Palmitic acid increases HCK protein and gene expression levels in vascular smoot muscle cells

Background and Aims: Some saturated fatty acids are known to involve in atherosclerosis through different biologic pathways. The aim of this study was to investigate the effects of palmitic acid on the HCK gene and protein expression levels in vascular smooth muscle cells (VSMCs). Methods and Results: The cells were treated with palmitic acid (0.5 mM, 24 hours) on the cell viability assays. The HCK gene and protein expression levels were measured by real time q-PCR and western blot techniques, respectively. Oil Red O staining method was used to determine the intracellular lipid values. The HCK gene expression level was increased signicantly in the PA-treated VSMCs (P 0.02). The total and phosphorylated HCK (p-HCK) protein expression levels increased in VSMCs. However, there was a signicant increase in p-HCK value (P 0.001). Conclusion: The results showed that the palmitic acid increases p-HCK function so that it may affect the VSMC proliferation.


Introduction
Over the past decades, obesity was considered as a potential risk factor for cardiovascular diseases through different mechanisms involved to adipokines, in ammation, oxidative stress and vessel cellular dysfunction (1,2). Many studies reported that the cardiovascular events relate to the biologic pathways involved in lipid homeostasis (2,3). On considering the life style, the atherogenic effects may improve due to the intake of fatty acids. Some reports have shown that the palmitic acid develops the risk of cardiovascular diseases (4)(5)(6). In another study, the serum palmitic acid-related phospholipid fraction was reported to involve with myocardial infarction (7).
In the other hand, it is well known that the endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are involved in the formation of atherosclerotic plaques. Regarding to some hypotheses, the contractile VSMCs are engaged through phenotypic alteration in order to organize the extracellular matrix (ECM) and to stabilize atherosclerotic plaques (8). Furthermore, the VSMC proliferation plays an important role in vessel stenosis and restenosis (9).
The SRC family (SRC, YES, FYN, LCK, FGR, BLK, LYN, YRK and HCK), known as non-receptor protein tyrosine kinase (SFK), expresses predominantly in mammalian cells. Considering to expression patterns, the SRC, YES and FYN have ubiquitously expressed in the all tissues and cells. Furthermore, the HCK is reported to relate with many cancers such as leukemia, pancreatic, breast, ovarian, prostate, colon and kidney. This protein triggers the cellular proliferation and has a key role in the cellular migration via the remodeling of extracellular matrix. Given its role, HCK therefore can be as a therapeutic target (10)(11)(12) Based on the known role of palmitic acid (PA) in the development of cardiovascular diseases (CVD) (13), the aim of this study was to investigate the effects of palmitic acid on the HCK gene and protein expression levels in vascular smooth muscle cells.

Cell viability assay
The effect of palmitic acid on the cell viability was carried out by microculture tetrazolium (MTT) colorimetric assay. Initially, the cells were grown in 96-well plate and were treated with the different concentrations of palmitate (0, 0.1, 0.5, 1, and 5 mM). After 24 and 48 hours' incubations, the cellular medium was removed and the cells were re-incubated for 2 hours in the presence of MTT solution (200 µl, 0.5 mg/ml). Then, the produced formazan crystals were dissolved in DMSO (150 µl for 4 hours) and light absorbance was measured at 570 nm using microplate reader.

Treatment
Palmitic acid was solved in ethanol (1%), and was added to cultured cells (Con uency, 70%; Dose, 0.5 mM) for 24 hours. Then, the treated cells were harvested to evaluate the gene and protein expression levels.

RNA extraction, cDNA synthesis and RT-qPCR techniques
Total RNA was isolated from VSMCs using GeneAll-Hybrid-R puri cation kit

Oil Red O staining technique
The intracellular lipid droplets were evaluated using Oil Red O staining method. Brie y, the cells were washed in phosphate-buffered saline (PBS) (2 times) and were xed in formalin (4%) at room temperature for 45 minutes. Then, the cells were washed with isopropanol (60%) for 3 minutes. After adding Oil Red solution (0.1%, Cat. No. 1320-06-5, Sigma-Aldrich, USA), the cells were incubated at room temperature for 30 minutes. Finally, the cells were rinsed with water (3 times) and were observed by model IX71 microscope.

Statistical Analysis
Graph Pad Prism statistical software (v 8.3.0.538, Graphpad, USA) were applied for the data analyses. The differences between groups were determined by using independent samples t-student and Mann-Whitney tests. The IC50 was calculated in the dose-response way. P value lower than 5% was considered signi cant.

Viability vs. Cytotoxicity
The results showed that the IC50 values are estimated 0.32 mM and 0.52 mM in VSMCs treated with palmitate in the periods of 48 and 24 hours, respectively (Fig. 1).

HCK gene expression level
The HCK expression level increased signi cantly up to 3.2 times in treated cells with palmitate as compared with control group (P 0.0286) (Fig. 2).

HCK protein expression level
In the converse with the total HCK protein expression level (P 0.8139) (Fig. 3. A), the phosphorylated HCK protein value increased signi cantly in the treated cells with palmitate (P 0.0012) (Fig. 3. B).

p-HCK/total HCK ratio
The p-HCK/HCK ratio in the palmitate-treated group increased signi cantly as compare to control (P 0.001) (Fig. 4, A vs. B).

Cellular lipid
The intracellular lipid distribution in VSMCs treated with palmitate (0.5 mM, 24 hours) increased as compared with controls (Fig. 5).

Discussion
Palmitic acid has the endogenous and exogenous sources in the body. This saturated fatty acid makes up about 44% of palm oil, 65% of butter, 53% of tallow, 15% of soybean, 13% of corn oil, and 17% of olive oil (9). It is also known as an atherogenic agent in the improvement of cardiovascular diseases in obesity (15). Thus, the role of palmitic acid in the proliferation and migration of VSMCs (14) maybe interesting. In this study, the HCK gene and protein expression levels was studied in the palmitate-treated VSMCs since the HCK is known to be involved in the cellular proliferative and adhesive pathways.
The many evidence indicated that some free fatty acids are able to proliferate VSMCs (16,17) resulted in the development of atherosclerosis process in the rat and human (18,19).
It is reported that PA causes the cardiomyocyte hypertrophy via phosphorylation of PCKζ and its interaction with STAT3 (20). Furthermore, it developed the melanoma by involving cellular proliferative pathways via the phosphorylation of Akt (21). The promotion of cancer invasiveness was also suggested by the activation of TLR4/ROS/NFκB/MMP9-related signaling pathways (22). It is suggested that PA modi es the protein sequences by phosphorylation of tyrosine residues (23). On the con rmation of above studies, this study showed that PA increases the HCK gene expression level improving of the cellular proliferation pathways. Furthermore, the p-HCK protein expression level increased in PA-treated VSMCs on the role of PA in the modi cation of amino acid residues. However, the HCK protein expression level increased in PA-treated VSMCs but it was not signi cantly as compared to control. It is proposed that the HCK protein synthesis is not in accordance with the gene expression level so that we suggested that the activation of HCK phosphorylation (p-HCK) prevents signi cantly from the total HCK synthesis. In the other hand, PA may affect the HCK-involved signaling pathways by the phosphorylation of HCK in a fast-response way. Since other studies reported that HCK elevates via the function of BCR/ABL, TEL/ABL, EGFR /PDGFR, and PI3K/Akt (10, 24) thus it is an impotent factor in the activation of the cellular proliferation and adhesive pathways.

Conclusion
This study suggested that palmitic acid increases the cellular proliferation pathway in VSMCs via the function of phosphorylated HCK (p-HCK).

Abbreviations
Phosphorylated HCK; p-HCK, Vascular smooth muscle cells; VSMCs, Endothelial cells; ECs, Extracellular matrix; ECM, Palmitic acid; PA, Cardiovascular diseases; CVD Declarations Ethics approval and consent to participate: It was approved by the Committee on the Ethics of IUMS.