The Role And Underlying Mechanism Of TRPC5Channel In The Development Of Early Atherosclerotic Plaques

BackgroundsCoronary heart disease (CHD) has become a major cause of mortality and morbidities worldwide. Atherosclerosis (AS) is the key pathophysiology basis of CHD. Smooth muscle cells (SMCs) play a key role in the formation and development of AS plaque through migration, proliferation, phenotype transformation and production of extracellular matrix. Therefore, it is very important for prevention and treatment of CHD to study the mechanism of function changes about SMCs in the AS.Previous studies found that intracellular Ca2+was a key second messenger and play an important role in regulating functions of SMCs. In recent years, calcium influx conducted by store-operated channels (SOCs) have been paid more and more attention in the cell function and diseases. Classical transient receptor potential (TRPC) channel is the main subtype of SOCs and can be activated by the activation of IP3leading to continuous calcium influx. So more and more studies investigate the role of TRPC channel in the regulation of SMCs function.Expression of TRPC channels was found in human coronary artery and cerebral artery; TRPC participates in the vein graft intimal hyperplasia. At the same time, studies have found that TRPC5play an important role in regulating cell function induced by lipids and is considered as a lipid receptor. Sphingosine1-phosphate (SIP) as one component of ox-LDL can activate TRPC5channel.As we known. both ox-LDL and SMCs are important effectors in the development of AS. especially TRPC channels participated in the vein intimal hyperplasia. we can conclude that TRPC channels may play an important role in AS. However, previous studies only investigated TRPC5in vitro expression system or its physiological effect, it is unclear whether TRPC5participate in the occurrence and development of AS plaque. Therefore, we hypothesized:TRPC5channel was upregulated in AS early plaque; blocking TRPC5channel could inhibit the development of AS early plaques by reducing numbers of SMCs and collagen content of plaques. We designed a series of in vivo and in vitro experiments to prove this hypothesis.Objectives1. To study expression changes of TRPC channel in AS:2. To investigate whether blocking TRPC5channels in vivo can inhibit development of AS early plaque and the underline mechanism.Methods1. Animal model of AS early plaque:ApoE-/-mice (male.8week old) were fed with high fat diet and underwent surgery with a perivascular collar around the right carotid artery to induce AS plaques.2. Blocking TRPC5channels in vivo:Mice were administrated200μl of different reagents the day before surgery, and were randomly divided into4groups (20per group):PBS group. IgG group(100μg). TRPC5T5E3Ab low-dose group(50μg) and TRPC5T5E3Ab high-dose group(100μg). Mice were given with drugs once a week, and were euthanized at the end of8weeks after surgery.3. General information of mice:At the end of8weeks, body weight, heart rate (HR) and blood pressure were measured by a noninvasive tail-cuff system. Serum levels of total cholesterol (TC). triglyceride (TG). low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured using an ELISA.4. Histological and immunohistochemical analysis:Serial transverse cryosections of carotid artery plaques were stained with hematoxylin and eosin, picosirius red. And oil-red O staining. Corresponding sections were used for immunohistochemical analysis with the following antibodies:anti-mouse monocyte/macrophage monoclonal antibodies(MOMA-2). anti-alpha smooth muscle actin antibodies(a-SM-actin).. anti-mouse TRPC5channel antibody. The plaque area, lipid area, collagen area and the intensive optical density (IOD) of immunostaining positive area for smooth muscle and macrophage were measured with an automated image analysis system and calculated as the proportion of plaque area.5. Real-time quantitative RT-PCR reaction was used to detect mRNA expression level of TRPC channels.6. Verification of the specificity of T5E3Ab blocking effect on TRPC5channel: TRPC5channel was stable expressed in HEK293cell line (TRPC5-HEK293cells), patch clamp technique was used to detect blocking effects of T5E3Ab on TRPC5channel current, calcium imaging was used to detect blocking effects of T5E3Ab on intracellular calcium increasing mediated by TRPC5channel.Results1. Verification of blocking effects of T5E3Ab on TRPC5channel:TRPC5channel currents were recorded in TRPC5-HEK293cells, patch clamp technique and calcium imaging showed that TRPC5channel activator Gd3+(100μM) could induce channel currents and intracellular calcium increase, which could be blocked by T5E3Ab.2. Comparing general data of mice:At the end of8weeks after surgery, there was no significant difference about body weight, SBP, DBP or serum levels of TC, TG, LDL-C, HDL-C and blood cell components among the4groups of ApoE-/-mice (P>0.05).3. Expression of TRPC channels in AS early plaques:At the end of8weeks after surgery, TRPC5channel mRNA expressions of plaque tissues were higher than the artery without perivascular collar (P<0.05). There were no significant difference about other types of TRPC channel mRNA expressiuon and about TRPC5channel mRNA among the4groups of ApoE-/-mice (P>0.05). 4. The relationship between TRPC5mRNA expression and characteristics of AS early plaque:At the end of8weeks after surgery, compared with the artery without perivascular collar in PBS group, plaques were obvious with more lipid content and TRPC5expression (P<0.05). The correlation analysis showed that TRPC5channel expression was positively correlated with plaque burden, lipid content, smooth muscle cells and collagen content (P<0.05). with no correlation with macrophage content of plaque (P>0.05).5. Effect of blocking TRPC5channels on the AS plaque burden:At the end of8weeks after surgery, compared with PBS and IgG (100μg) groups, blocking TRPC5channels significantly decreased plaque burden(P<0.05). Compared with low dose group (T5E3Ab50μg). plaque burden was further reduced in high dose group (T5E3Ab100μg,.P<0.05).6. Effect of blocking TRPC5channels on lipid content of AS plaques:At the end of8weeks after surgery, compared with PBS and IgG (100μg) groups, blocking TRPC5channels significantly decreased plaque lipid content (?<0.05). Compared with low dose group (T5E3Ab50μg). lipid content was further reduced in high dose group (T5E3Ab100ug) without statistically significance(P<O.O5).7. Effect of blocking TRPC5channels on collagen content of plaques:At the end of8weeks after surgery, compared with PBS and IgG (100μg) groups, blocking TRPC5channels significantly decreased collagen content of plaques (P<0.05), Compared with low dose group (T5E3Ab50μg). collagen content of plaques was further reduced in high dose group (T5E3Ab100μg, P<0.05).8. Effect of blocking TRPC5channels on smooth muscle cell content of plaques:At the end of8weeks after surgery, compared with PBS and IgG (100μg) groups, blocking TRPC5channels significantly decreased smooth muscle cell content of plaques (P<0.05). Compared with low dose group (T5E3Ab50μg). smooth muscle cell content of plaques was further reduced in high dose group (T5E3Ab100μg. p<0.05).9. Effect of blocking TRPC5channels on macrophage content of plaques:At the end of8weeks after surgery, there were no significant difference about macrophage content of plaques among the4groups of ApoE-/-mice(P>0.05).Conclusions1. TRPC5channel is upregulated in AS early plaque development. upregulated TRPC5channel is positively correlated with plaque burden, lipid content, smooth muscle cells and collagen content with no relation to plaque macrophage content.2. Blocking TRPC5channel with specific blocking antibody T5E3Ab can inhibit AS early plaque development. BackgroundsIn the development of AS early plaque, smooth muscle cells (SMCs) migrate to intima. proliferate, and phagocytize lipid resulting in formation of foam cells. Therefore it is very important for prevention and treatment of CHD to study the mechanism of functional regulation about SMCs in the AS.In SMCs. intracellular Ca2+not only participate in the regulation of contraction and relaxation, but also regulates various cellular functions, such as proliferation. secretion, and differentiation. Regulation of SMCs intracellular Ca2+is affected by a variety of ion channels. In recent years, classical transient receptor potential (TRPC) channel was found to lead to continuous calcium influx. So more and more studies investigate the role of TRPC channel in the regulation of SMCs function.Our above study found that TRPC5channel is upregulated in AS early plaque development, and that blocking TRPC5channel with specific blocking antibody T5E3Ab could inhibit AS early plaque development by decreasing plaque smooth muscle cells and collagen content. However, the underline mechanism is unclear. Previous studies have found that TRPC5channel can be used as a lipid receptor and that TRPC channels play an important role in the migration of SMCs. Therefore we assume that the underline mechanism of TRPC5channel in inhibiting AS plaque development:TRPC5channel participate in the regulation of ox-LDL induced intracellular calcium increasing and store-operated calcium influx, thus affect the proliferation and migration of smooth muscle cells.Objectives1. To investigate the role of TRPC5channels in the ox-LDL induced smooth muscle intracellular calcium increase and store-operated calcium influx;2. To investigate the role and underline mechanism of TRPC5channels in the proliferation and migration of smooth muscle cells induced by ox-LDLMethods1. Culture primary human aortic smooth muscle cells, stimulate SMCs with different concentrations of ox-LDL (25μg/ml,50μg/ml,100μg/ml) for24h, and treat SMCs with50μg/ml ox-LDL for different times (2h、6h、24h and48h). Real-time fluorescence quantitative RT-PCR was used to detect mRNA expression of TRPC5channel.2. Calcium imaging experiments:SMCs were cultured on13mm diameter coverslips and treated with50μg/ml ox-LDL for24h. Thapsigargin (Tg) was used to deplete intracellular calcium stores at free calcium bath solution following with restoring2mM calcium at bath solution, which is called store-operated calcium influx. With blocking antibody TRPC5T5E3Ab, we explore that TRPC5channel participated in the regulation of ox-LDL induced intracellular calcium increasing and store-operated calcium influx.3. Patch-clamp electrophysiology experiments:With blocking antibody TRPC5T5E3Ab, we explore that TRPC5channel participated in the regulation of ox-LDL induced calcium channel currents.4. BrdU cell proliferation assay:With blocking antibody TRPC5T5E3Ab, we explore that TRPC5channel participated in ox-LDL induced cell proliferation.5. Transwell cell migration experiment:With blocking antibody TRPC5T5E3Ab, we explore that TRPC5channel participated in ox-LDL induced cell migration. Results1. Effect of ox-LDL on the expression of TRPC5channel mRNA expression in SMCs:ox-LDL could significantly increase the expression of TRPC5channel mRNA with time-and concentration-dependence (P<0.05).2. The role of TRPC5channel in ox-LDL induced intracellular calcium increasing and store-operated calcium influx of SMCs:Compared with control.50μg/ml ox-LDL induced intracellular calcium increasing (P<0.05), which could be blocked by TRPC5blocking antibody T5E3Ab (P<0.05). Tg (1μM) is used to deplete intracellular calcium stores at free calcium bath solution following with restoring2mM calcium at bath solution, continuous calcium influx will occur, which is called store-operated calcium influx. When pretreated with50μg/ml ox-LDL for30min. store-operated calcium influx was higher than the control (P<0.05). which could be blocked by TRPC5blocking antibody T5E3Ab (P<0.05).3. The role of TRPC5channel in ox-LDL induced channel currents of SMCs: Compared with control.50μg/ml ox-LDL induced channel currents(P<0.05), which could be blocked by TRPC5blocking antibody T5E3Ab (P<0.05).4. The role of TRPC5channel in ox-LDL induced proliferation of SMCs: Compared with control,50μg/ml ox-LDL induced more proliferation of SMCs (P<0.05). which could be blocked by TRPC5blocking antibody T5E3Ab and calcium chelator EGTA (P<0.05).5. The role of TRPC5channel in ox-LDL induced migration of SMCs: Compared with control.50μg/ml ox-LDL induced more migration of SMCs (P<0.05), which could be blocked by TRPC5blocking antibody T5E3Ab and calcium chelator EGTA(P<0.05).Conclusions1. TRPC5channel is unregulated by ox-LDL in artery smooth muscle cells;2. TRPC5channel participated in the regulation of ox-LDL induced intracellular calcium increasing and store-operated calcium influx:3. Blocking TRPC5channel can reduce the proliferation and migration of smooth muscle cells induced by ox-LDL BackgroundOur above study found that TRPC5channel was upregulated in AS early plaque development, and that blocking TRPC5channel with specific blocking antibody T5E3Ab could inhibit AS early plaque development by decreasing plaque smooth muscle cells and collagen content. In vitro experiments, we revealed that TRPC5channel was unregulated by ox-LDL in artery smooth muscle cells, TRPC5channel participated in the regulation of ox-LDL induced intracellular calcium increasing and store-operated calcium influx and that blocking TRPC5channel can reduce the proliferation and migration of smooth muscle cells.Because ox-LDL is a key factor in the pathogenesis of AS, it is very important for revealing the special role of TRPC5channel in AS to study the regulatory effect and mechanism of ox-LDL on TRPC5channel. On the other hand, statins are one of the most important drugs for treatment of AS, previous studies have shown that statins have pleiotropic effects beyond their lowering lipid. However, it has not been reported whether statins can affect the TRPC5channel function.Meanwhile, studies found that intracellular reactive oxygen species (ROS) play an important role in the regulation of ion channels. Recent study has found that TRPC5is sensitive to antioxidant. A lot of studies have also proved that ox-LDL increase intracellular ROS and that statins reduce intracellular ROS. So we hypothesized that ox-LDL and statins regulate TRPC5channel function via affecting intracellular ROS. If this hypothesis is confirmed, we not only reveal a new regulation mechanism to regulating the TRPC5channel, but also further prove that the new target of statins pleiotropic effects.Objectives1. To study the effect of ox-LDL on stably expressed TRPC5channel:2. To investigate the effect of simvastatin on stably expressed TRPC5;3. To explore the possible mechanism of regulating ox-LDL and simvastatin effect on TRPC5channel function.Methods1. HEK293cell line with stable expression of TRPC5channel (HEK-TRPC5): cultured HEK-TRPC5cells in standard methods. Because the cell carries the tetracycline (Te) transcriptional repressor. TRPC5channel overexpression was induced by1μg/ml Te for24h.2. Calcium imaging experiments:Thapsigargin (Tg1μM) was used to deplete intracellular calcium stores at free calcium bath solution following with restoring2mM calcium at bath solution. which is called store-operated calcium influx. To study effects of different concentrations of ox-LDL (25μg/ml.50μg/ml.100μ g/ml) and simvastatin (10μM,20μM,50μM) on TRPC5channel-mediated calcium increasing and store-operated calcium influx.3. Patch-clamp electrophysiology experiments:we explore effects of different concentrations of ox-LDL (25μg/ml,50μg/ml,100μg/ml) and simvastatin (10μ M，20μM,50μM) on TRPC5channel currents.4. Intracellular ROS fluorescence probe experiments were used to detect effects of ox-LDL and simvastatin on the intracellular ROS in TRPC5channel stable expressed HEK293cells.Results1. Study of TRPC5channel function in HEK-TRPC5cells: TRPC5channel overexpression was induced by1μg/ml Te. In Te induced (Te+)cells. patch clamp technique and calcium imaging showed that TRPC5channel activator Gd3+(100μM) could induce channel currents (P<0.05) and intracellular calcium increase (P<0.05), which could be blocked by T5E3Ab(P<0.05). In non-induced (Te-) cells, there was no response to Gd33+(100μM).2. Store-operated calcium influx in HEK-TRPC5cells:Tg (1μM) is used to deplete intracellular calcium stores at free calcium bath solution following with restoring2mM calcium at bath solution, continuous calcium influx will occur, which is called store-operated calcium influx. Compared with Te-cells, store-operated calcium influx was higher in Te+cells (P<0.05).3. Effect of ox-LDL on stably expressed TRPC5channel:Different concentrations of ox-LDL (25μg/ml,50μg/ml,100μg/ml) increased TRPC5channel mediated intracellular calcium increase, store-operated calcium influx and TRPC5channel currents, which could be inhibited by specific TRPC5channel blocker T5E3Ab (P<0.05).4. Effect of simvastatin on stably expressed TRPC5channel:Different concentrations of simvastatin (10μM,20μM,50μM) could inhibit TRPC5channel mediated intracellular calcium increase, store-operated calcium influx and TRPC5channel currents (P<0.05).5. The possible mechanism of regulating TRPC5channel function by ox-LDL and simvastatin:Antioxidants (tiron,1mM and DPI,10μM) could inhibit TRPC5channel activator Gd3+induced intracellular calcium increase and TRPC5channel currents (P<0.05). Antioxidants could also block the activation of TRPC5channel induced by ox-LDL (P<0.05). Exogenous H2O2(100μM) could activate TRPC5channel and overcome the inhibiting effect of simvastatin on TRPC5channel (P<0.05). ox-LDL enhanced intracellular ROS which could be reduced by antioxidants (P<0.05):simvastatin could reduce the intracellular ROS which could be reversed by exogenous H2O2(P<0.05).Conclusions1. ox-LDL increased TRPC5channel mediated intracellular calcium increase, store-operated calcium influx and TRPC5channel currents.2. Simvastatin could inhibit TRPC5channel mediated intracellular calcium increase. store-operated calcium influx and TRPC5channel currents.3. ox-LDL and simvastatin regulated TRPC5channel function via affecting intracellular ROS.