Metabolic Syndrome Interferes with Packaging of Proteins within Porcine Mesenchymal Stem Cell-Derived Extracellular Vesicles

Abstract

Mesenchymal stem/stromal cells (MSCs) release extracellular vesicles (EVs), which shuttle proteins to recipient cells, promoting cellular repair. We hypothesized that cardiovascular risk factors may alter the pattern of proteins packed within MSC-derived EVs. To test this, we compared the protein cargo of EVs to their parent MSCs in pigs with metabolic syndrome (MetS) and Lean controls. Porcine MSCs were harvested from abdominal fat after 16 weeks of Lean- or MetS-diet (n = 5 each), and their EVs isolated. Following liquid chromatography mass spectrometry proteomic analysis, proteins were classified based on cellular component, molecular function, and protein class. Five candidate proteins were validated by Western blot. Clustering analysis was performed to identify primary functional categories of proteins enriched in or excluded from EVs. Proteomics analysis identified 6,690 and 6,790 distinct proteins in Lean- and MetS-EVs, respectively. Differential expression analysis revealed that 146 proteins were upregulated and 273 downregulated in Lean-EVs versus Lean-MSCs, whereas 787 proteins were upregulated and 185 downregulated in MetS-EVs versus MetS-MSCs. Proteins enriched in both Lean- and MetS-EVs participate in vesicle-mediated transport and cell-to-cell communication. Proteins enriched exclusively in Lean-EVs modulate pathways related to the MSC reparative capacity, including cell proliferation, differentiation, and activation, as well as transforming growth factor-β signaling. Contrarily, proteins enriched only in MetS-EVs are linked to proinflammatory pathways, including acute inflammatory response, leukocyte transendothelial migration, and cytokine production. Coculture with MetS-EVs increased renal tubular cell inflammation. MetS alters the protein cargo of porcine MSC-derived EVs, selectively packaging specific proinflammatory signatures that may impair their ability to repair damaged tissues. Stem Cells Translational Medicine 2019;8:430-440.

Keywords: Exosomes; Mesenchymal stem cells; Microvesicles; Proteomics; Vesicles.

Figure 1

(A): Volcano plot of identified proteins enriched in or excluded from Lean‐ (top) and metabolic syndrome‐extracellular vesicles (MetS‐EVs; bottom). A total of 146 proteins were enriched in lean‐ and 185 in MetS‐EVs compared with their parent mesenchymal stem/stromal cells (MSCs), whereas 273 proteins were excluded from Lean‐ and 787 from MetS‐EVs. (B): Venn diagrams showing distribution of proteins enriched in (left) or excluded from (right) Lean‐ and MetS‐EVs.

Figure 2

(A): Heat map of 55 proteins enriched both in Lean‐ and metabolic syndrome‐extracellular vesicles (MetS‐EVs) compared with their parent mesenchymal stem/stromal cells. (B): Panther analysis of cellular component, molecular function, and protein class of proteins enriched both in Lean‐ and MetS‐EVs. (C): Functional annotation clustering (using DAVID 6.8) of proteins enriched both in Lean‐ and MetS‐EVs.

Figure 3

(A): Heat map of 91 proteins enriched only in Lean‐extracellular vesicles (EVs). (B): Panther analysis of cellular component, molecular function, and protein class of proteins enriched only in Lean‐EVs. (C): Functional annotation clustering (using DAVID 6.8) of proteins enriched only in Lean‐EVs.

Figure 4

(A): Heat map of 130 proteins enriched only in metabolic syndrome‐extracellular vesicles (MetS‐EVs). (B): Panther analysis of cellular component, molecular function, and protein class of proteins enriched only in MetS‐EVs. (C): Functional annotation clustering (using DAVID 6.8) of proteins enriched only in MetS‐EVs.

Figure 5

Immunofluorescence staining showing subcellular distribution of nuclear factor (NF)‐kB in renal tubular epithelial cells (PK1) untreated or treated with lean‐ and metabolic syndrome‐extracellular vesicles (MetS‐EVs). MetS‐EVs induced a marked translocation of NFkB from the cytoplasm to the cellular nucleus (n = 5 each).

Figure 6

Expression of tumor necrosis factor (TNF)‐α, monocyte chemoattractant protein (MCP)‐1, interleukin (IL)‐6, and IL‐1β was similar in PK1 and PK1 + Lean‐extracellular vesicles (EVs), but higher in PK1 + metabolic syndrome‐EVs compared with PK1 or PK1 + Lean‐EVs (n = 5 each).