Focal adhesion kinase–related pathways may be suppressed by metformin in vascular smooth muscle cells in high glucose conditions

Abstract Introduction Cardiovascular diseases are known as one of the important causes of death in patients with diabetes mellitus. Metformin is used as an oral medication for reducing blood sugar. In this study, the effects of metformin were investigated on the FAK gene expression levels, pFAK protein values, cell viability and migration rate of VSMCs in high glucose conditions. Materials and methods The FAK gene expression levels and pFAK protein values were evaluated in VSMCs treated with different doses of metformin (1, 5 and 7 mM), based on cell viability using RT‐qPCR, western blotting and MTT techniques. The cellular migration was evaluated by scratch assay. Results The FAK gene expression levels reduced significantly in metformin‐treated VSMCs at 24 h and 48 h periods (p < .0008 and p < .0001, respectively). The pFAK protein values reduced significantly at 24 h (5 mM and 7 mM metformin doses) and 48 h periods (p < .001). In agreement with pFAK protein values, cellular migration reduced significantly at 24 h and 48 h periods (p < .001). Conclusion The results showed that metformin may suppress the proliferation and migration of VSMCs via FAK‐related pathways and may retard the progression of vessel stenosis in diabetes.


| INTRODUC TI ON
Cardiovascular diseases account for approximately 65 percent of deaths in patients with diabetes mellitus. 1 It is well known that the pathogenesis of atherosclerosis is related to the proliferation and migration of vascular smooth muscle cells (VSMCs) within the media layer of arteries, so that these events promote atherogenic plaques led to vessel stenosis and restenosis. Molecular mechanisms behind VSMC proliferation and migration remain unclear. 2,3 Hyperglycemia, however, is not the only factor involved in the progression of cardiovascular diseases in diabetics but it is involved in several pathways including the activation of protein kinase C, the formation of advanced glycation end-products (AGEs) and the stimulation of lipoxygenase synthesis pathway. 4 Metformin is an oral medication used commonly for patients with type 2 diabetes and other diseases related to insulin resistance. [5][6][7] Moreover, metformin affects the liver, skeletal muscle and adipose tissues via decreasing oxidative stress, improving insulin sensitivity, blocking gluconeogenesis and promoting glucose uptake and consumption. [5][6][7][8] Metformin also inhibits VSMC function by targeting the AMPK signalling pathway, which may retard the process of atherosclerosis in diabetes. 9 FAK is a tyrosine kinase that regulates cell proliferation and migration through some signalling pathways. 10 Several agents such as inflammatory factors, cytokines and growth factors activate the focal adhesion kinase. 11 Since pFAK (phosphorylated FAK) was considered as a central protein to transduce a signal into the cell nucleus in the cellular signalling pathways (Figure 1), thus in this study, the effects of metformin were investigated on the changes in FAK gene expression levels, pFAK protein values, cell viability and migration rate of VSMCs in high glucose conditions.

| Cell viability
Cell viability was carried out using the MTT (methyl thiazol tetrazolium, Sigma-Aldrich) method. Briefly, 4000 cells per well were seeded in the 96-well plate and were incubated with different doses of metformin for 24 and 48 h periods. Then, the medium was discarded and the seeded cells were incubated with MTT solution (0.5 mg/ml; Sigma-Aldrich) for 4 h at 37°C. After removing the MTT solution, 200 μl of DMSO (Dimethyl sulfoxide) was added to dissolve the formed crystals, and finally, the plate was shaken for 15 min at room temperature.
Afterwards, optical density was measured at 570 nm wavelength.

| Real-time qPCR technique
Total RNA was extracted from VSMCs by GeneAll-Hybrid-R RNA purification kit (Seoul, South Korea). cDNA synthesis was performed according to the SMOBIO kit protocol (Hsinchu, Taiwan). SYBR Green PCR Master Mix (Amplicon Denmark) was used for F I G U R E 1 Cellular proliferation and migration via FAK/pFAK axis. The FAK is phosphorylated by kinasefollowed signalling pathways or autophosphorylated by the cellular membrane receptors. Then, pFAK transduces the signals into the downstream proteins directly or by other the cross-talked signalling pathways led to the cellular proliferation and migration events Finally, the membrane was exposed to enhanced chemiluminescence (ECL) reagent (RPN2235; Amersham Biosciences). Beta-actin antibody (CAT.#:4967 s; 1:1000 v/v; Cell Signalling Technology) was used to normalize the protein values. The band densities were identified using Image-J software.

| Scratch assay
To investigate cellular migration, a scratch assay test was per- The analysis of images was accomplished by Image-J software. Then, data were compared using the ANOVA test. p < .05 was the subject to be considered significant.

| FAK gene expression levels
The metformin significantly reduced the FAK gene expression levels in all the treated groups in both 24 h (p < .0008) and 48 h (p < .001) periods as compared to the control high glucose group (Figure 3,1A,1B).

| pFAK protein values
The results showed that 5 and 7 mM metformin values significantly reduced the FAK values (p < .0008). However, metformin dose (1 mM) had no significant difference in the Fak protein at 24 h period (p > 0.8). Furthermore, the FAK protein values significantly decreased at 48 h after treatment with different doses of metformin (1, 5 and 7 Mm) (p < .0002) (Figure 3,2A-C).

| Cell migration
As shown in Figure 4, the metformin values 5 and 7 mM significantly decreased the migration in 24 h period (p < .05). Additionally, at 48 h period, a significant decrease in cellular migration was observed in all the concentrations of metformin (1, 5 and 7 mM) (p < .001) (Figure 4).

| DISCUSS ION
It is well known that some cardiovascular complications such as myocardial infarction, heart failure, and micro and macrovascular events are more prevalent in diabetes. 1,12 Some studies showed that hyperglycemia is a paraclinical characteristic of diabetes that has been related to atherosclerosis. 4,13,14 Atherosclerosis is a disorder in which atherogenic plaques are built within the heart's coronary artery walls, narrowing them and causing vessel stenosis.
One of the most important features involved in atherosclerosis is the proliferation and migration of VSMCs in vessel subendothelial space. 15,16 The studies showed that in the high glucose conditions, growth-promoting substances and cytokines activate the proliferation and migration of VSMCs in the intermediate layer of the vessel wall. 17,18 The FAK is known as one central protein of many signalling pathways involved in cellular proliferation and migration events. 19 Its phosphorylated form plays a crucial role in cell signalling transduction. Some studies showed that FAK affects VSMC migration via the RhoA/ROCK1 signalling pathway. 20,21 FAK also is activated by EGFR and integrin receptors through cytokine-stimulated pathways. 22 On the other hand, studies have shown that metformin has a glucose-lowering effect and affects some molecular pathways. Metformin is also suggested to have anti-inflammatory properties. 5,23 Other studies reported that metformin inhibits AMPK-mediated VSMC proliferation and migration. 24 Moreover, metformin has also been shown to inhibit vascular calcification via the AMPK/eNOS/NO signalling pathway, suggesting that it may have therapeutic potential for vascular calcification in type 2 diabetic complications. 25 In this study, the effects of metformin were investigated on the FAK protein values and its gene expression levels, and on the proliferation and migration of vascular smooth muscle cells in high glucose conditions.
The results showed that metformin reduces the FAK gene expression levels and protein values in VSMCs in a dose-dependent manner. Metformin also decreased cellular proliferation, so that the values above 16 mM of metformin affect effectively the VSMC viability. The results also showed that metformin decreases the VSMC migration according to the FAK protein changes, so that these data suggested that metformin may retard atherosclerosis in diabetes. In contrast with some studies, 26,27 the high glucose conditions did not affect cellular proliferation and migration. It was in agreement with the previous report that the cell viability and migration do not correlate with glucose values up to 50 mM. 28 It was proposed that the changes of VSMC phenotype in high glucose conditions are related to molecular intermediates produced by other cells, 4 so that it was proposed to study in animal models.

| CON CLUS ION
The results showed that metformin suppresses the VSMC proliferation and migration in high glucose conditions. However, the results may improve by focusing on the molecular mechanisms related to downstream genes of FAK-related signalling pathways.

CO N FLI C T O F I NTE R E S T S
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data were presented on the request.