HCO3 dependent impact of Na+,K+, 2Cl cotransport in vascular smooth muscle excitation-contraction coupling

In smooth muscles, inhibition of Na(+),K(+),2Cl(-) cotransport (NKCC) by bumetanide decreased intracellular Cl(-) content ([Cl(-)](i)) and suppressed the contractions triggered by diverse stimuli. This study examines whether or not bicarbonate, a regulator of several Cl(-) transporters, affects the impact of NKCC in excitation-contraction coupling. Addition of 25 mM NaHCO(3) attenuated the inhibitory action of bumetanide on mesenteric artery contractions evoked by 30 mM KCl and phenylephrine (PE) by 5 and 3-fold, respectively. In cultured vascular smooth muscle cells, NaHCO(3) almost completely abolished inhibitory actions of bumetanide on transient depolarization and [Ca(2+)](i) elevation triggered by PE. In bicarbonate-free medium, bumetanide decreased [Cl(-)](i) by approximately 15%; this effect was almost totally abrogated by NaHCO(3). The addition of NaHCO(3) resulted in 2-fold inhibition of NKCC activity and 3-fold attenuation of [Cl(-)](i). These data strongly suggest that extracellular HCO(3)(-) diminishes the NKCC-sensitive component of excitation-contraction coupling via suppression of this carrier

Plasma fatty acids and the risk of vascular disease and mortality outcomes in individuals with type 2 diabetes: results from the ADVANCE study

Aims/hypothesis: This biomarker study aimed to quantify the association of essential and other plasma fatty acid biomarkers with macrovascular disease, microvascular disease and death in individuals with type 2 diabetes. Methods: A case-cohort study (N = 3576), including 654 macrovascular events, 341 microvascular events and 631 deaths during 5 years of (median) follow-up, was undertaken as a secondary analysis of the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified-Release Controlled Evaluation (ADVANCE) study (full details of the study design and primary endpoints of the ADVANCE trial and its case-cohort have been published previously). This current study considers new data: fatty acids measured from baseline plasma samples by proton NMR analysis. The fatty acids measured were n-3, docosahexaenoic acid (DHA), n-6, linoleic acid, and polyunsaturated, monounsaturated and saturated fatty acids. HRs were modelled per SD higher (percentage) fatty acid. C statistics and continuous net reclassification improvement were used to test the added value of fatty acids compared with traditional cardiovascular risk factors. Results: After adjustment for traditional cardiovascular risk factors, an inverse association was observed for n-3 fatty acids and DHA with the risk of macrovascular events (HR [95% CI]: 0.87 [0.80, 0.95] and 0.88 [0.81, 0.96], respectively, per 1 SD higher percentage), and for n-3 fatty acids with the risk of death (HR 0.91 [95% CI 0.84, 0.99] per 1 SD higher percentage). Such associations were also evident when investigating absolute levels of fatty acids. There were no statistically significant associations between any fatty acids and microvascular disease after adjustment. However, there was limited improvement in the predictive ability of models when any fatty acid was added. Conclusions/interpretation: Plasma n-3 fatty acids and DHA were found to be inversely associated with macrovascular disease, while n-3 fatty acids were also inversely associated with death. These results support the cardioprotective effects of n-3 fatty acids and DHA and further merit testing the role of high-dose supplementation with n-3 fatty acids in individuals with type 2 diabetes. Trial registration: ClinicalTrials.gov NCT00145925

Circulating amino acids and the risk of macrovascular, microvascular and mortality outcomes in individuals with type 2 diabetes : results from the ADVANCE trial

Aims/hypotheses We aimed to quantify the association of individual circulating amino acids with macrovascular disease, microvascular disease and all-cause mortality in individuals with type 2 diabetes. Methods We performed a case-cohort study (N = 3587), including 655 macrovascular events, 342 microvascular events (new or worsening nephropathy or retinopathy) and 632 all-cause mortality events during follow-up, in a secondary analysis of the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) study. For this study, phenylalanine, isoleucine, glutamine, leucine, alanine, tyrosine, histidine and valine were measured in stored plasma samples by proton NMR metabolomics. Hazard ratios were modelled per SD increase in each amino acid. Results In models investigating associations and potential mechanisms, after adjusting for age, sex and randomised treatment, phenylalanine was positively, and histidine inversely, associated with macrovascular disease risk. These associations were attenuated to the null on further adjustment for extended classical risk factors (including eGFR and urinary albumin/creatinine ratio). After adjustment for extended classical risk factors, higher tyrosine and alanine levels were associated with decreased risk of microvascular disease (HR 0.78; 95% CI 0.67, 0.91 and HR 0.86; 95% CI 0.76, 0.98, respectively). Higher leucine (HR 0.79; 95% CI 0.69, 0.90), histidine (HR 0.89; 95% CI 0.81, 0.99) and valine (HR 0.79; 95% CI 0.70, 0.88) levels were associated with lower risk of mortality. Investigating the predictive ability of amino acids, addition of all amino acids to a risk score modestly improved classification of participants for macrovascular (continuous net reclassification index [NRI] +35.5%, p <0.001) and microvascular events (continuous NRI +14.4%, p = 0.012). Conclusions/interpretation We report distinct associations between circulating amino acids and risk of different major complications of diabetes. Low tyrosine appears to be a marker of microvascular risk in individuals with type 2 diabetes independently of fundamental markers of kidney function.Peer reviewe

Circulating amino acids and the risk of macrovascular, microvascular and mortality outcomes in individuals with type 2 diabetes : results from the ADVANCE trial

Aims/hypotheses We aimed to quantify the association of individual circulating amino acids with macrovascular disease, microvascular disease and all-cause mortality in individuals with type 2 diabetes. Methods We performed a case-cohort study (N = 3587), including 655 macrovascular events, 342 microvascular events (new or worsening nephropathy or retinopathy) and 632 all-cause mortality events during follow-up, in a secondary analysis of the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) study. For this study, phenylalanine, isoleucine, glutamine, leucine, alanine, tyrosine, histidine and valine were measured in stored plasma samples by proton NMR metabolomics. Hazard ratios were modelled per SD increase in each amino acid. Results In models investigating associations and potential mechanisms, after adjusting for age, sex and randomised treatment, phenylalanine was positively, and histidine inversely, associated with macrovascular disease risk. These associations were attenuated to the null on further adjustment for extended classical risk factors (including eGFR and urinary albumin/creatinine ratio). After adjustment for extended classical risk factors, higher tyrosine and alanine levels were associated with decreased risk of microvascular disease (HR 0.78; 95% CI 0.67, 0.91 and HR 0.86; 95% CI 0.76, 0.98, respectively). Higher leucine (HR 0.79; 95% CI 0.69, 0.90), histidine (HR 0.89; 95% CI 0.81, 0.99) and valine (HR 0.79; 95% CI 0.70, 0.88) levels were associated with lower risk of mortality. Investigating the predictive ability of amino acids, addition of all amino acids to a risk score modestly improved classification of participants for macrovascular (continuous net reclassification index [NRI] +35.5%, p <0.001) and microvascular events (continuous NRI +14.4%, p = 0.012). Conclusions/interpretation We report distinct associations between circulating amino acids and risk of different major complications of diabetes. Low tyrosine appears to be a marker of microvascular risk in individuals with type 2 diabetes independently of fundamental markers of kidney function.Peer reviewe

Activation of cAMP signaling transiently inhibits apoptosis in vascular smooth muscle cells in a site upstream of caspase-3

Intracellular signaling pathways that are involved in protection of vascular smooth muscle cells (VSMC) from apoptosis remain poorly understood. This study examines the effect of activators of cAMP/cGMP signaling on apoptosis in non-transfected VSMC and in VSMC transfected with c-myc (VSMC-MYC) or with its functional analogue, E1A-adenoviral protein (VSMC-E1A). Serum-deprived VSMC-E1A exhibited the highest apoptosis measured as the content of chromatin and low molecular weight DNA fragments, phosphatidylserine content in the outer surface of plasma membrane and caspase-3 activity (ten-, five-, four- and tenfold increase after 6 h of serum withdrawal, respectively). In VSMC-E1A, the addition of an activator of adenylate cyclase, forskolin, abolished chromatin cleavage, DNA laddering, caspase-3 activation and the appearance of morphologically-defined apoptotic cells triggered by 6 h of serum deprivation. In non-transfected VSMC and in VSMC-MYC, 6 h serum deprivation led to approximately six- and threefold activation of chromatin cleavage, respectively, that was also blocked by forskolin. In VSMC-E1A, inhibition of apoptosis was observed with other activators of cAMP signaling (cholera toxin, isoproterenol, adenosine, 8-Br-cAMP), whereas 6 h incubation with modulators of cGMP signaling (8-Br-cGMP, nitroprusside, atrial natriuretic peptide, L-NAME) did not affect the development of apoptotic machinery. The antiapoptotic effect of forskolin was abolished in 24 h of serum deprivation that was accompanied by normalization of intracellular cAMP content and protein kinase A (PKA) activity. Protection of VSMC-E1A from apoptosis by forskolin was blunted by PKA inhibitors (H-89 and KT5720), whereas transfection of cells with PKA catalytic subunit attenuated apoptosis triggered by serum withdrawal. The protection of VSMC-E1A by forskolin from apoptosis was insensitive to modulators of cytoskeleton assembly (cytochalasin B, colchicine). Neither acute (30 min) nor chronic (24 h) exposure of VSMC to forskolin modified basal and serum-induced phosphorylation of the MAP kinase ERK1/2. Thus, our results show that activation of cAMP signaling delays the development of apoptosis in serum-deprived VSMC at a site upstream of caspase-3 via activation of PKA and independently of cAMP-induced reorganization of the cytoskeleton network and the ERK1/2-terminated MAPK signaling cascade

Variability in estimated glomerular filtration rate and the risk of major clinical outcomes in diabetes:Post hoc analysis from the ADVANCE trial

There are limited data on whether estimated glomerular filtration rate (eGFR) variability modifies the risk of future clinical outcomes in type 2 diabetes (T2D). We assessed the association between 20-month eGFR variability and the risk of major clinical outcomes in T2D among 8241 participants in the ADVANCE trial. Variability in eGFR (coefficient of variation [CVeGFR]) was calculated from three serum creatinine measurements over 20 months. Participants were classified into three groups by thirds of CVeGFR: low (6.4 to 12.1). The primary outcome was the composite of major macrovascular events, new or worsening nephropathy and all-cause mortality. Cox regression models were used to estimate hazard ratios (HRs). Over a median follow-up of 2.9 years following the 20-month period, 932 (11.3%) primary outcomes were recorded. Compared with low variability, greater 20-month eGFR variability was independently associated with higher risk of the primary outcome (HR for moderate and high variability: 1.07, 95% CI: 0.91-1.27 and 1.22, 95% CI: 1.03-1.45, respectively) with evidence of a positive linear trend (p = .015). These data indicate that eGFR variability predict changes in the risk of major clinical outcomes in T2D