Background: The gut microbiome is essential for physiological host responses and development of immune functions. The impact of gut microbiota on blood pressure and systemic vascular function, processes that are determined by immune cell function, is unknown. Methods and Results: Unchallenged germ‐free mice ( GF ) had a dampened systemic T helper cell type 1 skewing compared to conventionally raised ( CONV ‐R) mice. Colonization of GF mice with regular gut microbiota induced lymphoid mRNA transcription of T‐box expression in T cells and resulted in mild endothelial dysfunction. Compared to CONV ‐R mice, angiotensin II (Ang II ; 1 mg/kg per day for 7 days) infused GF mice showed reduced reactive oxygen species formation in the vasculature, attenuated vascular mRNA expression of monocyte chemoattractant protein 1 ( MCP ‐1), inducible nitric oxide synthase (iNOS) and NADPH oxidase subunit Nox2, as well as a reduced upregulation of retinoic‐acid receptor‐related orphan receptor gamma t (Rorγt), the signature transcription factor for interleukin (IL)‐17 synthesis. This resulted in an attenuated vascular leukocyte adhesion, less infiltration of Ly6G ⁺ neutrophils and Ly6C ⁺ monocytes into the aortic vessel wall, protection from kidney inflammation, as well as endothelial dysfunction and attenuation of blood pressure increase in response to Ang II . Importantly, cardiac inflammation, fibrosis and systolic dysfunction were attenuated in GF mice, indicating systemic protection from cardiovascular inflammatory stress induced by AngII. Conclusion: Gut microbiota facilitate Ang II ‐induced vascular dysfunction and hypertension, at least in part, by supporting an MCP ‐1/ IL ‐17 driven vascular immune cell infiltration and inflammation.

Background— Hemodynamic load regulates myocardial function and gene expression. We tested the hypothesis that afterload and preload, despite similar average load, result in different phenotypes. Methods and Results— Afterload and preload were compared in mice with transverse aortic constriction (TAC) and aortocaval shunt (shunt). Compared with sham mice, 6 hours after surgery, systolic wall stress (afterload) was increased in TAC mice (+40%; P <0.05), diastolic wall stress (preload) was increased in shunt (+277%; P <0.05) and TAC mice (+74%; P <0.05), and mean total wall stress was similarly increased in TAC (69%) and shunt mice (67%) ( P =NS, TAC versus shunt; each P <0.05 versus sham). At 1 week, left ventricular weight/tibia length was significantly increased by 22% in TAC and 29% in shunt mice ( P =NS, TAC versus shunt). After 24 hours and 1 week, calcium/calmodulin-dependent protein kinase II signaling was increased in TAC. This resulted in altered calcium cycling, including increased L-type calcium current, calcium transients, fractional sarcoplasmic reticulum calcium release, and calcium spark frequency. In shunt mice, Akt phosphorylation was increased. TAC was associated with inflammation, fibrosis, and cardiomyocyte apoptosis. The latter was significantly reduced in calcium/calmodulin-dependent protein kinase IIδ-knockout TAC mice. A total of 157 mRNAs and 13 microRNAs were differentially regulated in TAC versus shunt mice. After 8 weeks, fractional shortening was lower and mortality was higher in TAC versus shunt mice. Conclusions— Afterload results in maladaptive fibrotic hypertrophy with calcium/calmodulin-dependent protein kinase II–dependent altered calcium cycling and apoptosis. Preload is associated with Akt activation without fibrosis, little apoptosis, better function, and lower mortality. This indicates that different loads result in distinct phenotype differences that may require specific pharmacological interventions.

Background— Extracellular matrix proteins, such as laminins, and endothelial cells are known to influence cardiomyocyte performance; however, the underlying molecular mechanisms remain poorly understood. Methods and Results— We used a forward genetic screen in zebrafish to identify novel genes required for myocardial function and were able to identify the lost-contact ( loc ) mutant, which encodes a nonsense mutation in the integrin-linked kinase ( ilk ) gene. This loc/ilk mutant is associated with a severe defect in cardiomyocytes and endothelial cells that leads to severe myocardial dysfunction. Additional experiments revealed the epistatic regulation between laminin-α4 (Lama4), integrin, and Ilk, which led us to screen for mutations in the human ILK and LAMA4 genes in patients with severe dilated cardiomyopathy. We identified 2 novel amino acid residue–altering mutations (2828C>T [Pro943Leu] and 3217C>T [Arg1073X]) in the integrin-interacting domain of the LAMA4 gene and 1 mutation (785C>T [Ala262Val]) in the ILK gene. Biacore quantitative protein/protein interaction data, which have been used to determine the equilibrium dissociation constants, point to the loss of integrin-binding capacity in case of the Pro943Leu ( K _d =5±3 μmol/L) and Arg1073X LAMA4 ( K _d =1±0.2 μmol/L) mutants compared with the wild-type LAMA4 protein ( K _d =440±20 nmol/L). Additional functional data point to the loss of endothelial cells in affected patients as a direct consequence of the mutant genes, which ultimately leads to heart failure. Conclusions— This is the first report on mutations in the laminin, integrin, and ILK system in human cardiomyopathy, which has consequences for endothelial cells as well as for cardiomyocytes, thus providing a new genetic basis for dilated cardiomyopathy in humans.

The evidence that active smoking is a risk factor for cardiovascular disease (CVD) and the leading cause of preventable death is overwhelming. However, numerous epidemiological findings indicate that even passive exposure to cigarette smoke may exert detrimental effects on vascular homoeostasis. Recent experimental data provide a deeper insight into the pathophysiological mechanisms linking secondhand smoke (SHS) to CVD. Importantly, most of these effects appear to be characterized by a rapid onset. For example, the relatively low doses of toxins inhaled by passive smoking are sufficient to elicit acute endothelial dysfunction, and these effects may be related, at least in part, to the inactivation of nitric oxide. Moreover, passive smoking may directly impair the viability of endothelial cells and reduce the number and functional activity of circulating endothelial progenitor cells. In addition, platelets of non-smokers appear to be susceptible to pro-aggregatory changes with every passive smoke exposure. Overall, SHS induces oxidative stress and promotes vascular inflammation. Apart from vascoconstriction and thrombus formation, however, the myocardial oxygen balance is further impaired by SHS-induced adrenergic stimulation and autonomic dysfunction. These data strongly suggest that passive smoking is capable of precipitating acute manifestations of CVD (atherothrombosis) and may also have a negative impact on the outcome of patients who suffer acute coronary syndromes.

Objectives— Human obesity is associated with elevated leptin levels and a high risk of death from cardiovascular disease. In the present study, we investigated the effects of leptin on vascular wound healing and arterial lesion growth in mice. Methods and Results— Wild-type mice placed on an atherogenic, high-fat diet had elevated (9-fold) leptin levels compared with their counterparts maintained on normal chow, and the former demonstrated significantly enhanced neointimal thickening after carotid artery injury with ferric chloride. The lesions forming in response to injury strongly expressed leptin receptor mRNA and protein. Unexpectedly, the atherogenic diet had no effect on injured vessels from leptin-deficient ob/ob mice despite aggravating obesity, diabetes, and hyperlipidemia in these animals. Daily administration of leptin to ob/ob mice during the 3-week period after injury reversed this phenotype, dramatically increasing neointimal thickness and the severity of luminal stenosis. Exogenous leptin also enhanced lesion growth and increased cellular proliferation in injured arteries from wild-type mice but had no effect on vessels from leptin receptor–deficient db/db mice. Conclusions— Our results raise the possibility that there might be a direct, leptin receptor-mediated link between the hyperleptinemia in human obesity and the increased risk for cardiovascular complications associated with this condition.

Objective: Reduced numbers of regulatory T (T_reg) cells have been observed in visceral adipose tissue of obese mice and humans. However, it is unknown whether human obesity affects circulating Treg cells and whether their number is associated with markers of systemic inflammation or glucose intolerance.Design and Methods: Peripheral blood mononuclear cells were isolated from venous blood of obese (BMI ≥ 27 kg/m²; n = 30) and nonobese (BMI ≥ 27 kg/m²; n = 13) individuals and analyzed using flow cytometry for the expression of CD4, CD25, and Foxp3.Results: Reduced circulating T_reg‐cell numbers were detected in obese compared with nonobese study participants (P = 0.038). Circulating CD4⁺CD25⁺CD127⁻Foxp3 T_reg cells inversely correlated with body weight (P = 0.009), BMI (P = 0.004) and plasma leptin levels (P = 0.004) and were reduced in subjects with hsCRP ≥ 3.0 mg/L (P = 0.034) or HbA1c ≥ 5.5% (P < 0.005). Receiver operating characteristic curve analysis revealed a cutoff of circulating Treg cells < 1.06% to be predictive for hsCRP levels ≥ 3.0 mg/L, and logistic regression showed that the risk of having hsCRP levels ≥ 3.0 mg/L was increased 9.6‐fold (P = 0.008), if T_reg cells were below this threshold. The T_reg cutoff for HbA1c levels ≥ 5.5% was 0.73%, and this cutoff also predicted an increased risk of having elevated levels of both hsCRP and HbA1c, if only obese subjects were examined.Conclusion: Our findings thus reveal an association between circulating T_reg cells and measures of adiposity, inflammation, and glucose intolerance. Although further prospective studies are needed, we present data suggesting that the determination of T_reg cells might be useful to identify obese subjects at increased risk of developing cardiovascular and/or metabolic complications.