Vascular stiffness mechanoactivates YAP/TAZ-dependent glutaminolysis to drive pulmonary hypertension
Thomas Bertero, William M. Oldham, Katherine A. Cottrill, Sabrina Pisano, Rebecca R. Vanderpool, Qiujun Yu, Jingsi Zhao, Yiyin Tai, Ying Tang, Ying-Yi Zhang, Sofiya Rehman, Masataka Sugahara, Zhi Qi, John Gorcsan III, Sara O. Vargas, Rajan Saggar, Rajeev Saggar, W. Dean Wallace, David J. Ross, Kathleen J. Haley, Aaron B. Waxman, Victoria N. Parikh, Teresa De Marco, Priscilla Y. Hsue, Alison Morris, Marc A. Simon, Karen A. Norris, Cedric Gaggioli, Joseph Loscalzo, Joshua Fessel, Stephen Y. Chan
Thomas Bertero, William M. Oldham, Katherine A. Cottrill, Sabrina Pisano, Rebecca R. Vanderpool, Qiujun Yu, Jingsi Zhao, Yiyin Tai, Ying Tang, Ying-Yi Zhang, Sofiya Rehman, Masataka Sugahara, Zhi Qi, John Gorcsan III, Sara O. Vargas, Rajan Saggar, Rajeev Saggar, W. Dean Wallace, David J. Ross, Kathleen J. Haley, Aaron B. Waxman, Victoria N. Parikh, Teresa De Marco, Priscilla Y. Hsue, Alison Morris, Marc A. Simon, Karen A. Norris, Cedric Gaggioli, Joseph Loscalzo, Joshua Fessel, Stephen Y. Chan
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Research Article Pulmonology Vascular biology

Vascular stiffness mechanoactivates YAP/TAZ-dependent glutaminolysis to drive pulmonary hypertension

Abstract

Dysregulation of vascular stiffness and cellular metabolism occurs early in pulmonary hypertension (PH). However, the mechanisms by which biophysical properties of the vascular extracellular matrix (ECM) relate to metabolic processes important in PH remain undefined. In this work, we examined cultured pulmonary vascular cells and various types of PH-diseased lung tissue and determined that ECM stiffening resulted in mechanoactivation of the transcriptional coactivators YAP and TAZ (WWTR1). YAP/TAZ activation modulated metabolic enzymes, including glutaminase (GLS1), to coordinate glutaminolysis and glycolysis. Glutaminolysis, an anaplerotic pathway, replenished aspartate for anabolic biosynthesis, which was critical for sustaining proliferation and migration within stiff ECM. In vitro, GLS1 inhibition blocked aspartate production and reprogrammed cellular proliferation pathways, while application of aspartate restored proliferation. In the monocrotaline rat model of PH, pharmacologic modulation of pulmonary vascular stiffness and YAP-dependent mechanotransduction altered glutaminolysis, pulmonary vascular proliferation, and manifestations of PH. Additionally, pharmacologic targeting of GLS1 in this model ameliorated disease progression. Notably, evaluation of simian immunodeficiency virus–infected nonhuman primates and HIV-infected subjects revealed a correlation between YAP/TAZ–GLS activation and PH. These results indicate that ECM stiffening sustains vascular cell growth and migration through YAP/TAZ-dependent glutaminolysis and anaplerosis, and thereby link mechanical stimuli to dysregulated vascular metabolism. Furthermore, this study identifies potential metabolic drug targets for therapeutic development in PH.

Authors

Thomas Bertero, William M. Oldham, Katherine A. Cottrill, Sabrina Pisano, Rebecca R. Vanderpool, Qiujun Yu, Jingsi Zhao, Yiyin Tai, Ying Tang, Ying-Yi Zhang, Sofiya Rehman, Masataka Sugahara, Zhi Qi, John Gorcsan III, Sara O. Vargas, Rajan Saggar, Rajeev Saggar, W. Dean Wallace, David J. Ross, Kathleen J. Haley, Aaron B. Waxman, Victoria N. Parikh, Teresa De Marco, Priscilla Y. Hsue, Alison Morris, Marc A. Simon, Karen A. Norris, Cedric Gaggioli, Joseph Loscalzo, Joshua Fessel, Stephen Y. Chan

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Figure 1

ECM stiffening activates glycolysis and glutaminolysis.

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ECM stiffening activates glycolysis and glutaminolysis. (A) By extracell...
(A) By extracellular flux analysis, PAECs cultivated in stiff matrix displayed decreased oxygen consumption rate (OCR) and increased extracellular acidification rate (ECAR), reflective of glycolysis. (B) PAECs cultivated in stiff matrix displayed increased basal glycolysis and corresponding decreased glycolytic reserve (as assessed by the difference between oligomycin A–induced ECAR and basal ECAR). (C) Basal OCR, ATP-dependent OCR (difference between basal OCR and oligomycin A–inhibited OCR), and respiratory reserve (maximal FCCP-induced OCR) were decreased in PAECs in stiff matrix. (D) MitoTracker (Thermo Fisher Scientific) labeling confirmed a decrease of mitochondrial activity in PAECs in stiff matrix. (E) Major metabolites in anaplerosis and glycolysis were measured in this study. GLS and PC generate the major anaplerotic metabolites (blue), feeding into the TCA cycle (black) and supporting the anabolic demand for biosynthesis (green). LDHA modulates glycolysis (red). α-KG, α-ketoglutarate; OAA, oxaloacetic acid. (F) In PAECs in stiff matrix, increased lactate/pyruvate ratio was observed, consistent with increased glycolysis and decreased oxidative phosphorylation. (G) In these same cells, glutamine, pyruvate, and succinate were decreased, while glutamate and aspartate were increased. (H) Released lactate was progressively increased in PAECs cultivated in an increasing gradient of matrix stiffness. (I–K) In PAECs, GLS1, LDHA, and PC expression was increased by matrix stiffening, as confirmed by RT–quantitative PCR (RT-qPCR) (CTGF, positive control) (I), and by immunoblot and densitometry (J and K). In all panels, mean expression in controls (soft matrix) was assigned a fold change of 1, to which relevant samples were compared. Data are expressed as the mean ± SEM (*P < 0.05, §P < 0.01, #P < 0.001) of at least 3 independent experiments performed in triplicate. Paired samples were compared by 2-tailed Student’s t test, while 1-way ANOVA and post-hoc Tukey’s tests were used for group comparisons. Scale bar: 20 μm. See also Supplemental Figure 1 (similar results were found in PASMCs).