Novel treatment and preventive strategies of pelvic floor disorders – Experimental studies

Vaginal birth is the most important risk factor for later pelvic floor dysfunction (PFD)(1). During vaginal delivery, the passage of the fetal head results in excessive muscle stretch, nerve injury(2) and causes also ischemia and reperfusion injury(3) mainly in the levator ani muscle, urethra and vagina. As the injury evolves, a myogenic damage is present which are characterized by atrophy, fibrosis(4) and increase of slow twitch muscle fiber type(5). These injuries can lead to stress urinary incontinence (SUI), Pelvic Organ Prolapse and fecal incontinence(6). Current treatment options include conservative management such as physiotherapy, pharmacotherapy, the use of pessaries or surgery. None of the current therapies can fully repair the underlying pathophysiology(7). Recently,stem cell (SC) therapy has gained attention as a potential treatment for PFD, particularly for SUI. Several groups have demonstrated functional and morphologic improvements following cell therapy (8-10), yet the mechanism of action, the best cell source, the administration route and dose still need to be further explored. Also, in view of clinical application, a number of translational aspects remain. The effects of vaginal birth can be experimentally mimicked in rats by Vaginal Distension (VD) and pudendal nerve crush (PNC). VD leads in rats to injury to the urethra, bladder, vagina and levator muscles, damages nerve fascicles and smooth and striated muscle(11-13). VD also reduces blood perfusion of urethra, bladder, urethra, and vagina, leading to hypoxic changes(14) and increased expression of hypoxia-inducible factor 1α expression in the urethra(15, 16). PNC is mainly characterized by neurodegeneration(17). PNC results in denervation injury of external urethral sphincter (EUS) eventually decreasing the leak point pressure (LPP) and EUS electromyography (EMG)(11). In this model, spontaneous functional recovery occurs by 3 weeks after injury(18). The combination of both VD and PNC has been suggested as a realistic mimic of what happens clinically. Restriction the trauma to PNC does not induce immediate muscle injury, whereas VD induces a myogenic damage without obvious neurogenic mechanism(19). This combination causes more severe and durable damage to the EUS than either PNC or VD alone(18). Although women with PFD present a myogenic damage in the late phase, no muscle morphometry or molecular assessment was performed in any of these models in the medium term yet. LPP, recovers 4- 6 weeks after PNC+VD and EUS EMG recovers only at 9 weeks. Despite functional recovery, neuromuscular recovery is still incomplete at this timepoint(20). Skeletal muscle can repair itself from injury by removing damaged myofibers and synthesizing new muscle fibers to restore functional contractile properties(21).After injury, an inflammatory response is initiated(22) leading to the phagocytosis of the injured myofibers and the activation of quiescent satellite cells(23) (1-3 days). The activated satellite cells proliferate, migrate to the site of injury, fuse and, differentiate to form new myofibers(23) (2-4 weeks). However, the regenerative capacity of injured skeletal muscle is limited and often, fibrotic tissue forms, limiting full functional recovery(24) (3-6 months). TGF-beta 1 has been shown to mediate this process of (excessive formation) of fibrotic tissue between skeletal muscle fibers(25). Earlier studies have documented the effects of different type of stem cells and administration routes. Adipose derived stem cell (ADSC) and bone marrow derived mesenchymal stem cells (BMSC) have already been studied in a rodent model for simulated birth injury, either by VD and/or PNC (9, 26-28). Administration of 1-2 X106 MSC after vaginal distension increased urinary leak point pressure as well as vessels, elastin and smooth muscle surrounding the urethra(26, 27). The effect of MSC on the vaginal smooth muscle layer or the levator ani muscle was not studied. The homing of the SC was demonstrated after intramuscular or intravenous administration (9, 29-31). Ligands critical for MSC homing have been shown to be increased after simulated birth injury. MMP-3 is significantly over expressed in rat urethral and vaginal tissues immediately following VD(32) whereas PNC results in greater SDF-1 and MCP-3 expression 10 days after injury(33). Several experiments have studied the effect of the route of administration(9, 34). Following local injection only 3% of cells survived (34), probably due to the limited diffusion of nutrients and oxygen (35). Since SC are known to home to sites of inflammation (36) systemic injection can be used as well. The intravenous administration is less invasive, yet is associated by capture of the majority of cells in non-target organs, mainly the lungs (37). The alternative is selective intra-arterial injection, i.e. in the feeding arteries, which results in a more efficient engraftment/lower cell dose in the target organ, and less off-target effects(38). This has been shown effective in many conditions, including ischemia-reperfusion and skeletal muscle injury (38, 39) Herein we propose to use mesangioblasts (MABs), a precursor cells for smooth muscle layer present in large blood vessels. Because of their potential to differentiate into the smooth and skeletal muscle lineage(40), these multipotent cells have been tested for their capacity to participate in regeneration of muscle (39, 41). Many of these cells express alkaline phosphatase (AP), a marker on pericytes in skeletal and cardiac muscle, indicating they might be pericytes(40). MABs from skeletal muscle infiltrate damaged muscle, engraft, and restore absent proteins in animal models of muscular dystrophy (39, 41, 42). Moreover, MABs express growth factors such as VEGF-B, IGF, FGF and HGF(31). Rat (autologous) MAB can be collected by punch needle biopsy, processed and injected intramuscular in the levator ani plate or administrated systemically by intra-arterial or intravenous route. Furthermore, VEGF overexpressed MAB injection will be investigated in the same model. VEGF is a growth factor known by its anti-inflammatory, angiogenic and mitogenic effect(43, 44). It is reasonable to postulate that an increased VEGF release by MABs may enhance the regeneration of the pelvic floor organs in a childbirth model since inflammation and ischemia play a role in the VD and PNC injury. Interesting VEGF accelerated regeneration of damaged neuromuscular junctions and inhibit fibrosis in an ischemic hind limb model (45). Moreover, the pro-angiogenic approaches seems to be pro-myogenic(46, 47) and could enhance muscle regeneration capacity through expansion of the satellite cell juxta vascular niche(47) 3 Herein we will explore whether MAB are effectively reversing the simulated birth trauma by a combination of VD and PNC. Once effective, we will explore the time course of events and determine early molecular read outs of later functional muscle repair. Further, we hypothesize that muscle regeneration may be improved by increased bioactivity, e.g. by overexpression of VEGF in the cells administered. Also, of relevance to later clinical application, we will explore the effect of changing the administration route, and compare results to those obtained by cell type that would be more practical from a clinical viewpoint (homologous/autologous ADSC). Lastly, in view of an eventual clinical translation, we will start working on a larger animal model.status: publishe