Author + information
- 1Department of Cardiology, Qilu Hospital, Shandong University, Jinan, 250012, China
- 2Department of Otorhinolaryngology, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan, 250014, China
- 3Department of Cardiology, Jinan Central Hospital Affiliated to Shandong University, Shandong, Jinan, 250013, China
Coronary heart disease (CHD) has become a major cause of mortality and morbidities worldwide. Smooth muscle cells (SMCs) play a key role in the formation and development of atherosclerosis (AS), which is the key pathophysiology basis of CHD. We aimed to investigate expression changes of classical transient receptor potential (TRPC) channel in AS and whether blocking TRPC5 channels in vivo cbould inhibit development of AS and the underlining mechanism.
We established AS early plaque model in apoE-/- mice fed with high fat diet and underwent surgery with a perivascular collar around the right carotid artery. Mice were then administrated with 200μl of different reagents the day before surgery, and were randomly divided into 4 groups (20 per group): PBS group, IgG group(100μg), TRPC5 T5E3Ab low-dose group(50 μg) and TRPC5 T5E3Ab high-dose group(100 μg). Mice were given with drugs once a week, and were euthanized at the end of 8 weeks after surgery. The mRNA and protein expression of TRPC5 channel was detected by quantitative real-time PCR and immunohistochemical analysis. The plaque area, lipid area, collagen area and the intensive optical density (IOD) of immunostaining positive area for smooth muscle and macrophage were measured by histological and immunohistochemical analysis. In vitro, primary human aortic smooth muscle cells (SMCs) were cultured, stimulated with ox-LDL (50 μ g/ml) and treated with T5E3. Calcium imaging and patch clamp experiments were used to determine intracellular calcium, store-operated calcium influx and currents. BrdU cell proliferation assay and transwell cell migration experiment were used to detect ox-LDL induced SMCs proliferation and migration.
TRPC5 mRNA was increased significantly in early plaque and other subtypes of TRPC channels were not changed. TRPC5 channel expression was also found to be positively related to plaque burden, lipid content, smooth muscle cells and collagen content, with no relation to the content of macrophages. We further blocked TRPC5 channel in vivo using specific blocking antibody: the E3-targeted, externally acting, anti-TRPC5 blocking antibody (T5E3). Compared with control group, plaque burden, lipid content, SMCs and collagen content were significantly inhibited by T5E3. However, plaque content of macrophages was not changed by blocking antibody T5E3. We found that TRPC5 channel mRNA and protein expression were unregulated by ox-LDL in artery SMCs. Calcium imaging and patch clamp experiments revealed that ox-LDL induced intracellular calcium increasing and store-operated calcium influx, which could be inhibited by TRPC5 blocking antibody T5E3. Blocking TRPC5 channel by T5E3 Ab and calcium chelating agent EGTA also reduced the proliferation and migration of smooth muscle cells.
TRPC5 channel is upregulated in AS early plaque development, blocking TRPC5 channel can inhibit AS plaque development via reducing proliferation and migration of SMCs. TRPC5 channel conducted ox-LDL induced continuous calcium influx of SMCs.