. 2025 Jan 24;387(6732):eadn7277.

doi: 10.1126/science.adn7277. Epub 2025 Jan 24.

Lysosomal dysfunction and inflammatory sterol metabolism in pulmonary arterial hypertension

Lloyd D Harvey^{1

2

3}, Mona Alotaibi^{4

5

6}, Yi-Yin Tai^{2

3}, Ying Tang^{2

3}, Hee-Jung J Kim^{2

3}, Neil J Kelly^{2

3

7}, Wei Sun^{2

3}, Chen-Shan C Woodcock^{2

3}, Sanya Arshad^{2

3}, Miranda K Culley^{1

2

3}, Wadih El Khoury^{2

3}, Rong Xie^{2

3}, Yassmin Al Aaraj^{2

3}, Jingsi Zhao^{2

3}, Neha Hafeez^{2

3}, Rashmi J Rao^{2

3}, Siyi Jiang^{2

3}, Vinny Negi^{2

3}, Anna Kirillova^{2

3}, Dror Perk^{2

3}, Annie M Watson^{2

3}, Claudette M St Croix⁸, Donna B Stolz⁸, Ji Young Lee^{9

10}, Mary Hongying Cheng^{9

10}, Manling Zhang^{2

3

7}, Samuel Detmer¹¹, Edward Guzman^{12

13}, Rajith S Manan¹⁴, Rajan Saggar^{15

16}, Kathleen J Haley¹⁷, Aaron B Waxman¹⁷, Satoshi Okawa^{2

3

18

19}, Tae-Hwi Schwantes-An^{20

21}, Michael W Pauciulo²², Bing Wang^{2

3}, Amy Webb²³, Caroline Chauvet²⁴, Daniel G Anderson¹², William C Nichols²², Ankit A Desai²¹, Robert Lafyatis²⁵, S Mehdi Nouraie²⁶, Haodi Wu^{2

3}, Jeffrey G McDonald^{27

28}, Susan Cheng²⁹, Ivet Bahar^{9

10}, Thomas Bertero²⁴, Raymond L Benza³⁰, Mohit Jain^{5

6}, Stephen Y Chan^{2

3}

Affiliations

¹ Medical Scientist Training Program, University of Pittsburgh, Pittsburgh, PA, USA.
² Center for Pulmonary Vascular Biology and Medicine, Pittsburgh, Heart, Lung, and Blood Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA.
³ Division of Cardiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
⁴ Division of Pulmonary and Critical Care Medicine, University of California San Diego, La Jolla, CA, USA.
⁵ Department of Medicine, University of California, San Diego, La Jolla, CA, USA.
⁶ Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA.
⁷ Division of Cardiology, Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, PA, USA.
⁸ Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA, USA.
⁹ Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY, USA.
¹⁰ Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA.
¹¹ Department of Chemistry, Massachusetts Institute of Technology, Boston, MA, USA.
¹² Institute of Medical Engineering and Science, Massachusetts Institute of Technology, Boston, MA, USA.
¹³ School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA.
¹⁴ Department of Chemical Engineering, Massachusetts Institute of Technology, Boston, MA, USA.
¹⁵ Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles (UCLA), Los Angeles, CA, USA.
¹⁶ Department of Pathology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
¹⁷ Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
¹⁸ Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, USA.
¹⁹ McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
²⁰ Department of Medical and Molecular Genetics, Indiana University, Indianapolis, IN, USA.
²¹ Department of Medicine, Indiana University, Indianapolis, IN, USA.
²² Division of Human Genetics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
²³ Department of Biomedical Informatics, Ohio State University, Columbus, OH, USA.
²⁴ Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia-Antipolis, Valbonne, France.
²⁵ Division of Rheumatology and Clinical Immunology, University of Pittsburgh, Pittsburgh, PA, USA.
²⁶ Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
²⁷ Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX, USA.
²⁸ Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
²⁹ Department of Cardiology, Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
³⁰ Division of Cardiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

PMID: 39847635
PMCID: PMC12087357
DOI: 10.1126/science.adn7277

Lysosomal dysfunction and inflammatory sterol metabolism in pulmonary arterial hypertension

Lloyd D Harvey et al. Science. 2025.

. 2025 Jan 24;387(6732):eadn7277.

doi: 10.1126/science.adn7277. Epub 2025 Jan 24.

Authors

Affiliations

¹ Medical Scientist Training Program, University of Pittsburgh, Pittsburgh, PA, USA.
² Center for Pulmonary Vascular Biology and Medicine, Pittsburgh, Heart, Lung, and Blood Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA.
³ Division of Cardiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
⁴ Division of Pulmonary and Critical Care Medicine, University of California San Diego, La Jolla, CA, USA.
⁵ Department of Medicine, University of California, San Diego, La Jolla, CA, USA.
⁶ Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA.
⁷ Division of Cardiology, Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, PA, USA.
⁸ Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA, USA.
⁹ Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY, USA.
¹⁰ Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA.
¹¹ Department of Chemistry, Massachusetts Institute of Technology, Boston, MA, USA.
¹² Institute of Medical Engineering and Science, Massachusetts Institute of Technology, Boston, MA, USA.
¹³ School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA.
¹⁴ Department of Chemical Engineering, Massachusetts Institute of Technology, Boston, MA, USA.
¹⁵ Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles (UCLA), Los Angeles, CA, USA.
¹⁶ Department of Pathology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
¹⁷ Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
¹⁸ Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, USA.
¹⁹ McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
²⁰ Department of Medical and Molecular Genetics, Indiana University, Indianapolis, IN, USA.
²¹ Department of Medicine, Indiana University, Indianapolis, IN, USA.
²² Division of Human Genetics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
²³ Department of Biomedical Informatics, Ohio State University, Columbus, OH, USA.
²⁴ Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia-Antipolis, Valbonne, France.
²⁵ Division of Rheumatology and Clinical Immunology, University of Pittsburgh, Pittsburgh, PA, USA.
²⁶ Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
²⁷ Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX, USA.
²⁸ Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
²⁹ Department of Cardiology, Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
³⁰ Division of Cardiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

PMID: 39847635
PMCID: PMC12087357
DOI: 10.1126/science.adn7277

Abstract

Vascular inflammation regulates endothelial pathophenotypes, particularly in pulmonary arterial hypertension (PAH). Dysregulated lysosomal activity and cholesterol metabolism activate pathogenic inflammation, but their relevance to PAH is unclear. Nuclear receptor coactivator 7 (NCOA7) deficiency in endothelium produced an oxysterol and bile acid signature through lysosomal dysregulation, promoting endothelial pathophenotypes. This oxysterol signature overlapped with a plasma metabolite signature associated with human PAH mortality. Mice deficient for endothelial Ncoa7 or exposed to an inflammatory bile acid developed worsened PAH. Genetic predisposition to NCOA7 deficiency was driven by single-nucleotide polymorphism rs11154337, which alters endothelial immunoactivation and is associated with human PAH mortality. An NCOA7-activating agent reversed endothelial immunoactivation and rodent PAH. Thus, we established a genetic and metabolic paradigm that links lysosomal biology and oxysterol processes to endothelial inflammation and PAH.

PubMed Disclaimer

Conflict of interest statement

S.Y.C. has served as a consultant for Merck, Janssen, and United Therapeutics. S.Y.C. is a director, officer, and shareholder in Synhale Therapeutics. S.Y.C. has held grants from Bayer and United Therapeutics. S.Y.C. and T.B. have filed patent applications regarding metabolism and next-generation therapeutics in pulmonary hypertension (US 10,925,869 B2, “Compositions and methods for treating pulmonary vascular disease”; PCT/US2015/029286, “Coordinate control of pathogenic signaling by the MIR-130-301 family in pulmonary hypertension and fibroproliferative diseases”; PCT/US2018/062013, “Compositions and methods for administering a YAP1/WWRT1 inhibiting composition and a GLS1 inhibiting composition”). S.Y.C., L.D.H., and I.B. have filed patent applications regarding the therapeutic targeting of NCOA7 (US 11,773,391 B2, “Therapeutic and diagnostic target for SARS-CoV-2 and COVID-19”; PCT/IB2023/055431, “Methods and compositions for treatment of inflammation and inflammatory conditions”). The other authors declare that they have no competing interests. M.J. is an employee of and holds equity in Sapient Bioanalytics for work unrelated to the current manuscript.

Figures

**Fig. 1.. Convergent inflammatory regulation of NCOA7 across cellular, animal, and human instances of PAH.**
(A) Transcriptomic analysis of human PAECs under control or IL-1β (n = 3 replicates per group). Z-score is indicated as positive in blue and negative in gold. Genes listed have a false discovery rate (FDR)–corrected P value < 0.05. (B) *NCOA7*_short expression by means of RT-qPCR (n = 3 replicates per group). (C) *NCOA7*_full expression by means of RT-qPCR (n = 3 replicates per group). (D) Immunofluorescent (IF) staining of NCOA7 (red with yellow arrowheads), CD31⁺ ECs (green), α-SMA⁺ smooth muscle cells (white), and 4′,6-diamidino-2-phenylindole (DAPI)–stained nuclei (blue) in pulmonary vessels of wild-type versus *Il6* Tg⁺ mice. (E) Quantification of NCOA7 intensity in pulmonary vessels (n = 8 mice per group). (F) IF staining of NCOA7 (red with yellow arrowheads), CD31⁺ ECs (green), α-SMA⁺ smooth muscle cells (white), and DAPI-stained nuclei (blue) in pulmonary vessels of healthy human controls versus PAH patients. (G) Quantification of NCOA7 intensity in pulmonary vessels (n = 6 to 7 patients per group). (H) *NCOA7* expression in ECs identified by means of single-cell RNA-sequencing from lungs of healthy human controls or PAH patients (n > 3 patients per group). (I) *NCOA7* expression in fibroblasts identified by means of single-cell RNA-sequencing from lungs of healthy human controls or PAH patients (n > 3 patients per group). (J) *NCOA7* expression in smooth muscle cells identified by means of single-cell RNA-sequencing from lungs of healthy human controls or PAH patients (n > 3 patients per group). Cells were identified as expressing *NCOA7* if the transformed expression value was >0.2. (K) *NCOA7*_short expression under RNA interference (RNAi) against *RELA* (n = 3 replicates per group). Two-way ANOVA. (L) *NCOA7*_full expression under RNAi against *RELA* (n = 3 replicates per group). Two-way ANOVA. (M) ChIP-qPCR against p65/RelA binding to full- and short-length promoter regions (n =3 replicates per group). All data were analyzed by means of Student’s t test unless otherwise specified and presented as mean ± SD.

**Fig. 2.. NCOA7 deficiency results in lysosomal dysfunction and lipid accumulation under proinflammatory conditions.**
(A) Transcriptomic analysis of PAECs under IL-1β subjected to RNAi against control or *NCOA7* (n = 3 replicates per group). Z-score is indicated as positive in blue and negative in gold. Identified lysosomal genes have an FDR-corrected P value < 0.05. (B) Expression of *ATP6V1B2* under siNC or siNCOA7 by means of RT-qPCR (n = 3 replicates per group). (C) Expression of *ATP6V1B2* with lentiviral delivery of control [lentiviral green fluorescent protein (LV-GFP)], NCOA7_short, or NCOA7_full isoforms (n = 3 replicates per group). Data analyzed by means of one-way ANOVA. (D) Association of the V-ATPase subunit ATP6V1B2 with NCOA7 measured by means of proximity ligation assay (orange). (Bottom) Control images of ATP6V1B2, NCOA7, or neither antibody. (Top) Dual incubation of ATP6V1B2 and NCOA7 antibodies with lentiviral transduction of GFP control, NCOA7_short, or NCOA7_full. (E) Quantification of amplified signal per cell (n =5 cells per group). Data were analyzed by means of one-way ANOVA. (F) LysoLive probe (green) reflecting β-galactosidase activity and thus lysosomal acidification. (G) Quantified as fluorescence per cell (n = 10 cells per group). (H) Silicone rhodamine (SiR)–Lysosome dye targeting active cathepsin D (purple) and indicating low lysosomal pH. (I) Quantified as fluorescence per cell (n = 10 cells per group). (J) Live-cell confocal microscopy of lysosomes (LysoTracker Deep Red) and lysosomal acidification (LysoSensor Green DND-189). (K) Median fluorescence intensity (MFI) ratio of LysoSensor Yellow/Blue DND-160 dye by means of flow cytometry indicating lysosomal acidification (n = 3 replicates per group). (L) Representative images of lysosomal size in transmission electron micrographs. Yellow arrowheads indicate lamellar-like inclusions reflecting lipid accumulation within lysosomes. (M) Quantification of lysosomal area (n = 20 electron micrographs per group). (N) IF staining of colocalization (yellow) of neutral sterols (BOPIDY in green) and acidic lysosomes (LysoTracker in red). (O) Colocalization was measured using EzColocalization and plotted as Pearson’s correlation coefficient (n = 15 cells per group). All data were analyzed by means of two-way ANOVA unless otherwise specified and presented as mean ± SD.

**Fig. 3.. NCOA7 deficiency reprograms sterol metabolism to up-regulate oxysterols and bile acids.**
(A) Transcriptomic analysis of PAECs under IL-1β subjected to RNAi against control or *NCOA7* (n = 3 replicates per group). Z-score is indicated as positive in blue and negative in gold. Identified cholesterol metabolism genes have an FDR-corrected P value < 0.05. (B) Gene set enrichment analysis of top 15 pathways by FDR-adjusted P value with a majority related to sterol metabolism and homeostasis (indicated with red arrows). (C) Expression of *LDLR* under IL-1β subjected to RNAi against control or *NCOA7* (n = 3 replicates per group). (D) Fluorescence intensity of tagged NBD-cholesterol uptake (n = 3 replicates per group). (E) Flow cytometric plot of fluorescently tagged nitrobenzoxadiazole (NBD)–cholesterol uptake. (F) Total cholesterol content under IL-1β subjected to RNAi against control or *NCOA7* (n = 3 replicates per group). (G) Total cholesterol content under IL-1β subjected to lentiviral overexpression of GFP, NCOA7_short, or NCOA7_full. Data analyzed by means of one-way ANOVA (n = 6 replicates per group). (H) Expression of *CH25H* in PAECs under IL-1β subjected to RNAi against control or *NCOA7* by means of RT-qPCR (n = 3 replicates per group). (I) IF staining for CH25H (red with yellow arrowheads), CD31⁺ ECs (green), α-SMA⁺ smooth muscle cells (white), and DAPI-stained nuclei (blue) in the pulmonary vessels of wild-type versus *Il6* Tg⁺ mice. (J) Quantification of CH25H in pulmonary vessels (n = 8 mice per group). Student’s t test. (K) IF staining for CH25H (red with yellow arrowheads), CD31⁺ ECs (green), α-SMA⁺ smooth muscle cells (white), and DAPI-stained nuclei (blue) in the pulmonary vessels of control versus monocrotaline rats. (L) Quantification of CH25H in pulmonary vessels (n = 5 rats per group). Student’s t test. (M) IF staining for CH25H (red with yellow arrowheads), CD31⁺ ECs (green), α-SMA⁺ smooth muscle cells (white), and DAPI-stained nuclei (blue) in the pulmonary vessels of healthy human controls versus PAH patients. (N) Quantification of CH25H in pulmonary vessels (n = 6 to 7 patients per group). Student’s t test. (O) Targeted quantification of 25-hydroxycholesterol by means of LC-MS (n = 5 replicates per group). (P) Targeted quantification of 7-hydroxycholesterol by means of LC-MS (n = 5 replicates per group). (Q) Targeted quantification of 27-hydroxycholesterol by means of LC-MS (n =5 replicates per group). (R) Unbiased bile acid quantification of 5-cholesten-3β−7α−25-triol by means of LC-MS (n = 4 to 6 replicates per group). (S) Unbiased bile acid quantification of 5α-cholestane-3α,7α,12α-triol by means of LC-MS (n = 4 to 6 replicates per group). (T) Unbiased bile acid quantification of 5α-cholestane-3α,7α,12α,24,25-pentol by means of LC-MS (n = 4 to 6 replicates per group). (U) Unbiased bile acid quantification of 3α,7α,12α-trihydroxy-5β−24E-cholesten-26-oic acid by means of LC-MS (n = 4 to 6 replicates per group). (V) Unbiased bile acid quantification of 3β,7α-dihydroxy-5-cholestenoate by means of LC-MS (n = 4 to 6 replicates per group). (W) Unbiased bile acid quantification of 7α-hydroxy-3-oxo-4-cholestenoic acid (7HOCA) by means of LC-MS (n = 4 to 6 replicates per group). Metabolites are organized by proposed pathways in the shaded boxes. Black arrows indicate proposed sequential metabolite pathways. All data were analyzed by means of two-way ANOVA unless otherwise specified and presented as mean ± SD.

**Fig. 4.. The NCOA7-CH25H axis drives pulmonary endothelial immunoactivation.**
(A) *VCAM1* expression by means of RT-qPCR under RNAi against *NCOA7* (n = 3 replicates per group). (B) VCAM1 expression by menas of immunoblot under RNAi against *NCOA7* (n = 3 replicates per group). (C) *VCAM1* expression by means of RT-qPCR under lentiviral delivery of NCOA7_short or NCOA7_full (n = 3 replicates per group). (D) VCAM1 expression by means of immunoblot under lentiviral delivery of NCOA7_short or NCOA7_full (n = 3 replicates per group). (E) *VCAM1* expression by means of RT-qPCR under RNAi against *NCOA7* and *CH25H* (n = 3 replicates per group). (F) VCAM1 expression by means ofimmunoblot under RNAi against *NCOA7* and *CH25H* (n = 3 replicates per group). (G) Leukocyte adhesion under RNAi against *NCOA7* (n = 6 replicates per group). (H) Leukocyte adhesion under lentiviral delivery of NCOA7_short or NCOA7_full (n = 6 replicates per group). (I) Leukocyte adhesion under RNAi against *NCOA7* and *CH25H* (n = 6 replicates per group). (J) Monocyte adhesion under RNAi against *NCOA7* (n = 6 replicates per group). (K) Monocyte adhesion under lentiviral delivery of NCOA7_short or NCOA7_full (n = 6 replicates per group). (L) Monocyte adhesion under RNAi against *NCOA7* and *CH25H* (n = 6 replicates per group). (M) *VCAM1* expression by means of RT-qPCR under control (ethanol) versus 7HOCA (50 μM) for 24 hours (n = 3 replicates per group; Student’s t test). (N) VCAM1 expression by means of immunoblot under control (ethanol) versus 7HOCA (50 μM) for 24 hours (n = 3 replicates per group; Student’s t test). (O) Leukocyte adhesion in 7HOCA-treated PAECs compared with ethanol controls (n = 6 replicates per group; Student’s t test). (P) Monocyte adhesion in 7HOCA-treated PAECs compared with ethanol controls (n = 6 replicates per group; Student’s t test). All data were analyzed by means of two-way ANOVA unless otherwise specified and presented as mean ± SD.

**Fig. 5.. Genetic loss of *Ncoa7* and the orotracheal delivery of 7HOCA worsens PAH in vivo.**
(A) *Ncoa7*-null mice crossed onto the *Il6* Tg⁺ PAH model. (B) Pulmonary arterioles from *Il6* Tg⁺ versus *Ncoa7*^−/− × *Il6* Tg⁺ mice stained for a target protein (CH25H, VCAM1, or CD11b; red with yellow arrowheads), the endothelial layer (CD31; green), the smooth muscle layer (α-SMA; white), and nuclear counterstain (DAPI; blue). (C to F) Quantification of (C) CH25H relative intensity, (D) VCAM1 relative intensity, (E) CD11b⁺ cells, and (F) vessel remodeling by means of percent muscularization in the pulmonary arterioles of *Il6* Tg⁺ versus *Ncoa7*^−/− × *Il6* Tg⁺ mice (n = 8 mice per group). (G) 7HOCA fold change, (H) Fulton’s Index, (I) RVFAC, (J) TAPSE, and (K) RVSP of *Il6* Tg⁺ versus *Ncoa7*^−/− × *Il6* Tg⁺ mice (n =6 to 10 mice per group). (L) *Il6* Tg⁺ mice were serially injected in the tail vein with either 7C1 nanoparticle encapsulated mouse siRNA against negative control or *Ncoa7* (1 mg/kg) starting at 10 weeks of age every 5 days for a total of five doses and were euthanized at 16 weeks. (M) Pulmonary arterioles from siNC:7C1 *Il6* Tg⁺ versus siNCOA7:7C1 *Il6* Tg⁺ mice stained for a target protein (NCOA7, CH25H, VCAM1, or CD11b; red with yellow arrowheads), the endothelial layer (CD31; green), the smooth muscle layer (α-SMA; white), and nuclear counterstain (DAPI; blue). (N to S) Quantification of the relative intensity of (N) NCOA7 in endothelium, (O) NCOA7 in smooth muscle cells, (P) CH25H, (Q) VCAM1, (R) the number of CD11b⁺ cells per vessel, or (S) the degree of vessel muscularization defined by α-SMA layer thickness to total vessel diameter. (n =6 mice per group). (T) Fulton’s Index, (U) RVFAC, (V) TAPSE, and (W) RVSP of 7C1:siNc *Il6* Tg⁺ versus 7C1:siNcoa7 *Il6* Tg⁺ mice (n =9 to 18 mice per group). (X) Mice received orotracheal delivery of either PBS or 7HOCA (10 mg/kg) for 4 weeks under hypoxic (10% O₂) conditions and were euthanized at 16 weeks. (Y) Pulmonary arterioles from PBS versus 7HOCA mice stained for a target protein (VCAM1, or CD11b; red with yellow arrowheads), the endothelial layer (CD31; green), the smooth muscle layer (α-SMA; white), and nuclear counterstain (DAPI; blue). (Z to AB) Quantification of the relative intensity of (Z) VCAM1, (AA) the number of CD11b⁺ cells per vessel, or (AB) the degree of vessel muscularization defined by α-SMA layer thickness to total vessel diameter (n = 4 mice per group). (AC) Fulton’s Index, (AD) RVFAC, (AE) TAPSE, and (AF) RVSP of mice receiving orotracheal PBS or 7HOCA (n = 3 to 7 mice per group). All data were analyzed by means of Student’s t test unless otherwise specified and presented as mean ± SD.

**Fig. 6.. The G allele of SNP rs11154337 prevents lysosomal lipid accumulation and attenuates oxysterol-mediated immunoactivation in iPSC-ECs.**
(A) Metabolites identified in *NCOA7*-deficit PAECs or in the plasma of *Ncoa7*-deficient mice individually were associated with higher mortality in PAH patients in the UPMC-a cohort (n = 116 patients; Cox regression with multivariate adjustment; P value cutoff < 0.01). (B and C) Allelic variants of SNP rs11154337 and their clinical readouts of 6-min walk distance (6MWD) and survival in PAH patients in the UPMC-b cohort (n = 93 patients) and STRIDE+ cohort (n = 630 patients). (D) Schematic of iPSC-EC production. (E) *NCOA7*_short and (F) *NCOA7*_full expression by means of RT-qPCR in iPSC-ECs under IL-1β (n = 3 replicates per group). (G) IF staining with SiR-Lysosome dye against active cathepsin D and (H) quantification of fluorescence in iPSC-ECs under control or IL-1β (purple; n = 10 cells per group). (I) *ATP6V1B2* expression by means of RT-qPCR in iPSC-ECs under control or IL-1β (n = 3 replicates per group). (J) Transmission electron micrograph quantification of lysosomal area in iPSC-ECs under control or IL-1β. (K) Total cholesterol content as measured by means of relative luminescence in iPSC-ECs under control or IL-1β (n =6 replicates per group). (L) IF staining with BODIPY dye against neutral lipids and (M) quantification of fluorescence in iPSC-ECs under control or IL-1β (green; n = 10 cells per group). (N) *CH25H* expression by means of RT-qPCR in iPSC-ECs under control or IL-1β (n = 3 replicates per group). (O) Targeted 25HC quantification by means of LC-MS in iPSC-ECs under control or IL-1β (n =5 replicates per group). (P) *VCAM1* expression by means of RT-qPCR or (Q) immunoblot in iPSC-ECs under control or IL-1β (n = 3 replicates per group). (R) Leukocyte and (S) monocyte adhesion to iPSC-EC monolayer under control or IL-1β (n =6 replicates per group). All data were analyzed by means of two-way ANOVA unless otherwise specified and presented as mean ± SD.

**Fig. 7.. Computational modeling identifies 958_ami as an NCOA7 activator that abrogates endothelial immunoactivation and PAH.**
(A to C) Computational protocol for identifying small-molecule modulators of NCOA7, composed of three major components: (A) druggability simulations, (B) pharmacophore modeling, and (C) virtual screening. (D and E) Refinement of the identified compound 958 after molecular dynamics simulations into its analog 958_ami. Interactions between compound atoms and NCOA7 residues are shown in two dimensions. Stronger interactions are indicated with orange dashed lines, and weaker interactions are indicated with gray dashed lines. Orange solid lines are strong or persistent interactions with more than 0.3 μs cumulative duration per interaction over 0.6 μs total simulations. (F) Association of the V-ATPase subunit ATP6V1B2 with NCOA7 measured by means of proximity ligation assay (orange). The images demonstrate dual incubation of ATP6V1B2 and NCOA7 antibodies with DMSO or 958_ami (20 μM). (G) Quantification of amplified signal per cell (n = 15 cells per group). (H) Lysosomal pH assessed by means of LysoSensor Green DND-189, and immunofluorescence intensity quantified per cell (n = 20 cells per group). (I) *CH25H* expression by means of RT-qPCR (n = 3 replicates per group; two-way ANOVA). (J) Expression of VCAM1 by means of RT-qPCR and (K) immunoblot in PAECs treated with DMSO versus 958_ami (n = 3 replicates per group; two-way ANOVA). (L) Leukocyte and (M) monocyte adhesion in 958_ami-treated PAECs compared with DMSO control (n = 6 replicates per group). (N) Rats were administered monocrotaline (80 mg/kg intraperitoneally) 1 week before initiation of a daily dose of 958_ami (7.5 mg/kg intraperitoneally) for 10 days. (O) Pulmonary arterioles from DMSO-versus 958_ami-injected rats stained for a target protein (CH25H, VCAM1, or CD11b; red with yellow arrowheads), the endothelial layer (CD31; green), the smooth muscle layer (α-SMA; white), and nuclear counterstain (DAPI; blue). (P to S) Quantification of the relative intensity of (P) CH25H or (Q) VCAM1, (R) the number of CD11b⁺ cells per vessel, or (S) the degree of vessel muscularization defined by α-SMA layer thickness to total vessel diameter (n = 6 to 7 rats per group). (T) Fulton’s Index, (U) RVFAC, (V) TAPSE, and (W) RVSP of monocrotaline rats receiving intraperitoneal DMSO or 958_ami (n = 5 to 15 rats per group). All data were analyzed by means of Student’s t test unless otherwise specified and presented as mean ± SD.

See this image and copyright information in PMC

Update of

Genetic regulation and targeted reversal of lysosomal dysfunction and inflammatory sterol metabolism in pulmonary arterial hypertension.
Harvey LD, Alotaibi M, Kim HJ, Tai YY, Tang Y, Sun W, El Khoury W, Woodcock CC, Aaraj YA, St Croix CM, Stolz DB, Lee J, Cheng MH, Schwantes-An TH, Desai AA, Pauciulo MW, Nichols WC, Webb A, Lafyatis R, Nouraie M, Wu H, McDonald JG, Chauvet C, Cheng S, Bahar I, Bertero T, Benza RL, Jain M, Chan SY. Harvey LD, et al. bioRxiv [Preprint]. 2024 Mar 1:2024.02.26.582142. doi: 10.1101/2024.02.26.582142. bioRxiv. 2024. Update in: Science. 2025 Jan 24;387(6732):eadn7277. doi: 10.1126/science.adn7277. PMID: 38464060 Free PMC article. Updated. Preprint.

Comment in

Decoding lysosome communication.
Pullamsetti SS, Savai R. Pullamsetti SS, et al. Science. 2025 Jan 24;387(6732):359-361. doi: 10.1126/science.adv1201. Epub 2025 Jan 23. Science. 2025. PMID: 39847645

References

1. Bäck M, Yurdagul A Jr, Tabas I, Öörni K, Kovanen PT, Inflammation and its resolution in atherosclerosis: Mediators and therapeutic opportunities. Nat. Rev. Cardiol 16, 389–406 (2019). doi: 10.1038/s41569-019-0169-2 - DOI - PMC - PubMed
1. Guzik TJ, Touyz RM, Oxidative stress, inflammation, and vascular aging in hypertension. Hypertension 70, 660–667 (2017). doi: 10.1161/HYPERTENSIONAHA.117.07802 - DOI - PubMed
1. Kelly PJ, Lemmens R, Tsivgoulis G, Inflammation and stroke risk: A new target for prevention. Stroke 52, 2697–2706 (2021). doi: 10.1161/STROKEAHA.121.034388 - DOI - PubMed
1. Hattori Y, Hattori K, Machida T, Matsuda N, Vascular endotheliitis associated with infections: Its pathogenetic role and therapeutic implication. Biochem. Pharmacol 197, 114909 (2022). doi: 10.1016/j.bcp.2022.114909 - DOI - PMC - PubMed
1. Jones JH, Minshall RD, Endothelial transcytosis in acute lung injury: Emerging mechanisms and therapeutic approaches. Front. Physiol 13, 828093 (2022). doi: 10.3389/fphys.2022.828093 - DOI - PMC - PubMed

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions
Actions
Actions
Actions

Grants and funding

LinkOut - more resources

Full Text Sources
Molecular Biology Databases
- Mouse Genome Informatics (MGI)
- NIAID Data Ecosystem - Find datasets on Infectious and Immune-mediated Diseases

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Lysosomal dysfunction and inflammatory sterol metabolism in pulmonary arterial hypertension

Affiliations

Lysosomal dysfunction and inflammatory sterol metabolism in pulmonary arterial hypertension

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Update of

Comment in

References

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Molecular Biology Databases