Nervonic acid improves fat transplantation by promoting adipogenesis and angiogenesis

Abstract

Adipose tissue engraftment has become a promising strategy in the field of regenerative surgery; however, there are notable challenges associated with it, such as resorption of 50‑90% of the transplanted fat or cyst formation due to fat necrosis after fat transplantation. Therefore, identifying novel materials or methods to improve the engraftment efficiency is crucial. The present study investigated the effects of nervonic acid (NA), a monounsaturated very long‑chain fatty acid, on adipogenesis and fat transplantation, as well as its underlying mechanisms. To assess this, NA was used to treat cells during adipogenesis in vitro, and the expression levels of markers, including PPARγ and CEBPα, and signaling molecules were detected through reverse transcription‑quantitative PCR and western blotting. In addition, NA was mixed with fat grafts in in vivo fat transplantation, followed by analysis through Oil Red O staining, hematoxylin & eosin staining and immunohistochemistry. It was demonstrated that NA treatment accelerated adipogenesis through activation of the Akt/mTOR pathway and inhibition of Wnt signaling. NA treatment enriched the expression of Akt/mTOR signaling‑related genes, and increased the expression of genes involved in angiogenesis and fat differentiation in human mesenchymal stem cells (MSCs). Additionally, NA effectively improved the outcome of adipose tissue engraftment in mice. Treatment of grafts with NA at transplantation reduced the resorption of transplanted fat and increased the proportion of perilipin‑1⁺ adipocytes with a lower portion of vacuoles in mice. Moreover, the NA‑treated group exhibited a reduced pro‑inflammatory response and had more CD31⁺ vessel structures, which were relatively evenly distributed among viable adipocytes, facilitating successful engraftment. In conclusion, the present study demonstrated that NA may not only stimulate adipogenesis by regulating signaling pathways in human MSCs, but could improve the outcome of fat transplantation by reducing inflammation and stimulating angiogenesis. It was thus hypothesized that NA could serve as an adjuvant strategy to enhance fat engraftment in regenerative surgery.

Keywords: adipocyte viability; adipogenesis; adjuvant; angiogenesis; fat transplantation; inflammation; nervonic acid.

Figure 1

Effect of NA on adipogenesis. (A) Cell viability of AD-MSCs as measured by O.D. values at day 3 and 7 after treatment with DMSO or serial doses of NA. (B) Representative images of AD-MSCs after 72 h of NA treatment. Yellow arrows indicate the oil droplets formed after NA treatment in the normal proliferation condition. Scale bars: 100 μm. (C) Representative images of Oil Red O staining of AD-MSCs on day 7 after adipogenic differentiation and treatment with DMSO or NA. Scale bars: 100 μm. (D) Relative amounts of lipid accumulation after 7 days of adipogenic differentiation in cells treated with DMSO or NA. Statistical significance was assessed by one-way ANOVA: ^***P<0.001. (E) Representative images of Oil Red O staining of AD-MSCs after 7, 14 and 21 days of adipogenic differentiation and treatment with DMSO or 160 μM NA. Scale bars: 100 μm. (F and G) Relative amounts of lipid accumulation after 7, 14 and 21 days of adipogenic differentiation and treatment with DMSO or 160 μM NA. (F) Increase in lipid accumulation by differentiation period and NA treatment. Relative lipid accumulation was normalized to the DMSO group at day 7. Statistical significance was assessed by one-way ANOVA: ^***P<0.001. (G) Change in lipid accumulation at each differentiation timepoint (7, 14 and 21 days) in the NA group versus the DMSO group. Relative lipid accumulation was normalized to the DMSO group at each time point. Statistical significance was assessed by unpaired Student's t-test: ^***P<0.001. (H) Reverse transcription-quantitative PCR analysis of adipogenic markers after 7, 14 and 21 days of adipogenic differentiation and treatment with DMSO or 160 μM NA. Statistical significance was assessed by unpaired Student's t-test: ^*P<0.05, ^**P<0.01 and ^***P<0.001. Data are presented as the mean ± SD. AD-MSCs, adipose-derived mesenchymal stem cells; DMSO, dimethyl sulfoxide; LPL, lipoprotein lipase; NA, nervonic acid; O.D., optical density.

Figure 2

Signaling pathways regulating adipogenesis are affected by NA. (A) Western blot analysis of adipogenic markers after 14 days of adipogenic differentiation and treatment with DMSO or 160 μM NA. (B) Western blot analysis of Akt and mTOR phosphorylation. (C) Western blot analysis of GSK3β expression and β-catenin phosphorylation. (D) Western blot analysis of Smad1/5 and ERK1/2 phosphorylation. (E) Graphical representation of signaling pathways affected by NA during adipogenesis. Created with BioRender.com. Statistical significance was assessed using unpaired Student's t-test: ^*P<0.05, ^**P<0.01 and ^***P<0.001. Data are presented as the mean ± SD. DMSO, dimethyl sulfoxide; LPL, lipoprotein lipase; NA, nervonic acid; p-, phosphorylated.

Figure 3

Transcriptomic changes in differentiating AD-MSCs after NA treatment. (A) Principal component analysis of AD-MSCs treated with DMSO or NA during adipogenesis. (B) Gene Ontology analysis of the biological processes associated with differentially expressed genes in the NA-treated group against the DMSO-treated group at day 7 of adipogenesis. (C) Heatmap of the expression of genes involved in 'mTOR signaling, 'positive regulation of mTOR signaling' (left) and 'adipose tissue development' (right) in the NA-treated group versus the DMSO-treated group. (D) Heatmap of the expression of genes involved in 'angiogenesis' (left) and 'positive regulation of vascular endothelial growth factor production' (right) in the NA-treated group versus the DMSO-treated group. AD-MSCs, adipose-derived mesenchymal stem cells; DMSO, dimethyl sulfoxide; NA, nervonic acid.

Figure 4

Effects of NA on adipose tissue engraftment. (A) Images of allogeneic fat transplantation in C57BL/6 mice. Peritoneal fat was processed for adipocyte isolation, implanted subcutaneously and treated with 160 μM NA or an equal volume of DMSO. The grafts were harvested after 5 weeks and were further analyzed. Created with BioRender.com. (B) Images of fat grafts 5 weeks after fat transplantation and treatment with DMSO (left) or NA (right). (C) Images and relative sizes of the isolated fat grafts treated with DMSO or NA after transplantation. (D) Axes lengths, (E) fat volume and (F) fat weight of isolated fat grafts treated with DMSO or NA. (G) Oil Red O staining of fat grafts treated with DMSO or NA. Black boxes in the upper images represent the lower images. Scale bars: 800 μm (upper), 200 μm (lower). (H) Oil Red O-positive area within whole fat grafts after transplantation. (I) Reverse transcription-quantitative PCR analysis of Pparg, Lep and Lpl in the fat grafts. Data are presented as the mean ± SD. Statistical significance was assessed using unpaired Student's t-test: ^*P<0.05, ^**P<0.01 and ^***P<0.001. DMSO, dimethyl sulfoxide; H&E, hematoxylin and eosin; Lpl, lipoprotein lipase; NA, nervonic acid.

Figure 5

NA functions in fat engraftment by improving inflammation and angiogenesis. (A) H&E staining within grafts treated with DMSO or NA. The black asterisks indicate formed vacuoles or oil cysts within the grafts and the black arrows indicate the vessels within the grafts. Scale bars: 200 μm. Immunohistochemistry analysis of (B) perilipin-1 and (C) CD31 within grafts treated with DMSO or NA. Black arrows indicate the vessel structures within the grafts. Scale bars: 200 μm. The proportion of (D) vacuole area, (E) perilipin-1⁺ area and (F) CD31⁺ area measured within the whole grafts. (G) RT-qPCR analysis of Tnf and Il10. (H) RT-qPCR analysis of Angpt1, Tek and Vegfa. Data are presented as the mean ± SD. Statistical significance was assessed using unpaired Student's t-test: ^*P<0.05 and ^**P<0.01. DMSO, dimethyl sulfoxide; H&E, hematoxylin and eosin; NA, nervonic acid; RT-qPCR, reverse transcription-quantitative PCR.

Figure 6

NA is a potential adjuvant strategy for supporting fat grafts in regenerative surgery. The addition of NA to adipose tissue transplantation could accelerate the differentiation of adipocytes through the activation of Akt/mTOR signaling and inhibition of Wnt signaling, and could facilitate the reconstruction of the microenvironment within the transplanted adipose tissue by promoting angiogenesis. Created with BioRender.com. NA, nervonic acid.