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This review highlights the gaps in knowledge and methodological challenges discussed during the Experimental Biology 2019 expert panel session titled "Moving the Needle on Hypertension: What Knowledge Is Needed?" Hypertension is a critical public health burden. Despite a demonstrated benefit of blood pressure reduction on measures of hypertension-related morbidity and mortality, rates for successful blood pressure control remain low. Dietary sodium reduction has been shown to reduce both systolic blood pressure by approximately 3.2 mm Hg and diastolic blood pressure by 2.3 mm Hg, depending on baseline blood pressure and degree of sodium reduction. The updated Dietary Reference Intakes for adults released by the National Academies of Sciences, Engineering, and Medicine include a Chronic Disease Risk Reduction sodium intake level of 2300 mg/d, highlighting the importance of dietary sodium intake in reducing elevated blood pressure and indicating that reducing intakes to this level is expected to reduce blood pressure and risk of cardiovascular disease. The average US daily sodium intake of 3400 mg/d is well above the Chronic Disease Risk Reduction of 2300 mg/d, suggesting that dietary sodium reduction has the potential to significantly improve public health. Although the National Academies of Sciences, Engineering, and Medicine report presents intake recommendations based on a systematic, comprehensive, and thorough evaluation of the evidence, several challenges to moving the needle on hypertension remain. Success will require a more advanced understanding of sodium and potassium physiology, as well as development of the tools needed to effectively address existing research gaps and reduce barriers to sodium intake reduction.

The mechanisms that sense alterations in total body sodium content to facilitate sodium homeostasis in response to an acute sodium challenge that does not increase blood pressure have not been fully elucidated. We hypothesized that the renal sympathetic nerves are critical to mediate natriuresis via α1- or β-adrenoceptors signal transduction pathways to maintain sodium balance in the face of acute increases in total body sodium content that do not activate the pressure-natriuresis mechanism. To address this hypothesis, we used acute bilateral renal denervation (RDNX), an anteroventral third ventricle (AV3V) lesion and α1- or β-antagonism during an acute 1M NaCl sodium challenge in conscious male Sprague Dawley rats. An acute 1M NaCl infusion did not alter blood pressure and evoked profound natriuresis and sympathoinhibition. Acute bilateral RDNX attenuated the natriuretic and sympathoinhibitory responses evoked by a 1M NaCl infusion [peak natriuresis (μeq/min) sham 14.5 ± 1.3 vs. acute RDNX: 9.2 ± 1.4, p < 0.05; plasma NE (nmol/L) sham control: 44 ± 4 vs. sham 1M NaCl infusion 11 ± 2, p < 0.05; acute RDNX control: 42 ± 6 vs. acute RDNX 1M NaCl infusion 25 ± 3, p < 0.05]. In contrast, an AV3V lesion did not impact the cardiovascular, renal excretory or sympathoinhibitory responses to an acute 1M NaCl infusion. Acute i.v. α1-adrenoceptor antagonism with terazosin evoked a significant drop in baseline blood pressure and significantly attenuated the natriuretic response to a 1M NaCl load [peak natriuresis (μeq/min) saline 17.2 ± 1.4 vs. i.v. terazosin 7.8 ± 2.5, p < 0.05]. In contrast, acute β-adrenoceptor antagonism with i.v. propranolol infusion did not impact the cardiovascular or renal excretory responses to an acute 1M NaCl infusion. Critically, the natriuretic response to an acute 1M NaCl infusion was significantly blunted in rats receiving a s.c. infusion of the α1-adrenoceptor antagonist terazosin at a dose that did not lower baseline blood pressure [peak natriuresis (μeq/min) sc saline: 18 ± 1 vs. sc terazosin 7 ± 2, p < 0.05]. Additionally, a s.c. infusion of the α1-adrenoceptor antagonist terazosin further attenuated the natriuretic response to a 1M NaCl infusion in acutely RDNX animals. Collectively these data indicate a specific role of a blood pressure-independent renal sympathetic nerve-dependent α1-adrenoceptor-mediated pathway in the natriuretic and sympathoinhibitory responses evoked by acute increases in total body sodium.

New findings • What is the central question of this study? We hypothesized that central inflammatory processes that involve activation of microglia and astrocytes contribute to the development of Gαi2 protein-dependent, salt-sensitive hypertension. • What is the main finding and its importance? The main finding is that PVN-specific inflammatory processes, driven by microglial activation, appear to be linked to the development of Gαi2 protein-dependent, salt-sensitive hypertension in Sprague-Dawley rats. This finding might reveal new mechanistic targets in the treatment of hypertension. Abstract The central mechanisms underlying salt-sensitive hypertension, a significant public health issue, remain to be established. Researchers in our laboratory have reported that hypothalamic paraventricular nucleus (PVN) Gαi2 proteins mediate the sympathoinhibitory and normotensive responses to high sodium intake in salt-resistant rats. Given the recent evidence of central inflammation in animal models of hypertension, we hypothesized that PVN inflammation contributes to Gαi2 protein-dependent, salt-sensitive hypertension. Male Sprague-Dawley rats received chronic intracerebroventricular infusions of a targeted Gαi2 or control scrambled oligodeoxynucleotide (ODN) and were maintained for 7 days on a normal-salt (NS; 0.6% NaCl) or high-salt (HS; 4% NaCl) diet; in subgroups on HS, intracerebroventricular minocycline (microglial inhibitor) was co-infused with ODNs. Radiotelemetry was used in subgroups of rats to measure mean arterial pressure (MAP) chronically. In a separate group of rats, plasma noradrenaline, plasma renin activity, urinary angiotensinogen and mRNA levels of the PVN pro-inflammatory cytokines TNFα, IL-1β and IL-6 and the anti-inflammatory cytokine IL-10 were assessed. In additional groups, immunohistochemistry was performed for markers of PVN and subfornical organ microglial activation and cytokine levels and PVN astrocyte activation. High salt intake evoked salt-sensitive hypertension, increased plasma noradrenaline, PVN pro-inflammatory cytokine mRNA upregulation, anti-inflammatory cytokine mRNA downregulation and PVN-specific microglial activation in rats receiving a targeted Gαi2 but not scrambled ODN. Minocycline co-infusion significantly attenuated the increase in MAP and abolished the increase in plasma noradrenaline and inflammation in Gαi2 ODN-infused animals on HS. Our data suggest that central Gαi2 protein prevents microglial-mediated PVN inflammation and the development of salt-sensitive hypertension.

NEW FINDINGS What is the central question of this study? What are the differential roles of the mechanosensitive and chemosensitive afferent renal nerves in the reno-renal reflex that promotes natriuresis, sympathoinhibition and normotension during acute and chronic challenges to sodium homeostasis? What is the main finding and its importance? The mechanosensitive afferent renal nerves contribute to an acute natriuretic sympathoinhibitory reno-renal reflex that may be integrated within the paraventricular nucleus of the hypothalamus. Critically, the afferent renal nerves are required for the maintenance of salt resistance in Sprague-Dawley and Dahl salt-resistant rats and attenuate the development of Dahl salt-sensitive hypertension. ABSTRACT These studies tested the hypothesis that in normotensive salt-resistant rat phenotypes the mechanosensitive afferent renal nerve (ARN) reno-renal reflex promotes natriuresis, sympathoinhibition and normotension during acute and chronic challenges to fluid and electrolyte homeostasis. Selective ARN ablation was conducted prior to (1) an acute isotonic volume expansion (VE) or 1 m NaCl infusion in Sprague-Dawley (SD) rats and (2) chronic high salt intake in SD, Dahl salt-resistant (DSR), and Dahl salt-sensitive (DSS) rats. ARN responsiveness following high salt intake was assessed ex vivo in response to noradrenaline and sodium concentration (SD, DSR and DSS) and via in vivo manipulation of renal pelvic pressure and sodium concentration (SD and DSS). ARN ablation attenuated the natriuretic and sympathoinhibitory responses to an acute VE [peak natriuresis (µeq min-1 ) sham 52 ± 5 vs. ARN ablation 28 ± 3, P

Increased sympathoexcitation and renal sodium retention during high salt intake are hallmarks of the salt sensitivity of blood pressure. The mechanism(s) by which excessive sympathetic nervous system release of norepinephrine influences renal sodium reabsorption is unclear. However, studies demonstrate that norepinephrine can stimulate the activity of the NCC (sodium chloride cotransporter) and promote the development of SSH (salt-sensitive hypertension). The adrenergic signaling pathways governing NCC activity remain a significant source of controversy with opposing studies suggesting a central role of upstream α 1 - and β-adrenoceptors in the canonical regulatory pathway involving WNKs (with-no-lysine kinases), SPAK (STE20/SPS1-related proline alanine-rich kinase), and OxSR1 (oxidative stress response 1). In our previous study, α 1 -adrenoceptor antagonism in norepinephrine-infused male Sprague-Dawley rats prevented the development of norepinephrine-evoked SSH in part by suppressing NCC activity and expression. In these studies, we used selective adrenoceptor antagonism in male Dahl salt–sensitive rats to test the hypothesis that norepinephrine-mediated activation of the NCC in Dahl SSH occurs via an α 1 -adrenoceptor dependent pathway. A high-salt diet evoked significant increases in NCC activity, expression, and phosphorylation in Dahl salt–sensitive rats that developed SSH. Increases were associated with a dysfunctional WNK1/4 dynamic and a failure to suppress SPAK/OxSR1 activity. α 1 -adrenoceptor antagonism initiated before high-salt intake or following the establishment of SSH attenuated blood pressure in part by suppressing NCC activity, expression, and phosphorylation. Collectively, our findings support the existence of a norepinephrine-activated α 1 -adrenoceptor gated pathway that relies on WNK/SPAK/OxSR1 signaling to regulate NCC activity in SSH.

Aim: Age-dependent hypertension (HTN) develops at different rates in men versus women. These studies tested the hypothesis that in a model of normal aging, the Sprague Dawley rat, alterations in fluid and electrolyte balance contribute to age-dependent HTN in a sex-dependent manner. Methods: Three, 8 and 16-month old male and female SD rats on a normal salt (NS; 0.6% NaCl) diet underwent an acute IV volume expansion (VE; 5% body weight) and mean arterial pressure (MAP), diuresis and natriuresis were assessed. In separate groups of aged rats fed a 21-day NS or high salt (HS; 4% NaCl) diet in vivo MAP and NCC activity (ΔUNaV to IV hydrochlorothiazide, 2mg/kg) were assessed. N=4-6/gp. Results: The diuretic and natriuretic responses to an acute VE are profoundly blunted in aged male, but not female, rats. In contrast to female rats, male rats exhibit age-dependent HTN on a lifelong NS intake and develop age-dependent salt sensitivity of blood pressure. Female rats exhibit higher baseline sodium chloride cotransporter (NCC) activity and both young male and female rats exhibit dietary sodium evoked suppression of the NCC that is lost with age. Conclusion: Aging in the Sprague Dawley rat is associated with the sex specific development of age dependent HTN and the salt sensitivity of blood pressure in the male vs. female rat. We speculate that this sex specific difference is related to the impaired ability of male rats to maintain fluid and electrolyte homeostasis - potentially via a mechanism involving increased age-dependent baseline NCC-mediated sodium retention.

Aim: Failure to upregulate hypothalamic paraventricular nucleus (PVN) Gαi 2 proteins promotes salt sensitive hypertension (ssHTN) and central inflammation plays a role in HTN. We hypothesize that 1) PVN-specific inflammation contributes to Gαi 2 protein dependent ssHTN, and 2) GNAI2 SNPs correlate with HTN. Methods: 3-month-old male Sprague Dawley rats were implanted with I.C.V. cannulas fitted to osmotic minipumps and infused with Gαi 2 or control scrambled (SCR) oligodeoxynucleotides (ODN) (25μg/5μl/day), or ODN + minocycline (MINO) (120μg/5μl/day, HS only) and placed on a 7-day normal (NS; 0.6% NaCl) or high (HS; 4% NaCl) salt diet. Day 7 BP (femoral cannulation) and Plasma NE (ELISA) were measured. PVN and subfornical organ microglia and astrocyte activation (IHC), and PVN pro-inflammatory (PIC) and anti-inflammatory (AIC) cytokine mRNA and protein levels were analyzed. GNAI2 SNPs were examined in the UK BioBank for associations with HTN. Results: In contrast to SCR ODN infusion, Gαi 2 ODN infusion and HS evoked HTN, elevated plasma NE, PVN-specific microgliosis, upregulation of PVN PICs and downregulation of PVN IL-10. MINO co-infusion with Gαi 2 ODN and HS diet restored PIC and AIC mRNA and protein levels. Furthermore, MINO attenuated microgliosis and reduced the magnitude of ssHTN. GNAI2 SNPs rs2298952 (p=0.0411, MAF=0.103) and rs4547694 (p=0.0117, MAF=0.381) are both associated with essential HTN in BioBank subjects. Conclusion: PVN-specific microgliosis and inflammation may drive Gαi 2 protein dependent ssHTN and GNAI2 SNPs represent a potential biomarker of essential HTN.

Aim The afferent renal nerves (ARN) mediate reno-renal reflexes that influence efferent renal sympathetic nerve activity, sodium homeostasis, and blood pressure. These studies tested the hypothesis...

Summary: Dendritic mislocalization of microtubule associated protein tau is a hallmark of tauopathies, but the role of dendritic tau is unknown. We now report that tau interacts with the RNA-binding protein (RBP) TIA1 in brain tissue, and we present the brain-protein interactome network for TIA1. Analysis of the TIA1 interactome in brain tissue from wild-type (WT) and tau knockout mice demonstrates that tau is required for normal interactions of TIA1 with proteins linked to RNA metabolism, including ribosomal proteins and RBPs. Expression studies show that tau regulates the distribution of TIA1, and tau accelerates stress granule (SG) formation. Conversely, TIA1 knockdown or knockout inhibits tau misfolding and associated toxicity in cultured hippocampal neurons, while overexpressing TIA1 induces tau misfolding and stimulates neurodegeneration. Pharmacological interventions that prevent SG formation also inhibit tau pathophysiology. These studies suggest that the pathophysiology of tauopathy requires an intimate interaction with RNA-binding proteins. : Vanderweyde et al. show that the interaction of microtubule associated protein tau with the RNA binding protein (RBP) TIA1 regulates stress granule (SG) formation as well as misfolding and aggregation of tau. TIA1 knockdown prevents tau misfolding and tau-mediated toxicity, which points to RBPs as potential targets for therapy of tauopathies.

Aim: Hypertension (HTN) is positively correlated with age and sympathetic tone in humans. We hypothesize that sympathetically driven sodium chloride cotransporter (NCC)-mediated sodium retention contributes to age-related HTN. Methods: Three, 8 and 16 month old male SD rats on a normal salt (NS; 0.6% NaCl) diet underwent an acute IV volume expansion (VE; 5% body weight – mechanoreceptor stimulus) or 1M NaCl infusion (20μL/min, 2hr – chemoreceptor stimulus) and mean arterial pressure (MAP), natriuresis (UNaV) and paraventricular nucleus (PVN) neuronal activation (c-Fos expression) were assessed. In separate groups of rats fed a 21 day NS or high salt (HS; 4% NaCl) diet, 1) ex vivo afferent renal nerve (ARN) activity (norepinephrine-evoked substance P release) or 2) in vivo MAP, NCC activity (ΔUNaV to IV hydrochlorothiazide, 2mg/kg) and sympathetic tone were assessed. N=4-6/gp. Results: The natriuretic and PVN sympathoinhibitory parvocellular neuronal responses to VE, but not 1M NaCl infusion, are blunted in aged rats. Aged rats exhibit reduced ARN activity on a NS diet and fail to increase ARN activity on a HS diet. Aged rats develop salt sensitive HTN accompanied by elevated NCC activity and sympathetic tone on a NS diet and impaired suppression of NCC activity and sympathetic tone on a HS diet. Conclusion: Aging is associated with a selective impairment in the mechanosensitive ARN and salt sensitive HTN. We speculate that an age-related decrease in the mechanosensitive ARN sympathoinhibitory reno-renal reflex promotes sympathoexcitation, perhaps via reduced activity of PVN sympathoinhibitory parvocellular neurons, driving NCC-mediated sodium retention and salt sensitive HTN.

The prevalence of hypertension rises with age to approximately two out of three adults over the age of 60 in the United States. Although the mechanisms underlying age-related hypertension are incompletely understood, sodium homeostasis is critical to the long-term regulation of blood pressure and there is strong evidence that aging is associated with alterations in renal sodium handling. This minireview focuses on recent advancements in our understanding of the vascular, neurohumoral, and renal mechanisms that influence sodium homeostasis and promote age-related hypertension.

Hypothesis: Sympathoexcitation and sodium retention contribute to age-related hypertension (HTN). Methods: Three, 8, and 16 month old male SD rats underwent an IV volume expansion (VE; 5% BW) and MAP, HR, urine output and paraventricular nucleus (PVN) neuronal activation (c-Fos expression) were assessed. In separate groups of rats fed a 21 day normal (NS; 0.6% NaCl) or high salt (HS; 4% NaCl) diet, measures of 1) ex vivo afferent renal nerve (ARN) activity (norepinephrine (NE)-evoked substance P release) or 2) MAP, HR, NCC activity (ΔUNaV to IV HCTZ, 2mg/kg) and sympathetic tone (plasma and renal NE content, ΔMAP to IV hexamethonium) were assessed. Separate groups of rats underwent bilateral renal denervation (RDNX) or chronic NCC antagonism (SC HCTZ, 4mg/kg/d, 14d) and MAP was assessed (N=4/gp). Results: Acute VE-evoked natriuresis, diuresis, and PVN parvocellular neuronal activation were impaired in aged rats. Aged rats exhibited reduced ARN activity on a NS diet and failed to increase ARN activity during a HS diet. In aged rats, basal MAP, NCC activity and sympathetic tone were increased and HS-evoked suppression of NCC activity and sympathetic tone was impaired. Chronic NCC antagonism and RDNX attenuated age-related HTN (MAP [mmHg] 16mo 149±3 vs RDNX 139±1 vs SC HCTZ 136±5, P Conclusion: Our data suggest that the ARN, which have been implicated in the regulation of sympathetic outflow and renal sodium excretion, are impaired in age-related HTN. We speculate that an age-related decrease in ARN activity promotes sympathoexcitation, perhaps in part via reduced activation of PVN sympathoinhibitory parvocellular neurons, resulting in NCC-mediated sodium retention and HTN.

Hypothesis: Excess SNS release of norepinephrine (NE) increases NCC activity, via an α1 adrenoceptor pathway, to drive the development and maintenance of salt-sensitive and neurogenic hypertension (HTN). Methods: Male Sprague-Dawley (SD) rats receiving a continuous s.c. saline or NE (600ng/min) infusion alone or in combination with terazosin (10mg/kg/day) were fed a 14-day 0.6% (NS) or 4% NaCl (HS) diet. Separate groups of male SD rats received a continuous s.c. NE infusion and 28-day NS or HS intake, on day 14 of HS a sub group of rats were switched to a co NE-terazosin s.c. infusion. Groups of male SHR rats received s.c. saline or terazosin for 14 days. Endpoint measurements (day 14 or 28) were basal MAP and NCC activity (peak natriuresis to iv hydrochlorothiazide (HCTZ; 2mg/kg) infusion and phosphoNCCT58 immunoblotting) and expression (via immunoblotting) was assessed (N=4/gp). Results: NE infused SD rats exhibit HTN and fail to suppress NCC expression and activity during HS-intake. α1-adreoceptor antagonism (confirmed pharmacologically) abolished the salt-sensitive component of NE HTN and restored dietary sodium evoked suppression of the NCC in NE infused SD rats. Critically, α 1 -adrenoceptor antagonism lowers BP and reduces NCC activity in established SHR HTN and restores sodium-evoked suppression of NCC activity and abolishes the salt sensitivity of BP in established NE-evoked SS HTN. Conclusion: SNS activation of the NCC by NE occurs in rat models of neurogenic and SS HTN. Our data demonstrates antagonism of α 1 -adrenoceptors lowers BP and NCC activity in established SS and neurogenic HTN and suggests α 1 -antagonists as a therapeutic option in sympathetically mediated HTN.

Salt sensitivity of blood pressure (BP) increases hypertension risk and associated adverse cardiovascular outcomes. At present, there are no validated rapid tests or diagnostic markers to identify salt sensitivity of BP in clinical practice. Based on our prior animal studies that report a role for brain Gαi2 proteins in the salt sensitivity of BP and evidence that GNAI2 single nucleotide polymorphisms (SNPs) associate with hypertension risk, we investigated the hypothesis that GNAI2 SNPs associate with salt sensitivity of BP in humans. Our data provide the first evidence that a GNAI2 SNP ( rs10510755 ) positively associates with salt sensitivity of BP in the Genetic Epidemiology of Salt Sensitivity data set (continuous phenotype P = 0.049, case-control phenotype P = 0.039; n = 968), independently of subject sex or age. These observations suggest that genotyping at GNAI2 may be a useful biomarker in identifying individuals at risk for developing salt-sensitive BP and related complications or in identifying salt sensitivity within the hypertensive population.

Purpose of Review The etiology of hypertension, a critical public health issue affecting one in three US adults, involves the integration of the actions of multiple organ systems, including the renal sympathetic nerves. The renal sympathetic nerves, which are comprised of both afferent (sensory input) and efferent (sympathetic outflow) arms, have emerged as a major potential therapeutic target to treat hypertension and disease states exhibiting excess renal sympathetic activity. Recent Findings This review highlights recent advances in both clinical and basic science that have provided new insight into the distribution, function, and reinnervation of the renal sympathetic nerves, with a focus on the renal afferent nerves, in hypertension and hypertension-evoked disease states including salt-sensitive hypertension, obesity-induced hypertension, and chronic kidney disease. Summary Increased understanding of the differential role of the renal afferent versus efferent nerves in the pathophysiology of hypertension has the potential to identify novel targets and refine therapeutic interventions designed to treat hypertension.

Aim: Hypertension is strongly correlated with increased age in human subjects. These studies tested the hypothesis that impaired natriuretic responses to acute and chronic challenges to fluid and electrolyte homeostasis contribute to age-dependent hypertension. Methods: Two-month old (young) and 6-month old (aged) male Sprague-Dawley rats underwent an acute IV volume expansion (VE; 5% BW) and MAP, HR, urine output, and PVN neuronal activation (c-Fos expression) were assessed (n=4 per group). In a separate study, naïve 2 and 6-month old male Sprague-Dawley rats were maintained on a normal (0.6% NaCl) or high salt (4% NaCl) diet, and on day 21, MAP and NCC activity (peak natriuresis to IV HCTZ, 2mg/kg) were assessed (n=4 per group). Results: Renal excretion of sodium and water after acute VE was impaired in aged rats (total % sodium load excreted; young 78±6 vs aged 60±7: total % water load excreted; young 96±7 vs aged 66±5, P Conclusion: Activation of sympathoinhibitory PVN parvocellular neurons is a well-characterized response to acute VE. Our data suggest there are attenuated acute PVN sympathoinhibitory responses to alterations in fluid and electrolyte balance in aged rats. In aged animals, HS intake increased NCC-mediated sodium reabsorption and promoted the development of sodium-dependent hypertension. We speculate that this is driven by blunted HS-evoked sympathoinhibition, likely due to reduced PVN neuronal activation.

Aim: We hypothesize that challenges to sodium homeostasis differentially activate chemo- vs. mechanosensitive afferent renal nerves to evoke sympathoinhibition, sodium homeostasis and normotension in the Sprague-Dawley (SD) rat. Methods: Conscious SD rats, post sham (S) or afferent renal nerve ablation (Renal-CAP; capsaicin 33 mM) underwent IV volume expansion (VE; 5% BW) or IV sodium loading (1M NaCl Infusion – constant infusion volume) and HR, MAP, natriuresis and PVN neuronal activation (c-Fos expression) were assessed (N=4/gp). Naïve SD rats were fed a 0.6% (NS) or 4% NaCl (HS) diet for 21 days and afferent renal nerve activity was assessed as norepinephrine (NE) (1250 pmol) and NaCl-evoked (450mM) substance P (SP) release in a renal pelvic assay (N=4/gp). Radiotelemetered SD rats post S or Renal-CAP immediately prior a 0.6% (NS) or 4% NaCl (HS) diet underwent continuous MAP monitoring. On day-21 plasma and renal NE content was assessed (N=5/group). Results: Renal-CAP attenuated the natriuretic and PVN parvocellular responses to IV VE (peak UNaV [μeq/min]; S 43±4 vs Renal-CAP 26±6, P Conclusion: The mechanosensitive afferent renal nerves mediate acute natriuresis and blood pressure regulation via activation of PVN sympathoinhibitory neurons. During HS intake the afferent renal nerves counter the development of salt-sensitive hypertension via a mechanism involving increased mechano but not chemosensitive afferent nerve responsiveness to potentiate sympathoinhibition.

Background Endoplasmic reticulum ( ER ) stress and the subsequent unfolded protein response may initially be protective, but when prolonged, have been implicated in atherogenesis in diabetic conditions. Triglycerides and free fatty acids ( FFAs ) are elevated in patients with diabetes and may contribute to ER stress. We sought to evaluate the effect of acute FFA elevation on ER stress in endothelial and circulating white cells. Methods and Results Twenty‐one healthy subjects were treated with intralipid (20%; 45 mL/h) plus heparin (12 U/kg/h) infusion for 5 hours. Along with increased triglyceride and FFA levels, intralipid/heparin infusion reduced the calf reactive hyperemic response without a change in conduit artery flow‐mediated dilation consistent with microvascular dysfunction. To investigate the short‐term effects of elevated triglycerides and FFA , we measured markers of ER stress in peripheral blood mononuclear cells ( PBMC s) and vascular endothelial cells ( VEC s). In VEC s, activating transcription factor 6 ( ATF 6) and phospho‐inositol requiring kinase 1 ( pIRE 1) proteins were elevated after infusion (both P PBMC s, ATF 6 and spliced X‐box‐binding protein 1 ( XBP ‐1) gene expression increased by 2.0‐ and 2.5‐fold, respectively (both P CHOP and GADD 34 decreased by ≈67% and 74%, respectively (both P ATF 6 and pIRE 1 protein levels also increased (both P ATF 6 after infusion, suggesting activation. Conclusions Along with microvascular dysfunction, intralipid infusion induced an early protective ER stress response evidenced by activation of ATF 6 and IRE 1 in both leukocytes and endothelial cells. Our results suggest a potential link between metabolic disturbances and ER stress that may be relevant to vascular disease.

Background— Endothelial dysfunction contributes to the development of atherosclerosis in patients with diabetes mellitus, but the mechanisms of endothelial dysfunction in this setting are incompletely understood. Recent studies have shown altered mitochondrial dynamics in diabetes mellitus with increased mitochondrial fission and production of reactive oxygen species. We investigated the contribution of altered dynamics to endothelial dysfunction in diabetes mellitus. Methods and Results— We observed mitochondrial fragmentation ( P =0.002) and increased expression of fission-1 protein (Fis1; P Conclusion— These findings implicate increased mitochondrial fission as a contributing mechanism for endothelial dysfunction in diabetic states.

Recent studies have shown mitochondrial dysfunction and increased production of reactive oxygen species in peripheral blood mononuclear cells (PBMCs) and endothelial cells from patients with diabetes mellitus. Mitochondria oxygen consumption is coupled to adenosine triphosphate (ATP) production and also occurs in an uncoupled fashion during formation of reactive oxygen species by components of the electron transport chain and other enzymatic sites. We therefore hypothesized that diabetes would be associated with higher total and uncoupled oxygen consumption in PBMCs that would correlate with endothelial dysfunction. We developed a method to measure oxygen consumption in freshly isolated PBMCs and applied it to 26 patients with type 2 diabetes mellitus and 28 non-diabetic controls. Basal (192 ± 47 vs 161 ± 44 pmoles/min, p = 0.01), uncoupled (64 ± 16 vs 53 ± 13 pmoles/min, p = 0.007), and maximal (795 ± 87 vs 715 ± 128 pmoles/min, p=0.01) oxygen consumption rates were higher in diabetic patients compared to controls. There were no significant correlations between oxygen consumption rates and endothelium-dependent flow-mediated dilation measured by vascular ultrasound. Non-endothelium-dependent nitroglycerin-mediated dilation was lower in diabetics (10.1 ± 6.6 vs 15.8 ± 4.8%, p = 0.03) and correlated with maximal oxygen consumption (r = -0.64, p=0.001). In summary, we found that diabetes mellitus is associated with a pattern of mitochondrial oxygen consumption consistent with higher production of reactive oxygen species. The correlation between oxygen consumption and nitroglycerin-mediated dilation may suggest a link between mitochondrial dysfunction and vascular smooth muscle cell dysfunction that merits further study. Finally, the described method may have utility for the assessment of mitochondrial function in larger scale observational and interventional studies in humans.

Background— Abnormal endothelial function promotes atherosclerotic vascular disease in diabetes. Experimental studies indicate that disruption of endothelial insulin signaling, through the activity of protein kinase C-β (PKCβ) and nuclear factor κB, reduces nitric oxide availability. We sought to establish whether similar mechanisms operate in the endothelium in human diabetes mellitus. Methods and Results— We measured protein expression and insulin response in freshly isolated endothelial cells from patients with type 2 diabetes mellitus (n=40) and nondiabetic controls (n=36). Unexpectedly, we observed 1.7-fold higher basal endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 in patients with diabetes mellitus ( P =0.007) without a difference in total eNOS expression. Insulin stimulation increased eNOS phosphorylation in nondiabetic subjects but not in diabetic patients ( P =0.003), consistent with endothelial insulin resistance. Nitrotyrosine levels were higher in diabetic patients, indicating endothelial oxidative stress. PKCβ expression was higher in diabetic patients and was associated with lower flow-mediated dilation (r=−0.541, P =0.02). Inhibition of PKCβ with LY379196 reduced basal eNOS phosphorylation and improved insulin-mediated eNOS activation in patients with diabetes mellitus. Endothelial nuclear factor κB activation was higher in diabetes mellitus and was reduced with PKCβ inhibition. Conclusions— We provide evidence for the presence of altered eNOS activation, reduced insulin action, and inflammatory activation in the endothelium of patients with diabetes mellitus. Our findings implicate PKCβ activity in endothelial insulin resistance.

Background: Consumption of flavonoid-containing foods may be useful for the management of hypertension. Objective: We investigated whether 100% Concord grape juice lowers blood pressure in patients with prehypertension and stage 1 hypertension. Design: We conducted a double-blind crossover study to compare the effects of grape juice (7 mL · kg−1 · d−1) and matched placebo beverage on 24-h ambulatory blood pressure, stress-induced changes in blood pressure, and biochemical profile. Participants consumed each beverage for 8 wk with a 4-wk rest period between beverages. They ceased consumption of grapes and other flavonoid-containing beverages throughout the study. Results: We enrolled 64 otherwise healthy patients taking no antihypertensive medications (31% women, 42% black, age 43 ± 12 y). Baseline mean (±SD) cuff blood pressure was 138 ± 7 (systolic)/82 ± 7 (diastolic) mm Hg. No effects on the primary endpoint of 24-h mean systolic blood pressure, diastolic blood pressure, or stress-induced changes in blood pressure were observed. A secondary endpoint was nocturnal dip in systolic pressure. At baseline, nocturnal pressure was 8.3 ± 7.1% lower at night than during daytime. The mean nocturnal dip increased 1.4 percentage points after grape juice and decreased 2.3 percentage points after placebo (P = 0.005). Fasting blood glucose was 91 ± 10 mg/dL at baseline for the entire cohort. Glucose decreased 2 mg/dL after consumption of grape juice and increased 1 mg/dL after consuming the placebo (P = 0.03). Conclusions: We observed no effect of grape juice on ambulatory blood pressure in this cohort of relatively healthy individuals with modestly elevated blood pressure. Secondary analyses suggested favorable effects on nocturnal dip and glucose homeostasis that may merit further investigation. This trial was registered at clinicaltrials.gov as NCT00302809.