Adipose-Derived Stem Cells from Type 2 Diabetic Rats Retain Positive Effects in a Rat Model of Erectile Dysfunction

Abstract

Erectile dysfunction is a common complication associated with type 2 diabetes mellitus (T2DM) and after prostatectomy in relation to cancer. The regenerative effect of cultured adipose-derived stem cells (ASCs) for ED therapy has been documented in multiple preclinical trials as well as in recent Pase 1 trials in humans. However, some studies indicate that diabetes negatively affects the mesenchymal stem cell pool, implying that ASCs from T2DM patients could have impaired regenerative capacity. Here, we directly compared ASCs from age-matched diabetic Goto-Kakizaki (ASC_GK) and non-diabetic wild type rats (ASC_WT) with regard to their phenotypes, proteomes and ability to rescue ED in normal rats. Despite ASC_GK exhibiting a slightly lower proliferation rate, ASC_GK and ASC_WT proteomes were more or less identical, and after injections to corpus cavernosum they were equally efficient in restoring erectile function in a rat ED model entailing bilateral nerve crush injury. Moreover, molecular analysis of the corpus cavernosum tissue revealed that both ASC_GK and ASC_WT treated rats had increased induction of genes involved in recovering endothelial function. Thus, our finding argues that T2DM does not appear to be a limiting factor for autologous adipose stem cell therapy when correcting for ED.

Keywords: adipose derived stem/stromal cells; bilateral nerve crush injury; erectile dysfunction; type 2 diabetes mellitus.

Figure 1

Characterization of ASC from WT and GK rats. (A) Blood glucose levels were measured in tail vein blood from 17-week-old wildtype (WT) and diabetic Goto–Kakizaki (GK) rats to validate their non-diabetic/diabetic status, respectively. (B) The primary yield of nucleated cells in SVF from SAT of WT and GK expressed per mg of adipose tissue (cells/mg of tissue). (C) The frequency of stromal progenitors in the original SVF was determined by CFU-F assays of parent SVF_WT and SVF_GK cells. (D) Proliferation of ASC_WT and ASC_GK were followed by cell number assessment after 2, 4, 6, 8, 10 and 12 days of culture. (E) The presence of well-known mesenchymal stem cell surface markers of ASC_WT and ASC_GK cells were analyzed by flow cytometry. For each marker, the percentage of positive cells was obtained by gating using the appropriate isotype control. CD31 and CD34 expression were not detected (ND). Not significant (ns). Data are shown as mean ± SD, n = 3 and statistical significance was tested using a nonparametric Mann–Whitney test in (A–C) and by two-way ANOVA test with Sidak’s multiple comparison test in (D,E). * p < 0.001, ** p < 0.0001.

Figure 2

Quantitative proteomic analysis on ASCs from WT and GK rats. (A) Schematic representation of the TMT proteomic study. (B,C) Cluster analysis of proteins expressed in ASC_WT (n = 3) and ASC_GK (n = 3) with heatmap analysis was performed in “R”. Heatmap (B) and PCA plot (C) showing no association between samples. (D) Heatmap with no dendrogram clustering of samples. The legend color bar in (B,D) indicates the relation between scaled ratios and colors.

Figure 3

Assessment of erectile function following ASC_WT or ASC_GK treatment. (A) Schematic representation of the in vivo experiment. Rats were subjected to bilateral nerve crush injury (BNCI) followed by penile injection of vehicle (PBS) or ASC_WT or ASC_GK and compared with sham controls (n = 9 in each group). 28 days after surgery the erectile function was evaluated by intracavernosal pressure (ICP) (mmHg) recordings during electro-stimulation of 2, 4, 6 or 8V. (B) Representative ICP and mean arterial pressure (MAP, mmHg) traces in response to 8V electro-stimulation for 50 s (marked by a line below the x-axis) in Sham, BNCI+vehicle, BNCI+ASC_WT and BNCI+ASC_GK treated rats are shown. (C) The maximum ICP increase (MICP) was normalized to MAP by calculating the MICP/MAP ratio in order to compare erectile function between animals. Data are represented as mean ± SD, and statistical significance was tested by mixed-effect model (REML) with Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01.

Figure 4

ASCs derived from both WT and GK rats induce endothelial gene expression in vivo: mRNA expression of endothelial markers Cd31 (Pecam1) and Nos3 (eNOS), neuronal marker Nos1 (nNOS) and fibrotic marker Col1a1 (Pro-collagen 1) were analyzed by qRT-PCR of corpus cavernous tissue from rats as indicated by treatment group (n = 9 in each). All data were normalized to two stably expressed reference genes, Gapdh and Actb (β-actin). Data is represented as mean ± SD, and statistical significance was tested by ordinary one-way ANOVA with Tukey’s multiple comparison test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.