and endothelial dysfunction. Considering that oxidative stress is a key mediator of vascular dysfunction, antioxidant therapy with classic antioxidants seemed to be a promising alternative for the treatment of vascular diseases. In this sense, some commonly used drugs for the treatment of cardiovascular diseases such as Angiotensin Converting Enzyme (ACE) inhibitors or angiotensin receptor AT1 antagonists showed antioxidant effects that might have contributed, at least in part, to the beneficial effects of these drugs on the treatment of cardiovascular diseases. The effectiveness of these drugs shows that ROS are in fact important mediators of vascular dysfunction and that angiotensin II plays a critical role in such response.

Extracellular vesicles: biomarkers and regulators of vascular function during extracorporeal circulation.

Extracellular vesicles (EVs) are generated at increased rates from parenchymal and circulating blood cells during exposure of the circulation to abnormal flow conditions and foreign materials associated with extracorporeal circuits (ExCors). This review describes types of EVs produced in different ExCors and extracorporeal life support (ECLS) systems including cardiopulmonary bypass circuits, extracorporeal membrane oxygenation (ECMO), extracorporeal carbon dioxide removal (ECCO2R), apheresis, dialysis and ventricular assist devices. Roles of EVs not only as biomarkers of adverse events during ExCor/ECLS use, but also as mediators of vascular dysfunction are explored. Manipulation of the number or subtypes of circulating EVs may prove a means of improving vascular function for individuals requiring ExCor/ECLS support. Strategies for therapeutic manipulation of EVs during ExCor/ECLS use are discussed such as accelerating their clearance, preventing their genesis or pharmacologic options to reduce or select which and how many EVs circulate. Strategies to reduce or select for specific types of EVs may prove beneficial in preventing or treating other EV-related diseases such as cancer.

Asymmetric dimethylarginine as a mediator of vascular dysfunction in cirrhosis.

Cirrhosis is associated with marked abnormalities in the circulatory function that involve a reduction in systemic vascular resistance. An important cause of this vasodilatation is the increased production or activity of nitric oxide (NO) in the splanchnic circulation. During portal hypertension and cirrhosis an increased endothelial NO synthase (eNOS) activity is demonstrated in splanchnic vessels. In contrast, the activity of eNOS in the cirrhotic liver is decreased, which suggests a different regulation of eNOS in the liver and in the splanchnic vessels. Asymmetric dimethylarginine (ADMA) is an endogenous NO inhibitor and higher plasma levels of ADMA are related to increased cardiovascular risk in both the general population and among patients with cirrhosis. It has been demonstrated that the liver is a key player in the metabolism of ADMA. This observation was further supported by investigations in human patients, showing a close correlation between ADMA plasma levels and the degree of hepatic dysfunction. ADMA is degraded to citrulline and dimethylamine by dimethylarginine dimethylaminohydrolases (DDAHs). DDAHs are expressed as type 1 and 2 isoforms and are widely distributed in various organs and tissues, including the liver. In this review, we discuss experimental and clinical data that document the effects of dimethylarginines on vascular function in cirrhosis. Our increasing understanding of the routes of synthesis and metabolism of methylarginines is beginning to provide insights into novel mechanisms of liver disease and allowing us to identify potential therapeutic opportunities.

Biochip Array Analysis of Various Mediators of Inflammation in Disseminated Intravascular Coagulation

Abstract 2302Disseminated intravascular coagulation is a polypathologic syndrome which involves blood, endothelial and target organ dysfunction resulting in the generation of various mediators of vascular dysfunction, hemostatic aberrations and hemodynamic disturbances. In addition, various disorders of target organs such as kidney, liver, heart and brain are observed. Uncontrolled protease generation results in the formation of various mediators of inflammation such as neuron specific enolase (NSE), neutrophil gelatinase associated lipocalin (NGal) and soluble tumor necrosis factor receptor 1 (TNF R1). Endothelial damage results in the generation of thrombomodulin (TM) and endogenous coagulation/fibrinolysis results in D- Dimer formation. In order to study the circulating levels of these mediators, a Biochip array method (Randox, Oceanside, CA) was utilized with samples obtained from clinically diagnosed DIC (n=100) and normal individuals (n=10). The results are summarized in the following table. Circulating levels of these mediators were markedly increased in DIC patients in comparison to normals. The most striking increase was noted in NGal (4–6 fold) and TNF R1 (10 fold). Other mediators were also increased in the DIC group, however the data was broadly scattered. These results indicate that beside the aberration in the hemostatic system NGal and TNF R1 along with other inflammatory mediators may play a major role in the pathophysiology of DIC.MarkerNormal ControlsDIC BaselineCRP (ug/ml)3.47 + 0.913.11 + 0.2*NSE (ng/ml)6.11 + 8.610.78 + 1.6*NGAL(ng/ml)306.45 + 25.51376.56 + 48.5*TNFR1 (ng/ml)0.35 + 0.013.89 + 0.3*DD (ng/ml)232.8 + 74.41318.94 + 80.5*TM (ng/ml)3.4 + 0.33.16 + 0.2P=<0.05 Disclosures:No relevant conflicts of interest to declare.

Biochip Array Analysis of Various Mediators of Inflammation in Disseminated Intravascular Coagulation

Disseminated intravascular coagulation is a polypathologic syndrome which involves blood, endothelial and target organ dysfunction resulting in the generation of various mediators of vascular dysfunction, hemostatic aberrations and hemodynamic disturbances. In addition, various disorders of target organs such as kidney, liver, heart and brain are observed. Uncontrolled protease generation results in the formation of various mediators of inflammation such as neuron specific enolase (NSE), neutrophil gelatinase associated lipocalin (NGal) and soluble tumor necrosis factor receptor 1 (TNF R1). Endothelial damage results in the generation of thrombomodulin (TM) and endogenous coagulation/fibrinolysis results in D‐ Dimer formation. In order to study the circulating levels of these mediators, a Biochip array method (Randox, Oceanside, CA) was utilized with samples obtained from clinically diagnosed DIC (n=100) and normal individuals (n=10). Circulating levels of these mediators were markedly increased in DIC patients in comparison to normals. The most striking increase was noted in NGal (4–6 fold) and TNF R1 (10 fold). Other mediators were also increased in the DIC group, however the data was broadly scattered. These results indicate that NGal and TNF R1 along with other mediators play a major role in the pathophysiology of DIC.

Review article: Asymmetric dimethylarginine as a mediator of vascular dysfunction and a marker of cardiovascular disease and mortality: an intriguing interaction with diabetes mellitus

Asymmetric dimethylarginine (ADMA) has evolved as an important regulator of nitric oxide (NO) synthesis in recent years. Elevated levels of ADMA have been reported in many conditions associated with a high cardiovascular risk. Moreover, ADMA is a biomarker for major cardiovascular events and mortality in cohorts with high, intermediate and low overall cardiovascular risk. Discrepant data have been reported on cardiovascular risk in people with and without diabetes mellitus, and the association of ADMA with diabetes mellitus per se has also remained controversial, possibly relating to type and stage of diabetes. Clinical and experimental data suggest that there is a multifaceted link between ADMA and insulin metabolism and action on one hand, and ADMA and glucose utilisation on the other. This interplay may be regulated by the enzyme involved in the metabolic degradation of ADMA, dimethylarginine dimethylaminohydrolase (DDAH). Recent data from prospective clinical studies suggest that whilst ADMA may be a marker for total mortality in patients without diabetes, elevated ADMA may exert beneficial effects in patients with diabetes. In this respect, ADMA could serve as a re-coupling agent overcoming endothelial nitric oxide synthase (eNOS) uncoupling in patients with diabetes. Anticipated advances in clinical and experimental investigation will help us to better understand this complex interrelationship between diabetes, eNOS, DDAH and ADMA.

Deletion of Protein Tyrosine Phosphatase 1b Improves Peripheral Insulin Resistance and Vascular Function in Obese, Leptin-Resistant Mice via Reduced Oxidant Tone

Obesity is a risk factor for cardiovascular dysfunction, yet the underlying factors driving this impaired function remain poorly understood. Insulin resistance is a common pathology in obese patients and has been shown to impair vascular function. Whether insulin resistance or obesity, itself, is causal remains unclear. The present study tested the hypothesis that insulin resistance is the underlying mediator for impaired NO-mediated dilation in obesity by genetic deletion of the insulin-desensitizing enzyme protein tyrosine phosphatase (PTP)1B in db/db mice. The db/db mouse is morbidly obese, insulin-resistant, and has tissue-specific elevation in PTP1B expression compared to lean controls. In db/db mice, PTP1B deletion improved glucose clearance, dyslipidemia, and insulin receptor signaling in muscle and fat. Hepatic insulin signaling in db/db mice was not improved by deletion of PTP1B, indicating specific amelioration of peripheral insulin resistance. Additionally, obese mice demonstrate an impaired endothelium dependent and independent vasodilation to acetylcholine and sodium nitroprusside, respectively. This impairment, which correlated with increased superoxide in the db/db mice, was corrected by superoxide scavenging. Increased superoxide production was associated with increased expression of NAD(P)H oxidase 1 and its molecular regulators, Noxo1 and Noxa1. Deletion of PTP1B improved both endothelium dependent and independent NO-mediated dilation and reduced superoxide generation in db/db mice. PTP1B deletion did not affect any vascular function in lean mice. Taken together, these data reveal a role for peripheral insulin resistance as the mediator of vascular dysfunction in obesity.

Tissue-specific downregulation of dimethylarginine dimethylaminohydrolase in hyperhomocysteinemia

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, has been proposed to be a mediator of vascular dysfunction during hyperhomocysteinemia. Levels of ADMA are regulated by dimethylarginine dimethylaminohydrolase (DDAH). Using both in vitro and in vivo approaches, we tested the hypothesis that hyperhomocysteinemia causes downregulation of the two genes encoding DDAH (Ddah1 and Ddah2). In the MS-1 murine endothelial cell line, the addition of homocysteine decreased NO production but did not elevate ADMA or alter levels of Ddah1 or Ddah2 mRNA. Mice heterozygous for cystathionine beta-synthase (Cbs) and their wild-type littermates were fed either a control diet or a high-methionine/low-folate (HM/LF) diet to produce varying degrees of hyperhomocysteinemia. Maximal relaxation of the carotid artery to the endothelium-dependent dilator acetylcholine was decreased by approximately 50% in Cbs(+/-) mice fed the HM/LF diet compared with Cbs(+/+) mice fed the control diet (P < 0.001). Compared with control mice, hyperhomocysteinemic mice had lower levels of Ddah1 mRNA in the liver (P < 0.001) and lower levels of Ddah2 mRNA in the liver, lung, and kidney (P < 0.05). Downregulation of DDAH expression in hyperhomocysteinemic mice did not result in an increase in plasma ADMA, possibly due to a large decrease in hepatic methylation capacity (S-adenosylmethionine-to-S-adenosylhomocysteine ratio). Our findings demonstrate that hyperhomocysteinemia causes tissue-specific decreases in DDAH expression without altering plasma ADMA levels in mice with endothelial dysfunction.

Abstract 1630: Uric Acid Revisited: Interrelationships with Low-Grade Inflammation, Adiponectin and Arterial Stiffness in Essential Hypertension

Introduction: Increased uric acid (UA) levels are associated with high cardiovascular risk, while subclinical inflammation, arterial stiffening and hypoadiponectinemia contribute to atherosclerosis progression. Hypothesis: Serum UA levels may be related to high-sensitivity C-reactive protein (hs-CRP), adiponectin and arterial stiffness in essential hypertensives. Methods: In our population of 292 newly diagnosed untreated non-diabetic patients with stage I to II essential hypertension [190 men, aged 50 years, office blood pressure (BP)=148/95 mmHg], the distribution of UA was split by the median (5.3 mg/dl) and accordingly subjects were classified into those with high and low values. In all participants, arterial stiffness was evaluated on the basis of carotid to femoral pulse wave velocity (PWV), by means of a computerized method (Complior SP). Results: Patients with high UA (n=143) compared to those with low UA (n=149) exhibited higher 24-h systolic BP (139±12 vs 132±11 mmHg, p&lt;0.0001), while did not differ regarding age, body mass index and lipid profile (p=NS). Those with high UA compared to those with low UA had increased levels of hs-CRP (3.1±1.4 vs 2.3±1.2 mg/l, p&lt;0.05) and PWV (8.7±1.2 vs 7.9±1.2 m/sec, p&lt;0.05), whereas exhibited lower adiponectin (8.1±2.4 vs 9.7±2.8 μg/ml, p&lt;0.05). In the entire population, UA was positively associated with 24-h systolic BP (r=0.297, p&lt;0.0001), hs-CRP (r=0.204, p&lt;0.001) and PWV (r=0.165, p=0.009), while it was negatively related to adiponectin (r=−0.218, p&lt;0.0001). In multiple regression analysis, 24-h systolic BP (b=0.246, p&lt;0.001), hs-CRP (b=0.142, p&lt;0.05) and adiponectin (b=−0.154, p≥0.05) were independent predictors of UA. Analysis of covariance revealed that hs-CRP, PWV and adiponectin values were significantly different between groups after adjustment for confounders (p&lt;0.05). Conclusions: Increased UA levels in essential hypertension are paralleled by a state of pronounced inflammatory activation, hypoadiponectinemia and accelerated arterial stiffening. These findings suggest that UA is interrelated with diverse mediators of vascular dysfunction, underscoring its pluripotent nature in the progression of the hypertensive atherosclerotic disease.

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

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 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-κB 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-κB inhibitor, SOD, catalase, or siRNA against Nox1. This study establishes that AGE activate iNOS in VSMC through a ROS-sensitive, NF-κB-dependent mechanism involving ROS generation by a Nox1-based oxidase.

Gene-targeted mice reveal a critical role for inducible nitric oxide synthase in vascular dysfunction during diabetes.

Inducible nitric oxide synthase (iNOS) is a mediator of vascular dysfunction during inflammation. The purpose of this study was to test the hypothesis that vascular dysfunction during diabetes is dependent on expression of iNOS. Diabetes was produced in mice with streptozotocin (150 mg/kg IP). After 4 to 6 months of diabetes, vasomotor function was examined in vitro in carotid arteries from mice with targeted disruption of the gene for iNOS (iNOS-deficient mice) and from normal, wild-type (WT) mice. Contractile responses of carotid arteries to U46619, a thromboxane A2 analogue, were not altered by diabetes in WT mice. Responses to U46619 were increased in arteries from diabetic iNOS-deficient mice compared with diabetic WT and nondiabetic mice (iNOS-deficient and WT mice). These results indicate that expression of iNOS inhibits an increased vasoconstrictor response during diabetes. Arteries from nondiabetic WT mice relaxed 83+/-2% (mean+/-SE) in response to acetylcholine (1 micromol/L) compared with 58+/-6% in arteries from diabetic WT mice (P<0.05 versus nondiabetic mice). In contrast, relaxation of carotid arteries to acetylcholine was similar (81+/-4% versus 76+/-6%; P>0.05) in iNOS-deficient mice under nondiabetic and diabetic conditions, respectively. Thus, diabetes produced impairment of endothelium-dependent relaxation in arteries from WT but not iNOS-deficient mice. Endothelium-independent relaxation in response to nitroprusside was similar in arteries from all mice. These results provide the first direct evidence that impairment of endothelium-dependent relaxation during diabetes is dependent on expression of iNOS.

Endothelial dysfunction and elevation of S-adenosylhomocysteine in cystathionine beta-synthase-deficient mice.

Hyperhomocysteinemia is associated with increased risk for cardiovascular events, but it is not certain whether it is a mediator of vascular dysfunction or a marker for another risk factor. Homocysteine levels are regulated by folate bioavailability and also by the methyl donor S-adenosylmethionine (SAM) and its metabolite S-adenosylhomocysteine (SAH). We tested the hypotheses that endothelial dysfunction occurs in hyperhomocysteinemic mice in the absence of folate deficiency and that levels of SAM and SAH are altered in mice with dysfunction. Heterozygous cystathionine beta-synthase-deficient (CBS(+/-)) and wild-type (CBS(+/+)) mice were fed a folate-replete, methionine-enriched diet. Plasma levels of total homocysteine were elevated in CBS(+/-) mice compared with CBS(+/+) mice after 7 weeks (27.1+/-5.2 versus 8.8+/-1.1 micromol/L; P<0.001) and 15 weeks (23.9+/-3.0 versus 13.0+/-2.3 micromol/L; P<0.01). After 15 weeks, but not 7 weeks, relaxation of aortic rings to acetylcholine was selectively impaired by 35% (P<0.05) and thrombomodulin anticoagulant activity was decreased by 20% (P<0.05) in CBS(+/-) mice. Plasma levels of folate did not differ between groups. Levels of SAH were elevated approximately 2-fold in liver and brain of CBS(+/-) mice, and correlations were observed between plasma total homocysteine and SAH in liver (r=0.54; P<0.001) and brain (r=0.67; P<0.001). These results indicate that endothelial dysfunction occurs in hyperhomocysteinemic mice even in the absence of folate deficiency. Endothelial dysfunction in CBS(+/-) mice was associated with increased tissue levels of SAH, which suggests that altered SAM-dependent methylation may contribute to vascular dysfunction in hyperhomocysteinemia.

Management of systemic sclerosis: the art and science.

There have been substantial strides in the therapy of systemic sclerosis (SSc) in recent years, particularly in the management of individual organ manifestations. Effective treatments are available for SSc renal crisis and many of the gastrointestinal manifestations of the disease. Raynaud's phenomenon, a nearly universal problem in SSc, also may be effectively managed. Treatment of the pulmonary complications, pulmonary hypertension and interstitial lung disease, remains difficult. Patients with early, diffuse SSc are the best candidates for experimental therapies intended to modify the overall disease process. Most disease-modifying agents have been directed at the fibrotic and inflammatory processes characteristic of SSc and have achieved little success. Future therapies may target mediators of vascular dysfunction in SSc. The success of future therapeutic trials will depend on collaborative efforts between treatment centers.