Liver sinusoidal endothelial cells rely on oxidative phosphorylation but avoid processing long-chain fatty acids in their mitochondria.

BACKGROUND: It is generally accepted that endothelial cells (ECs), primarily rely on glycolysis for ATP production, despite having functional mitochondria. However, it is also known that ECs are heterogeneous, and their phenotypic features depend on the vascular bed. Emerging evidence suggests that liver sinusoidal ECs (LSECs), located in the metabolically rich environment of the liver, show high metabolic plasticity. However, the substrate preference for energy metabolism in LSECs remains unclear. METHODS: Investigations were conducted in primary murine LSECs in vitro using the Seahorse XF technique for functional bioenergetic assays, untargeted mass spectrometry-based proteomics to analyse the LSEC proteome involved in energy metabolism pathways, liquid chromatography-tandem mass spectrometry-based analysis of acyl-carnitine species and Raman spectroscopy imaging to track intracellular palmitic acid. RESULTS: This study comprehensively characterized the energy metabolism of LSECs, which were found to depend on oxidative phosphorylation, efficiently fuelled by glucose-derived pyruvate, short- and medium-chain fatty acids and glutamine. Furthermore, despite its high availability, palmitic acid was not directly oxidized in LSEC mitochondria, as evidenced by the acylcarnitine profile and etomoxir's lack of effect on oxygen consumption. However, together with L-carnitine, palmitic acid supported mitochondrial respiration, which is compatible with the chain-shortening role of peroxisomal ß-oxidation of long-chain fatty acids before further degradation and energy generation in mitochondria. CONCLUSIONS: LSECs show a unique bioenergetic profile of highly metabolically plastic ECs adapted to the liver environment. The functional reliance of LSECs on oxidative phosphorylation, which is not a typical feature of ECs, remains to be determined.

Endothelial function in patients with chronic myeloid leukemia treated with tyrosine kinase inhibitors is not related to cardiovascular risk assessed by SCORE2 algorithm.

INTRODUCTION: Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia (CML) but are endowed with negative effects on endothelial function. OBJECTIVES: To characterize endothelial function in patients with CML treated with various TKIs. PATIENTS AND METHODS: Forty-eight patients diagnosed with chronic phase CML treated with TKIs, such as imatinib, bosutinib, nilotinib, ponatinib, and asciminib were included. Endothelial function was assessed in the brachial artery and microcirculation based on flow-mediated dilation (FMD), reactive hyperemia peripheral arterial tonometry (RH-PAT) and Flow Mediated Skin Fluorescence (FMSF). RESULTS: Reactive Hyperemia Index (RHI), FMD [%], Reactive Hyperemia Response (RHR [%]), Normoxia Oscillatory index (NOI [%]) and Hyperemic Response index (HR index [%]) did not differentiate between the group of patients with low / moderate risk in the Systemic Coronary Risk Estimation 2 (SCORE2), SCORE2-Older Persons (SCORE2-OP) and those with high / very high-risk scores. Among patients with low/intermediate risk based on the SCORE2 algorithms, some had lower (

Protein disulfide isomerase A1 regulates fenestration dynamics in primary mouse liver sinusoidal endothelial cells (LSECs).

Protein disulfide isomerases (PDIs) are involved in many intracellular and extracellular processes, including cell adhesion and cytoskeletal reorganisation, but their contribution to the regulation of fenestrations in liver sinusoidal endothelial cells (LSECs) remains unknown. Given that fenestrations are supported on a cytoskeleton scaffold, this study aimed to investigate whether endothelial PDIs regulate fenestration dynamics in primary mouse LSECs. PDIA3 and PDIA1 were found to be the most abundant among PDI isoforms in LSECs. Taking advantage of atomic force microscopy, the effects of PDIA1 or PDIA3 inhibition on the fenestrations in LSECs were investigated using a classic PDIA1 inhibitor (bepristat) and novel aromatic N-sulfonamides of aziridine-2-carboxylic acid derivatives as PDIA1 (C-3389) or PDIA3 (C-3399) inhibitors. The effect of PDIA1 inhibition on liver perfusion was studied in vivo using dynamic contrast-enhanced magnetic resonance imaging. Additionally, PDIA1 inhibitors were examined in vitro in LSECs for effects on adhesion, cytoskeleton organisation, bioenergetics, and viability. Inhibition of PDIA1 with bepristat or C-3389 significantly reduced the number of fenestrations in LSECs, while inhibition of PDIA3 with C-3399 had no effect. Moreover, the blocking of free thiols by the cell-penetrating N-ethylmaleimide, but not by the non-cell-penetrating 4-chloromercuribenzenesulfonate, resulted in LSEC defenestration. Inhibition of PDIA1 did not affect LSEC adhesion, viability, and bioenergetics, nor did it induce a clear-cut rearrangement of the cytoskeleton. However, PDIA1-dependent defenestration was reversed by cytochalasin B, a known fenestration stimulator, pointing to the preserved ability of LSECs to form new pores. Importantly, systemic inhibition of PDIA1 in vivo affected intra-parenchymal uptake of contrast agent in mice consistent with LSEC defenestration. These results revealed the role of intracellular PDIA1 in the regulation of fenestration dynamics in LSECs, and in maintaining hepatic sinusoid homeostasis.

Early microvascular coronary endothelial dysfunction precedes pembrolizumab-induced cardiotoxicity. Preventive role of high dose of atorvastatin.

Immune checkpoint inhibitors (ICIs) exhibit remarkable antitumor activity and immune-related cardiotoxicity of unknown pathomechanism. The aim of the study was to investigate the ICI class-dependent cardiotoxicity in vitro and pembrolizumab's (Pem's) cardiotoxicity in vivo, seeking for translational prevention means. Cytotoxicity was investigated in primary cardiomyocytes and splenocytes, incubated with ipilimumab, Pem and avelumab. Pem's cross-reactivity was assessed by circular dichroism (CD) on biotechnologically produced human and murine PD-1 and in silico. C57BL6/J male mice received IgG4 or Pem for 2 and 5 weeks. Echocardiography, histology, and molecular analyses were performed. Coronary blood flow velocity mapping and cardiac magnetic resonance imaging were conducted at 2 weeks. Human EA.hy926 endothelial cells were incubated with Pem-conditioned media from human mononuclear cells, in presence and absence of statins and viability and molecular signaling were assessed. Atorvastatin (20 mg/kg, daily) was administered in vivo, as prophylaxis. Only Pem exerted immune-related cytotoxicity in vitro. Pem's cross-reactivity with the murine PD-1 was confirmed by CD and docking. In vivo, Pem initiated coronary endothelial and diastolic dysfunction at 2 weeks and systolic dysfunction at 5 weeks. At 2 weeks, Pem induced ICAM-1 and iNOS expression and intracardiac leukocyte infiltration. At 5 weeks, Pem exacerbated endothelial activation and triggered cardiac inflammation. Pem led to immune-related cytotoxicity in EA.hy926 cells, which was prevented by atorvastatin. Atorvastatin mitigated functional deficits, by inhibiting endothelial dysfunction in vivo. We established for the first time an in vivo model of Pem-induced cardiotoxicity. Coronary endothelial dysfunction precedes Pem-induced cardiotoxicity, whereas atorvastatin emerges as a novel prophylactic therapy.

Chronic heart failure induces early defenestration of liver sinusoidal endothelial cells (LSECs) in mice.

AIM: Chronic heart failure (CHF) is often linked to liver malfunction and systemic endothelial dysfunction. However, whether cardio-hepatic interactions in heart failure involve dysfunction of liver sinusoidal endothelial cells (LSECs) is not known. Here we characterize LSECs phenotype in early and end stages of chronic heart failure in a murine model. METHODS: Right ventricle (RV) function, features of congestive hepatopathy, and the phenotype of primary LSECs were characterized in Tgαq*44 mice, with cardiomyocyte-specific overexpression of the Gαq protein, at the age of 4- and 12-month representative for early and end-stage phases of CHF, respectively. RESULTS: 4- and 12-month-old Tgαq*44 mice displayed progressive impairment of RV function and alterations in hepatic blood flow velocity resulting in hepatic congestion with elevated GGT and bilirubin plasma levels and decreased albumin concentration without gross liver pathology. LSECs isolated from 4- and 12-month-old Tgαq*44 mice displayed significant loss of fenestrae with impaired functional response to cytochalasin B, significant changes in proteome related to cytoskeleton remodeling, and altered vasoprotective function. However, LSECs barrier function and bioenergetics were largely preserved. In 4- and 12-month-old Tgαq*44 mice, LSECs defenestration was associated with prolonged postprandial hypertriglyceridemia and in 12-month-old Tgαq*44 mice with proteomic changes of hepatocytes indicative of altered lipid metabolism. CONCLUSION: Tgαq*44 mice displayed right-sided HF and altered hepatic blood flow leading to LSECs dysfunction involving defenestration, shift in eicosanoid profile, and proteomic changes. LSECs dysfunction appears as an early and persistent event in CHF, preceding congestive hepatopathy and contributing to alterations in lipoprotein transport and CHF pathophysiology.

Myoglobin deficiency impairs maximal oxygen uptake and exercise performance: a lesson from Mb-/- mice.

Myoglobin (Mb) plays an important role at rest and during exercise as a reservoir of oxygen and has been suggested to regulate NO• bioavailability under hypoxic/acidic conditions. However, its ultimate role during exercise is still a subject of debate. We aimed to study the effect of Mb deficiency on maximal oxygen uptake ( V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ ) and exercise performance in myoglobin knockout mice (Mb-/- ) when compared to control mice (Mb+/+ ). Furthermore, we also studied NO• bioavailability, assessed as nitrite (NO2 - ) and nitrate (NO3 - ) in the heart, locomotory muscle and in plasma, at rest and during exercise at exhaustion both in Mb-/- and in Mb+/+ mice. The mice performed maximal running incremental exercise on a treadmill with whole-body gas exchange measurements. The Mb-/- mice had lower body mass, heart and hind limb muscle mass (P < 0.001). Mb-/- mice had significantly reduced maximal running performance (P < 0.001). V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ expressed in ml min-1 in Mb-/ - mice was 37% lower than in Mb+/+ mice (P < 0.001) and 13% lower when expressed in ml min-1  kg body mass-1 (P = 0.001). Additionally, Mb-/- mice had significantly lower plasma, heart and locomotory muscle NO2 - levels at rest. During exercise NO2 - increased significantly in the heart and locomotory muscles of Mb-/- and Mb+/+ mice, whereas no significant changes in NO2 - were found in plasma. Our study showed that, contrary to recent suggestions, Mb deficiency significantly impairs V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ and maximal running performance in mice. KEY POINTS: Myoglobin knockout mice (Mb-/- ) possess lower maximal oxygen uptake ( V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ ) and poorer maximal running performance than control mice (Mb+/+ ). Respiratory exchange ratio values at high running velocities in Mb-/- mice are higher than in control mice suggesting a shift in substrate utilization towards glucose metabolism in Mb-/- mice at the same running velocities. Lack of myoglobin lowers basal systemic and muscle NO• bioavailability, but does not affect exercise-induced NO2 - changes in plasma, heart and locomotory muscles. The present study demonstrates that myoglobin is of vital importance for V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ and maximal running performance as well as explains why previous studies have failed to prove such a role of myoglobin when using the Mb-/- mouse model.

Functional deterioration of vascular mitochondrial and glycolytic capacity in the aortic rings of aged mice.

Vascular ageing is associated with increased arterial stiffness and cardiovascular mortality that might be linked to altered vascular energy metabolism. The aim of this study was to establish a Seahorse XFe96 Analyzer-based methodology for the reliable, functional assessment of mitochondrial respiration and glycolysis in single murine aortic rings and to validate this functional assay by characterising alterations in vascular energy metabolism in aged mice. Healthy young and old C57BL/6 mice were used for the analyses. An optimised setup consisting of the Seahorse XFe96 Analyzer and Seahorse Spheroid Microplates was applied for the mitochondrial stress test and the glycolysis stress test on the isolated murine aortic rings, supplemented with analysis of NAD content in the aorta. To confirm the age-dependent stiffness of the vasculature, pulse wave velocity was measured in vivo. In addition, the activity of vascular nitric oxide synthase and vascular wall morphology were analysed ex vivo. The vascular ageing phenotype in old mice was confirmed by increased aortic stiffness, vascular wall remodelling, and nitric oxide synthase activity impairment. The rings of the aorta taken from old mice showed changes in vascular energy metabolism, including impaired spare respiratory capacity, maximal respiration, glycolysis, and glycolytic capacity, as well as a fall in the NAD pool. In conclusion, optimised Seahorse XFe96-based analysis to study energy metabolism in single aortic rings of murine aorta revealed a robust impairment of functional vascular respiratory and glycolytic capacity in old mice linked to NAD deficiency that coincided with age-related aortic wall remodelling and stiffness.

Epigenetic clock in the aorta and age-related endothelial dysfunction in mice.

While epigenetic age (EA) of mouse blood can be determined using DNA methylation analysis at three CpG sites in the Prima1, Hsf4 and Kcns1 genes it is not known whether this approach is useful for predicting vascular biological age. In this study we validated the 3-CpG estimator for age prediction in mouse blood, developed a new predictive model for EA in mouse aorta, and assessed whether epigenetic age acceleration (EAA) measured with blood and aorta samples correlates with age-dependent endothelial dysfunction. Endothelial function was characterized in vivo by MRI in 8-96-week-old C57BL/6 mice. Arterial stiffness was measured by USG-doppler. EA-related changes within 41 CpG sites in Prima1, Kcns1 and Hsf4 loci, were analyzed in the aorta and blood using bisulfite amplicon high-throughput sequencing. Progressive age-dependent endothelial dysfunction and changes in arterial stiffness were observed in 36-96-week-old C57BL/6 mice. Methylation levels of the investigated loci correlated with chronological age in blood and the aorta. The new model for EA estimation in aorta included three cytosines located in the Kcns1 and Hsf4, explained R2 = 87.8% of the variation in age, and predicted age with an mean absolute error of 9.6 weeks in the independent test set. EAA in the aorta was associated with endothelial dysfunction in the abdominal aorta and femoral artery what was consistent with the EAA direction estimated in blood samples. The rate of vascular biological ageing in mice, reflected by the age-dependent systemic endothelial dysfunction, could be estimated using DNA methylation measurements at three loci in aorta and blood samples.

Vascular protein disulfide isomerase A1 mediates endothelial dysfunction induced by angiotensin II in mice.

AIM: Protein disulfide isomerases (PDIs) are involved in platelet aggregation and intravascular thrombosis, but their role in regulating endothelial function is unclear. Here, we characterized the involvement of vascular PDIA1 in angiotensin II (Ang II)-induced endothelial dysfunction in mice. METHODS: Endothelial dysfunction was induced in C57BL/6JCmd male mice via Ang II subcutaneous infusion, and PDIA1 was inhibited with bepristat. Endothelial function was assessed in vivo with magnetic resonance imaging and ex vivo with a myography, while arterial stiffness was measured as pulse wave velocity. Nitric oxide (NO) bioavailability was measured in the aorta (spin-trapping electron paramagnetic resonance) and plasma (NO2 - and NO3 - levels). Oxidative stress, eNOS uncoupling (DHE-based aorta staining), and thrombin activity (thrombin-antithrombin complex; calibrated automated thrombography) were evaluated. RESULTS: The inhibition of PDIA1 by bepristat in Ang II-treated mice prevented the impairment of NO-dependent vasodilation in the aorta as evidenced by the response to acetylcholine in vivo, increased systemic NO bioavailability and the aortic NO production, and decreased vascular stiffness. Bepristat's effect on NO-dependent function was recapitulated ex vivo in Ang II-induced endothelial dysfunction in isolated aorta. Furthermore, bepristat diminished the Ang II-induced eNOS uncoupling and overproduction of ROS without affecting thrombin activity. CONCLUSION: In Ang II-treated mice, the inhibition of PDIA1 normalized the NO-ROS balance, prevented endothelial eNOS uncoupling, and, thereby, improved vascular function. These results indicate the importance of vascular PDIA1 in regulating endothelial function, but further studies are needed to elucidate the details of the mechanisms involved.

Retrospectively gated ultrashort-echo-time MRI T1 mapping reveals compromised pulmonary microvascular NO-dependent function in a murine model of acute lung injury.

This study sought to develop noninvasive, in vivo imaging schemes that allow for quantitative assessment of pulmonary microvascular functional status based on the combination of pulmonary T1 mapping and dynamic contrast-enhanced (DynCE) imaging. Ultrashort-echo-time (UTE) imaging at 9.4 T of lung parenchyma was performed. Retrospective gating was based on modulation of the first point in each recorded spoke. T1 maps were obtained using a series of five consecutive images with varying RF angles and analyzed with the variable flip angle approach. The obtained mean T1 lung value of 1078 ± 38 ms correlated well with previous reports. Improved intersession variability was observed, as evident from a decreased standard deviation of motion-resolved T1 mapping (F-test = 0.051). Animals received lipopolysaccharide (LPS) and were imaged at t = 2, 6, and 12 h after administration. The nitric oxide (NO)-dependent function was assessed according to changes in lung T1 after L-NAME injection, while microvascular perfusion and oxidant stress were assessed with contrast-enhanced imaging after injection of gadolinium or 3-carbamoyl-proxyl nitroxide radical, respectively. Retrospectivel gated UTE allowed robust, motion-compensated imaging that could be used for T1 mapping of lung parenchyma. Changes in lung T1 after L-NAME injection indicated that LPS induced overproduction of NO at t = 2 and 6 h after LPS, but NO-dependent microvascular function was impaired at t = 12 h after LPS. DynCE imaging at t = 6 h after LPS injection revealed decreased microvascular perfusion, with increased vascular permeability and oxidant stress. MRI allows to visualize and quantify lung microvascular NO-dependent function and its concomitant impairment during acute respiratory distress syndrome development with high sensitivity. UTE T1 mapping appears to be sensitive and useful in probing pulmonary microvascular functional status.

TNF-α protects from exacerbated myocarditis and cardiac death by suppressing expansion of activated heart-reactive CD4+ T cells.

AIMS: 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. METHODS AND RESULTS: 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 (Teff) cells effectively activated the adhesive properties of cardiac microvascular endothelial cells (cMVECs). Our data suggested that TNF-α produced by endothelial in addition to Teff cells promoted leucocyte adhesion to activated cMVECs. Analysis of CD4+ T lymphocytes from both models of myocarditis showed a strongly increased fraction of Teff cells in hearts, spleens, and in the blood of Tnf+/- and Tnf-/- mice. Indeed, antigen-activated Tnf-/- Teff cells showed prolonged long-term survival and TNF-α cytokine-induced cell death of heart-reactive Teff. CONCLUSION: 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 Teff. 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.

Chymase-independent vascular Ang-(1-12)/Ang II pathway and TXA2 generation are involved in endothelial dysfunction in the murine model of heart failure.

The angiotensin (Ang)-(1-12)/Ang II pathway contributes to cardiac pathology. However, its involvement in the development of peripheral endothelial dysfunction associated with heart failure (HF) remains unknown. Therefore, this study aimed to characterise the effect of exogenous Ang-(1-12) and its conversion to Ang II on endothelial function using the murine model of HF (Tgαq*44 mice), focusing on the role of chymase and vascular-derived thromboxane A2 (TXA2). Ex vivo myographic assessments of isolated aorta showed impaired endothelium-dependent vasodilation in late-stage HF in 12-month-old Tgαq*44 mice. However, endothelium-dependent vasodilation was fully preserved in the early stage of HF in 4-month-old Tgαq*44 mice and 4- and 12-month-old FVB control mice. Ang-(1-12) impaired endothelium-dependent vasodilation in 4- and 12-month-old Tgαq*44 mice, that was associated with increased Ang II production. The chymase inhibitor chymostatin did not inhibit this response. Interestingly, TXA2 production reflected by TXB2 measurement was upregulated in response to Ang-(1-12) and Ang II in aortic rings isolated from 12-month-old Tgαq*44 mice but not from 4-month-old Tgαq*44 mice or age-matched FVB mice. Furthermore, in vivo magnetic resonance imaging showed that Ang-(1-12) impaired endothelium-dependent vasodilation in the aorta of Tgαq*44 mice and FVB mice. However, this response was inhibited by angiotensin I converting enzyme (ACE) inhibitor; perindopril, angiotensin II receptor type 1 (AT1) antagonist; losartan and TXA2 receptor (TP) antagonist-picotamide in 12-month-old-Tgαq*44 mice only. In conclusion, the chymase-independent vascular Ang-(1-12)/Ang II pathway and subsequent TXA2 overactivity contribute to systemic endothelial dysfunction in the late stage of HF in Tgαq*44 mice. Therefore, the vascular TXA2 receptor represents a pharmacotherapeutic target to improve peripheral endothelial dysfunction in chronic HF.

Training-induced impairment of endothelial function in track and field female athletes.

Professional athletes are often exposed to high training loads that may lead to overfatigue, overreaching and overtraining that might have a detrimental effects on vascular health. We determined the effects of high training stress on endothelial function assessed by the flow-mediated dilation (FMD) and markers of glycocalyx shedding. Vascular examination as well as broad biochemical, hormonal and cardiometabolic evaluation of sprint and middle-distance female runners were performed after 2 months of preparatory training period and compared to age-matched control group of women. Female athletes presented with significantly reduced FMD (p < 0.01) and higher basal serum concentrations of hyaluronan (HA) and syndecan-1 (SDC-1) (p < 0.05 and p < 0.001, respectively), that was accompanied by significantly lower basal serum testosterone (T) and free testosterone (fT) concentrations (p < 0.05) and higher cortisol (C) concentration (p < 0.05). It resulted in significantly lower T/C and fT/C ratios in athletes when compared to controls (p < 0.01). Moreover, fT/C ratio were significantly positively correlated to FMD and negatively to HA concentrations in all studied women. Accordingly, the training load was significantly negatively correlated with T/C, fT/C and FMD and positively with the concentrations of HA and SDC-1. We concluded that young female track and field athletes subjected to physical training developed impairment of endothelial function that was associated with anabolic-catabolic hormone balance disturbances. Given that training-induced impairment of endothelial function may have a detrimental effects on vascular health, endothelial status should be regularly monitored in the time-course of training process to minimalize vascular health-risk in athletes.

Vascular lipid droplets formed in response to TNF, hypoxia, or OA: biochemical composition and prostacyclin generation.

Biogenesis of lipid droplets (LDs) in various cells plays an important role in various physiological and pathological processes. However, the function of LDs in endothelial physiology and pathology is not well understood. In the present work, we investigated the formation of LDs and prostacyclin (PGI2) generation in the vascular tissue of isolated murine aortas following activation by proinflammatory factors: tumor necrosis factor (TNF), lipopolysaccharides (LPS), angiotensin II (AngII), hypoxic conditions, or oleic acid (OA). The abundance, size, and biochemical composition of LDs were characterized based on Raman spectroscopy and fluorescence imaging. We found that blockade of lipolysis by the adipose triglyceride lipase (ATGL) delayed LDs degradation and simultaneously blunted PGI2 generation in aorta treated with all tested proinflammatory stimuli. Furthermore, the analysis of Raman spectra of LDs in the isolated vessels stimulated by TNF, LPS, AngII, or hypoxia uncovered that these LDs were all rich in highly unsaturated lipids and had a negligible content of phospholipids and cholesterols. Additionally, by comparing the Raman signature of endothelial LDs under hypoxic or OA-overload conditions in the presence or absence of ATGL inhibitor, atglistatin (Atgl), we show that Atgl does not affect the biochemical composition of LDs. Altogether, independent of whether LDs were induced by pro-inflammatory stimuli, hypoxia, or OA and of whether they were composed of highly unsaturated or less unsaturated lipids, we observed LDs formation invariably associated with ATGL-dependent PGI2 generation. In conclusion, vascular LDs formation and ATGL-dependent PGI2 generation represent a universal response to vascular proinflammatory insult.

Accelerated ageing and coronary microvascular dysfunction in chronic heart failure in Tgαq*44 mice.

Age represents a major risk factor in heart failure (HF). However, the mechanisms linking ageing and HF are not clear. We aimed to identify the functional, morphological and transcriptomic changes that could be attributed to cardiac ageing in a model of slowly progressing HF in Tgαq*44 mice in reference to the cardiac ageing process in FVB mice. In FVB mice, ageing resulted in the impairment of diastolic cardiac function and in basal coronary flow (CF), perivascular and interstitial fibrosis without changes in the cardiac activity of angiotensin-converting enzyme (ACE) or aldosterone plasma concentration. In Tgαq*44 mice, HF progression was featured by the impairment of systolic and diastolic cardiac function and in basal CF that was associated with a distinct rearrangement of the capillary architecture, pronounced perivascular and interstitial fibrosis, progressive activation of cardiac ACE and systemic angiotensin-aldosterone-dependent pathways. Interestingly, cardiac ageing genes and processes were represented in Tgαq*44 mice not only in late but also in early phases of HF, as evidenced by cardiac transcriptome analysis. Thirty-four genes and 8 biological processes, identified as being ageing related, occurred early and persisted along HF progression in Tgαq*44 mice and were mostly associated with extracellular matrix remodelling and fibrosis compatible with perivascular fibrosis resulting in coronary microvascular dysfunction (CMD) in Tgαq*44 mice. In conclusion, accelerated and persistent cardiac ageing contributes to the pathophysiology of chronic HF in Tgαq*44 mice. In particular, prominent perivascular fibrosis of microcirculation resulting in CMD represents an accelerated cardiac ageing phenotype that requires targeted treatment in chronic HF.

Age-related decrease in serum dihydrotestosterone concentration is accompanied by impaired vascular status.

INTRODUCTION: The effect of androgens on the cardiovascular system in humans is ambiguous. Moreover, still little is known about the effects of the most potent androgen, dihydrotestosterone, on arterial stiffness and endothelial function. The aim of this study was to evaluate whether age-dependent alterations in serum concentration of dihydrotestosterone and its circulating metabolite are accompanied by changes in endothelial function and arterial stiffness. METHODS: In 12 young and 11 older men, basal serum concentrations of testosterone, dehydroepiandrosterone sulfate (DHAE-S), androstenedione (AE), dihydrotestosterone (DHT) and androstanediol glucuronide (ADG) were analyzed in relation to vascular status including cIMT - carotid intima media thickness, cAI - central augmentation index, crPWV - carotid radial pulse wave velocity, SI - stiffness index, endothelial and inflammatory markers. RESULTS: Although concentration of testosterone was not different between young and older group, it was demonstrated that DHT, DHEA-S, AE and ADG were significantly lower in older men in comparison to young men (p < 0.01). Interestingly the most surprising difference was found for DHT concentration, that was as much as 61 % lower in aged men that displayed significantly higher values of cIMT, AI, crPWV and SI (p < 10-4), suggestive of arterial stiffness. Furthermore, DHT was negatively correlated to all arterial wall parameters (cAI, crPWV, SI and cIMT), c-reactive protein (CRP) and hyaluronic acid (HA) concentration, as well as positively correlated to markers of endothelial function (MNA and 6-keto-PGF1α) in all studied individuals (n = 23). CONCLUSIONS: We have shown that ageing leads to a significant decrease in DHT concentration that is accompanied by impaired arterial wall characteristics and worsened endothelial function. Therefore more attention should be paid to the DHT, DHEA-S and ADG concentrations as a biomarkers for vascular dysfunction in ageing men.

Platelets in COVID-19 disease: friend, foe, or both?

Immuno-thrombosis of COVID-19 results in the activation of platelets and coagulopathy. Antiplatelet therapy has been widely used in COVID-19 patients to prevent thrombotic events. However, recent analysis of clinical trials does not support the major effects of antiplatelet therapy on mortality in hospitalized COVID-19 patients, despite the indisputable evidence for an increased risk of thrombotic complications in COVID-19 disease. This apparent paradox calls for an explanation. Platelets have an important role in sensing and orchestrating host response to infection, and several platelet functions related to host defense response not directly related to their well-known hemostatic function are emerging. In this paper, we aim to review the evidence supporting the notion that platelets have protective properties in maintaining endothelial barrier integrity in the course of an inflammatory response, and this role seems to be of particular importance in the lung. It might, thus, well be that the inhibition of platelet function, if affecting the protective aspect of platelet activity, might diminish clinical benefits resulting from the inhibition of the pro-thrombotic phenotype of platelets in immuno-thrombosis of COVID-19. A better understanding of the platelet-dependent mechanisms involved in the preservation of the endothelial barrier is necessary to design the antiplatelet therapeutic strategies that inhibit the pro-thrombotic activity of platelets without effects on the vaso-protective function of platelets safeguarding the pulmonary endothelial barrier during multicellular host defense in pulmonary circulation.

Endothelial-mesenchymal transition induced by metastatic 4T1 breast cancer cells in pulmonary endothelium in aged mice.

Ageing is a major risk factor for cancer metastasis but the underlying mechanisms remain unclear. Here, we characterised ageing effects on cancer-induced endothelial-mesenchymal transition (EndMT) in the pulmonary circulation of female BALB/c mice in a metastatic 4T1 breast cancer model. The effect of intravenously injected 4T1 cells on pulmonary endothelium, pulmonary metastasis, lung tissue architecture, and systemic endothelium was compared between 40-week-old and 20-week-old mice. The 40-week-old mice showed features of ongoing EndMT in their lungs before 4T1 breast cancer cell injection. Moreover, they had preexisting endothelial dysfunction in the aorta detected by in vivo magnetic resonance imaging (MRI) compared to 20-week-old mice. The injection of 4T1 breast cancer cells into 40-week-old mice resulted in rapid EndMT progression in their lungs. In contrast, injection of 4T1 breast cancer cells into 20-week-old mice resulted in initiation and less pronounced EndMT progression. Although the number of metastases did not differ significantly between 20-week-old and 40-week-old mice, the lungs of older mice displayed altered lung tissue architecture and biochemical content, reflected in higher Amide II/Amide I ratio, higher fibronectin levels, and hypoxia-inducible factor 1 subunit alpha (HIF1α) levels as well as lower nitric oxide (NO) production. Our results indicate that age-dependent pre-existing endothelial dysfunction in the pulmonary endothelium of 40-week-old mice predisposed them to rapid EndMT progression in the presence of circulating 4T1 breast cancer cells what might contribute to a more severe metastatic breast cancer phenotype in these ageing mice compared to younger mice.

Receptor-independent fluid-phase macropinocytosis promotes arterial foam cell formation and atherosclerosis.

Accumulation of lipid-laden foam cells in the arterial wall plays a central role in atherosclerotic lesion development, plaque progression, and late-stage complications of atherosclerosis. However, there are still fundamental gaps in our knowledge of the underlying mechanisms leading to foam cell formation in atherosclerotic arteries. Here, we investigated the role of receptor-independent macropinocytosis in arterial lipid accumulation and pathogenesis of atherosclerosis. Genetic inhibition of fluid-phase macropinocytosis in myeloid cells (LysMCre+ Nhe1fl/fl) and repurposing of a Food and Drug Administration (FDA)-approved drug that inhibits macrophage macropinocytosis substantially decreased atherosclerotic lesion development in low-density lipoprotein (LDL) receptor-deficient and Apoe-/- mice. Stimulation of macropinocytosis using genetic (H-RASG12V) and physiologically relevant approaches promoted internalization of unmodified native (nLDL) and modified [e.g., acetylated (ac) and oxidized (ox) LDL] lipoproteins in both wild-type and scavenger receptor (SR) knockout (Cd36-/-/Sra-/-) macrophages. Pharmacological inhibition of macropinocytosis in hypercholesterolemic wild-type and Cd36-/-/Sra-/- mice identified an important role of macropinocytosis in LDL uptake by lesional macrophages and development of atherosclerosis. Furthermore, serial section high-resolution imaging, LDL immunolabeling, and three-dimensional (3D) reconstruction of subendothelial foam cells provide visual evidence of lipid macropinocytosis in both human and murine atherosclerotic arteries. Our findings complement the SR paradigm of atherosclerosis and identify a therapeutic strategy to counter the development of atherosclerosis and cardiovascular disease.