Introduction/Purpose:During the septic cardiomyopathy, the mechanism and relationship to outcome of changes in left ventricular (LV) end diastolic volume (EDV) and ejection fraction (EF) remains obscure. We compared serial changes in LVEF and LVEDV to successive alterations in LV wall ultrastructure, water content, and total mass to investigate whether these measures can explain their basis. Methods:We performed cardiac magnetic resonance imaging at 0,6,18,30,42,54, and 92h post-bacterial challenge in a large-animal model (n=57) that mimics human septic cardiomyopathy. LV tissue was obtained for electron microscopy (EM) upon death and 66h in sacrificed survivors. Results:Between 0-6h post-challenge, LV compliance and EDV reached its greatest decline. Non-survivors (n=18) exhibited significantly greater reductions in LVEDV, along with more myocyte edema, mitochondrial swelling and myofilament fragmentation on EM. This increased tissue damage may explain why non-survivors developed worse LV compliance and a greater decline in LVEDV, which persisted until death. From 6-30h, LVEDV significantly improved to baseline in non-survivors, while survivors experienced ∼20% increases (n=39). Concurrently, there was significant LV mass loss and increases in percent water content that were significantly associated with increases in LVEDV. This is consistent with a passive mechanism for rapidly improving LV compliance and EDV. Full recovery of EF required additional days. We hypothesize the prolonged significant mass loss over 5d reflects an active process for remodeling fragmented myofilaments, eliminating myocyte edema, and mitochondrial swelling, ultimately restoring contractile function. Conclusion:The septic cardiomyopathy constitutes a diffuse ultrastructural injury to myocytes with three phases. Initially, there is a decrease in LVEDV, and EF due to myocyte damage within 6h of bacterial challenge; next, the patient sees a passive LVEDV recovery from 6-30h, where LV mass loss increases relative wall percent water content, which facilitates wall compliance and LVEDV; and lastly, the patient sees mass loss beyond 30h consistent with an active repair mechanism of myocytes, returning systolic function to normal. Therefore, EDV changes are a pathophysiological biomarker for sepsis outcomes. A lower LVEDV indicates persistent unrepairable ultrastructure damage with worsening wall compliance and poorer outcomes. LVEDV dilation is a sign of near-full recovery of ultrastructure injury, augmenting wall compliance and improving outcomes. Clinical Implications:We explain herein why septic cardiomyopathy findings don't have clinical implications like heart failure. Septic patients who exhibit signs of heart failure, low LVEF with high EDVs, are doing well - reflecting mild myocyte injury, effective damaged tissues clearance, increased relative LV wall water content, and compliance. This augments the LVEDV, lowering the LVEF. Septic patients who deteriorate rapidly, contrary to heart failure patients, show high/normal LVEF and low/normal LVEDV. Here, the myocyte damage is severe, leading to insufficient wall repair, and this decreased wall compliance persists, preventing the LV from dilating and making LVEDV low which ultimately raises the LVEF.

RATIONALE: Constitutive activation of PI3K/AKT, as observed in many types of cancer, was identified as a prominent signaling abnormality in BMPR2-silenced primary human pulmonary artery endothelial cells (PAECs). Inhibition of PI3K/AKT activation in BMPR2-silenced PAECs decreased proliferation and induced apoptosis (Awad 2024). In PH animal models, a PI3K-alpha specific inhibitor improved vascular remodeling (Berghausen 2021). Collectively these findings suggest that blockade of the PI3K/AKT pathway is a potential therapeutic strategy in PAH. Unlike PI3K/AKT inhibitors approved or under development for the treatment of cancer, use in PAH will likely require long-term drug exposure with a much lower tolerance for drug-associated toxicities. Thus, we sought to determine relative expression of PI3K isoforms in BMPR2-deficient PAECs to better understand the potential efficacy of isoform specific inhibitors. METHODS: Normal PAECs were transfected with either gene specific or non-targeting control siRNA (siCTRL) for 72h. PAECs isolated from PAH patients or failed donor (FD) control lung tissue were obtained from the PHBI. Gene expression changes were assessed by quantitative real-time PCR and changes in protein expression were analyzed by Western blot. Statistical analyses were performed in GraphPad Prism. RESULTS: Normal PAECs predominantly express PI3KCA, PI3KCB and PI3KCD isoforms of p110, the catalytic subunit of PI3K, whereas PI3KCG is expressed at significantly lower levels (mean Ct relative to beta-actin: 4.29, 4.82, 6.46 and 10.28, respectively; p<0.01 for pairwise comparisons of gamma vs. other isoforms). Compared to siCTRL transfected PAECs, PI3KCD mRNA expression was increased in BMPR2-silenced PAECs (p=0.047), whereas PI3KCA tended to be lower (p=0.16) and PI3KCB was unchanged (p=0.33). mRNA expression of PI3KCA, PI3KCB, and PI3KCD were all increased in PAECs from PAH patients with BMPR2 mutations compared to FD controls (p<0.05 for each pairwise comparison). Similar to previous findings in BMPR2-silenced PAECs, AKT was activated (phosphorylation of Ser 473) in PAECs from PAH patients with BMPR2 mutations compared to FD controls (p=0.04), and treatment with either a PI3K-alpha, -beta or -delta isoform specific inhibitor significantly reduced AKT phosphorylation (p<0.05 compared to vehicle control). CONCLUSIONS: Human PAECs principally express 3 different p110/PI3K isoforms. PI3KCD is further upregulated following BMPR2 silencing and expression of PI3KCA, PI3KCB and PI3KCD are all increased in PAECs from PAH patients with BMPR2 mutations. Given their varied clinical side effects and relative expression in human PAECs, pre-clinical studies comparing the efficacy of PI3K isoform specific inhibitors are warranted.

Introduction: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), has both acute and chronic manifestations. Severe acute disease is associated with multiorgan failure and small vessel vasculopathy characterized by widespread microthrombi. Survivors, who have otherwise recovered from the acute disease, have an increased risk for cardiovascular complications including stroke and myocardial infarction (MI). Notably, this extended period of endothelial dysfunction is not only poorly understood, but specific diagnostic and therapeutic approaches are absent. We hypothesize that SARS-CoV-2-induced endothelial cell senescence contributes to pulmonary vascular microthrombi in acute disease and possibly increases the risk for later cardiovascular complications. Methods: Primary human pulmonary arterial endothelial cells (PAECs) were exposed to recombinant SARS-CoV-2 spike (S1) or nucleocapsid (N) protein, viral components that may circulate for weeks after acute COVID-19. The senescent cellular phenotype associated with S1 exposure was compared to three well-characterized inducers of senescence (etoposide, H 2 O 2 , and ionizing radiation). Senescence was assessed using a flow cytometric assay of SA-b-galactosidase activity, quantitative PCR determination of p21, Ki67, LAMB3, H2AX, and PURPL expression, and apoptosis resistance to serum withdraw as measured by caspase 3/7 activity. Results: SARS-CoV-2 S1 exposure, as well as all three known inducers of senescence, increased SA-b-galactosidase activity in PAECs. mRNA expression of p21, Ki67, LAMB3, H2AX, and PURPL was variable across the different inducers of senescence, with the effects of S1 protein being most similar to H 2 O 2 . Both S1 and N-protein exposure for 24h dose dependently resulted in significant apoptosis resistance. Conclusions: Our data support that SARS-CoV-2 protein-induced cellular senescence may contribute to COVID-19 vasculopathy and endothelial dysfunction during acute and convalescent illness. These findings suggest that therapeutically targeting endothelial senescence might mitigate early and late cardiovascular complications of COVID-19.

Introduction: Pulmonary arterial hypertension (PAH) is a rare, fatal disease affecting the distal precapillary pulmonary arterioles. BMPR2 mutations cause 70% of heritable PAH and BMPR2 expression is significantly reduced in plexiform lesions of patients with idiopathic PAH. In addition to genetic and other causes, obesity has been investigated as a PAH risk factor. Obesity increases leptin levels, resulting in oxidative stress, chronic inflammation, and endothelial dysfunction, which may exacerbate PAH. Notably, leptin can regulate growth differentiation factor-15 (GDF15), a pro-inflammatory, stress-induced cytokine and adipokine associated with PAH severity. Here, leptin effects on BMPR2 and GDF15 expression was investigated in human pulmonary artery endothelial cells (PAECs) with and without BMPR2 deficiency. Methods: Human PAECs were transfected with non-targeting siRNA or siRNA targeting BMPR2 for 48h, followed by incubation with vehicle or recombinant human leptin (10 ng/mL) for 24h. RNA and protein were harvested, and quantitative PCR and Western blotting were performed to determine the mRNA and protein expression of BMPR2, leptin receptor (LEPR), and GDF15. Secreted GDF15 was measured in cell culture supernatants by ELISA. Samples have been collected for genome-wide expression profiling to determine impact of leptin treatment in the presence and absence of BMPR2 silencing. Results: Human PAECs were found to express a functional LEPR, but as expected, not leptin. Leptin treatment induced LEPR mRNA expression and upregulated BMPR2 mRNA and protein expression. These effects were reduced in LEPR-silenced cells. Additionally, BMPR2 silencing was partially rescued by leptin exposure, suggesting that leptin signaling might exert beneficial effects on plexogenic pulmonary arteriopathy. BMPR2 loss alone significantly induced GDF15 expression and secretion, which was suppressed by leptin treatment. In contrast, leptin induced GDF15 in control PAECs, somewhat supporting the notion that leptin is harmful to vascular homeostasis. Recombinant human GDF15 decreased BMPR2 and LEPR mRNA and protein expression. Silencing GDF15 had no significant effect on BMPR2 but significantly upregulated LEPR expression. Conclusion: Leptin, acting via the LEPR, upregulates BMPR2 and suppresses GDF15 in a BMPR2 loss model of PAH. However, leptin suppression of the stress-induced cytokine/adipokine GDF15 was context-dependent and only seen in the setting of BMPR2 deficiency.

Background High levels of catecholamines are cardiotoxic and associated with stress‐induced cardiomyopathies. Using a septic shock model that reproduces the reversible cardiomyopathy seen over 10 days associated with human septic shock, we investigated the effects of catecholamines on microcirculatory perfusion and cardiac dysfunction. Methods and Results Purpose‐bred beagles received intrabronchial Staphylococcus aureus (n=30) or saline (n=6). The septic animals were than randomized to epinephrine (1 μg/kg per minute, n=15) or saline (n=15) infusions from 4 to 44 hours. Serial cardiac magnetic resonance imaging, catecholamine levels, and troponins were collected over 92 hours. Serial adenosine‐stress perfusion cardiac magnetic resonance imaging was performed on septic animals randomized to receive saline (n=8 out of 15) or epinephrine (n=8 out of 15). High‐dose sedation was given to suppress endogenous catecholamine release. Despite catecholamine levels largely remaining within the normal range throughout, by 48 hours, septic animals receiving saline versus nonseptic animals still developed significant worsening of left ventricular ejection fraction, circumferential strain, and ventricular‐aortic coupling. In septic animals that received epinephrine versus saline infusions, plasma epinephrine levels increased 800‐fold, but epinephrine produced no significant further worsening of left ventricular ejection fraction, circumferential strain, or ventricular‐aortic coupling. Septic animals receiving saline had a significant increase in microcirculatory reserve without troponin elevations. Septic animals receiving epinephrine had decreased edema, blunted microcirculatory perfusion, and elevated troponin levels that persisted for hours after the epinephrine infusion stopped. Conclusions Cardiac dysfunction during sepsis is not primarily due to elevated endogenous or exogenous catecholamines nor due to decreased microvascular perfusion‐induced ischemia. However, epinephrine itself has potentially harmful long‐lasting ischemic effects during sepsis including impaired cardiac microvascular perfusion that persists after stopping the infusion.

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, activates NOTCH1 signaling and plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in PAECs activated AKT and decreased DLL4 expression. DLL4 loss was also seen in lungs of patients with IPAH and HPAH. Over-expression of DLL4 in PAECs induced BMPR2 promoter activity and exogenous DLL4 increased BMPR2 mRNA through NOTCH1 activation. Furthermore, DLL4/NOTCH1 signaling blocked AKT activation, decreased proliferation and reversed EndoMT in BMPR2-silenced PAECs and ECs from IPAH patients. PPARγ, suppressed by BMPR2 loss, was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH PAECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Finally, leniolisib, a well-tolerated oral PI3Kδ/AKT inhibitor, decreased cell proliferation, induced apoptosis and reversed markers of EndoMT in BMPR2-silenced PAECs. Restoring DLL4/NOTCH1/PPARγ signaling and/or suppressing AKT activation may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.

ABSTRACT BACKGROUND In hypoxic and pseudohypoxic rodent models of pulmonary arterial hypertension (PAH), hypoxia-inducible factor (HIF) inhibition reduces disease severity. However, HIF activation alone, due to genetic alterations or use of inhibitors of prolyl hydroxylase domain (PHD) enzymes, has not been definitively shown to cause PAH in humans, indicating the involvement of other mechanisms. METHODS Pseudohypoxia was investigated in primary human lung endothelial cells by silencing PHD2, and in Tie2-Cre / Phd2 knockout mice, a rodent model of PAH. Lung vascular endothelial cells from PAH patients, and lung tissue from both SU5416/hypoxia PAH rats and PAH patients, were examined for validation. RESULTS PHD2 silencing or inhibition, while activating HIF2α, induces apoptosis-resistance, hypo-proliferation, and IFN/STAT activation in endothelial cells, independent of HIF signaling. Mechanistically, PHD2 deficiency activates AKT and ERK, inhibits JNK, and reduces AIP1 (ASK1-interacting protein 1), all independent of HIF2α. Like PHD2, AIP1 silencing affects these same kinase pathways and produces a similar dysfunctional endothelial cell phenotype, which can be partially reversed by AKT inhibition. These findings are corroborated in lung tissues of rodent PAH models and pulmonary vascular endothelial cells and tissues from PAH patients. CONCLUSIONS PHD2 deficiency in lung vascular endothelial cells induces an apoptosis-resistant, inflammatory, and hypo-proliferative phenotype. AKT activation and AIP1 loss, but not HIF signaling, drive these aberrant phenotypic changes. Our study suggests that HIF blockade alone may not suffice for PAH therapy; targeting PHD2, AKT, and AIP1 has the potential for developing more effective treatment. GRAPHIC ABSTRACT Highlights PHD2 silencing in human lung vascular endothelial cells suppresses apoptosis, inhibits proliferation, and activates STAT signaling, effects that persist despite HIF2α inhibition or knockdown. PHD2 silencing activates AKT and ERK, inhibits JNK, and decreases AIP1, all independently of HIF2α Like PHD2, AIP1 silencing led to similar alterations in kinase signaling and endothelial cell phenotypes, which are partially reversed by ATK inhibition. These in vitro findings align with observations in lung vascular endothelial cells and tissues from rodent models of PAH as well as PAH patients.

Background: BMPR2 gene silencing in primary human pulmonary artery endothelial cells (PAECs) produces a proliferative, hypermigratory PAH-like cellular phenotype. Genome-wide expression profiling in BMPR2 -silenced cells uncovered CD44 , a cancer stem cell marker associated with tumor progression and metastasis, among the top upregulated transcripts. Therapeutic strategies that block CD44 or reduce its expression are currently in various stages of development for cancer. Thus, mechanistic studies investigating the contribution of CD44 to vascular remodeling in PAH may reveal new therapeutic targets. Hypothesis: Upregulation of CD44 contributes to the abnormal phenotype of BMPR2-deficient PAECs and thus may contribute to vascular remodeling in PAH. Aims: Determine the effect of CD44 -silencing on gene expression and cell proliferation in BMPR2-deficient PAECs. Methods: Commercially available primary, human PAECs were transfected with scrambled control (siCTRL) or gene specific siRNA(s). PAH patient-derived (N=24) and failed donor-derived (N=11) PAECs were obtained from the PHBI. mRNA and protein expression were determined by quantitative RT-PCR and Western blotting, respectively. Cell proliferation was assessed by BrdU incorporation 96h after siRNA transfection. Data was analyzed using t-tests or ANOVA with post hoc pairwise comparisons. Results: Consistent with our previous findings, CD44 protein expression was increased 4-fold in BMPR2 -silenced PAECs 48h following siRNA transfection (P<0.0001 vs siCTRL; N=10 unique donors). Genes involved in endothelial-mesenchymal transition and cell proliferation were also increased in BMPR2 -silenced PAECs including HMGA1 , ID1 , SNAI1, and SNAI2 (P<0.01 for all vs siCTRL; N=5 independent experiments) and co-silencing CD44 attenuated their upregulation (P<0.05 for all vs siBMPR2 alone). Importantly, CD44 knockdown reduced PAEC proliferation in the absence (P=0.01 vs siCTRL) and presence of BMPR2 deficiency (P=0.002 vs siBMPR2 alone). Like BMPR2 -silenced PAECs, CD44 mRNA levels were higher in PAH patient-derived versus failed donor control PAECs (P=0.07). Conclusions: CD44 knockdown corrected aberrant gene expression associated with endothelial-mesenchymal transition and reduced cellular proliferation in BMPR2-deficient PAECs. Thus, endothelial CD44 upregulation may contribute to pathologic vascular remodeling in PAH and represents an unexplored therapeutic target.

Background:Septic shock, in humans and in our well-established animal model, is associated with increases in biventricular end diastolic volume (EDV) and decreases in ejection fraction (EF). These abnormalities occur over 2 days and reverse within 10 days. Septic non-survivors do not develop an increase in EDV. The mechanism for this cardiac dysfunction and EDV differences is unknown. Methods:Purpose-bred beagles randomized to receive intrabronchial Staphylococcus aureus (n=27) or saline (n=6) were provided standard ICU care including sedation, mechanical ventilation, and fluid resuscitation to a pulmonary arterial occlusion pressure of over 10mmHg. No catecholamines were administered. Over 96h, cardiac magnetic resonance imaging, echocardiograms, and invasive hemodynamics were serially performed, and laboratory data was collected. Tissue was obtained at 66h from six septic animals. Results:From 0-96h after bacterial challenge, septic animals vs. controls had significantly increased left ventricular wall edema (6%) and wall thinning with loss of mass (15%) which was more pronounced at 48h in non-survivors than survivors. On histology, edema was located predominantly in myocytes, the interstitium, and endothelial cells. Edema was associated with significantly worse biventricular function (lower EFs), ventricular-arterial coupling, and circumferential strain. In septic animals, from 0-24h, the EDV decreased from baseline and, despite cardiac filling pressures being similar, decreased significantly more in non-survivors. From 24-48h, all septic animals had increases in biventricular chamber sizes. Survivors biventricular EDVs were significantly greater than baseline and in non-survivors, where biventricular EDVs were not different from baseline. Preload, afterload, or HR differences did not explain these differential serial changes in chamber size. Conclusion:Systolic and diastolic cardiac dysfunction during sepsis is associated with ventricular wall edema. Rather than differences in preload, afterload, or heart rate, structural alterations to the ventricular wall best account for the volume changes associated with outcome during sepsis. In non-survivors, from 0-24h, sepsis induces a more severe diastolic dysfunction, further decreasing chamber size. The loss of left ventricular mass with wall thinning in septic survivors may, in part explain, the EDV increases from 24-48h. However, these changes continued and even accelerated into the recovery phase consistent with a reparative process rather than ongoing injury.

Background Septic shock is associated with increases in end‐diastolic volume (EDV) and decreases in ejection fraction that reverse within 10 days. Nonsurvivors do not develop EDV increases. The mechanism is unknown. Methods and Results Purpose‐bred beagles (n=33) were randomized to receive intrabronchial Staphylococcus aureus or saline. Over 96 hours, cardiac magnetic resonance imaging and echocardiograms were performed. Tissue was obtained at 66 hours. From 0 to 96 hours after bacterial challenge, septic animals versus controls had significantly increased left ventricular wall edema (6%) and wall thinning with loss of mass (15%). On histology, the major finding was nonocclusive microvascular injury with edema in myocytes, the interstitium, and endothelial cells. Edema was associated with significant worsening of biventricular ejection fractions, ventricular‐arterial coupling, and circumferential strain. Early during sepsis, (0–24 hours), the EDV decreased; significantly more in nonsurvivors (ie, greater diastolic dysfunction). From 24 to 48 hours, septic animals' biventricular chamber sizes increased; in survivors significantly greater than baseline and nonsurvivors, whose EDVs were not different from baseline. Preload, afterload, or heart rate differences did not explain these differential changes. Conclusions The cardiac dysfunction of sepsis is associated with wall edema. In nonsurvivors, at 0 to 24 hours, sepsis induces a more severe diastolic dysfunction, further decreasing chamber size. The loss of left ventricular mass with wall thinning in septic survivors may, in part, explain the EDV increases from 24 to 48 hours because of a potentially reparative process removing damaged wall tissue. Septic cardiomyopathy is most consistent with a nonocclusive microvascular injury resulting in edema causing reversible systolic and diastolic dysfunction with more severe diastolic dysfunction being associated with a decreased EDV and death.

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by pathologic vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in pulmonary artery endothelial cells (PAECs) activated AKT and suppressed the expression of DLL4. Consistent with these in vitro findings, increased AKT activation and reduced DLL4 expression was found in the small pulmonary arteries of patients with PAH. Increased NOTCH1 activation through exogenous DLL4 blocked AKT activation, decreased proliferation and reversed EndoMT. Exogenous and overexpression of DLL4 induced BMPR2 and PPRE promoter activity, and BMPR2 and PPARG mRNA in idiopathic PAH (IPAH) ECs. PPARγ, a nuclear receptor associated with EC homeostasis, suppressed by BMPR2 loss was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH ECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Directly blocking AKT or restoring DLL4/NOTCH1/PPARγ signaling may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.

BACKGROUND:Comparative effectiveness research is meant to determine which commonly employed medical interventions are most beneficial, least harmful, and/or most costly in a real-world setting. While the objectives for comparative effectiveness research are clear, the field has failed to develop either a uniform definition of comparative effectiveness research or an appropriate set of recommendations to provide standards for the design of critical care comparative effectiveness research trials, spurring controversy in recent years. The insertion of non-representative control and/or comparator arm subjects into critical care comparative effectiveness research trials can threaten trial subjects' safety. Nonetheless, the broader scientific community does not always appreciate the importance of defining and maintaining critical care practices during a trial, especially when vulnerable, critically ill populations are studied. Consequently, critical care comparative effectiveness research trials sometimes lack properly constructed control or active comparator arms altogether and/or suffer from the inclusion of "unusual critical care" that may adversely affect groups enrolled in one or more arms. This oversight has led to critical care comparative effectiveness research trial designs that impair informed consent, confound interpretation of trial results, and increase the risk of harm for trial participants.METHODS/EXAMPLES:We propose a novel approach to performing critical care comparative effectiveness research trials that mandates the documentation of critical care practices prior to trial initiation. We also classify the most common types of critical care comparative effectiveness research trials, as well as the most frequent errors in trial design. We present examples of these design flaws drawn from past and recently published trials as well as examples of trials that avoided those errors. Finally, we summarize strategies employed successfully in well-designed trials, in hopes of suggesting a comprehensive standard for the field.CONCLUSION:Flawed critical care comparative effectiveness research trial designs can lead to unsound trial conclusions, compromise informed consent, and increase risks to research subjects, undermining the major goal of comparative effectiveness research: to inform current practice. Well-constructed control and comparator arms comprise indispensable elements of critical care comparative effectiveness research trials, key to improving the trials' safety and to generating trial results likely to improve patient outcomes in clinical practice.

NR2F2 is expressed in endothelial cells (ECs) and Nr2f2 knockout produces lethal cardiovascular defects. In humans, reduced NR2F2 expression is associated with cardiovascular diseases including congenital heart disease and atherosclerosis. Here, NR2F2 silencing in human primary ECs led to inflammation, endothelial-to-mesenchymal transition (EndMT), proliferation, hypermigration, apoptosis-resistance, and increased production of reactive oxygen species. These changes were associated with STAT and AKT activation along with increased production of DKK1. Co-silencing DKK1 and NR2F2 prevented NR2F2-loss-induced STAT and AKT activation and reversed EndMT. Serum DKK1 concentrations were elevated in patients with pulmonary arterial hypertension (PAH) and DKK1 was secreted by ECs in response to in vitro loss of either BMPR2 or CAV1, which are genetic defects associated with the development of PAH. In human primary ECs, NR2F2 suppressed DKK1, whereas its loss conversely induced DKK1 and disrupted endothelial homeostasis, promoting phenotypic abnormalities associated with pathologic vascular remodeling. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating chronic vascular diseases associated with EC dysfunction. NEW & NOTEWORTHY NR2F2 loss in the endothelial lining of blood vessels is associated with cardiovascular disease. Here, NR2F2-silenced human endothelial cells were inflammatory, proliferative, hypermigratory, and apoptosis-resistant with increased oxidant stress and endothelial-to-mesenchymal transition. DKK1 was induced in NR2F2-silenced endothelial cells, while co-silencing NR2F2 and DKK1 prevented NR2F2-loss-associated abnormalities in endothelial signaling and phenotype. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating vascular diseases associated with endothelial dysfunction.