Background
Mesenchymal stem/stromal cells (MSCs) are non-embryonic stem cells that have the potential to proliferate and differentiate into several cell types, and can be isolated from the umbilical cord, bone marrow, adipose tissue, and other tissues [
1]. Because MSCs possess important pro-angiogenic, immunomodulatory, and anti-inflammatory properties, they are a promising source for cell-based strategies that support tissue regeneration [
2]. Indeed, over 200 clinical trials are currently testing the efficacy of MSC therapy for treating a variety of diseases [
3].
MSCs exert their reparative actions in part by releasing extracellular vesicles (EVs), membrane microparticles including microvesicles and exosomes that shuttle their genetic and protein cargo to damaged tissues [
4,
5]. Indeed, studies using a pig model of renal vascular disease have shown that EVs may activate an endogenous repair program in parenchymal cells [
6,
7]. We have previously shown that adipose tissue-derived MSCs release EVs that transport gene and protein-based regulatory information to modulate angiogenesis, apoptosis, extracellular matrix remodeling and other cellular pathways in recipient cells [
8‐
10], underscoring the reparative potential of MSCs. For example, MSC-derived EVs are selectively enriched with mRNAs encoding for transcription factors, Golgi apparatus components, and proteins involved in transforming growth factor (TGF)-β signaling. However, comorbidities and cardiovascular risk factors that alter the microenvironment from which these cells are harvested may potentially impact on the cargo packed within their EV progeny [
11‐
13].
The metabolic syndrome (MetS) is a constellation of cardiovascular risk factors, including obesity, hypertension, dyslipidemia, increased serum triglycerides, and insulin resistance, that increases cardiovascular morbidity and mortality. In a recently developed porcine model of MetS and fat inflammation [
14], we found that adipose tissue-derived MSCs show increased propensity for adipogenesis and for premature senescence compared to Lean-MSCs [
15]. In the current study, we addressed the key question whether this phenotypic change is accompanied by altered packaging of mRNA cargo in their EV paracrine vectors. We tested the hypothesis that MetS affects the phenotype of MSCs, which in turn modifies the mRNA expression profile in EVs derived from MSCs. Expression profiles of mRNAs were obtained in EVs and their parent MSCs, using high-throughput RNA sequencing (RNA-seq). We show that the mRNA cargo of MetS-EVs is characterized by genes primarily involved in inflammation. Yet, mRNAs enriched in Lean-EVs are involved in tissue repair and TGF-β signaling, and these mRNAs appear to be excluded from MetS-EVs. Lastly, microRNAs enriched in EVs have the potential to target a significant proportion of genes enriched in Lean and MetS EVs, implying post-transcriptional regulatory mechanisms. These observations may help refine cell-based therapy and support development of adequate strategies to improve the efficacy of MSCs and their EVs.
Discussion
Our study employed high-throughput mRNA sequencing to interrogate mRNA expression in EVs and their parent MSCs, and explore the putative function of enriched or excluded genes in Lean- vs. MetS-EVs. The current study demonstrates that MetS modulates the mRNA cargo packed within porcine adipose tissue MSC-derived EVs, as the mRNA content of Lean- and MetS-EVs is substantially different. Lean-EVs are enriched with mRNAs primarily involved in transcription regulation and TGF-β signaling pathway, but not in genes related to regulation of inflammation. In contrast, MetS-EVs contain mRNAs involved in translational regulation and modulation of inflammation, but lack mRNAs related TGF-β signaling. Furthermore, we found that co-culture with MetS-EVs increases renal tubular cell inflammation in-vitro. Therefore, these observations demonstrate that MetS alters the genetic cargo of MSC-derived EVs, which might interfere with their ability to repair damaged tissues.
MSCs constitute a promising approach for cell therapy, but their function might be altered in a cardiovascular disease milieu, compromising MSC function. For example, MSCs from patients with MetS and diabetes show increased oxidative stress and autophagy, and fail to differentiate in functional adipocytes [
11,
29]. Likewise, MSCs isolated from horses with MetS show impaired viability, oxidative stress, and senescence [
12]. In agreement, we have previously shown that adipose tissue-derived MSCs obtained from pigs with MetS show enhanced adipogenic and osteogenic propensity and senescence compared to their Lean counterparts, which was partly mediated by adipose tissue inflammation [
15]. More recently, Saleh et al. [
30] summarized findings from preclinical studies testing the efficacy of adipose tissue-derived MSCs on obesity and MetS. They found promising beneficial effects of MSC transplantation on obesity and MetS, and conclude that paracrine effects of MSCs may constitute their most biologically significant role.
The paracrine function of MSCs is partly mediated by releasing EVs. As therapeutic tools, EVs may have a superior safety profile compared to their parent cells [
31], because unlike cells they can be safely stored without losing function. In previous studies in healthy pigs [
8‐
10], we found that adipose tissue-derived MSCs release EVs that transport genes and proteins capable of modulating several cellular pathways, particularly high levels of TGF-β-related genes and pro-angiogenic genes. The current study extends our previous findings, and underscores the selective content of genes encoding regulators of transcription, TGF-β signaling, and angiogenesis in Lean-EVs. TGF-β is an anti-inflammatory factor that regulates cell growth, differentiation, and fibrosis, as well as regulatory T-cells. Among its signaling partners are growth/differentiation factor-7 (or bone morphogenetic protein-12), which stimulates MSC differentiation [
32] and induces tenogenesis in bone-marrow MSCs [
33], and Activin receptor type-2B (ACVR2B), a member of bone morphogenetic protein signaling involved in regulating adult bone mass [
34]. Wnt signaling is also one of the top functional categories of genes enriched in Lean-EVs, and has a close link to TGF-β signaling. They synergistically modulate embryonic development, fibrotic disease, and tumor progression through interactions among Smad, Axin, Dvl and β-catenin [
35]. For example, Adenomatous Polyposis Coli (Apc), enriched in Lean-EVs (×1.5 vs. MSCs), is a Wnt pathway regulator that in conjunction with TGF-β regulates epithelial progenitor cell fate [
36]. Hence, EVs may be endowed with functions linked to repair and regeneration of tissues and suppression of inflammation.
Compared to Lean-EVs, MetS EVs were found be enriched with mRNAs associated with inflammation, such as those encoding the integrin family proteins. Integrins are a family of transmembrane receptors that activate signal transduction pathways, inducing inflammation and early atherosclerosis. For example, ITGA2 (integrin alpha-2) triggers inflammation and endothelial dysfunction in patients with cardiovascular disease, chronic kidney disease, and type-2 diabetes [
37‐
39], and joint inflammation in mouse models of rheumatoid arthritis [
40]. Other pro-inflammatory genes enriched in MetS-EVs (Fig.
1c) included the phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (PIK3CB) [
41] and several members of the ubiquitin-proteasome pathway involved in development of inflammatory and autoimmune diseases [
42]. For example, the ubiquitin conjugating enzyme E2-C (UBE2C), which activates several other genes (SLC2A1-CCNB2-HMMR-KIF11-NUSAP1-PRC1-UBE2C), triggering inflammation [
43], and the UCHL5 (or ubiquitin C-terminal hydrolase UCH37), which plays an important role in inflammation and host defense [
44]. Lastly, mRNAs enriched in MetS-EVs encode proteins involved in FGF signaling, a family of proteins which can contribute to pathological conditions by modulating expression of inflammatory cytokines [
45]. Hence, MetS-EVs have specific pro-inflammatory signatures that may impair the ability of MSCs to repair damaged tissues.
Genes that modulate apoptosis were similarly enriched in Lean- and MetS-EVs, but further analysis showed that those enriched in Lean-EVs encoded anti-apoptotic proteins, such as CFLAR (CASP8 and FADD-like apoptosis regulator) and heat-shock-related 70-kDa protein-2 (HSPA2). CFLARL-deficient T-cells undergo severe cell death upon T-cell receptor stimulation, involving apoptosis and necroptosis [
46]. Likewise, HSPA2 increases long-term survival of cells subjected to heat shock and proteasome inhibitors, and can be a part of a system protecting cells against cytotoxic stimuli inducing proteotoxic stress [
47]. In contrast, apoptosis-related genes that were enriched in MetS-EVs promote autophagy and apoptosis, including EIF2S1 and MAP3K5. EIF2S1 is an ER stress sensor that induces mitophagy through ER stress activation due to reactive oxygen species (ROS) accumulation, and MAP3K5 (also known as apoptosis signal-regulating kinase-1; ASK1) is a key component of ROS-induced JNK and p38 activation that activates apoptosis.
Our analysis also identified several mRNAs related to insulin signaling enriched in EVS, which were different between Lean-EVs and MetS-EVs. Interestingly, two mRNAs enriched in MetS-EVs were also enriched in MetS-MSCs, as we observed in a recent study [
25]. The nuclear receptor subfamily-5 group-A member-1 (NR5A1, or steroidogenic factor-1), which modulates the expression of insulin-like factor 3, relaxin-like factor [
48], and the oligopeptide transporter PepT1 solute carrier family-15 member-1 (SLC15A1), which is highly regulated by insulin [
49]. These observations suggest that MetS alters mRNA expression related to insulin signaling in adipose tissue-derived MSCs and their daughter EVs.
To test whether MetS-induced changes in the cargo of MSC-derived EVs impact their anti-inflammatory and immunomodulatory potential, we compared the effects of Lean- and MetS-EVs in M1-polarized macrophages and renal tubular epithelial cells in vitro. We have previously demonstrated the ability of porcine adipose tissue-derived MSCs to switch macrophages from a pro-inflammatory M1 phenotype to M2, a repair-linked phenotype [
17]. In the current study, we found that co-culture of M1-polarized macrophages with either Lean- or MetS-MSC-derived EVs similarly decreased the expression of the M1 markers and increased expression of M2 markers, in accordance with the similar capacity of Lean- and MetS-MSCs to restore an M2 phenotype in activated macrophages [
15]. Contrarily, we found that expression of the pro-inflammatory cytokines TNF-α and MCP-1 was higher in renal tubular cells co-cultured with MetS-EVs compared to untreated cells and cells co-cultured with Lean-EVs, consistent with our previous observation that TNF-α magnified MSC dysfunction in MetS [
15]. Taken together, these observations reflect the complex regulation of inflammatory responses, and suggest that MetS-MSCs and their EVs induce pro-inflammatory cytokines, but do not directly alter macrophage phenotype.
These observations have important clinical implications. Autologous transplantation is preferred for MSC therapy in patients, many of who have comorbidities and cardiovascular risk factors. The primary mechanism of action of MSCs is the delivery of EVs carrying mRNAs, microRNAs, and proteins that are transferred to damaged cells. Thus, our observation that MetS induces changes in the mRNA cargo of MSC-derived EVs that affects their immunomodulatory properties in vitro suggests that the reparative capacity of these cells may be limited in subjects with MetS.
Our study has a number of strengths, including a novel swine model of MetS, the comprehensive characterization of the mRNA cargo of Lean- and MetS-EVs, and in vitro studies in macrophages and renal tubular cells to elucidate the impact of MetS-induced changes in the cargo of EVs on their immunomodulatory function. On the other hand, our study is limited by the modest number of animals and short duration of MetS. Nevertheless, MetS pigs developed several features of human MetS, and we detected a clear difference in the mRNA content of their EVs compared to their Lean counterparts. The MSC mRNA profile can change with the passage of cells [
50], thus we opted to study the 3rd passage cells that are relatively stable [
51]. Additional studies are needed to explore the other functional ramification of the distinctive expression profile in MetS-EVs, and whether their mRNA content imposes a differential effect compared to Lean-EV on the phenotype of their recipient cells.
Authors’ contributions
YM, AE: Collection and/or assembly of data, data analysis and interpretation, manuscript writing. XYZ, DRO: Collection and/or assembly of data, data analysis and interpretation. AL, AJV: Data analysis and interpretation, manuscript writing. LOL: conception and design, financial support, provision of study material, manuscript writing. All authors read and approved the final manuscript.