Challenges in interpreting cytokine data in COVID-19 affect patient care and management.

Challenges in using cytokine data are limiting Coronavirus Disease 2019 (COVID-19) patient management and comparison among different disease contexts. We suggest mitigation strategies to improve the accuracy of cytokine data, as we learn from experience gained during the COVID-19 pandemic.

Liver injury in COVID-19 and IL-6 trans-signaling-induced endotheliopathy.

BACKGROUND AND AIMS: COVID-19 is associated with liver injury and elevated interleukin-6 (IL-6). We hypothesized that IL-6 trans-signaling in liver sinusoidal endothelial cells (LSECs) leads to endotheliopathy (a proinflammatory and procoagulant state) and liver injury in COVID-19. METHODS: Coagulopathy, endotheliopathy, and alanine aminotransferase (ALT) were retrospectively analyzed in a subset (n = 68), followed by a larger cohort (n = 3,780) of patients with COVID-19. Liver histology from 43 patients with COVID-19 was analyzed for endotheliopathy and its relationship to liver injury. Primary human LSECs were used to establish the IL-6 trans-signaling mechanism. RESULTS: Factor VIII, fibrinogen, D-dimer, von Willebrand factor (vWF) activity/antigen (biomarkers of coagulopathy/endotheliopathy) were significantly elevated in patients with COVID-19 and liver injury (elevated ALT). IL-6 positively correlated with vWF antigen (p = 0.02), factor VIII activity (p = 0.02), and D-dimer (p <0.0001). On liver histology, patients with COVID-19 and elevated ALT had significantly increased vWF and platelet staining, supporting a link between liver injury, coagulopathy, and endotheliopathy. Intralobular neutrophils positively correlated with platelet (p <0.0001) and vWF (p <0.01) staining, and IL-6 levels positively correlated with vWF staining (p <0.01). IL-6 trans-signaling leads to increased expression of procoagulant (factor VIII, vWF) and proinflammatory factors, increased cell surface vWF (p <0.01), and increased platelet attachment in LSECs. These effects were blocked by soluble glycoprotein 130 (IL-6 trans-signaling inhibitor), the JAK inhibitor ruxolitinib, and STAT1/3 small-interfering RNA knockdown. Hepatocyte fibrinogen expression was increased by the supernatant of LSECs subjected to IL-6 trans-signaling. CONCLUSION: IL-6 trans-signaling drives the coagulopathy and hepatic endotheliopathy associated with COVID-19 and could be a possible mechanism behind liver injury in these patients. LAY SUMMARY: Patients with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection often have liver injury, but why this occurs remains unknown. High levels of interleukin-6 (IL-6) and its circulating receptor, which form a complex to induce inflammatory signals, have been observed in patients with COVID-19. This paper demonstrates that the IL-6 signaling complex causes harmful changes to liver sinusoidal endothelial cells and may promote blood clotting and contribute to liver injury.

Intermediate-dose anticoagulation, aspirin, and in-hospital mortality in COVID-19: A propensity score-matched analysis.

Thrombotic complications occur at high rates in hospitalized patients with COVID-19, yet the impact of intensive antithrombotic therapy on mortality is uncertain. We examined in-hospital mortality with intermediate- compared to prophylactic-dose anticoagulation, and separately with in-hospital aspirin compared to no antiplatelet therapy, in a large, retrospective study of 2785 hospitalized adult COVID-19 patients. In this analysis, we established two separate, nested cohorts of patients (a) who received intermediate- or prophylactic-dose anticoagulation ("anticoagulation cohort", N = 1624), or (b) who were not on home antiplatelet therapy and received either in-hospital aspirin or no antiplatelet therapy ("aspirin cohort", N = 1956). To minimize bias and adjust for confounding factors, we incorporated propensity score matching and multivariable regression utilizing various markers of illness severity and other patient-specific covariates, yielding treatment groups with well-balanced covariates in each cohort. The primary outcome was cumulative incidence of in-hospital death. Among propensity score-matched patients in the anticoagulation cohort (N = 382), in a multivariable regression model, intermediate- compared to prophylactic-dose anticoagulation was associated with a significantly lower cumulative incidence of in-hospital death (hazard ratio 0.518 [0.308-0.872]). Among propensity-score matched patients in the aspirin cohort (N = 638), in a multivariable regression model, in-hospital aspirin compared to no antiplatelet therapy was associated with a significantly lower cumulative incidence of in-hospital death (hazard ratio 0.522 [0.336-0.812]). In this propensity score-matched, observational study of COVID-19, intermediate-dose anticoagulation and aspirin were each associated with a lower cumulative incidence of in-hospital death.

Chronic Thromboembolic Pulmonary Hypertension: the Bedside.

PURPOSE OF REVIEW: Chronic thromboembolic pulmonary hypertension (CTEPH), included in group 4 PH, is an uncommon complication of acute pulmonary embolism (PE), in which emboli in the pulmonary vasculature do not resolve but rather form into an organized scar-like obstruction which can result in right ventricular (RV) failure. Here we provide an overview of current diagnosis and management of CTEPH. RECENT FINDINGS: CTEPH management is complex with treatments that range from surgery, percutaneous interventions, to medical therapies. Current CTEPH medical therapies have largely been repurposed from pulmonary arterial hypertension (PAH). The diagnosis of CTEPH can be challenging, requiring a multimodality approach to differentiate from disease mimics. While these treatments improve symptoms, they may not reverse the underlying pathology of CTEPH.

Chronic Thromboembolic Pulmonary Hypertension: the Bench.

PURPOSE OF REVIEW: Chronic thromboembolic pulmonary hypertension (CTEPH) is an uncommon complication of acute pulmonary embolism (PE), in which the red, platelet-rich thrombus does not resolve but forms into an organized yellow, fibrotic scar-like obstruction in the pulmonary vasculature. Here we review the pathobiology of CTEPH. RECENT FINDINGS: Our current knowledge has predominantly been informed by studies of human samples and animal models that are inherently limited in their ability to recapitulate all aspects of the disease. These studies have identified alterations in platelet biology and inflammation in the formation of a scar-like thrombus that comprised endothelial cells, myofibroblasts, and immune cells, along with a small vessel pulmonary arterial hypertension-like vasculopathy. The development of CTEPH-specific therapies is currently hindered by a limited knowledge of its pathobiology. The development of new CTEPH medical therapies will require new insights into its pathobiology that bridge the gap from bench to bedside.

Modulation of Endothelial Bone Morphogenetic Protein Receptor Type 2 Activity by Vascular Endothelial Growth Factor Receptor 3 in Pulmonary Arterial Hypertension.

BACKGROUND: Bone morphogenetic protein (BMP) signaling has multiple roles in the development and function of the blood vessels. In humans, mutations in BMP receptor type 2 (BMPR2), a key component of BMP signaling, have been identified in the majority of patients with familial pulmonary arterial hypertension (PAH). However, only a small subset of individuals with BMPR2 mutation develops PAH, suggesting that additional modifiers of BMPR2 function play an important role in the onset and progression of PAH. METHODS: We used a combination of studies in zebrafish embryos and genetically engineered mice lacking endothelial expression of Vegfr3 to determine the interaction between vascular endothelial growth factor receptor 3 (VEGFR3) and BMPR2. Additional in vitro studies were performed by using human endothelial cells, including primary lung endothelial cells from subjects with PAH. RESULTS: Attenuation of Vegfr3 in zebrafish embryos abrogated Bmp2b-induced ectopic angiogenesis. Endothelial cells with disrupted VEGFR3 expression failed to respond to exogenous BMP stimulation. Mechanistically, VEGFR3 is physically associated with BMPR2 and facilitates ligand-induced endocytosis of BMPR2 to promote phosphorylation of SMADs and transcription of ID genes. Conditional, endothelial-specific deletion of Vegfr3 in mice resulted in impaired BMP signaling responses, and significantly worsened hypoxia-induced pulmonary hypertension. Consistent with these data, we found significant decrease in VEGFR3 expression in pulmonary arterial endothelial cells from human PAH subjects, and reconstitution of VEGFR3 expression in PAH pulmonary arterial endothelial cells restored BMP signaling responses. CONCLUSIONS: Our findings identify VEGFR3 as a key regulator of endothelial BMPR2 signaling and a potential determinant of PAH penetrance in humans.

Rac2 Modulates Atherosclerotic Calcification by Regulating Macrophage Interleukin-1ß Production.

OBJECTIVE: The calcium composition of atherosclerotic plaque is thought to be associated with increased risk for cardiovascular events, but whether plaque calcium itself is predictive of worsening clinical outcomes remains highly controversial. Inflammation is likely a key mediator of vascular calcification, but immune signaling mechanisms that promote this process are minimally understood. APPROACH AND RESULTS: Here, we identify Rac2 as a major inflammatory regulator of signaling that directs plaque osteogenesis. In experimental atherogenesis, Rac2 prevented progressive calcification through its suppression of Rac1-dependent macrophage interleukin-1ß (IL-1ß) expression, which in turn is a key driver of vascular smooth muscle cell calcium deposition by its ability to promote osteogenic transcriptional programs. Calcified coronary arteries from patients revealed decreased Rac2 expression but increased IL-1ß expression, and high coronary calcium burden in patients with coronary artery disease was associated with significantly increased serum IL-1ß levels. Moreover, we found that elevated IL-1ß was an independent predictor of cardiovascular death in those subjects with high coronary calcium burden. CONCLUSIONS: Overall, these studies identify a novel Rac2-mediated regulation of macrophage IL-1ß expression, which has the potential to serve as a powerful biomarker and therapeutic target for atherosclerosis.

Enhancing Insights into Pulmonary Vascular Disease through a Precision Medicine Approach. A Joint NHLBI-Cardiovascular Medical Research and Education Fund Workshop Report.

The Division of Lung Diseases of the NHLBI and the Cardiovascular Medical Education and Research Fund held a workshop to discuss how to leverage the anticipated scientific output from the recently launched "Redefining Pulmonary Hypertension through Pulmonary Vascular Disease Phenomics" (PVDOMICS) program to develop newer approaches to pulmonary vascular disease. PVDOMICS is a collaborative, protocol-driven network to analyze all patient populations with pulmonary hypertension to define novel pulmonary vascular disease (PVD) phenotypes. Stakeholders, including basic, translational, and clinical investigators; clinicians; patient advocacy organizations; regulatory agencies; and pharmaceutical industry experts, joined to discuss the application of precision medicine to PVD clinical trials. Recommendations were generated for discussion of research priorities in line with NHLBI Strategic Vision Goals that include: (1) A national effort, involving all the stakeholders, should seek to coordinate biosamples and biodata from all funded programs to a web-based repository so that information can be shared and correlated with other research projects. Example programs sponsored by NHLBI include PVDOMICS, Pulmonary Hypertension Breakthrough Initiative, the National Biological Sample and Data Repository for PAH, and the National Precision Medicine Initiative. (2) A task force to develop a master clinical trials protocol for PVD to apply precision medicine principles to future clinical trials. Specific features include: (a) adoption of smaller clinical trials that incorporate biomarker-guided enrichment strategies, using adaptive and innovative statistical designs; and (b) development of newer endpoints that reflect well-defined and clinically meaningful changes. (3) Development of updated and systematic variables in imaging, hemodynamic, cellular, genomic, and metabolic tests that will help precisely identify individual and shared features of PVD and serve as the basis of novel phenotypes for therapeutic interventions.

Restoration of impaired endothelial myocyte enhancer factor 2 function rescues pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary arterioles, characterized by increased pulmonary arterial pressure and right ventricular failure. The cause of PAH is complex, but aberrant proliferation of the pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells is thought to play an important role in its pathogenesis. Understanding the mechanisms of transcriptional gene regulation involved in pulmonary vascular homeostasis can provide key insights into potential therapeutic strategies. METHODS AND RESULTS: We demonstrate that the activity of the transcription factor myocyte enhancer factor 2 (MEF2) is significantly impaired in the PAECs derived from subjects with PAH. We identified MEF2 as the key cis-acting factor that regulates expression of a number of transcriptional targets involved in pulmonary vascular homeostasis, including microRNAs 424 and 503, connexins 37, and 40, and Kruppel Like Factors 2 and 4, which were found to be significantly decreased in PAH PAECs. The impaired MEF2 activity in PAH PAECs was mediated by excess nuclear accumulation of 2 class IIa histone deacetylases (HDACs) that inhibit its function, namely HDAC4 and HDAC5. Selective, pharmacological inhibition of class IIa HDACs led to restoration of MEF2 activity in PAECs, as demonstrated by increased expression of its transcriptional targets, decreased cell migration and proliferation, and rescue of experimental pulmonary hypertension models. CONCLUSIONS: Our results demonstrate that strategies to augment MEF2 activity hold potential therapeutic value in PAH. Moreover, we identify selective HDAC IIa inhibition as a viable alternative approach to avoid the potential adverse effects of broad spectrum HDAC inhibition in PAH.

Apelin-APJ signaling is a critical regulator of endothelial MEF2 activation in cardiovascular development.

RATIONALE: The peptide ligand apelin and its receptor APJ constitute a signaling pathway with numerous effects on the cardiovascular system, including cardiovascular development in model organisms such as xenopus and zebrafish. OBJECTIVE: This study aimed to characterize the embryonic lethal phenotype of the Apj-/- mice and to define the involved downstream signaling targets. METHODS AND RESULTS: We report the first characterization of the embryonic lethality of the Apj-/- mice. More than half of the expected Apj-/- embryos died in utero because of cardiovascular developmental defects. Those succumbing to early embryonic death had markedly deformed vasculature of the yolk sac and the embryo, as well as poorly looped hearts with aberrantly formed right ventricles and defective atrioventricular cushion formation. Apj-/- embryos surviving to later stages demonstrated incomplete vascular maturation because of a deficiency of vascular smooth muscle cells and impaired myocardial trabeculation and ventricular wall development. The molecular mechanism implicates a novel, noncanonical signaling pathway downstream of apelin-APJ involving Gα13, which induces histone deacetylase (HDAC) 4 and HDAC5 phosphorylation and cytoplasmic translocation, resulting in activation of myocyte enhancer factor 2. Apj-/- mice have greater endocardial Hdac4 and Hdac5 nuclear localization and reduced expression of the myocyte enhancer factor 2 (MEF2) transcriptional target Krüppel-like factor 2. We identify a number of commonly shared transcriptional targets among apelin-APJ, Gα13, and MEF2 in endothelial cells, which are significantly decreased in the Apj-/- embryos and endothelial cells. CONCLUSIONS: Our results demonstrate a novel role for apelin-APJ signaling as a potent regulator of endothelial MEF2 function in the developing cardiovascular system.

Essential role of Apelin signaling during lymphatic development in zebrafish.

OBJECTIVE: Apelin and its cognate receptor Aplnr/Apj are essential for diverse biological processes. However, the function of Apelin signaling in lymphatic development remains to be identified, despite the preferential expression of Apelin and Aplnr within developing blood and lymphatic endothelial cells in vertebrates. In this report, we aim to delineate the functions of Apelin signaling during lymphatic development. APPROACH AND RESULTS: We investigated the functions of Apelin signaling during lymphatic development using zebrafish embryos and found that attenuation of Apelin signaling substantially decreased the formation of the parachordal vessel and the number of lymphatic endothelial cells within the developing thoracic duct, indicating an essential role of Apelin signaling during the early phase of lymphatic development. Mechanistically, we found that abrogation of Apelin signaling selectively attenuates lymphatic endothelial serine-threonine kinase Akt 1/2 phosphorylation without affecting the phosphorylation status of extracellular signal-regulated kinase 1/2. Moreover, lymphatic abnormalities caused by the reduction of Apelin signaling were significantly exacerbated by the concomitant partial inhibition of serine-threonine kinase Akt/protein kinase B signaling. Apelin and vascular endothelial growth factor-C (VEGF-C) signaling provide a nonredundant activation of serine-threonine kinase Akt/protein kinase B during lymphatic development because overexpression of VEGF-C or apelin was unable to rescue the lymphatic defects caused by the lack of Apelin or VEGF-C, respectively. CONCLUSIONS: Taken together, our data present compelling evidence suggesting that Apelin signaling regulates lymphatic development by promoting serine-threonine kinase Akt/protein kinase B activity in a VEGF-C/VEGF receptor 3-independent manner during zebrafish embryogenesis.

Evaluation of the in vitro and in vivo angiogenic effects of exendin-4.

Exendin-4, an analog of glucagon-like peptide (GLP)-1, has beneficial effects on cardiovascular disease induced by diabetes mellitus (DM). Recently, exendin-4 was reported to induce the proliferation of endothelial cells. However, its angiogenic effect on endothelial cells has not been clearly evaluated. Therefore, we investigated the effects of exendin-4 on the angiogenic process with respect to migration, sprouting, and neovascularization using in vitro and in vivo assays. Treatment with exendin-4 increased the migration of human umbilical vein endothelial cells (HUVECs) in in vitro scratch wound assays, as well as the number of lumenized vessels sprouting from HUVECs in in vitro 3D bead assays. These responses were abolished by co-treatment with exendin (9-39), a GLP-1 receptor antagonist, which suggests that exendin-4 regulates endothelial cell migration and tube formation in a GLP-1 receptor-dependent manner. In an ex vivo assay, treatment of aortic rings with exendin-4 increased the sprouting of endothelial cells. Exendin-4 also significantly increased the number of new vessels and induced blood flow in Matrigel plugs in in vivo assays. Our results provide clear evidence for the angiogenic effect of exendin-4 in in vitro and in vivo assays and provide a mechanism underlying the cardioprotective effects of exendin-4.

Apelin/APJ signaling is a critical regulator of statin effects in vascular endothelial cells--brief report.

OBJECTIVE: The endothelial response elicited by the G-protein-coupled receptor pathway involving apelin and APJ predicts an overall vasoprotective effect. As a number of downstream endothelial targets of apelin/APJ signaling are also known to be targeted by statins (3-hydroxy-3-methyl-glutaryl [HMG]-CoA reductase inhibitors) as potential mediators of their known pleiotropic effects, we evaluated for the involvement of apelin/APJ signaling in statin endothelial effects. METHODS AND RESULTS: We found that disruption of apelin/APJ signaling in endothelial cells leads to significantly decreased expression of Kruppel-like factor 2, endothelial nitric oxide synthase, and thrombomodulin. We found that statin-mediated induction of Kruppel-like factor 2, endothelial nitric oxide synthase, and thrombomodulin expression, as well as inhibition of monocyte-endothelial adhesion, was abrogated by concurrent apelin knockdown. Moreover, we found that statins can transcriptionally regulate APJ in a Kruppel-like factor 2-dependent manner, demonstrating the presence of a positive-feedback loop. CONCLUSIONS: Our findings provide a novel mechanism by which the apelin/APJ pathway serves as a critical intermediary that links statin to its pleiotropic effects in regulating endothelial gene targets and function.

Severe obstructive sleep apnea increases mortality in patients with ischemic heart disease and myocardial injury.

OBJECTIVES: We hypothesized that obstructive sleep apnea(OSA) has a dose-dependent impact on mortality in those with ischemic heart disease or previous myocardial injury. METHODS: We performed a retrospective cohort study of 281 consecutive OSA patients with a history of myocardial injury as determined by elevated troponin levels or with known existing ischemic heart disease. We compared survival between those with severe OSA [apnea­hypopneaindex (AHI) ≥30] and those with mild to moderate OSA(AHI >5 and <30). RESULTS: Of the 281 patients (mean age 65 years, mean BMI34, 98% male, 58% with diabetes), 151 patients had mild moderate OSA and 130 had severe OSA. During a mean follow-up of 4.1 years, there were significantly greater deaths in the severe OSA group compared to the mild moderate OSA group [53 deaths (41%) vs. 44 deaths(29%), respectively, p00.04]. The adjusted hazard ratio for mortality with severe OSA was 1.72 (95% confidence interval1.01­2.91, p00.04). CONCLUSIONS: The severity of obstructive sleep apnea is associated with increased risk of death, and risk stratification based on OSA severity is relevant even in the diseased cardiac patient.

Proteomic profiling demonstrates inflammatory and endotheliopathy signatures associated with impaired cardiopulmonary exercise hemodynamic profile in Post Acute Sequelae of SARS-CoV-2 infection (PASC) syndrome.

Approximately 50% of patients who recover from the acute SARS-CoV-2 experience Post Acute Sequelae of SARS-CoV-2 infection (PASC) syndrome. The pathophysiological hallmark of PASC is characterized by impaired system oxygen extraction (EO2) on invasive cardiopulmonary exercise test (iCPET). However, the mechanistic insights into impaired EO2 remain unclear. We studied 21 consecutive iCPET in PASC patients with unexplained exertional intolerance. PASC patients were dichotomized into mildly reduced (EO2peak-mild) and severely reduced (EO2peak-severe) EO2 groups according to the median peak EO2 value. Proteomic profiling was performed on mixed venous blood plasma obtained at peak exercise during iCPET. PASC patients as a group exhibited depressed peak exercise aerobic capacity (peak VO2; 85 ± 18 vs. 131 ± 45% predicted; p = 0.0002) with normal systemic oxygen delivery, DO2 (37 ± 9 vs. 42 ± 15 mL/kg/min; p = 0.43) and reduced EO2 (0.4 ± 0.1 vs. 0.8 ± 0.1; p < 0.0001). PASC patients with EO2peak-mild exhibited greater DO2 compared to those with EO2peak-severe [42.9 (34.2-41.2) vs. 32.1 (26.8-38.0) mL/kg/min; p = 0.01]. The proteins with increased expression in the EO2peak-severe group were involved in inflammatory and fibrotic processes. In the EO2peak-mild group, proteins associated with oxidative phosphorylation and glycogen metabolism were elevated. In PASC patients with impaired EO2, there exist a spectrum of PASC phenotype related to differential aberrant protein expression and cardio-pulmonary physiologic response. PASC patients with EO2peak-severe exhibit a maladaptive physiologic and proteomic signature consistent with persistent inflammatory state and endothelial dysfunction, while in the EO2peak-mild group, there is enhanced expression of proteins involved in oxidative phosphorylation-mediated ATP synthesis along with an enhanced cardiopulmonary physiological response.

BMPR1A promotes ID2-ZEB1 interaction to suppress excessive endothelial to mesenchymal transition.

AIMS: Components of bone morphogenetic protein (BMP) signalling have been implicated in both pathogenesis of pulmonary arterial hypertension (PAH) and endothelial-mesenchymal transition (EndoMT). In particular, the importance of BMP type 2 receptor in these processes has been extensively analysed. However, the contribution of BMP type 1 receptors (BMPR1s) to the onset of PAH and EndoMT remains poorly understood. BMPR1A, one of BMPR1s, was recently implicated in the pathogenesis of PAH, and was found to be down-regulated in the lungs of PAH patients, neither the downstream mechanism nor its contribution to EndoMT has been described. Therefore, we aim to delineate the role of endothelial BMPR1A in modulating EndoMT and pathogenesis of PAH. METHODS AND RESULTS: We find that BMPR1A knockdown in endothelial cells (ECs) induces hallmarks of EndoMT, and deletion of endothelial Bmpr1a in adult mice (Bmpr1aiECKO) leads to development of PAH-like symptoms due to excessive EndoMT. By lineage tracing, we show that endothelial-derived smooth muscle cells are increased in endothelial Bmpr1a-deleted mice. Mechanistically, we identify ZEB1 as a primary target for BMPR1A in this setting; upon BMPR1A activation, ID2 physically interacts and sequesters ZEB1 to attenuate transcription of Tgfbr2, which in turn lowers the responses of ECs towards transforming growth factor beta (TGFß) stimulation and prevents excessive EndoMT. In Bmpr1aiECKO mice, administering endothelial targeting lipid nanoparticles containing siRNA against Tgfbr2 effectively ameliorate PAH, reiterating the importance of BMPR1A-ID2/ZEB1-TGFBR2 axis in modulating progression of EndoMT and pathogenesis of PAH. CONCLUSIONS: We demonstrate that BMPR1A is key to maintain endothelial identity and to prevent excessive EndoMT. We identify BMPR1A-induced interaction between ID2 and ZEB1 is the key regulatory step for onset of EndoMT and pathogenesis of PAH. Our findings indicate that BMPR1A-ID2/ZEB1-TGFBR2 signalling axis could serve as a potential novel therapeutic target for PAH and other EndoMT-related vascular disorders.

Disruption of the apelin-APJ system worsens hypoxia-induced pulmonary hypertension.

OBJECTIVE: The G-protein-coupled receptor APJ and its ligand apelin are highly expressed in the pulmonary vasculature, but their function in this vascular bed is unclear. We hypothesized that disruption of apelin signaling would lead to worsening of the vascular remodeling associated with pulmonary hypertension (PH). METHODS AND RESULTS: We found that apelin-null mice developed more severe PH compared with wild-type mice when exposed to chronic hypoxia. Micro-computed tomography of the pulmonary arteries demonstrated significant pruning of the microvasculature in the apelin-null mice. Apelin-null mice had a significant reduction of serum nitrate levels. This was secondary to downregulation of endothelial nitric oxide synthase (eNOS), which was associated with reduced expression of Kruppel-like factor 2 (KLF2), a known regulator of eNOS expression. In vitro knockdown studies targeting apelin in human pulmonary artery endothelial cells demonstrated decreased eNOS and KLF2 expression, as well as impaired phosphorylation of AMP-activated kinase and eNOS. Moreover, serum apelin levels of patients with PH were significantly lower than those of controls. CONCLUSIONS: These data demonstrate that disruption of apelin signaling can exacerbate PH mediated by decreased activation of AMP-activated kinase and eNOS, and they identify this pathway as a potentially important therapeutic target for treatment of this refractory human disease.