Hypertension is an underlying risk factor for cardiovascular disease. Despite this, its pathogenesis remains unknown in most cases. Recently, the transient receptor potential (TRP) channel family was associated with the development of several cardiovascular diseases linked to hypertension. The melastatin TRP channels TRPM4 and TRPM5 have distinct properties within the TRP channel family: they form nonselective cation channels activated by intracellular calcium ions. Here we report the identification of TRPM4 proteins in endothelial cells, heart, kidney, and chromaffin cells from the adrenal gland, suggesting that they have a role in the cardiovascular system. Consistent with this hypothesis, Trpm4 gene deletion in mice altered long-term regulation of blood pressure toward hypertensive levels. No changes in locomotor activity, renin-angiotensin system function, electrolyte and fluid balance, vascular contractility, and cardiac contractility under basal conditions were observed. By contrast, inhibition of ganglionic transmission with either hexamethonium or prazosin abolished the difference in blood pressure between Trpm4 -/-and wild-type mice. Strikingly, plasma epinephrine concentration as well as urinary excretion of catecholamine metabolites were substantially elevated in Trpm4 -/-mice. In freshly isolated chromaffin cells, lack of TRPM4 was shown to cause markedly more acetylcholineinduced exocytotic release events, while neither cytosolic calcium concentration, size, nor density of vesicles were different. We therefore conclude that TRPM4 proteins limit catecholamine release from chromaffin cells and that this contributes to increased sympathetic tone and hypertension.

Non-alcoholic fatty liver disease (NAFLD) results from increased hepatic lipid accumulation and steatosis, and is closely linked to liver one-carbon (C1) metabolism. We assessed in C57BL6/N mice whether NAFLD induced by a high-fat (HF) diet over 8 weeks can be reversed by additional 4 weeks of a dietary methyl-donor supplementation (MDS). MDS in the obese mice failed to reverse NAFLD, but prevented the progression of hepatic steatosis associated with major changes in key hepatic C1-metabolites, e.g. S-adenosyl-methionine and S-adenosyl-homocysteine. Increased phosphorylation of AMPK-α together with enhanced β-HAD activity suggested an increased flux through fatty acid oxidation pathways. This was supported by concomitantly decreased hepatic free fatty acid and acyl-carnitines levels. Although HF diet changed the hepatic phospholipid pattern, MDS did not. Our findings suggest that dietary methyl-donors activate AMPK, a key enzyme in fatty acid β-oxidation control, that mediates increased fatty acid utilization and thereby prevents further hepatic lipid accumulation.

Whereas it is generally perceived to be harmful, enhanced coagulation activation can also convey salutary effects. The high prevalence of the prothrombotic factor V Leiden (FVL) mutation in whites has been attributed to a positive selection pressure (eg, resulting from reduced blood loss or improved survival in sepsis). The consequences of enhanced coagulation activation, as observed in FVL carriers, on microvascular diabetic complications remain unknown. We therefore investigated the role of FVL in diabetic nephropathy. In heterozygous or homozygous diabetic FVL mice, albuminuria and indices of diabetic nephropathy were reduced compared with diabetic wild-type mice. This was associated with reduced glomerular apoptosis and preservation of podocytes in diabetic FVL-positive mice. In vitro, low-dose thrombin (50pM) prevented, whereas high-dose thrombin (20nM) aggravated, glucose-induced apoptosis in podocytes. In diabetic patients, the FVL mutation, but not the plasminogen activator inhibitor-1 4G/5G polymorphism, is associated with reduced albuminuria, which is consistent with a nephroprotective role of low but sustained thrombin generation. Consistently, anticoagulation of diabetic FVL-positive mice with hirudin abolished the nephroprotective effect. These results identify a nephroprotective function of low but sustained thrombin levels in FVL carriers, supporting a dual, context-dependent function of thrombin in chronic diseases. (Blood. 2011;117(19):5231-5242) IntroductionThe coagulation system provides an "on-demand" vascular repair system. After vascular injury, the coagulation system is activated, inducing fibrin formation and platelet activation, thus sealing the vascular leak and initiating a healing process leading in most cases to a restitutio ad integrum. As is evident in patients with severe hemophilia, this beneficial function of the hemostatic system is required for normal survival. Enhanced coagulation activation, however, is generally assumed to be disadvantageous for an individual's health, because some genetic polymorphisms associated with increased thrombin generation mediate an increased risk of venous thrombosis. 1 Considering the impending health risks associated with prothrombotic genetic polymorphisms, the high prevalence of some of them, in particular the factor V Leiden (FVL) mutation, led to the assumption that these genetic polymorphisms must be coupled with a positive selection pressure during evolution. 2 The FVL mutation is a missense mutation in the factor V gene (R506Q), resulting in the resistance of activated factor V to inactivation by activated protein C (aPC). The prevalence of the FVL mutation in whites is 4%-6%. 1 The potential benefits associated with FVL include a higher embryonic implantation rate, reduced puerperal maternal blood loss, and improved survival from severe infections. [3][4][5] The latter has been a matter of debate, because not all studies were able to reproduce the improved survival of FVL carriers in sepsis. [6][7][8][9][10][11] We have previously shown...

Obesity is an underlying risk factor in the development of cardiovascular disease, dyslipidemia and non-alcoholic fatty liver disease (NAFLD). Increased hepatic lipid accumulation is a hallmark in the progression of NAFLD and impairments in liver phosphatidylcholine (PC) metabolism may be central to the pathogenesis. Hepatic PC biosynthesis, which is linked to the one-carbon (C1) metabolism by phosphatidylethanolamine N-methyltransferase, is known to be important for hepatic lipid export by VLDL particles. Here, we assessed the influence of a high-fat (HF) diet and NAFLD status in mice on hepatic methyl-group expenditure and C1-metabolism by analyzing changes in gene expression, protein levels, metabolite concentrations, and nuclear epigenetic processes. In livers from HF diet induced obese mice a significant downregulation of cystathionine β-synthase (CBS) and an increased betaine-homocysteine methyltransferase (BHMT) expression were observed. Experiments in vitro, using hepatoma cells stimulated with peroxisome proliferator activated receptor alpha (PPARα) agonist WY14,643, revealed a significantly reduced Cbs mRNA expression. Moreover, metabolite measurements identified decreased hepatic cystathionine and L-α-amino-n-butyrate concentrations as part of the transsulfuration pathway and reduced hepatic betaine concentrations, but no metabolite changes in the methionine cycle in HF diet fed mice compared to controls. Furthermore, we detected diminished hepatic gene expression of de novo DNA methyltransferase 3b but no effects on hepatic global genomic DNA methylation or hepatic DNA methylation in the Cbs promoter region upon HF diet. Our data suggest that HF diet induces a PPARα-mediated downregulation of key enzymes in the hepatic transsulfuration pathway and upregulates BHMT expression in mice to accommodate to enhanced dietary fat processing while preserving the essential amino acid methionine.

Human monocytes are a heterogeneous cell population consisting of 3 subsets: classical CD14++CD16-, intermediate CD14++CD16+ and nonclassical CD14+CD16++ monocytes. Via poorly characterized mechanisms, intermediate monocyte counts rise in chronic inflammatory diseases, among which chronic kidney disease is of particular epidemiologic importance. DNA methylation is a central epigenetic feature that controls hematopoiesis. By applying next-generation Methyl-Sequencing we now tested how far the 3 monocyte subsets differ in their DNA methylome and whether uremia induces DNA methylation changes in differentiating monocytes. We found that each monocyte subset displays a unique phenotype with regards to DNA methylation. Genes with differentially methylated promoter regions in intermediate monocytes were linked to distinct immunological processes, which is in line with results from recent gene expression analyses. In vitro, uremia induced dysregulation of DNA methylation in differentiating monocytes, which affected several transcription regulators important for monocyte differentiation (e.g., FLT3, HDAC1, MNT) and led to enhanced generation of intermediate monocytes. As potential mediator, the uremic toxin and methylation inhibitor S-adenosylhomocysteine induced shifts in monocyte subsets in vitro, and associated with monocyte subset counts in vivo. Our data support the concept of monocyte trichotomy and the distinct role of intermediate monocytes in human immunity. The shift in monocyte subsets that occurs in chronic kidney disease, a proinflammatory condition of substantial epidemiological impact, may be induced by accumulation of uremic toxins that mediate epigenetic dysregulation.