INTRODUCTION:

During the last decade, there have been several epidemiologic research present that plasma ADMA related to the cardiovascular disease (CVD) risk factors and chronic renal disease (CKD).^1-5 Enhancement of ADMA in the circulating bloodstream and in cells be correlated with endothelial dysfunction by inhibiting nitric oxide (NO) production.^6,7

Tissue is the main source of ADMA synthesis followed by transport of ADMA to blood circulation, but it is able to diffuse through the cell membrane mediated by cationic amino acids transporters (CAT)^8-10, the impact of plasma reuptake of ADMA by mononuclear cells has not been widely explored. Like ADMA was correlated with high all-cause mortality and late mortality in metabolic disease, Hydrogen peroxide (H₂O₂) indicate a stable reactive oxygen species (ROS), the notable molecule in vascular cells signaling, and have the ability to diffuse accros the cellular membranes^11-13, allowing for cellular signaling^14-16 and also induced cell damage.¹²

Cellular performance relies on the alignment of various important processes that induce constant internal stresses, that must occure precisely to ensure their integrity is maintained.¹⁷ Enhancement of intracellular ADMA and H2O2 diffusion causes a supraphysiological atmosphere in the cell, followed by a deviation of intracellular signaling to form a broad range of molecular shiftness that cells encounter reaction to environmental stressors, including mechanical vulnerability.¹⁸ It is interesting to verify how the interplay of a singlular protein can play many different roles in various biological circumtances as well as equally intriguing stress signals and response pathways can assembly into one distinct biological outcome.¹⁹

A cell use various strategies to respond to different type of stresses, but there is a common pattern of sensing the damage, transducing the signal, selecting the suitable pathway, and arrange the decision to recovery or reprogram (or in some cases die), based on severity of the stress.¹⁸ Exploring the molecular principle for these prominent steps will continue to be a useful field of research. It is challenging, to ask how a cell executes transient, spatial, and stress-specific regulation in response to myriad stresses at a time.¹⁹ Determining how different stress pathways are integrated in cells requires a special approach that is being explored in the realm of biology.

Cellular stress responses are key for sustaining genomic stability such as DNA damage include activation of cell cycle arrest.^20,21 The p53 tumor suppressor gene is a valuable sensor of cellular stress that transcriptionally activates genes implicated in enhance and responding to various types of damage.²² The elevation of p16^INK4a serves as a discovering presence of oxidative stress,²³ intracellular ROS and oxidative DNA damage. The p16^INK4a tumor suppressor protein usually prevents the kinase activity of cyclin-dependent kinases (CDK) 4 and 6, furthermore suppressing the hyperphosphorylation of retinoblastoma (Rb)-related pocket proteins required for cell-cycle progression.^24,25 p16^INK4a functionally serves to restrict unsuitable division of damaged cells by keeping them in the late G1-S transition and may encourage irreversible exit from the cell cycle into a senescent state.²⁶ The p16^INK4a tumor suppressor is known to function by inducing cell-cycle arrest or senescence when cells undergo potentially oncogenic DNA damage.^27-29

Accordingly, the present study was designed to determine the contribution of exogenous ADMA and H2O2 levels exposure to the increased tumor suppressor p16^INK4a in human circulating mononuclear cells. We hypothesized that adding exogenous ADMA and H2O2 exposure can increase the number of p16^INK4apositive mononuclear cells.

MATERIAL AND METHODS:

Subjects:

The true experimental study was designed to describe cellular stress response after exogenous ADMA and oxidative stress exposure for 24h through quantification of p16^INK4a. Study subjects were recruited from the Medical Faculty of Brawijaya University, Indonesia with approval by the head of the medical study program. Subject with no history of cardiovascular disease, diabetes mellitus, have no sedentary lifestyle, BMI in the normal range were included. The participants received consent information which contains the objectives and benefits of the research as well as makes sure their readiness for having peripheral blood sample collection. The ethical approval is required from Reseach Ethics Committee of Medical Faculty, Brawijaya University, Indonesia, by registered number: 1206-KEP-UB/2019.

Peripheral blood mononuclear cells isolation:

Peripheral venous blood (15 mL) were collected in the vacutainer-ACD tube (Becton Dickinson Inc). MNCs were isolated from each subjects by using Ficoll-Hypaque density gradient centrifugation as previously reported³⁰ was Lympoperm^TMwith a density of 1.077 g/mL (catalog#07801/07811, Germany) in falcon tube 15ml were added Peripheral blood with an equal amount. Centrifugation procedure at 1600rpm for 30 minutes to obtained buffy coat layer. Buffy coats were collected in a 15ml falcon. Then, the washing phosphate-buffered saline (PBS) process was done twice with 1200rpm centrifugation for 10 minutes to obtain the pellet. Mononuclear cells were collected and remaining erythrocytes lysed. Cell viability assessed by trypan blue (Sigma) exclusion was always >95%. The isolation procedure and all subsequent experiments were carried out at the laminar space at room temperature.

Cell culture:

Primary cells in this study are intended to describe circulating cells without genetic modification or surface markers that will affect the expression of the p16INK4a parameter measured in this study design. The isolated PBMNCs were cultured in medium EBM-2 (Lonza Inc) supplemented with 10% activated fetal bovine serum (FBS) (Gibco-Brl) and 1% L-glutamine 2 µM and 1% penicillin-streptomycin (PS) into 24-well culture plates until 80% confluent. After 5 days, confluent PBMNCs were seeding in EBM with 300µM N^G, N^G′-dimethylarginine (Santacruz) containing 10% FBS, 1% L-glutamine, and 1% PS for 24h (5%CO₂at 37⁰C). In the other PBMNCs group were treated with ADMA at a final concentration of, 500µM, on the other well plate PBMNCs were treated with H2O2 at the same dose. Treated cells were incubated for 24hours. EBM containing 10% FBS, 1% L-glutamine, and 1% PS alone was set as a control group. All treatments were performed in triplicate.

Sample Preparation for p16^INK4ameasurement:

After co-culture with ADMA and H₂O₂, PBMNCs were harvested by trypsinization, washed once with PBS, and fixed with 80% paraformaldehyde (PFA) 4% (5 min), and then permeabilized with 0.1% PBS tween for 20 min. The cells were then incubated in 1x PBS followed 1x PBS / 10% normal goat serum/0.3M glycine to block non-specific protein-protein interactions followed by the anti-CDKN2A/p16^INK4aantibody (ab54210, 1/50 dilution) for 30 min at 22°C. The secondary antibody used DyLight 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at room themperature. The expression of p16^INK4aquantity was measured by FACS with the acquisition of > 5,000 events was performed.

Flow cytometry assay:

To characterize the predominant mononuclear cell expression of p16^INK4a, we analyzed adherent cells by using FACS, Becton Dickinson, Inc). Dead cells detection were performed by propidium iodide and exclude from our analysis. Mononuclear cells were analyzed by electronic gating. The intracellular expression of p16^INK4apositive cells, which in ADMA treated cell compared in mononuclear cells were treated with H₂O₂.

Data analysis:

All data are described as mean±SD. For P16^INK4astudies, data were expressed as the percentage of added cells (total mononuclear cells) that were bound relative to cells from control and each experimental treatment method was used. In this manner, the difference in the number of cells with positive p16^INK4a expression after ADMA administration at different doses was analyzed by the Mann-Whitney test, as well as the analysis differences in groups of cells by administering H2O2 with 2 different doses. The difference of p16^INK4a positive cells in the 4 types of treatment was analyzed using the Kruskal-Wallis test. P.value ≤ 0.01 was considered statistically significant.

RESULT:

P16^INK4arepresentation in ADMA treated cells:

The p16 tumor suppressor were established inducing cell-cycle arrest or senescence when cells undergoes DNA damage.²⁷ We confirmed that p16^INK4apositive cells are elevated in PBMNCs following ADMA exposure, detected in 300µM and 500µM doses (Fig. 1; Table 1).

Table 1: Mean of p16^INK4aexpression in circulating human mononuclear cells after ADMA and H₂O₂ exposure.

Kelompok perlakuan	Percentage of p16^INK4apositive cells Mean±SD (n=18)	Mann whitney test	Kruskal-Wallis test
Control cells	0.34 ± 0.125		0.001
ADMA 300µM	1.43 ± 0.21	0.000
ADMA 500 µM	2.76 ± 1.39	0.000
H2O2 300µM	0.65 ± 0.12	0.000
H2O2 500 µM	1.22 ± 0.33	0.000

S.D. =Standard Deviation

PBMNCs exposed to ADMA at a dose of 300µM caused a higher number of p16^INK4a positive cells (1.43%±0.21) than control cells (0.34%±0.125) after 24 hours, the number of p16INK4a positive cells tended to increase after the ADMA exposure dose was increased at a dose of 500µM (2.76). % ± 1.39) (Fig. 1; Table 1). The number of p16^INK4a positive cells was significantly different in cells given ADMA exposure at a dose of 300 µM and 500 µM with a significant value p=0.000 (Table 1)

Fig. 1: Representation means percentage gate of p16^INK4aby FACS analysis in PBMNCs were treated with ADMA for 24 hours.

P16^INK4arepresentation in H₂O₂ treated cells:

Presence of oxidative stress had comparable impact on p16^INK4a, as MNCs treated with H₂O₂ indicate similar upregulation of p16^INK4a with ADMA exposure, p16^INK4a upregulation following H₂O₂ treatment was associated with dose-dependent increased positive staining cells. This p16^INK4a response to oxidative stress may be particularly notable in MNCs (Fig. 2).

MNCs exposed to H₂O₂ at a dose of 300µM caused a higher number of p16^INK4a positive cells (0.65% ± 0.12) than control cells (0.34%±0.125) after 24 hours, total of p16^INK4a positive cells tended to increase after the H₂O₂ exposure dose was further increased by 500µM (1.22 % ±0.33). The amount of p16^INK4a positive cells was significantly different in cells given 300µM and 500 µM H₂O₂exposure with a significant value p=0.000 (Fig. 2; Table 1)

Fig. 2: Representation means percentage gate of p16^INK4aby FACS analysis in PBMNCs were treated with Exogenous oxidative stress for 24 hours.

Comparison total p16^INK4apositive cells in ADMA and H₂O₂ treated cells:

The results of this study indicated that the p16^INK4apositive cells was seen both in untreated cells and under exogenous stress exposure. This suggests that p16^INK4a has both physiological and pathological functions. The total of p16^INK4a positive cells tended to be higher in treated cells compared to untreated cells. The highest of p16^INK4a positive cells was seen in the 500µM ADMA exposure, and the lowest was seen in the cells were treated with 300µM of H₂O₂. In Fig.3 it appears that ADMA exposure tends to induce an increase in p16^INK4a compared to H₂O₂ induction, and the Kruskal-Wallis test results indicate that the number of p16^INK4a positive cells is different in the five cell groups we observed with a significance value p=0.001 (Table 1).

Fig. 3: Representation means percentage gate of p16^INK4aby FACS analysis in MNCs treated and untreated cells. ^*Statistically significant difference between groups within the given experimental condition (P ˂ 0.001).

DISCUSSION:

The major finding in the circulating human mononuclear cells fraction after cellular stress exposure is 1) intracellular p16^INK4apositive cells are higher in treated cells than control; 2) intracellular p16^INK4apositive cells are higher in ADMA treated cells than H₂O₂ treated cells; 3) increasing number p16^INK4apositive cells occurred with the addition of treatment doses both on cells exposed to ADMA or cells treated with H₂O₂. Together, these findings point related to increased p16^INK4aas cellular stress response after exogenous stress exposure.

Fig. 4: Flow cytometry analysis result of p16^INK4afluorescence in circulating human mononuclear treated cells and untreated cells with exogenous stress exposure. (A) Side scatter plot (SSC) for gating mononuclear cells; (B) percentage gate p16^INK4apositive cells in control cells; (C) treated cells with ADMA 300µM for 24h; (D) treated cells with ADMA 500µM for 24h; (E) treated cells with H2O2 300µM for 24h; (F) treated cells with H2O2 500µM for 24h.

Protein p16^INK4ahas been described to be a tumor suppressor gene that suppresses the proliferation of cells through the cell cycle inhibition.³¹ In general, the increase in p16^INK4a is associated with the aging process or age-related disease, and inhibition of cell proliferation. The protein N-arginine methyltransferases (PRMTs) is referred to as a regulator of p16^INK4aactivity to prevent cell proliferation through downregulation of p16^INK4a function.³¹ PRMTs enzymatic activity counteracts the regulation of ADMA level.^32-35 ADMA is present in plasma and also we can identify ADMA in the cells. As an endogenous inhibitor of nitric oxide synthase, elevation plasma ADMA levels are correlated with the severity of endothelial dysfunction and atherosclerosis.^36-38,9

The rapid exchange of ADMA between in intracellular and plasma pool provides limited information. It is also recognized that role CAT as a transporter of amino acids into cells or clearance of amino acids from the circulation^8,39, resulting in an interplay that regulates ADMA re-uptake by cells with subsequently degrading it by dimethylarginine dimethylaminohydrolase (DDHA).^40-42 Accumulation of intracellular ADMA lead uncouple NOS isoenzymes to produce superoxide, subsequently contributing to the oxidative stress loading.⁴³ Accordingly, oxidative stress is a factor that causes decreased DDHA expression and activity. Another study states that exogenous ADMA treatment with micromolar concentrations significantly upregulates the levels of TNF-α, IL-8, MCP-1, chemokine receptors including CCR₂ and CXCR₂, and also NF-κB activation³², it can modulate inducible NO (iNOS) which is able to activate the cell cycle arrest pathway through p21 and p16^INK4aactivities.⁴⁴ In addition, the unphysiological amino acid composition of culture medium due to the addition of glutamine and high lysine content may cause an imbalance of the CAT system and a decrease in intracellular NO production.^45,46

As well as ADMA-mediated cell signaling disrupted, H₂O₂ mediates chemical shiftness of specific cysteine residues in functionally relevant regions of proteins.⁴⁷ DNA damage after H₂O₂ exposure triggers a complex network were called of DNA damage response (DDR) pathways that may initiate DNA repair, halt cell cycle progression, and stir apoptosis.^48,49,22 Elevation of p16^INK4a following H₂O₂ treatment related with dose-dependent were in line with previous studies which states that exogenous oxidative stress upregulates p16 in human skin cells acutely.^23,50-52Thus ROS-induced p38 phosphorylation is needful for the upregulation of p16^INK4a, describe the function of ROS-dependent p38 SAPK-p16^INK4a signaling pathway. It is assigned that p16^INK4a is a negative regulator of the cell cycle, and cells with p16^INK4a mutations lead rapid proliferation rates and the diminished fraction of cells in the G1 phase.^53,12 Increased proliferation is associated to mitochondrial respiration and enhanced ROS leakage into the cytoplasm.^54-57

It is widely accepted that p16^INK4a-mediated cell cycle arrest provide time to improve DNA damage before replication, thereby reducing the potential for mutations expansion.²³ We present evidence that ADMA and exogenous oxidative exposure lead to the accumulation of intracellular p16^INK4a in MNCs. Our finding further suggests that compromise of p16^INK4amay be apart of DNA damage response and it seems plausible that upregulation of p16^INK4a allows cells to progress to repairing DNA lesions after cellular stress exposure. Based on the above statement, the results of this study provide an overview of the potential damage to circulating human mononuclear cells from metabolic diseases represented by ADMA and H₂O₂. So it is necessary to appropriate leadership⁵⁸ for the management of metabolic diseases that are contributors to organ degenerative progression.^59,60

CONCLUSION:

Administration ADMA and H₂O₂ for 24 hours up-regulate the intracellular expression of p16^INK4ain human PBMNCs compared with control cells. Augment dose of ADMA and H₂O₂ were lead an enhancement of p16^INK4a expression in treated cells. Expression of p16^INK4ahas been proven higher in ADMA treated cells compared with H₂O₂ treated cells. It is well known that p16^INK4a serve as negative regulator of the cell cycle, Finally, we show the possibility that exposure to ADMA or H₂O₂ causes accelerated re-uptake of ADMA and H₂O₂ by cells followed by a series of intracellular signaling deviations, and that ultimately p16^INK4a upregulation may be associated with inhibition of the continuation of the cell cycle in circulating cells to further prevent cell division that has been damaged.

ACKNOWLEDGEMENT:

The authors are grateful to the Directorate General of Education and Culture of the Republic of Indonesia for supporting research funding in 2020.

CONFLICT OF INTEREST:

We have no conflict of interest to declare.

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Received on 14.02.2021 Modified on 19.04.2021

Research J. Pharm.and Tech 2022; 15(2):707-712.

DOI: 10.52711/0974-360X.2022.00117