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Acute Respiratory Distress Syndrome (ARDS) is a severe lung condition with a high mortality rate for which there are no effective therapeutics. The failure of the alveolar–capillary barrier, composed of lung endothelial (EC) and alveolar epithelial (AEC) cells, is a critical factor leading to excessive inflammation and edema characteristic of acute lung injury (ALI) pathophysiology. Phosphodiesterases (PDE) are enzymes well-recognized for their roles in regulating endothelial permeability and inflammation. Although PDE inhibitors are used as therapeutics for inflammatory diseases like COPD (chronic obstructive pulmonary disease), their efficacy in treating ARDS has not yet been established. In this study, we investigated the effects of ensifentrine, an FDA-approved novel dual PDE 3/4 inhibitor, on lung endothelial and epithelial dysfunction caused by methicillin-resistant S. aureus (MRSA), a pathogen involved in bacterial ARDS. Human primary lung endothelial cells and alveolar epithelial cell lines (A549 and immortalized AEC) were treated with heat-killed MRSA, and their responses were assessed in the presence or absence of ensifentrine. Ensifentrine given either pre- or post-exposure attenuated MRSA-induced increased lung endothelial permeability. VE-cadherin junctions, which serve to stabilize the EC barrier, were disrupted by MRSA; however, ensifentrine effectively prevented this disruption. Pre-treatment with ensifentrine protected against MRSA-induced EC pro-inflammatory signaling by inhibiting the expression of VCAM-1, ICAM-1, and by reducing the IL-6 and IL-8 release. In AEC, MRSA caused the upregulation of ICAM-1, the activation of NF-kB, and the production of IL-8, all of which were inhibited by ensifentrine. These results indicate that the dual inhibition of phosphodiesterases 3 and 4 by ensifentrine is barrier protective and attenuates MRSA-induced inflammation in both lung endothelial and epithelial cells. The PDE3/4 inhibitor ensifentrine may represent a promising novel strategy for the treatment of MRSA-induced ARDS. [ABSTRACT FROM AUTHOR]

Endothelial dysfunction is an early predictor of atherosclerosis and cardiovascular disease. Flow-mediated dilation (FMD) is the gold standard to assess endothelial function in humans. FMD reproducibility has been mainly assessed in the brachial artery (BA) with limited research in lower limb arteries. The purpose of this study was to compare FMD reproducibility in the upper limb BA and lower limb superficial femoral artery (SFA) in young healthy adults. Fifteen young healthy adults (nine males; six females) underwent FMD, resting diameter, velocity, and shear rate measurements on three occasions to determine intra-and inter-day reproducibility in both BA and SFA, assessed by coefficient of variation (CV), intraclass correlation coefficient (ICC), and Bland–Altman plots. BA FMD CVs (intra-day: 4.2%; inter-day: 8.7%) and ICCs (intra-day: 0.967; inter-day: 0.903) indicated excellent reproducibility and reliability, while for SFA FMD, both CVs (intra-day: 11.6%; inter-day: 26.7%) and ICCs (intra-day: 0.898; inter-day: 0.651) showed good/moderate reproducibility and reliability. BA FMD was significantly more reproducible than SFA FMD (p < 0.05). Diameter reproducibility was excellent and similar between arteries, while resting velocity and shear rate have lower reproducibility in the BA compared to SFA. Bland–Altman plots displayed no proportional and fixed bias between measurements. In summary, SFA FMD is less reproducible than BA FMD, with identical volume of ultrasound training. Given the increasing interest in using SFA FMD to test the efficacy of interventions targeting lower limb's vascular health and as a potential biomarker for peripheral arterial disease risk, future studies should ensure higher levels of training for adequate reproducibility. [ABSTRACT FROM AUTHOR]

Methylglyoxal (MGO) triggers oxidative stress responses in vascular endothelial cells, leading to apoptosis linked to diabetic vascular complications. Total flavonoids of Eucommia ulmoides leaves (TFEL) display antioxidant activity, yet its prevention of MGO‐induced apoptosis and mechanisms are unclear. Our study used western blotting and ELISA to evaluate protein levels and enzyme activities. Cell viability and apoptosis were evaluated using CCK8 assay and PE Annexin V/7‐AAD double staining. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured using fluorescence probes. Vascular pathological changes and apoptosis were analyzed through H&E and TUNEL staining. In vitro, MGO‐stimulated human umbilical vein endothelial cells (HUVECs) were treated with varying TFEL concentrations. Our results demonstrated that TFEL significantly enhanced cell viability, reduced apoptosis, downregulated caspase‐3 activity, and Bax/Bcl‐2 ratio. Moreover, TFEL markedly suppressed MGO‐induced ROS and malondialdehyde (MDA) production while restoring antioxidant enzyme activity and MMP. TFEL pretreatment promoted the expression of p‐Akt, Nrf2, and HO‐1 proteins. Pharmacological inhibition of p‐Akt significantly suppressed the upregulation of Nrf2 and HO‐1 protein levels mediated by TFEL. Consistently, pharmacological inhibition of Nrf2 or p‐Akt partially abrogated the protective effects of TFEL against MGO‐induced damage in HUVECs. In vivo studies revealed that TFEL (100 and 200 mg/kg) partially restored antioxidant capacity and reduced aortic thickness and apoptosis in MGO‐injured mice. In conclusion, the findings indicate that TFEL mitigates MGO‐induced apoptosis via activation of p‐Akt/Nrf2/HO‐1 and scavenging of oxidative stress, highlighting its potential in diabetic vascular complication management. [ABSTRACT FROM AUTHOR]

The delivery of nutrients to the brain is provided by a 600 km network of capillaries and microvessels. Indeed, the brain is highly energy demanding and, among a total amount of 100 billion neurons, each neuron is located just 10–20 μm from a capillary. This vascular network also forms part of the blood–brain barrier (BBB), which maintains the brain's stable environment by regulating chemical balance, immune cell transport, and blocking toxins. Typically, brain microvascular endothelial cells (BMECs) have low turnover, indicating a stable cerebrovascular structure. However, this structure can adapt significantly due to development, aging, injury, or disease. Temporary neural activity changes are managed by the expansion or contraction of arterioles and capillaries. Hypoxia leads to significant remodeling of the cerebrovascular architecture and pathological changes have been documented in aging and in vascular and neurodegenerative conditions. These changes often involve BMEC proliferation and the remodeling of capillary segments, often linked with local neuronal changes and cognitive function. Cerebrovascular plasticity, especially in arterioles, capillaries, and venules, varies over different time scales in development, health, aging, and diseases. Rapid changes in cerebral blood flow (CBF) occur within seconds due to increased neural activity. Prolonged changes in vascular structure, influenced by consistent environmental factors, take weeks. Development and aging bring changes over months to years, with aging-associated plasticity often improved by exercise. Injuries cause rapid damage but can be repaired over weeks to months, while neurodegenerative diseases cause slow, varied changes over months to years. In addition, if animal models may provide useful and dynamic in vivo information about vascular plasticity, humans are more complex to investigate and the hypothesis of glymphatic system together with Magnetic Resonance Imaging (MRI) techniques could provide useful clues in the future. [ABSTRACT FROM AUTHOR]

Fluid shear stress (FSS) from blood flow, sensed by the vascular endothelial cells (ECs) that line all blood vessels, regulates vascular development during embryogenesis, controls adult vascular physiology and determines the location of atherosclerotic plaque formation. Although a number of papers have reported a crucial role for cell-cell adhesions or adhesion receptors in these processes, a recent publication has challenged this paradigm, presenting evidence that ECs can very rapidly align in fluid flow as single cells without cell–cell contacts. To address this controversy, four independent laboratories assessed EC alignment in fluid flow across a range of EC cell types. These studies demonstrate a strict requirement for cell-cell contact in shear stress sensing over timescales consistent with previous literature and inconsistent with the newly published data. [ABSTRACT FROM AUTHOR]

Summary: There have been increasing concerns about the great risks posed by petroleum products which contribute to the progression endothelial renal vasoconstriction and hypertension. This study aims to investigate the modulatory effects of Roasted cashew nut on rats induced with endothelial renal vasoconstriction syndrome upon exposure to mixed fractionated petroleum products (MFPP). Seventy male albino rats were randomized into seven (n=10) groups. Group I (normal control) was given distilled water. Group II was administered with MFPP (20 %/day). Group III was administered with MFPP (20 %/day) + atenolol (50 mg/kg). Group IV was administered with MFPP (20 %/day) + 10 % roasted cashew nut (10 %RCN). Group V was administered with MFPP (20 %/day) + (20 %RCN). Group VI was treated with 10 %RCN while Group VII was treated with 20 %RCN. Data were analyzed using one-way analysis of variance (ANOVA) and Duncan's multiple range test with P

The adult mammalian heart has been demonstrated to be endowed with low but real turnover capacity, especially for cardiomyocytes, the key functional cell type. The source, however, of that turnover capacity remains controversial. In this regard, we have defined and characterized a resident multipotent cardiac mouse progenitor population, Bmi1+DR (for Bmi1+ Damage-Responsive cells). Bmi1+DR is one of the cell types with the lowest ROS (Reactive Oxygen Species) levels in the adult heart, being particularly characterized by their close relationship with cardiac vessels, most probably involved in the regulation of proliferation/maintenance of Bmi1+DR. This was proposed to work as their endothelial niche. Due to the scarcity of Bmi1+DR cells in the adult mouse heart, we have generated an immortalization/dis-immortalization model using Simian Vacuolating Virus 40-Large Antigen T (SV40-T) to facilitate their in vitro characterization. We have obtained a heterogeneous population of immortalized Bmi1+DR cells (Bmi1+DRIMM) that was validated attending to different criteria, also showing a comparable sensitivity to strong oxidative damage. Then, we concluded that the Bmi1-DRIMM population is an appropriate model for primary Bmi1+DR in vitro studies. The co-culture of Bmi1+DRIMM cells with endothelial cells protects them against oxidative damage, showing a moderate depletion in non-canonical autophagy and also contributing with a modest metabolic regulation. [ABSTRACT FROM AUTHOR]

Chronic exercise training is associated with an "athlete's artery" phenotype in young adults and an attenuated age-related decline in endothelium-dependent arterial function. Adolescence is associated with an influx of sex-specific hormones that may exert divergent effects on endothelial function, but whether training adaptations interact with biological maturation to produce a "youth athlete's artery" has not been explored. We investigated the influence of exercise-training status on endothelium-dependent arterial function during childhood and adolescence. Brachial artery flow-mediated dilation (FMD) was assessed in n = 102 exercise-trained (males, n = 25; females, n = 29) and untrained (males, n = 23; females, n = 25) youths, characterized as pre (males, n = 25; females, n = 26)- or post (males, n = 23; females, n = 28)-predicted age at peak height velocity (PHV). Baseline brachial artery diameter was larger in post- compared with pre-PHV youths (P ≤ 0.001), males compared with females (P ≤ 0.001), and trained compared with untrained youths (3.26 ± 0.51 vs. 3.11 ± 0.42 mm; P = 0.041). Brachial FMD was similar in pre- and post-PHV youths (P = 0.298), and males and females (P = 0.946). However, exercise-trained youths demonstrated higher FMD when compared with untrained counterparts (5.3 ± 3.3 vs. 3.0 ± 2.6%; P ≤ 0.001). Furthermore, brachial artery diameter (r2 = 0.142; P = 0.007 vs. r2 = 0.004; P = 0.652) and FMD (r2 = 0.138; P = 0.008 vs. r2 = 0.003; P = 0.706) were positively associated with cardiorespiratory fitness in post-, but not pre-PHV youths, respectively. Collectively, our data indicate that exercise training is associated with brachial artery remodeling and enhanced endothelial function during youth. However, arterial remodeling and endothelium-dependent function are only associated with elevated cardiorespiratory fitness during later stages of adolescence. NEW & NOTEWORTHY: We report preliminary evidence of the "youth athlete's artery," characterized by training-related arterial remodeling and elevated endothelium-dependent arterial function in children and adolescents. However, training-related adaptations in brachial artery diameter and flow-mediated dilation (FMD) were associated with cardiorespiratory fitness in adolescents, but not in children. Our findings indicate that endothelium-dependent arterial function is modifiable with chronic exercise training during childhood, but the association between FMD and elevated cardiorespiratory fitness is only apparent during later stages of adolescence. [ABSTRACT FROM AUTHOR]

Background: Doxorubicin (DOX) is a potent chemotherapy widely used in treating various neoplastic diseases. However, the clinical use of DOX is limited due to its potential toxic effect on the cardiovascular system. Thus, identifying the pathway involved in this toxicity may help minimize chemotherapy risk and improve cancer patients' quality of life. Recent studies suggest that Endothelial-to-Mesenchymal transition (EndMT) and endothelial toxicity contribute to the pathogenesis of DOX-induced cardiovascular toxicity. However, the molecular mechanism is yet unknown. Given that arachidonic acid and associated cytochrome P450 (CYP) epoxygenase have been involved in endothelial and cardiovascular function, we aimed to examine the effect of suppressing CYP epoxygenases on DOX-induced EndMT and cardiovascular toxicity in vitro and in vivo. Methods and Results: To test this, human endothelial cells were treated with DOX, with or without CYP epoxygenase inhibitor, MSPPOH. We also investigated the effect of MSPPOH on the cardiovascular system in our zebrafish model of DOX-induced cardiotoxicity. Our results showed that MSPPOH exacerbated DOX-induced EndMT, inflammation, oxidative stress, and apoptosis in our endothelial cells. Furthermore, we also show that MSPPOH increased cardiac edema, lowered vascular blood flow velocity, and worsened the expression of EndMT and cardiac injury markers in our zebrafish model of DOX-induced cardiotoxicity. Conclusion: Our data indicate that a selective CYP epoxygenase inhibitor, MSPPOH, induces EndMT and endothelial toxicity to contribute to DOX-induced cardiovascular toxicity. [ABSTRACT FROM AUTHOR]

Studies on the bench and at bedside have demonstrated that the process of epileptogenesis is involved in neuroinflammatory responses. As the receptor of proinflammatory cytokine IL-1β, IL-1β type 1 receptor (IL-1R1) is reported to express abundantly in the endothelial cells in epileptic brains, which is deemed to be implicated in the epileptogenic process. However, whether and how endothelial IL-1R1 modulates neuroinflammatory responses in the pathological process of epileptic seizures and/or status epilepticus (SE) remains obscure. Here, we indicated endothelial IL-1R1 is involved in neuroinflammation, facilitating epilepsy progress via Nrf2/HO-1/NLRP3. In vitro, we observed upregulation of inflammatory cytokines in co-culture model under IL-1β challenge, as well as in BV2 cells after stimulation with conditional medium (CM) from IL-1β-stimulated bEnd.3 cells. In vivo, mice with conditional knockout of endothelial IL-1R1 (IL-1R1-CKO) were generated by hybrid IL-1R1flox/flox mice with Tek-Cre mice. IL-1R1-CKO reduced seizure susceptibility in kainic acid (KA)-induced SE model. In addition, IL-1R1-CKO KA mice exhibited lessened hippocampal neuroinflammation, mitigated neuronal damage, and decreased abnormal neurogenesis. In cognitive behavioral tests, IL-1R1-CKO KA mice presented improvement in learning and memory. Furthermore, we also indicated blockage of endothelial IL-1R1 downregulated the expressions of Nrf2/HO-1/NLRP3 pathway-related proteins. Nrf2-siRNA reversed the downregulation of HO-1, NLRP3, caspase-1, and IL-1β. These results demonstrated CKO of endothelial IL-1R1 reduces seizure susceptibility and attenuates SE-related neurobehavioral damage by suppressing hippocampal neuroinflammation via Nrf2/HO-1/NLRP3. [ABSTRACT FROM AUTHOR]

It has remained unclear how aging of endothelial cells (EC) contributes to pathophysiology of individual organs. Cell senescence results in part from inactivation of telomerase (TERT). Here, we analyzed mice with Tert knockout specifically in EC. Tert loss in EC induced transcriptional changes indicative of senescence and tissue hypoxia in EC and in other cells. We demonstrate that EC‐Tert‐KO mice have leaky blood vessels. The blood–brain barrier of EC‐Tert‐KO mice is compromised, and their cognitive function is impaired. EC‐Tert‐KO mice display reduced muscle endurance and decreased expression of enzymes responsible for oxidative metabolism. Our data indicate that Tert‐KO EC have reduced mitochondrial content and function, which results in increased dependence on glycolysis. Consistent with this, EC‐Tert‐KO mice have metabolism changes indicative of increased glucose utilization. In EC‐Tert‐KO mice, expedited telomere attrition is observed for EC of adipose tissue (AT), while brain and skeletal muscle EC have normal telomere length but still display features of senescence. Our data indicate that the loss of Tert causes EC senescence in part through a telomere length‐independent mechanism undermining mitochondrial function. We conclude that EC‐Tert‐KO mice is a model of expedited vascular senescence recapitulating the hallmarks aging, which can be useful for developing revitalization therapies. [ABSTRACT FROM AUTHOR]

Understanding the factors which control endothelial cell (EC) function and angiogenesis is crucial for developing the horse as a disease model, but equine ECs remain poorly studied. In this study, we have optimised methods for the isolation and culture of equine aortic endothelial cells (EAoECs) and characterised their angiogenic functions in vitro. Mechanical dissociation, followed by magnetic purification using an anti-VE-cadherin antibody, resulted in EC-enriched cultures suitable for further study. Fibroblast growth factor 2 (FGF2) increased the EAoEC proliferation rate and stimulated scratch wound closure and tube formation by EAoECs on the extracellular matrix. Pharmacological inhibitors of FGF receptor 1 (FGFR1) (SU5402) or mitogen-activated protein kinase (MEK) (PD184352) blocked FGF2-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and functional responses, suggesting that these are dependent on FGFR1/MEK-ERK signalling. In marked contrast, vascular endothelial growth factor-A (VEGF-A) had no effect on EAoEC proliferation, migration, or tubulogenesis and did not promote ERK1/2 phosphorylation, indicating a lack of sensitivity to this classical pro-angiogenic growth factor. Gene expression analysis showed that unlike human ECs, FGFR1 is expressed by EAoECs at a much higher level than both VEGF receptor (VEGFR)1 and VEGFR2. These results suggest a predominant role for FGF2 versus VEGF-A in controlling the angiogenic functions of equine ECs. Collectively, our novel data provide a sound basis for studying angiogenic processes in horses and lay the foundations for comparative studies of EC biology in horses versus humans. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are ubiquitously expressed cell surface receptors that mediate numerous physiological responses and are highly druggable. Upon activation, GPCRs rapidly couple to heterotrimeric G proteins and are then phosphorylated and internalized from the cell surface. Recent studies indicate that GPCRs not only localize at the plasma membrane but also exist in intracellular compartments where they are competent to signal. Intracellular signaling by GPCRs is best described to occur at endosomes. Several studies have elegantly documented endosomal GPCR-G protein and GPCR-β-arrestin signaling. Besides phosphorylation, GPCRs are also posttranslationally modified with ubiquitin. GPCR ubiquitination has been studied mainly in the context of receptor endosomal-lysosomal trafficking. However, new studies indicate that ubiquitination of endogenous GPCRs expressed in endothelial cells initiates the assembly of an intracellular p38 mitogen-activated kinase signaling complex that promotes inflammatory responses from endosomes. In this mini-review, we discuss emerging discoveries that provide critical insights into the function of ubiquitination in regulating GPCR inflammatory signaling at endosomes. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cell transplantation (HSCT) and chimeric antigen receptor-T (CAR-T) immunotherapy are widely used for the management of hematological malignancies. HSCT can be complicated by endothelial injury syndromes, such as HSCT-thrombotic microangiopathy (HSCT-TMA) and sinusoidal obstructive syndrome/veno-occlusive disease (SOS/VOD), which are life-threatening. Moreover, venous thromboembolic events (VTEs) are common in HSCT recipients due to endothelial injury, use of central venous catheters, prolonged hospitalization, and the development of a procoagulant state. VTEs have also been reported post-CAR-T infusion. The management of thrombotic events in these patients is challenging, due to the high risk of bleeding that is present. CAR-T immunotherapy might be followed by toxicities, such as cytokine release syndrome (CRS) and immune effector cell-associated neuro-toxicity syndrome (ICANS). Endothelial dysfunction is implicated in the pathogenesis of these syndromes. Early recognition and management of the above complications are crucial for better patient outcomes.

Sodium alendronate (ALN) is a very hydrosoluble and poorly permeable molecule used as an antiresorptive agent and with vascular anticalcifying capacity. Loaded into targeted nanovesicles, its anti-inflammatory activity may be amplified towards extra-osseous and noncalcified target cells, such as severely irritated vascular endothelium. Here cytotoxicity, mitochondrial membrane potential, ATP content, and membrane fluidity of human endothelial venous cells (HUVECs) were determined after endocytosis of ALN-loaded nanoarchaeosomes (nanoARC-Chol(ALN), made of polar lipids from Halorubrum tebenquichense: cholesterol 7:3 w/w, 166 ± 5 nm, 0.16 ± 0.02 PDI, −40.8 ± 5.4 mV potential, 84.7 ± 21 µg/mg ALN/total lipids, TL). The effect of nanoARC-Chol(ALN) was further assessed on severely inflamed HUVECs. To that aim, HUVECs were grown on a porous barrier on top of a basal compartment seeded either with macrophages or human foam cells. One lighter and one more pronounced inflammatory context was modelled by adding lipopolysaccharide (LPS) to the apical or the apical and basal compartments. The endocytosis of nanoARC-Chol(ALN), was observed to partly reduce the endothelial-mesenchymal transition of HUVECs. Besides, while 10 mg/mL dexamethasone, 7.6 mM free ALN and ALN-loaded liposomes failed, 50 μg/mL TL + 2.5 μg/mL ALN (i.e., nanoARC-Chol(ALN)) reduced the IL-6 and IL-8 levels by, respectively, 75% and 65% in the mild and by, respectively, 60% and 40% in the pronounced inflammation model. This is the first report showing that the endocytosis of nanoARC-Chol(ALN) by HUVECs magnifies the anti-inflammatory activity of ALN even under conditions of intense irritation, not only surpassing that of free ALN but also that of dexamethasone.

Hematopoietic stem cell transplantation (HSCT) remains a cornerstone in the management of patients with hematological malignancies. Endothelial injury syndromes, such as HSCT-associated thrombotic microangiopathy (HSCT-TMA), veno-occlusive disease/sinusoidal obstruction syndrome (SOS/VOD), and capillary leak syndrome (CLS), constitute complications after HSCT. Moreover, endothelial damage is prevalent after immunotherapy with chimeric antigen receptor-T (CAR-T) and can be manifested with cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). Our literature review aims to investigate the genetic susceptibility in endothelial injury syndromes after HSCT and CAR-T cell therapy. Variations in complement pathway- and endothelial function-related genes have been associated with the development of HSCT-TMA. In these genes, CFHR5, CFHR1, CFHR3, CFI, ADAMTS13, CFB, C3, C4, C5, and MASP1 are included. Thus, patients with these variations might have a predisposition to complement activation, which is also exaggerated by other factors (such as acute graft-versus-host disease, infections, and calcineurin inhibitors). Few studies have examined the genetic susceptibility to SOS/VOD syndrome, and the implicated genes include CFH, methylenetetrahydrofolate reductase, and heparinase. Finally, specific mutations have been associated with the onset of CRS (PFKFB4, CX3CR1) and ICANS (PPM1D, DNMT3A, TE2, ASXL1). More research is essential in this field to achieve better outcomes for our patients.

Abstract Background Diabetic cardiomyopathy (DCM) poses a growing health threat, elevating heart failure risk in diabetic individuals. Understanding DCM is crucial, with fibroblasts and endothelial cells playing pivotal roles in driving myocardial fibrosis and contributing to cardiac dysfunction. Advances in Multimodal single-cell profiling, such as scRNA-seq and scATAC-seq, provide deeper insights into DCM’s unique cell states and molecular landscape for targeted therapeutic interventions. Methods Single-cell RNA and ATAC data from 10x Multiome libraries were processed using Cell Ranger ARC v2.0.1. Gene expression and ATAC data underwent Seurat and Signac filtration. Differential gene expression and accessible chromatin regions were identified. Transcription factor activity was estimated with chromVAR, and Cis-coaccessibility networks were calculated using Cicero. Coaccessibility connections were compared to the GeneHancer database. Gene Ontology analysis, biological process scoring, cell-cell communication analysis, and gene-motif correlation was performed to reveal intricate molecular changes. Immunofluorescent staining utilized various antibodies on paraffin-embedded tissues to verify the findings. Results This study integrated scRNA-seq and scATAC-seq data obtained from hearts of WT and DCM mice, elucidating molecular changes at the single-cell level throughout the diabetic cardiomyopathy progression. Robust and accurate clustering analysis of the integrated data revealed altered cell proportions, showcasing decreased endothelial cells and macrophages, coupled with increased fibroblasts and myocardial cells in the DCM group, indicating enhanced fibrosis and endothelial damage. Chromatin accessibility analysis unveiled unique patterns in cell types, with heightened transcriptional activity in myocardial cells. Subpopulation analysis highlighted distinct changes in cardiomyocytes and fibroblasts, emphasizing pathways related to fatty acid metabolism and cardiac contraction. Fibroblast-centered communication analysis identified interactions with endothelial cells, implicating VEGF receptors. Endothelial cell subpopulations exhibited altered gene expressions, emphasizing contraction and growth-related pathways. Candidate regulators, including Tcf21, Arnt, Stat5a, and Stat5b, were identified, suggesting their pivotal roles in DCM development. Immunofluorescence staining validated marker genes of cell subpopulations, confirming PDK4, PPARγ and Tpm1 as markers for metabolic pattern-altered cardiomyocytes, activated fibroblasts and endothelial cells with compromised proliferation. Conclusion Our integrated scRNA-seq and scATAC-seq analysis unveils intricate cell states and molecular alterations in diabetic cardiomyopathy. Identified cell type-specific changes, transcription factors, and marker genes offer valuable insights. The study sheds light on potential therapeutic targets for DCM.

Abstract Aims Regular transient limb ischemia (RTLI) can prevent atherosclerosis (AS) progression in hypercholesterolemic rabbits. This study aimed to investigate the minimum effective intensity and possible mechanisms of RTLI for preventing atherosclerosis. Methods Eighty rabbits were divided into eight groups: normal (N), high cholesterol (H), three RTLI [three RTLI cycles every other day (R3qod), three RTLI cycles daily (R3qd), and six RTLI cycles daily (R6qd), each cycle of RTLI included 5 min of limb ischemia followed by 5 min limb reperfusion], and three correlated sham RTLI [sham ischemia for 30 min once every other day (S3qod), sham ischemia for 30 min once daily (S3qd), and sham ischemia for 60 min once daily (S6qd)]. Rabbits in group N were kept normally, while the others were fed 1% cholesterol diet for 12 weeks. The RTLI and sham RTLI groups were received RTLI or sham RTLI procedure, respectively. The plaque area in the thoracic aorta was determined by oil red O staining, and quantifying the ratio of plaque area to intimal area (PA/IA). Endothelium-dependent and -independent relaxation were also determined. Endothelial cell were isolated from abdominal aorta of rabbits, and the apoptosis ratio was detected using flow cytometry. Results The PA/IA and early apoptotic cell ratio was significantly lower as well as the endothelium-dependent relaxation response was higher in group R6qd than those in groups H and S6qd, while those in the R3qod group was not significantly different from those in groups H and S3qod, as well as those in the R3qd group showed no significant difference compared to those in groups H and S3qd. Conclusions Six cycles of RTLI daily was the optimal effective intensity to prevent AS progression in rabbits. Endothelial function improvement and apoptosis inhibition might contribute to the anti-AS effects.

Abstract Background The active transport of molecules into the brain from blood is regulated by receptors, transporters, and other cell surface proteins that are present on the luminal surface of endothelial cells at the blood–brain barrier (BBB). However, proteomic profiling of proteins present on the luminal endothelial cell surface of the BBB has proven challenging due to difficulty in labelling these proteins in a way that allows efficient purification of these relatively low abundance cell surface proteins. Methods Here we describe a novel perfusion-based labelling workflow: in vivo glycocapture. This workflow relies on the oxidation of glycans present on the luminal vessel surface via perfusion of a mild oxidizing agent, followed by subsequent isolation of glycoproteins by covalent linkage of their oxidized glycans to hydrazide beads. Mass spectrometry-based identification of the isolated proteins enables high-confidence identification of endothelial cell surface proteins in rats and mice. Results Using the developed workflow, 347 proteins were identified from the BBB in rat and 224 proteins in mouse, for a total of 395 proteins in both species combined. These proteins included many proteins with transporter activity (73 proteins), cell adhesion proteins (47 proteins), and transmembrane signal receptors (31 proteins). To identify proteins that are enriched in vessels relative to the entire brain, we established a vessel-enrichment score and showed that proteins with a high vessel-enrichment score are involved in vascular development functions, binding to integrins, and cell adhesion. Using publicly-available single-cell RNAseq data, we show that the proteins identified by in vivo glycocapture were more likely to be detected by scRNAseq in endothelial cells than in any other cell type. Furthermore, nearly 50% of the genes encoding cell-surface proteins that were detected by scRNAseq in endothelial cells were also identified by in vivo glycocapture. Conclusions The proteins identified by in vivo glycocapture in this work represent the most complete and specific profiling of proteins on the luminal BBB surface to date. The identified proteins reflect possible targets for the development of antibodies to improve the crossing of therapeutic proteins into the brain and will contribute to our further understanding of BBB transport mechanisms.

Endothelial cells (ECs) within the vascular system encounter fluid shear stress (FSS). High, laminar FSS promotes vasodilation and anti-inflammatory responses, whereas low or disturbed FSS induces dysfunction and inflammation. However, the adaptation of endothelial cells (ECs) to dynamically changing FSS patterns remains underexplored. Here, by combining traction force microscopy with a custom flow chamber, we examined human umbilical vein endothelial cells adapting their traction during transitions from short-term low shear to long-term high shear stress. We discovered that the initial low FSS elevates the traction by only half of the amount in response to direct high FSS even after flow changes to high FSS. However, in the long term under high FSS, the flow started with low FSS triggers a substantial second rise in traction for over 10 h. In contrast, the flow started directly with high FSS results in a quick traction surge followed by a huge reduction below the baseline traction in <30 min. Importantly, we find that the orientation of traction vectors is steered by initial shear exposure. Using Granger causality analysis, we show that the traction that aligns in the flow direction under direct high FSS functionally causes cell alignment toward the flow direction. However, EC traction that orients perpendicular to the flow that starts with temporary low FSS functionally causes cell orientation perpendicular to the flow. Taken together, our findings elucidate the significant influence of initial short-term low FSS on lasting changes in endothelial traction that induces EC alignment. NEW & NOTEWORTHY In our study, we uncover that preconditioning with low shear stress yields enduring impacts on endothelial cell traction and orientation, persisting even after transitioning to high-shear conditions. Using Granger causality analysis, we demonstrate a functional link between the direction of cell traction and subsequent cellular alignment across varying shear environments. [ABSTRACT FROM AUTHOR]

Atherosclerosis, a chronic disease of arteries, results in high mortality worldwide as the leading cause of cardiovascular disease. The development of clinically relevant atherosclerosis involves the dysfunction of endothelial cells and vascular smooth muscle cells. A large amount of evidence indicates that noncoding RNAs, such as microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), are involved in various physiological and pathological processes. Recently, noncoding RNAs were identified as key regulators in the development of atherosclerosis, including the dysfunction of endothelial cells, and vascular smooth muscle cells and it is pertinent to understand the potential function of noncoding RNAs in atherosclerosis development. In this review, the latest available research relates to the regulatory role of noncoding RNAs in the progression of atherosclerosis and the therapeutic potential for atherosclerosis is summarized. This review aims to provide a comprehensive overview of the regulatory and interventional roles of ncRNAs in atherosclerosis and to inspire new insights for the prevention and treatment of this disease. [ABSTRACT FROM AUTHOR]

Regulation of subcellular messenger (m)RNA localization is a fundamental biological mechanism, which adds a spatial dimension to the diverse layers of post- transcriptional control of gene expression. The cellular compartment in which mRNAs are located may define distinct aspects of the encoded proteins, ranging from production rate and complex formation to localized activity. Despite the detailed roles of localized mRNAs that have emerged over the past decades, the identity of factors anchoring mRNAs to subcellular domains remains ill- defined. Here, we used an unbiased method to profile the RNA- bound proteome in migrating endothelial cells (ECs) and discovered that the plasma membrane (PM)--associated scaffolding protein A- kinase anchor protein (AKAP)12 interacts with various mRNAs, including transcripts encoding kinases with Actin remodeling activity. In particular, AKAP12 targets a transcript coding for the kinase Abelson Tyrosine- Protein Kinase 2 (ABL2), which we found to be necessary for adequate filopodia formation and angiogenic sprouting. Moreover, we demonstrate that AKAP12 is necessary for anchoring ABL2 mRNA to the PM and show that in the absence of AKAP12, the translation efficiency of ABL2 mRNA is reduced. Altogether, our work identified a unique post- transcriptional function for AKAP12 and sheds light into mechanisms of spatial control of gene expression. [ABSTRACT FROM AUTHOR]

Background: Diabetic cardiomyopathy (DCM) poses a growing health threat, elevating heart failure risk in diabetic individuals. Understanding DCM is crucial, with fibroblasts and endothelial cells playing pivotal roles in driving myocardial fibrosis and contributing to cardiac dysfunction. Advances in Multimodal single-cell profiling, such as scRNA-seq and scATAC-seq, provide deeper insights into DCM's unique cell states and molecular landscape for targeted therapeutic interventions. Methods: Single-cell RNA and ATAC data from 10x Multiome libraries were processed using Cell Ranger ARC v2.0.1. Gene expression and ATAC data underwent Seurat and Signac filtration. Differential gene expression and accessible chromatin regions were identified. Transcription factor activity was estimated with chromVAR, and Cis-coaccessibility networks were calculated using Cicero. Coaccessibility connections were compared to the GeneHancer database. Gene Ontology analysis, biological process scoring, cell-cell communication analysis, and gene-motif correlation was performed to reveal intricate molecular changes. Immunofluorescent staining utilized various antibodies on paraffin-embedded tissues to verify the findings. Results: This study integrated scRNA-seq and scATAC-seq data obtained from hearts of WT and DCM mice, elucidating molecular changes at the single-cell level throughout the diabetic cardiomyopathy progression. Robust and accurate clustering analysis of the integrated data revealed altered cell proportions, showcasing decreased endothelial cells and macrophages, coupled with increased fibroblasts and myocardial cells in the DCM group, indicating enhanced fibrosis and endothelial damage. Chromatin accessibility analysis unveiled unique patterns in cell types, with heightened transcriptional activity in myocardial cells. Subpopulation analysis highlighted distinct changes in cardiomyocytes and fibroblasts, emphasizing pathways related to fatty acid metabolism and cardiac contraction. Fibroblast-centered communication analysis identified interactions with endothelial cells, implicating VEGF receptors. Endothelial cell subpopulations exhibited altered gene expressions, emphasizing contraction and growth-related pathways. Candidate regulators, including Tcf21, Arnt, Stat5a, and Stat5b, were identified, suggesting their pivotal roles in DCM development. Immunofluorescence staining validated marker genes of cell subpopulations, confirming PDK4, PPARγ and Tpm1 as markers for metabolic pattern-altered cardiomyocytes, activated fibroblasts and endothelial cells with compromised proliferation. Conclusion: Our integrated scRNA-seq and scATAC-seq analysis unveils intricate cell states and molecular alterations in diabetic cardiomyopathy. Identified cell type-specific changes, transcription factors, and marker genes offer valuable insights. The study sheds light on potential therapeutic targets for DCM. [ABSTRACT FROM AUTHOR]

Aims: Regular transient limb ischemia (RTLI) can prevent atherosclerosis (AS) progression in hypercholesterolemic rabbits. This study aimed to investigate the minimum effective intensity and possible mechanisms of RTLI for preventing atherosclerosis. Methods: Eighty rabbits were divided into eight groups: normal (N), high cholesterol (H), three RTLI [three RTLI cycles every other day (R3qod), three RTLI cycles daily (R3qd), and six RTLI cycles daily (R6qd), each cycle of RTLI included 5 min of limb ischemia followed by 5 min limb reperfusion], and three correlated sham RTLI [sham ischemia for 30 min once every other day (S3qod), sham ischemia for 30 min once daily (S3qd), and sham ischemia for 60 min once daily (S6qd)]. Rabbits in group N were kept normally, while the others were fed 1% cholesterol diet for 12 weeks. The RTLI and sham RTLI groups were received RTLI or sham RTLI procedure, respectively. The plaque area in the thoracic aorta was determined by oil red O staining, and quantifying the ratio of plaque area to intimal area (PA/IA). Endothelium-dependent and -independent relaxation were also determined. Endothelial cell were isolated from abdominal aorta of rabbits, and the apoptosis ratio was detected using flow cytometry. Results: The PA/IA and early apoptotic cell ratio was significantly lower as well as the endothelium-dependent relaxation response was higher in group R6qd than those in groups H and S6qd, while those in the R3qod group was not significantly different from those in groups H and S3qod, as well as those in the R3qd group showed no significant difference compared to those in groups H and S3qd. Conclusions: Six cycles of RTLI daily was the optimal effective intensity to prevent AS progression in rabbits. Endothelial function improvement and apoptosis inhibition might contribute to the anti-AS effects. [ABSTRACT FROM AUTHOR]

Background: The active transport of molecules into the brain from blood is regulated by receptors, transporters, and other cell surface proteins that are present on the luminal surface of endothelial cells at the blood–brain barrier (BBB). However, proteomic profiling of proteins present on the luminal endothelial cell surface of the BBB has proven challenging due to difficulty in labelling these proteins in a way that allows efficient purification of these relatively low abundance cell surface proteins. Methods: Here we describe a novel perfusion-based labelling workflow: in vivo glycocapture. This workflow relies on the oxidation of glycans present on the luminal vessel surface via perfusion of a mild oxidizing agent, followed by subsequent isolation of glycoproteins by covalent linkage of their oxidized glycans to hydrazide beads. Mass spectrometry-based identification of the isolated proteins enables high-confidence identification of endothelial cell surface proteins in rats and mice. Results: Using the developed workflow, 347 proteins were identified from the BBB in rat and 224 proteins in mouse, for a total of 395 proteins in both species combined. These proteins included many proteins with transporter activity (73 proteins), cell adhesion proteins (47 proteins), and transmembrane signal receptors (31 proteins). To identify proteins that are enriched in vessels relative to the entire brain, we established a vessel-enrichment score and showed that proteins with a high vessel-enrichment score are involved in vascular development functions, binding to integrins, and cell adhesion. Using publicly-available single-cell RNAseq data, we show that the proteins identified by in vivo glycocapture were more likely to be detected by scRNAseq in endothelial cells than in any other cell type. Furthermore, nearly 50% of the genes encoding cell-surface proteins that were detected by scRNAseq in endothelial cells were also identified by in vivo glycocapture. Conclusions: The proteins identified by in vivo glycocapture in this work represent the most complete and specific profiling of proteins on the luminal BBB surface to date. The identified proteins reflect possible targets for the development of antibodies to improve the crossing of therapeutic proteins into the brain and will contribute to our further understanding of BBB transport mechanisms. [ABSTRACT FROM AUTHOR]

The discovery of an inhibitor for acyl-CoA synthetase long-chain family member 4 (ACSL4), a protein involved in the process of cell injury through ferroptosis, has the potential to ameliorate cell damage. In this study, we aimed to investigate the potential of berberine (BBR) as an inhibitor of ACSL4 in order to suppress endothelial ferroptosis and provide protection against atherosclerosis. An atherosclerosis model was created in ApoE-/- mice by feeding a high fat diet for 16 weeks. Additionally, a mouse model with endothelium-specific overexpression of ACSL4 was established. BBR was administered orally to assess its potential therapeutic effects on atherosclerosis. Human umbilical vein endothelial cells (HUVECs) were exposed to oxidized low density lipoprotein (ox-LDL) to simulate atherosclerotic endothelial damage in vitro. The interaction between ACSL4 and BBR has been confirmed, with BBR playing a role in inhibiting erastin-induced ferroptosis by regulating ACSL4. Additionally, BBR has been found to inhibit lipid deposition, plaque formation, and collagen deposition in the aorta, thereby delaying the progression of atherosclerosis. It also restored the abnormal expression of ferroptosis-related proteins in atherosclerotic vascular endothelial cells both in vivo and in vitro. In conclusion, BBR, acting as an ACSL4 inhibitor, can improve atherosclerosis by inhibiting ferroptosis in endothelial cells. This highlights the potential of targeted inhibition of vascular endothelial ACSL4 as a strategy for treating atherosclerosis, with BBR being a candidate for this purpose.

Cardiotoxicity can be defined as “chemically induced heart disease”, which can occur with many different drug classes treating a range of diseases. It is the primary cause of drug attrition during pre-clinical development and withdrawal from the market. Drug induced cardiovascular toxicity can result from both functional effects with alteration of the contractile and electrical regulation in the heart and structural changes with morphological changes to cardiomyocytes and other cardiac cells. These adverse effects result in conditions such as arrhythmia or a more serious reduction in left ventricular ejection fraction (LVEF), which can lead to heart failure and death. Anticancer drugs can adversely affect cardiomyocyte function as well as cardiac fibroblasts and cardiac endothelial cells, interfering in autocrine and paracrine signalling between these cell types and ultimately altering cardiac cellular homeostasis. This review aims to highlight potential toxicity mechanisms involving cardiomyocytes and non-cardiomyocyte cells by first introducing the physiological roles of these cells within the myocardium and secondly, identifying the physiological pathways perturbed by anticancer drugs in these cells.

The delivery of nutrients to the brain is provided by a 600 km network of capillaries and microvessels. Indeed, the brain is highly energy demanding and, among a total amount of 100 billion neurons, each neuron is located just 10–20 μm from a capillary. This vascular network also forms part of the blood–brain barrier (BBB), which maintains the brain’s stable environment by regulating chemical balance, immune cell transport, and blocking toxins. Typically, brain microvascular endothelial cells (BMECs) have low turnover, indicating a stable cerebrovascular structure. However, this structure can adapt significantly due to development, aging, injury, or disease. Temporary neural activity changes are managed by the expansion or contraction of arterioles and capillaries. Hypoxia leads to significant remodeling of the cerebrovascular architecture and pathological changes have been documented in aging and in vascular and neurodegenerative conditions. These changes often involve BMEC proliferation and the remodeling of capillary segments, often linked with local neuronal changes and cognitive function. Cerebrovascular plasticity, especially in arterioles, capillaries, and venules, varies over different time scales in development, health, aging, and diseases. Rapid changes in cerebral blood flow (CBF) occur within seconds due to increased neural activity. Prolonged changes in vascular structure, influenced by consistent environmental factors, take weeks. Development and aging bring changes over months to years, with aging-associated plasticity often improved by exercise. Injuries cause rapid damage but can be repaired over weeks to months, while neurodegenerative diseases cause slow, varied changes over months to years. In addition, if animal models may provide useful and dynamic in vivo information about vascular plasticity, humans are more complex to investigate and the hypothesis of glymphatic system together with Magnetic Resonance Imaging (MRI) techniques could provide useful clues in the future.

Adrenal cysts are uncommon and usually asymptomatic, and therefore are usually incidentally discovered adrenal lesions. They have a broad pathohistological spectrum that includes pseudocysts and endothelial (vascular), parasitic, and epithelial (mesothelial) cysts. Although most adrenal cysts are benign and hormonally non-functional lesions, some can have ambiguous imaging appearances and mimic malignant adrenal neoplasms. On the other hand, the actual malignant neoplasms could undergo cystic transformation. Additionally, immune cell infiltrations, thrombosis, or haemorrhage seen in sepsis can frequently cause adrenal cyst development, raising a question about the possible connection between severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and adrenal cystic lesions. Due to the disease's rarity, the likelihood of malignancy, and the lack of specific guidelines, the management of adrenal cysts is always challenging especially in a young person. This review discusses the important diagnostic and the current treatment possibilities for adrenal cystic lesions. Aiming to emphasize clinical dilemmas and help clinicians navigate the challenges when encountering a patient with an adrenal cyst in everyday practice, we based our review on a practical question–answer framework centred around the case of a young woman with an incidentally discovered large adrenal cyst. [ABSTRACT FROM AUTHOR]

Inspiratory resistance training (IRT) yields significant reductions in resting blood pressure and improves vascular endothelial function. Our objective was to quantify the acute effects of IRT on brachial artery flow‐mediated dilation (FMD) and shear rates (SRs) in healthy men and women. Twenty young adults (22.9 ± 3.4 years; 10 male, 10 female) completed a single bout of IRT or Rest condition in a randomized crossover design. Brachial artery FMD was performed before, 10 min after, and 40 min after the assigned condition. Brachial artery blood flow velocities were collected during IRT, separated by breathing cycle phase, and converted into SRs. FMD improved 10 min post‐IRT (+1.86 ± 0.61%; p = 0.025) but returned to baseline by 40 min post‐IRT (p = 0.002). Anterograde SR decreased by 10% and retrograde SR increased 102% during resisted inspiration, relative to baseline SR (p < 0.001). Anterograde SR increased by 7% in men and women (p < 0.001) and retrograde SR decreased by 12% in women but not men (p = 0.022) during unresisted expiration, relative to baseline SR. A single bout of IRT elicits a transient enhancement in FMD in both men and women. Acute IRT‐related enhancements in SRs may contribute to sustained improvements in FMD that have been reported previously. [ABSTRACT FROM AUTHOR]

Objective: Increased O-linked β-N-acetylglucosamine (O-GlcNAc) stimulation has been reported to protect against sepsis associated mortality and cardiovascular derangement. Previous studies, including our own research, have indicated that gasdermin-D(GSDMD)-mediated endothelial cells pyroptosis contributes to sepsis-associated endothelial injury. This study explored the functions and mechanisms of O-GlcNAc modification on lipopolysaccharide (LPS)-induced pyroptosis and its effects on the function of GSDMD. Methods: A LPS-induced septic mouse model administrated with O-GlcNAcase (OGA) inhibitor thiamet-G (TMG) was used to assess the effects of O-GlcNAcylation on sepsis-associated vascular dysfunction and pyroptosis. We conducted experiments on human umbilical vein endothelial cells (HUVECs) by challenging them with LPS and TMG to investigate the impact of O-GlcNAcylation on endothelial cell pyroptosis and implications of GSDMD. Additionally, we identified potential O-GlcNAcylation sites in GSDMD by utilizing four public O-GlcNAcylation site prediction database, and these sites were ultimately established through gene mutation. Results: Septic mice with increased O-GlcNAc stimulation exhibited reduced endothelial injury, GSDMD cleavage (a marker of pyroptosis). O-GlcNAc modification of GSDMD mitigates LPS-induced pyroptosis in endothelial cells by preventing its interaction with caspase-11 (a human homologous of caspases-4/5). We also identified GSDMD Serine 338 (S338) as a novel site of O-GlcNAc modification, leading to decreased association with caspases-4 in HEK293T cells. Conclusions: Our findings identified a novel post-translational modification of GSDMD and elucidated the O-GlcNAcylation of GSDMD inhibits LPS-induced endothelial injury, suggesting that O-GlcNAc modification-based treatments could serve as potential interventions for sepsis-associated vascular endothelial injury. [ABSTRACT FROM AUTHOR]

This article examines the impact of endothelial cell senescence on aging and disease by studying mice with a specific gene knockout of telomerase. The researchers found that the loss of telomerase led to the upregulation of genes associated with cell senescence and endothelial dysfunction in adipose tissue and skeletal muscle. These findings contribute to our understanding of the mechanisms behind aging and may have implications for the development of interventions for metabolic and degenerative diseases. The article also provides information on the availability of the datasets and the ethical approval for the animal study. [Extracted from the article]

As a p53-regulated gene, Wip1 regulates proliferation, migration, apoptosis, and senescence of several type cells, but its biological functions in keratinocytes and endothelial cells which are involved wound healing are not fully understood. This study aims to reveal the function and underlying mechanism of Wip1 in wound healing using models of transgenic animal, keratinocytes, and endothelial cells. Using Wip1 knockout C57 BL/6 mice, we investigated effect of Wip1 deficiency on wound healing and angiogenesis; And using HaCaT and HUVEC as keratinocytes and endothelial cells, combined using primary keratinocytes from Wip1 knockout mice, we studied the effects of Wip1 knockdown/knockout or overexpression on proliferation, migration, and protein expressions of signaling components in ATM-p53 and mTOR pathway. Wip1 deficiency in mice impaired the wound repair and endothelial angiogenesis, reduced the thickness of granulation tissue, and decreased the number of Ki67-positive cells and CD31 positive vessels in granulation tissue. Knockdown of Wip1 by shRNAs suppressed the proliferation and migration of HaCaT and HUVEC cells and induced notably apoptosis in the two cells. In western blot, Wip1 knockdown enriched p53 and ATM proteins, while decreased activated AKT, mTOR and activated S6 ribosomal protein (pS6) levels in HaCaT and HUVEC cells. Ectopic expression of Wip1 decreased the p53 and ATM proteins, while increased activated AKT, mTOR and pS6 levels in HaCaT and HUVEC cells. And in primary keratinocytes from mice tail skin, Wip1 knockout increased p53 and ATM, while decreased activated AKT, mTOR and pS6 protein levels. Our study directly supports that Wip1 regulated skin wound healing possibly by affecting bioactivities including proliferation, migration and apoptosis of keratinocytes and endothelial cells at least through by modulating ATM-p53 and mTOR signaling. • Wip1 participated in mice wound repair and endothelial angiogenesis. • Wip1 regulated thickness of granulation tissue and the number of Ki67-positive cells and CD31 positive vessels in granulation tissue. • Wip1 affected the proliferation, migration and apoptosis of keratinocytes and endothelial cells by modulating ATM-p53 and mTOR signaling. [ABSTRACT FROM AUTHOR]

• Endothelial dysfunction related to Ca2+ signaling loss in VEGF 165 , bFGF, PlGF, TNFα, or IL-1β treated HUVEC is improved by the Src kinase inhibor PP2. • Src kinase inhibitor PP2 also improves TNFα-mediated losses in HUVEC monolayer integrity. • The MEK/ERK signaling inhibitor U0126 offers no improvement to growth factor or cytokine-mediated endothelial dysfunction in HUVEC. Endothelial dysfunction is known to be a key characteristic of preeclampsia (PE) and can contribute to progression of symptoms and injury to multiple organ systems. Delivery is the only treatment for progression of PE, but development of an endothelial-based therapy for PE presents a promising strategy. Growth factors and cytokines are dysregulated in PE and can impact endothelial function, manifesting changes in Ca2+ signaling and interruptions in monolayer barrier function that contribute to symptoms of hypertension, proteinuria, and edema. In this study, we highlight Src kinase as a partial mediator of growth factor and cytokine mediated endothelial dysfunction. Fura-2 Ca2+ imaging and Electrical Cell Impedance Sensing (ECIS) assays are performed on growth factor or cytokine exposed human umbilical vein endothelial cells (HUVECs). Inhibitors to MEK/ERK (U0126) or Src (PP2) are used to determine the contribution of kinase signaling pathways. Decreases in HUVEC Ca2+ signaling or monolayer resistance measure endothelial dysfunction. Reversal of endothelial dysfunction by kinase inhibitors reveals the respective contibutions of MEK/ERK and Src kinase. We show that Src inhibition protects Ca2+ signaling responses against insults induced by VEGF 165 , bFGF, PlGF, TNFα, and IL-1β. Additionally, we show that Src inhibition protects the endothelial monolayer from the full impact of TNFα insult. Further, we find that MEK/ERK inhibition does not offer protection from growth factor-mediated endothelial dysfunction. The results of this study suggest cytokine and growth factor-stimulated Src kinase plays a partial role on promoting endothelial dysfunction in HUVECs. [ABSTRACT FROM AUTHOR]

BACKGROUND Both diabetic and hypertensive nephropathy eventually progress to glomerulosclerosis. Previous studies revealed a potential role of endothelial-to-mesenchymal transition (EndMT) in the pathophysiology of glomerulosclerosis in diabetic rats. Therefore, we hypothesized that EndMT was also involved in the development of glomerulosclerosis in salt-sensitive hypertension. We aimed to explore the effects of high-salt diet on endothelial-to-mesenchymal transition (EndMT) in glomerulosclerosis in Dahl salt-sensitive (Dahl-SS) rats. METHODS Eight-week-old male rats were fed high-salt (8%NaCl; DSH group) or normal salt (0.3%NaCl; DSN group) for eight weeks, with systolic blood pressure (SBP), serum creatinine, urea, 24-hour urinary protein/sodium, renal interlobar artery blood flow, and pathological examination measured. We also examined endothelial-(CD31) and fibrosis-related protein(α-SMA) expressions in glomeruli. RESULTS High-salt diet increased SBP (DSH vs. DSN, 205.2 ± 8.9 vs. 135.4 ± 7.9 mm Hg, P  < 0.01), 24-hour urinary protein (132.55 ± 11.75 vs. 23.52 ± 5.94 mg/day, P  < 0.05), urine sodium excretions (14.09 ± 1.49 vs. 0.47 ± 0.06 mmol/day, P  < 0.05), and renal interlobar artery resistance. Glomerulosclerosis increased (26.1 ± 4.6 vs. 7.3 ± 1.6%, P  < 0.05), glomerular CD31 expressions decreased while α-SMA expression increased in DSH group. Immunofluorescence staining showed that CD31 and α-SMA co-expressed in glomeruli of the DSH group. The degree of glomerulosclerosis negatively correlated with CD31 expressions (r  = −0.823, P  < 0.01) but positively correlated with α-SMA expressions (r  = 0.936, P  < 0.01). CONCLUSIONS We demonstrated that a high-salt diet led to glomerulosclerosis involving the EndMT process, which played an essential role in glomerulosclerosis in hypertensive Dahl-SS rats. [ABSTRACT FROM AUTHOR]

A biotechnological platform consisting of two-color 3D super-resolution readout and a microfluidic system was developed to investigate platelet interaction with a layer of perfused endothelial cells under flow conditions. Platelet activation has been confirmed via CD62P clustering on the membrane and mitochondrial morphology of ECs at the single cell level were examined using 3D two-color single-molecule localization microscopy and classified applying machine learning. To compare binding of activated platelets to intact or stressed ECs, a femtosecond laser was used to induced damage to single ECs within the perfused endothelial layer. We observed that activated platelets bound to the perfused ECs layer preferentially in the proximity to single stressed ECs. Platelets activated under flow were ∼6 times larger compared to activated ones under static conditions. The CD62P expression indicated more CD62P proteins on membrane of dynamically activated platelets, with a tendency to higher densities at the platelet/EC interface. Platelets activated under static conditions showed a less pronounced CD62P top/bottom asymmetry. The clustering of CD62P in the platelet membrane differs depending on the activation conditions. Our results confirm that nanoscopic analysis using two-color 3D super-resolution technology can be used to assess platelet interaction with a stressed endothelium under dynamic conditions.

Abstract Nanomedicine has emerged as a promising therapeutic approach, but its translation to the clinic has been hindered by the lack of cellular models to anticipate how tumor cells will respond to therapy. Three-dimensional (3D) cell culture models are thought to more accurately recapitulate key features of primary tumors than two-dimensional (2D) cultures. Heterotypic 3D tumor spheroids, composed of multiple cell types, have become more popular than homotypic spheroids, which consist of a single cell type, as a superior model for mimicking in vivo tumor heterogeneity and physiology. The stromal interactions demonstrated in heterotypic 3D tumor spheroids can affect various aspects, including response to therapy, cancer progression, nanomedicine penetration, and drug resistance. Accordingly, to design more effective anticancer nanomedicinal therapeutics, not only tumor cells but also stromal cells (e.g., fibroblasts and immune cells) should be considered to create a more physiologically relevant in vivo microenvironment. This review aims to demonstrate current knowledge of heterotypic 3D tumor spheroids in cancer research, to illustrate current advances in utilizing these tumor models as a novel and versatile platform for in vitro evaluation of nanomedicine-based therapeutics in cancer research, and to discuss challenges, guidelines, and future directions in this field. Graphical Abstract

Abstract Inspiratory resistance training (IRT) yields significant reductions in resting blood pressure and improves vascular endothelial function. Our objective was to quantify the acute effects of IRT on brachial artery flow‐mediated dilation (FMD) and shear rates (SRs) in healthy men and women. Twenty young adults (22.9 ± 3.4 years; 10 male, 10 female) completed a single bout of IRT or Rest condition in a randomized crossover design. Brachial artery FMD was performed before, 10 min after, and 40 min after the assigned condition. Brachial artery blood flow velocities were collected during IRT, separated by breathing cycle phase, and converted into SRs. FMD improved 10 min post‐IRT (+1.86 ± 0.61%; p = 0.025) but returned to baseline by 40 min post‐IRT (p = 0.002). Anterograde SR decreased by 10% and retrograde SR increased 102% during resisted inspiration, relative to baseline SR (p

Our understanding of the pathophysiology of pulmonary arterial hypertension (PAH) has evolved over recent years, with the recognition that endothelial cell (EC) dysfunction and inflammation play an integral role in the development of this disease. ECs within the pulmonary vasculature play a unique role in maintaining vascular integrity and barrier function, regulating gas exchange, and contributing to vascular tone. Using single-cell transcriptomics, research has shown that there are multiple, unique EC subpopulations with different phenotypes. In response to injury or certain stressors such as hypoxia, there can be a dysregulated response with aberrant endothelial injury repair involving other pulmonary vascular cells and even immune cells. This aberrant signaling cascade is potentially a primary driver of pulmonary arterial remodeling in PAH. Recent studies have examined the role of EC clonal expansion, immune dysregulation, and genetic mutations in the pathogenesis of PAH. This review summarizes the existing literature on EC subpopulations and the intricate mechanisms through which ECs develop aberrant physiologic phenotypes and contribute to PAH. Our goal is to provide a framework for understanding the unique pulmonary EC biology and pathophysiology that is involved in the development of PAH.

The endothelium is a monolayer of flatten cells named endothelial cells that form the inner layer of the heart, blood, and lymphatic vessels. Its function is not just as a barrier, but it is a regulator of vascular permeability and tone, hemostasis, inflammation, and angiogenesis. This review is about the general aspects of vascular endothelium and endothelial dysfunction that leads to increased vascular risk. Activation and dysfunction are discussed, considering the endothelial activation as a self-limiting process, necessary to promote inflammation and hemostasis. Endothelial dysfunction is a pathological process in which the endothelium loses its ability for self-regulation and acquires a prothrombotic and proinflammation phenotype. Endothelial dysfunction is the initial step for atherosclerosis and increased cardiovascular risk, so the main biomarkers of endothelial dysfunction are reviewed. As basic knowledge about endothelium increases, preventive or therapeutic measures can be designed as treatment or prevention the risk of its dysfunction. [ABSTRACT FROM AUTHOR]

Patients with chronic kidney disease (CKD) are at increased risk of cardiovascular disease. This increased risk cannot be fully explained by traditional risk factors such as hypertension. Endothelial dysfunction and arterial stiffness have been suggested as factors that explain some of the increased risk and are independently associated with important cardiovascular outcomes in patients with CKD. Studies in other disease populations have shown the positive effects of exercise on vascular dysfunction. The aim of this review was to determine whether exercise training interventions improve measures of vascular function and morphology in patients across the spectrum of CKD and which exercise training interventions are most efficacious. A systematic search of Medline, Embase, and the Cochrane Central Register identified 25 randomized controlled trials. Only randomized control trials using an exercise intervention with a nonexercising control group and at least one measure of vascular function or morphology were included. Participants were patients with nondialysis CKD or transplant patients or those requiring dialysis therapy. A systematic review was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A meta-analysis was completed for pulse wave velocity, augmentation index, and measures of endothelium-dependent vasodilation. Data from 25 studies with 872 participants showed that exercise training reduced pulse wave velocity and augmentation index but had no effect on endothelium-dependent vasodilation. Subgroup analyses suggested that exercise interventions of at least moderate intensity were more likely to be effective. Limitations included the absence of observational studies or other interventions aimed at increasing habitual physical activity. Further studies are warranted to investigate which are the most effective exercise interventions. [ABSTRACT FROM AUTHOR]

Although robustly expressed in the disease-free (DF) breast stroma, CD36 is consistently absent from the stroma surrounding invasive breast cancers (IBCs). In this study, we primarily observed CD36 expression in adipocytes and intralobular capillaries within the DF breast. Larger vessels concentrated in interlobular regions lacked CD36 and were instead marked by the expression of CD31. When evaluated in perilesional capillaries surrounding ductal carcinoma in situ, a nonobligate IBC precursor, CD36 loss was more commonly observed in lesions associated with subsequent IBC. Peroxisome proliferator-activated receptor γ (PPARγ) governs the expression of CD36 and genes involved in differentiation, metabolism, angiogenesis, and inflammation. Coincident with CD36 loss, we observed a dramatic suppression of PPARγ and its target genes in capillary endothelial cells (ECs) and pericytes, which typically surround and support the stability of the capillary endothelium. Factors present in conditioned media from malignant cells repressed PPARγ and its target genes not only in cultured ECs and pericytes but also in adipocytes, which require PPARγ for proper differentiation. In addition, we identified a role for PPARγ in opposing the transition of pericytes toward a tumor-supportive myofibroblast phenotype. In mouse xenograft models, early intervention with rosiglitazone, a PPARγ agonist, demonstrated significant antitumor effects; however, following the development of a palpable tumor, the antitumor effects of rosiglitazone were negated by the repression of PPARγ in the mouse stroma. In summary, PPARγ activity in healthy tissues places several stromal cell types in an antitumorigenic state, directly inhibiting EC proliferation, maintaining adipocyte differentiation, and suppressing the transition of pericytes into tumor-supportive myofibroblasts. [ABSTRACT FROM AUTHOR]

Glomerular endothelial cell (GEnC) fenestrations are a critical component of the glomerular filtration barrier. Their unique non-diaphragmed structure is key to their function in glomerular hydraulic permeability, and their aberration in disease can contribute to loss of glomerular filtration function. This review provides a comprehensive update of current understanding of the regulation and biogenesis of fenestrae. We consider diseases in which GEnC fenestration loss is recognized or may play a role and discuss methods with potential to facilitate the study of these critical structures. Literature is drawn from GEnCs as well as other fenestrated cell types such as liver sinusoidal endothelial cells that most closely parallel GEnCs. [ABSTRACT FROM AUTHOR]

The endothelial glycocalyx is a dynamic signaling surface layer that is involved in the maintenance of cellular homeostasis. The glycocalyx has a very diverse composition, with glycoproteins, proteoglycans, and glycosaminoglycans interacting with each other to form a mesh-like structure. Due to its highly interactive nature, little is known about the relative contribution of each glycocalyx constituent to its overall function. Investigating the individual roles of the glycocalyx components to cellular functions and system physiology is challenging, as the genetic manipulation of animals that target specific glycocalyx components may result in the development of a modified glycocalyx. Thus, it is crucial that genetically modified animal models for glycocalyx components are characterized and validated before the development of mechanistic studies. Among the glycocalyx components, glypican 1, which acts through eNOS-dependent mechanisms, has recently emerged as a player in cardiovascular diseases. Whether glypican 1 regulates eNOS in physiological conditions is unclear. Herein, we assessed how the deletion of glypican 1 affects the development of the pulmonary endothelial glycocalyx and the impact on eNOS activity and endothelial function. Male and female 5–9-week-old wild-type and glypican 1 knockout mice were used. Transmission electron microscopy, immunofluorescence, and immunoblotting assessed the glycocalyx structure and composition. eNOS activation and content were assessed by immunoblotting; nitric oxide production was assessed by the Griess reaction. The pulmonary phenotype was evaluated by histological signs of lung injury, in vivo measurement of lung mechanics, and pulmonary ventilation. Glypican 1 knockout mice showed a modified glycocalyx with increased glycocalyx thickness and heparan sulfate content and decreased expression of syndecan 4. These alterations were associated with decreased phosphorylation of eNOS at S1177. The production of nitric oxides was not affected by the deletion of glypican 1, and the endothelial barrier was preserved in glypican 1 knockout mice. Pulmonary compliance was decreased, and pulmonary ventilation was unaltered in glypican 1 knockout mice. Collectively, these data indicate that the deletion of glypican 1 may result in the modification of the glycocalyx without affecting basal lung endothelial function, validating this mouse model as a tool for mechanistic studies that investigate the role of glypican 1 in lung endothelial function. [ABSTRACT FROM AUTHOR]

Venous hypoxia is considered as the major pathogenetic mechanism linking blood flow stagnancy with deep vein thrombosis (DVT). Our previous study showed that activating SIRT1 may attenuate inferior vena cava (IVC) stenosis-induced DVT in rats. This study was aimed to investigate the role of endothelial SIRT1 in DVT and hypoxia-induced endothelial dysfunction as well as the underlying mechanism. Protein profiling of IVCs and blood plasma of DVT rats induced by IVC stenosis was analysed by 4D Label free proteomics analysis. To verify the independent role of SIRT1 in DVT and oxygen–glucose deprivation (OGD)-induced endothelial dysfunction, SIRT1 specific activator SRT1720 and SIRT1 knockdown in both local IVCs and endothelial cells were employed. Moreover, the role of the NF-κB were investigated using NF-κB inhibitor caffeic acid phenethyl ester (CAPE). SRT1720 significantly inhibited thrombus burden, leukocytes infiltration, protein expressions of cell adhesion molecules and chemokines, as well as acetylation level of NF-κB/p65 in wild DVT rats, while these protective effects of SRT1720 were abolished in rats with SIRT1 knockdown in local IVCs. In vitro, SRT1720 protected endothelial cells against OGD-induced dysfunction characterized with enhanced adhesion of monocytes as well as the protein expressions of cell adhesion molecules and chemokines, whereas these protective effects of SRT1720 were vanished by SIRT1 stable knockdown. Furthermore, CAPE attenuated endothelial cell dysfunction and abolished these effects of SIRT1 knockdown. Collectively, these data suggested that endothelial SIRT1 plays an independent role in ameliorating hypoxia-induced endothelial dysfunction and thrombotic inflammation in DVT, and this effect is mediated by NF-κB deacetylation. [ABSTRACT FROM AUTHOR]

PIEZO1 is a mechanosensitive cation channel implicated in shear stress-mediated endothelial-dependent vasorelaxation. Since altered shear stress patterns induce a pro-inflammatory endothelial environment, we analyzed transcriptional profiles of human endothelial cells to determine the effect of altered shear stress patterns and subsequent prooxidant and inflammatory conditions on PIEZO1 and mechanosensitive-related genes (MRG). In silico analyses were validated in vitro by assessing PIEZO1 transcript levels in both the umbilical artery (HUAEC) and vein (HUVEC) endothelium. Transcriptional profiling showed that PIEZO1 and some MRG associated with the inflammatory response were upregulated in response to high (15 dyn/cm2) and extremely high shear stress (30 dyn/cm2) in HUVEC. Changes in PIEZO1 and inflammatory MRG were paralleled by p65 but not KLF or YAP1 transcription factors. Similarly, PIEZO1 transcript levels were upregulated by TNF-alpha (TNF-α) in diverse endothelial cell types, and pre-treatment with agents that prevent p65 translocation to the nucleus abolished PIEZO1 induction. ChIP-seq analysis revealed that p65 bonded to the PIEZO1 promoter region, an effect increased by the stimulation with TNF-α. Altogether this data showed that NF-kappa B activation via p65 signaling regulates PIEZO1 expression, providing a new molecular link for prooxidant and inflammatory responses and mechanosensitive pathways in the endothelium. [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension (PAH) is a rare cardiovascular disorder leading to pulmonary hypertension and, often fatal, right heart failure. Sex differences in PAH are evident, which primarily presents with a female predominance and increased male severity. Disturbed signalling of the transforming growth factor-β (TGFβ) family and gene mutations in the bone morphogenetic protein receptor 2 (BMPR2) are risk factors for PAH development, but how sex-specific cues affect the TGFβ family signalling in PAH remains poorly understood. In this review, we aim to explore the sex bias in PAH by examining sex differences in the TGFβ signalling family through mechanistical and translational evidence. Sex hormones including oestrogens, progestogens, and androgens, can determine the expression of receptors (including BMPR2), ligands, and soluble antagonists within the TGFβ family in a tissue-specific manner. Furthermore, sex-related genetic processes, i.e. Y-chromosome expression and X-chromosome inactivation, can influence the TGFβ signalling family at multiple levels. Given the clinical and mechanistical similarities, we expect that the conclusions arising from this review may apply also to hereditary haemorrhagic telangiectasia (HHT), a rare vascular disorder affecting the TGFβ signalling family pathway. In summary, we anticipate that investigating the TGFβ signalling family in a sex-specific manner will contribute to further understand the underlying processes leading to PAH and likely HHT. [ABSTRACT FROM AUTHOR]

Brewster, L. Madden, Anthony R. Bain, Vinicius P. Garcia, Noah M. DeSouza, Michael M. Tymko, Jared J. Greiner, and Philip N. Ainslie. Global REACH 2018: high altitude-related circulating extracellular microvesicles promote a proinflammatory endothelial phenotype in vitro. High Alt Med Biol. 24:223–229, 2023. Introduction: Ascent to high altitude (HA) can induce vascular dysfunction by promoting a proinflammatory endothelial phenotype. Circulating microvesicles (MVs) can mediate the vascular endothelium and inflammation. It is unclear whether HA-related MVs are associated with endothelial inflammation. Objectives: We tested the hypothesis that MVs derived from ascent to HA induce a proinflammatory endothelial phenotype. Methods: Ten healthy adults (8 M/2 F; age: 28 ± 2 years) residing at sea level (SL) were studied before and 4–6 days after rapid ascent to HA (4,300 m). MVs were isolated and enumerated from plasma by centrifugation and flow cytometry. Human umbilical vein endothelial cells were treated with MVs collected from each subject at SL (MV-SL) and at HA (MV-HA). Results: Circulating MV number significantly increased at HA (26,637 ± 3,315 vs. 19,388 ± 1,699). Although intracellular expression of total nuclear factor kappa beta (NF-κB; 83.4 ± 6.7 arbitrary units [AU] vs. 90.2 ± 6.9 AU) was not affected, MV-HA resulted in ∼55% higher (p < 0.05) active NF-κB (129.6 ± 19.8 AU vs. 90.7 ± 10.5 AU) expression compared with MV-SL. In addition, MV-HA induced higher interleukin (IL)-6 (63.9 ± 3.9 pg/ml vs. 53.3 ± 3.6 pg/ml) and IL-8 (140.2 ± 3.6 pg/ml vs. 120.7 ± 3.8 pg/ml) release compared with MV-SL, which was blunted with NF-κB blockade. Conclusions: Circulating extracellular MVs increase at HA and induce endothelial inflammation, potentially contributing to altitude-related vascular dysfunction. [ABSTRACT FROM AUTHOR]

Proper wound closure requires the functional coordination of endothelial cells (ECs) and keratinocytes. In the late stages of wound healing, keratinocytes become activated and ECs promote the maturation of nascent blood vessels. In diabetes mellitus, decreased keratinocyte activation and impaired angiogenic action of ECs delay wound healing. Porcine urinary bladder matrix (UBM) improves the rate of wound healing, but the effect of exposure to UBM under diabetic conditions remains unclear. We hypothesized that keratinocytes and ECs isolated from both diabetic and non‐diabetic donors would exhibit a similar transcriptome representative of the later stages of wound healing following incubation with UBM. Human keratinocytes and dermal ECs isolated from non‐diabetic and diabetic donors were incubated with and without UBM particulate. RNA‐Seq analysis was performed to identify changes in the transcriptome of these cells associated with exposure to UBM. While diabetic and non‐diabetic cells exhibited different transcriptomes, these differences were minimized following incubation with UBM. ECs exposed to UBM exhibited changes in the expression of transcripts suggesting an increase in the endothelial–mesenchymal transition (EndoMT) associated with vessel maturation. Keratinocytes incubated with UBM demonstrated an increase in markers of activation. Comparison of the whole transcriptomes with public datasets suggested increased EndoMT and keratinocyte activation following UBM exposure. Both cell types exhibited loss of pro‐inflammatory cytokines and adhesion molecules. These data suggest that application of UBM may accelerate healing by promoting a transition to the later stages of wound healing. This healing phenotype is achieved in cells isolated from both diabetic and non‐diabetic donors. [ABSTRACT FROM AUTHOR]