Combined Raman- and AFM-based detection of biochemical and nanomechanical features of endothelial dysfunction in aorta isolated from ApoE/LDLR-/- mice.

Endothelial dysfunction is recognized as a critical condition in the development of cardiovascular disorders. This multifactorial process involves changes in the biochemical and mechanical properties of endothelial cells leading to disturbed release of vasoprotective mediators. Hypercholesterolemia and increased stiffness of the endothelial cortex are independently shown to result in reduced release of nitric oxide and thus endothelial dysfunction. However, direct evidence linking these parameters to each other is missing. Here, a novel method combining Raman spectroscopy for biochemical analysis and Atomic Force Microscopy (AFM) for analyzing the endothelial nanomechanics was established. Using this dual approach, the same areas of native ex vivo aortas were investigated, either derived from mice with endothelial dysfunction (ApoE/LDLR-/-) or wild type mice. In particular an increased intracellular lipid content and elevated cortical stiffness/elasticity were shown in ApoE/LDLR-/- aortas, demonstrating a direct link between endothelial dysfunction, the biochemical composition and the nanomechanical properties of endothelial cells.

Short-Chain Fatty Acid Propionate Protects From Hypertensive Cardiovascular Damage.

BACKGROUND: Arterial hypertension and its organ sequelae show characteristics of T cell-mediated inflammatory diseases. Experimental anti-inflammatory therapies have been shown to ameliorate hypertensive end-organ damage. Recently, the CANTOS study (Canakinumab Antiinflammatory Thrombosis Outcome Study) targeting interleukin-1ß demonstrated that anti-inflammatory therapy reduces cardiovascular risk. The gut microbiome plays a pivotal role in immune homeostasis and cardiovascular health. Short-chain fatty acids (SCFAs) are produced from dietary fiber by gut bacteria and affect host immune homeostasis. Here, we investigated effects of the SCFA propionate in 2 different mouse models of hypertensive cardiovascular damage. METHODS: To investigate the effect of SCFAs on hypertensive cardiac damage and atherosclerosis, wild-type NMRI or apolipoprotein E knockout-deficient mice received propionate (200 mmol/L) or control in the drinking water. To induce hypertension, wild-type NMRI mice were infused with angiotensin II (1.44 mg·kg-1·d-1 subcutaneous) for 14 days. To accelerate the development of atherosclerosis, apolipoprotein E knockout mice were infused with angiotensin II (0.72 mg·kg-1·d-1 subcutaneous) for 28 days. Cardiac damage and atherosclerosis were assessed using histology, echocardiography, in vivo electrophysiology, immunofluorescence, and flow cytometry. Blood pressure was measured by radiotelemetry. Regulatory T cell depletion using PC61 antibody was used to examine the mode of action of propionate. RESULTS: Propionate significantly attenuated cardiac hypertrophy, fibrosis, vascular dysfunction, and hypertension in both models. Susceptibility to cardiac ventricular arrhythmias was significantly reduced in propionate-treated angiotensin II-infused wild-type NMRI mice. Aortic atherosclerotic lesion area was significantly decreased in propionate-treated apolipoprotein E knockout-deficient mice. Systemic inflammation was mitigated by propionate treatment, quantified as a reduction in splenic effector memory T cell frequencies and splenic T helper 17 cells in both models, and a decrease in local cardiac immune cell infiltration in wild-type NMRI mice. Cardioprotective effects of propionate were abrogated in regulatory T cell-depleted angiotensin II-infused mice, suggesting the effect is regulatory T cell-dependent. CONCLUSIONS: Our data emphasize an immune-modulatory role of SCFAs and their importance for cardiovascular health. The data suggest that lifestyle modifications leading to augmented SCFA production could be a beneficial nonpharmacological preventive strategy for patients with hypertensive cardiovascular disease.

The endothelial αENaC contributes to vascular endothelial function in vivo.

The Epithelial Sodium Channel (ENaC) is a key player in renal sodium homeostasis. The expression of α ß Î³ ENaC subunits has also been described in the endothelium and vascular smooth muscle, suggesting a role in vascular function. We recently demonstrated that endothelial ENaC is involved in aldosterone-modulated endothelial stiffness. Here we explore the functional role of the endothelial αENaC subunit in vascular function in vivo. Compared to littermates, mice with conditional αENaC subunit gene inactivation in the endothelium only (endo-αENaC Knock Out mice) had no difference in their physiological parameters such as systolic blood pressure or heart rate. Acute and long-term renal Na+ handlings were not affected, indicating that endothelial αENaC subunit is not involved in renal sodium balance. Pharmacological inhibition of ENaC with benzamil blunted acetylcholine-induced nitric oxide production in mesenteric arteries from wild type mice but not in endo-αENaC KO mice, suggesting a critical role of endothelial ENaC in agonist-induced nitric oxide production. In endo-αENaC KO mice, compensatory mechanisms occurred and steady state vascular function was not altered except for flow-mediated dilation. Our data suggest that endothelial αENaC contributes to vascular endothelial function in vivo.

Nanomechanics of the endothelial glycocalyx contribute to Na+-induced vascular inflammation.

High dietary salt (NaCl) is a known risk factor for cardiovascular pathologies and inflammation. High plasma Na+ concentrations (high Na+) have been shown to stiffen the endothelial cortex and decrease nitric oxide (NO) release, a hallmark of endothelial dysfunction. Here we report that chronic high Na+ damages the endothelial glycocalyx (eGC), induces release of inflammatory cytokines from the endothelium and promotes monocyte adhesion. Single cell force spectroscopy reveals that high Na+ enhances vascular adhesion protein-1 (VCAM-1)-dependent adhesion forces between monocytes and endothelial surface, giving rise to increased numbers of adherent monocytes on the endothelial surface. Mineralocorticoid receptor antagonism with spironolactone prevents high Na+-induced eGC deterioration, decreases monocyte-endothelium interactions, and restores endothelial function, indicated by increased release of NO. Whereas high Na+ decreases NO release, it induces endothelial release of the pro-inflammatory cytokines IL-1ß and TNFα. However, in contrast to chronic salt load (hours), in vivo and in vitro, an acute salt challenge (minutes) does not impair eGC function. This study identifies the eGC as important mediator of inflammatory processes and might further explain how dietary salt contributes to endothelialitis and cardiovascular pathologies by linking endothelial nanomechanics with vascular inflammation.

Aldosterone synthase knockout mouse as a model for sodium-induced endothelial sodium channel up-regulation in vascular endothelium.

Recently, a novel feedforward activation of the endothelial epithelial sodium channel (ENaC) [endothelial sodium channel (EnNaC)] by sodium was reported that counteracts ENaC function in kidney. In the absence of aldosterone, a rise in extracellular sodium (>145 mM) increases EnNaC surface abundance, thereby stiffening the cortex of vascular endothelial cells (ECs) in vitro. The latter reduces the release of NO-the hallmark of endothelial dysfunction. Here, we test whether high extracellular sodium per se increases EnNaC expression and cortical stiffness in an aldosterone synthase (Cyp11b2)-deficient (AS(-/-)) mouse model. Therefore, we employed in situ ECs of ex vivo aorta preparations from wild-type (WT) and AS(-/-). EnNaC surface expression (-16%) and cortical stiffness (-22%) were reduced in AS(-/-), compared with WT, whereas NO secretion was exclusively detectable in AS(-/-). EnNaC inhibition with benzamil decreased stiffness in both, while mineralocorticoid receptor antagonism diminished stiffness only in the WT. In the absence of aldosterone, high sodium (150 mM) increased EnNaC surface expression ex vivo (plus 19%) and cortical stiffness ex vivo (plus 41%) and in vivo (plus 44%). Application of aldosterone adjusted the stiffness of AS(-/-) to the WT level. We conclude that high sodium per se determines EnNaC expression and consequently endothelial cortical nanomechanics, thus likely contributing to endothelial dysfunction.

Soluble adenylyl cyclase in vascular endothelium: gene expression control of epithelial sodium channel-α, Na+/K+-ATPase-α/ß, and mineralocorticoid receptor.

The Ca(2+)- and bicarbonate-activated soluble adenylyl cyclase (sAC) has been identified recently as an important mediator of aldosterone signaling in the kidney. Nuclear sAC has been reported to stimulate cAMP response element-binding protein 1 phosphorylation via protein kinase A, suggesting an alternative cAMP pathway in the nucleus. In this study, we analyzed the sAC as a potential modulator of endothelial stiffness in the vascular endothelium. We determined the contribution of sAC to cAMP response element-mediated transcriptional activation in vascular endothelial cells and kidney collecting duct cells. Inhibition of sAC by the specific inhibitor KH7 significantly reduced cAMP response element-mediated promoter activity and affected cAMP response element-binding protein 1 phosphorylation. Furthermore, KH7 and anti-sAC small interfering RNA significantly decreased mRNA and protein levels of epithelial sodium channel-α and Na(+)/K(+)-ATPase-α. Using atomic force microscopy, a nano-technique that measures stiffness and deformability of living cells, we detected significant endothelial cell softening after sAC inhibition. Our results suggest that the sAC is a regulator of gene expression involved in aldosterone signaling and an important regulator of endothelial stiffness. Additional studies are warranted to investigate the protective action of sAC inhibitors in humans for potential clinical use.