Near-Infrared II Photobiomodulation Preconditioning Ameliorates Stroke Injury via Phosphorylation of eNOS.

BACKGROUND: The current management of patients with stroke with intravenous thrombolysis and endovascular thrombectomy is effective only when it is timely performed on an appropriately selected but minor fraction of patients. The development of novel adjunctive therapy is highly desired to reduce morbidity and mortality with stroke. Since endothelial dysfunction is implicated in the pathogenesis of stroke and is featured with suppressed endothelial nitric oxide synthase (eNOS) with concomitant nitric oxide deficiency, restoring endothelial nitric oxide represents a promising approach to treating stroke injury. METHODS: This is a preclinical proof-of-concept study to determine the therapeutic effect of transcranial treatment with a low-power near-infrared laser in a mouse model of ischemic stroke. The laser treatment was performed before the middle cerebral artery occlusion with a filament. To determine the involvement of eNOS phosphorylation, unphosphorylatable eNOS S1176A knock-in mice were used. Each measurement was analyzed by a 2-way ANOVA to assess the effect of the treatment on cerebral blood flow with laser Doppler flowmetry, eNOS phosphorylation by immunoblot analysis, and stroke outcomes by infarct volumes and neurological deficits. RESULTS: Pretreatment with a 1064-nm laser at an irradiance of 50 mW/cm2 improved cerebral blood flow, eNOS phosphorylation, and stroke outcomes. CONCLUSIONS: Near-infrared II photobiomodulation could offer a noninvasive and low-risk adjunctive therapy for stroke injury. This new modality using a physical parameter merits further consideration to develop innovative therapies to prevent and treat a wide array of cardiovascular diseases.

Near-infrared II photobiomodulation augments nitric oxide bioavailability via phosphorylation of endothelial nitric oxide synthase.

There is solid evidence of the beneficial effect of photobiomodulation (PBM) with low-power near-infrared (NIR) light in the NIR-I window in increasing bioavailable nitric oxide (NO). However, it is not established whether this effect can be extended to NIR-II light, limiting broader applications of this therapeutic modality. Since we have demonstrated PBM with NIR laser in the NIR-II window, we determined the causal relationship between NIR-II irradiation and its specific biological effects on NO bioavailability. We analyzed the impact of NIR-II irradiation on NO release in cultured human endothelial cells using a NO-sensitive fluorescence probe and single-cell live imaging. Two distinct wavelengths of NIR-II laser (1064 and 1270 nm) and NIR-I (808 nm) at an irradiance of 10 mW/cm2 induced NO release from endothelial cells. These lasers also enhanced Akt phosphorylation at Ser 473, endothelial nitric oxide synthase (eNOS) phosphorylation at Ser 1177, and endothelial cell migration. Moreover, the NO release and phosphorylation of eNOS were abolished by inhibiting mitochondrial respiration, suggesting that Akt activation caused by NIR-II laser exposure involves mitochondrial retrograde signaling. Other inhibitors that inhibit known Akt activation pathways, including a specific inhibitor of PI3K, Src family PKC, did not affect this response. These two wavelengths of NIR-II laser induced no appreciable NO generation in cultured neuronal cells expressing neuronal NOS (nNOS). In short, NIR-II laser enhances bioavailable NO in endothelial cells. Since a hallmark of endothelial dysfunction is suppressed eNOS with concomitant NO deficiency, NIR-II laser technology could be broadly used to restore endothelial NO and treat or prevent cardiovascular diseases.

Photobiomodulation and nitric oxide signaling.

Nitric oxide (NO) is a well-known gaseous mediator that maintains vascular homeostasis. Extensive evidence supports that a hallmark of endothelial dysfunction, which leads to cardiovascular diseases, is endothelial NO deficiency. Thus, restoring endothelial NO represents a promising approach to treating cardiovascular complications. Despite many therapeutic agents having been shown to augment NO bioavailability under various pathological conditions, success in resulting clinical trials has remained elusive. There is solid evidence of diverse beneficial effects of the treatment with low-power near-infrared (NIR) light, defined as photobiomodulation (PBM). Although the precise mechanisms of action of PBM are still elusive, recent studies consistently report that PBM improves endothelial dysfunction via increasing bioavailable NO in a dose-dependent manner and open a feasible path to the use of PBM for treating cardiovascular diseases via augmenting NO bioavailability. In particular, the use of NIR light in the NIR-II window (1000-1700 nm) for PBM, which has reduced scattering and minimal tissue absorption with the largest penetration depth, is emerging as a promising therapy. In this review, we update recent findings on PBM and NO.

Involvement of Endothelial Nitric Oxide Synthase in Cerebral Microcirculation and Oxygenation in Traumatic Brain Injury.

Traumatic brain injury (TBI) leads to cerebral microvascular dysfunction and cerebral ischemia. Endothelial nitric oxide synthase (eNOS) is a key regulator of vascular homeostasis. We aimed to assess the role of eNOS in cerebral blood flow (CBF) changes after TBI. Moderate TBI was induced in eNOS knockout (KO) and wild-type (WT) mice (8 per group). Cerebral microvascular tone, microvascular CBF (mCBF) and tissue oxygenation (NADH) were measured by two-photon laser scanning microscopy (2PLSM) before and 1 h, 1 day and 3 days after TBI. Cerebrovascular reactivity (CVR) was evaluated by the hypercapnia test. Laser Doppler cortical flux (cLDF) was simultaneously measured in the perilesional area. One hr after TBI, cLDF was 59.4 ± 8.2% and 60.3 ± 9.1% from the baseline (p < 0.05) in WT and eNOS KO, respectively. 2PLSM showed decreased arteriolar diameter, the number of functioning capillaries, mCBF and tissue oxygenation (p < 0.05). At 1 day, cLDF increased to 65.2 ± 6.4% in the WT group, while it decreased to 56.1 ± 7.2% in the eNOS KO mice. 2PLSM revealed a further decrease in the number of functioning capillaries, mCBF, and oxygen supply which was slightly milder in WT mice (p < 0.05 from the baseline). On the third day after TBI, cLDF increased to 72 ± 5.2% in the WT, while it stayed the same in the eNOS KO group (55.9 ± 6.4%, p < 0.05 from the WT). 2PLSM showed reduction in arterioles with vasospasm, increase in the number of functioning capillaries, and improvement in mCBF and tissue oxygen supply in WT, while no significant changes were observed in eNOS KO (p < 0.05). CVR was impaired in both groups 1 h after TBI, and improved by the third day in the WT, while staying impaired in eNOS KO. In the subacute TBI period, the significance of eNOS in maintaining cerebral microcirculation and oxygen supply increases with time after the injury.

Discovery of novel heart rate-associated loci using the Exome Chip.

Resting heart rate is a heritable trait, and an increase in heart rate is associated with increased mortality risk. Genome-wide association study analyses have found loci associated with resting heart rate, at the time of our study these loci explained 0.9% of the variation. This study aims to discover new genetic loci associated with heart rate from Exome Chip meta-analyses.Heart rate was measured from either elecrtrocardiograms or pulse recordings. We meta-analysed heart rate association results from 104 452 European-ancestry individuals from 30 cohorts, genotyped using the Exome Chip. Twenty-four variants were selected for follow-up in an independent dataset (UK Biobank, N = 134 251). Conditional and gene-based testing was undertaken, and variants were investigated with bioinformatics methods.We discovered five novel heart rate loci, and one new independent low-frequency non-synonymous variant in an established heart rate locus (KIAA1755). Lead variants in four of the novel loci are non-synonymous variants in the genes C10orf71, DALDR3, TESK2 and SEC31B. The variant at SEC31B is significantly associated with SEC31B expression in heart and tibial nerve tissue. Further candidate genes were detected from long-range regulatory chromatin interactions in heart tissue (SCD, SLF2 and MAPK8). We observed significant enrichment in DNase I hypersensitive sites in fetal heart and lung. Moreover, enrichment was seen for the first time in human neuronal progenitor cells (derived from embryonic stem cells) and fetal muscle samples by including our novel variants.Our findings advance the knowledge of the genetic architecture of heart rate, and indicate new candidate genes for follow-up functional studies.

Inhibition of Marfan Syndrome Aortic Root Dilation by Losartan: Role of Angiotensin II Receptor Type 1-Independent Activation of Endothelial Function.

Marfan syndrome (MFS) is a genetic disorder that frequently leads to aortic root dissection and aneurysm. Despite promising preclinical and pilot clinical data, a recent large-scale study using antihypertensive angiotensin II (AngII) receptor type 1 (ATR1) blocker losartan has failed to meet expectations at preventing MFS-associated aortic root dilation, casting doubts about optimal therapy. To study the deleterious role of normal ATR1 signaling in aortic root widening, we generated MFS mice lacking ATR1a expression in an attempt to preserve protective ATR2 signaling. Despite being hypotensive and resistant to AngII vasopressor effects, MFS/ATR1a-null mice showed unabated aortic root enlargement and remained fully responsive to losartan, confirming that blood pressure lowering is of minor therapeutic value in MFS and that losartan's antiremodeling properties may be ATR1 independent. Having shown that MFS causes endothelial dysfunction and that losartan can activate endothelial function in mice and patients, we found that nitric oxide synthase (NOS) inhibition renders losartan therapeutically inactive, whereas multiple transgenic and pharmacologic models of endothelial NOS activation block aortic root dilation by correcting extracellular signal-regulated kinase signaling. In vitro, losartan can increase endothelial NO release in the absence of AngII and correct MFS NO levels in vivo. Our data suggest that increased protective endothelial function, rather than ATR1 inhibition or blood pressure lowering, might be of therapeutic significance in preventing aortic root disease in MFS.

Genetic Evidence That Carbohydrate-Stimulated Insulin Secretion Leads to Obesity.

BACKGROUND: A fundamental precept of the carbohydrate-insulin model of obesity is that insulin secretion drives weight gain. However, fasting hyperinsulinemia can also be driven by obesity-induced insulin resistance. We used genetic variation to isolate and estimate the potentially causal effect of insulin secretion on body weight. METHODS: Genetic instruments of variation of insulin secretion [assessed as insulin concentration 30 min after oral glucose (insulin-30)] were used to estimate the causal relationship between increased insulin secretion and body mass index (BMI), using bidirectional Mendelian randomization analysis of genome-wide association studies. Data sources included summary results from the largest published metaanalyses of predominantly European ancestry for insulin secretion (n = 26037) and BMI (n = 322154), as well as individual-level data from the UK Biobank (n = 138541). Data from the Cardiology and Metabolic Patient Cohort study at Massachusetts General Hospital (n = 1675) were used to validate genetic associations with insulin secretion and to test the observational association of insulin secretion and BMI. RESULTS: Higher genetically determined insulin-30 was strongly associated with higher BMI (ß = 0.098, P = 2.2 × 10-21), consistent with a causal role in obesity. Similar positive associations were noted in sensitivity analyses using other genetic variants as instrumental variables. By contrast, higher genetically determined BMI was not associated with insulin-30. CONCLUSIONS: Mendelian randomization analyses provide evidence for a causal relationship of glucose-stimulated insulin secretion on body weight, consistent with the carbohydrate-insulin model of obesity.

Loss of nNOS inhibits compensatory muscle hypertrophy and exacerbates inflammation and eccentric contraction-induced damage in mdx mice.

Approaches targeting nitric oxide (NO) signaling show promise as therapies for Duchenne and Becker muscular dystrophies. However, the mechanisms by which NO benefits dystrophin-deficient muscle remain unclear, but may involve nNOSß, a newly discovered enzymatic source of NO in skeletal muscle. Here we investigate the impact of dystrophin deficiency on nNOSß and use mdx mice engineered to lack nNOSµ and nNOSß to discern how the loss of nNOS impacts dystrophic skeletal muscle pathology. In mdx muscle, nNOSß was mislocalized and its association with the Golgi complex was reduced. nNOS depletion from mdx mice prevented compensatory skeletal muscle cell hypertrophy, decreased myofiber central nucleation and increased focal macrophage cell infiltration, indicating exacerbated dystrophic muscle damage. Reductions in muscle integrity in nNOS-null mdx mice were accompanied by decreases in specific force and increased susceptibility to eccentric contraction-induced muscle damage compared with mdx controls. Unexpectedly, muscle fatigue was unaffected by nNOS depletion, revealing a novel latent compensatory mechanism for the loss of nNOS in mdx mice. Together with previous studies, these data suggest that localization of both nNOSµ and nNOSß is disrupted by dystrophin deficiency. They also indicate that nNOS has a more complex role as a modifier of dystrophic pathology and broader therapeutic potential than previously recognized. Importantly, these findings also suggest nNOSß as a new drug target and provide a new conceptual framework for understanding nNOS signaling and the benefits of NO therapies in dystrophinopathies.

Hydrogen sulfide cytoprotective signaling is endothelial nitric oxide synthase-nitric oxide dependent.

Previous studies have demonstrated that hydrogen sulfide (H2S) protects against multiple cardiovascular disease states in a similar manner as nitric oxide (NO). H2S therapy also has been shown to augment NO bioavailability and signaling. The purpose of this study was to investigate the impact of H2S deficiency on endothelial NO synthase (eNOS) function, NO production, and ischemia/reperfusion (I/R) injury. We found that mice lacking the H2S-producing enzyme cystathionine γ-lyase (CSE) exhibit elevated oxidative stress, dysfunctional eNOS, diminished NO levels, and exacerbated myocardial and hepatic I/R injury. In CSE KO mice, acute H2S therapy restored eNOS function and NO bioavailability and attenuated I/R injury. In addition, we found that H2S therapy fails to protect against I/R in eNOS phosphomutant mice (S1179A). Our results suggest that H2S-mediated cytoprotective signaling in the setting of I/R injury is dependent in large part on eNOS activation and NO generation.

Macula Densa Nitric Oxide Synthase 1ß Protects against Salt-Sensitive Hypertension.

Nitric oxide (NO) is an important negative modulator of tubuloglomerular feedback responsiveness. We recently found that macula densa expresses α-, ß-, and γ-splice variants of neuronal nitric oxide synthase 1 (NOS1), and NOS1ß expression in the macula densa increases on a high-salt diet. This study tested whether upregulation of NOS1ß expression in the macula densa affects sodium excretion and salt-sensitive hypertension by decreasing tubuloglomerular feedback responsiveness. Expression levels of NOS1ß mRNA and protein were 30- and five-fold higher, respectively, than those of NOS1α in the renal cortex of C57BL/6 mice. Furthermore, macula densa NO production was similar in the isolated perfused juxtaglomerular apparatus of wild-type (WT) and nitric oxide synthase 1α-knockout (NOS1αKO) mice. Compared with control mice, mice with macula densa-specific knockout of all nitric oxide synthase 1 isoforms (MD-NOS1KO) had a significantly enhanced tubuloglomerular feedback response and after acute volume expansion, significantly reduced GFR, urine flow, and sodium excretion. Mean arterial pressure increased significantly in MD-NOS1KO mice (P<0.01) but not NOS1flox/flox mice fed a high-salt diet. After infusion of angiotensin II, mean arterial pressure increased by 61.6 mmHg in MD-NOS1KO mice versus 32.0 mmHg in WT mice (P<0.01) fed a high-salt diet. These results indicate that NOS1ß is a primary NOS1 isoform expressed in the macula densa and regulates the tubuloglomerular feedback response, the natriuretic response to acute volume expansion, and the development of salt-sensitive hypertension. These findings show a novel mechanism for salt sensitivity of BP and the significance of tubuloglomerular feedback response in long-term control of sodium excretion and BP.

Endothelial dysfunction abrogates the efficacy of normobaric hyperoxia in stroke.

Hyperoxia has been uniformly efficacious in experimental focal cerebral ischemia. However, pilot clinical trials have showed mixed results slowing its translation in patient care. To explain the discordance between experimental and clinical outcomes, we tested the impact of endothelial dysfunction, exceedingly common in stroke patients but under-represented in experimental studies, on the neuroprotective efficacy of normobaric hyperoxia. We used hyperlipidemic apolipoprotein E knock-out and endothelial nitric oxide synthase knock-out mice as models of endothelial dysfunction, and examined the effects of normobaric hyperoxia on tissue perfusion and oxygenation using high-resolution combined laser speckle and multispectral reflectance imaging during distal middle cerebral artery occlusion. In normal wild-type mice, normobaric hyperoxia rapidly and significantly improved tissue perfusion and oxygenation, suppressed peri-infarct depolarizations, reduced infarct volumes, and improved neurological function. In contrast, normobaric hyperoxia worsened perfusion in ischemic brain and failed to reduce infarct volumes or improve neurological function in mice with endothelial dysfunction. These data suggest that the beneficial effects of hyperoxia on ischemic tissue oxygenation, perfusion, and outcome are critically dependent on endothelial nitric oxide synthase function. Therefore, vascular risk factors associated with endothelial dysfunction may predict normobaric hyperoxia nonresponders in ischemic stroke. These data may have implications for myocardial and systemic circulation as well.

eNOS-derived nitric oxide regulates endothelial barrier function through VE-cadherin and Rho GTPases.

Transient disruption of endothelial adherens junctions and cytoskeletal remodeling are responsible for increases in vascular permeability induced by inflammatory stimuli and vascular endothelial growth factor (VEGF). Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is crucial for VEGF-induced changes in permeability in vivo; however, the molecular mechanism by which endogenous NO modulates endothelial permeability is not clear. Here, we show that the lack of eNOS reduces VEGF-induced permeability, an effect mediated by enhanced activation of the Rac GTPase and stabilization of cortical actin. The loss of NO increased the recruitment of the Rac guanine-nucleotide-exchange factor (GEF) TIAM1 to adherens junctions and VE-cadherin (also known as cadherin 5), and reduced Rho activation and stress fiber formation. In addition, NO deficiency reduced VEGF-induced VE-cadherin phosphorylation and impaired the localization, but not the activation, of c-Src to cell junctions. The physiological role of eNOS activation is clear given that VEGF-, histamine- and inflammation-induced vascular permeability is reduced in mice bearing a non-phosphorylatable knock-in mutation of the key eNOS phosphorylation site S1176. Thus, NO is crucial for Rho GTPase-dependent regulation of cytoskeletal architecture leading to reversible changes in vascular permeability.

Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages.

The generation and expansion of diverse cardiovascular cell lineages is a critical step during human cardiogenesis, with major implications for congenital heart disease. Unravelling the mechanisms for the diversification of human heart cell lineages has been hampered by the lack of genetic tools to purify early cardiac progenitors and define their developmental potential. Recent studies in the mouse embryo have identified a multipotent cardiac progenitor that contributes to all of the major cell types in the murine heart. In contrast to murine development, human cardiogenesis has a much longer onset of heart cell lineage diversification and expansion, suggesting divergent pathways. Here we identify a diverse set of human fetal ISL1(+) cardiovascular progenitors that give rise to the cardiomyocyte, smooth muscle and endothelial cell lineages. Using two independent transgenic and gene-targeting approaches in human embryonic stem cell lines, we show that purified ISL1(+) primordial progenitors are capable of self-renewal and expansion before differentiation into the three major cell types in the heart. These results lay the foundation for the generation of human model systems for cardiovascular disease and novel approaches for human regenerative cardiovascular medicine.

Endothelial nitric oxide synthase controls the expression of the angiogenesis inhibitor thrombospondin 2.

Injury- and ischemia-induced angiogenesis is critical for tissue repair and requires nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS). We present evidence that NO induces angiogenesis by modulating the level of the angiogenesis inhibitor thrombospondin 2 (TSP2). TSP2 levels were higher than WT in eNOS KO tissues in hind-limb ischemia and cutaneous wounds. In vitro studies confirmed that NO represses TSP2 promoter activity. Moreover, double-eNOS/TSP2 KO mice were generated and found to rescue the phenotype of eNOS KO mice. Studies in mice with knock-in constitutively active or inactive eNOS on the Akt-1 KO background showed that eNOS activity correlates with TSP2 levels. Our observations of NO-mediated regulation of angiogenesis via the suppression of TSP2 expression provide a description of improved eNOS KO phenotype by means other than restoring NO signaling.

Deficient eNOS phosphorylation is a mechanism for diabetic vascular dysfunction contributing to increased stroke size.

BACKGROUND AND PURPOSE: Phosphorylation of eNOS, an important post-translational modulator of its enzymatic activity, is reduced in diabetes mellitus. We hypothesized that modulation of eNOS phosphorylation could overcome diabetic vascular dysfunction and improves the outcome to stroke. METHODS: We used the db/db mouse model of type 2 diabetes mellitus. We mated db/db mice with eNOS knock-in mice that carry single amino acid mutations at the S1176 phosphorylation site; the phosphomimetic SD mutation (serine replaced by aspartate) shows increased eNOS enzymatic activity, whereas the unphosphorylatable SA mutation (serine replaced by alanine) shows decreased eNOS activity. We characterized the vascular anatomy, baseline physiological parameters, and vascular reactivity. We used the middle cerebral artery occlusion model of stroke and measured infarct volume and neurological deficits. RESULTS: db/db mice showed diminished eNOS phosphorylation at S1176. eNOS SD and SA mutations do not change the vascular anatomy at the Circle of Willis, brain capillary density, heart rate, or arterial blood gases of db/db mice. The eNOS SD mutation, but not the SA mutation, lowers blood pressure and improves vascular reactivity to acetylcholine in db/db mice. The eNOS SD mutation reduces stroke size and neurological deficit after middle cerebral artery occlusion. CONCLUSIONS: Diminished eNOS phosphorylation is a mechanism of vascular dysfunction in db/db mice. We show here that modulation of the eNOS S1176 phosphorylation site in db/db mice is associated with improved vascular reactivity and improved outcome to stroke after middle cerebral artery occlusion.

eNOS phosphorylation on serine 1176 affects insulin sensitivity and adiposity.

Phosphorylation of endothelial nitric oxide synthase (eNOS) is an important regulator of its enzymatic activity. We generated knockin mice expressing phosphomimetic (SD) and unphosphorylatable (SA) eNOS mutations at S1176 to study the role of eNOS phosphorylation. The single amino acid SA mutation is associated with hypertension and decreased vascular reactivity, while the SD mutation results in increased basal and stimulated endothelial NO production. In addition to these vascular effects, modulation of the S1176 phosphorylation site resulted in unanticipated effects on metabolism. The eNOS SA mutation results in insulin resistance, hyperinsulinemia, adiposity, and increased weight gain on high fat. In contrast, the eNOS SD mutation is associated with decreased insulin levels and resistance to high fat-induced weight gain. These results demonstrate the importance of eNOS in regulation of insulin sensitivity, energy metabolism, and bodyweight regulation, and suggest eNOS phosphorylation as a novel target for the treatment of obesity and insulin resistance.

The ribosome-inactivating proteins MAP30 and Momordin inhibit SARS-CoV-2.

The continuing emergence of SARS-CoV-2 variants has highlighted the need to identify additional points for viral inhibition. Ribosome inactivating proteins (RIPs), such as MAP30 and Momordin which are derived from bitter melon (Momordica charantia), have been found to inhibit a broad range of viruses. MAP30 has been shown to potently inhibit HIV-1 with minimal cytotoxicity. Here we show that MAP30 and Momordin potently inhibit SARS-CoV-2 replication in A549 human lung cells (IC50 ~ 0.2 µM) with little concomitant cytotoxicity (CC50 ~ 2 µM). Both viral inhibition and cytotoxicity remain unaltered by appending a C-terminal Tat cell-penetration peptide to either protein. Mutation of tyrosine 70, a key residue in the active site of MAP30, to alanine completely abrogates both viral inhibition and cytotoxicity, indicating the involvement of its RNA N-glycosylase activity. Mutation of lysine 171 and lysine 215, residues corresponding to those in Ricin which when mutated prevented ribosome binding and inactivation, to alanine in MAP30 decreased cytotoxicity (CC50 ~ 10 µM) but also the viral inhibition (IC50 ~ 1 µM). Unlike with HIV-1, neither Dexamethasone nor Indomethacin exhibited synergy with MAP30 in the inhibition of SARS-CoV-2. From a structural comparison of the two proteins, one can explain their similar activities despite differences in both their active-sites and ribosome-binding regions. We also note points on the viral genome for potential inhibition by these proteins.

Rhesus macaques develop metabolic syndrome with reversible vascular dysfunction responsive to pioglitazone.

BACKGROUND: The metabolic syndrome (MetS) is a constellation of clinical features that include central obesity, hypertension, atherogenic dyslipidemia, and insulin resistance. However, the concept remains controversial; it has been debated whether MetS represents nothing more than simultaneous co-occurrence of individual risk factors or whether there are common shared pathophysiological mechanisms that link the individual components. METHODS AND RESULTS: To investigate the emergence of metabolic and cardiovascular components during the development of MetS, we identified MetS-predisposed animals (n=35) in a large population of rhesus macaques (Macaca mulatta, 12.7±2.9 years old, n=408), acclimated them to standardized conditions, and monitored the progression of individual component features over 18 months. In 18 MetS animals with recently developed fasting hyperinsulinemia, central obesity, hypertension, and atherogenic dyslipidemia, we found that individual metabolic and cardiovascular components track together during the transition from pre-MetS to onset of MetS; MetS was associated with a 60% impairment of flow-mediated dilation, establishing the mechanistic link with vascular dysfunction. Pioglitazone treatment (3 mg/kg body weight/d for 6 weeks), a peroxisome proliferator-activated receptor γ agonist, reversibly improved atherogenic dyslipidemia and insulin resistance and fully restored flow-mediated dilation with persistent benefits. CONCLUSIONS: Coemergence of metabolic and cardiovascular components during MetS progression and complete normalization of vascular dysfunction with peroxisome proliferator-activated receptor γ agonists suggest shared underlying mechanisms rather than separate processes, arguing for the benefit of early intervention of MetS components. Predictive nonhuman primate (NHP) models of MetS should be highly valuable in mechanistic and translational studies on the pathogenesis of MetS in relation to cardiovascular disease and diabetes mellitus.

Aldosterone regulates vascular gene transcription via oxidative stress-dependent and -independent pathways.

OBJECTIVE: Aldosterone (Aldo) antagonism prevents cardiovascular mortality by unclear mechanisms. Aldo binds to the mineralocorticoid receptor (MR), a ligand-activated transcription factor, which is expressed in human vascular cells. Here we define the early Aldo-regulated vascular transcriptome and investigate the mechanisms of gene regulation by Aldo in the vasculature that may contribute to vascular disease. METHODS AND RESULTS: Gene expression profiling of Aldo-treated mouse aortas identified 72 genes regulated by Aldo. These genes are overrepresented in Gene Ontology categories involved in vascular function and disease. Quantitative reverse transcription-polymerase chain reaction was used to confirm and further explore mechanisms of vascular gene regulation by Aldo. Aldo-regulated vascular gene expression was inhibited by actinomycin D and MR antagonists supporting a transcriptional MR-dependent mechanism. Aldo regulation of a subset of genes was enhanced in the setting of vascular endothelial denudation and blocked by the free radical scavenger Tempol, supporting synergy between Aldo and vascular injury that is oxidative stress dependent. In the aortic arch, a region predisposed to atherosclerosis, the injury-enhanced genes also demonstrated enhanced expression compared with the descending aorta, both at baseline and after Aldo exposure. Furthermore, the clinically beneficial MR antagonist spironolactone inhibited expression of the identified genes in aortic tissue from humans with atherosclerosis. CONCLUSIONS: This study defines the Aldo-regulated vascular transcriptome and characterizes a subset of proatherogenic genes with enhanced Aldo-stimulated, oxidative stress-dependent expression in the setting of vascular injury and in areas predisposed to atherosclerosis. Inhibition of MR regulation of these genes may play a role in the protective effects of Aldo antagonists in patients with vascular disease, and these pathways may provide novel drug targets to prevent atherosclerosis in humans.