Characterization of USCs and USC-Exo
The cell colonies were observed around 3-5 days after initial seeding of cells isolated from urine samples (Fig. 1A). The culture medium was changed regularly, and the USCs were confirmed with a fibroblast-like morphology after contact inhibition under light microscopy (Fig. 1A). After 14 days, cell colonies contained an increasing number of cells and reached more than 70% confluence. Flow cytometry analysis showed that USCs were positive for CD29, CD44, CD73, and CD90 and negative for CD31, CD45, and HLA-DR (Fig. 1B), which are common characteristics of pluripotent MSCs. In addition, CD146, a marker of renal pericytes, was highly expressed. These demonstrates that the characteristics of the cells used in this experiment meet the current main identification standards of USCs.
The morphology of the negatively stained USC-Exo sample was observed by TEM, which showed that USC-Exos were spherical vesicles with uneven sizes and a complete membrane structure (Fig. 1C). The diameter of USC-Exo was measured to be about 40–130 nm by NTA analysis (Fig. 1D). In addition, Western blot analysis demonstrated that the collected USC-Exo expressed exosomal specific surface markers CD9, CD63 and TSG-101 (Fig. 1E).
USC-Exo enhanced the proliferation, migration and tube formation of HUVECs in vitro
In order to explore the in vitro pro-angiogenic potential of USC-Exo under high glucose and the most suitable dosage for in vivo experiments, we designed a series of experiments using HUEVCs. HUVECs were treated with normal (5.5 mM, NG) or high (33 mM, HG) concentrations of glucose in combination with different concentrations (108 or 109 particles/ml) of USC-Exo. After 48 h, the absorbance of high glucose-treated HUVECs were significantly reduced compared with the normal group (P<0.01; Fig. 2A). With the normal glucose, 109 group had a certain proliferation effect on cells (P<0.001; Fig. 2A) while 108 group did not. In addition, USC-Exo significantly reversed the deleterious effects of high glucose on HUVECs (P<0.001; Fig. 2A), and 109 group were better than 108 group. Therefore, 109 particles/ml USC-Exo was subsequently selected as the experimental group.
The scratch wound healing assay showed that compared with NG group, HG group had an inhibitory effect on migration of HUVECs, and the difference appeared in a short time (4h, 6h; P<0.001; Fig. 2B, C and D). In HG group, the simultaneous addition of USC-Exo reversed this phenomenon and promoted the healing process (P<0.001; Fig. 2C and D). As shown in Figure 2E, after incubation with matrigel for 16 h, the lumen of the control group was uniform, with many vascular connection nodes and abundant long branches. In contrast, the HG group formed less intact lumina which were thinner and irregular with more unconnected areas (P<0.05; Fig. 2F and G). The addition of USC-Exo to the HG group restored the impaired angiogenesis ability, formed more complete and uniform lumen, and returned to normal levels in the number of junctions and branches (P<0.01; Fig. 2F and G). The results of qRT-PCR in Figure 2H, I and J showed that compared with NG group, the expression of BAX in HUVECs was up-regulated and the expression of Bcl-2 and SOD2 was down-regulated after HG treatment (P<0.001). However, adding USC-Exo at the same time in HG group can down-regulate the expression of BAX and up-regulate that of Bcl-2 and SOD2 (P<0.01 or P<0.001). These results indicated that USC-Exo could improve high glucose-induced oxidation of HUVECs, and promote their proliferation and angiogenesis in vitro.
Modeling and administration workflow for DM2ED rats
Before the injection of STZ and the assessment of erectile function, we analyzed the body weight and fasting blood glucose of all the rats, respectively. After modeling, the body weight of rats in the HA group and the two Exo groups was significantly lower than that in the NC group (P<0.001; Fig. 3A). In addition, the fasting blood glucose levels in the HA group and the two Exo groups were significantly higher than those in the NC group (P<0.01; Fig. 3B). In the insulin challenge test, blood glucose levels in NC rats decreased significantly at 15, 30, 45, 60, 90, and 120 minutes after intraperitoneal insulin injection. DM2 rats were less responsive to insulin compared to NC rats, which means the modeling was successful (P<0.01; Fig. 3C). We performed APO testing 8 weeks after STZ injection and excluded individuals in the diabetic group which still had erectile responses without stimulation, as shown in Fig. 3D. The topical administration of the hydrogel (Fig. 3F and G) was carried out with the aid of a 1 ml syringe. The foreskin is turned over and the hydrogel is applied in a circle around the glans. After topical absorption for 15 minutes, the foreskin is re-covered with the glans. Fig. 3E shows the workflow of the whole animal research study.
USC-Exo-HA restored erectile function in DM2ED rats and reduced apoptosis in corpus cavernosum
Four weeks after topical administration, to evaluate erectile function in each group of rats, erection stiffness was measured by recording ICP and MAP during electrical stimulation of the cavernous nerve under anesthesia (Fig. 3H). As shown in Fig. 4A to D, max ICP, max ICP/MAP and ICP integral were used to evaluate erectile function in each rat. The NC group exhibited high max ICP (91.35±11.35) and max ICP/MAP (0.79±0.11) with large area under the curve (AUC). After diabetes modeled, the HA group consistently resulted in ED, with lower max ICP (26.13±3.61) and ICP/MAP (0.26±0.09). These metrics were significantly increased after administration of USC-Exo-HA (P<0.01), and the 10 treatments group improved erectile function more effectively than 5 treatments group (P < 0.05).
Apoptosis in corpus cavernosum of each rat was detected by TUNEL. Fluorescence images were taken according to the site in Fig. 4F. As shown in Fig. 4E, apoptotic cells in 4 groups of corpus cavernosum exhibited green fluorescence. Apparently, apoptosis was reduced after USC-Exo-HA. Fig. 4G indicated the apoptotic index for each group. After USC-Exo-HA treatment, cell apoptosis was less than that in HA group (P<0.01).
USC-Exo-HA alleviates fibrosis in the corpus cavernosum of DM2ED rats
As shown in Fig. 5A, the corpus cavernosum of the rats in the DM2ED group exhibited severe fibrosis. According to the results of Masson trichrome staining, Western blot analysis and immunohistochemistry (Fig. 5A, B and 6A), smooth muscle content was reduced in the HA group compared to the NC group. Exosome-treated groups exhibited partial inhibition of fibrosis. Significantly, USC-Exo-HA increased smooth muscle content to a greater extent than the HA group (P < 0.05; Fig. 5B and C). TGF-β1 is an important profibrotic factor in the corpus cavernosum, and was significantly increased in the HA group. However, the levels of TGF-β1 in the Exo X5 and Exo X10 group were significantly lower than in the HA group (P < 0.01; Fig. 5B and D). The above results indicated that USC-Exo-HA could inhibit the fibrosis of corpus cavernosum in DM2ED rats.
Improvement from USC-Exo-HA in endothelium of diabetic penis tissues
To determine the effect of topical treatment on the endothelium of the rat corpus cavernosum, we observed the expression of endothelial markers eNOS and CD31 by immunohistochemistry and Western blot analysis (Fig. 6A and C). According to the schematic screenshot in Figure 6B, the expressions of eNOS and CD31 in the NC and Exo X10 groups were significantly higher than those in the other two groups, indicating improved vascular function. Consistent with the immunohistochemical results, Western blot analysis of eNOS and nNOS also showed that the expression was decreased in the HA group and partially increased after USC-Exo-HA treatment (P < 0.05; Figure 6D, E). All above findings demonstrated that the treatment of USC-Exo-HA improved endothelium function, which may be better with prolonged use.