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

Background and Aims: 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 (PA) on the tyrosine-protein kinase (HCK) gene and protein expression levels in vascular smooth muscle cells (VSMCs). Methods: The human Vascular Smooth Muscle Cells (VSMC) were treated with palmitic acid (0.5 mM, 24 hours) based on the cellular viability studies. The HCK gene and protein expression levels were measured by real-time qRT-PCR and western blotting techniques, respectively. Oil Red O staining method was used to determine the intracellular lipid values. Results: 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. There was a signicant increase in p-HCK value (P=0.001). Conclusion: The results showed that the palmitic acid increases intracellular p-HCK value so that it may affect the HCK-mediated pathways in VSMCs.


Introduction
Over the past decades, obesity was considered as a potential risk factor for cardiovascular diseases through different mechanisms involved in adipokines, in ammation, oxidative stress, and vessel cellular dysfunction (1, 2). On considering the lifestyle, the atherogenic effects may improve due to the intake of fatty acids. Some reports have shown that palmitic acid develops the risk of cardiovascular diseases (3)(4)(5). In another study, the serum palmitic acid-related phospholipid fraction was reported to involve with myocardial infarction (6). Many studies reported that cardiovascular events relate to the cellular biological pathways involved with lipid homeostasis (1, 7). It is well known that endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are involved in the formation of atherosclerotic plaques. Regarding 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, 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 expression patterns, the SRC, YES, and FYN have ubiquitously expressed in all tissues and cells. Furthermore, the HCK is reported to relate to many cancers such as leukemia, pancreatic, breast, ovarian, prostate, colon, and kidney. This protein triggers cellular proliferation and has a key role in cellular migration via the remodeling of the extracellular matrix. Given its role, HCK, therefore, can be a therapeutic target (10)(11)(12)(13).
Based on the known roles of palmitic acid in the development of cardiovascular diseases (CVD) (14), the aim of this study was to investigate the effects of palmitic acid on the HCK gene and protein expression humidi ed incubator (CO2, 5%; T, 37°C). In this study, the cultured cells were divided into two groups including the control and treated groups which were grown in DMEM-F12 containing ethanol 0.1% (as the palmitic acid vehicle) and DMEM-F12 containing palmitic acid, respectively.

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 different concentrations of palmitate (0, 0.1, 0.5, 1, and 5 mM). After 24 and 48 hours, the cellular media were 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 a microplate reader.

Treatment
Palmitic acid was dissolved in ethanol (0.1%), and was added to cultured cells (Con uency, 70%; Concentration, 0.5 mM) for 24 hours. Then, the treated cells were harvested to evaluate the gene and protein expression levels.

Gene Selection
Based on the in ammatory events, a protein complex (containing FGR, HCK, and LYN, SRC) was considered to have a central role in the chemokine signaling pathway (KEGG, hsa04062). The gene data related to this protein complex were improved on the reactome server (https://reactome.org) and were the subjects on the primary network in string (https://string-db.org). Then, the protein network was trimmed on the string score (more than 0.9) and was enriched with gene ontology and pathway data. The HCK gene was considered as a high-edge node with Phosphothyrosine residue binding function involved in the CB2 pathway ( Figure 1).

Oil Red O staining technique
The intracellular lipid was evaluated using the Oil Red O staining method. Brie y, the cells were washed in phosphate-buffered saline (PBS) 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 the 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) was 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 a dose-response way. A p-value less than 5% was considered to be signi cant.

Viability and Cytotoxicity
The results showed that the IC50 values are estimated at 0.32 mM and 0.52 mM in VSMCs treated with palmitate in the periods of 48 and 24 hours, respectively (Figure 2).

HCK gene expression level
The HCK gene expression level increased signi cantly up to 3.2 times in the treated cells with palmitate as compared with the control group (p=0.0286) (Figure 3, B).

HCK protein expression level
The total HCK protein expression level was not signi cant (p= 0.8139) (Figure 3. A, C) but the phosphorylated HCK value increased signi cantly in the treated VSMCs with palmitate (p= 0.0012) (Figure 3. A, D).

p-HCK/total HCK ratio
The p-HCK/HCK ratio in the palmitate-treated cell group increased signi cantly as compared to the control (P=0.001) (Figure 3, E).

Intracellular lipid Intensity
As shown in Figure 4, intracellular lipid intensity (red color) was highly observed in the palmitate-treated VSMCs which were (0.5mM, 24 hours) as compared with control.

Discussion
Palmitic acid has 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 (15). Thus, the role of palmitic acid in the function of VSMCs (16) may be interesting.
In this study, the HCK gene and protein expression levels were studied in the palmitate-treated VSMCs since the HCK is contributed in central complex involved in some cellular signaling pathways. Some studies reported that free fatty acids can proliferate VSMCs (17,18) resulted in the development of atherosclerosis process in the rat and human (19,20). It is reported that PA causes cardiomyocyte hypertrophy (21) and develops the cellular apoptosis (22)(23)(24)(25). Furthermore, it developed melanoma by involving cellular proliferative pathways via the phosphorylation of Akt (26). The promotion of cancer invasiveness was also suggested by the activation of TLR4/ROS/NFκB/MMP9-related signaling pathways (27). It is suggested that PA modi es the protein sequences by phosphorylation of tyrosine residues (28). On the con rmation of the above studies, this study showed that PA increases the HCK gene expression level improving the cellular HCK-mediated pathways. Furthermore, the p-HCK value 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 cant as compared to control. We suggested that the activation of HCK phosphorylation (p-HCK) may diminish the total HCK synthesis. On the other hand, PA may affect the HCK-mediated 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 (29) thus it is an impotent factor in the activation of the cellular proliferation and adhesive pathways. Moreover, HCK may co-expresses with other genes in cellular pathways such as apoptosis (30).

Conclusion
This study showed that palmitic acid increases the total p-HCK/HCK ratio in VSMCs. Since HCK contributes to the intracellular SRC complex thus it mediates some cellular signaling pathways via the in ammatory stimulators and down-regulatory factors. We suggested that the role of HCK in PA-treated VSMCs can more be evaluated by the combined effects with chemokines and the changes of downstream proteins in HCK-mediated pathways.

Declarations
Ethics approval and consent to participate: It was approved by Committee on the Ethics of IUMS.

Consent for publication: No
Availability of data and material: It is presented on the request from the corresponding author. Gene Selection. A, Gene network is categorized on the enrichment of nodes with B, Gene ontology and pathway data. The gene data improved on the reactome. Then, the gene network was made and trimmed using string, and was subject for enrichment with gene ontology and pathway data. The HCK considered as a high-edge node and involved in some pathways.  HCK expression assessment. A, Western blotting images. B, HCK gene expression levels. Palmitic acid increased the HCK gene expression level as compared to the control (p=0.0286). C, HCK protein expression levels. Although the HCK protein expression level increased in the palmitate-treated group but it was not statistically signi cant (p= 0.8139). D, p-HCK gene expression levels. Palmitate increased signi cantly p-HCK value in the palmitate-treated group (p= 0.0012). E, p-HCK/total HCK ratio. p-HCK/HCK ratio elevated signi cantly as compared to control (p<0.001). Control group treated with ethanol 0.1% which was used as the palmitic acid vehicle.