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Treatment of erectile dysfunction following cavernous nerve injury with adipose-tissue derived stem cells: efficacy and insights into the mechanisms of action of stem cell therapy.

Radical prostatectomy is for most urologists the preferred treatment option for localized prostate carcinoma in young patients. During this surgery, irrespective of the extent of neurovascular budle preservation, neuropraxia of the cavernous nerves occurs, and Wallerian degeneration of these nerves ensues, resulting in fibrosis in the corpus cavernosum, anda loss in smooth muscle mass. These changes in turn result in changed compliance of the erectile tissue and thus a defective veno-occlusive mechanism. As a result of this denervation and the secondary changes to theerectile tissue, often severe and difficult-to-treat erectile dysfunction develops. This erectile dysfunction can be temporary, and resolve when reinnervation of the corpus cavernosum occurs, or, in a large proportion of men, definitive.

Clinical attempts to preserve erectile function following radical prostatectomy with the use of so-called penile rehabilitation schemes, has resulted in disappointing outcomes. Therefore, researchers have been searching for alternative treatment options aimed at preservation of smooth muscle in the penis, protection of the cavernous nerve from degenerating after injury, or enhancing the endogenousneuroregenerative process. With the initial presumption that stem cellscould engraft in the erectile tissue and replace apoptotic smooth muscle cells, preclinical experiments directed at improving erectile functionwith stem cells have been set up. 

In this PhD, our primaryobjective was to test the feasibility of the intracavernous injection of adipose tissue-derived stem cells (ADSC) in an animal model of cavervous nerve crush injury. Crushing the nerve resembles a state of neuropraxia such as occurs during radical prostatectomy. In an initial study, we observed a beneficial effect of injection of ADSC on recovery of erectile function, as illustrated by intracorporal pressure increase upon electrostimulation of the cavernous nerve 4 weeks after injury and injection of cells. This functional observation was accompanied by a restoration of neuronal nitric oxide synthase (nNOS; the enzyme that produces NO, themain pro-erectile neurotransmitter in the corpus cavernosum) expressionin the penile nerves. Furthermore, smooth muscle content in the penis was preserved, and fibrosis was diminished. 

Four weeks following injection of labeled ADSC in the corpus cavernosum, scarce ADSC were found in the erectile tissue, in numbers too low to possibly explain treatment effects. Furthermore, the injection of lysate of an equivalent number of ADSC into the corpus could partially replicate the functional and structural effects of stem cell treatment. With this approach, we exposed the injured tissues to soluble factors contained in ADSC, without allowing live cells to directly act on the host tissue. Therefore we suggested that ADSC may not act by engraftment in the erectile tissue and transdifferentiation into smooth muscle cells, but rather may exert their effects by a paracrine interaction with the injured host tissue. 

As ADSC did not engraft in the penis, we were intrigued by the fate of these cells after injection. ADSC were therefore labeled withEdU, a DNA marker, and injected in both injured and non-injured animals. When traced, we observed that cells dissapear from the injection site rather quickly, to appear in the major pelvic ganglion, from which the cavernous nerve arises. At 28 days, few ADSC were found in the penis as well as in the ganglia. In injured rats treated with ADSC, we further observed a time-dependent increase in nNOS indicating neuroregeneration, rather than neuroprotection as a treatment effect of stem cells. 

As it is known that chemokines, a class of small signaling molecules, are involved in chemo-attraction, the migration of cells towards a gradient of a certain molecule, we tested which chemokines may play a role in recruitment of ADSC towards the major pelvic ganglion following cavernous nerve injury. We identified the ligand/chemokine receptor pairs CCL2/CCR4, CX3CL1/CX3CR1 and XCL1/XCR1 as potentially responsible for ADSC recruitment in this nerve injury model by a thorough multistep analysis of chemokiine receptor expression in ADSC. Surprisingly in the light of what was previously proposed, CXCR4-CXCL12 (SDF-1) interaction is not likely a major homing factor for ADSC. 

Conversely, we wondered what the effect would be of early blocking of the inflammatory cascade that leads to chemokine release in injured tissues, as this inflammatory reaction draws macrophages into the neural tissue, which are much needed for clearing debris, but often cause more damage than desired by the release of reactive oxygen species. Therefore, we blocked the inflammatory response at the level of TNFA  and TGF-β signalling with orally administered Pentoxifylline. Rats that underwent cavernous nerve crush injury and were treated daily with Pentoxifylline for four weeks had better recovery of erectile function than untreated rats, and this effect was dose dependent. Furthermore there was enhancement of nervegrowth in vitro, implying that besides anti-inflammatory effects, Pentoxifylline may induce neurotrophic signalling via other pathways. 

In conclusion, this work shows that ADSC have the capacity to induce neuroregeneration in rats with cavernous nerve injury. These effects are mediated by a neuro-inflammation induced chemoattraction of ADSC towards the major pelvic ganglion where they induce nerve regeneration. These insights may help translation of this autologous cellular therapy into clinical practice, but can aso be helpful in future experiments aimed at potentiating the effects of ADSC therapy. 
Date:3 Aug 2009  →  30 Sep 2012
Keywords:Radical prostatectomy, Erectile dysfunction, Stem cell, Apoptosis, Adipose-tissue stem cell, Nerve regeneration, Cavernous nerve injury, Fibrosis
Disciplines:Orthopaedics, Surgery, Nursing, Endocrinology and metabolic diseases, Gynaecology and obstetrics, Urology and nephrology
Project type:PhD project