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Using the combined application of L-arginine with HMG-Co-A reductase inhibitors simvastatin, atorvastatin, rosuvastatin and nanoparticulated rosuvastatin on the background of modeling of sepsis-induced disease through the introduction of strain 603 Staphylococcus aureus shows endotelio- and cardioprotective effects, manifesting itself in preventing the proliferation of endothelial dysfunction coefficient (CED), adrenoreactivity, maintenance of myocardial reserve and the normalization of biochemical markers values (Total NO, eNOS expression, C-reactive protein, IL-6, TNF). In this case, the combined therapy was so effective that the values obtained thereunder did not differ from those obtained from control animals.

International audience; Reaching the corneal endothelium through the topical administration of therapeutic drugs remains a challenge in ophthalmology. Besides, endothelial cells are not able to regenerate, and diseases at this site can lead to corneal blindness. Targeting the corneal endothelium implies efficient penetration through the three corneal layers, which still remains difficult for small molecules. Carbon quantum dots (CQDs) have demonstrated great potential for ocular nanomedicine. This study focuses on the corneal penetration abilities of differently charged CQDs and their use as permeation enhancers for drugs. Excised whole bovine eyes were used as an ex vivo model to investigate corneal penetration of CQDs derived from glucosamine using β-alanine, ethylenediamine, or spermidine as a passivation agent. It was found that negatively charged CQDs have limited corneal penetration ability, while positively charged CQDs derived from glucosamine hydrochloride and spermidine (CQD-S) penetrate the entire corneal epithelium all the way down to the endothelium. CQD-S were shown to enhance the penetration of FITC-dextran 150 kDa, suggesting that they could be used as efficient penetration enhancers for therapeutic delivery to the corneal endothelium.

The central nervous system is lined by meninges, classically known as dura, arachnoid, and pia mater. We show the existence of a fourth meningeal layer that compartmentalizes the subarachnoid space in the mouse and human brain, designated the subarachnoid lymphatic-like membrane (SLYM). SLYM is morpho- and immunophenotypically similar to the mesothelial membrane lining of peripheral organs and body cavities, and it encases blood vessels and harbors immune cells. Functionally, the close apposition of SLYM with the endothelial lining of the meningeal venous sinus permits direct exchange of small solutes between cerebrospinal fluid and venous blood, thus representing the mouse equivalent of the arachnoid granulations. The functional characterization of SLYM provides fundamental insights into brain immune barriers and fluid transport.

At the moment, many researchers are using in vitro techniques to investigate chemokine-driven leukocyte adhesion/recruitment, for example, by using a transwell or flow chamber system. Here we describe a more physiologically relevant, sophisticated, and highly flexible method to study leukocyte adhesion ex vivo in fresh murine carotid arteries under arterial flow conditions. This model mimics an in vivo situation and allows the combination of leukocytes and arteries isolated from different donors in one experiment, generating information on both vascular and leukocyte adhesive properties of both donors. This method provides a versatile, highly physiologically relevant model to investigate leukocyte adhesion.

Posterior reversible encephalopathy syndrome (PRES) is an acute neurological disorder characterized by variable symptoms and radiological images characteristic of vasogenic parietal-occipital edema. It is associated with clinical conditions such as high blood pressure, infection / sepsis, or cytotoxic / immunosuppressive drugs, among others. It is characterized pathophysiologically by endothelial damage with breakdown of blood-brain barrier (BBB), cerebral hypoperfusion, and vasogenic edema.The cases are presented on two critical COVID-19 patients who were admitted to pneumonia requiring mechanical ventilation and who, after removing sedation, developed acute and reversible neurological symptoms consisting of epilepsy and encephalopathy, associated with hyperintense subcortical lesions on brain magnetic resonance imaging (MRI) compatible with PRES.SARS-CoV-2 coronavirus would activate an inflammatory response that would damage brain endothelium. It could be triggered by cytokine release, as well as by direct viral injury, given that endothelium expresses ACE2 receptors. It could explain the possible association between PRES and COVID-19.

Lymphatic vessel contractions generate net antegrade pulsatile lymph flow. By contrast, impaired lymphatic vessels are often associated with lymphoedema and altered lymph flow. The effect of lymphoedema on the lymph flow field and endothelium is not completely known. Here, we characterized the lymphatic flow field of a platelet-specific receptor C-type lectin-like receptor 2 (CLEC2) deficient lymphoedema mouse model. In regions of lymphoedema, collecting vessels were significantly distended, vessel contractility was greatly diminished and pulsatile lymph flow was replaced by quasi-steady flow.

Rationale: Jacobsen syndrome is a rare chromosomal disorder caused by deletions in the long arm of human chromosome 11, resulting in multiple developmental defects including congenital heart defects. Combined studies in humans and genetically engineered mice implicate that loss of ETS1 (E26 transformation specific 1) is the cause of congenital heart defects in Jacobsen syndrome, but the underlying molecular and cellular mechanisms are unknown. Objective: To determine the role of ETS1 in heart development, specifically its roles in coronary endothelium and endocardium and the mechanisms by which loss of ETS1 causes coronary vascular defects and ventricular noncompaction. Methods and Results: ETS1 global and endothelial-specific knockout mice were used. Phenotypic assessments, RNA sequencing, and chromatin immunoprecipitation analysis were performed together with expression analysis, immunofluorescence and RNAscope in situ hybridization to uncover phenotypic and transcriptomic changes in response to loss of ETS1. Loss of ETS1 in endothelial cells causes ventricular noncompaction, reproducing the phenotype arising from global deletion of ETS1. Endothelial-specific deletion of ETS1 decreased the levels of Alk1 (activin receptor-like kinase 1), Cldn5 (claudin 5), Sox18 (SRY-box transcription factor 18), Robo4 (roundabout guidance receptor 4), Esm1 (endothelial cell specific molecule 1) and Kdr (kinase insert domain receptor), 6 important angiogenesis-relevant genes in endothelial cells, causing a coronary vasculature developmental defect in association with decreased compact zone cardiomyocyte proliferation. Downregulation of ALK1 expression in endocardium due to the loss of ETS1, along with the upregulation of TGF (transforming growth factor)-β1 and TGF-β3, occurred with increased TGFBR2/TGFBR1/SMAD2 signaling and increased extracellular matrix expression in the trabecular layer, in association with increased trabecular cardiomyocyte proliferation. Conclusions: These results demonstrate the importance of endothelial and endocardial ETS1 in cardiac development. Delineation of the gene regulatory network involving ETS1 in heart development will enhance our understanding of the molecular mechanisms underlying ventricular and coronary vascular developmental defects and will lead to improved approaches for the treatment of patients with congenital heart disease.

Women with a history of gestational diabetes mellitus (GDM) are twice as likely to develop cardiovascular disease (CVD) and ∼7 times as likely to develop type 2 diabetes as their age-matched counterparts. However, the mechanism(s) mediating these associations remain unclear. We hypothesized that endothelium- and (nitric oxide) NO-dependent dilation would be attenuated through oxidant stress mechanisms in the microvasculature of women with a history of GDM compared with control women with a history of uncomplicated pregnancy (HC). Ten HC (35 ± 4 yr) and 10 GDM (34 ± 4 yr) underwent a standard local heating protocol (42°C; 0.1°C·s

Alteration in endothelial function during sepsis is thought to play a key role in the progression of organ failure. We herein compared plasma concentrations of endothelial activation biomarkers vascular endothelial growth factor (VEGF), hyaluronan (HA), plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF), as well as inflammatory mediator concentrations (IL-6, IL-8, IL-10, C-reactive protein and monocyte chemoattractant protein-1) in dogs with sepsis to healthy dogs. This study was a multicenter observational clinical trial conducted at two university teaching hospitals from February 2016 until July 2017. Thestudy included 18 client-owned dogs hospitalized with sepsis and at least one distant organ dysfunction, as well as 20 healthy dogs.Plasma biomarker concentrations were measured using ELISA. Severity of illness in dogs with sepsis was calculated using the 5-variable acute physiologic and laboratory evaluation (APPLEFAST) score. Biomarker concentrations were compared between septic and healthy dogs using linear models. Septic peritonitis was the most frequent source of sepsis (11/18; 61%), followed by pneumonia (4/18; 22%). Ten dogs (56%) had only 1 organ dysfunction, whereas 3 dogs (17%) had 2, 3 (17%) had 3, 1 (6%) had 4 and 1 (6%) had 5 organ dysfunctions. The median APPLEFAST score in the septic dogs was 28.5 (Q1-Q3, 24-31). Mean plasma concentrations of all endothelial and inflammatory biomarkers, except vWF, were higher in the sepsis cohort than in controls. The mean endothelial biomarker concentrations in the septic cohort ranged from ~2.7-fold higher for HA (difference in means; 118.2 ng/mL, (95% credible limit; 44.5 – 221.7) to ~150-fold for VEGF (difference in means; 76.6 pg/mL, 95% credible limit; 33.0-143.4), compared to the healthy cohort. Fifteen dogs with sepsis (83%) died; 7 (46%) were euthanized and 8 (53%) died during hospitalization. Dogs with naturally occurring sepsis and organ dysfunction had higher mean concentrations of biomarkers of endothelial activation and inflammation compared to healthy dogs, broadening our understanding of the pathophysiology of sepsis secondary to endothelialdysfunction.

Studies on inflammatory markers, endothelial activation, and bleeding during extracorporeal membrane oxygenation (ECMO) are lacking. Blood samples were prospectively collected after ECMO initiation from 150 adult patients who underwent ECMO for respiratory failure between 2018 and 2021. After excluding patients who died early (within 48 h), 132 patients were finally included. Their tumor necrosis factor-alpha (TNF-α), tissue factor (TF), soluble thrombomodulin (sTM), and E-selectin levels were measured. A Cox proportional hazards regression model was used to estimate the hazard ratio for hemorrhagic complications during ECMO. The 132 patients were divided into hemorrhagic (n = 23, H group) and non-complication (n = 109, N group) groups. The sequential organ failure assessment score, hemoglobin level, and ECMO type were included as covariates in all Cox models to exclude the effects of clinical factors. After adjusting for these factors, initial TNF-α, TF, sTM, E-selectin, and activated protein C levels were significantly associated with hemorrhagic complications (all p < 0.001). TNF-α, TF, and E-selectin better predicted hemorrhagic complications than the model that included only the aforementioned clinical factors (clinical factors only (area under the curve [AUC]: 0.804), reference; TNF-α (AUC: 0.914); TF (AUC: 0.915); E-selectin (AUC: 0.869)). Conclusions: TNF-α levels were significantly predictive of hemorrhagic complications during ECMO.

Purpose of review: The current review aims to present the latest scientific updates on the role of Sortilin in the pathophysiology of hypertension. Recent findings: The main focus of this systematic overview is on the functional contribution of Sortilin to the pathogenesis of hypertension. Sortilin is a glycoprotein mostly known for its actions as a trafficking molecule directing proteins to specific secretory or endocytic compartments of the cell. Emerging evidence indicates that Sortilin is associated with pathological conditions, including inflammation, arteriosclerosis, dyslipidemia, insulin resistance, and vascular calcification. Most recently, Sortilin has been shown to finely control endothelial function and to drive hypertension by modulating sphingolipid/ceramide homeostasis and by triggering oxidative stress. Summary: The latest findings linking Sortilin and hypertension that are herein discussed can inspire novel areas of research which could eventually lead to the discovery of new therapeutic strategies in cardiovascular medicine.

Purpose of reviewAging is an important risk factor for cardiovascular disease and is associated with increased vessel wall stiffness. Pathophysiological stiffening, notably in arteries, disturbs the integrity of the vascular endothelium and promotes permeability and transmigration of immune cells, thereby driving the development of atherosclerosis and related vascular diseases. Effective therapeutic strategies for arterial stiffening are still lacking.Recent findingsHere, we overview the literature on age-related arterial stiffening, from patient-derived data to preclinical in-vivo and in-vitro findings. First, we overview the common techniques that are used to measure stiffness and discuss the observed stiffness values in atherosclerosis and aging. Next, the endothelial response to stiffening and possibilities to attenuate this response are discussed.SummaryFuture research that will define the endothelial contribution to stiffness-related cardiovascular disease may provide new targets for intervention to restore endothelial function in atherosclerosis and complement the use of currently applied lipid-lowering, antihypertensive, and anti-inflammatory drugs.

Endothelial dysfunction contributes to cardiometabolic disorders, including hypertension, obesity, and type 2 diabetes. Esaxerenone is a selective, nonsteroidal, high-affinity mineralocorticoid receptor blocker recently approved in Japan for the treatment of hypertension. Although imbalanced signaling between vasorelaxant and vasocontractile factors induced by endothelial stimulation is often observed in type 2 diabetic vessels, the effects of esaxerenone on endothelium-dependent responses in type 2 diabetes remain unclear. The aim of this study was to investigate the effect of esaxerenone on endothelium-dependent responses in superior mesenteric arteries isolated from type 2 diabetic Goto-Kakizaki (GK) rats. It was found that esaxerenone (3 mg/kg/d for 4 weeks, per os (p.o.)) partially ameliorated acetylcholine (ACh)-induced endothelium-derived hyperpolarizing factor (EDHF)-type relaxation and NS309, a potent activator of small- and intermediate-conductance Ca

Microvascular disease plays a critical role in systemic end-organ dysfunction, and treatment of microvascular pathologies may greatly reduce cardiovascular morbidity and mortality. The Call for Papers collection: New Developments in Translational Microcirculatory Research highlights key advances in our understanding of the role of microvessels in the development of chronic diseases as well as therapeutic strategies to enhance microvascular function. This Mini Review provides a concise summary of these advances and draws from other relevant research to provide the most up-to-date information on the influence of cutaneous, cerebrovascular, coronary, and peripheral microcirculation on the pathophysiology of obesity, hypertension, cardiovascular aging, peripheral artery disease, and cognitive impairment. In addition to these disease- and location-dependent research articles, this Call for Papers includes state-of-the-art reviews on coronary endothelial function and assessment of microvascular health in different organ systems, with an additional focus on establishing rigor and new advances in clinical trial design. These articles, combined with original research evaluating cellular, exosomal, pharmaceutical, exercise, heat, and dietary interventional therapies, establish the groundwork for translating microcirculatory research from bench to bedside. Although numerous studies in this collection are focused on human microcirculation, most used robust preclinical models to probe mechanisms of pathophysiology and interventional benefits. Future work focused on translating these findings to humans are necessary for finding clinical strategies to prevent and treat microvascular dysfunction.

The present study provides the ultrastructure of the pneumocytes types II and I in piglets living in the trash environment. Samples of the lungs of twelve piglets two months old were used. By light microscopy, the pneumocytes type I were squamous and somewhat flattened cells, while pneumocytes type II was cuboidal to spherical projected within the alveolar lumen and characterized by a spherical nucleus and foamy cytoplasm, it contained vacuolated bodies that were polygonal with variable size manly at the poles of the cell. The electron microscopy investigation showed blood air barrier between the endothelial lining of pneumocytes type I and therefore the endothelial lining of blood capillary and their nucleus were irregular in shape varied from nearly irregular triangular to polygon rough endoplasmic reticulum represented at their cytoplasm. The pneumocytes type II were frequently covered by pneumocytes type I extensions and united to them by a tight junction. It had been characterized by a high number of mitochondria within the cytoplasm and vacuolated bodies encircled the nucleus and at the two extremities of the cell. The lamellar vacuolated bodies were connected to the endoplasmic reticulum membranes and therefore the intravascular macrophages were attached to the endothelial cells within the pulmonary capillaries until two months old piglets. The occurrence of the intravascular macrophages could be attributed to the higher resistance to the respiratory diseases of the piglets.

Evaluating the vascular function in HIV-infected compared with HIV uninfected with assessment of body composition, inflammation, and gut integrity markers.A noninvasive test that measures the endothelial function.We included participants at least 18 years old, with peripheral arterial tonometry testing (EndoPAT2000) between 2014 and 2022. Persons with HIV (PWH) had documented infection, a stable ART regimen, and a viral load less than 400 copies/ml. We measured the vessel's function with the reactive hyperemia index (RHI) (normal1.67) and Augmentation Index. Lower Augmentation Index reflect better arterial elasticity. We assessed markers of systemic inflammation, immune activation, and gut integrity. We used linear mixed models to estimate endothelial dysfunction with a significant P value less than 0.05.Overall, 511 participants (296 HIV-infected; 215 HIV-uninfected controls) were included. Estimated RHI among PWH was 13% lower (P = 0.01) compared with persons without HIV. In nonwhite race, the estimated RHI was 9% lower (P = 0.001) than white race. For every 1% increase in BMI, we would expect RHI to increase 0.17% (P = 0.01). At the time of EndoPAT, the estimated RHI was 8% lower (P = 0.04) among protease inhibitor users compared with PWH who were not taking protease inhibitors. The estimated odds of abnormal RHI ≤1.67) is 1.56 times greater [95% confidence interval (CI) 1.05-2.31] in nonwhite race compared with white race, independent of HIV status [OR = 1.4 (95% CI 0.94-2.13)]. There was not enough evidence to suggest that inflammation, gut, or monocyte markers, current or nadir CD4+ cell count, or duration of HIV were associated with endothelial dysfunction.HIV, nonwhite race, and protease inhibitor use are independently associated with endothelial dysfunction.

Background: Autosomal dominant polycystic kidney disease is the most frequent hereditary kidney disease and is generally due to mutations in PKD1 and PKD2 , encoding polycystins 1 and 2. In autosomal dominant polycystic kidney disease, hypertension and cardiovascular disorders are highly prevalent, but their mechanisms are partially understood. Methods: Since endothelial cells express the polycystin complex, where it plays a central role in the mechanotransduction of blood flow, we generated a murine model with inducible deletion of Pkd1 in endothelial cells ( Cdh5-Cre ERT2 ; Pkd1 fl/fl ) to specifically determine the role of endothelial polycystin-1 in autosomal dominant polycystic kidney disease. Results: Endothelial deletion of Pkd1 induced endothelial dysfunction, as demonstrated by impaired flow-mediated dilatation of resistance arteries and impaired relaxation to acetylcholine, increased blood pressure and prevented the normal development of arteriovenous fistula. In experimental chronic kidney disease induced by subtotal nephrectomy, endothelial deletion of Pkd1 further aggravated endothelial dysfunction, vascular remodeling, and heart hypertrophy. Conclusions: Altogether, this study provides the first in vivo demonstration that specific deletion of Pkd1 in endothelial cells promotes endothelial dysfunction and hypertension, impairs arteriovenous fistula development, and potentiates the cardiovascular alterations associated with chronic kidney disease.

Tumor metastasis via the circulation requires crossing the vascular barrier twice: first, during intravasation when tumor cells disseminate from the primary site through proximal vasculature, and second, during extravasation, when tumor cells exit the circulation to form distant metastatic seeds. During these key metastatic events, chemomechanical signaling between tumor cells and endothelial cells elicits reciprocal changes in cell morphology and behavior that are necessary to breach the vessel wall. Existing experimental systems have provided a limited understanding of the diverse mechanisms underlying tumor-endothelial interactions during intravasation and extravasation. Recent advances in microphysiological systems have revolutionized the ability to generate miniaturized human tissues with tailored three-dimensional architectures, physiological cell interfaces, and precise chemical and physical microenvironments. By doing so, microphysiological systems enable experimental access to complex morphogenic processes associated with human tumor progression with unprecedented resolution and biological control. Here, we discuss recent examples in which microphysiological systems have been leveraged to reveal new mechanistic insight into cellular and molecular control systems operating at the tumor-endothelial interface during intravasation and extravasation.

The endothelium is a critical mediator of homeostasis on blood-contacting surfaces in the body, serving as a selective barrier to regulate processes such as clotting, immune cell adhesion, and cellular response to fluid shear stress. Implantable cardiovascular devices including stents, vascular grafts, heart valves, and left ventricular assist devices are in direct contact with circulating blood and carry a high risk for platelet activation and thrombosis without a stable endothelial cell (EC) monolayer. Development of a healthy endothelium on the blood-contacting surface of these devices would help ameliorate risks associated with thrombus formation and eliminate the need for long-term anti-platelet or anti-coagulation therapy. Although ECs have been seeded onto or recruited to these blood-contacting surfaces, most ECs are lost upon exposure to shear stress due to circulating blood. Many investigators have attempted to generate a stable EC monolayer by improving EC adhesion using surface modifications, material coatings, nanofiber topology, and modifications to the cells. Despite some success with enhanced EC retention in vitro and in animal models, no studies to date have proven efficacious for routinely creating a stable endothelium in the clinical setting. This review summarizes past and present techniques directed at improving the adhesion of ECs to blood-contacting devices.

Bleeding disorders are a major group of hematological disorders, which are highly prevalent in the world. Excessive bleeding can result in serious consequences including hypoperfusion and cardiac arrest. The body has its selfmechanism to control excessive bleeding which is termed hemostasis. Hemostasis is achieved in two major steps, the formation of the primary and secondary hemostatic plugs. Endothelium, platelets, and coagulation factors are three components involved in hemostasis. Endothelium and platelets have a major role in forming the primary hemostatic plug. Consequently, the first step in investigating a bleeding disorder is platelet count. Despite normal platelet count, abnormality in the primary hemostatic plug may arise due to functional defects of the platelets including adhesion, activation, and aggregation. Von Willebrand disease (VWD) is an endothelial defect and the most prevalent inherited defect in coagulation. Abnormalities in the secondary hemostatic plug are largely due to coagulation factor deficiencies, and, to a lesser extent, the presence of inhibitors. Techniques involving viscoelastics have been aiding in rapid diagnosis and are useful in point-of-care testing. This article discusses the investigation of bleeding disorders from the perspective of the endothelium, platelet, and coagulation factor physiology. These three components should be properly investigated to achieve the definitive diagnosis of bleeding disorders.

Discovering new nephroprotectants may provide therapeutic strategies in AKI. This study provides the first evidence that KLF11, a member of the Krüppel-like factor (KLF) family of proteins, protects against AKI. In the absence of KLF11, exaggerated induction of endothelin-1 and IL-6 occurs after ischemic renal injury and may contribute to worse AKI.

Chronic kidney disease (CKD) affects 10% of the world’s population. Uremic toxins, such as indoxyl sulfate (IS), p-Cresylsulfate (PCS) and indole acetic acid (IAA), are not sufficiently removed by conventional hemodialysis (HD) and have been associated with inflammation, poor quality of life, bone mineral disease (BMD) and endothelial injury. Online hemodiafiltration (OL-HDF) may promote greater clearance of uremic toxins than HD. However, there are few studies evaluating the effect of OL-HDF on serum levels of IS, PCS, IAA, and biomarkers associated with inflammatory, endothelial, and bone and mineral disorder in the elderly population. We evaluated the effect of 6 months of OL-HDF on the serum concentration of uremic toxins, biomarkers of inflammation, endothelial and bone mineral disorder in older patients on OL-HDF. IS, PCS, and IAA were measured by high-performance liquid chromatography. We included 31 patients (77.4 ± 7.1 years, 64.5% male, 35.5% diabetic, on maintenance dialysis for 45 ± 20 days). From baseline to 6 months there was a decrease in serum concentration of IS but not PCS and IAA. We found no change in serum concentration of inflammatory, endothelial, or mineral and bone biomarkers. In summary, OL-HDF was capable to reduce IS in older patients. Whether this reduction may have an impact on clinical outcomes deserves further evaluation.

COVID-19 is associated to an increased risk of thrombosis, as a result of a complex process that involves the activation of vascular and circulating cells, the release of soluble inflammatory and thrombotic mediators and blood clotting activation.This article reviews the pathophysiological role of platelets, neutrophils, and the endothelium, and of their interactions, in the thrombotic complications of COVID-19 patients, and the current and future therapeutic approaches targeting these cell types.Virus-induced platelet, neutrophil, and endothelial cell changes are crucial triggers of the thrombotic complications and of the adverse evolution of COVID-19. Both the direct interaction with the virus and the associated cytokine storm concur to trigger cell activation in a classical thromboinflammatory vicious circle. Although heparin has proven to be an effective prophylactic and therapeutic weapon for the prevention and treatment of COVID-19-associated thrombosis, it acts downstream of the cascade of events triggered by SARS-CoV-2. The identification of specific molecular targets interrupting the thromboinflammatory cascade upstream, and more specifically acting either on the interaction of SARS-CoV-2 with blood and vascular cells or on the specific signaling mechanisms associated with their COVID-19-associated activation, might theoretically offer greater protection with potentially lesser side effects.

Increased circulating levels of soluble interleukin (IL)-6 receptor α (sIL-6Rα) are commonly observed during inflammatory responses, allowing for IL-6 signaling in cells that express the ubiquitous receptor subunit gp130 but not IL-6Rα, such as endothelial cells. Activation of Toll-like receptor (TLR)-4 or the tumor necrosis factor (TNF) receptor leads to NF-κB-dependent increases in endothelial IL-6 expression. Thus, we hypothesize that danger signals may induce autocrine IL-6 signaling within the endothelium via sIL-6Rα-mediated trans-signaling. In support of this hypothesis, we recently demonstrated that conditional deletion in the endothelium of the IL-6 signaling inhibitor SOCS3 leads to rapid mortality in mice challenged with the TLR-4 agonist endotoxin through increases in vascular leakage, thrombosis, leukocyte adhesion, and a type I-like interferon response. Here, we sought to directly test a role for sIL-6Rα in LPS-treated human umbilical vein and dermal blood microvascular endothelial cells. We show that cotreatment with sIL-6Rα dramatically increases the loss of barrier function and the expression of COX2 and tissue factor mRNA levels induced by LPS. This cotreatment led to strong activation of STAT1 and STAT3 while not affecting LPS-induced activation of p38 and NF-κB signaling. Similar results were obtained when sIL-6Rα was added to a TNF challenge. JAK inhibition by pretreatment with ruxolitinib or by SOCS3 overexpression blunted LPS and sIL-6R synergistic effects, whereas SOCS3 knockdown further increased the response. Together, these findings demonstrate that IL-6 signaling downstream of NF-κB activation leads to a strong endothelial activation and may explain the acute endotheliopathy observed during critical illness.

To generate sufficient numbers of transplantable hematopoietic stem cells (HSCs) in vitro, a detailed understanding of how this process takes place in vivo is essential. The endothelial-to-hematopoietic transition (EHT), which culminates in the production of the first HSCs, is a highly complex process during which key regulators are switched on and off at precise moments, and that is embedded into a myriad of microenvironmental signals from surrounding cells and tissues. We have previously demonstrated an HSC-supportive function for GATA3 within the sympathetic nervous system and the sub-aortic mesenchyme, but show here that it also plays a cell-intrinsic role during the EHT. It is expressed in hemogenic endothelial cells and early HSC precursors, where its expression correlates with a more quiescent state. Importantly, endothelial-specific deletion of Gata3 shows that it is functionally required for these cells to mature into HSCs, placing GATA3 at the core of the EHT regulatory network.

Pulmonary arterial hypertension (PAH) is a deadly disease, characterized by increased vascular resistance, pulmonary arteriolar loss, and occlusive arterial remodeling, leading to eventual right heart failure. Evidence increasingly points to the pulmonary endothelium as a central actor in PAH. Endothelial cell apoptosis can result directly in distal lung arteriolar pruning and indirectly in the formation of complex and occlusive arterial lesions, reflecting an imbalance between endothelial injury and repair in the development and progression of PAH. Many of the mutations implicated in PAH are in genes, which are predominantly, or solely, expressed in endothelial cells, and the endothelium is a major target for therapeutic interventions to restore BMP signaling. We explore how arterial pruning can promote the emergence of occlusive arterial remodeling mediated by ongoing endothelial injury secondary to hemodynamic perturbation and pathological increases in luminal shear stress. The emerging role of endothelial cell senescence is discussed in the transition from reversible to irreversible arterial remodeling in advanced PAH, and we review the sometimes conflicting evidence that female sex hormones can both protect or promote vascular changes in disease. Finally, we explore the contribution of the endothelium to metabolic changes and the altered inflammatory and immune state in the PAH lung, focusing on the role of excessive TGFβ signaling. Given the complexity of the endothelial pathobiology of PAH, we anticipate that emerging technologies that allow the study of molecular events at a single cell level will provide answers to many of the questions raised in this review.

Diabetic vascular endothelial impairment is one of the main causes of death in patients with diabetes lacking adequately defined mechanisms or effective treatments. REGγ, the 11S proteasome activator known to promote the degradation of cellular proteins in a ubiquitin- and ATP-independent manner, emerges as a new regulator in the cardiovascular system. Here, we found that REGγ was upregulated in streptozocin (STZ)-induced diabetic mouse aortic endothelium in vivo and high glucose (HG)-treated vascular endothelial cells (ECs) in vitro. REGγ deficiency ameliorated endothelial impairment in STZ-induced diabetic mice by protecting against a decline in cellular glucose uptake and associated vascular ECs dysfunction by suppressing high mobility group AT-hook 2 (HMGA2) decay. Mechanically, REGγ interacted with and degraded the transcription factor HMGA2 directly, leading to decreased HMGA2 transcriptional activity, subsequently lowered expression of glucose transporter type 1 (GLUT1), and reduced cellular glucose uptake, vascular endothelial dysfunction, and impaired diabetic endothelium. Ablation of endogenous GLUT1 or HMGA2 or overexpressing exogenous HMGA2 in vascular ECs significantly blocked or reestablished the REGγ-dependent action on cellular glucose uptake and vascular endothelial functions of HG stimulation in vitro. Furthermore, exogenously introducing HMGA2 improved diabetic mice endothelial impairment features caused by REGγ in vivo, thereby substantiating a REGγ-HMGA2-GLUT1 pathway in diabetic endothelial impairment. Our findings indicate that modulating REGγ-proteasome activity may be a potential therapeutic approach for diabetic disorders with endothelial impairment.

Introduction To report a late onset, deep stromal and endothelial corneal scar in a keratoconus patient after corneal collagen cross-linking (CXL). Case description Observational case report. A 29-year-old man with bilateral keratoconus received an accelerated (A-CXL 10*9) epithelium-off CXL procedure in the left eye. 6-months postoperatively, a 2.2 × 1.2 mm inferocentral corneal scar was detected, which was located in the posterior stroma ranging from approximately 350 µm until the endothelium, therefore was situated below the demarcation line. A topical corticosteroid treatment did not influence the magnitude or configuration of the scar. Visual acuity was never affected, which includes the examination 12 months postoperatively. Conclusions We report a case of a late onset deep stromal and endothelial corneal scar 6 months after accelerated CXL as postoperative complication without affecting visual acuity.

Von Willebrand factor (VWF) is classically associated with primary hemostasis and platelet-rich arterial thromboses, but recently has also been implicated in fibrin clotting and venous thrombosis. Direct interaction between fibrin and VWF may mediate these processes, although prior reports are conflicting.We combined two complementary platforms to characterize VWF-fibrin(ogen) interactions and identify their potential physiologic significance.Engineered microvessels were lined with human endothelial cells, cultured under flow, and activated to release VWF and form transluminal VWF fibers. Fibrinogen, fibrin monomers, or polymerizing fibrin were then perfused, and interactions with VWF evaluated. Thrombin and fibrinogen were perfused into living versus paraformeldahyde-fixed microvessels and the pressure drop across microvessels monitored. Separately, protein binding to tethered VWF was assessed on a single-molecule level using total internal reflection fluorescence (TIRF) microscopy.Within microvessels, VWF fibers colocalized with polymerizing fibrin, but not fibrinogen. TIRF microscopy showed no colocalization between VWF and fibrinogen or fibrin monomers in a microfluidic flow chamber across a range of shear rates and protein concentrations. Thrombin-mediated fibrin polymerization within living microvessels triggered endothelial VWF release, increasing the rate and amount of microvessel obstruction compared to fixed vessels with an inert endothelium.We did not identify specific binding between fibrin(ogen) and VWF at a single-molecule level. Despite this, our results suggest that rapid release of endothelial VWF during clotting may provide a physical support for fibrin polymerization and accelerate thrombosis. This interaction may be of fundamental importance for the understanding and treatment of human thrombotic disease.

Introduction Although capsaicin has long been used as food additive and medication worldwide, its actions on gastrointestinal tract as its most delivery pathway have not been well addressed. Objectives In the present study, we aimed to study GI actions of capsaicin on mesenteric arterioles in normal and colitis mice and to elucidate the underlying mechanisms. Methods Vasorelaxation of human submucosal arterioles and the mesenteric arterioles from wide-type (WT) mice, TRPV1−/− and TRPV4−/− (KO) mice were measured. The expression and function of TRPV channels in endothelial cells were examined by q-PCR, immunostaining, Ca2+ imaging and membrane potential measurements. Results Capsaicin dose-dependently induced vasorelaxation of human submucosal arterioles and mouse mesenteric arterioles in vitro and in vivo through endothelium-dependent hyperpolarization (EDH), nitric oxide (NO), and prostacyclin (PGI2). Using TRPV1 and TRPV4 KO mice, we found that capsaicin-induced vasorelaxation was predominately through TRPV4/EDH, but marginally through TRPV1/NO/PGI2. Capsaicin induced hyperpolarization through activation of endothelial TRPV4 channels and intermediate-conductance of Ca2+-activated K+ channels to finally stimulate vasorelaxation. Importantly, capsaicin exerted anti-colitis action by rescuing the impaired ACh-induced vasorelaxation in WT colitis mice but not in TRPV4 KO colitis mice. Conclusions Capsaicin increases intestinal mucosal blood perfusion to potentially prevent/treat colitis through a novel TRPV4/EDH-dependent vasorelaxation of submucosal arterioles in health and colitis. This study further supports our previous notion that TRPV4/EDH in mesenteric circulation plays a critical role in the pathogenesis of colitis.

Objective Vascular findings in preeclampsia are usually attributed to increased vascular tone. Recently, however, important studies have improved the understanding of the main pathophysiological events in this condition, especially vascular brain remodeling, impaired autoregulation, and damage of the blood-brain barrier, which are well recognized features of cerebral overperfusion. Methods In this study, the association between choriocapillaris ischemia with ophthalmic artery blood flow parameters on orbital Doppler ultrasound is reported for the first time using multivariate logistic models. Multivariate logistic models with ophthalmic artery blood flow parameters, as well as major clinical and laboratory predictive variables were established for choriocapillaris ischemia and choriocapillaris ischemia with retinal detachment. Results In a series of 165 patients, 46 (28%) presented choriocapillaris ischemia; among them, 20 (12%) presented associated retinal detachment. The ophthalmic artery resistive index was the main predictor for choriocapillaris ischemia and choriocapillaris ischemia with retinal detachment in multivariate logistic models. Ophthalmic artery resistance lower than 0.56 was associated with a significantly high incidence of both outcomes. Conclusion This study supports that the branching pattern of choroidal arterioles and the lobular organization of choriocapillaris are the major morphological aspects underlying endothelial damage and lobular ischemia in the context of choroidal overperfusion. Overperfused lobules bordering areas of choriocapillaris ischemia produce a perfusion pressure gradient, with lobular reperfusion, leakage from reperfused choriocapillaris, and retinal detachment. Ophthalmic artery-resistive index lower than 0.56 is proposed as a major predictor of the overperfusion-related choriocapillaris ischemia and choriocapillaris ischemia with retinal detachment in preeclampsia. RESUMO Objetivo Os achados vasculares na pré-eclâmpsia são usualmente atribuídos ao aumento do tônus vascular. Recentemente, no entanto, importantes estudos têm melhorado a compreensão dos principais eventos fisiopatológicos nessa condição, especialmente o remodelamento vascular cerebral, a perda de autorregulação e a ruptura da barreira hematoencefálica, características bem reconhecidas de hiperperfusão cerebral. Métodos Neste estudo, a associação entre a isquemia da coriocapilar e parâmetros de fluxo sanguíneo da artéria oftálmica no Doppler orbitário é relatada pela primeira vez por meio de modelos logísticos multivariados. Modelos logísticos multivariados com parâmetros de fluxo sanguíneo de artéria oftálmica, assim como os principais preditores clínicos e laboratoriais, foram estabelecidos para isquemia da coriocapilar e coriocapilar associada a descolamento de retina. Resultados Em uma série de 165 pacientes, 46 (28%) apresentaram isquemia da coriocapilar; dentre eles, 20 (12%) apresentaram descolamento de retina associado. O índice de resistência da artéria oftálmica foi o principal preditor para isquemia da coriocapilar e isquemia da coriocapilar associada a descolamento de retina em modelos logísticos multivariados, e índice de resistência da artéria oftálmica menor que 0,56 foi associado a uma incidência significativamente elevada de ambos os desfechos. Conclusão Este estudo sustenta que o padrão de ramificação das arteríolas coroidianas e a organização lobular da coriocapilar são os principais aspectos morfológicos subjacentes ao dano endotelial e à isquemia lobular no contexto do hiperfluxo coroidiano. O hiperfluxo de lóbulos adjacentes às áreas de isquemia da coriocapilar estabelece um gradiente de pressão de perfusão, o que produz reperfusão lobular, extravasamento a partir de coriocapilares reperfundidos e descolamento da retina. O índice de resistência da artéria oftálmica inferior a 0,56 é proposto como um importante preditor de isquemia da coriocapilar e isquemia da coriocapilar associada a descolamento de retina relacionados ao hiperfluxo na pré-eclâmpsia.

The objective of this study was to determine the endothelial cell density (ECD) and hexagonality of the cornea in the different regions of healthy swine corneal endothelium using specular microscopy. Twenty-four eyeballs from 12 male, 6-month-old Large White pigs (Sus scrofa domesticus) were studied. Contact specular microscopy was performed in the central, superior, inferior, lateral and medial regions. The corneal parameters analysed in this study were ECD and hexagonality. The ECD in the central region was 1865 cells/mm2; in the upper region, it was 1877 cells/mm2, in the lower region, it was 1854 cells/mm2, in the lateral region, it was 1847 cells/mm2, in the medial region, it was 1831 cells/mm2. Hexagonality in the central region, was 53%; in the upper region, it was 54%, in the lower region, it was 54%, in the lateral region, it was 54%, in the medial region, it was 54%. There was no significant difference regarding to the evaluated parameters in all corneal regions evaluated. No statistically significantly differences were observed in ECD and hexagonality between the left and the right eyes. This study demonstrates that ECD and hexagonality of the central cornea area represent the entire endothelial mosaic. Resumo O objetivo do presente estudo foi avaliar a densidade endotelial e a hexagonalidade das células endoteliais nas diferentes regiões da córnea de suínos utilizando a microscopia especular de contato. Foram estudados 24 bulbos oculares de 12 suínos (Sus scrofa domesticus), machos, com seis meses de idade e da raça Large White. A microscopia especular de contato foi realizada nas regiões central, superior, inferior, lateral e medial da córnea. A densidade endotelial média na região central foi de 1865 células/mm2, na região superior foi de 1877 células/mm2, na região inferior foi de 1854 células/mm2, na região lateral foi de 1847 células/mm2 e na região medial foi de 1831 células/mm2. Na região central, a hexagonalidade foi de 53%, na região superior foi de 54%, na região inferior foi de 54%, na região lateral foi de 54%, na região medial foi de 54%. Não foram observadas diferenças significativas na densidade celular e na hexagonalidade nas diferentes regiões da córnea analisadas. Este estudo demonstrou que a densidade endotelial e a hexagonalidade da área central da córnea representam todo o mosaico endotelial.

Background: Acute cardiovascular stress increases systemic wall shear stress (WSS)–a frictional force exerted by the flow of blood on vessel walls–which raises plasma nitrite concentration due to enhanced endothelial nitric oxide synthase (eNOS) activity. Upstream eNOS inhibition modulates distal perfusion, and autonomic stress increases both the consumption and vasodilatory effects of endogenous nitrite. Plasma nitrite maintains vascular homeostasis during exercise and disruption of nitrite bioavailability can lead to intermittent claudication. Hypothesis: During acute cardiovascular stress or strenuous exercise, we hypothesize enhanced production of nitric oxide (NO) by vascular endothelial cells raises nitrite concentrations in near-wall layers of flowing blood, resulting in cumulative NO concentrations in downstream arterioles sufficient for vasodilation. Confirmation and implications: Utilizing a multiscale model of nitrite transport in bifurcating arteries, we tested the hypothesis for femoral artery flow under resting and exercised states of cardiovascular stress. Results indicate intravascular transport of nitrite from upstream endothelium could result in vasodilator-active levels of nitrite in downstream resistance vessels. The hypothesis could be confirmed utilizing artery-on-a-chip technology to measure NO production rates directly and help validate numerical model predictions. Further characterization of this mechanism may improve our understanding of symptomatic peripheral artery occlusive disease and exercise physiology.

The cornea is a transparent and avascular tissue located in front of the eye. Its inner surface is lined by a monolayer of corneal endothelial cells (CECs), which maintain the cornea transparency. CECs remain arrested in a non-proliferative state and damage to these cells can compromise their function leading to corneal opacity. The primary culture of donor-derived CECs is a promising cell therapy. It confers the potential to treat multiple patients from a single donor, alleviating the global donor shortage. Nevertheless, this approach has limitations preventing its adoption, particularly culture protocols allow limited expansion of CECs and there is a lack of clear parameters to identify therapy-grade CECs. To address this limitation, a better understanding of the molecular changes arising from the primary culture of CECs is required. Using single-cell RNA sequencing on primary cultured CECs, we identify their variable transcriptomic fingerprint at the single cell level, provide a pseudo-temporal reconstruction of the changes arising from primary culture, and suggest markers to assess the quality of primary CEC cultures. This research depicts a deep transcriptomic understanding of the cellular heterogeneity arising from the primary expansion of CECs and sets the basis for further improvement of culture protocols and therapies.

PurposeThere is a pressing need to investigate the impact of type II diabetes mellitus on the posterior cornea in donor tissues given its increasing prevalence and potential impact on endothelial keratoplasty surgical outcomes.MethodsImmortalized human cultured corneal endothelial cells (CECs; HCEC-B4G12) were grown in hyperglycemic media for 2 weeks. Extracellular matrix (ECM) adhesive glycoprotein expression and advanced glycation end products (AGEs) in cultured cells and corneoscleral donor tissues, as well as the elastic modulus for the Descemet membrane (DMs) and CECs of diabetic and nondiabetic donor corneas, were measured.ResultsIn CEC cultures, increasing hyperglycemia resulted in increased transforming growth factor beta-induced (TGFBI) protein expression and colocalization with AGEs in the ECM. In donor corneas, the thicknesses of the DM and the interfacial matrix (IFM) between the DM and stroma both increased from 8.42 ± 1.35 µm and 0.504 ± 0.13 µm in normal corneas, respectively, to 11.13 ± 2.91 µm (DM) and 0.681 ± 0.24 µm (IFM) in non-advanced diabetes (P = 0.013 and P = 0.075, respectively) and 11.31 ± 1.76 µm (DM) and 0.744 ± 0.18 µm (IFM) in advanced diabetes (AD; P = 0.0002 and P = 0.003, respectively). Immunofluorescence in AD tissues versus controls showed increased AGEs (P < 0.001) and markedly increased labeling intensity for adhesive glycoproteins, including TGFBI, that colocalized with AGEs. The elastic modulus significantly increased between AD and control tissues for the DMs (P < 0.0001) and CECs (P < 0.0001).ConclusionsDiabetes and hyperglycemia alter human CEC ECM structure and composition, likely contributing to previously documented complications of endothelial keratoplasty using diabetic donor tissue, including tearing during graft preparation and reduced graft survival. AGE accumulation in the DM and IFM may be a useful biomarker for determining diabetic impact on posterior corneal tissue.

OBJECTIVEFailure to close the ductus arteriosus immediately post-birth, patent ductus arteriosus (PDA), accounts for up to 10% of all congenital heart defects. Despite significant advances in PDA management options, including pharmacological treatment targeting the prostaglandin pathway, a proportion of patients fail to respond and must undergo surgical intervention. Thus, further refinement of the cellular and molecular mechanisms that govern vascular remodeling of this vessel is required.APPROACH AND RESULTSAs anticipated, single-cell RNA sequencing on the ductus arteriosus in mouse embryos at E18.5, P0.5, and P5, revealed broad transcriptional alterations in the endothelial, smooth muscle, and fibroblast cell compartments. Making use of these data sets, vimentin emerged as an interesting candidate for further investigation. Subsequent studies demonstrated that, in fact, mice with genetic deletion of vimentin fail to complete vascular remodeling of the ductus arteriosus, as per presence of a functional lumen.CONCLUSIONSThrough single-cell RNA-sequencing and by tracking closure of the ductus arteriosus postnatally in mice, we uncovered the unexpected contribution of vimentin in driving complete closure of the ductus arteriosus potentially through regulation of the Notch signaling pathway.HIGHLIGHTSSingle-cell RNA-sequencing on the ductus arteriosus at E18.5, P0.5, and P5 reveals how the ductus arteriosus undergoes drastic transcriptional changes at the single-cell level.Endothelial cells increase levels of Vimentin, Notch1 and Jag1 transcripts soon after birth (P0.5), concurrent with ductus arteriosus closure.Loss of vimentin, the major intermediate filament protein of endothelial cells, prevents proper permanent closure of the ductus arteriosus.

Endothelial dysfunction is characterized by disturbances in nitric oxide (NO) bioavailability and increased circulating asymmetric dimethylarginine (ADMA) due to the enormous release of free radicals. Increased circulating ADMA may cause endothelial dysfunction and a variety of clinical disorders, such as liver and kidney disease. Young male Sprague-Dawley rats at postnatal day 17 ± 1 received continuous ADMA infusion via an intraperitoneal pump to induce endothelial dysfunction. Four groups of rats (n = 10 per group) were allocated: control, control and resveratrol, ADMA infusion, and ADMA infusion and resveratrol groups. Spatial memory, NLR family pyrin-domain-containing 3 (NLRP3) inflammasome, cytokine expression, tight junction proteins in the ileum and dorsal hippocampus, and microbiota composition were examined. We found cognitive deficits; increased NLRP3 inflammasome in the plasma, ileum, and dorsal hippocampus; decreased ileum and dorsal hippocampal cytokine activation and tight junction proteins; and microbiota composition alterations in the ADMA-infusion young male rats. Resveratrol had beneficial effects in this context. In conclusion, we observed NLRP3 inflammasome activation in peripheral and central dysbiosis in young male rats with increased circulating ADMA, and found that resveratrol had beneficial effects. Our work adds to the mounting evidence that inhibiting systemic inflammation is a promising therapeutic avenue for cognition impairment, probably via the gut-brain axis.

Uvod: Kronična HCV infekcija je povezana sa sistemskim upalnim odgovorom i metaboličkim komplikacijama koje mogu pogodovati razvoju ateroskleroze i većem riziku od cerebrovaskularne bolesti (CVB). Cilj: Cilj ove disertacije bio je procijeniti cerebrovaskularnu reaktivnosti (CVR) u bolesnika s kroničnim hepatitisom C te odrediti serumske razine E-selektina, VCAM-1, ICAM-1 i VEGF-A i njihovu korelaciju s CVR. Metode: Ispitanicima s kroničnim hepatitisom C, kao i zdravim kontrolama učinjena je procjena CVR sukladno etabliranom testu zadržavanja daha koji koristi transkranijski color Doppler (TCCD) za mjerenje brzina protoka krvi i indeksa zadržavanja daha (BHI). Uzet je uzorak krvi ispitanicima u hepatitis i kontrolnoj skupini radi analize biljega endotelne disfunkcije i njihove korelacije s CVR procijenjene kroz ultrazvučno određen BHI. Rezultati: Rezultati dobiveni tijekom zadržavanja daha su otkrili značajno niže vrijednosti prosječne vršne sistoličke brzine (AvPS start, P = 0.018), krajnje dijastoličke (AvED start, P = 0.031), srednje brzine na samom početku postupka zadržavanja daha (AvmeanV start, P = 0.02), kao i niže srednje vršne sistoličke brzine na kraju zadržavanja daha (AvPS max, P = 0.02) u hepatitis skupini. Vrijednosti CVR, procijenjene kroz BHI, su bile također značajno niže (P = 0.045) u HCV ispitanika. Dobivene su značajne razlike između testiranih skupina u svim endotelnim markerima izuzev E-selektina. Dok su ICAM-1 i sVCAM-1 bili značajno viši u hepatitis skupini, VEGF-A je bio značajno niži. Nije nađena značajna povezanost između BHI i bilo kojeg od biljega endotelne disfunkcije u kontrolnoj skupini, dok u skupini s hepatitisom dijagram rasapa ICAM-1 u odnosu na BHI sugerira moguću povezanost. Zaključak: Rezultati ove disertacije potvrđuju povezanost kroničnog hepatitisa C s promijenjenom CVR i povišenim biljezima endotelne aktivacije (ICAM-1, VCAM-1) kao mogućim indikatorima povećanog rizika od cerebrovaskularne bolesti u bolesnika s HCV-om., Background: Hepatitis C viral (HCV) infection is associated with systemic inflammation and metabolic complications that might predispose patients to atherosclerosis and increased risk of ischemic cerebrovascular disease. Aim: This doctoral thesis is aimed to assess cerebrovascular reactivity (CVR) in patients with chronic hepatitis C, and also to determine E-selectin, VCAM-1, ICAM-1 and VEGF-A serum levels in patients with chronic HCV infection in order to correlate them with CVR. Methods: Patients with chronic hepatitis C infection, as well as healthy blood donors in the control group, were assessed for CVR according to the well-established breath-holding test that uses the transcranial color Doppler (TCCD) for measurement of blood flow velocity and breath-holding index (BHI). A blood sample was taken from examinees in both groups to assess markers of endothelial dysfunction and to correlate them with CVR that was expressed as BHI determined by TCCD. Results: Results obtained during the breath-holding revealed significantly lower average peak systolic (AvPS start, P = 0.018), end-diastolic (AvED start, P = 0.031) and mean velocity values at the very beginning of the breath-holding procedure (AvmeanV start, P = 0.02), as well as a lower mean peak systolic velocity at the end of the breath-holding test (AvPS max, P = 0.02) in the hepatitis C group. CVR values, calculated as the BHI, were also significantly lower (P = 0.045) in the hepatitis C group. The obtained results revealed significant differences between the groups in all endothelial markers with the exception of E selectin. While the ICAM-1 and sVCAM-1 were significantly increased in the hepatitis group, VEGF-A was significantly decreased. No significant association between the BHI and any of the endothelial markers was found in the control group, while in the hepatitis group the scatter plot of ICAM-1 vs BHI suggested possible association. Conclusions: the results of this doctoral thesis confirm an association between a chronic HCV infection and altered CVR as well as higher levels of markers of endothelial activation (ICAM-1, VCAM-1) as possible indicators of an increased cerebrovascular disease risk.

Type 1 diabetes mellitus is related to the vascular oxidative and nitrosative stress, the trigger for atherosclerosis and cardiovascular complications. The effects of moderate swimming training associated with quercetin oral administration were evaluated in aorta of rats with experimentally induced type 1 diabetes mellitus (T1DM), by analysing the nitric oxide-endothelial dependent relaxation (NO-EDR). T1DM rats received daily quercetin 30 mg/kg and followed the protocol of 5-weeks swimming exercise (30 min/day; 5 days/week). Aorta relaxation to acetylcholine (Ach) and sodium nitroprusside (SNP) were measured at the end of the experiment. Ach-induced endothelial dependent relaxation was significantly decreased in phenylephrine (PE) pre-contracted aorta of diabetic rats. Swimming exercise with quercetin administration preserved Ach-induced EDR but did not have any impact on SNP-induced endothelium-independent relaxation in the diabetic aorta. These findings suggest that quercetin administration associated with moderate swimming exercise could improve the endothelial NO-dependent relaxation in the aorta of rats with experimentally induced type 1 diabetes mellitus, showing that this therapeutical combination may improve and even prevent the vascular complications that occur in diabetic patients.

This study aimed to investigate the specific role of nitric oxide (NO) in micro- and macrovascular response to a 7-day high-salt (HS) diet, specifically by measuring skin microvascular local thermal hyperemia and the flow-mediated dilation of the brachial artery, as well as serum NO and three NO synthase enzyme (NOS) isoform concentrations in healthy individuals. It also aimed to examine the concept of non-osmotic sodium storage in the skin following the HS diet by measuring body fluid status and systemic hemodynamic responses, as well as serum vascular endothelial growth factor C (VEGF-C) concentration. Forty-six young, healthy individuals completed a 7-day low-salt diet, followed by a 7-day HS diet protocol. The 7-day HS diet resulted in impaired NO-mediated endothelial vasodilation in peripheral microcirculation and conduit arteries, in increased eNOS, decreased nNOS, and unchanged iNOS concentration and NO serum level. The HS diet did not change the volume of interstitial fluid, the systemic vascular resistance or the VEGF-C serum level. These results indicate that the 7-day HS-diet induces systemic impairment of NO-mediated endothelial vasodilation, while dissociation in the eNOS and nNOS response indicates complex adaptation of main NO-generating enzyme isoforms to HS intake in healthy individuals. Our results failed to support the concept of non-osmotic sodium storage.

Sirtuin 6 (SIRT6) is a member of the mammalian NAD+-dependent deac(et)ylase sirtuin family. SIRT6’s anti-inflammatory roles are emerging increasingly often in different diseases and cell types, including endothelial cells. In this study, the role of SIRT6 in pro-inflammatory conditions was investigated by engineering human umbilical vein endothelial cells to overexpress SIRT6 (SIRT6+ HUVECs). Our results showed that SIRT6 overexpression affected the levels of adhesion molecules and sustained megakaryocyte proliferation and proplatelet formation. Interestingly, the pro-inflammatory activation of the ATP/purinergic axis was reduced in SIRT6+ HUVECs. Specifically, the TNFα-induced release of ATP in the extracellular space and the increase in pannexin-1 hemichannel expression, which mediates ATP efflux, were hampered in SIRT6+ cells. Instead, NAD+ release and Connexin43 expression were not modified by SIRT6 levels. Moreover, the Ca2+ influx in response to ATP and the expression of the purinergic receptor P2X7 were decreased in SIRT6+ HUVECs. Contrary to extracellular ATP, extracellular NAD+ did not evoke pro-inflammatory responses in HUVECs. Instead, NAD+ administration reduced endothelial cell proliferation and motility and counteracted the TNFα-induced angiogenesis. Altogether, our data reinforce the view of SIRT6 activation as an anti-inflammatory approach in vascular endothelium.

Background Pulmonary arterial hypertension (PAH) is a complex, fatal disease where disease severity has been associated with the single nucleotide polymorphism (SNP) rs2856830, located near the human leukocyte antigen DPA1 (HLA‐DPA1) gene. We aimed to define the genetic architecture of functional variants associated with PAH disease severity by identifying allele‐specific binding transcription factors and downstream targets that control endothelial pathophenotypes and PAH. Methods and Results Electrophoretic mobility shift assays of oligonucleotides containing SNP rs2856830 and 8 SNPs in linkage disequilibrium revealed functional SNPs via allele‐imbalanced binding to human pulmonary arterial endothelial cell nuclear proteins. DNA pulldown proteomics identified SNP‐binding proteins. SNP genotyping and clinical correlation analysis were performed in 84 patients with PAH at University of Pittsburgh Medical Center and in 679 patients with PAH in the All of Us database. SNP rs9277336 was identified as a functional SNP in linkage disequilibrium ( r 2 >0.8) defined by rs2856830, and the minor allele was associated with decreased hospitalizations and improved cardiac output in patients with PAH, an index of disease severity. SNP pulldown proteomics showed allele‐specific binding of nuclear ACTN4 (alpha actinin 4) protein to rs9277336 minor allele. Both ACTN4 and HLA‐DPA1 were downregulated in pulmonary endothelium in human patients and rodent models of PAH. Via transcriptomic and phenotypic analyses, knockdown of HLA‐DPA1 phenocopied knockdown of ACTN4, both similarly controlling cell structure pathways, immune pathways, and endothelial dysfunction. Conclusions We defined the pathogenic activity of functional SNP rs9277336, entailing the allele‐specific binding of ACTN4 and controlling expression of the neighboring HLA‐DPA1 gene. Through inflammatory or genetic means, downregulation of this ACTN4‐HLA‐DPA1 regulatory axis promotes endothelial pathophenotypes, providing a mechanistic explanation for the association between this SNP and PAH outcomes.

PurposeWe sought to define the role of Wwtr1 in murine ocular structure and function and determine the role of mechanotransduction in Fuchs' endothelial corneal dystrophy (FECD), with emphasis on interactions between corneal endothelial cells (CEnCs) and Descemet's membrane (DM).MethodsA Wwtr1 deficient mouse colony was established, and advanced ocular imaging, atomic force microscope (AFM), and histology/immunofluorescence were performed. Corneal endothelial wound healing was assessed using cryoinjury and phototherapeutic keratectomy in Wwtr1 deficient mice. Expression of WWTR1/TAZ was determined in the corneal endothelium from normal and FECD-affected patients; WWTR1 was screened for coding sequence variants in this FECD cohort.ResultsMice deficient in Wwtr1 had reduced CEnC density, abnormal CEnC morphology, softer DM, and thinner corneas versus wildtype controls by 2 months of age. Additionally, CEnCs had altered expression and localization of Na/K-ATPase and ZO-1. Further, Wwtr1 deficient mice had impaired CEnC wound healing. The WWTR1 transcript was highly expressed in healthy human CEnCs comparable to other genes implicated in FECD pathogenesis. Although WWTR1 mRNA expression was comparable between healthy and FECD-affected patients, WWTR1/TAZ protein concentrations were higher and localized to the nucleus surrounding guttae. No genetic associations were found in WWTR1 and FECD in a patient cohort compared to controls.ConclusionsThere are common phenotypic abnormalities seen between Wwtr1 deficient and FECD-affected patients, suggesting that Wwtr1 deficient mice could function as a murine model of late-onset FECD. Despite the lack of a genetic association between FECD and WWTR1, aberrant WWTR1/TAZ protein subcellular localization and degradation may play critical roles in the pathogenesis of FECD.

The heterogeneity of endothelium across different organs was recently explored using single-cell RNA-sequencing analysis. Compared to other organs, the lung exhibits a distinct structure composed of a thin layer of capillary for efficient gas exchange. In this study, we demonstrate that Tmem100 is a lung-specific endothelium gene.

Hepatocellular carcinoma (HCC) is a malignant tumor associated with high morbidity and mortality rates. In many non-prostate solid tumors such as HCC, prostate-specific membrane antigens (PSMA) are overexpressed in tumor-associated endothelial cells. Therefore, the aim of this study was to evaluate the performance of [sup68/supGa]Ga-PSMA-617 PET imaging on HCC with different animal models, including cell line-derived xenografts (CDX) and patient-derived xenografts (PDX), and to explore its mechanisms of function.[sup68/supGa]Ga-PSMA-617 was prepared. The expression level of PSMA in two human hepatocellular cancer cells (HepG2 and HuH-7) was evaluated, and the cellular uptakes of [sup68/supGa]Ga-PSMA-617 were assayed. HepG2 and HuH-7 subcutaneous xenograft models, HepG2 orthotopic xenograft models, and four different groups of PDX models were prepared. Preclinical pharmacokinetics and performance of [sup68/supGa]Ga-PSMA-617 were evaluated in different types of HCC xenografts models using small animal PET and biodistribution studies.Low PSMA expression level of HepG2 and HuH-7 cells was observed, and the cellular uptake and blocking study confirmed the non-specificity of the PSMA-targeted probe binding to HepG2 and HuH-7 cells. In the subcutaneous xenograft models, the tumor uptakes at 0.5 h were 0.76 ± 0.12%ID/g (HepG2 tumors) and 0.78 ± 0.08%ID/g (HuH-7 tumors), respectively, which were significantly higher than those of the blocking groups (0.23 ± 0.04%ID/g and 0.20 ± 0.04%ID/g, respectively). In the orthotopic xenograft models, PET images clearly displayed the tumor locations based on the preferential accumulation of [sup68/supGa]Ga-PSMA-617 in tumor tissue versus normal liver tissue, suggesting the possibility of using [sup68/supGa]Ga-PSMA-617 PET imaging to detect primary HCC lesions in deep tissue. In the four different groups of HCC PDX models, PET imaging with [sup68/supGa]Ga-PSMA-617 provided clear tumor uptakes with prominent tumor-to-background contrast, further demonstrating its potential for the clinical imaging of PSMA-positive HCC lesions. The staining of tumor tissue sections with CD31- and PSMA-specific antibodies visualized the tumor-associated blood vessels and PSMA expression on endothelial cells in subcutaneous, orthotopic tissues, and PDX tissues, confirming the imaging with [sup68/supGa]Ga-PSMA-617 might be mediated by targeting tumor associated endothelium.In this study, in vivo PET on different types of HCC xenograft models illustrated high uptake within tumors, which confirmed that [sup68/supGa]Ga-PSMA-617 PET may be a promising imaging modality for HCC by targeting tumor associated endothelium.

Obesity changes gut microbial ecology and is related to endothelial dysfunction. Although the correlation between gut microbial ecology and endothelial dysfunction has been studied in obese persons, the underlying mechanisms by which exercise enhances endothelial function in this group remain unclear. This study investigated whether exercise improves endothelial function and alters gut microbiome composition in rats fed a high-fat diet (HFD).Obesity was induced by an HFD for 11 weeks. Whole-body composition and endothelium-dependent relaxation of mesenteric arteries were measured. Blood biochemical tests were performed, and gut microbiomes were characterized by 16S rRNA gene sequencing on an Illumina HiSeq platform.Exercise training for 8 weeks improved body composition in HFD-fed rats. Furthermore, compared with the untrained/HFD group, aerobic exercise significantly increased acetylcholine-induced, endothelium-dependent relaxation in mesenteric arteries (P0.05) and circulating vascular endothelial growth factor levels (P0.01) and decreased circulating C-reactive protein levels (P0.05). In addition, exercise and HFD resulted in alterations in the composition of the gut microbiome; exercise reduced the relative abundance of Clostridiales and Romboutsia. Moreover, 12 species of bacteria, including Romboutsia, were significantly associated with parameters of endothelial function in the overall sample.These results suggest that aerobic exercise enhances endothelial function in HFD-fed rats by altering the composition of the gut microbiota. These findings provide new insights on the application of physical exercise for improving endothelial function in obese persons.

Hemophilia A is an inherited bleeding disorder caused by defective or deficient coagulation factor VIII (FVIII) activity. Until recently, the only treatment for prevention of bleeding involved IV administration of FVIII. Gene therapy with adeno-associated vectors (AAVs) has shown some efficacy in patients with hemophilia A. However, limitations persist due to AAV-induced cellular stress, immunogenicity, and reduced durability of gene expression. Herein, we examined the efficacy of liver-directed gene transfer in FVIII knock-out mice by AAV8-GFP. Surprisingly, compared with control mice, FVIII knockout (F8TKO) mice showed significant delay in AAV8-GFP transfer in the liver. We found that the delay in liver-directed gene transfer in F8TKO mice was associated with absence of liver sinusoidal endothelial cell (LSEC) fenestration, which led to aberrant expression of several sinusoidal endothelial proteins, causing increased capillarization and decreased permeability of LSECs. This is the first study to link impaired liver-directed gene transfer to liver-endothelium maladaptive structural changes associated with FVIII deficiency in mice.

Phosphomannomutase 2 (PMM2) deficiency is the most prevalent congenital disorder of glycosylation. It is associated with coagulopathy, including protein C deficiency. Since all components of the anticoagulant and cytoprotective protein C system are glycosylated, we sought to investigate the impact of an N-glycosylation deficiency on this system as a whole. To this end, we developed a PMM2 knockdown model in the brain endothelial cell line hCMEC/D3. The resulting PMM2low cells were less able to generate activated protein C (APC), due to lower surface expression of thrombomodulin and endothelial protein C receptor. The low protein levels were due to downregulated transcription of the corresponding genes (THBD and PROCR, respectively), which itself was related to downregulation of transcription regulators Krüppel-like factors 2 and 4 and forkhead box C2. PMM2 knockdown was also associated with impaired integrity of the endothelial cell monolayer—partly due to an alteration in the structure of VE-cadherin in adherens junctions. The expression of protease-activated receptor 1 (involved in the cytoprotective effects of APC on the endothelium) was not affected by PMM2 knockdown. Thrombin stimulation induced hyperpermeability in PMM2low cells. However, pretreatment of cells with APC before thrombin simulation was still associated with a barrier-protecting effect. Taken as a whole, our results show that the partial loss of PMM2 in hCMEC/D3 cells is associated with impaired activation of protein C and a relative increase in barrier permeability.

Pathological changes in the brain endothelium play an important role in the progression of ischemic stroke and the compromised BBB under ischemic stroke conditions cause neuronal damage. However, the pathophysiological mechanisms of the BBB under normal conditions and under ischemic stroke conditions have not been fully elucidated. The present study demonstrated that knockdown of TAR DNA-binding protein 43 (TDP-43) or overexpression of TDP43-CTFs35 inhibited tight junction protein expression, and mammalian sterile-20-like 1/2 (MST1/2) and YES-associated protein (YAP) phosphorylation in brain ECs and suppressed brain EC migration in vitro. The cytoplasmic TDP43-CTFs35 level was increased in brain ECs 24 h and 72 h after MCAO, but it disappeared 1 week after cerebral ischemia. The expression of tight junction proteins was also significantly deceased 24 h after MCAO and then gradually recovered at 72 h and 1 week after MCAO. The level of YAP phosphorylation was first significantly decreased 24 h after MCAO and then increased 72 h and 1 week after MCAO, accompanied by nuclear YAP translocation. The underlying mechanism is TDP43-CTFs35-mediated inhibition of Hippo signaling pathway activity through the dephosphorylation of MST1/2, which leads to the inhibition of YAP phosphorylation and the subsequent impairment of brain EC migration and tight junction protein expression. This study provides new insights into the mechanisms of brain vascular EC regulation, which may impact on BBB integrity after cerebral ischemic injury.