Chronic mTOR activation induces a degradative smooth muscle cell phenotype
Guangxin Li, … , Jay D. Humphrey, George Tellides
Guangxin Li, … , Jay D. Humphrey, George Tellides
Published February 10, 2020
Citation Information: J Clin Invest. 2020;130(3):1233-1251. https://doi.org/10.1172/JCI131048.
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Research Article Vascular biology

Chronic mTOR activation induces a degradative smooth muscle cell phenotype

Abstract

Smooth muscle cell (SMC) proliferation has been thought to limit the progression of thoracic aortic aneurysm and dissection (TAAD) because loss of medial cells associates with advanced disease. We investigated effects of SMC proliferation in the aortic media by conditional disruption of Tsc1, which hyperactivates mTOR complex 1. Consequent SMC hyperplasia led to progressive medial degeneration and TAAD. In addition to diminished contractile and synthetic functions, fate-mapped SMCs displayed increased proteolysis, endocytosis, phagocytosis, and lysosomal clearance of extracellular matrix and apoptotic cells. SMCs acquired a limited repertoire of macrophage markers and functions via biogenesis of degradative organelles through an mTOR/β-catenin/MITF–dependent pathway, but were distinguishable from conventional macrophages by an absence of hematopoietic lineage markers and certain immune effectors even in the context of hyperlipidemia. Similar mTOR activation and induction of a degradative SMC phenotype in a model of mild TAAD due to Fbn1 mutation greatly worsened disease with near-uniform lethality. The finding of increased lysosomal markers in medial SMCs from clinical TAAD specimens with hyperplasia and matrix degradation further supports the concept that proliferation of degradative SMCs within the media causes aortic disease, thus identifying mTOR-dependent phenotypic modulation as a therapeutic target for combating TAAD.

Authors

Guangxin Li, Mo Wang, Alexander W. Caulk, Nicholas A. Cilfone, Sharvari Gujja, Lingfeng Qin, Pei-Yu Chen, Zehua Chen, Sameh Yousef, Yang Jiao, Changshun He, Bo Jiang, Arina Korneva, Matthew R. Bersi, Guilin Wang, Xinran Liu, Sameet Mehta, Arnar Geirsson, Jeffrey R. Gulcher, Thomas W. Chittenden, Michael Simons, Jay D. Humphrey, George Tellides

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Figure 8

SMCs gain degradative functions.

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SMCs gain degradative functions. SMCs were cultured from thoracic aortas...
SMCs were cultured from thoracic aortas of 24-week-old, tamoxifen-treated Myh11-CreERT2 mT/mG (Tsc1+/+) and Tsc1fl/fl Myh11-CreERT2 mT/mG (Tsc1−/−) mice. Confocal microscopy of GFP+ SMCs (green) incubated with (A) LysoTracker Deep Red (white) that accumulates in perinuclear vesicles or (B) DQ Red–conjugated BSA (red) that is visualized after hydrolysis of the probe. See supplemental methods for details. Scale bars: 50 μm. (C) Fluorescence intensity of the cells by flow cytometry. (D) Flow cytometry of GFP+ SMCs cultured with rhodamine-labeled fibronectin (Fn) with or without chloroquine (CQ) to distinguish endocytosis-dependent uptake (n = 4–6) from lysosomal degradation (n = 3). (E) Flow cytometry of GFP+ SMCs cultured with or without rhodamine-labeled elastin and (F) confocal microscopy of cells sorted for dim or bright rhodamine fluorescence revealing elastin fragments (red, arrows) confirmed as intracellular by Z‑stack image. Scale bar: 10 μm. (G) Confocal microscopy of GFP+ SMCs (green) cultured with PKH26-labeled, heat-damaged erythrocytes (red, arrows) confirmed as intracellular by Z-stack image (scale bars: 25 μm); percentage GFP+ SMCs containing erythrocytes (n = 10). (H) Flow cytometry of GFP+ SMCs cultured without or with erythrocytes in the presence of control IgG, Axl-neutralizing Ab, or Axl:Fc chimeric protein. (I) Immunofluorescence microscopy of ascending aorta for erythrocyte antigen, TER-119 (white, arrow) with GFP (green), RFP (red), and DAPI (blue) overlay (scale bar: 100 μm); number of medial TER-119+ cells per cross section (cs) (n = 10). Data are represented as individual values with mean ± SEM bars. *P < 0.05, **P < 0.01, ***P < 0.001 for Tsc1−/− vs. Tsc1+/+ by t test.