Upregulated tumor necrosis factor-α transcriptome and proteome in adipose tissue-derived mesenchymal stem cells from pigs with metabolic syndrome
Introduction
The prevalence of obesity has doubled in the past few decades, and it is estimated that nearly one third of the world population is either overweight or obese [1]. Obesity is associated with multiple comorbidities, such as cardiovascular disease and chronic kidney disease (CKD), and can involve multiple organs. Adipose tissue is a major endocrine organ secreting various cytokines, which induces a state of chronic low-grade inflammation. In particular, adipose tissue in obesity has upregulated expression of the inflammatory cytokine tumor necrosis factor (TNF)-α, and its levels correlate with the degree of obesity and insulin resistance [2].
With the concomitant rise in the prevalence of CKD and its associated comorbidities, novel therapeutic approaches are needed. For example, delivery of mesenchymal stem cells (MSCs) has shown promise in clinical trials in patients with renovascular disease [3]. These multipotent stromal cells have the ability for self-renewal, and are characterized by expression of several surface marker, such as CD44, CD73, CD90, CD105, CD166, CD271 and absence of CD14, CD34, CD45 and HLA DR [4]. Additionally, they show plasticity and are able to differentiate into mesodermal cell types.
However, in patients with obesity, MSCs are exposed to an aberrant microenvironment that may impact their phenotype and function. Adipose tissue-derived MSCs harvested from animal models of obesity show altered mRNA, micro-RNA, and protein cargo compared with Lean-MSCs, with upregulation of genes involved in inflammation [5]. Moreover, this inflammatory microenvironment favors MSC differentiation into adipocytes and increases senescence, which is prominently mediated by TNF-α [6]. Furthermore, an inflammatory milieu can abolish the MSC immunomodulatory capacity [7]. A range of MSC tissue sources for MSC collection, as well as diverse microenvironments at both the harvest and implantation sites, may therefore partly account for inconsistent effects of autologous MSCs delivered exogenously.
MSC regenerative potential relies primarily on their paracrine function which is executed by release of soluble mediators and extracellular vesicles (EVs). EVs carry cargo from their parent cells consisting of proteins, mRNAs, and microRNAs, and activate several repair mechanisms to ameliorate injury in target organs or tissues. However, in addition to its influence on MSCs [5], [8], metabolic syndrome (MetS) may also alter the cargo of their EV progeny. Therefore, changes in the cargo of EVs derived from MetS-MSCs may impact MSC paracrine function and reparative potential [9]
TNF-α is a pivotal mediator of inflammation, and its systemic level is of the earliest to rise during development of obesity [10]. Therefore, it might be involved in alterations in MSCs in experimental obesity and MetS. TNF-α belongs to the TNF superfamily (TNFSF), and signals through its receptors, TNF-R1 and TNF-R2. TNFSF is involved in many processes, and constitutes a potential target area for therapies against disease processes like atherosclerosis and ischemia [11]. TNF-R1 is expressed on most cells, whereas the expression of TNF-R2 is more limited, yet includes MSC [12]. TNF-R1 is associated with inflammation and apoptosis via activation of the adaptor proteins TNF-R1 associated death-domain and Fas-associated death-domain (FADD), and TNF-R2 signaling is dependent on TNF receptor-associated factor-2 (TRAF2) activation and associated with pro-survival [13], [14].
Notably, EVs from MetS-MSCs can upregulate TNF-α in target cells and target insulin signaling and metabolic complications [15], [16], [17]. However, whether MetS alters TNF-α signaling pathways in MSCs and their EVs remains unclear. We therefore hypothesized that MetS upregulates TNF-α signaling in adipose tissue-derived MSCs, and this alteration would be reflected in their EVs.
Section snippets
Materials and methods
Mayo Clinic Animal Care and Use Committee approved this study. Three-month-old female domestic pigs were randomized into Lean and MetS groups (n = 4 each) for 16 weeks. MetS pigs were fed a high-cholesterol/carbohydrate diet (ether extract fat 43.0%, carbohydrates 40.8%, and protein 16.1%, 5B4L, Purina Test Diet, Richmond, IN) [18] and Lean pigs were fed a standard chow (13% protein, 2% fat, 6% fiber, Purina Animal Nutrition LLC, MN).
Systemic characteristics
At sixteen weeks after commencing the diet, body weight and blood pressure were both elevated in MetS pigs in comparison to Lean pigs (Table 1). Levels of insulin, HOMA-IR, and lipid fractions were greater in MetS pigs versus Lean, whereas fasting glucose levels were comparable between the groups. These findings were consistent with development of MetS.
mRNAs and proteins upregulated in MetS-MSCs
Annotated genes (n = 10,413) were filtered for mRNAs involved in TNF-α pathways, including TNF-R1 and TNF-R2. Of these, we found 13 mRNAs
Discussion
The current study shows that MetS upregulates TNF-α signaling-related transcriptome and proteome in swine adipose tissue-derived MSCs, and may thereby activate pro-inflammatory signaling via the NF-kB pathway. Similarly, modest alterations were propagated to paracrine function of MSCs reflected by upregulated TNF-α genes in MetS-EVs. These findings suggest that the MetS microenvironment may modulate the impact of endogenous MSCs on target cells, and increase the inflammatory profile not only in
Credit authorship contribution statement
Aditya S. Pawar: Conceptualization, Investigation, Formal analysis, Methodology, Software, Writing - original draft. Alfonso Eirin: Investigation, Formal analysis, Methodology, Software. Hui Tang: Investigation, Formal analysis, Methodology, Software. Xiang-Yang Zhu: Investigation, Formal analysis, Methodology, Software. Amir Lerman: Investigation, Formal analysis, Methodology, Software. Lilach O. Lerman: Investigation, Formal analysis, Methodology, Software.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
None.
Funding
Dr. Lerman receives grant funding from Novo Nordisk, and is an advisor to Weijian Technologies and AstraZeneca. This study was partly supported by NIH grant numbers DK120292, DK122734, DK102325, DK122137, and DK106427.
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Author’s current address: Transplant Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States.