Erectile dysfunction (ED) is one of the major complications of diabetes mellitus (18). Erectile function is accompanied by relaxation of penile smooth muscle and dilatation of the arteries, leading to increased blood flow to cavernous spaces (19). Therefore, integrity of intracavernous structures like smooth muscles, endothelium and nerve terminals are greatly required to provide a normal erection (20). Interest in SC treatment strategy for erectile restoration is increasing aiming to deal with the multifactorial pathogenesis of ED (21). Stem cells act either by differentiation and direct integration within the recipient tissue or by their paracrine effect through secretion of growth factors and cytokines4. In the current study we have tested the effect of USCs in restoration the structure and the ultrastructure of the CC of a rat model of DED along with functional improving of erection. The USC-L was also used for the same purpose trying to confirm USCs paracrine effect.
In this study, rats were subjected to induction of diabetes. After which, there were significant decreases in weight. Furthermore, biochemical evaluation of insulin tolerance test, fast blood sugar, total cholesterol & triglyceride was indicative for DM. Development of DED was confirmed using Apomorphine hydrochloride. There was also obvious alteration on the structure and ultrastructure of the CC of diabetic rats besides significant deterioration of copulatory function.
Light microscopic examination of diabetic group showed histopathological changes. In H&E-stained sections, there was marked narrowing of the cavernous spaces which appeared to be invaded by bands of thick collagen fibers and penile fibrosis. These findings were confirmed by Masson trichrome-stained histological sections and their quantitative analysis which revealed that the collagen/smooth muscle ratio increased significantly in diabetic rat. The same results were obtained by Ouyang et al. and Gou et al., (7 &22). The immunohistochemically stained sections of the same group revealed marked decreased for immunopositivity of α-SMA in CC. Using a-SMA as a marker of smooth muscles (SMs) contractility is reasonable being the first protein found to be expressed in contractile SMs and it was proved to be down regulated in the erectile dysfunction (23 & 24).
These findings were documented in many studies (25 &26) which proved that the vascular SMs maintain plasticity and can change from a contractile (differentiated) to a synthetic (dedifferentiated) state. The synthetic state is characterized by a high level of proliferation, migration, extracellular matrix production, vimentin overexpression and decreased expression of contractile cytoskeletal proteins such as α-SMA, SM myosin heavy chain (SMMHC), and desmin. Cultured SMs of diabetic rat were also found to exhibit significantly less contractility compared with those of non-diabetics and decrease expression of α-SMA and SMMHC under hyperglycemic conditions, indicating that they could have a key role in the pathogenesis of DED (27). Moreover, Musicki and Burnett (28) demonstrated that several factors have been implicated with the onset of ED, including changes in SMs, collagen and elastic fibres, which are major penile structural components accountable for erection.
In our study, electron microscopy of diabetic group showed CS narrowing due to protrusion of the endothelium towards the lumen may be due to thickening and splitting of their basal lamina and marked irregularity of their luminar surface. They also exhibited pinocytotic vesicles. The same findings were noticed in the aortic endothelium of diabetic rats after 10 weeks of diabetic induction along with decrease expression of their eNOSmRNA content (29). During studying of the ultrastructure of the CC endothelium in a rat model of type II diabetes Parikh et al., (30) proved a significant decrease in their caveolae; site of localization of endothelial NOS which play a major role in the penile hemodynamic required for maintain the intracavernous pressure and erection. The pinocytotic vesicles in the endothelium of diabetic group increased as a result of increase autophagic activity into the cells due to presence of some metabolites resulting from hyperglycemia and active oxidative stress (31 &32) the basal lamina thickening, and splitting may alter the diffusion between the CS and the surrounding tissues and affect the cell-to-cell side talks between the endothelium and SM of the CC which is essential for erection (33).
Many active fibroblasts with multiple cytoplasmic processes were seen among the CS. They laid immature collagen fibres that appeared adjacent to their cell membrane and loss the normal striation of the mature fibres. indicating newly synthesized collagen fibres leading to penile fibrosis (21 &29).
The histological sections of USCs and USCs-L groups showed obvious less fibrosis and a significant preservation of smooth muscle content compared to the diabetic group. They exhibited apparent restoration of the CS regarding their size and structure. However, few thick collagen fibres were still seen obliterating them in USCs group. Quantitative analysis of Masson trichrome-stained sections showed that the collagen/smooth muscle ratio decreased significantly compared to the diabetic group. Similar results were obtained by using human urine-derived stem cells either alone or genetically modified with fibroblast growth factor 2 (FGF2) (12), this therapeutic strategy was thought to induce improvement of erectile function in type II diabetic rats by recruiting resident cells and increasing the endothelial expression and contents of smooth muscle. Hence restore the smooth muscle/total collagen ratio, a key factor in the relaxation and nutrition of endothelium cells and SM in the CC. The decreased smooth muscle/total collagen ratio decreased the ability of the sinusoids to expand, resulting in veno-occlusive dysfunction (34&35).
Marked ultrastructural repair of CC of USCs and USCs-L group was evident in comparison to the diabetic group regarding the intracavernous structures and the lining endothelium of the CS. USCs were proved to secrete proangiogenic trophic factors and immune-modulatory factors and can be differentiated into endothelial cells in vitro (12).
There were multiple mast cells nearby the CS and in between the cellular component of the CC. Mast cells (MCs) are bone marrow progenitor–derived immune cells that complete their maturity in tissues. MCs are considerably affected by the local microenvironment and can share in numerous biological processes, including inflammation and neovascularization, through the release of several mediators and interaction with macrophages, endothelial cells, and fibroblasts (36). We may explain the strong presence of those cells in the treated groups by the paracrine effect of both USCs and their lysate. The paracrine mechanism of stem cell-based therapy is believed to involve secretion of trophic factors which activate endogenous stem cells to share in tissue repair. Growth factors produced by stem cells demonstrate mitogenic, reparative, anti-apoptotic, and anti-inflammatory properties, it also promotes angiogenesis (4 & 37).
Regarding function assessment, we used the APO test (12) as a confirmation of DED and to follow up the animal 4 and 8 weeks after transplantation. Assessment of copulatory function was performed and showed significant improvement of all parameters in both USCs and USCs-L groups in comparison to the DED group. Sexual behaviour, sperm quantity and quality were proved to be deteriorated even after short term streptozotocin-induced hyperglycaemia in rats (38) and different types of stem cell therapies were used and proved to restore the erectile function based on measuring the intracavernous pressure in relation to the main arterial pressure (4, 12 & 13).
In the current study, urine was used as an easy and available source of USCs which can be obtained via a non-invasive, simple, and low-cost approach (12 & 39). The isolated USCs exhibited the same morphology and phenotypic character of MSCs and were proved to have high efficiency to be differentiated (39).
Although there was almost no significant difference between the USCs and USCs lysate groups regarding repair penile structure and function, we could prefer using USCs-L rather than USCs based on the following reasons: In our study, stem cells numbers were detected by fluorescence microscopic sections showing recruitment of PKH26-labeled USCs in the corpora cavernosa of group III rats, 4 weeks and 8 weeks after transplantation. but their number was significantly decreased after 8 weeks indicating decreased survivability of stem cells after transplantation which is considered one of the limitations of their use as a cytotherapy (37). Utilizing stem cell-cell free therapy by using their lysate containing rich amounts of growth factors and bioactive components or isolation of their exosomes may be benefit cell growth and tissue repair without taking the risk of the unknown behaviour of the transplanted cells on the long run or their unwanted pathway of differentiation. However, further studies aimed to identify the proteomic characteristics, the exact components of the cell-free lysate and their mode of action is highly recommended.