Endothelial SIRT1 as a Target for the Prevention of Arterial Aging: Promises and Challenges.

ABSTRACT: SIRT1, a member of the sirtuin family of longevity regulators, possesses potent activities preventing vascular aging. The expression and function of SIRT1 in endothelial cells are downregulated with age, in turn causing early vascular aging and predisposing various vascular abnormalities. Overexpression of SIRT1 in the vascular endothelium prevents aging-associated endothelial dysfunction and senescence, thus the development of hypertension and atherosclerosis. Numerous efforts have been directed to increase SIRT1 signaling as a potential strategy for different aging-associated diseases. However, the complex mechanisms underlying the regulation of SIRT1 have posed a significant challenge toward the design of specific and effective therapeutics. This review aimed to provide a summary on the regulation and function of SIRT1 in the vascular endothelium and to discuss the different approaches targeting this molecule for the prevention and treatment of age-related cardiovascular and cerebrovascular diseases.

Rho GTPases in Retinal Vascular Diseases.

The Rho family of small GTPases (Rho GTPases) act as molecular switches that transduce extrinsic stimuli into cytoskeletal rearrangements. In vascular endothelial cells (ECs), Cdc42, Rac1, and RhoA control cell migration and cell-cell junctions downstream of angiogenic and inflammatory cytokines, thereby regulating vascular formation and permeability. While these Rho GTPases are broadly expressed in various types of cells, RhoJ is enriched in angiogenic ECs. Semaphorin 3E (Sema3E) releases RhoJ from the intracellular domain of PlexinD1, by which RhoJ induces actin depolymerization through competition with Cdc42 for their common effector proteins. RhoJ further mediates the Sema3E-induced association of PlexinD1 with vascular endothelial growth factor receptor (VEGFR) 2 and the activation of p38. Upon stimulation with VEGF-A, RhoJ facilitates the formation of a holoreceptor complex comprising VEGFR2, PlexinD1, and neuropilin-1, leading to the prevention of VEGFR2 degradation and the maintenance of intracellular signal transduction. These pleiotropic roles of RhoJ are required for directional EC migration in retinal angiogenesis. This review highlights the latest insights regarding Rho GTPases in the field of vascular biology, as it will be informative to consider their potential as targets for the treatment of aberrant angiogenesis and hyperpermeability in retinal vascular diseases.

Pathophysiological functions of Rnd proteins.

Rnd proteins constitute a subfamily of Rho GTPases represented in mammals by Rnd1, Rnd2 and Rnd3. Despite their GTPase structure, their specific feature is the inability to hydrolyse GTP-bound nucleotide. This aspect makes them atypical among Rho GTPases. Rnds are regulated for their expression at the transcriptional or post-transcriptional levels and they are activated through post-translational modifications and interactions with other proteins. Rnd proteins are mainly involved in the regulation of the actin cytoskeleton and cell proliferation. Whereas Rnd3 is ubiquitously expressed, Rnd1 and 2 are tissue-specific. Increasing data has described their important role during development and diseases. Herein, we describe their involvement in physiological and pathological conditions with a focus on the neuronal and vascular systems, and summarize their implications in tumorigenesis.

Physiology and role of PCSK9 in vascular disease: Potential impact of localized PCSK9 in vascular wall.

Proprotein convertase subtilisin/kexin type-9 (PCSK9), a member of the proprotein convertase family, is an important drug target because of its crucial role in lipid metabolism. Emerging evidence suggests a direct role of localized PCSK9 in the pathogenesis of vascular diseases. With this in our consideration, we reviewed PCSK9 physiology with respect to recent development and major studies (clinical and experimental) on PCSK9 functionality in vascular disease. PCSK9 upregulates low-density lipoprotein (LDL)-cholesterol levels by binding to the LDL-receptor (LDLR) and facilitating its lysosomal degradation. PCSK9 gain-of-function mutations have been confirmed as a novel genetic mechanism for familial hypercholesterolemia. Elevated serum PCSK9 levels in patients with vascular diseases may contribute to coronary artery disease, atherosclerosis, cerebrovascular diseases, vasculitis, aortic diseases, and arterial aging pathogenesis. Experimental models of atherosclerosis, arterial aneurysm, and coronary or carotid artery ligation also support PCSK9 contribution to inflammatory response and disease progression, through LDLR-dependent or -independent mechanisms. More recently, several clinical trials have confirmed that anti-PCSK9 monoclonal antibodies can reduce systemic LDL levels, total nonfatal cardiovascular events, and all-cause mortality. Interaction of PCSK9 with other receptor proteins (LDLR-related proteins, cluster of differentiation family members, epithelial Na+ channels, and sortilin) may underlie its roles in vascular disease. Improved understanding of PCSK9 roles and molecular mechanisms in various vascular diseases will facilitate advances in lipid-lowering therapy and disease prevention.

NT5E Genetic Mutation Is a Rare But Important Cause of Intermittent Claudication and Chronic Limb-Threatening Ischemia.

BACKGROUND: NT5Egenetic mutations are known to result in calcification of joints and arteries (CALJA), and worldwide, 14 patients from 7 families have been reported.Methods and Results:A total of 5 patients from 2 independent families with CALJA were found in Japan. Of them, 3 complained of intermittent claudication (IC), and 1 suffered from bilateral chronic limb-threatening ischemia (CLTI). Whole-exome sequencing analysis revealed an identical mutation pattern (c.G3C on the exon 1 start codon) that was unique compared withNT5Emutations reported in other countries. CONCLUSIONS: Vascular specialists need to recognize CALJA as a rare cause of ischemic IC and CLTI.

Biochemical analysis of ectonucleotidases on primary rat vascular smooth muscle cells and in silico investigation of their role in vascular diseases.

Vascular smooth muscle cells (VSMCs) exhibit a high degree of plasticity when they undergo the progression from a normal to a disease condition, which makes them a potential target for evaluating early markers and for the development of new therapies. Purinergic signalling plays a key role in vascular tonus control, ATP being an inductor of vasoconstriction, whereas adenosine mediates a vasodilation effect antagonising the ATP actions. The control of extracellular ATP and adenosine levels is done by ectonucleotidases, which represent a potential target to be evaluated in the progression of cardiovascular diseases. In this study, we analysed the basal activity and expression of the ectonucleotidases in aortic rat VSMCs, and we further performed in silico analysis to determine the expression of those enzymes in conditions that mimicked vascular diseases. Cultured in vitro VSMCs showed a prominent expression of Entpd1 followed by Entpd2 and Nt5e (CD73) and very low levels of Entpd3. Slightly faster AMP hydrolysis was observed when compared to ATP and ADP nucleotides. In silico analysis showed that the ectonucleotidases were modulated after induction of conditions that can lead to vascular diseases such as, hypertensive and hypotensive mice models (Nt5e); exposition to high-fat (Entpd1 and Entpd2) or high-phosphate (Nt5e) diet; mechanical stretch (Entpd1, Entpd2 and Nt5e); and myocardial infarction (Entpd1). Our data show that VSMCs are able to efficiently metabolise the extracellular nucleotides generating adenosine. The modulation of Entpd1, Entdp2 and Nt5e in vascular diseases suggests these ectoenzymes as potential targets or markers to be investigated in future studies.

Underexpression of endothelial nitric oxide synthase leads to more severe pulmonary complex vascular lesions associated with HIV patients / La baja expresión de oxido nítrico sintetasa provoca mayor severidad en las lesiones vasculares complejas asociadas al VIH

Abstract Background: Despite increase in survival of human immunodeficiency virus (HIV) patients due to highly active antiretroviral therapy, non-infectious complications are still prevalent such as presentation of lung vasculopathy, even in asymptomatic patients. Endothelial nitric oxide synthase (eNOS) is necessary to produce nitric oxide that causes pulmonary endothelial vasodilation. Participation of this protein in the pulmonary circulation in HIV patients has not been elucidated. This work studied the presence and expression of eNOS in pulmonary complex vascular lesions associated with HIV (PCVL/HIV). Methods: In lung tissues from patients who died from complications of HIV, we used immunohistochemistry and immune chemiluminescence (imageJ) to determine the different degrees of expression of eNOS in PCVL-HIV in comparison with non-PCVL/HIV. Reagents used were anti-eNOS and an automated system. All data are presented as mean and standard deviation. Differences were analyzed with Wilcoxon; p < 0.05 was accepted as statistically significant. Results: In 57 tissues, the histological evidence of pulmonary vasculopathy was showed as different types (proliferative, obliterative, and plexiform) and severe presentation of vasculopathy than non-PCVL/HIV. A statistically significant decrease of eNOS was observed in all PCVL/HIV tissue samples. Conclusion: eNOS has a relevant role in the pathogenesis of pulmonary vasculopathy in acquired immunodeficiency syndrome patients. It is necessary to determine in the future the participation of eNOS and other mechanisms involved in PCVL/HIV.

Resumen Antecedentes: A pesar del incremento en la sobrevivencia del paciente con virus de inmunodeficiencia humana (VIH) debido al uso del tratamiento antiretroviral altamente efectivo, las complicaciones no infecciosas siguen ocasionando vasculopatía pulmonar, aun en pacientes asintomáticos. La óxido nítrico sintetasa (ONSe) es necesaria para la producción de óxido nítrico la cual provoca vasodilatación pulmonar. La participación de esta proteína en la circulación pulmonar en los pacientes con VIH aún no se ha dilucidado. Este trabajo estudia la presencia y la expresión de ONSe en las lesiones vasculares pulmonares complejas asociadas al VIH (LVPC/VIH). Métodos: En tejidos pulmonares de pacientes que fallecieron por complicaciones del VIH, se utilizó inmunohistoquímica e inmunoquimioluminescencia (imageJ) para determinar los diferentes grados de expresión de la ONSe en LVPC/VIH. Los reactivos utilizados son anti-ONSe en sistema automatizado. Todos los datos son presentados en media y desviación estándar. Las diferencias son analizadas con la prueba de Wilcoxon; se aceptó como estadísticamente significativa una p < 0.05. Resultados: En 57 pacientes, la histología de la vasculopatía pulmonar mostró diferentes tipos (proliferativo, obliterativo y plexiforme) además de varias presentaciones de vasculopatía en tejidos no-LVPC/VIH. Se observó diferencia estadística en la disminución de ONSe en todos los tejidos LVPC/VIH. Conclusiones: La ONSe tiene un papel relevante en la patogénesis de la vasculopatía pulmonar en el VIH. Es necesario determinar en el futuro la participación de ONSe y otros mecanismos involucrados en LVPC/VIH.

Underexpression of endothelial nitric oxide synthase leads to more severe pulmonary complex vascular lesions associated with HIV patients.

Background: Despite increase in survival of human immunodeficiency virus (HIV) patients due to highly active antiretroviral therapy, non-infectious complications are still prevalent such as presentation of lung vasculopathy, even in asymptomatic patients. Endothelial nitric oxide synthase (eNOS) is necessary to produce nitric oxide that causes pulmonary endothelial vasodilation. Participation of this protein in the pulmonary circulation in HIV patients has not been elucidated. This work studied the presence and expression of eNOS in pulmonary complex vascular lesions associated with HIV (PCVL/HIV). Methods: In lung tissues from patients who died from complications of HIV, we used immunohistochemistry and immune chemiluminescence (imageJ) to determine the different degrees of expression of eNOS in PCVL-HIV in comparison with non-PCVL/HIV. Reagents used were anti-eNOS and an automated system. All data are presented as mean and standard deviation. Differences were analyzed with Wilcoxon; p < 0.05 was accepted as statistically significant. Results: In 57 tissues, the histological evidence of pulmonary vasculopathy was showed as different types (proliferative, obliterative, and plexiform) and severe presentation of vasculopathy than non-PCVL/HIV. A statistically significant decrease of eNOS was observed in all PCVL/HIV tissue samples. Conclusion: eNOS has a relevant role in the pathogenesis of pulmonary vasculopathy in acquired immunodeficiency syndrome patients. It is necessary to determine in the future the participation of eNOS and other mechanisms involved in PCVL/HIV.

Antecedentes: A pesar del incremento en la sobrevivencia del paciente con virus de inmunodeficiencia humana (VIH) debido al uso del tratamiento antiretroviral altamente efectivo, las complicaciones no infecciosas siguen ocasionando vasculopatía pulmonar, aun en pacientes asintomáticos. La óxido nítrico sintetasa (ONSe) es necesaria para la producción de óxido nítrico la cual provoca vasodilatación pulmonar. La participación de esta proteína en la circulación pulmonar en los pacientes con VIH aún no se ha dilucidado. Este trabajo estudia la presencia y la expresión de ONSe en las lesiones vasculares pulmonares complejas asociadas al VIH (LVPC/VIH). Métodos: En tejidos pulmonares de pacientes que fallecieron por complicaciones del VIH, se utilizó inmunohistoquímica e inmunoquimioluminescencia (imageJ) para determinar los diferentes grados de expresión de la ONSe en LVPC/VIH. Los reactivos utilizados son anti-ONSe en sistema automatizado. Todos los datos son presentados en media y desviación estándar. Las diferencias son analizadas con la prueba de Wilcoxon; se aceptó como estadísticamente significativa una p < 0.05. Resultados: En 57 pacientes, la histología de la vasculopatía pulmonar mostró diferentes tipos (proliferativo, obliterativo y plexiforme) además de varias presentaciones de vasculopatía en tejidos no-LVPC/VIH. Se observó diferencia estadística en la disminución de ONSe en todos los tejidos LVPC/VIH. Conclusiones: La ONSe tiene un papel relevante en la patogénesis de la vasculopatía pulmonar en el VIH. Es necesario determinar en el futuro la participación de ONSe y otros mecanismos involucrados en LVPC/VIH.

Increased Mortality and Vascular Phenotype in a Knock-In Mouse Model of Retinal Vasculopathy With Cerebral Leukoencephalopathy and Systemic Manifestations.

Background and Purpose- Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) is an autosomal dominant small vessel disease caused by C-terminal frameshift mutations in the TREX1 gene that encodes the major mammalian 3' to 5' DNA exonuclease. RVCL-S is characterized by vasculopathy, especially in densely vascularized organs, progressive retinopathy, cerebral microvascular disease, white matter lesions, and migraine, but the underlying mechanisms are unknown. Methods- Homozygous transgenic RVCL-S knock-in mice expressing a truncated Trex1 (three prime repair exonuclease 1) protein (similar to what is seen in patients) and wild-type littermates, of various age groups, were subjected to (1) a survival analysis, (2) in vivo postocclusive reactive hyperemia and ex vivo Mulvany myograph studies to characterize the microvascular and macrovascular reactivity, and (3) experimental stroke after transient middle cerebral artery occlusion with neurological deficit assessment. Results- The mutant mice show increased mortality starting at midlife (P=0.03 with hazard ratio, 3.14 [95% CI, 1.05-9.39]). The mutants also show a vascular phenotype as evidenced by attenuated postocclusive reactive hyperemia responses (across all age groups; F[1, 65]=5.7, P=0.02) and lower acetylcholine-induced relaxations in aortae (in 20- to 24-month-old mice; RVCL-S knock-in: Emax: 37±8% versus WT: Emax: 65±6%, P=0.01). A vascular phenotype is also suggested by the increased infarct volume seen in 12- to 14-month-old mutant mice at 24 hours after infarct onset (RVCL-S knock-in: 75.4±2.7 mm3 versus WT: 52.9±5.6 mm3, P=0.01). Conclusions- Homozygous RVCL-S knock-in mice show increased mortality, signs of abnormal vascular function, and increased sensitivity to experimental stroke and can be instrumental to investigate the pathology seen in patients with RVCL-S.

The Role of Deubiquitinases in Vascular Diseases.

Deubiquitinases (DUBs) are a major component of the ubiquitin-proteasome system in eukaryotic cells; the system plays a crucial role in many biological processes, such as inflammation, oxidative stress, and cell apoptosis, which are important in vascular biology and pathology. DUBs have drawn significant attention in recent years, and their function is increasingly linked with vascular diseases. In this review, we summarize the indirect and direct evidence for the effects of DUBs on atherosclerosis, abdominal aortic aneurysm, angiogenesis, and hypertension, and point out pathways that could be pursued for investigating DUBs. Intervention in the function of DUBs in vascular diseases has potential therapeutic value.

NADPH oxidase 4 mediates the protective effects of physical activity against obesity-induced vascular dysfunction.

AIMS: Physical activity is one of the most potent strategies to prevent endothelial dysfunction. Recent evidence suggests vaso-protective properties of hydrogen peroxide (H2O2) produced by main endothelial NADPH oxidase isoform 4 (Nox4) in the vasculature. Therefore, we hypothesized that Nox4 connects physical activity with vaso-protective effects. METHODS AND RESULTS: Analysis of the endothelial function using Mulvany Myograph showed endothelial dysfunction in wild-type (WT) as well as in C57BL/6J/ Nox4-/- (Nox4-/-) mice after 20 weeks on high-fat diet (HFD). Access to running wheels during the HFD prevented endothelial dysfunction in WT but not in Nox4-/- mice. Mechanistically, exercise led to an increased H2O2 release in the aorta of WT mice with increased phosphorylation of eNOS pathway member AKT serine/threonine kinase 1 (AKT1). Both H2O2 release and phosphorylation of AKT1 were diminished in aortas of Nox4-/- mice. Deletion of Nox4 also resulted in lower intracellular calcium release proven by reduced phenylephrine-mediated contraction, whilst potassium-induced contraction was not affected. H2O2 scavenger catalase reduced phenylephrine-induced contraction in WT mice. Supplementing H2O2 increased phenylephrine-induced contraction in Nox4-/- mice. Exercise-induced peroxisome proliferative-activated receptor gamma, coactivator 1 alpha (Ppargc1a), as key regulator of mitochondria biogenesis in WT but not Nox4-/- mice. Furthermore, exercise-induced citrate synthase activity and mitochondria mass were reduced in the absence of Nox4. Thus, Nox4-/- mice became less active and ran less compared with WT mice. CONCLUSIONS: Nox4 derived H2O2 plays a key role in exercise-induced adaptations of eNOS and Ppargc1a pathway and intracellular calcium release. Hence, loss of Nox4 diminished physical activity performance and vascular protective effects of exercise.

Emerging role of 12/15-Lipoxygenase (ALOX15) in human pathologies.

12/15-lipoxygenase (12/15-LOX) is an enzyme, which oxidizes polyunsaturated fatty acids, particularly omega-6 and -3 fatty acids, to generate a number of bioactive lipid metabolites. A large number of studies have revealed the importance of 12/15-LOX role in oxidative and inflammatory responses. The in vitro studies have demonstrated the ability of 12/15-LOX metabolites in the expression of various genes and production of cytokine related to inflammation and resolution of inflammation. The studies with the use of knockout and transgenic animals for 12/15-LOX have further shown its involvement in the pathogenesis of a variety of human diseases, including cardiovascular, renal, neurological and metabolic disorders. This review summarizes our current knowledge on the role of 12/15-LOX in inflammation and various human diseases.

Hepatic injury induced by thioacetamide causes aortic endothelial dysfunction by a cyclooxygenase-dependent mechanism.

Liver cirrhosis is associated with a wide range of cardiovascular abnormalities including hyperdynamic circulation and cirrhotic cardiomyopathy. The pathogenic mechanisms of these cardiovascular changes are multifactorial and include vascular dysregulations. AIM: The present study tested the hypothesis that the systemic vascular hyporesponsiveness in thioacetamide (TAA)-induced liver injury model is dependent on nitric oxide (NO) and cyclooxygenase (COX) derivatives. MAIN METHODS: Wistar rats were treated with TAA for eight weeks to induce liver injury. KEY FINDINGS: The maximal contractile response in concentration-effect curves to phenylephrine was decreased in aorta from TAA-treated rats, but no differences were found in aorta without endothelium, suggesting an endothelium-dependent mechanism in decreased contractile response. There was no difference in the contractile response with and without L-NAME (N(ω)-nitro-l-arginine methyl ester) in rats with liver injury, showing that the TAA treatment impairs NO synthesis. Pre-incubation of the aorta with indomethacin, a COX-inhibitor, normalized the reduced contractile response to phenylephrine in arteries from TAA group. Also, COX-2 and iNOS (inducible nitric oxide syntase) protein expression was increased in aorta from TAA group compared to control group. Animals submitted to TAA treatment had a reduction in systolic blood pressure. Our findings demonstrated that liver injury induced by TAA caused a decrease in aortic contractile response by a COX-dependent mechanism but not by NO release. Also, it was demonstrated an inflammatory process in the aorta of TAA-treated rats by increased expression of COX-2 and iNOS. SIGNIFICANCE: Therefore, there is an essential contribution of COX-2 activation in extra-hepatic vascular dysfunction and inflammation present in cirrhosis induced by TAA.

HO-1hi patrolling monocytes protect against vaso-occlusion in sickle cell disease.

Patients with sickle cell disease (SCD) suffer from intravascular hemolysis associated with vascular injury and dysfunction in mouse models, and painful vaso-occlusive crisis (VOC) involving increased attachment of sickle erythrocytes and activated leukocytes to damaged vascular endothelium. Patrolling monocytes, which normally scavenge damaged cells and debris from the vasculature, express higher levels of anti-inflammatory heme oxygenase 1 (HO-1), a heme degrading enzyme. Here, we show that HO-1-expressing patrolling monocytes protect SCD vasculature from ongoing hemolytic insult and vaso-occlusion. We found that a mean 37% of patrolling monocytes from SCD patients express very high levels of HO-1 (HO-1hi) vs 6% in healthy controls and demonstrated that HO-1hi expression was dependent on uptake of heme-exposed endothelium. SCD patients with a recent VOC episode had lower numbers of HO-1hi patrolling monocytes. Heme-mediated vaso-occlusion by mouse SCD red blood cells was exacerbated in mice lacking patrolling monocytes, and reversed following transfer of patrolling monocytes. Altogether, these data indicate that SCD patrolling monocytes remove hemolysis-damaged endothelial cells, resulting in HO-1 upregulation and dampening of VOC, and that perturbation in patrolling monocyte numbers resulting in lower numbers of HO-1hi patrolling monocyte may predispose SCD patients to VOC. These data suggest that HO-1hi patrolling monocytes are key players in VOC pathophysiology and have potential as therapeutic targets for VOC.

CaMKII in Vascular Signalling: "Friend or Foe"?

Signalling mechanisms within and between cells of the vasculature enable function and maintain homeostasis. However, a number of these mechanisms also contribute to the pathophysiology of vascular disease states. The multifunctional signalling molecule calcium/calmodulin-dependent kinase II (CaMKII) has been shown to have critical functional effects in many tissue types. For example, CaMKII is known to have a dual role in cardiac physiology and pathology. The function of CaMKII within the vasculature is incompletely understood, but emerging evidence points to potential physiological and pathological roles. This review discusses the evidence for CaMKII signalling within the vasculature, with the aim to better understand both positive and potentially deleterious effects of CaMKII activation in vascular tissue.

Matrix Metalloproteinases, Vascular Remodeling, and Vascular Disease.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that degrade various proteins in the extracellular matrix (ECM). Typically, MMPs have a propeptide sequence, a catalytic metalloproteinase domain with catalytic zinc, a hinge region or linker peptide, and a hemopexin domain. MMPs are commonly classified on the basis of their substrates and the organization of their structural domains into collagenases, gelatinases, stromelysins, matrilysins, membrane-type (MT)-MMPs, and other MMPs. MMPs are secreted by many cells including fibroblasts, vascular smooth muscle (VSM), and leukocytes. MMPs are regulated at the level of mRNA expression and by activation through removal of the propeptide domain from their latent zymogen form. MMPs are often secreted in an inactive proMMP form, which is cleaved to the active form by various proteinases including other MMPs. MMPs degrade various protein substrates in ECM including collagen and elastin. MMPs could also influence endothelial cell function as well as VSM cell migration, proliferation, Ca2+ signaling, and contraction. MMPs play a role in vascular tissue remodeling during various biological processes such as angiogenesis, embryogenesis, morphogenesis, and wound repair. Alterations in specific MMPs could influence arterial remodeling and lead to various pathological disorders such as hypertension, preeclampsia, atherosclerosis, aneurysm formation, as well as excessive venous dilation and lower extremity venous disease. MMPs are often regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs), and the MMP/TIMP ratio often determines the extent of ECM protein degradation and tissue remodeling. MMPs may serve as biomarkers and potential therapeutic targets for certain vascular disorders.

Nonmuscle Myosin Light Chain Kinase: A Key Player in Intermittent Hypoxia-Induced Vascular Alterations.

BACKGROUND: Obstructive sleep apnea is characterized by repetitive pharyngeal collapses during sleep, leading to intermittent hypoxia (IH), the main contributor of obstructive sleep apnea-related cardiovascular morbidity. In patients and rodents with obstructive sleep apnea exposed to IH, vascular inflammation and remodeling, endothelial dysfunction, and circulating inflammatory markers are linked with IH severity. The nonmuscle myosin light chain kinase (nmMLCK) isoform contributes to vascular inflammation and oxidative stress in different cardiovascular and inflammatory diseases. Thus, in the present study, we hypothesized that nmMLCK plays a key role in the IH-induced vascular dysfunctions and inflammatory remodeling. METHODS AND RESULTS: Twelve-week-old nmMLCK+/+ or nmMLCK-/- mice were exposed to 14-day IH or normoxia. IH was associated with functional alterations characterized by an elevation of arterial blood pressure and stiffness and perturbations of NO signaling. IH caused endothelial barrier dysfunction (ie, reduced transendothelial resistance in vitro) and induced vascular oxidative stress associated with an inflammatory remodeling, characterized by an increased intima-media thickness and an increased expression and activity of inflammatory markers, such as interferon-γ and nuclear factor-κB, in the vascular wall. Interestingly, nmMLCK deletion prevented all IH-induced functional and structural alterations, including the restoration of NO signaling, correction of endothelial barrier integrity, and reduction of both oxidative stress and associated inflammatory response. CONCLUSIONS: nmMLCK is a key mechanism in IH-induced vascular oxidative stress and inflammation and both functional and structural remodeling.

Involvement of bilitranslocase and beta-glucuronidase in the vascular protection by hydroxytyrosol and its glucuronide metabolites in oxidative stress conditions.

Olive oil vascular benefits have been attributed to hydroxytyrosol (HT). However, HT biological actions are still debated because it is extensively metabolized into glucuronides (GCs). The aim of this study was to test HT and GC vasculoprotective effects and the underlying mechanisms using aorta rings from 8-week-old male Wistar rats. In the absence of oxidative stress, incubation with 100 µM HT or GC for 5 min did not exert any vasorelaxing effect and did not influence the vascular function. Conversely, in condition of oxidative stress [upon incubation with 500 µM tert-butylhydroperoxide (t-BHP) for 30 min], preincubation with HT or GC improved acetylcholine-induced vasorelaxation compared with untreated samples (no t-BHP). This protective effect was lost for GC, but not for HT, when a washing step (15 min) was introduced between preincubation with HT or GC and t-BHP addition, suggesting that only HT enters the cells. In agreement, bilitranslocase inhibition with 100 µM phenylmethanesulfonyl fluoride for 20 min reduced significantly HT, but not GC, effect on the vascular function upon stress induction. Moreover, GC protective effect (improvement of endothelium-dependent relaxation in response to acetylcholine) in oxidative stress conditions was reduced by preincubation of aorta rings with 300 µM D-saccharolactone to inhibit ß-glucuronidase, which can deconjugate polyphenols. Finally, only HT was detected by high-pressure liquid chromatography in aorta rings incubated with GC and t-BHP. These results suggest that, in conditions of oxidative stress, GC can be deconjugated into HT that is transported through the cell membrane by bilitranslocase to protect vascular function.

Obesity-induced vascular dysfunction and arterial stiffening requires endothelial cell arginase 1.

AIMS: Elevation of arginase activity has been linked to vascular dysfunction in diabetes and hypertension by a mechanism involving decreased nitric oxide (NO) bioavailability due to L-arginine depletion. Excessive arginase activity also can drive L-arginine metabolism towards the production of ornithine, polyamines, and proline, promoting proliferation of vascular smooth muscle cells and collagen formation, leading to perivascular fibrosis. We hypothesized that there is a specific involvement of arginase 1 expression within the vascular endothelial cells in this pathology. METHODS AND RESULTS: To test this proposition, we used models of type 2 diabetes and metabolic syndrome. Studies were performed using wild type (WT), endothelial-specific arginase 1 knockout (EC-A1-/-) and littermate controls(A1con) mice fed high fat-high sucrose (HFHS) or normal diet (ND) for 6 months and isolated vessels exposed to palmitate-high glucose (PA/HG) media. Some WT mice or isolated vessels were treated with an arginase inhibitor, ABH [2-(S)-amino-6-boronohexanoic acid. In WT mice, the HFHS diet promoted increases in body weight, fasting blood glucose, and post-prandial insulin levels along with arterial stiffening and fibrosis, elevated blood pressure, decreased plasma levels of L-arginine, and elevated L-ornithine. The HFHS diet or PA/HG treatment also induced increases in vascular arginase activity along with oxidative stress, reduced vascular NO levels, and impaired endothelial-dependent vasorelaxation. All of these effects except obesity and hypercholesterolemia were prevented or significantly reduced by endothelial-specific deletion of arginase 1 or ABH treatment. CONCLUSION: Vascular dysfunctions in diet-induced obesity are prevented by deletion of arginase 1 in vascular endothelial cells or arginase inhibition. These findings indicate that upregulation of arginase 1 expression/activity in vascular endothelial cells has an integral role in diet-induced cardiovascular dysfunction and metabolic syndrome.

The emerging role of NADPH oxidase NOX5 in vascular disease.

Oxidative stress is a consequence of up-regulation of pro-oxidant enzyme-induced reactive oxygen species (ROS) production and concomitant depletion of antioxidants. Elevated levels of ROS act as an intermediate and are the common denominator for various diseases including diabetes-associated macro-/micro-vascular complications and hypertension. A range of enzymes are capable of generating ROS, but the pro-oxidant enzyme family, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs), are the only enzymes known to be solely dedicated to ROS generation in the vascular tissues, kidney, aortas and eyes. While there is convincing evidence for a role of NOX1 in vascular and eye disease and for NOX4 in renal injury, the role of NOX5 in disease is less clear. Although NOX5 is highly up-regulated in humans in disease, it is absent in rodents. Thus, so far it has not been possible to study NOX5 in traditional mouse or rat models of disease. In the present review, we summarize and critically analyse the emerging evidence for a pathophysiological role of NOX5 in disease including the expression, regulation and molecular and cellular mechanisms which have been demonstrated to be involved in NOX5 activation.