Thoracic aortic disease in tuberous sclerosis complex: molecular pathogenesis and potential therapies in Tsc2+/- mice

Abstract

Tuberous sclerosis complex (TSC) is a genetic disorder with pleiotropic manifestations caused by heterozygous mutations in either TSC1 or TSC2. One of the less investigated complications of TSC is the formation of aneurysms of the descending aorta, which are characterized on pathologic examination by smooth muscle cell (SMC) proliferation in the aortic media. SMCs were explanted from Tsc2(+/-) mice to investigate the pathogenesis of aortic aneurysms caused by TSC2 mutations. Tsc2(+/-) SMCs demonstrated increased phosphorylation of mammalian target of rapamycin (mTOR), S6 and p70S6K and increased proliferation rates compared with wild-type (WT) SMCs. Tsc2(+/-) SMCs also had reduced expression of SMC contractile proteins compared with WT SMCs. An inhibitor of mTOR signaling, rapamycin, decreased SMC proliferation and increased contractile protein expression in the Tsc2(+/-) SMCs to levels similar to WT SMCs. Exposure to alpha-elastin fragments also decreased proliferation of Tsc2(+/-) SMCs and increased levels of p27(kip1), but failed to increase expression of contractile proteins. In response to artery injury using a carotid artery ligation model, Tsc2(+/-) mice significantly increased neointima formation compared with the control mice, and the neointima formation was inhibited by treatment with rapamycin. These results demonstrate that Tsc2 haploinsufficiency in SMCs increases proliferation and decreases contractile protein expression and suggest that the increased proliferative potential of the mutant cells may be suppressed in vivo by interaction with elastin. These findings provide insights into the molecular pathogenesis of aortic disease in TSC patients and identify a potential therapeutic target for treatment of this complication of the disease.

Figure 1.

MRA and pathological examination of the aorta of a patient with TSC2 mutation. (A) A coronal maximum intensity pixel reconstruction from contrast-enhanced MRA demonstrates a multilobulated aneurysm (arrows) involving the distal thoracic and abdominal aorta, with extension below the renal arteries. The distal thoracic aorta harbored the most proximal aneurysm dilation, measuring 2.8 cm in length and 2.4 cm at its greatest diameter. The second fusiform dilation was located in the proximal abdominal aorta, which was 5.3 cm in length, and from which the celiac artery arose. The third fusiform dilation involved the origins of the superior mesenteric artery and both renal arteries. The distal abdominal aorta was normal in caliber, with no involvement of the common iliac arteries. (B) A chest CT with contrast shows two aneurysms in the general region of the proximal and distal graft anastomosis. The first aneurysm is located in the descending thoracic aorta extending anteriorly from the aorta/graft at the level of T8 to L1 and measuring about 6.5 cm in transverse and 3.5 cm in AP dimension. There is a thrombotic layer around the aneurysm that measures about 1.2 cm in thickness. The second aneurysm originates at the level of L1–L2 and extends anteriorly from the abdominal aorta, measuring 3.2 cm in AP dimension × 3 cm transversely, and involves the origin of the right renal artery. (C) H&E, SMC α-actin and Movat pentachrome staining of aortas from control and TSC patient. H&E staining demonstrates SMC disarray and hyperplasia in the subintimal layer of the media in patient with TSC2 mutation. α-Actin staining confirms the nodules of cells in the subintimal layer are SMCs in patient's aorta. SMCs in the affected areas of the aorta and from control aorta were quantified by images collection from five fields of vision at ×400 magnification. Counting of the SMCs confirmed a significant increase of SMCs in patient's affected aorta (P < 0.005). Movat staining shows medial disruption in the aorta of the TSC patient that is characterized by nodules of SMCs (red), accumulation of collagen (yellow) and loss and fragmentation of elastic fibers (black). Arrowheads indicate the location of the IEL (stained black), confirming that the pathology occurs primarily in the subintimal layer. Magnification is indicated on each set of panels.

Figure 2.

Tsc2^+/− SMCs demonstrate increased proliferation and decreased expression of contractile proteins. (A) BrdU assay demonstrates that cell proliferation in Tsc2^+/− mouse SMCs was increased compared with WT SMCs. Proliferation of Tsc2^+/− SMCs explanted from descending aorta is significantly higher than SMCs explanted from the ascending aorta. (B) Effects of Tsc2^+/− on cell cycle distribution. After staining with PI, cell cycle distribution was analyzed using a flow cytometer. The data indicated that the number of Tsc2^+/− SMCs were significantly decreased in G0/G1 phase and significantly increased in S and G2/M phases compared with WT SMCs. Data are reported as means ± SD of three independent experiments. (C) Increase of the phosphorylation of proteins involved in mTOR signaling pathway in cell lysates from the Tsc2^+/− SMCs compared with WT SMCs. (D) qPCR analysis of mRNA isolated from SMCs from WT and Tsc2^+/− aorta. The SMCs explanted from ascending and descending aorta demonstrate that Tsc2^+/− SMCs have significantly reduced expression of SMC contractile genes, including Acta2, Cnn1 and Actg2. In contrast, SMCs from both Tsc2^+/− and WT mice express similar amounts of cytoskeletal genes, including Actg1 and Actb. Tsc2^+/− SMCs significantly increase expression of SMC de-differentiation marker, S100A4, particularly SMCs explanted from the descending aorta. Gene expression levels are standardized to Gapdh. *P < 0.05, **P < 0.01, ***P < 0.001. (E) Immunoblot analysis of SMC lysates confirms reduced levels of SMC contractile protein in SMCs explanted from Tsc2^+/− aorta compared with those from WT mice. Protein levels are normalized to Gapdh.

Figure 3.

Phenotypic abnormalities in Tsc2^+/− SMCs are reversed by blocking mTOR signaling with rapamycin treatment. (A) Rapamycin treatment inhibited the phosphorylation of pS6 and p70 S6K in both mutant and WT cells. (B) BrdU assay demonstrates that rapamycin significantly inhibits SMC proliferation, especially the SMCs explanted from Tsc2^+/− aorta. (C) SMC cell counts by FACS analysis demonstrate that the number of Tsc2^+/− SMCs in G0/G1 phase is decreased and cells in S and G2/M phases increased compared with WT SMCs. Quantitative assessment of the percentage of SMCs at G0/G1, S and G2/M phases after treatment with rapamycin indicates that rapamycin induces cell cycle arrest at G0/G1 phase. Data are reported as means ± SD of three independent experiments. (D) qPCR analysis of mRNA isolated from SMCs from WT and Tsc2^+/− aorta indicates that expression of SMC contractile genes (Acta2, Cnn1, Myh11, Actg2) significantly increases after treatment with rapamycin, and expression of S100A4 significantly decreases. RNA levels were normalized to Gapdh. (E) Protein levels of SMC contractile proteins, Cnn1 and Acta2, in SMCs explanted from control and Tsc2^+/− after treated with rapamycin. Protein levels were normalized to Gapdh. (F) Immunofluorescence analysis of α-actin and stress fibers in cultured SMCs from control and Tsc2^+/− aorta. SMC nuclei were counterstained with DAPI (blue). Magnification ×600.

Figure 4.

Histological and morphometric analysis of injured carotid arteries from WT and Tsc2^+/− mice. (A) Representative photomicrographs of carotid arteries from WT (top panels) and Tsc2^+/− (bottom panels) mice stained with H&E for 21 days following no injury (left panels) or injury (right panels). Scale bars represent 50 μm. Results are representative of seven independent experiments. (B) Neointima area of uninjured and injured carotid artery cross-sections from WT and Tsc2^+/− mice. Data represent mean neointima area of seven cross-sections ± SD, n = 7. For Tsc2^+/− uninjured versus injured, P < 0.01, and for WT injured versus Tsc2^+/− injured, P < 0.01. I/M ratio of uninjured and injured carotid artery cross-sections from WT and Tsc2^+/− mice. Data represent mean I/M ratio of seven cross-sections ± SD, n = 7. For Tsc2^+/− uninjured versus injured, P < 0.01, and for WT injured versus Tsc2^+/− injured, P < 0.01. Percentage of carotid artery stenosis 21 days following injury in WT and Tsc2^+/− mice. Data represent mean percent stenosis of seven cross-sections ± SD, n = 7, P<0.05. (C) Representative photomicrographs of H&E-stained carotid arteries from WT (top panels) and Tsc2^+/− (bottom panels) mice 21 days following no injury and vehicle treatment (left panels), injury and vehicle treatment (middle panels) or injury and rapamycin treatment (right panels). Scale bars represent 50 μm. Results are representative of seven independent experiments. (D) I/M ratio of injured carotid artery cross-sections from vehicle and rapamycin-treated WT and Tsc2^+/− mice. Data represent mean I/M ratio of seven cross-sections ± SD, n = 7 mice. For Tsc2^+/− uninjured versus injured with vehicle treatment, P< 0.01; for Tsc2^+/− injured with vehicle versus injured with rapamycin treatment, P< 0.01; for WT injured with vehicle treatment versus Tsc2^+/− injured with vehicle treatment, P < 0.05.

Figure 5.

Effect of α-elastin exposure on cell proliferation and protein levels in Tsc2^+/− SMCs. (A) SMCs explanted from WT and Tsc2^+/− mice were cultured at α-elastin concentration of 0, 0.5, 1 and 5 mg/ml, respectively, for 72 h. BrdU assay demonstrated that proliferation of Tsc2^+/− SMCs was inhibited in a dose-dependent manner. A specific Rho inhibitor (Y27632) could prevent the reduction of Tsc2^+/− SMC proliferation by α-elastin. (B) Western blot assay demonstrated that α-elastin-treated Tsc2^+/− SMCs increased expression level of p27^kip1, inhibited mTOR signaling and decreased SMC proliferation. In addition, α-elastin did not alter expression level of contractile proteins in Tsc2^+/− SMCs.