Nitration-mediated activation of the small GTPase RhoA stimulates cellular glycolysis through enhanced mitochondrial fission.

Mitochondrial fission and a Warburg phenotype of increased cellular glycolysis are involved in the pathogenesis of pulmonary hypertension (PH). The purpose of this study was to determine whether increases in mitochondrial fission are involved in a glycolytic switch in pulmonary arterial endothelial cells (PAECs). Mitochondrial fission is increased in PAEC isolated from a sheep model of PH induced by pulmonary overcirculation (Shunt PAEC). In Shunt PAEC we identified increases in the S616 phosphorylation responsible for dynamin-related protein 1 (Drp1) activation, the mitochondrial redistribution of Drp1, and increased cellular glycolysis. Reducing mitochondrial fission attenuated cellular glycolysis in Shunt PAEC. In addition, we observed nitration-mediated activation of the small GTPase RhoA in Shunt PAEC, and utilizing a nitration-shielding peptide, NipR1 attenuated RhoA nitration and reversed the Warburg phenotype. Thus, our data identify a novel link between RhoA, mitochondrial fission, and cellular glycolysis and suggest that targeting RhoA nitration could have therapeutic benefits for treating PH.

Simvastatin restores pulmonary endothelial function in the setting of pulmonary over-circulation.

Statin therapy is a cornerstone in the treatment of systemic vascular diseases. However, statins have failed to translate as therapeutics for pulmonary vascular disease. Early pulmonary vascular disease in the setting of congenital heart disease (CHD) is characterized by endothelial dysfunction, which precedes the more advanced stages of vascular remodeling. These features make CHD an ideal cohort in which to re-evaluate the potential pulmonary vascular benefits of statins, with a focus on endothelial biology. However, it is critical that the full gamut of the pleiotropic effects of statins in the endothelium are uncovered. The purpose of this investigation was to evaluate the therapeutic potential of simvastatin for children with CHD and pulmonary over-circulation, and examine mechanisms of simvastatin action on the endothelium. Our data demonstrate that daily simvastatin treatment preserves endothelial function in our shunt lamb model of pulmonary over-circulation. Further, using pulmonary arterial endothelial cells (PAECs) isolated from Shunt and control lambs, we identified a new mechanism of statin action mediated by increased expression of the endogenous Akt1 inhibitor, C-terminal modifying protein (CTMP). Increases in CTMP were able to decrease the Akt1-mediated mitochondrial redistribution of endothelial nitric oxide synthase (eNOS) which correlated with increased enzymatic coupling, identified by increases in NO generation and decreases in NOS-derived superoxide. Together our data identify a new mechanism by which simvastatin enhances NO signaling in the pulmonary endothelium and identify CTMP as a potential therapeutic target to prevent the endothelial dysfunction that occurs in children born with CHD resulting in pulmonary over-circulation.

Three-Year Outcomes of the Abre Venous Self-Expanding Stent System in Patients with Symptomatic Iliofemoral Venous Outflow Obstruction.

PURPOSE: To report 36-month outcomes and subgroup analysis of the ABRE study evaluating the safety and effectiveness of the Abre venous self-expanding stent system for the treatment of symptomatic iliofemoral venous outflow obstruction disease. METHODS: The ABRE study was a prospective, multicenter, nonrandomized study that enrolled and implanted Abre venous stents in 200 participants (mean age 51.5 years [SD ± 15.9], 66.5% women) with symptomatic iliofemoral venous outflow obstruction at 24 global sites. Outcomes assessed through 36 months included patency, major adverse events, stent migration, stent fracture, and quality-of-life changes. Adverse events and imaging studies were adjudicated by independent clinical events committee and core laboratories, respectively. RESULTS: Primary, primary-assisted, and secondary patency through 36 months by Kaplan-Meier estimates were 81.6%, 84.8%, and 86.3%, respectively. The cumulative incidence of major adverse events through 36 months was 10.2%, mainly driven by 12 thrombosis events. Subgroup analyses demonstrated a primary patency of 76.5% in the acute deep vein thrombosis group, 70.4% in the postthrombotic syndrome group, and 97.1% in the nonthrombotic iliac vein lesion group through 36 months. The overall mean lesion length was 112.4 mm (SD ± 66.1). There were no stent fractures or migrations in this study. Quality of life and venous functional assessments demonstrated significant improvements from baseline to 36 months across all patient subsets. CONCLUSIONS: Results from the ABRE study demonstrated sustained patency with a good safety profile after implantation of a dedicated venous stent in patients with symptomatic iliofemoral venous outflow obstruction disease.

Cost Effectiveness of Early Endovenous Thrombus Removal for Acute Iliofemoral Deep Vein Thrombosis in the United Kingdom.

OBJECTIVE: Early clot removal using endovascular intervention aims to reduce post-thrombotic syndrome (PTS) following iliofemoral deep venous thrombosis (DVT). This may reduce long term morbidity but incurs a higher initial cost. This study examined the cost effectiveness of catheter directed thrombolysis (CDT) and pharmacochemical thrombectomy (PMT) compared with oral anticoagulation (OAC) alone for treatment of acute iliofemoral DVT in the United Kingdom. METHODS: A combined decision tree (acute DVT complications) and Markov model (long term complications [PTS]) was used for decision analytic modelling with five states: no PTS, mild PTS, moderate PTS, severe PTS, and dead. All patients started with acute DVT. Patients who survived acute complications transitioned into the Markov model. Cycle time was six months. A healthcare payer perspective and lifetime horizon was used, adjusting for excess mortality due to history of thrombosis. Data for probabilities, transition probabilities, mortality, and utilities were obtained from the published literature. Cost data were obtained from UK NHS tariffs and published literature. Outcomes were mean lifetime cost, quality adjusted life years (QALYs), and cost effectiveness. RESULTS: Over a patient's lifetime, OAC was more costly (£37 206) than CDT (£32 043) and PMT (£36 288). Mean lifetime QALYs for OAC (12.9) were lower than CDT (13.5) and PMT (13.3). Therefore, in the incremental cost effectiveness analysis, both CDT and PMT were dominant: CDT was less costly (-£5 163) and more effective (+0.6 QALYs) than OAC, and PMT was also less costly (-£917) and more effective (+0.3 QALYs) than OAC. Results were robust to univariable sensitivity analyses, but probabilistic sensitivity analyses suggested considerable parameter uncertainty. CONCLUSION: Early interventional treatment of iliofemoral DVT is cost effective in the UK. Future clinical and epidemiological studies are needed to characterise parameter uncertainty. Further analysis of modern practice, alternative treatments, and optimised care models is warranted.

Fatigue Test Method to Evaluate the 50 Year Durability of Venous Stents.

OBJECTIVE: Iliofemoral venous obstructive disease can result in significant, potentially debilitating symptoms that can negatively affect quality of life. Unlike arterial disease, patients with deep venous disease have a significantly lower median age, therefore the need for long term stent patency becomes a matter of decades rather than years. Furthermore, iliofemoral lesions frequently require stent placement across the inguinal ligament. Such stents are subject to dynamic stress from leg movement and associated concerns for device fatigue, resulting in stent fracture. The aim of this study was to describe an in vitro 50 year stent fatigue test method designed to assess durability against dynamic stress induced device fracture. METHODS: Through literature review, cadaver studies, and computer modelling, the most challenging loading was confirmed to be hip flexion across the inguinal ligament. This occurs when the patient adjusts between a seated and standing position. Sit to stand hip flexion at the inguinal ligament was effectively simulated on the bench in this in vitro experimental study. RESULTS: When tested under challenge parameters, hip flexion was reliably found to cause fractures in non-venous nitinol stents. However, a dedicated self expanding nitinol venous stent, engineered for improved durability, underwent up to 50 years of simulated loading on the bench with 15% (3/20) of stents experiencing fractures at 50 years, compared with fractures in 35% (14/40) of non-venous stents tested to 1.4 years; no statistical testing was performed as durations do not match and the objective was to demonstrate the test method). CONCLUSION: The presented fatigue test method is a suitable approach for evaluating the durability of stents intended for venous use. Venous stents demonstrated superior fatigue resistance compared with non-venous stents via in vitro hip flexion testing.

Editor's Choice - Management of Lower Extremity Venous Outflow Obstruction: Results of an International Delphi Consensus.

OBJECTIVE: The endovascular treatment of venous obstruction has expanded significantly in recent years. Best practices for optimal patient outcomes are not well established and the evidence base is poor. The purpose of this study was to obtain consensus on management criteria for patients with lower extremity venous outflow obstruction. METHODS: The study was conducted as a two round Delphi consensus. Statements addressed imaging, symptoms and other baseline measures, differential diagnosis, treatment algorithm, indications for stenting, inflow and outflow assessment, successful procedural outcomes, post-procedure therapies and stent surveillance, and clinical success factors. Statements were prepared by six expert physicians (round 1, 40 statements) and an expanded panel of 24 physicians (round 2, 80 statements) and sent to a pre-identified group of venous experts who met qualifying criteria. A 9 point Likert scale was used and consensus was defined as ≥ 70% of respondents rating a statement between 7 and 9 (agreement) or between 1 and 3 (disagreement). Round 1 results were used to guide rewording and splitting compound statements for greater clarity in round 2. RESULTS: In round 1, 75 of 110 (68%) experts responded, and 91 of 121 (75%) experts responded in round 2. Round 1 achieved consensus in 32/40 (80%) statements. Consensus was not reached in the treatment algorithm section. Round 2 achieved consensus in 50/80 (62.5%). Statements reaching consensus were imaging (2/3, 66%), symptoms and other baseline measures (12/24, 50%), differential diagnosis (2/8, 25%), treatment algorithm (10/17, 59%), indications for stenting (10/10, 100%), inflow and outflow assessment (2/2, 100%), procedural outcomes (2/2, 100%), post-procedure therapies and stent surveillance, (5/7, 71%), and clinical success factors (5/7, 71%). CONCLUSION: This study demonstrated that considerable consensus was achieved between venous experts on the optimal management of lower extremity venous outflow obstruction. There were multiple domains where consensus is lacking, highlighting important areas for further investigation and research.

SOX17 Deficiency Mediates Pulmonary Hypertension: At the Crossroads of Sex, Metabolism, and Genetics.

Rationale: Genetic studies suggest that SOX17 (SRY-related HMG-box 17) deficiency increases pulmonary arterial hypertension (PAH) risk. Objectives: On the basis of pathological roles of estrogen and HIF2α (hypoxia-inducible factor 2α) signaling in pulmonary artery endothelial cells (PAECs), we hypothesized that SOX17 is a target of estrogen signaling that promotes mitochondrial function and attenuates PAH development via HIF2α inhibition. Methods: We used metabolic (Seahorse) and promoter luciferase assays in PAECs together with the chronic hypoxia murine model to test the hypothesis. Measurements and Main Results: Sox17 expression was reduced in PAH tissues (rodent models and from patients). Chronic hypoxic pulmonary hypertension was exacerbated by mice with conditional Tie2-Sox17 (Sox17EC-/-) deletion and attenuated by transgenic Tie2-Sox17 overexpression (Sox17Tg). On the basis of untargeted proteomics, metabolism was the top pathway altered by SOX17 deficiency in PAECs. Mechanistically, we found that HIF2α concentrations were increased in the lungs of Sox17EC-/- and reduced in those from Sox17Tg mice. Increased SOX17 promoted oxidative phosphorylation and mitochondrial function in PAECs, which were partly attenuated by HIF2α overexpression. Rat lungs in males displayed higher Sox17 expression versus females, suggesting repression by estrogen signaling. Supporting 16α-hydroxyestrone (16αOHE; a pathologic estrogen metabolite)-mediated repression of SOX17 promoter activity, Sox17Tg mice attenuated 16αOHE-mediated exacerbations of chronic hypoxic pulmonary hypertension. Finally, in adjusted analyses in patients with PAH, we report novel associations between a SOX17 risk variant, rs10103692, and reduced plasma citrate concentrations (n = 1,326). Conclusions: Cumulatively, SOX17 promotes mitochondrial bioenergetics and attenuates PAH, in part, via inhibition of HIF2α. 16αOHE mediates PAH development via downregulation of SOX17, linking sexual dimorphism and SOX17 genetics in PAH.

Free Radical-Associated Gene Signature Predicts Survival in Sepsis Patients.

Sepsis continues to overwhelm hospital systems with its high mortality rate and prevalence. A strategy to reduce the strain of sepsis on hospital systems is to develop a diagnostic/prognostic measure that identifies patients who are more susceptible to septic death. Current biomarkers fail to achieve this outcome, as they only have moderate diagnostic power and limited prognostic capabilities. Sepsis disrupts a multitude of pathways in many different organ systems, making the identification of a single powerful biomarker difficult to achieve. However, a common feature of many of these perturbed pathways is the increased generation of reactive oxygen species (ROS), which can alter gene expression, changes in which may precede the clinical manifestation of severe sepsis. Therefore, the aim of this study was to evaluate whether ROS-related circulating molecular signature can be used as a tool to predict sepsis survival. Here we created a ROS-related gene signature and used two Gene Expression Omnibus datasets from whole blood samples of septic patients to generate a 37-gene molecular signature that can predict survival of sepsis patients. Our results indicate that peripheral blood gene expression data can be used to predict the survival of sepsis patients by assessing the gene expression pattern of free radical-associated -related genes in patients, warranting further exploration.

Inflammation and vascular remodeling in COVID-19 hearts.

A wide range of cardiac symptoms have been observed in COVID-19 patients, often significantly influencing the clinical outcome. While the pathophysiology of pulmonary COVID-19 manifestation has been substantially unraveled, the underlying pathomechanisms of cardiac involvement in COVID-19 are largely unknown. In this multicentre study, we performed a comprehensive analysis of heart samples from 24 autopsies with confirmed SARS-CoV-2 infection and compared them to samples of age-matched Influenza H1N1 A (n = 16), lymphocytic non-influenza myocarditis cases (n = 8), and non-inflamed heart tissue (n = 9). We employed conventional histopathology, multiplexed immunohistochemistry (MPX), microvascular corrosion casting, scanning electron microscopy, X-ray phase-contrast tomography using synchrotron radiation, and direct multiplexed measurements of gene expression, to assess morphological and molecular changes holistically. Based on histopathology, none of the COVID-19 samples fulfilled the established diagnostic criteria of viral myocarditis. However, quantification via MPX showed a significant increase in perivascular CD11b/TIE2 + -macrophages in COVID-19 over time, which was not observed in influenza or non-SARS-CoV-2 viral myocarditis patients. Ultrastructurally, a significant increase in intussusceptive angiogenesis as well as multifocal thrombi, inapparent in conventional morphological analysis, could be demonstrated. In line with this, on a molecular level, COVID-19 hearts displayed a distinct expression pattern of genes primarily coding for factors involved in angiogenesis and epithelial-mesenchymal transition (EMT), changes not seen in any of the other patient groups. We conclude that cardiac involvement in COVID-19 is an angiocentric macrophage-driven inflammatory process, distinct from classical anti-viral inflammatory responses, and substantially underappreciated by conventional histopathologic analysis. For the first time, we have observed intussusceptive angiogenesis in cardiac tissue, which we previously identified as the linchpin of vascular remodeling in COVID-19 pneumonia, as a pathognomic sign in affected hearts. Moreover, we identified CD11b + /TIE2 + macrophages as the drivers of intussusceptive angiogenesis and set forward a putative model for the molecular regulation of vascular alterations.

Leg Ulcer Pathway Acceleration (LUPA) study.

BACKGROUND: Leg ulcers are common, costly, and significantly impair quality of life, but their management is variable and associated with considerable delays in healing. The aim of this study was to design an accelerated leg ulcer care pathway in a community and hospital setting to improve patient outcomes. METHODS: A new referral pathway was developed using a series of healthcare professional and patient interviews, focus groups, and stakeholder workshops. The referral pathway, investigation and treatment protocols were further informed by clinical guidelines to develop the Leg Ulcer Pathway Acceleration care pathway. The outcomes of a consecutive series of patients enrolled in the Leg Ulcer Pathway Acceleration care pathway were compared with the outcomes of patients from a historical leg ulcer cohort from the same community and hospital setting. RESULTS: A total of 110 eligible patients were enrolled and followed prospectively through the Leg Ulcer Pathway Acceleration care pathway. Their outcomes were compared with those of 183 patients with venous leg ulcers identified from existing hospital and community ulcer centres, and acting as the historical control group. The 110 patients in the Leg Ulcer Pathway Acceleration group consisted of 73 (66 per cent) men, had a mean(s.d.) age of 55.7(17.2) years, and had a median initial ulcer duration of 14.5 (i.q.r. 6-30) months. The 183 patients in the historical control group consisted of 119 (65 per cent) men, had a mean(s.d.) age of 56.4(17.2) years, and had a median initial ulcer duration of 13.5 (i.q.r. 6-47) months. Venous disease was treated in 67/110 (61 per cent) and deep venous disease was treated in 33/110 (30 per cent) of patients in the Leg Ulcer Pathway Acceleration cohort. In contrast, only 16/183 (8 per cent) of patients in the control group were treated for superficial venous insufficiency and 4/183 (2 per cent) of patients in the control group were treated for deep venous disease. Ulcer healing rates at 12 months were 80 and 20 per cent in the Leg Ulcer Pathway Acceleration group and the control group respectively (P < 0.001). Adjusted for baseline characteristics, the OR for a healed ulcer at 12 months was 21.21 (95 per cent c.i.. 11.32 to 42.46) (P < 0.001). CONCLUSION: The introduction of an accelerated leg ulcer care pathway significantly improves ulcer healing when compared with historical controls.

PTK2-associated gene signature could predict the prognosis of IPF.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with a poor prognosis. Current/available clinical prediction tools have limited sensitivity and accuracy when evaluating clinical outcomes of IPF. Research has shown that focal adhesion kinase (FAK), produced by the protein tyrosine kinase 2 (PTK2) gene, is crucial in IPF development. FAK activation is a characteristic of lesional fibroblasts; Thus, FAK may be a valuable therapeutic target or prognostic biomarker for IPF. This study aimed to create a gene signature based on PTK2-associated genes and microarray data from blood cells to predict disease prognosis in patients with IPF. PTK2 levels were found to be higher in lung tissues of IPF patients compared to healthy controls, and PTK2 inhibitor Defactinib was found to reduce TGFß-induced FAK activation and increase α-smooth muscle actin. Although the blood PTK2 levels were higher in IPF patients, blood PTK level alone could not predict IPF prognosis. From 196 PTK2-associated genes, 11 genes were prioritized to create a gene signature (PTK2 molecular signature) and a risk score system using univariate and multivariate Cox regression analysis. Patients were divided into high-risk and low-risk groups using PTK2 molecular signature. Patients in the high-risk group experienced decreased survival rates compared to patients in the low-risk group across all discovery and validation cohorts. Further functional enrichment and immune cell proportion analyses revealed that the PTK2 molecular signature strongly reflected the activation levels of immune pathways and immune cells. These findings suggested that PTK2 is a molecular target of IPF and the PTK2 molecular signature is an effective IPF prognostic biomarker.

Endothelin-1 acutely increases nitric oxide production via the calcineurin mediated dephosphorylation of Caveolin-1.

Endothelin (ET)-1 is an endothelial-derived peptide that exerts biphasic effects on nitric oxide (NO) levels in endothelial cells such that acute exposure stimulates-while sustained exposure attenuates-NO production. Although the mechanism involved in the decrease in NO generation has been identified but the signaling involved in the acute increase in NO is still unresolved. This was the focus of this study. Our data indicate that exposing pulmonary arterial endothelial cells (PAEC) to ET-1 led to an increase in NO for up to 30min after which levels declined. These effects were attenuated by ET receptor antagonists. The increase in NO correlated with significant increases in pp60Src activity and increases in eNOS phosphorylation at Tyr83 and Ser1177. The ET-1 mediated increase in phosphorylation and NO generation were attenuated by the over-expression of a pp60Src dominant negative mutant. The increase in pp60Src activity correlated with a reduction in the interaction of Caveolin-1 with pp60Src and the calcineurin-mediated dephosphorylation of caveolin-1 at three previously unidentified sites: Thr91, Thr93, and Thr95. The calcineurin inhibitor, Tacrolimus, attenuated the acute increase in pp60Src activity induced by ET-1 and a calcineurin siRNA attenuated the ET-1 mediated increase in eNOS phosphorylation at Tyr83 and Ser1177 as well as the increase in NO. By using a Caveolin-1 celluSpot peptide array, we identified a peptide targeting a sequence located between aa 41-56 as the pp60Src binding region. This peptide fused to the TAT sequence was found to decrease caveolin-pp60Src interaction, increased pp60Src activity, increased eNOS pSer1177 and NO levels in PAEC and induce vasodilation in isolated aortic rings in wildtype but not eNOS knockout mice. Together, our data identify a novel mechanism by which ET-1 acutely increases NO via a calcineurin-mediated dephosphorylation of caveolin-1 and the subsequent stimulation of pp60Src activity, leading to increases in phosphorylation of eNOS at Tyr83 and Ser1177.

A Systematic Review of the Safety and Efficacy of Inferior Vena Cava Stenting.

OBJECTIVE: Inferior vena cava (IVC) stenting may provide benefit to patients with symptomatic obstruction; however, there are no devices currently licensed for use in the IVC and systematic reviews on the topic are lacking. The aim of this study was to carry out a systematic review of the literature and meta-analysis to investigate the safety and efficacy of IVC stenting in all adult patient groups. DATA SOURCES: The Medline and Embase databases were searched for studies reporting outcomes for safety and effectiveness of IVC stenting for any indication in series of 10 or more patients. REVIEW METHODS: A systematic review of the literature was carried out according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. RESULTS: Thirty-three studies were included describing 1 575 patients. Indications for stenting were malignant IVC syndrome (229 patients), thrombotic disease (807 patients), Budd-Chiari syndrome (501 patients), and IVC stenosis post liver transplantation (47 patients). The male:female ratio was 2:1 and the median age ranged from 30 to 61 years. The studies included were not suitable for formal meta-analysis as 30/33 were single centre retrospective studies with no control groups and there was considerable inconsistency in outcome reporting. There was significant risk of bias in 94% of studies. Median reported technical success was 100% (range 78 - 100%), primary patency was 75% (38 - 98%), and secondary patency was 91.5% (77 - 100%). Major complications were pulmonary embolism (three cases), stent migration (12 cases), and major bleeding (15 cases), and there were three deaths in the immediate post-operative period. Most studies reported improvement in clinical symptoms but formal reporting tools were not used consistently. CONCLUSION: The evidence base for IVC stenting consists of predominantly single centre, retrospective, observational studies that have a high risk of bias. Nonetheless the procedure appears safe with few major adverse events, and studies that reported clinical outcomes demonstrate improvement in symptoms and quality of life. Randomised controlled trials and prospective registry based studies with larger patient numbers and standardised outcome are required to improve the evidence base for this procedure.

Circadian-Coupled Genes Expression and Regulation in HIV-Associated Chronic Obstructive Pulmonary Disease (COPD) and Lung Comorbidities.

People living with HIV (PLWH) have an elevated risk of chronic obstructive pulmonary disease (COPD) and are at a higher risk of asthma and worse outcomes. Even though the combination of antiretroviral therapy (cART) has significantly improved the life expectancy of HIV-infected patients, it still shows a higher incidence of COPD in patients as young as 40 years old. Circadian rhythms are endogenous 24 h oscillations that regulate physiological processes, including immune responses. Additionally, they play a significant role in health and diseases by regulating viral replication and its corresponding immune responses. Circadian genes play an essential role in lung pathology, especially in PLWH. The dysregulation of core clock and clock output genes plays an important role in chronic inflammation and aberrant peripheral circadian rhythmicity, particularly in PLWH. In this review, we explained the mechanism underlying circadian clock dysregulation in HIV and its effects on the development and progression of COPD. Furthermore, we discussed potential therapeutic approaches to reset the peripheral molecular clocks and mitigate airway inflammation.

Sphingosine-1-Phosphate Receptor 3 Induces Endothelial Barrier Loss via ADAM10-Mediated Vascular Endothelial-Cadherin Cleavage.

Mechanical ventilation (MV) is a life-supporting strategy employed in the Intensive Care Unit (ICU). However, MV-associated mechanical stress exacerbates existing lung inflammation in ICU patients, resulting in limited improvement in mortality and a condition known as Ventilator-Induced Lung Injury (VILI). Sphingosine-1-phosphate (S1P) is a circulating bioactive lipid that maintains endothelial integrity primarily through S1P receptor 1 (S1PR1). During VILI, mechanical stress upregulates endothelial S1PR3 levels. Unlike S1PR1, S1PR3 mediates endothelial barrier disruption through Rho-dependent pathways. However, the specific impact of elevated S1PR3 on lung endothelial function, apart from Rho activation, remains poorly understood. In this study, we investigated the effects of S1PR3 in endothelial pathobiology during VILI using an S1PR3 overexpression adenovirus. S1PR3 overexpression caused cytoskeleton rearrangement, formation of paracellular gaps, and a modified endothelial response towards S1P. It resulted in a shift from S1PR1-dependent barrier enhancement to S1PR3-dependent barrier disruption. Moreover, S1PR3 overexpression induced an ADAM10-dependent cleavage of Vascular Endothelial (VE)-cadherin, which hindered endothelial barrier recovery. S1PR3-induced cleavage of VE-cadherin was at least partially regulated by S1PR3-mediated NFκB activation. Additionally, we employed an S1PR3 inhibitor TY-52156 in a murine model of VILI. TY-52156 effectively attenuated VILI-induced increases in bronchoalveolar lavage cell counts and protein concentration, suppressed the release of pro-inflammatory cytokines, and inhibited lung inflammation as assessed via a histological evaluation. These findings confirm that mechanical stress associated with VILI increases S1PR3 levels, thereby altering the pulmonary endothelial response towards S1P and impairing barrier recovery. Inhibiting S1PR3 is validated as an effective therapeutic strategy for VILI.

The Pulmonary Endothelial Glycocalyx Modifications in Glypican 1 Knockout Mice Do Not Affect Lung Endothelial Function in Physiological Conditions.

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.

Novel Relationship between Mitofusin 2-Mediated Mitochondrial Hyperfusion, Metabolic Remodeling, and Glycolysis in Pulmonary Arterial Endothelial Cells.

The disruption of mitochondrial dynamics has been identified in cardiovascular diseases, including pulmonary hypertension (PH), ischemia-reperfusion injury, heart failure, and cardiomyopathy. Mitofusin 2 (Mfn2) is abundantly expressed in heart and pulmonary vasculature cells at the outer mitochondrial membrane to modulate fusion. Previously, we have reported reduced levels of Mfn2 and fragmented mitochondria in pulmonary arterial endothelial cells (PAECs) isolated from a sheep model of PH induced by pulmonary over-circulation and restoring Mfn2 normalized mitochondrial function. In this study, we assessed the effect of increased expression of Mfn2 on mitochondrial metabolism, bioenergetics, reactive oxygen species production, and mitochondrial membrane potential in control PAECs. Using an adenoviral expression system to overexpress Mfn2 in PAECs and utilizing 13C labeled substrates, we assessed the levels of TCA cycle metabolites. We identified increased pyruvate and lactate production in cells, revealing a glycolytic phenotype (Warburg phenotype). Mfn2 overexpression decreased the mitochondrial ATP production rate, increased the rate of glycolytic ATP production, and disrupted mitochondrial bioenergetics. The increase in glycolysis was linked to increased hypoxia-inducible factor 1α (HIF-1α) protein levels, elevated mitochondrial reactive oxygen species (mt-ROS), and decreased mitochondrial membrane potential. Our data suggest that disrupting the mitochondrial fusion/fission balance to favor hyperfusion leads to a metabolic shift that promotes aerobic glycolysis. Thus, therapies designed to increase mitochondrial fusion should be approached with caution.

Nitration of protein kinase G-Iα modulates cyclic nucleotide crosstalk via phosphodiesterase 3A: Implications for acute lung injury.

Phosphodiesterase 3A (PDE3A) selectively cleaves the phosphodiester bond of cAMP and is inhibited by cGMP, making it an important regulator of cAMP-cGMP signaling crosstalk in the pulmonary vasculature. In addition, the nitric oxide-cGMP axis is known to play an important role in maintaining endothelial barrier function. However, the potential role of protein kinase G-Iα (PKG-Iα) in this protective process is unresolved and was the focus of our study. We describe here a novel mechanism regulating PDE3A activity, which involves a PKG-Iα-dependent inhibitory phosphorylation of PDE3A at serine 654. We also show that this phosphorylation is critical for maintaining intracellular cAMP levels in the pulmonary endothelium and endothelial barrier integrity. In an animal model of acute lung injury (ALI) induced by challenging mice with lipopolysaccharide (LPS), an increase in PDE3 activity and a decrease in cAMP levels in lung tissue was associated with reduced PKG activity upon PKG-Iα nitration at tyrosine 247. The peroxynitrite scavenger manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin prevented this increase in PDE3 activity in LPS-exposed lungs. In addition, site-directed mutagenesis of PDE3A to replace serine 654 with alanine yielded a mutant protein that was insensitive to PKG-dependent regulation. Taken together, our data demonstrate a novel functional link between nitrosative stress induced by LPS during ALI and the downregulation of barrier-protective intracellular cAMP levels. Our data also provide new evidence that PKG-Iα is critical for endothelial barrier maintenance and that preservation of its catalytic activity may be efficacious in ALI therapy.

Midterm and Long-Term Outcomes following Dedicated Endovenous Nitinol Stent Placement for Symptomatic Iliofemoral Venous Obstruction: Three- to 5-Year Results of the VIRTUS Study.

PURPOSE: To assess the midterm patency and long-term safety of placement of a dedicated venous stent (Vici Venous Stent System) for the treatment of venous lesions of the iliofemoral outflow tract. MATERIALS AND METHODS: Patients with unilateral obstructive disease of the iliofemoral veins and a Clinical, Etiological, Anatomical, Pathophysiological class of 3 or higher or a Venous Clinical Severity Score of 2 or greater were enrolled in this prospective, multicenter, single-arm study at 23 sites in the United States and Europe. The patients were followed up for 36 months after the index procedure for the assessment of patency and up to 60 months for the assessment of safety. The clinical outcomes in 11 patients with a stent fracture were assessed. RESULTS: A total of 200 patients enrolled in 2 cohorts (ie, feasibility cohort, n = 30; pivotal cohort, n = 170) were combined for this analysis. The overall 36-month primary patency rate was 71.7% (86/120), and the 36-month primary patency rate was 96.4% (27/28) for the nonthrombotic group and 64.1% (59/92) for the postthrombotic group. The freedom from major adverse events was 81.2% (53/65) through 60 months. The 60-month Kaplan-Meier estimate of freedom from target vessel revascularization (TVR) was 84.3%. In 9 of the 11 patients who had a stent fracture (1 patient with nonthrombotic etiology and 10 patients with postthrombotic etiology) identified at 12 months, the stents extended into the common femoral vein. The TVR rates and clinical outcomes were similar between patients with and without a stent fracture. CONCLUSIONS: The results of the VIRTUS study demonstrated good midterm patency and long-term safety following the placement of a dedicated venous stent for iliofemoral obstruction.

Performance of Open and Closed Cell Laser Cut Nitinol Stents for the Treatment of Chronic Iliofemoral Venous Outflow Obstruction in Patients Treated at a Single Centre.

OBJECTIVE: A number of dedicated self expanding nitinol stents have been developed for use in the venous system, with both open cell (OC) and closed cell (CC) designs available. Data comparing these different designs are lacking. The objective of this study was to evaluate outcomes in patients treated with open and closed cells for unilateral chronic iliac vein obstruction. METHODS: A single centre retrospective cohort study was conducted, including all patients treated with a dedicated nitinol venous stent between 2014 and 2019. Stent patency and details of re-interventions (including lysis, venoplasty, reinforcement, extension, arteriovenous fistula formation) were examined in the first post-operative year. Subgroup analysis described outcomes for patients treated with OC and CC stents ending above the inguinal ligament and those who required extension into the common femoral vein. Cox regression analysis was used to identify factors associated with loss of primary patency. RESULTS: A total of 207 patients were included (OC 100 patients, CC 107 patients). There was no significant difference between the groups for age (OC 42 years, CC 44 years); gender (OC and CC 67% female); presence of post-thrombotic lesions (OC 71%, CC 73%); stenting across the inguinal ligament (OC 58%, CC 56%), or presence of inflow disease (OC 49%, CC 47%). Primary and cumulative patency at 12 months were similar between groups (primary: OC 63%, CC 65%; cumulative: OC 93%, CC 90%). Patients with a CC stent across the inguinal ligament had a greater risk of needing multiple re-interventions at one year compared with those with an OC stent (odds ratio 2.84, 95% confidence interval [CI] 1.16 - 6.9) but overall, the only factor significantly associated with loss of primary patency was inflow vessel disease (hazard ratio 3.39, 95% CI 1.73 - 6.62, p < .001). CONCLUSION: OC and CC dedicated nitinol venous stents were observed to perform similarly in terms of patency and symptom improvement at one year. Disease of the inflow vessels was the most important factor associated with a loss of stent patency irrespective of stent design.