161 Low Intensity Extracorporeal Shock Wave Therapy Activates Endogenous Progenitor/Stem Cells as Indicated by Histone 3 Phosphorylation

Abstract

Background and significance Low Intensity Extracorporeal Shock Wave Therapy (Li-ESWT) has been employed for many years to treat ED yet its mechanism of action remains poorly defined. We have previously demonstrated that the therapeutic effects from Li-ESWT may be partially related to the activation of endogenous progenitor/stem cells and we are endeavoring to further describe these pathways. Aims To determine the mechanism by which Li-ESWT activates endogenous stem cells by interrogating histone 3 phosphorylation, which occurs only during the mitotic phase of the cell cycle. Materials and Methods Male Sprague-Dawley (SD) rats were treated with 5-ethynyl-2'-deoxyuridine (EdU) pulse followed by Li-ESWT, and cohort analysis was performed. Additionally, rat adipose derived stem cells (RADSCs) and rat penile cavernous smooth muscle cells (RCSMCs) were also treated with Li-ESWT. Fifty male SD rats at 12 weeks old were grouped into three cohorts: 1). Sham; 2). Sub-acute: the animals underwent bilateral cavernous nerve injury (BCNI) then were separated into control and Li-ESWT groups. One week after BCNI, the Li-ESWT group were injected with EdU (50mg/kg, i.p) 30 minutes before Li-ESWT (0.02mJ/mm2, 3 Hz, 300 pulses). One week later, ICP/MAP were measured. 3). Chronic: the animals underwent BCNI then grouped into control and Li-ESWT groups. One week after BCNI, the animals were treated with Li-ESWT (0.02mJ/mm2, 3 Hz, 300 pulses) twice a week for 4 weeks. EdU was injected 30 min before the first and last Li-ESWT. One week after the last Li-ESWT, ICP/MAP were measured. The penile erectile tissues and major pelvic ganglion (MPG) were harvested for histological study to assess smooth muscle/collagen content and endothelium content in the corpora cavernosa. EdU as marker of cell activation and phosphorylated histone 3 (H3P) as marker of the mitotic phase of the cell cycle were also assessed. To confirm the effect of Li-ESWT on the phosphorylation of H3 in vitro, RADSCs and RCSMCs were treated with Li-ESWT (0.02mJ/mm2, 3 Hz, 100 pulses), and EdU incorporation rate and the expression of H3P was assessed. Results Erectile function in the sub-acute and chronic groups was assessed by measuring ICP/MAP. Erectile function was severely impaired by BCNI. In the sub-acute group, Li-ESWT slightly improved ICP/MAP, without statistical significance (P=0.24). In the chronic group, Li-ESWT improved erectile function significantly (P<0.05) over the BCNI only group. Penile smooth muscle content was examined with alpha-SMA immunofluorescence and trichrome staining. In both the sub-acute and chronic groups, there was more penile smooth muscle after Li-ESWT, especially in the chronic group (P<0.01). Penile endothelium was damaged in both the sub-acute and chronic groups; Li-ESWT enhanced the regeneration of endothelial cells (P<0.01). Li-ESWT also enhanced BiP expression, a marker for the unfolded protein reaction in the endoplasmic reticulum, in both the penile tissue and MPG significantly over the controls. Li-ESWT also increased EdU+ cells in both the sub-acute and chronic groups. Strikingly, Li-ESWT significantly enhanced the phosphorylation level of Histone 3 (H3P) at Ser-10. Most H3P positive cells were localized in penile smooth muscle, and some localized at endothelium, sub-tunical space, para-sinusoid area, penile vessel, and dorsal nerve. In vitro, Li-ESWT enhanced the phosphorylation level of H3 from 4 hr after treatment and peaked at 24 hr in both RADSCs and RCSMCs. More H3P+ cells were noted in Li-ESWT treated groups with a time-dependent response. Conclusion Li-ESWT is a noninvasive therapeutic approach to improve erectile function through activation of endogenous stem/progenitor cells. The increases in EdU, BIP expression, and histone 3 phosphorylation after Li-ESWT treatment reveal that Li-ESWT activates cells and induces cell mitosis, which is a likely mechanism of tissue regeneration occurring after Li-ESWT treatment.