Oxidative stress modulates vascular smooth muscle cell phenotype via CTGF in thoracic aortic aneurysm

Abstract

Aims: Dissection and rupture of the ascending aorta are life-threatening conditions resulting in 80% mortality. Ascending aortic replacement in patients presenting with thoracic aortic aneurysm (TAA) is determined by metric measurement. However, a significant number of dissections occur outside of the parameters suggested by the current guidelines. We investigate the correlation among altered haemodynamic condition, oxidative stress, and vascular smooth muscle cell (VSMC) phenotype in controlling tissue homoeostasis.

Methods and results: We demonstrate using finite element analysis (FEA) based on computed tomography geometries that TAA patients have higher wall stress in the ascending aorta than non-dilated patients. We also show that altered haemodynamic conditions are associated with increased levels of reactive oxygen species (ROS), direct regulators of the VSMC phenotype in the microregional area of the ascending aorta. Using in vitro and ex vivo studies on human tissues, we show that ROS accumulation correlates with media layer degeneration and increased connective tissue growth factor (CTGF) expression, which modulate the synthetic VSMC phenotype. Results were validated by a murine model of TAA (C57BL/6J) based on Angiotensin II infusion showing that medial thickening and luminal expansion of the proximal aorta is associated with the VSMC synthetic phenotype as seen in human specimens.

Conclusions: Increased peak wall stress correlates with change in VSMC towards a synthetic phenotype mediated by ROS accumulation via CTGF. Understanding the molecular mechanisms that regulate VSMC towards a synthetic phenotype could unveil new regulatory pathways of aortic homoeostasis and impact the risk-stratification tool for patients at risk of aortic dissection and rupture.

Keywords: CTGF; ROS; Thoracic aortic aneurysm; VSMC phenotype.

Figure 1

Altered wall stress correlates with accumulation of ROS and dysfunctional microstructure of the aorta. (A) Stress contour plots of a patient with a normal ascending aorta (left) compared with one with an aneurysmal ascending aorta (right). Concave and Convex sides are indicated by CV and CX, respectively. (B) Graph showing walls stress values (C) Modified Movat Pentachrome staining and Verhoeff-Van Gieson staining on ascending aorta tissues from non-dilated and aneurysmal patients. Images are representative of staining performed on n = 3 tissues/group of patients; ×10 and ×40 magnification. (D) Fold change gene expression for superoxide dismutase 1, and 2 (SOD1, 2). qPCR data were normalized against actinB (E) Nitrotyrosine staining in aortic media from patients with and without aneurysm (magnification ×10 and ×40).

Figure 2

Synthetic VSMC phenotype and thoracic aortic aneurysm. (A) Western blotting for smoothelin B, smooth muscle actin (SMA) and vimentin (VIM) from dilated and non-dilated patients. GAPDH as loading control. (B) Densitometry analysis over ascending aorta diameter (measured by CT scan) of the relative patient. (C) Immunohistochemistry for smoothelin B and vimentin on tissue sections. Images are representatives of staining performed on n = 6 tissues/group of patients. (*P < 0.05). Magnification ×40. (D) Fold change gene expression for SMA, OPN, MMP-9 in ascending aorta tissues (n = 4 patients/group) normalized against actinB. (E) Myocardin and Elk-1 expression tested by western blotting in the ascending aorta tissues from non-dilated and dilated patients and (F) relative densitometry. (G) Immunofluorescence staining for Elk-1 (green) and SRF (red) on isolated cells from two groups of patients. Magnification ×60.

Figure 3

Overexpression of connective tissue growth factor is associated with thoracic aortic aneurysm. (A) RT² PCR Array performed in n = 4 tissue/group for ECM and cellular adhesion molecules. Table represents genes up or down-regulated more than two fold in aneurismal tissues compared with non-dilated. (B) Immunohistochemistry showing CTGF protein levels in non-dilated and aneurismal tissues; ×40 magnification. (C) CTGF expression validation by qPCR performed on n = 10 tissues/group of patients normalized against actinB. (D) Nitrotyrosine dot blot and CTGF western blotting of whole cell extracts (E) Dot blot for nitrotyrosine and CTGF using tissue extracts from the ascending aorta [convex (CX) and concave (CV) side] and (F) relative densitometry analysis over ascending aorta diameter of each patient.

Figure 4

CTGF modulates VSMC synthetic phenotype in vitro. (A) vimentin and smoothelin B expression in VSMCs treated with hrCTGF in the presence or absence of CTGF neutralizing antibody and relative densitometry. (B) vimentin and smoothelin B expression assayed by qPCR (*P < 0.05). (C and D) In vitro hrCTGF treatment of VSMCs on ECM components analysed by qPCR (*P < 0.05). (E) Smoothelin B and vimentin expression in VSMC isolated from non-dilated and dilated tissues treated with 80 ng/mL of hrCTGF. GAPDH as a loading control. Data are means ± SD.

Figure 5

Oxidative stress mediates the expression of synthetic VSCM markers via CTGF. (A) Western blotting and densitometry showing CTGF expression in VSMC treated with H₂O₂ in the presence or absence of MnTMPyP. GAPDH was used as a loading control. (B and C) CTGF, vimentin, MMP-2, and MMP-9 expression in VSMC treated with 100 or 200 μM H₂O₂ assayed by qPCR. Fold change were normalized against actinB (*P < 0.05). (D) Western blotting and densitometry of VSMCs treated with 100 μM H₂O₂ assayed for vimentin expression in the presence or absence of CTGF neutralizing antibody. Data are means ± SD.

Figure 6

Angiotensin II infusion provokes media thickness, formation of aortic aneurysms, and vimentin accumulation. (A) Ang II-induced ascending aortic aneurysm (B) AA expansion (in mm) in mice with Ang II-induced TAA compared with saline-infused controls measured by TEE. (C) Histology of the ascending aorta showing medial thickness and measure of luminal diameter (mm). (D) CTGF and vimentin quantification by qPCR performed using RNA obtained from the ascending (AA) and descending (DA) aorta. (E and F) Immunohistochemistry showing CTGF, nitrotyrosine and vimentin expression in saline vs. ANGII-induced aneurysmal aorta. Magnification ×10–40. Data are means ± SD.*P < 0.05.