Mitochondrial Activity Regulates Skin-derived Mesenchymal Stem Cell Differentiation Into Brown Adipocyte Contributing to Hypertension

Background: Brown adipocytes (BAs) are the major component of brown adipose tissue (BAT) that is closely related to systemic hypertension. BAs are derived from multiple progenitors including PDGFRα + adipose-derived stem cells (ASCs). Skin-derived Mesenchymal Stem Cells (S-MSCs) have the capacity to differentiate into adipocytes. However, the differentiation of S-MSCs into BAs remains unexplored. We aim to study the ability and regulation mechanism of S-MSCs differentiation into BAs, and the direct role of BAT in blood pressure regulation. Methods: Protein expression was measured by Flow Cytometry or Western blotting, and gene mRNA levels were detected by real-time quantitative PCR (RT-PCR). For the BA differentiation of S-MSCs, S-MSCs were stimulated with a brown adipogenic cocktail containing insulin, IBMX, dexamethasone, triiodothyronine (T3), and rosiglitazone for the indicated periods. The oxygen consumption rate (OCR) was measured with an XF24 Extracellular Flux Analyzer. Mitochondrial mass was checked by ow cytometry and uorescence staining. Hypertensive mouse model was induced in WT mice by infusion with angiotensin II (Ang II) and measured SBP using tail-cuff. The interscapular brown adipose tissue (iBAT)-deciency mice were gotten by surgically removing the iBAT depot and allowed to recover for 6 days. Aorta, iBAT or heart tissue sections were examined by hematoxylin and eosin (HE) staining. Results: We found that S-MSCs isolated from the mouse dermis expressed the stem cell markers CD90/105 and PDGFRα, and readily differentiated into BAs. Mitochondrial biogenesis and oxygen consumption were markedly increased during BA differentiation of S-MSCs in vitro. Another, Ang II-induced hypertensive mice carried the change of iBAT to white adipose tissue (WAT), the enhanced Ang II-induced blood pressure and vascular remodeling were observed in BAT-decient mice generated by surgically removing iBAT comparing with C57BL/6 (wild type-WT) mice. Conclusions: S-MSCs hypertension in we rstly detected and compared the changes of iBAT in hypertensive mice to WT control mice, the results showed extensive brown-to-white adipose transformation in the iBAT extracted from the Ang II-infused mice but no change in the control mice. And in vitro, we also found that Ang II stimulation caused the decreased expression of UCP-1 and PGC-1α in BAs. Further, we generated iBAT-decient mice by surgically removing the iBAT depot in C57BL/6 mice followed with Ang II infusion, and the results demonstrated that the direct deciency of iBAT was able to accelerate Ang II–induced BP elevation and target organ damage compared to control. Overall, this study provides direct evidence that


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The adipose organ is composed of WAT and BAT. WAT serves as an energy store for the body, whereas BAT utilizes chemical energy through uncoupled respiration and thermogenesis in all mammals [1]. BAT plays a critical role in energy homeostasis and body weight control via fat thermogenesis and is highly metabolically active, raising the possibility that it may serve as a potential therapeutic target for metabolic diseases [2]. Transgenic ablation of BAT is associated with not only obesity but also systemic hypertension and cardiac brosis as shown in transgenic mice with reduced brown fat [3,4]. Fibroblast growth factor 21 (FGF21) derived from BAT plays an endocrine protective role against hypertensive cardiac remodeling in mice [5]. BAs are the major component of BAT and arise from distinct developmental origins, including adipogenic progenitors, myogenic factor 5 (Myf5) + progenitors and neuronal cell differentiation [6,7]. However, the underlying origin of BAs is not completely understood.
Mesenchymal stem cells (MSCs) can be expanded and differentiated into a variety of mesenchymal cell types such as adipocytes, osteoblasts and chondrocytes [8]. Human fetal mesenchymal stem cells (fMSCs) have been shown to differentiate into BAs [9]. Previous studies have demonstrated that the PDGFRα + ASCs isolated from WAT could differentiate into BAs in vitro and in vivo [10]. S-MSCs isolated from the largest lymphoid organ-skin, share functional similarities to MSCs and consistently differentiate into adipocytes, osteocytes, and chondrocytes as well [11,12]. S-MSCs have the ability to migrate to the in amed tissues and perform immunosuppressive activity to inhibit the development of atherosclerosis, EAE and hypertension in mouse models, which indicate that S-MSCs represent a promising cell source for stem cell-based therapies of chronic in ammatory diseases and possibly transplantation [12][13][14][15].
However, it is unknown whether S-MSCs can differentiate into BAs. Understanding of the speci c mechanisms that regulate the differentiation of S-MSCs into BAs is very important to the development feasible clinical treatments.
Recent studies demonstrate that mitochondrial regulation is increasingly recognized as an important determining factor in stem cell biology and function. There has been mounting evidence that mitochondrial metabolism is implicated in the adipogenic differentiation of MSCs [16,17]. In addition, the differentiation and homeostatic function of the adipose tissue are supported by mitochondrial biogenesis [18]. It is also worthwhile to note that BAs are rich in mitochondrial content, and the BAT has been known to play important roles in lipid metabolism and energy expenditure, with a rich expression of thermogenic markers which participate signi cantly in mitochondrial biogenesis [19,20]. Studies have demonstrated that mitochondria play an important regulatory role in determining the differentiation capacity of MSCs [17].
Skin provides an easily accessible and ideal source of tissue for the isolation of S-MSCs. Thus far, no study has reported whether the S-MSCs located in the dermis can differentiate into BAs, or the effect of mitochondrial biogenesis and activity during the BA differentiation of S-MSCs. In our study, we demonstrated that S-MSCs readily differentiated into BAs regulated by mitochondrial activity, and the iBAT directly contributed to improving the blood pressure in Ang II-induced hypertensive mice. Brown adipocytes differentiation of S-MSCs Preparation, culture and characterization of S-MSCs were performed as described previously [12]. For in vitro BAs differentiation, S-MSCs were plated in complete medium at a density of 2 × 10 4 cells per square centimeter in 6-well culture plates and re-fed every 2 days until they were 100% con uent or post con uent. BAs differentiation was induced by treating cells for 48 hours in medium containing 10% FBS, 0.5 mM isobuylmethylxanthine (IBM), 1 µM dexamethasone (DEX), 240 IU/µM insulin, 1 nM T3 and 1 µM rosiglitazone (Alexis Biochemicals). After 48 hours, cells were switched to medium containing 10% FBS (Gibco, #10099-141, Australia), 240 IU/µM insulin, 1 nM T3 and 1 µM rosiglitazone for another 6-8 days, and the differentiation medium was replaced every 2 days. Images of cells were obtained at 0, 2, 4, 6, 8, and 10 days after stimulation by light microscopy (Zeiss, Germany). Original magni cations 100x or 400x.

Mice
Oxygen Consumption S-MSCs were seeded at a density of 4 × 10 4 cells per well in XF 96-well plates. After BAs induction, OCR was detected with a Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA) following the manufacturer's manual. Oligomycin and Rotenone were used as inhibitors and the uncoupler FCCP was used as agonist. The results were calculated and represented as the basal respiration, ATP production, proton leakage, maximal respiration and spare capacity.

Flow Cytometry
Flow cytometry were carried out to assess mitochondrial mass using MitoTracker Green FM (MTGFM), a uorescent mitochondrion-selective probe (Invitrogen,#M7514, USA). S-MSCs were seeded at a density of 4 × 10 5 cells per well on 12-well plates. After BAs differentiation, cells were suspended by 0.125% trypsin, centrifuged, washed with PBS twice and stained with 100 nM MTGFM at 37℃ for 30 minutes. Then the cells were washed with PBS and followed by staining with Propidium Iodide at room temperature for 15 minutes to label the dead cells. The intensity of uorescence was measured on a Beckman Coulter CytoFLEX instrument and the mean uorescence intensity (MFI) was analyzed using a FlowJo 10.2 software.

RNA Extraction and Real-time RT-PCR
S-MSCs were plated at a density of 4 × 10 5 cells per well on 12-well plates for total RNA extraction. Total RNA was extracted with TRIzol reagent (Gibco Life Technologies) following the manufacturer's manual.
Total RNA (2 µg) was reversely transcribed (RT) to cDNA and 0.5 µl RT product was used for Real-time RT-PCR to determine the mRNA level of each gene. Real-time RT-PCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems, USA) using the SYBR Green PCR Master Mix (TaKaRa). β-actin was used as internal control. All gene expression results were normalized to the house keeping gene βactin expression. Cycle threshold (CT) was used for data analysis. All cytokines primers were ordered from Invitrogen (Shanghai). The Primers sequences were listed as follows: PPARG coactivator 1 alpha Immunoblotting S-MSCs were plated at a density of 8 × 10 5 cells per well on 6-well plates. After BAs differentiation, cells were lysed in cell lysis buffer (10 mmol/L Tris-HCl PH7.5, 150 mmol/L NaCl, 0.1% SDS, 1%TritonX-100, 2µ g/ml Aprotinin, 2µ g/ml Leupeptin and 1 mmol/L PMSF) (Beyotime, Shanghai, China). Protein concentrations were determined using the bicinchoninic acid protein assay (Thermo Fisher Scienti c).

Hypertension induction and blood pressure measurement
Minipumps (1004, Alzet, Cupertino, California) were implanted subcutaneously in mice to deliver Ang II (Sigma) or 0.9% NaCl. Blood pressure (BP) was measured by tail-cuff using the BP-2000 Blood Pressure Analysis System (Visitech Systems, Napa Place Apex, North Carolina). Starting from 10 weeks of age, 12 male C57BL/6 mice were randomly divided into 2 groups: WT-C (Control group, infused with 0.9% NaCl, n = 6), WT-A (Ang II-infused group, infused with Ang II for 28 days-750 ng/kg per minute, n = 6), this induction is only for investigating change of interscapular brown adipose tissue (iBAT) to white adipose tissue (WAT) in angiotensin II(Ang II)-induced hypertensive mice. Starting from 10 weeks of age, 24 male C57BL/6 mice were randomly divided into 4 groups respectively: WT-C (infused with 0.9% NaCl), -BAT-34-C (iBAT-de ciency mice infused with 0.9% NaCl, the iBAT depot were surgically removed and allowed to recover for 6 days, and then treated with 0.9% NaCl for another 28 days), WT-Ang II (infused with 750 ng/kg per minute Ang II for 28 days), -BAT-34-Ang II (iBAT-de ciency mice infused with Ang II, the iBAT depot were surgically removed and allowed to recover for 6 days, and then treated with 750 ng/kg per minute Ang II for 28 days). Noninvasive tail-cuff monitoring of systolic blood pressure (SBP) of above 4 groups of mice (n = 6).

Histological Analysis
Aorta, iBAT or heart tissue xed by Paraformaldehyde was embedded in para n and 6-µm sections were stained with hematoxylin and eosin (HE) staining [13]. Images of tissues were obtained by using the Axio Imager 2 upright microscope (Zeiss, Germany), and the images were acquired with ZEN Imaging software

Statistical Analysis
The data were analyzed with GraphPad Prism 5 and were presented as the mean ± SD. Student's t-test was used when two conditions were compared, and analysis of variance (ANOVA) with Bonferroni or Newman-Keuls correction was used for multiple comparisons. Probability values of < 0.05 were considered signi cant; two-sided Student's t-tests or ANOVA were performed. *p<0.05; **p<0.01; ***p < 0.001; ****p < 0.0001; ns, not signi cant. Error bars depicted SD.

PDGFRα + S-MSCs readily differentiated into brown-type adipocytes in vitro
BAs are known to be derived from the multipotent progenitor cells, such as PDGFRα + ASCs [10]. S-MSCs are a type of easily attainable MSCs recently favored in stem cell research and the development of tissue therapies [12][13][14][15]. We reasoned that, S-MSCs could also be induced to differentiate into BAs. Here, we demonstrated that S-MSCs isolated from the dermis of mouse skin expressed the stem cell markers CD90/105 and PDGFRα by FACS analysis (Fig. 1A). In order to initiate BA induction, S-MSCs were stimulated with an adipogenic cocktail containing insulin, IBMX, dexamethasone, triiodothyronine (T3), and rosiglitazone for the indicated periods. Lipid droplets became discernible in the cells with 2 days of stimulation and could be observed in about 90% of the cells by day 8, indicating the transition of the cells into BAs (Fig. 1B). Thermogenic markers mitochondrial UCP-1 and PGC-1α have been dubbed the hallmark of BAs [9]. We measured the expression of UCP-1 and PGC-1α in the cells during the course of induced differentiation by RT-PCR and found that the levels of UCP-1 and PGC-1α transcripts steadily increased during the 10-day period of differentiation, with a slight drop in PGC-1α level at day10 (Fig. 1C). This mRNA expression pattern was largely correlated with their protein levels during differentiation, in which both markers showed a continual increase with a minor drop on day 10 (Fig. 1D). These results showed that S-MSCs expressed PDGFRα and readily differentiated into BAs upon adipogenic induction in vitro.

BA differentiation of S-MSCs is accompanied by enhanced oxygen consumption
Increased mitochondrial activity is a prerequisite for MSC differentiation into adipocytes [17]. Using the Seahorse XFe96 analyzer, we found that oxygen consumption of S-MSCs was highly increased during the differentiation periods from day 2 to day 8 ( Fig. 2A), indicating that BA differentiation of S-MSCs is a process that requires enhanced mitochondrial function and energy supply. Mitochondrial function as indicated by ATP levels and mitochondrial complex activities such as basal mitochondrial respiration, proton leak, maximal respiratory capacity, and spare capacity were also signi cantly enhanced during the adipogenic differentiation process (Fig. 2B-F), suggesting that S-MSCs not only acquire higher baseline oxygen consumption, but also exhibit more activated mitochondrial function during BA differentiation.

Mitochondrial biogenesis was increased during BA differentiation of S-MSCs
To further examine the role of mitochondria in brown adipogenic differentiation of S-MSCs, we measured mitochondrial mass in the S-MSCs stimulated with brown adipogenic cocktail for the indicated times by staining with MTGFM and subsequent analysis by ow cytometry. Results demonstrated that mitochondrial mass was signi cantly increased in the S-MSCs after 2 days of BA differentiation and remained at a relatively high level throughout the BA differentiation process (Fig. 3A). Consistently, uorescence microscopy of cells stained with MTGFM and DAPI also showed the increased mitochondrial content in cells at day 4 after stimulation compared with unstimulated S-MSCs (Fig. 3B). Immunoblot analysis showed that expression of mitochondrial complex-1 and complex-4 proteins was robustly enhanced during the BA differentiation process (Fig. 3C). Further, real-time RT-PCR revealed that the mRNA levels of mitochondrial TFAM and NRF1, the key regulating factors of mitochondrial biogenesis, were up-regulated signi cantly during the BA differentiation of S-MSCs (Fig. 3D). These data con rmed that the mitochondrial biogenesis is boosted during the BA differentiation of S-MSCs.
Change of interscapular brown fat tissue to white fat tissue in Ang II-induced hypertensive mice BAT is mainly composed of BAs. Animal studies has displayed that transgenic ablation of BAT is associated with systemic hypertension [3]. However, the change of BAT in Ang II-induced hypertensive mice is rarely investigated. Here, we employed the Ang II-induced hypertensive mouse model. During 28 days of Ang II infusion at 750 ng/kg/min administered by ALZET osmotic pumps, the Ang II-infused mice showed a signi cant increase of systolic blood pressure (SBP) as well as remarkable vascular injury compared with the control mice ( Fig. 4A and 4B). Subsequent histological analysis revealed extensive brown-to-white adipose transformation in the iBAT extracted from the Ang II-infused mice compared with the control mice (Fig. 4C). For further in vitro assessment, we used Ang II to stimulate BAs either induced from S-MSC differentiation or isolated from the iBAT of mice, and found that expression of brown fatspeci c marker UCP-1 and adipogenic marker PGC-1α was decreased in a dose-dependent manner after Ang II stimulation (Fig. 4D and 4E). Together, these data suggest that Ang II causes the whitening of BAT in hypertensive mice and induced BA dysfunction in vitro.
Brown adipose tissue-de ciency enhanced Ang II-induced hypertension and vascular remodeling Prompted by these results, we next aimed to determine the direct role of BAT in blood pressure (BP) regulation by using iBAT-de cient mice (generated by surgically removing the iBAT depot in C57BL/6 mice and allowing to recover for 6 days, -BAT) and WT mice. At day 7, -BAT and WT mice were further administered Ang II or 0.9% NaCl infusion at 750 ng/kg per minute for 28 days and BP was monitored using the noninvasive tail cuff method. We found that iBAT-de cient mice manifested drastically increased SBP elevation compared with WT mice (Fig. 5A). The histological analysis of aorta sections was used to examine the aorta structural changes, and HE staining showed that 4 weeks of Ang II infusion caused hypertrophy of aortas (intima and media), which was aggravated in iBAT-de cient mice (Fig. 5B). The aortic intima and media thickness were quanti ed (Fig. 5C). Ang II-induced brosis of heart was signi cantly aggravated in iBAT-de cient mice compared with WT mice (Fig. 5D). Collectively, these data suggested that the direct de ciency of iBAT was able to facilitate Ang II-induced BP elevation and target organ damage.

Discussion
The ability, molecular mechanism and role of the differentiation of S-MSCs into BAs have not been demonstrated before. Although it is well documented that BAs are derived from distinct developmental origins, such as PDGFRα + ASCs located in WAT that express the common stem cell markers Sca1 and are shown to differentiate into BAs in vitro and in vivo [10]. BAs play a key role in the endocrine protection of hypertensive cardiac remodeling by activating A2AR/FGF21 pathway [5]. In the present study, we revealed that S-MSCs isolated in the dermis of mouse skin which expressed PDGFRα and readily differentiated into BAs regulated by mitochondrial activity in vitro. S-MSCs could be another promising developmental origin of BAs. Functionally, Ang II causes the iBAT to turn into WAT in Ang II-induced hypertensive mice and decreased the thermogenic functional genes UCP-1 and PGC-1α expression in vitro. The direct de ciency of iBAT markedly facilitated Ang II-induced BP elevation and target organ damage.
Recent studies have revealed that BAs could be derived from the Myf5 + progenitor cells located in the perirenal and interscapular regions [21], the Sca-1 + adipogenic progenitor cells residing in murine skeletal muscle and subcutaneous white fat after bone morphogenetic protein 7 (BMP7) stimulation [22]. The BAs emerging in white fat in response to β3-adrenergica stimulation in abdominal WAT that arose from PDGFRα + ASCs [10]. Human fetal mesenchymal stem cells have been proved to differentiate into BAs [9]. Previous study had con rmed that S-MSCs could migrate to the in amed tissues and inhibited the development of atherosclerosis, EAE and hypertension in mouse models, which could be a promising cell source for stem cell-based therapies of chronic in ammatory diseases [12,13,15]. To our knowledge, no prior study has addressed the ability of S-MSCs located in the dermis to differentiate into BAs. The present study is the rst to demonstrate that S-MSCs are able to readily differentiate into BAs in vitro. It could be another promising developmental origin of BAs.
Mitochondria are considered multifaceted organelles that regulate stem cell fate decisions [20]. Generally, mitochondria in MSCs are maintained at a low activity level, and after MSC induction, mitochondrial DNA (mtDNA) copy number, OCR, mitochondrial biogenesis related genes (NRF1 and TFAM) expression and intracellular ATP content are enhanced [23]. Mitochondrial elongation is critical during embryonic stem cell (ESC) differentiation to cardiomyocytes and for the normal cardiac development and function [24]. The robust mitochondrial activity is a prerequisite for hMSC differentiation into adipocytes [17]. The zincnger containing protein PRDM16 initiates differentiation of myoblasts or white preadipocytes into functional BAs [7]. The differentiated BAs augment the expression of mitochondrial function genes (NRF1 and TFAM) and ultimately of UCP1 [25]. In agreement with the previous studies described in this article, we found the increased mitochondrial oxygen consumption and expression of thermogenic markers (UCP1 and PGC1-α) and mitochondrial biogenesis markers (NRF1 and TFAM) during BAs differentiation of S-MSCs.
PDGFRα + S-MSCs could be a novel BA progenitors. BAs are the major component of BAT. Transgenic ablation of BAT is associated with cardiovascular abnormalities and systemic hypertension in the UCP-DTA mice [3]. BAT plays a potential endocrine role against hypertensive cardiac remodeling in DOCA-salt induced hypertension [5]. Tissue-grafting of converted BAT has been proposed as a direct approach to increase endogenous brown fat in vivo [26]. In order to directly con rm the role of BAT in AngII-induced hypertension in mice, we rstly detected and compared the changes of iBAT in hypertensive mice to WT control mice, the results showed extensive brown-to-white adipose transformation in the iBAT extracted from the Ang II-infused mice but no change in the control mice. And in vitro, we also found that Ang II stimulation caused the decreased expression of UCP-1 and PGC-1α in BAs. Further, we generated iBATde cient mice by surgically removing the iBAT depot in C57BL/6 mice followed with Ang II infusion, and the results demonstrated that the direct de ciency of iBAT was able to accelerate Ang II-induced BP elevation and target organ damage compared to control. Overall, this study provides direct evidence that BAT plays a pivotal role in Ang II-induced hypertension and target organ remodeling and further supports previous studies [3,5].
In conclusion, we provide direct evidence that S-MSCs have a potential ability to easily differentiate into BAs, which is regulated by mitochondrial activity. Functionally, the combination of our in vitro and in vivo experiments illustrate that Ang II causes the whitening of the iBAT in hypertensive mice and the dysfunction of BAs. And the direct de ciency of iBAT markedly facilitated Ang II-induced BP elevation and target organ damage (Fig. 6). Our results suggest that S-MSCs can readily differentiate into BAs which are potential therapeutic cell type for the treatment of hypertension.