Arginase-1 Deletion in Erythrocytes Promotes Vascular Calcification via Enhanced GSNOR (S-Nitrosoglutathione Reductase) Expression and NO Signaling in Smooth Muscle Cells.

BACKGROUND: Erythrocytes (red blood cells) participate in the control of vascular NO bioavailability. The purpose of this study was to determine whether and how genetic deletion of ARG1 (arginase-1) affects vascular smooth muscle cell NO signaling, osteoblastic differentiation, and atherosclerotic lesion calcification. METHODS: Atherosclerosis-prone mice with conditional, erythrocyte-restricted deletion of ARG1 (apoE-/- red blood cell.ARG1 knockout) were generated and vascular calcification studied using molecular imaging of the osteogenic activity agent OsteoSense, Alizarin staining or immunohistochemistry, qPCR of osteogenic markers and ex vivo assays. RESULTS: Atherosclerotic lesion size at the aortic root did not differ, but calcification was significantly more pronounced in apoE-/- mice lacking erythrocyte ARG1. Incubation of murine and human VSMCs with lysed erythrocyte membranes from apoE-/- red blood cell. ARG1-knockout mice accelerated their osteogenic differentiation, and mRNA transcripts of osteogenic markers decreased following NO scavenging. In addition to NO signaling via sGC (soluble guanylyl cyclase), overexpression of GSNOR (S-nitrosoglutathione reductase) enhanced degradation of S-nitrosoglutathione to glutathione and reduced protein S-nitrosation of HSP (heat shock protein)-70 were identified as potential mechanisms of vascular smooth muscle cell calcification in mice lacking ARG1 in erythrocytes, and calcium phosphate deposition was enhanced by heat shock and prevented by GSNOR inhibition. Messenger RNA levels of enzymes metabolizing the arginase products L-ornithine and L-proline also were elevated in VSMCs, paralleled by increased proliferation, myofibroblast marker and collagen type 1 expression. CONCLUSIONS: Our findings support an important role of erythrocyte ARG1 for NO bioavailability and L-arginine metabolism in VSMCs, which controls atherosclerotic lesion composition and calcification.

Cardiac MRI-based right-to-left ventricular blood pool T2 relaxation times ratio correlates with exercise capacity in patients with chronic heart failure.

BACKGROUND: MRI T2 mapping has been proven to be sensitive to the level of blood oxygenation. We hypothesized that impaired exercise capacity in chronic heart failure is associated with a greater difference between right (RV) to left ventricular (LV) blood pool T2 relaxation times due to a higher level of peripheral blood desaturation, compared to patients with preserved exercise capacity and to healthy controls. METHODS: Patients with chronic heart failure (n = 70) who had undergone both cardiac MRI (CMR) and a 6-min walk test (6MWT) were retrospectively identified. Propensity score matched healthy individuals (n = 35) served as control group. CMR analyses included cine acquisitions and T2 mapping to obtain blood pool T2 relaxation times of the RV and LV. Following common practice, age- and gender-adjusted nominal distances and respective percentiles were calculated for the 6MWT. The relationship between the RV/LV T2 blood pool ratio and the results from 6MWT were evaluated by Spearman's correlation coefficients and regression analyses. Inter-group differences were assessed by independent t-tests and univariate analysis of variance. RESULTS: The RV/LV T2 ratio moderately correlated with the percentiles of nominal distances in the 6MWT (r = 0.66) while ejection fraction, end-diastolic and end-systolic volumes showed no correlation (r = 0.09, 0.07 and - 0.01, respectively). In addition, there were significant differences in the RV/LV T2 ratio between patients with and without significant post-exercise dyspnea (p = 0.001). Regression analyses showed that RV/LV T2 ratio was an independent predictor of the distance walked and the presence of post-exercise dyspnea (p < 0.001). CONCLUSION: The proposed RV/LV T2 ratio, obtained by two simple measurements on a routinely acquired four-chamber T2 map, was superior to established parameters of cardiac function to predict exercise capacity and the presence of post-exercise dyspnea in patients with chronic heart failure.

Evaluation of right atrial function by two-dimensional echocardiography and strain imaging in patients with RCA CTO recanalization.

OBJECTIVES: The right heart is mainly supplied with blood by the right coronary artery (RCA). The impact of RCA chronic total occlusion (CTO) on the function of the right heart [right atrium (RA) and ventricle (RV)] and whether successful recanalization of a RCA CTO improves the function of the right heart is not clearly understood yet. We aimed to evaluate right atrial function after recanalization of the RCA using transthoracic echocardiography with additional strain imaging. METHODS AND RESULTS: Fifty-five patients undergoing RCA CTO recanalization at the University Medical Center of Mainz were included in the study. Right atrial strain was assessed before and 6 months after successful CTO revascularization. The median age of the total collective was 66 (50-90) years. We did not find difference in our analysis of RA Volume (p 0.086), RA area (p 0.093), RA major dimension (p 0.32) and RA minor dimension (p 0.139) at baseline and follow-up. Mean RA reservoir strain at baseline was 30.9% (21.1-43.0) vs. 33.4% (20.7-47.7) at follow up (p < 0.001). Mean RA conduit strain was - 17.5% (- 10.7-(- 29.7)) at baseline vs. - 18.2% (- 9.6-(- 31.7)) at follow-up (p = 0.346). Mean RA contraction strain was - 12.9% (- 8.0- (- 21.3)) at baseline vs. - 15.5% (- 8.7-(- 26.6)) at follow-up (p < 0.001). CONCLUSION: Right atrial function was altered in patients with RCA CTO. Successful revascularisation of an RCA CTO improved RA function assessed by strain imaging at follow-up.

Tubulin-folding cofactor E deficiency promotes vascular dysfunction by increased endoplasmic reticulum stress.

AIMS: Assessment of endothelial function in humans by measuring flow-mediated dilation (FMD) risk-stratifies individuals with established cardiovascular disease, whereas its predictive value is limited in primary prevention. We therefore aimed to establish and evaluate novel markers of FMD at the population level. METHODS AND RESULTS: In order to identify novel targets that were negatively correlated with FMD and investigate their contribution to vascular function, we performed a genome-wide association study (GWAS) of 4175 participants of the population based Gutenberg Health Study. Subsequently, conditional knockout mouse models deleting the gene of interest were generated and characterized. GWAS analysis revealed that single-nucleotide polymorphisms (SNPs) in the tubulin-folding cofactor E (TBCE) gene were negatively correlated with endothelial function and TBCE expression. Vascular smooth muscle cell (VSMC)-targeted TBCE deficiency was associated with endothelial dysfunction, aortic wall hypertrophy, and endoplasmic reticulum (ER) stress-mediated VSMC hyperproliferation in mice, paralleled by calnexin up-regulation and exacerbated by the blood pressure hormone angiotensin II. Treating SMMHC-ERT2-Cre+/-TBCEfl/fl mice with the ER stress modulator tauroursodeoxycholic acid amplified Raptor/Beclin-1-dependent autophagy and reversed vascular dysfunction. CONCLUSION: TBCE and tubulin homeostasis seem to be novel predictors of vascular function and offer a new drug target to ameliorate ER stress-dependent vascular dysfunction.

TNF-α blockade may lead to improvement of vascular function in psoriasis patients.

Psoriasis is one of the most common chronic inflammatory skin diseases and at the same time a risk factor for cardiovascular disease. Interleukin-17A (IL-17A)-mediated inflammation in psoriasis may lead to vascular dysfunction. This study aimed at investigating whether anti-inflammatory treatment by tumor necrosis factor (TNF)-α blockade alters vascular function in psoriasis patients. A total of 11 patients with psoriasis who underwent treatment with either adalimumab (n = 8) or etanercept (n = 3), 10 healthy control individuals and 14 patients with coronary artery disease (CAD) were included in this study. Treatment response was assessed using the Psoriasis Area and Severity Index (PASI) score. Endothelial reactivity and resting endothelium-dependent vascular tone were assessed by ultrasound measurement of flow-mediated dilation (FMD) and low-flow-mediated constriction (l-FMC), respectively. FMD was slightly impaired in psoriasis patients compared to healthy controls. Anti-TNF-α treatment did not significantly change FMD levels. Psoriasis patients showed a trend towards increased baseline vascular activity compared to healthy controls. Anti-TNF-α treatment significantly improved l-FMC in psoriasis patients. Noteworthy, both FMD and l-FMC in psoriasis patients were comparable to those in patients with CAD; however, an important influence of age differences between the groups or co-existent classical cardiovascular risk factors on FMD and l-FMC cannot be ruled out by our small study. The results suggest that anti-inflammatory treatment with TNF-α blockade improves vascular function in patients with psoriasis, mainly by altering baseline vascular tone. Further studies will be necessary to establish the potentially protective impact of anti-inflammatory therapy on vascular function in patients with chronic inflammatory diseases.

Acute mental stress drives vascular inflammation and promotes plaque destabilization in mouse atherosclerosis.

AIMS: Mental stress substantially contributes to the initiation and progression of human disease, including cardiovascular conditions. We aim to investigate the underlying mechanisms of these contributions since they remain largely unclear. METHODS AND RESULTS: Here, we show in humans and mice that leucocytes deplete rapidly from the blood after a single episode of acute mental stress. Using cell-tracking experiments in animal models of acute mental stress, we found that stress exposure leads to prompt uptake of inflammatory leucocytes from the blood to distinct tissues including heart, lung, skin, and, if present, atherosclerotic plaques. Mechanistically, we found that acute stress enhances leucocyte influx into mouse atherosclerotic plaques by modulating endothelial cells. Specifically, acute stress increases adhesion molecule expression and chemokine release through locally derived norepinephrine. Either chemical or surgical disruption of norepinephrine signalling diminished stress-induced leucocyte migration into mouse atherosclerotic plaques. CONCLUSION: Our data show that acute mental stress rapidly amplifies inflammatory leucocyte expansion inside mouse atherosclerotic lesions and promotes plaque vulnerability.

Intramyocardial Inflammation after COVID-19 Vaccination: An Endomyocardial Biopsy-Proven Case Series.

Myocarditis in response to COVID-19 vaccination has been reported since early 2021. In particular, young male individuals have been identified to exhibit an increased risk of myocardial inflammation following the administration of mRNA-based vaccines. Even though the first epidemiological analyses and numerous case reports investigated potential relationships, endomyocardial biopsy (EMB)-proven cases are limited. Here, we present a comprehensive histopathological analysis of EMBs from 15 patients with reduced ejection fraction (LVEF = 30 (14-39)%) and the clinical suspicion of myocarditis following vaccination with Comirnaty® (Pfizer-BioNTech) (n = 11), Vaxzevria® (AstraZenica) (n = 2) and Janssen® (Johnson & Johnson) (n = 2). Immunohistochemical EMB analyses reveal myocardial inflammation in 14 of 15 patients, with the histopathological diagnosis of active myocarditis according the Dallas criteria (n = 2), severe giant cell myocarditis (n = 2) and inflammatory cardiomyopathy (n = 10). Importantly, infectious causes have been excluded in all patients. The SARS-CoV-2 spike protein has been detected sparsely on cardiomyocytes of nine patients, and differential analysis of inflammatory markers such as CD4+ and CD8+ T cells suggests that the inflammatory response triggered by the vaccine may be of autoimmunological origin. Although a definitive causal relationship between COVID-19 vaccination and the occurrence of myocardial inflammation cannot be demonstrated in this study, data suggest a temporal connection. The expression of SARS-CoV-2 spike protein within the heart and the dominance of CD4+ lymphocytic infiltrates indicate an autoimmunological response to the vaccination.

Role of angiopoietin-2 in venous thrombus resolution and chronic thromboembolic disease.

BACKGROUND: Defective angiogenesis, incomplete thrombus revascularisation and fibrosis are considered critical pathomechanisms of chronic thromboembolic pulmonary hypertension (CTEPH) after pulmonary embolism. Angiopoietin-2 (ANGPT2) has been shown to regulate angiogenesis, but its importance for thrombus resolution and remodelling is unknown. METHODS: ANGPT2 plasma concentrations were measured in patients with CTEPH (n=68) and acute pulmonary embolism (n=84). Tissue removed during pulmonary endarterectomy (PEA) for CTEPH was analysed (immuno)histologically. A mouse model of inferior vena cava ligation was used to study the kinetics of venous thrombus resolution in wild-type mice receiving recombinant ANGPT2 via osmotic pumps, and in transgenic mice overexpressing ANGPT2 in endothelial cells. RESULTS: Circulating ANGPT2 levels were higher in CTEPH patients compared to patients with idiopathic pulmonary arterial hypertension and healthy controls, and decreased after PEA. Plasma ANGPT2 levels were elevated in patients with pulmonary embolism and diagnosis of CTEPH during follow-up. Histological analysis of PEA specimens confirmed increased ANGPT2 expression, and low levels of phosphorylated TIE2 were observed in regions with early-organised pulmonary thrombi, myofibroblasts and fibrosis. Microarray and high-resolution microscopy analysis could localise ANGPT2 overexpression to endothelial cells, and hypoxia and transforming growth factor-ß1 were identified as potential stimuli. Gain-of-function experiments in mice demonstrated that exogenous ANGPT2 administration and transgenic endothelial ANGPT2 overexpression resulted in delayed venous thrombus resolution, and thrombi were characterised by lower TIE2 phosphorylation and fewer microvessels. CONCLUSION: Our findings suggest that ANGPT2 delays venous thrombus resolution and that overexpression of ANGPT2 contributes to thrombofibrosis and may thus support the transition from pulmonary embolism to CTEPH.

Ablation of lysozyme M-positive cells prevents aircraft noise-induced vascular damage without improving cerebral side effects.

Aircraft noise induces vascular and cerebral inflammation and oxidative stress causing hypertension and cardiovascular/cerebral dysfunction. With the present studies, we sought to determine the role of myeloid cells in the vascular vs. cerebral consequences of exposure to aircraft noise. Toxin-mediated ablation of lysozyme M+ (LysM+) myeloid cells was performed in LysMCreiDTR mice carrying a cre-inducible diphtheria toxin receptor. In the last 4d of toxin treatment, the animals were exposed to noise at maximum and mean sound pressure levels of 85 and 72 dB(A), respectively. Flow cytometry analysis revealed accumulation of CD45+, CD11b+, F4/80+, and Ly6G-Ly6C+ cells in the aortas of noise-exposed mice, which was prevented by LysM+ cell ablation in the periphery, whereas brain infiltrates were even exacerbated upon ablation. Aircraft noise-induced increases in blood pressure and endothelial dysfunction of the aorta and retinal/mesenteric arterioles were almost completely normalized by ablation. Correspondingly, reactive oxygen species in the aorta, heart, and retinal/mesenteric vessels were attenuated in ablated noise-exposed mice, while microglial activation and abundance in the brain was greatly increased. Expression of phagocytic NADPH oxidase (NOX-2) and vascular cell adhesion molecule-1 (VCAM-1) mRNA in the aorta was reduced, while NFκB signaling appeared to be activated in the brain upon ablation. In sum, we show dissociation of cerebral and peripheral inflammatory reactions in response to aircraft noise after LysM+ cell ablation, wherein peripheral myeloid inflammatory cells represent a dominant part of the pathomechanism for noise stress-induced cardiovascular effects and their central nervous counterparts, microglia, as key mediators in stress responses.

Vitamin K antagonism impairs the bone marrow microenvironment and hematopoiesis.

Vitamin K antagonists (VKAs) have been used in 1% of the world's population for prophylaxis or treatment of thromboembolic events for 64 years. Impairment of osteoblast function and osteoporosis has been described in patients receiving VKAs. Given the involvement of cells of the bone marrow microenvironment (BMM), such as mesenchymal stem cells (MSCs) and macrophages, as well as other factors such as the extracellular matrix for the maintenance of normal hematopoietic stem cells (HSCs), we investigated a possible effect of VKAs on hematopoiesis via the BMM. Using various transplantation and in vitro assays, we show here that VKAs alter parameters of bone physiology and reduce functional HSCs 8-fold. We implicate impairment of the functional, secreted, vitamin K-dependent, γ-carboxylated form of periostin by macrophages and, to a lesser extent, MSCs of the BMM and integrin ß3-AKT signaling in HSCs as at least partly causative of this effect, with VKAs not being directly toxic to HSCs. In patients, VKA use associates with modestly reduced leukocyte and monocyte counts, albeit within the normal reference range. VKAs decrease human HSC engraftment in immunosuppressed mice. Following published examples that alteration of the BMM can lead to hematological malignancies in mice, we describe, without providing a causal link, that the odds of VKA use are higher in patients with vs without a diagnosis of myelodysplastic syndrome (MDS). These results demonstrate that VKA treatment impairs HSC function via impairment of the BMM and the periostin/integrin ß3 axis, possibly associating with increased MDS risk.

Compressed sensing acceleration of cardiac cine imaging allows reliable and reproducible assessment of volumetric and functional parameters of the left and right atrium.

OBJECTIVES: To compare volumetric and functional parameters of the atria derived from highly accelerated compressed sensing (CS)-based cine sequences in comparison to conventional (Conv) cine imaging. METHODS: CS and Conv cine sequences were acquired in 101 subjects (82 healthy volunteers (HV) and 19 patients with heart failure with reduced ejection fraction (HFrEF)) using a 3T MR scanner in this single-center study. Time-volume analysis of the left (LA) and right atria (RA) were performed in both sequences to evaluate atrial volumes and function (total, passive, and active emptying fraction). Inter-sequence and inter- and intra-reader agreement were analyzed using correlation, intraclass correlation (ICC), and Bland-Altman analysis. RESULTS: CS-based cine imaging led to a 69% reduction of acquisition time. There was significant difference in atrial parameters between CS and Conv cine, e.g., LA minimal volume (LAVmin) (Conv 24.0 ml (16.7-32.7), CS 25.7 ml (19.2-35.2), p < 0.0001) or passive emptying fraction (PEF) (Conv 53.9% (46.7-58.4), CS 49.0% (42.0-54.1), p < 0.0001). However, there was high correlation between the techniques, yielding good to excellent ICC (0.76-0.99) and small mean of differences in Bland-Altman analysis (e.g. LAVmin - 2.0 ml, PEF 3.3%). Measurements showed high inter- (ICC > 0.958) and intra-rater (ICC > 0.934) agreement for both techniques. CS-based parameters (PEF AUC = 0.965, LAVmin AUC = 0.864) showed equivalent diagnostic ability compared to Conv cine imaging (PEF AUC = 0.989, LAVmin AUC = 0.859) to differentiate between HV and HFrEF. CONCLUSION: Atrial volumetric and functional evaluation using CS cine imaging is feasible with relevant reduction of acquisition time, therefore strengthening the role of CS in clinical CMR for atrial imaging. KEY POINTS: • Reliable assessment of atrial volumes and function based on compressed sensing cine imaging is feasible. • Compressed sensing reduces scan time and has the potential to overcome obstacles of conventional cine imaging. • No significant differences for subjective image quality, inter- and intra-rater agreement, and ability to differentiate healthy volunteers and heart failure patients were detected between conventional and compressed sensing cine imaging.

Stabilization of Perivascular Mast Cells by Endothelial CNP (C-Type Natriuretic Peptide).

OBJECTIVE: Activated perivascular mast cells (MCs) participate in different cardiovascular diseases. Many factors provoking MC degranulation have been described, while physiological counterregulators are barely known. Endothelial CNP (C-type natriuretic peptide) participates in the maintenance of vascular barrier integrity, but the target cells and mechanisms are unclear. Here, we studied whether MCs are regulated by CNP. Approach and Results: In cultured human and murine MCs, CNP activated its specific GC (guanylyl cyclase)-B receptor and cyclic GMP signaling. This enhanced cyclic GMP-dependent phosphorylation of the cytoskeleton-associated VASP (vasodilator-stimulated phosphoprotein) and inhibited ATP-evoked degranulation. To elucidate the relevance in vivo, mice with a floxed GC-B (Npr2) gene were interbred with a Mcpt5-CreTG line to generate mice lacking GC-B in connective tissue MCs (MC GC-B knockout). In anesthetized mice, acute ischemia-reperfusion of the cremaster muscle microcirculation provoked extensive MC degranulation and macromolecule extravasation. Superfusion of CNP markedly prevented MC activation and endothelial barrier disruption in control but not in MC GC-B knockout mice. Notably, already under resting conditions, such knockout mice had increased numbers of degranulated MCs in different tissues, together with elevated plasma chymase levels. After transient coronary occlusion, their myocardial areas at risk and with infarction were enlarged. Moreover, MC GC-B knockout mice showed augmented perivascular neutrophil infiltration and deep vein thrombosis in a model of inferior vena cava ligation. CONCLUSIONS: CNP, via GC-B/cyclic GMP signaling, stabilizes resident perivascular MCs at baseline and prevents their excessive activation under pathological conditions. Thereby CNP contributes to the maintenance of vascular integrity in physiology and disease.

Endothelial GLP-1 (Glucagon-Like Peptide-1) Receptor Mediates Cardiovascular Protection by Liraglutide In Mice With Experimental Arterial Hypertension.

OBJECTIVE: Cardiovascular outcome trials demonstrated that GLP-1 (glucagon-like peptide-1) analogs including liraglutide reduce the risk of cardiovascular events in type 2 diabetes mellitus. Whether GLP-1 analogs reduce the risk for atherosclerosis independent of glycemic control is challenging to elucidate as the GLP-1R (GLP-1 receptor) is expressed on different cell types, including endothelial and immune cells. Approach and Results: Here, we reveal the cardio- and vasoprotective mechanism of the GLP-1 analog liraglutide at the cellular level in a murine, nondiabetic model of arterial hypertension. Wild-type (C57BL/6J), global (Glp1r-/-), as well as endothelial (Glp1rflox/floxxCdh5cre) and myeloid cell-specific knockout mice (Glp1rflox/floxxLysMcre) of the GLP-1R were studied, and arterial hypertension was induced by angiotensin II. Liraglutide treatment normalized blood pressure, cardiac hypertrophy, vascular fibrosis, endothelial dysfunction, oxidative stress, and vascular inflammation in a GLP-1R-dependent manner. Mechanistically, liraglutide reduced leukocyte rolling on the endothelium and infiltration of myeloid Ly6G-Ly6C+ and Ly6G+Ly6C+ cells into the vascular wall. As a consequence, liraglutide prevented vascular oxidative stress, reduced S-glutathionylation as a marker of eNOS (endothelial NO synthase) uncoupling, and increased NO bioavailability. Importantly, all of these beneficial cardiovascular effects of liraglutide persisted in myeloid cell GLP-1R-deficient (Glp1rflox/floxxLysMcre) mice but were abolished in global (Glp1r-/-) and endothelial cell-specific (Glp1rflox/floxxCdh5cre) GLP-1R knockout mice. CONCLUSIONS: GLP-1R activation attenuates cardiovascular complications of arterial hypertension by reduction of vascular inflammation through selective actions requiring the endothelial but not the myeloid cell GLP-1R.

Angiotensin II Infusion Leads to Aortic Dissection in LRP8 Deficient Mice.

Myeloid cells are crucial for the development of vascular inflammation. Low-density lipoprotein receptor-related protein 8 (LRP8) or Apolipoprotein E receptor 2 (ApoER2), is expressed by macrophages, endothelial cells and platelets and has been implicated in the development of cardiovascular diseases. Our aim was to evaluate the role of LRP8, in particular from immune cells, in the development of vascular inflammation. METHODS: LRP8+/+ and LRP8-/- mice (on B6;129S background) were infused with angiotensin II (AngII, 1 mg/kg/day for 7 to 28 day) using osmotic minipumps. Blood pressure was recorded using tail cuff measurements. Vascular reactivity was assessed in isolated aortic segments. Leukocyte activation and infiltration were assessed by flow cytometry of aortic tissue and intravital videomicroscopy imaging. Histological analysis of aortic sections was conducted using sirius red staining. RESULTS: AngII infusion worsened endothelial-dependent vascular relaxation and immune cells rolling and adherence to the carotid artery in both LRP8+/+ as well as LRP8-/- mice. However, only LRP8-/- mice demonstrated a drastically increased mortality rate in response to AngII due to aortic dissection. Bone marrow transplantation revealed that chimeras with LRP8 deficient myeloid cells phenocopied LRP8-/- mice. CONCLUSION: AngII-infused LRP8 deficient mice could be a useful animal model to study aortic dissection reflecting the lethality of this disease in humans.

Investigating the Vascular Toxicity Outcomes of the Irreversible Proteasome Inhibitor Carfilzomib.

BACKGROUND: Carfilzomib's (Cfz) adverse events in myeloma patients include cardiovascular toxicity. Since carfilzomib's vascular effects are elusive, we investigated the vascular outcomes of carfilzomib and metformin (Met) coadministration. METHODS: Mice received: (i) saline; (ii) Cfz; (iii) Met; (iv) Cfz+Met for two consecutive (acute) or six alternate days (subacute protocol). Leucocyte-derived reactive oxygen species (ROS) and serum NOx levels were determined and aortas underwent vascular and molecular analyses. Mechanistic experiments were recapitulated in aged mice who received similar treatment to young animals. Primary murine (prmVSMCs) and aged human aortic smooth muscle cells (HAoSMCs) underwent Cfz, Met and Cfz+Met treatment and viability, metabolic flux and p53-LC3-B expression were measured. Experiments were recapitulated in AngII, CoCl2 and high-glucose stimulated HAoSMCs. RESULTS: Acutely, carfilzomib alone led to vascular hypo-contraction and increased ROS release. Subacutely, carfilzomib increased ROS release without vascular manifestations. Cfz+Met increased PGF2α-vasoconstriction and LC3-B-dependent autophagy in both young and aged mice. In vitro, Cfz+Met led to cytotoxicity and autophagy, while Met and Cfz+Met shifted cellular metabolism. CONCLUSION: Carfilzomib induces a transient vascular impairment and oxidative burst. Cfz+Met increased vascular contractility and synergistically induced autophagy in all settings. Therefore, carfilzomib cannot be accredited for a permanent vascular dysfunction, while Cfz+Met exert vasoprotective potency.

Endothelial α1AMPK modulates angiotensin II-mediated vascular inflammation and dysfunction.

Mice with a global deletion of α1AMPK are characterized by endothelial dysfunction and NADPH oxidase subunit 2 (NOX-2)-mediated vascular oxidative stress. However, the underlying mechanisms are incompletely understood and may involve endothelial NOX-2 upregulation or facilitated vascular infiltration of phagocytic cells. Therefore, the current study was designed to investigate the vascular effects of chronic angiotensin II (AngII) infusion in mice with an endothelial-specific α1AMPK deletion. A mouse strain with endothelial-specific α1AMPK deletion was generated by breeding α1AMPKflox/flox mice with TekCre+ or Cadh5Cre+ mice. Chronic AngII infusion (0.5 mg/kg/day for 7day) caused mild endothelial dysfunction in wild-type mice that was significantly aggravated in endothelial α1AMPK knockout mice. Aortic NOX-2 and CD68 expression were increased, indicating that infiltrating leukocytes may significantly contribute to enhanced vascular oxidative stress. Flow cytometry revealed a higher abundance of aortic CD90.2+ T-cells, CD11b+F4/80+ macrophages and Ly6G-Ly6C+ monocytes. Vascular mRNA expression of monocyte chemoattractant protein 1, CCL5 and vascular cell adhesion molecule 1 was enhanced in AngII-infused mice lacking endothelial α1AMPK, facilitating the recruitment of inflammatory cells to the vessel wall. In addition, AngII-induced upregulation of cytoprotective heme oxygenase 1 (HO-1) was blunted in mice with endothelial α1AMPK deletion, compatible with an impaired antioxidant defense in these animals. In summary, endothelial expressed α1AMPK limits the recruitment of inflammatory cells to the vessel wall and maintains HO-1 mediated antioxidant defense. Both mechanisms reduce vascular oxidative damage and preserve endothelial function during chronic AngII treatment.

Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development.

Expanding evidence indicates multiple interactions between the hemostatic system and innate immunity, and the coagulation and complement cascades. Here we show in a tissue factor (TF)-dependent model of flow restriction-induced venous thrombosis that complement factors make distinct contributions to platelet activation and fibrin deposition. Complement factor 3 (C3) deficiency causes prolonged bleeding, reduced thrombus incidence, thrombus size, fibrin and platelet deposition in the ligated inferior vena cava, and diminished platelet activation in vitro. Initial fibrin deposition at the vessel wall over 6 hours in this model was dependent on protein disulfide isomerase (PDI) and TF expression by myeloid cells, but did not require neutrophil extracellular trap formation involving peptidyl arginine deiminase 4. In contrast to C3-/- mice, C5-deficient mice had no apparent defect in platelet activation in vitro, and vessel wall platelet deposition and initial hemostasis in vivo. However, fibrin formation, the exposure of negatively charged phosphatidylserine (PS) on adherent leukocytes, and clot burden after 48 hours were significantly reduced in C5-/- mice compared with wild-type controls. These results delineate that C3 plays specific roles in platelet activation independent of formation of the terminal complement complex and provide in vivo evidence for contributions of complement-dependent membrane perturbations to prothrombotic TF activation on myeloid cells.

Effect of a Strategy of Comprehensive Vasodilation vs Usual Care on Mortality and Heart Failure Rehospitalization Among Patients With Acute Heart Failure: The GALACTIC Randomized Clinical Trial.

Importance: Short-term infusions of single vasodilators, usually given in a fixed dose, have not improved outcomes in patients with acute heart failure (AHF). Objective: To evaluate the effect of a strategy that emphasized early intensive and sustained vasodilation using individualized up-titrated doses of established vasodilators in patients with AHF. Design, Setting, and Participants: Randomized, open-label blinded-end-point trial enrolling 788 patients hospitalized for AHF with dyspnea, increased plasma concentrations of natriuretic peptides, systolic blood pressure of at least 100 mm Hg, and plan for treatment in a general ward in 10 tertiary and secondary hospitals in Switzerland, Bulgaria, Germany, Brazil, and Spain. Enrollment began in December 2007 and follow-up was completed in February 2019. Interventions: Patients were randomized 1:1 to a strategy of early intensive and sustained vasodilation throughout the hospitalization (n = 386) or usual care (n = 402). Early intensive and sustained vasodilation was a comprehensive pragmatic approach of maximal and sustained vasodilation combining individualized doses of sublingual and transdermal nitrates, low-dose oral hydralazine for 48 hours, and rapid up-titration of angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, or sacubitril-valsartan. Main Outcomes and Measures: The primary end point was a composite of all-cause mortality or rehospitalization for AHF at 180 days. Results: Among 788 patients randomized, 781 (99.1%; median age, 78 years; 36.9% women) completed the trial and were eligible for primary end point analysis. Follow-up at 180 days was completed for 779 patients (99.7%). The primary end point, a composite of all-cause mortality or rehospitalization for AHF at 180 days, occurred in 117 patients (30.6%) in the intervention group (including 55 deaths [14.4%]) and in 111 patients (27.8%) in the usual care group (including 61 deaths [15.3%]) (absolute difference for the primary end point, 2.8% [95% CI, -3.7% to 9.3%]; adjusted hazard ratio, 1.07 [95% CI, 0.83-1.39]; P = .59). The most common clinically significant adverse events with early intensive and sustained vasodilation vs usual care were hypokalemia (23% vs 25%), worsening renal function (21% vs 20%), headache (26% vs 10%), dizziness (15% vs 10%), and hypotension (8% vs 2%). Conclusions and Relevance: Among patients with AHF, a strategy of early intensive and sustained vasodilation, compared with usual care, did not significantly improve a composite outcome of all-cause mortality and AHF rehospitalization at 180 days. Trial Registration: ClinicalTrials.gov Identifier: NCT00512759.

Telmisartan Lowers Elevated Blood Pressure in Psoriatic Mice without Attenuating Vascular Dysfunction and Inflammation.

BACKGROUND: Psoriasis is hallmarked by vascular dysfunction, arterial hypertension, and an increased risk for cardiovascular diseases. We have shown recently that skin-driven interleukin (IL)-17A expression promotes psoriasis-like disease in mice, and this is associated with vascular inflammation, vascular dysfunction, and hypertension. As an intensive risk-factor reduction is recommended for psoriasis patients, we aimed to elucidate the impact of the angiotensin II receptor type 1 (AT1) antagonist telmisartan in a mouse model of severe psoriasis-like skin disease. METHODS AND RESULTS: Elevated blood pressure measured by tail-cuff plethysmography in mice with keratinocyte-specific IL-17A overexpression (K14-IL-17Aind/+ mice) was significantly reduced in response to telmisartan. Importantly, vascular dysfunction, as assessed by isometric tension studies of isolated aortic rings, vascular inflammation measured by flow cytometry analysis of CD45+CD11b+ immune cells, as well as the increased peripheral oxidative stress levels assessed by L-012-enhanced chemiluminescence were not attenuated by telmisartan treatment of K14-IL-17Aind/+ mice, nor was the persisting skin inflammation. CONCLUSION: We provide first evidence for an effective antihypertensive treatment in experimental psoriasis by AT1 blockade, but without any impact on vascular inflammation and dysfunction in our mouse model of severe psoriasis-like skin disease. This suggests that vascular function and inflammation in psoriasis might not be attenuated as long as skin inflammation persists.

Distinct Regulatory Effects of Myeloid Cell and Endothelial Cell NAPDH Oxidase 2 on Blood Pressure.

BACKGROUND: Hypertension caused by increased renin-angiotensin system activation is associated with elevated reactive oxygen species production. Previous studies implicate NADPH oxidase (Nox) proteins as important reactive oxygen species sources during renin-angiotensin system activation, with different Nox isoforms being potentially involved. Among these, Nox2 is expressed in multiple cell types, including endothelial cells, fibroblasts, immune cells, and microglia. Blood pressure (BP) is regulated at the central nervous system, renal, and vascular levels, but the cell-specific role of Nox2 in BP regulation is unknown. METHODS: We generated a novel mouse model with a floxed Nox2 gene and used Tie2-Cre, LysM Cre, or Cdh5-CreERT2 driver lines to develop cell-specific models of Nox2 perturbation to investigate its role in BP regulation. RESULTS: Unexpectedly, Nox2 deletion in myeloid but not endothelial cells resulted in a significant reduction in basal BP. Both Tie2-CreNox2 knockout (KO) mice (in which Nox2 was deficient in both endothelial cells and myeloid cells) and LysM CreNox2KO mice (in which Nox2 was deficient in myeloid cells) had significantly lower BP than littermate controls, whereas basal BP was unaltered in Cdh5-CreERT2 Nox2KO mice (in which Nox2 is deficient only in endothelial cells). The lower BP was attributable to an increased NO bioavailability that dynamically dilated resistance vessels in vivo under basal conditions without a change in renal function. Myeloid-specific Nox2 deletion had no effect on angiotensin II-induced hypertension, which, however, was blunted in Tie2-CreNox2KO mice, along with preservation of endothelium-dependent relaxation during angiotensin II stimulation. CONCLUSIONS: We identify a hitherto unrecognized modulation of basal BP by myeloid cell Nox2, whereas endothelial cell Nox2 regulates angiotensin II-induced hypertension. These results identify distinct cell-specific roles for Nox2 in BP regulation.