Myeloid Cell Derived IL1ß Contributes to Pulmonary Hypertension in HFpEF.

BACKGROUND: Pulmonary hypertension (PH) in heart failure with preserved ejection fraction (HFpEF) is a common and highly morbid syndrome, but mechanisms driving PH-HFpEF are poorly understood. We sought to determine whether a well-accepted murine model of HFpEF also displays features of PH, and we sought to identify pathways that might drive early remodeling of the pulmonary vasculature in HFpEF. METHODS: Eight-week-old male and female C57BL/6J mice received either Nγ-nitro-L-arginine methyl ester and high-fat diet or control water and diet for 2, 5, and 12 weeks. The db/db mice were studied as a second model of HFpEF. Early pathways regulating PH were identified by bulk and single-cell RNA sequencing. Findings were confirmed by immunostain in lungs of mice or lung slides from clinically performed autopsies of patients with PH-HFpEF. ELISA was used to verify IL-1ß (interleukin-1 beta) in mouse lung, mouse plasma, and also human plasma from patients with PH-HFpEF obtained at the time of right heart catheterization. Clodronate liposomes and an anti-IL-1ß antibody were utilized to deplete macrophages and IL-1ß, respectively, to assess their impact on pulmonary vascular remodeling in HFpEF in mouse models. RESULTS: Nγ-nitro-L-arginine methyl ester/high-fat diet-treated mice developed PH, small vessel muscularization, and right heart dysfunction. Inflammation-related gene ontologies were overrepresented in bulk RNA sequencing analysis of whole lungs, with an increase in CD68+ cells in both murine and human PH-HFpEF lungs. Cytokine profiling showed an increase in IL-1ß in mouse and human plasma. Finally, clodronate liposome treatment in mice prevented PH in Nγ-nitro-L-arginine methyl ester/high-fat diet-treated mice, and IL-1ß depletion also attenuated PH in Nγ-nitro-L-arginine methyl ester/high-fat diet-treated mice. CONCLUSIONS: We report a novel model for the study of PH and right heart remodeling in HFpEF, and we identify myeloid cell-derived IL-1ß as an important contributor to PH in HFpEF.

BMPR2 dysfunction impairs insulin signaling and glucose homeostasis in cardiomyocytes.

Insulin resistance and right ventricular (RV) dysfunction are associated with lipotoxicity in heritable forms of pulmonary arterial hypertension (PAH), commonly due to mutations in bone morphogenetic protein receptor type 2 (BMPR2). How BMPR2 dysfunction in cardiomyocytes alters glucose metabolism and the response of these cells to insulin are unknown. We hypothesized that BMPR2 mutation in cardiomyocytes alters glucose-supported mitochondrial respiration and impairs cellular responses to insulin, including glucose and lipid uptake. We performed metabolic assays, immunofluorescence and Western analysis, RNA profiling, and radioactive isotope uptake studies in H9c2 cardiomyocyte cell lines with and without patient-derived BMPR2 mutations (mutant cells), with and without insulin. Unlike control cells, BMPR2 mutant cardiomyocytes have reduced metabolic plasticity as indicated by reduced mitochondrial respiration with increased mitochondrial superoxide production. These mutant cells show enhanced baseline phosphorylation of insulin-signaling protein as indicated by increased Akt, AMPK, and acetyl-CoA carboxylase phosphorylation that may negatively influence fatty acid oxidation and enhance lipid uptake, and are insulin insensitive. Furthermore, mutant cells demonstrate an increase in milk fat globule-EGF factor-8 protein (MFGE8), which influences the insulin-signaling pathway by phosphorylating AktSer473 via phosphatidylinositol 3-kinase and mammalian target of rapamycin. In conclusion, BMPR2 mutant cardiomyocytes have reduced metabolic plasticity and fail to respond to glucose. These cells have enhanced baseline insulin-signaling pattern favoring insulin resistance with failure to augment this pattern in response to insulin. BMPR2 mutation possibly blunts glucose uptake and enhances lipid uptake in these cardiomyocytes. The MFGE8-driven signaling pathway may suggest a new mechanism underlying RV lipotoxicity in PAH.

Isolation and characterization of endothelial-to-mesenchymal transition cells in pulmonary arterial hypertension.

Endothelial-to-mesenchymal transition (EndMT) is a process in which endothelial cells lose polarity and cell-to cell contacts, and undergo a dramatic remodeling of the cytoskeleton. It has been implicated in initiation and progression of pulmonary arterial hypertension (PAH). However, the characteristics of cells which have undergone EndMT cells in vivo have not been reported and so remain unclear. To study this, sugen5416 and hypoxia (SuHx)-induced PAH was established in Cdh5-Cre/Gt(ROSA)26Sortm4(ACTB-tdTomato,EGFP)Luo/J double transgenic mice, in which GFP was stably expressed in pan-endothelial cells. After 3 wk of SuHx, flow cytometry and immunohistochemistry demonstrated CD144-negative and GFP-positive cells (complete EndMT cells) possessed higher proliferative and migratory activity compared with other mesenchymal cells. While CD144-positive and α-smooth muscle actin (α-SMA)-positive cells (partial EndMT cells) continued to express endothelial progenitor cell markers, complete EndMT cells were Sca-1-rich mesenchymal cells with high proliferative and migratory ability. When transferred in fibronectin-coated chamber slides containing smooth muscle media, α-SMA robustly expressed in these cells compared with cEndMT cells that were grown in maintenance media. Demonstrating additional paracrine effects, conditioned medium from isolated complete EndMT cells induced enhanced mesenchymal proliferation and migration and increased angiogenesis compared with conditioned medium from resident mesenchymal cells. Overall, these findings show that EndMT cells could contribute to the pathogenesis of PAH both directly, by transformation into smooth muscle-like cells with higher proliferative and migratory potency, and indirectly, through paracrine effects on vascular intimal and medial proliferation.

Mechanisms of Lipid Accumulation in the Bone Morphogenetic Protein Receptor Type 2 Mutant Right Ventricle.

RATIONALE: In heritable pulmonary arterial hypertension with germline mutation in the bone morphogenetic protein receptor type 2 (BMPR2) gene, right ventricle (RV) dysfunction is associated with RV lipotoxicity; however, the underlying mechanism for lipid accumulation is not known. OBJECTIVES: We hypothesized that lipid accumulation in cardiomyocytes with BMPR2 mutation occurs owing to alterations in lipid transport and impaired fatty acid oxidation (FAO), which is exacerbated by a high-lipid (Western) diet (WD). METHODS: We used a transgenic mouse model of pulmonary arterial hypertension with mutant BMPR2 and generated a cardiomyocyte cell line with BMPR2 mutation. Electron microscopy and metabolomic analysis were performed on mouse RVs. MEASUREMENTS AND MAIN RESULTS: By metabolomics analysis, we found an increase in long-chain fatty acids in BMPR2 mutant mouse RVs compared with controls, which correlated with cardiac index. BMPR2-mutant cardiomyocytes had increased lipid compared with controls. Direct measurement of FAO in the WD-fed BMPR2-mutant RV showed impaired palmitate-linked oxygen consumption, and metabolomics analysis showed reduced indices of FAO. Using both mutant BMPR2 mouse RVs and cardiomyocytes, we found an increase in the uptake of (14)C-palmitate and fatty acid transporter CD36 that was further exacerbated by WD. CONCLUSIONS: Taken together, our data suggest that impaired FAO and increased expression of the lipid transporter CD36 are key mechanisms underlying lipid deposition in the BMPR2-mutant RV, which are exacerbated in the presence of dietary lipids. These findings suggest important features leading to RV lipotoxicity in pulmonary arterial hypertension and may point to novel areas of therapeutic intervention.

Myeloid Cell Derived IL1ß Contributes to Pulmonary Vascular Remodeling in Heart Failure with Preserved Ejection Fraction.

Background: Pulmonary hypertension (PH) in heart failure with preserved ejection fraction (HFpEF) is a common and highly morbid syndrome, but mechanisms driving PH-HFpEF are not well understood. We sought to determine whether a well-accepted murine model of HFpEF also displays features of PH in HFpEF, and we sought to identify pathways that might drive early remodeling of the pulmonary vasculature in HFpEF. Methods: Eight week old male and female C57/BL6J mice were given either L-NAME and high fat diet (HFD) or control water/diet for 2,5, and 12 weeks. Bulk RNA sequencing and single cell RNA sequencing was performed to identify early and cell-specific pathways that might regulate pulmonary vascular remodeling in PH-HFpEF. Finally, clodronate liposome and IL1ß antibody treatments were utilized to deplete macrophages or IL1ß, respectively, to assess their impact on pulmonary vascular remodeling in HFpEF. Results: Mice given L-NAME/HFD developed PH, small vessel muscularization, and right heart dysfunction after 2 weeks of treatment. Inflammation-related gene ontologies were over-represented in bulk RNA sequencing analysis of whole lungs, with an increase in CD68+ cells in both murine and human PH-HFpEF lungs. Cytokine profiling of mouse lung and plasma showed an increase in IL1ß, which was confirmed in plasma from patients with HFpEF. Single cell sequencing of mouse lungs also showed an increase in M1-like, pro-inflammatory populations of Ccr2+ monocytes and macrophages, and transcript expression of IL1ß was primarily restricted to myeloid-type cells. Finally, clodronate liposome treatment prevented the development of PH in L-NAME/HFD treated mice, and IL1ß antibody treatment also attenuated PH in L-NAME/HFD treated mice. Conclusions: Our study demonstrated that a well-accepted model of HFpEF recapitulates features of pulmonary vascular remodeling commonly seen in patients with HFpEF, and we identified myeloid cell derived IL1ß as an important contributor to PH in HFpEF.

A multifaceted investigation into molecular associations of chronic thromboembolic pulmonary hypertension pathogenesis.

PURPOSE: Chronic thromboembolic pulmonary hypertension is characterized by incomplete thrombus resolution following acute pulmonary embolism, leading to pulmonary hypertension and right ventricular dysfunction. Conditions such as thrombophilias, dysfibrinogenemias, and inflammatory states have been associated with chronic thromboembolic pulmonary hypertension, but molecular mechanisms underlying this disease are poorly understood. We sought to characterize the molecular and functional features associated with chronic thromboembolic pulmonary hypertension using a multifaceted approach. METHODS: We utilized functional assays to compare clot lysis times between chronic thromboembolic pulmonary hypertension patients and multiple controls. We then performed immunohistochemical characterization of tissue from chronic thromboembolic pulmonary hypertension, pulmonary arterial hypertension, and healthy controls, and examined RNA expression patterns of cultured lymphocytes and pulmonary arterial specimens. We then confirmed RNA expression changes using immunohistochemistry, immunofluorescence, and Western blotting in pulmonary arterial tissue. RESULTS: Clot lysis times in chronic thromboembolic pulmonary hypertension patients are similar to multiple controls. Chronic thromboembolic pulmonary hypertension endarterectomized tissue has reduced expression of both smooth muscle and endothelial cell markers. RNA expression profiles in pulmonary arteries and peripheral blood lymphocytes identified differences in RNA transcript levels related to inflammation and growth factor signaling, which we confirmed using immunohistochemistry. Gene expression data also suggested significant alterations in metabolic pathways, and immunofluorescence and Western blot experiments confirmed that unglycosylated CD36 and adiponectin expression were increased in chronic thromboembolic pulmonary hypertension versus controls. CONCLUSIONS: Our data do not support impaired clot lysis underlying chronic thromboembolic pulmonary hypertension, but did demonstrate distinct molecular patterns present both in peripheral blood and in pathologic specimens of chronic thromboembolic pulmonary hypertension patients suggesting that altered metabolism may play a role in chronic thromboembolic pulmonary hypertension pathogenesis.

Whole exome sequencing to identify a novel gene (caveolin-1) associated with human pulmonary arterial hypertension.

BACKGROUND: Heritable and idiopathic pulmonary arterial hypertension (PAH) are phenotypically identical and associated with mutations in several genes related to transforming growth factor (TGF) beta signaling, including bone morphogenetic protein receptor type 2, activin receptor-like kinase 1, endoglin, and mothers against decapentaplegic 9. Approximately 25% of heritable cases lack identifiable mutations in any of these genes. METHODS AND RESULTS: We used whole exome sequencing to study a 3-generation family with multiple affected family members with PAH, but no identifiable TGF beta mutation. We identified a frameshift mutation in caveolin-1 (CAV1), which encodes a membrane protein of caveolae abundant in the endothelium and other cells of the lung. An independent de novo frameshift mutation was identified in a child with idiopathic PAH. Western blot analysis demonstrated a reduction in caveolin-1 protein, while lung tissue immunostaining studies demonstrated a reduction in normal caveolin-1 density within the endothelial cell layer of small arteries. CONCLUSIONS: Our study represents successful elucidation of a dominant Mendelian disorder using whole exome sequencing. Mutations in CAV1 are associated in rare cases with PAH. This may have important implications for pulmonary vascular biology, as well as PAH-directed therapeutic development.