Background: The impairment of endothelium‐dependent vasodilation, increased endothelial permeability, and glycocalyx degradation are all important pathophysiological components of endothelial dysfunction. However, it is still not clear whether in atherosclerosis, glycocalyx injury precedes other features of endothelial dysfunction or these events coincide. Methods and Results: Herein, we demonstrate that in 4‐ to 8‐week‐old apolipoprotein E/low‐density lipoprotein receptor‐deficient mice, at the stage before development of atherosclerotic plaques, impaired acetylcholine‐induced vasodilation, reduced NO production in aorta, and increased endothelial permeability were all observed; however, flow‐mediated dilation in the femoral artery was fully preserved. In 4‐week‐old mice, glycocalyx coverage was reduced and endothelial stiffness was increased, whereas glycocalyx length was significantly decreased at 8 weeks of age. Early changes in endothelial function were also featured by increased plasma concentration of biomarkers of glycocalyx disruption (endocan), biomarkers of endothelial inflammation (soluble vascular cell adhesion molecule 1), increased vascular permeability (angiopoietin 2), and alterations in hemostasis (tissue plasminogen activator and plasminogen activator inhibitor 1). In 28‐week‐old mice, at the stage of advanced atherosclerotic plaque development, impaired NO production and nearly all other features of endothelial dysfunction were changed to a similar extent, compared with the preatherosclerotic plaque phase. The exceptions were the occurrence of acetylcholine‐induced vasoconstriction in the aorta and brachiocephalic artery, impaired flow‐mediated vasodilation in the femoral artery, and further reduction of glycocalyx length and coverage with a concomitant further increase in endothelial permeability. Conclusions: In conclusion, even at the early stage before the development of atherosclerotic plaques, endothelial dysfunction is a complex multifactorial response that has not been previously appreciated.

Tumour necrosis factor α (TNF-α) represents a classical pro-inflammatory cytokine, and its increased levels positively correlate with the severity of many cardiovascular diseases. Surprisingly, some heart failure patients receiving high doses of anti-TNF-α antibodies showed serious health worsening. This work aimed to examine the role of TNF-α signalling on the development and progression of myocarditis and heart-specific autoimmunity. Mice with genetic deletion of TNF-α (Tnf^+/− and Tnf^−/−) and littermate controls (Tnf^+/+) were used to study myocarditis in the inducible and the transgenic T cell receptor (TCRM) models. Tnf^+/− and Tnf^−/− mice immunized with α-myosin heavy chain peptide (αMyHC) showed reduced myocarditis incidence, but the susceptible animals developed extensive inflammation in the heart. In the TCRM model, defective TNF-α production was associated with increased mortality at a young age due to cardiomyopathy and cardiac fibrosis. We could confirm that TNF-α as well as the secretome of antigen-activated heart-reactive effector CD4⁺ T (T_eff) cells effectively activated the adhesive properties of cardiac microvascular endothelial cells (cMVECs). Our data suggested that TNF-α produced by endothelial in addition to T_eff cells promoted leucocyte adhesion to activated cMVECs. Analysis of CD4⁺ T lymphocytes from both models of myocarditis showed a strongly increased fraction of T_eff cells in hearts, spleens, and in the blood of Tnf^+/− and Tnf^−/− mice. Indeed, antigen-activated Tnf^−/− T_eff cells showed prolonged long-term survival and TNF-α cytokine-induced cell death of heart-reactive T_eff. TNF-α signalling promotes myocarditis development by activating cardiac endothelial cells. However, in the case of established disease, TNF-α protects from exacerbating cardiac inflammation by inducing activation-induced cell death of heart-reactive T_eff. These data might explain the lack of success of standard anti-TNF-α therapy in heart failure patients and open perspectives for T cell–targeted approaches. A dual role of TNF-α in autoimmune myocarditis. The top panel illustrates that TNF-α can have a pro-inflammatory effect by activating cardiac endothelial cells and an anti-inflammatory effect by inducing activation-induced cell death of heart-reactive T cells. The bottom panel depicts two alternative scenarios in case of the absence of TNF-α.

The role of epithelial sodium channel (ENaC) activity in the regulation of endothelial function is not clear. Here, we analyze the role of ENaC in the regulation of endothelium-dependent vasodilation and endothelial permeability in vivo in mice with conditional αENaC subunit gene inactivation in the endothelium (endo-αENaC^KO mice) using unique MRI-based analysis of acetylcholine-, flow-mediated dilation and vascular permeability. Mice were challenged or not with lipopolysaccharide (LPS, from Salmonella typhosa, 10 mg/kg, i.p.). In addition, changes in vascular permeability in ex vivo organs were analyzed by Evans Blue assay, while changes in vascular permeability in perfused mesenteric artery were determined by a FITC-dextran-based assay. In basal conditions, Ach-induced response was completely lost, flow-induced vasodilation was inhibited approximately by half but endothelial permeability was not changed in endo-αENaC^KO vs. control mice. In LPS-treated mice, both Ach- and flow-induced vasodilation was more severely impaired in endo-αENaC^KO vs. control mice. There was also a dramatic increase in permeability in lungs, brain and isolated vessels as evidenced by in vivo and ex vivo analysis in endotoxemic endo-αENaC^KO vs. control mice. The impaired endothelial function in endotoxemia in endo-αENaC^KO was associated with a decrease of lectin and CD31 endothelial staining in the lung as compared with control mice. In conclusion, the activity of endothelial ENaC in vivo contributes to endothelial-dependent vasodilation in the physiological conditions and the preservation of endothelial barrier integrity in endotoxemia.

Endothelial dysfunction (ED) and red blood cell distribution width (RDW) are both prognostic factors in heart failure (HF), but the relationship between them is not clear. In this study, we used a unique mouse model of chronic HF driven by cardiomyocyte-specific overexpression of activated Gαq protein (Tgαq*44 mice) to characterize the relationship between the development of peripheral ED and the occurrence of structural nanomechanical and biochemical changes in red blood cells (RBCs). Systemic ED was detected in vivo in 8-month-old Tgαq*44 mice, as evidenced by impaired acetylcholine-induced vasodilation in the aorta and increased endothelial permeability in the brachiocephalic artery. ED in the aorta was associated with impaired nitric oxide (NO) production in the aorta and diminished systemic NO bioavailability. ED in the aorta was also characterized by increased superoxide and eicosanoid production. In 4- to 6-month-old Tgαq*44 mice, RBC size and membrane composition displayed alterations that did not result in significant changes in their nanomechanical and functional properties. However, 8-month-old Tgαq*44 mice presented greatly accentuated structural and size changes and increased RBC stiffness. In 12-month-old Tgαq*44 mice, the erythropathy was featured by severely altered RBC shape and elasticity, increased RDW, impaired RBC deformability, and increased oxidative stress (gluthatione (GSH)/glutathione disulfide (GSSG) ratio). Moreover, RBCs taken from 12-month-old Tgαq*44 mice, but not from 12-month-old FVB mice, coincubated with aortic rings from FVB mice, induced impaired endothelium-dependent vasodilation and this effect was partially reversed by an arginase inhibitor [2(S)-amino-6-boronohexanoic acid]. In the Tgαq*44 murine model of HF, systemic ED accelerates erythropathy and, conversely, erythropathy may contribute to ED. These results suggest that erythropathy may be regarded as a marker and a mediator of systemic ED in HF. RBC arginase and possibly other RBC-mediated mechanisms may represent novel therapeutic targets for systemic ED in HF.

Background: Long‐term feeding with a high‐fat diet (HFD) induces endothelial dysfunction in mice, but early HFD‐induced effects on endothelium have not been well characterized. Methods and Results: Using an magnetic resonance imaging‐based methodology that allows characterization of endothelial function in vivo, we demonstrated that short‐term (2 weeks) feeding with a HFD to C57BL/6 mice or to E3L.CETP mice resulted in the impairment of acetylcholine‐induced response in the abdominal aorta (AA), whereas, in the thoracic aorta (TA), the acetylcholine‐induced response was largely preserved. Similarly, HFD resulted in arterial stiffness in the AA, but not in the TA. The difference in HFD‐induced response was ascribed to distinct characteristics of perivascular adipose tissue in the TA and AA, related to brown‐ and white‐like adipose tissue, respectively, as assessed by histology, immunohistochemistry, and Raman spectroscopy. In contrast, short‐term HFD‐induced endothelial dysfunction could not be linked to systemic insulin resistance, changes in plasma concentration of nitrite, or concentration of biomarkers of glycocalyx disruption (syndecan‐1 and endocan), endothelial inflammation (soluble form of vascular cell adhesion molecule 1, soluble form of intercellular adhesion molecule 1 and soluble form of E‐selectin), endothelial permeability (soluble form of fms‐like tyrosine kinase 1 and angiopoietin 2), and hemostasis (tissue plasminogen activator and plasminogen activator inhibitor 1). Conclusions: Short‐term feeding with a HFD induces endothelial dysfunction in the AA but not in the TA, which could be ascribed to a differential response of perivascular adipose tissue to a HFD in the AA versus TA. Importantly, early endothelial dysfunction in the AA is not linked to elevation of classical systemic biomarkers of endothelial dysfunction.