Single-Cell and Spatial Transcriptomics Identified Fatty Acid-Binding Proteins Controlling Endothelial Glycolytic and Arterial Programming in Pulmonary Hypertension

Abstract

Background: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure and premature death. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for patients with PAH. Recent studies showed that FABP (fatty acid-binding protein) 4 and FABP5 are expressed in endothelial cells (ECs) across multiple tissues, and circulating FABP4 level is elevated in patients with PAH. However, the role of endothelial FABP4/5 in the pathogenesis of PAH remains undetermined.

Methods: FABP4/5 expression was examined in pulmonary arterial ECs and lung tissues from patients with idiopathic PAH and pulmonary hypertension (PH) rat models. Plasma proteome analysis was performed in human PAH samples. Echocardiography, hemodynamics, histology, and immunostaining were performed to evaluate the lung and heart PH phenotypes in Egln1^Tie2Cre (CKO) mice and Egln1^Tie2Cre/Fabp4/5^-/- (TKO) mice. Bulk RNA sequencing (RNA-seq), single-cell RNA sequencing analysis, and spatial transcriptomic analysis were performed to understand the cellular and molecular mechanisms of endothelial FABP4/5-mediated PAH pathogenesis.

Results: Both FABP4 and FABP5 were highly induced in ECs of CKO mice and pulmonary arterial ECs from patients with idiopathic PAH (IPAH) and in whole lungs of PH rats. Plasma levels of FABP4/5 were upregulated in patients with IPAH and directly correlated with severity of hemodynamics and biochemical parameters. Genetic deletion of both Fabp4 and Fabp5 in CKO mice caused a reduction of right ventricular systolic pressure and right ventricular hypertrophy, attenuated pulmonary vascular remodeling, and prevented the right heart failure secondary to PH. FABP4/5 deletion also normalized EC glycolysis and distal arterial programming, reduced reactive oxygen species and HIF (hypoxia-inducible factor)-2α expression, and decreased aberrant EC proliferation in CKO lungs.

Conclusions: PH causes aberrant expression of FABP4/5 in pulmonary ECs, which leads to enhanced EC glycolysis and distal arterial programming, contributing to the accumulation of arterial ECs and vascular remodeling and exacerbating the disease.

Keywords: glycolysis; heart failure; hypoxia; lung; pulmonary arterial hypertension.

Figure 1.

Upregulation of FABP (fatty acid–binding protein) 4 and FABP5 in the patients with pulmonary arterial hypertension (PAH) and pulmonary hypertension rodents. A, Single-cell RNA sequencing analysis showed that FABP4 and FABP5 were upregulated in the lung endothelial cells (ECs) of Egln1^Tie2Cre (CKO) mice compared with wild-type (WT) mice. Wilcoxon Rank-sum test, ****adjusted P<0.0001. B, Representative images of immunostaining against FABP4 and FABP5 showed that FABP4 and FABP5 were upregulated in the lung ECs of Egln1^Tie2Cre mice compared with WT mice. Lung tissues were costained with anti-FABP4 or anti-FABP5 and anti-CD31 (marker for ECs). Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). FABP4 and FABP5 expression was upregulated in pulmonary vascular ECs in CKO mice. More than 3 lungs from each group of mice were checked. Yellow arrows indicate the colocalization of FABP4 or 5 with CD31 staining. C, Representative Western blotting demonstrating upregulation of FABP4 and FABP5 protein expression in the lung lysate isolated from CKO lungs compared with WT mice. D, Representative Western blotting showing an increase of FABP4 and FABP5 protein expression in the lung lysate isolated from Sugen5416/hypoxia (SuHx)–exposed lungs or monocrotaline (MCT)-exposed lungs compared with basal rats. E and F, Quantitative RT-PCR (real time-polymerase chain reaction) analysis and Western blotting demonstrating an upregulation of FABP4 and FABP5 in the isolated pulmonary arterial ECs from patients with idiopathic PAH (IPAH) compared with healthy donor lungs. Each sample represents PAECs from individual patients or healthy controls (n=3). t test (C through E). Scale bar, 50 µm. AT1 indicates alveolar type 1 cells; AT2, alveolar type 2 cells; A.U., arbitrary unit; DC, dendritic cell; iMac, interstitial macrophage; inMono, inflammatory monocyte; LEC, lymphatic endothelial cell; NK, natural killer cell; PMN, neutrophil; SMC, smooth muscle cell; Treg, regulatory T cell; and V, vessel.

Figure 2.

Upregulation of the plasma FABP (fatty acid–binding protein) 4 and FABP5 in patients with pulmonary arterial hypertension (PAH). A and B, Plasma protein normalized protein expression (NPX) levels of the FABP4 (A) and FABP5 (B) in donors (CTRL) and patients with PAH from Canada cohorts. Scatter dot plots show individual values (n). According to the contemporary cardiac index (CI) measured by right heart catheterization at the time, the blood sample was drawn. Patients with PAH were categorized as compensated right ventricle (cRV, CI> 2.2 L/min per m²) and decompensated RV (dRV, CI≤2.2 L/min per m²). CTRL n=28, cRV n=31, dRV n=29. C, Plasma protein NPX level of FABP4 was correlated with plasma protein NPX level of FABP5 in the human subjects including both donors and patients with PAH. D through I, Pearson correlation coefficient of the levels of FABP4 and FABP5 and hemodynamics/biomedical parameters of patients with PAH with associated P value is shown in each graph. t test (left, A and B). ANOVA followed by Tukey post hoc analysis was used for statistical analysis (right, A and B). Pearson correlation test (C through I). eGFR indicates estimated glomerular filtration rate; MDRD, modification of diet in renal disease; mPAP, mean pulmonary arterial pressure; NT-proBNP, N-terminal pro-B-type natriuretic peptide; PVR, pulmonary vascular resistance; and SV, stroke volume.

Figure 3.

FABP (fatty acid–binding protein) 4 and FABP5 deletions protected from Egln1-deficiency–induced pulmonary hypertension in mice. A, A diagram showing the strategy for generation of FABP4 and FABP5 double knockouts in Egln1^Tie2Cre mice (TKO). B, Representative Western blotting demonstrating diminished FABP4 and FABP5 protein expression in lung lysate isolated from TKO lungs compared with CKO lungs. C, Dramatic reduction in right ventricular (RV) systolic pressure (RVSP) was seen in TKO mice compared with CKO mice. Bars represent the mean. D, Marked inhibition of RV hypertrophy in TKO mice compared with CKO mice. E and F, Representative images from Russel-Movat pentachrome staining and quantification of wall thickness of pulmonary artery (PA) exhibited reduced thickness in the intima, media, and adventitia layers of tissue of TKO mice compared with CKO mice. Occlusive lesions were diminished in TKO mice compared with CKO mice. G, Exemplary images showed a reduction in muscularization of distal pulmonary vessels in TKO mice. Lung sections were subjected to immunostaining using anti–α-SMA (α-smooth muscle actin). H, Muscularization quantification was performed by tallying SMA-positive vessels across 40 fields (at ×20 magnification) within every lung section. The results were presented as the mean value along with the SD (mean ± SD) based on a sample size of 5 to 10 mice per group. ANOVA followed by Tukey post hoc analysis was used for statistical analysis (B, C, D, F, and H). A.U. indicates arbitrary unit; Br, bronchus; DAPI, 4',6-diamidino-2-phenylindole; RV(LV+S), RV and the left ventricle plus septum; V, vessel; and WT, wild-type.

Figure 4.

Genetic deletion of FABP (fatty acid–binding protein) 4/5 improved right heart function in pulmonary hypertension mice. A, Representative echocardiography images revealed a reduction of right ventricular (RV) wall thickness during diastole (RVWTD) in Egln1Tie2Cre/Fabp4/5-/- mice (TKO) compared with Egln1^Tie2Cre (CKO) mice. B, Improved RV fraction area change (RVFAC), indicating enhancing RV contractility, in TKO mice compared with CKO mice. C, An increased ratio of pulmonary artery acceleration time to ejection time (pulmonary artery acceleration time to ejection time [PA AT/ET]) in TKO mice compared with CKO mice. D through F, There is no significant change in heart rate (HR), cardiac output (CO), and left ventricular fractional shortening (FS). ANOVA followed by Tukey post hoc analysis was used for statistical analysis (A through C). WT indicates wild-type. DKO indicates Egln1^f/f/Fabp4/5^−/−.

Figure 5.

FABP (fatty acid–binding protein) 4/5 regulated genes related to pulmonary hypertension (PH) and glycolysis. A, Principal component analysis showed that FABP4/5 deletion normalized the altered gene signature by Egln1 deficiency in mice. B, A representative heat map showed that Fabp4/5 deletion altered the overall gene signature of CKO lungs toward wild-type (WT) lungs. C, Venn diagrams indicated FABP4/5 controlled the expression of the dysregulated genes in CKO mice. The left Venn diagram shows that upregulated gene list in CKO lungs compared with WT lungs was mostly overlapped with downregulated gene list in TKO lungs compared with CKO lungs. The right Venn diagram shows that downregulated gene list in CKO lungs compared with WT lungs was mostly overlapped with upregulated gene list in TKO lungs compared with CKO lungs. D, The Kyoto Encyclopedia of Genes and Genomes pathway analysis of the genes upregulated in CKO lungs and normalized by FABP4/5 deletion. E, Gene Set Enrichment Analysis showed the enrichment of hypoxia and glycolysis pathways based on the gene sets regulated by FABP4/5. F, A panel of representative genes related to PH pathogenesis and glycolysis were normalized by FABP4/5 deletion in CKO mice. G, Quantitative polymerase chain reaction analysis confirmed the PH-causing genes and glycolysis-related genes were altered in CKO lungs and normalized in the TKO mice by RNA sequencing data. H, Western blotting showing FABP4/5 deletion reduced the protein level of glycolytic genes in CKO mice. ANOVA followed by Turkey post hoc analysis was used for statistical analysis (G and H). A.U. indicates arbitrary unit; FDR, false discovery rate; IL, interleukin; PC, principal components; PCA, principal component analysis; and Rep, replicate.

Figure 6.

Single-cell transcriptomics analysis on FABP (fatty acid–binding protein) 4/5 regulated pulmonary hypertension (PH) development. A and B, Single-cell RNA sequencing (scRNA-seq) analysis demonstrated that FABP4/5 deletion partially normalized the alteration of endothelial cell (EC) subpopulations in CKO mice. C, Venn diagrams indicated FABP4/5 controlled the expression of the dysregulated genes in ECs. The left Venn diagram shows that upregulated gene list in CKO ECs compared with wild-type (WT) ECs was partially overlapped with downregulated gene list in TKO EC compared with CKO ECs. The right Venn diagram shows that downregulated gene list in CKO ECs compared with WT ECs was partially overlapped with upregulated gene list in TKO ECs compared with CKO ECs. D, The Kyoto Encyclopedia of Genes and Genomes pathway analysis of the genes upregulated in CKO ECs and normalized by FABP4/5 deletion in ECs. E, A heat map analysis based on the scRNA-seq analysis on the EC population showed that the glycolytic genes were upregulated in CKO lungs and normalized in TKO ECs. F, The glycolytic score calculation showed that glycolysis is controlled by FABP4/5 in ECs. aMac indicates alveolar macrophage; AT1, alveolar type 1 cells; AT2, alveolar type 2 cells; DC, dendritic cell; DEG, differentially expressed gene; FDR, false discovery rate; IL, interleukin; iMac, interstitial macrophage; inMono, inflammatory monocyte; LEC, lymphatic endothelial cell; NK, natural killer cell; PMN, neutrophil; SMC, smooth muscle cell; Treg, regulatory T cell; and UMAP, Uniform Manifold Approximation and Projection.

Figure 7.

Single-cell and spatial transcriptomics analysis identified induction of distal arterialization by FABP (fatty acid–binding protein) 4/5 in pulmonary hypertension. A and B, A representative Uniform Manifold Approximation and Projection (UMAP) and a cellular proportion plot showing the endothelial cell (EC) subpopulation change between groups. Upregulation of arterial EC proportion in CKO lungs was inhibited in TKO lungs. C, Statistical analysis showed that an increase in arterial ECs and a reduction of aerocytes (aCap) in CKO lungs were rescued in TKO lungs. D, Integration of single-cell RNA sequencing (scRNA-seq) and Visium spatial data revealed increased arterial ECs and decreased general capillary (gCap) ECs in the distal capillary bed of CKO mice, which was normalized in the TKO mice. The same wild-type (WT) and CKO lungs were used in our previous publication. The visualization shows the predicted spatial distribution of arterial ECs (ArtECs) and capillary ECs within the lungs. E, A spatial plot showed that both gCap marker Gpihbp1 and aCap marker Car4 were reduced in the CKO lungs and restored in the TKO lungs. The frame size is 6.5 mm. F, A heat map showing that a panel of representative genes related to arterial EC markers were increased in the CKO ECs and normalized in TKO ECs based on the scRNA-seq data. G, A heat map based on the bulk RNA sequencing data showing the rescue of arterial gene programming in TKO lungs compared with CKO lungs. H, Western blotting demonstrated the classical arterial marker SOX17 was increased in the CKO lungs and inhibited in the TKO lungs. I, Immunostaining against SOX17 demonstrated the increase in distal arterial ECs in CKO lungs, which was rescued in the TKO lungs. ANOVA followed by Tukey post hoc analysis was used for statistical analysis (H and I). abs indicates absolute; A.U., arbitrary unit; Cap, capillary endothelial cells; FD, false discovery; FDR, false discovery rate; ns, no significance; and Rep, replicate.

Figure 8.

Overexpression of FABP (fatty acid–binding protein) 4/5–induced endothelial cell (EC) dysfunction. A, Overexpression of FABP4 and FABP5 fused with Flag tag in human pulmonary arterial ECs (hPAECs) using lentiviruses mediated gene overexpression. B and C, FABP4/5 overexpression promoted ECs glycolysis and proliferation assessed by 5-bromo-20-deoxyuridine (BrdU) incorporation assay. D, Glycolytic inhibitor 2-DG treatment inhibited FABP4/5–induced PAEC proliferation. E, FABP4/5 deletion in vivo reduced lung PAECs proliferation in TKO mice compared with CKO mice. F and G, Overexpression of FABP4/5 promoted fatty acid oxidation (FAO) in hPAECs in the presence of palmitic acid. The double arrows label the OCR generation contributed by FAO. H, Both CPT1α inhibitor etomoxir (ETO, 20 µmol/L) and DNA synthesis inhibitor (5-FU, 20 µmol/L) blocked FABP4/5–induced EC proliferation. I and J, A representative seahorse data showing upregulation of FAO in PAECs isolated from patients with idiopathic PAH (IPAH) compared with control donors. Six lines of control or PAECs of patients with IPAH were tested. C, D, and H, For BrdU assay, each dot represents a biological replicate. The experiments were performed at least 3 times. Student t test (D). ANOVA followed by Tukey post hoc analysis was used for statistical analysis (B, C, E, F, G, H, and J). Significance levels were denoted as P<0.05. abs indicates absolute; A.U., arbitrary unit; Cap, capillary endothelial cells; DAPI, 4',6-diamidino-2-phenylindole; FD, false discovery; FDR, false discovery rate; Rep, replicate; and WT, wild type.

Figure 9.

HIF (hypoxia-inducible factor)-2α–mediated glycolysis induced by FABP (fatty acid–binding protein) 4/5 in pulmonary endothelial cells (ECs). A, A diagram showing the predicted transcription factors based on the DEGs and literature. B, Western blotting demonstrated HIF-2α but not p53 or c-Myc was upregulated in CKO lungs and normalized in TKO lungs. The β-actin on c-Myc blot was shared with Figure 1C FABP5. C, A representative heat map showed that glycolytic genes were dependent on HIF-2α using wild-type (WT), CKO mice, and Egln1^Tie2Cre/Hif2a^Tie2Cre (EH2) mice. D, HIF-2α knockdown inhibited FABP4/5–induced endothelial proliferation. E, Nitrative stress assessed by protein nitrotyrosine modification was reduced in TKO lungs compared with CKO lungs. F, Overexpression of FABP4 and FABP5 promoted mitochondrial reactive oxygen species (ROS) levels in human pulmonary arterial EC (hPAECs). G, A diagram showing our proposed model. Our study addresses a novel role of lung endothelial FABP4/5 controlling PAEC accumulation through increased glycolysis in the pathogenesis of pulmonary arterial hypertension (PAH). By facilitating fatty acid uptake and translocation into mitochondria, FABP4/5 promote fatty acid oxidation (FAO) and ROS generation, which activates HIF-2α signaling to promote endothelial glycolysis. For 5-bromo-20-deoxyuridine (BrdU) assay in (D) and mitochondrial ROS assay in (F), each dot represents a biological replicate. The experiments were performed at least 3 times. ANOVA followed by Tukey post hoc analysis was used for statistical analysis (B, D, E, and F). Significance levels were denoted as P<0.05. α-NT indicates anti-nitrotyrosine; Adjp, adjusted P value; A.U., arbitrary unit; DAPI, 4',6-diamidino-2-phenylindole; DEG, differentially expressed gene; dNTP, deoxyribonucleotide triphosphate; ETO, etomoxir; 5-FU, 5-fluorouracil; siCTL, small interfering RNA control; siHIF, small interfering RNA targeting hypoxia-inducible factor; TF, transcription factor; and 2-DG, 2-deoxy-D-glucose.