Effect of uncultured adipose-derived stromal vascular fraction on preventing urethral stricture formation in rats

Abstract

Urethral stricture (US) remains a challenging disease without effective treatment options due to the high recurrence rate. This study aims to evaluate the preventive effect of uncultured adipose derived stromal vascular fraction (SVF) on urethral fibrosis in a rat model of US. Results demonstrated that US rats displayed hyperechogenic urethral wall with a narrowed lumen compared with sham rats, while SVF rats exhibited less extensive urethral changes. By histology, US rats showed obvious submucosal fibrosis in the urethral specimens, while SVF rats exhibited mild submucosal fibrosis with less extensive tissue changes. Furthermore, US rats showed increased gene and protein expression of collagen I (2.0 ± 0.2, 2.2 ± 0.2, all were normalized against GAPDH, including the following), collagen III (2.5 ± 0.3, 1.2 ± 0.1), and TGFβ1R (2.8 ± 0.3, 1.9 ± 0.2), while SVF cells administration contributed to decreased gene and protein expression of collagen I (1.6 ± 0.2, 1.6 ± 0.2), collagen III (1.8 ± 0.4, 0.9 ± 0.1), and TGFβ1R (1.8 ± 0.3, 1.3 ± 0.2), in parallel with the improvement of vascularization and increased expression of VEGF (1.7 ± 0.1) and bFGF (3.1 ± 0.3). Additionally, SVF served anti-inflammatory effect through regulation of inflammatory cytokines and cells, accompanied with conversion of the macrophage phenotype. Our findings suggested that uncultured SVF presented an inhibitory effect on stricture formation at an early stage of urethral fibrosis.

Figure 1

Representative flow cytometry histograms of uncultured adipose derived stromal vascular fraction (SVF). Uncultured SVF cells expressed hematopoietic (CD11b/c, CD34, CD45, and CD133), mesenchymal (CD90 and CD106), and endothelial (CD31 and VEGFR2) markers. The red lines represent isotype controls.

Figure 2

Representative microultrasound images of penile urethras in sham, US and SVF groups at 4 weeks after surgery. White arrows indicate sites of hyperechogenic tissues and narrowing of the urethral lumen in the US and SVF groups. CS corpus spongiosun, U urethral lumen.

Figure 3

Representative microscopic findings of hematoxylin and eosin (H&E) and Masson’s trichrome staining in rat urethral tissues in sham, US and SVF groups at 4 weeks after surgery. Scale bar = 100 μm. US rats showed densely packed collagen bundles and sparse smooth muscle in the urethral tissues. Black arrows indicate the sites of collagen deposition with few vascular. SVF rats exhibited mild submucosal fibrosis with less extensive tissue changes. Asterisk indicate the sites of less collagen deposition with visible vascular.

Figure 4

Protein expression of Collagen I, Collagen III and TGFβ1R in urethral tissues were measured by western blot analysis in sham, US, and SVF groups at 4 weeks after surgery. Western blots were quantified with data expressed as relative abundance of Collagen I/GAPDH, Collagen III/GAPDH, and TGFβ1R/GAPDH. Asterisk indicates p < 0.05, which was considered significant differences in protein expression.

Figure 5

Gene expression of collagen I, collagen III, and TGFβ1R in urethral tissues were measured by real-time PCR in sham, US, and SVF groups at 4 weeks after surgery. Asterisk indicates p < 0.05, which was considered significant differences in gene expression.

Figure 6

The angiogenic effect of SVF in urethral tissues. (A) CD31 and CD34 expression was evaluated by immunofluorescent staining of urethral tissues in sham, US, and SVF groups at 4 weeks after surgery. Scale bar = 100 μm. (B) Expression of VEGF and bFGF in urethral tissues were measured by western blot analysis. Western blots were quantified with data expressed as relative abundance of VEGF/GAPDH, and bFGF/GAPDH. Asterisk indicates p < 0.05.

Figure 7

Expression of inflammatory cytokines (TNF-α and IL-10) in the urethral tissues was evaluated by immunofluorescent staining in sham, US, and SVF groups at 4 weeks after surgery. Scale bar = 50 μm.

Figure 8

The infiltration of inflammatory cells in urethral tissues was evaluated by immunofluorescent staining (anti-CD68 for pan macrophages, anti-iNOS for M1 macrophages, anti-CD163 for M2 macrophages, anti-Ly6G for neutrophils, anti-CD3 for T cells) in sham, US, and SVF groups at 4 weeks after surgery. Scale bar = 50 μm.