5‐Azacytydine and resveratrol reverse senescence and ageing of adipose stem cells via modulation of mitochondrial dynamics and autophagy

Abstract Obesity and endocrine disorders have become prevalent issues in the field of both human and veterinary medicine. Equine metabolic syndrome is a complex disorder involving alternation in metabolism and chronic systemic inflammation. It has been shown that unfavourable microenvironment of inflamed adipose tissue negatively affects adipose stem cell population (ASC) residing within, markedly limiting their therapeutic potential. ASCsEMS are characterized by increased senescence apoptosis, excessive accumulation of reactive oxygen species (ROS), mitochondria deterioration and “autophagic flux.” The aim of the present study was to evaluate whether treatment of ASCsEMS with a combination of 5‐azacytydine (AZA) and resveratrol (RES) would reverse aged phenotype of these cells. For this reason, we performed the following analyzes: molecular biology (RT‐PCR), microscopic (immunofluorescence, TEM) and flow cytometry (JC‐1, ROS, Ki67). We evaluated the mitochondrial status, dynamics and clearance as well as autophagic pathways. Furthermore, we investigated epigenetic alternations in treated cells by measuring the expression of TET genes and analysis of DNA methylation status. We have demonstrated that AZA/RES treatment of ASCsEMS is able to rejuvenate these cells by modulating mitochondrial dynamics, in particular by promoting mitochondrial fusion over fission. After AZA/RES treatment, ASCsEMS were characterized by increased proliferation rate, decreased apoptosis and senescence and lower ROS accumulation. Our findings offer a novel approach and potential targets for the beneficial effects of AZA/RES in ameliorating stem cell dysfunctions.


| INTRODUCTION
Obesity and endocrine disorders have become common problems in the field of both human and veterinary medicine. It is estimated that approximately 19%-40% of the horse population is obese 1 and 22%-29% suffer from hyperinsulinemia. 2 Regional adiposity, obesity, hyperinsulinemia, insulin resistance (IR) and laminitis or susceptibility to laminitis are the major characteristics of equine metabolic syndrome (EMS). 3 It is worth noting that recent data have indicated that obesity should not be used as a diagnostic criterion 4 EMS is a complex disorder involving alternation in metabolism and chronic systemic inflammation. 5 However, due to its complexity, understanding EMS pathophysiology is still elusive. Adipose tissue is not only an energy storage, but also a highly active, endocrine organ secreting hormones and cytokines. In our previous study, we have shown increased expression of IL-6 and TNF-α in adipose tissue and serum of Welsh ponies suffering from EMS. Similarly, many other authors have described increased levels of proinflammatory cytokines, including IL-1 β, IL-6 and TNF-α in serum and adipose tissue in EMS individuals [6][7][8] It has been shown that TNF-α is directly implicated in IR development by interfering with insulin signalling. 5,9 This adverse microenvironment of adipose tissue negatively affects adipose stem cell population (ASC) residing within it.
Adipose stem cells are considered to be ideal for application in regenerative medicine, as they can differentiate into multiple lineages both in vitro and in vivo. Moreover, ASCs are well known for their immunosuppressive properties. They were proved to secrete a wide range of proteins in extracellular microvesicles (MVs). Robust secretion of trophic factors strongly correlates with clinical outcome in a wide range of applications. In addition, their huge advantage over mesenchymal stem cells (MSCs) isolated from other sources, like bone marrow, is that they are easily harvested and isolated in large quantities with minimal donor-site morbidity.
Due to their unique properties, ASCs hold promise in treating multiple disorders, including graft versus host disease, 10 multiple sclerosis, 11 diabetes mellitus 12 and autoimmune-induced diseases. 13,14 Moreover, their application in bone and cartilage regeneration as well as wound healing was widely investigated. [15][16][17][18] There has been a growing number of ASC-related studies demonstrating the ability of these cells to decrease IR, promote regeneration of pancreatic beta cells and suppress autoimmunity in the course of diabetes. 19 However, our previous data indicated a severe deterioration of ASCs isolated from EMS horses (ASC EMS ), which questions their therapeutic utility in MS treatment. According to our results, ASCs EMS are characterized by limited proliferation potential, increased senescence, apoptosis, excessive accumulation of ROS and mitochondria deterioration. 3,20,21 In consequence, "autophagic flux" was observed in those cellsa protective mechanism that helps metabolically impaired cells maintain multipotency and stemness. During chondrogenic and osteogenic differentiations, increased autophagy provides ASC EMS precursors to macromolecules, adenosine triphosphate and amino acid synthesis. 22,23 Accelerated degradation of heterochromatin associated with the inner nuclear membrane and increased amounts of 5-methylcytosine in DNA of ASCs EMS also indicate epigenetic alternation in these cells. Moreover, secretion of MVs, significant in regeneration and cellular communication, was markedly reduced in those cells.
Increased levels of ROS result in impairment of mitochondrial dynamics and endoplasmic reticulum stress. Elevated mitochondrial fission and decreased mitochondrial membrane potential (MMP) may be particularly responsible for the activation of the apoptotic pathway in those cells. All these phenomena strongly limit the therapeutic potential of ASCs EMS and bring serious consequences for their usefulness in regenerative medicine.
Increasing body of evidence suggests that allogeneic MSCs may elicit an immune response in a recipient animal and that allogeneic donor MSCs are not fully immune-privileged, as previously claimed.
The formation of anti-allogeneic MSC antibodies, following an intradermal allogeneic MSC injection, was observed in horses. 24,25 These results highlight the potential risk of allogeneic MSC application.
Given the aforementioned facts, it needs to be considered in EMSdiagnosed individuals whether autologous graft of ASCs will be therapeutically valuable and effective. Thus, alternative approaches, aiming to rejuvenate autologous ASCs in vitro to enhance their regenerative potential have become the centre of scientific attention.
The approach of ASC EMS rejuvenation presented by our group relies on two distinct features of these cells -ROS accumulation and epigenetic alternation. In our research, we decided to combine two molecules targeting mitochondria and DNA of ASCs EMS in order to reverse their aged phenotype.
Resveratrol (RES) has been shown to exert immunomodulatory, anti-inflammatory and antioxidative effects. 26,27 Moreover, RES has gained widespread attention, because of its ability to prolong lifespan and protect against age-related disorders in different animal models. 28  inducing PGC-1α and SIRT1 activity. Our own data 31 revealed that polyurethane/polylactide-based material doped with RES decreased senescence and oxidative stress of ASCs. The fabricated material directed ASCs towards an osteoblast-like phenotype, which indicated its potential application in regenerative medicine.
On the other hand, 5-azacytidine (AZA) is easily incorporated into DNA and inhibits methylation pattern of specific gene regions. 32 It was shown that AZA treatment of hepatocyte-like cells resulted in an enhancement of metabolic and enzymatic activities of these cells. 33 A study conducted by Yan et al 34 revealed that AZA improved osteogenic differentiation potential of human aged ASCs. Our recent research confirmed beneficial effects of AZA, as it reversed aged phenotype of ASCs by decreasing apoptosis and enhancing proliferation rate. 35 Taking into consideration biological role of micro RNAs (miR) in modulation of cell proliferation and senescence, in presented study we investigated the expression of miR-24 and miR-519d. MiR-24 plays important role in inflammation, cell migration and many diseases. Moreover, it was shown that oxidative stress leads to up-regulation of miR-24 an in consequence to apoptosis. 36 On the other hand, miR-519 was shown to block autophagy. 37 It exerts its role by targeting Beclin-1, ATG10, and ATG16L1.
The aim of this study was to investigate whether the treatment of ASCs EMS with a combination of AZA/RES would reverse aged phenotype of these cells. In order to investigate the effectiveness of ASC EMS rejuvenation, we performed both molecular biology and microscopic analyzes. We evaluated mitochondrial status, dynamics and clearance as well as autophagic pathways. Furthermore, we investigated epigenetic alternations in treated cells and evaluated whether AZA/RES treatment influenced surface antigen expression.

| MATERIALS AND METHODS
All reagents and chemicals used in this research were purchased from Sigma-Aldrich (Poznań, Poland), unless indicated otherwise.

| Experimental animals
The study involved thirty, mixed sex, agematched (8-12 years) horses, classified into the EMS groupconsisted of animals suffering from EMS (n = 15) and the control group of healthy individuals (n = 15). Qualification to the experimental groups was performed based on detailed interviews with owners, body weight, body condition score, cresty neck score, existing laminitis, resting insulin levels, blood glucose levels, combined glucoseinsulin test and leptin concentration. Comprehensive characteristic of the animals used in this study is presented in Table 1.

| Adipose tissue collection and ASC isolation
White, subcutaneous adipose tissue samples were collected from the horse tail base following the ethical rules and standard surgical procedures as presented elsewhere. The tissue fragments were placed in a sterile Hanks' balanced salt solution (HBSS) containing 1% of penicillin/streptomycin/amphotericin B (PSA) solution. In order to isolate ASCs harvested material was fragmented by mechanical mincing and digested enzymatically with collagenase type I in a concentration of 1 mg/mL for 40 minutes at 37°C. Obtained homogenate was centrifuged at 1200 g for 10 minutes at room temperature.
Cell pellet was extensively washed by centrifuging with HBSS (300 g, 4 minutes), resuspended in the culture medium and transferred to a T-25 culture flasks. In order to perform the experiments, cells were passaged three times using trypsin solution (TrypLETM; Life Technologies, Carlsbad, CA, USA).    BMG Labtech, Ortenberg, Germany). Reduction of the dye was measured spectrophotometrically at a wavelength of 600 nm for resazurin and 690 nm as a reference wavelength. In order to estimate the clonogenic potential of cells, they were seeded in a 6-well plate at an initial density of 1 × 102 as described elsewhere. 38 After 7 days in culture, biological material was fixed with 4% icecold paraformaldehyde For SEM analysis, cell cultures were fixed with 2.5% glutaraldehyde for 1 hour at room temperature, dehydrated in a graded ethanolwater mixtures, dried with air for 30 minutes and coated with gold (ScanCoat 6, Oxford). 39,40 Prepared samples were observed using an SE1 detector, at 10 kV filament tension To perform TEM analysis, the samples were collected and fixed with 2.5% glutaraldehyde for 24 hours at 4°C and proceed as described elsewhere. 22 Briefly, cells were incubated for 2 hours with 1% osmium tetroxide and counterstained with lead citrate and uranyl acetate. Next specimens were dehydrated in a graded series of acetone, embedded Prior to the analysis of LAMP2 and DNMT1-localization, cells were fixed in 4% PFA for 30 minutes and washed three times with HBSS. Whole procedure was conducted based on the protocol presented previously. 22 Briefly, cells' membranes were permeabilized with 0.5% Triton X-100 for 20 minutes at room temperature while unspecific binding sites were blocked with blocking buffer (10% Goat Serum, 0.2% Tween-20 in HBSS) for 45 minutes. Cells were then incubated overnight at 4°C with primary antibodies against LAMP2 (Abcam) or DNMT-1 (Abcam), diluted 1:500 in HBSS containing 10% Goat Serum. Cells were then washed again and

| Flow cytometric analysis
All flow cytometry analysis were performed after 24 hours of the experiment. To evaluate the expression of LAMP -2 and 5-mathylocytosine

| Oxidative stress factors and senescence
Oxidative stress and apoptosis were assessed after 24 hours of culture. Supernatants were collected from cultures and subjected to spectrophotometric analysis. Superoxide dismutase (SOD) activity was detected using SOD assay kit, nitric oxide concentration was assessed with the Griess reagent kit (Life Technologies) in accordance to manufacturer' protocols.  Table 2.
To determine miRNA expression, 500 ng of RNA was reverse-

| Identification of ASCs characteristics
Flow cytometer was used to assess immunophenotype of ASCs. Iso-  Results expressed as mean ± SD. Statistical significance indicated as asterisk (*) when comparing the result to ASC CTRL , and as hashtag (#) when comparing to ASC EMS . #, *P < 0.05; ##P < 0.01; ###, ***P < 0.001 F I G U R E 3 AZA/RES reversed aging and reduced apoptosis in ASC EMS . In order to evaluate apoptosis in culture cells were cultured for 24 hours in control or experimental (AZA/RES) condition. Next, cells were subjected to staining procedures and RT-PCR analysis. In order to visualize live and dead cells in culture Calcein A.M and propidium iodide staining was applied (A). Moreover, senescent cells in cultures were visualized by β-galactosidase staining (A). Furthermore, data obtained from representative photographs was quantified (B and C). Presented results displayed that AZA/RES treatment markedly reduced number of dead and senescent cells. Apoptosis incidence was also investigated with RT-PCR for p53 (D), p21 (E), and Caspase-9 (F). Representative graphs from cell cycle analysis (G). Western blot for caspase-3 (H) and ELISA for p53 (I), Magnification ×100, scale bars: 250 μm.
Results expressed as mean ± SD. Statistical significance indicated as asterisk (*) when comparing the result to ASC CTRL , and as hashtag (#) when comparing to ASC EMS . *P < 0.05; ##, **P < 0.01; ###, ***P < 0.  Figure 4F). There were also mitochondria with rounded, swollen shape. In case of ASC CTRL , elongate, bean shape organelles were noted without any significant aberrations. Based on those parameters we quantified TEM photographs as shown in Figure 4G. Obtained results indicated that AZA/RES diminished mitochondrial damage as its significantly reduced abnormalities in mitochondrial morphology. Thus, we next investigated, if those changes results from mitochondrial dynamic shift. Mitochondrial fission is protective mechanism by which deteriorated organelles are arrested to ultimately be removed by the mitophagy process.
Hence, we investigated the FIS expression using RT-PCR. ASC EMS were characterized by increased fission ( Figure 4H) and increased expression of PINK ( Figure 4I) and PARKIN ( Figure 4J), both crucial to initiate mitophagy. Moreover, expression of Mief1 ( Figure 4K) and Mief2 ( Figure 4L) was diminished. Furthermore, amount of MFF, MNF and PINK were visualized by western blot ( Figure 4M).
We opined that enhanced mitophagy is protective mechanism which enables cells to maintain their stemness and compromise with excessive ROS as no mitophagy induction was noted in control cells. What is interesting, we noted reduced mitochondrial F I G U R E 4 AZA/RES treatment improved mitochondrial condition and reduced ROS accumulation in ASC EMS . To investigate oxidative stress in cells, they were culture for 24 hours in control or experimental conditions and subjected to further analysis. MMP was measured using JC-1 probe by flow cytometry (A). MMP depolarization in ASC EMS was markedly reversed in ASC EMS II (B). Intracellular ROS level was stained with H2DCFDA and detected by flow cytometry. Results were respectively shown in the histograms (A). AZA/RES treatment significantly reduced ROS production in ASC EMS (C). Antioxidative properties of cells were analyzed by spectrophotometric measurement of SOD activity (D). ASC EMS II displayed enhanced enzyme activity. Using RT-PCR amount of miR-24 was evaluated (E). TEM analysis showed ultrastructural changes in mitochondria morphology in ASC EMS  in those groups as well ( Figure 5H).

| AZA/RES decreased ER stress in ASC EMS
Recently, it become more and more evident, that endoplasmic reticu- TEM photographs demonstrated that, ER of ASC EMS was characterized by enlarged lumen and swollen cisternae ( Figure 6A). On the contrary, ASC CTRL ER, was well developed, extended as a network that formed large ER sheets. Therefore we investigated using RT-PCR mRNA levels of CHOP and PERK. CHOP expression was significantly up-regulated ASC EMS in comparison to control cells, although AZA/RES treatment did not significantly reduced its expression (Figure 6B). Similarly, PERK was also up-regulated in ASC EMS , however AZA/RES markedly decreased its expression in experimental groups ( Figure 6C). Furthermore, analysis of IR expression revealed its decreased transcript amount in ASC EMS while AZA/RES increased its expression in ASC EMS I ( Figure 6D).

| AZA/RES diminish apoptosis and autophagy in ASC EMS by the inhibition of mitochondrial fission
To further investigate the mechanism underlying the beneficial effects of AZA/RES on ASC EMS , we used mitochondrial division inhibitor-Mdivi-1. In order to perform the experiments, cells were treated with AZA/RES at two different concentration and supplemented or not with the inhibitor for 24 hours. To evaluate its influence on cells, RT-PCR,TEM and confocal analysis were performed. ASC EMS were characterized by increased expression of p53 ( Figure 7A) and p21 ( Figure 7B) but this could be suppressed by AZA/RES and/or Mdivi-1 treatment. Similar, the expression of p62 was brought back to levels observed in control cells after AZA/RES and/or Mdivi-1 treatment ( Figure 7C). However, AZA/RES do not promote fission and PINK expression ( Figure 7D). It seems to be promoting mitochondrial fusion over fission as shown in Figure 7E. Deeper investigation on mitochondrial dynamics and it differences between groups was investigated with confocal and TEM microscope ( Figure 7F).  Figure 8A). Furthermore, using RT-PCR, expression of TET2 ( Figure 8C) and TET3 ( Figure 8D), both promoting demethylation. Although no differences were observed between ASC CTRL and ASC EMS , ASC EMS I were characterized by increased expression of both transcripts. No differences were noed in the expression of DNMT-1 between investigated groups on mRNA levels ( Figure 8E). Immunofluorescence staining for anti-DNMT-1 confirmed RT-PCR results ( Figure 8F).

| DISCUSSION
In our previous studies, EMS has been recognized as a factor that significantly contributes to the deterioration of ASC functionality. Many other studies confirmed the beneficial effects of RES on MSC biology, mainly by enhancing their differentiation potential. [48][49][50][51] The antioxidant properties of RES are well documented, as it is known to protect both proteins and organelles from oxidation in time-and concentration-dependent manner. Using oxidatively stressed erythrocytes, it has been shown that polyphenol exerts the best antioxidative protection between 30 and 60 minutes after addition. 52 However, RES is not only a free radical scavenger by itself, but it can also modulate the activity of antioxidative enzymes. Studies have proved that RES can increase the amount of SOD, glutathione peroxidase and glutathione reductase. 53  in ASCs EMS , because ER appeared to act directly as a negative regulator of insulin signalling pathway. 62 We have found that IR expression is significantly down-regulated in ASCs EMS , which may indicate that IR in EMS horses affects not only insulin-sensitive tissues, but also adipose-derived stem cells. However, the mechanism of IR in stem cells remains elusive and clearly needs to be further investigated. It should be noted that AZA/RES significantly increased IR expression in ASCs EMS II , which is consistent with the experiments conducted by other researchers, who found that RES considerably improved insulin sensitivity and glucose control in subjects with diabetes. 63 Mitochondrial morphology is mediated by the balance between We also thank Marta Jeleń and Damian Kacperski for technical support in molecular biology assays.

CONFLI CT OF INTEREST
The authors confirm that there are no conflicts of interest.