The Effect Of COX-10aa-PGIS On Erectile Dysfunction After Cavernous Nerve Injury In Rats

Chapter One:IntroductionPost radical prostatectomy erectile dysfunction is a very common complication which is almost unavoidable and without effective rescue method. Erectile function impaired immediately following RP is thought due to the damage to the cavernous nerves, which is known as neuropraxia. The most popular medication of PDE5-Is may not be effective in the early stage after RP due to the lack of cavernous nerve impulse to release nitric oxide to increase cGMP. Therefore, there is an acute need for exploration of novel rehabilitation approaches. Both PGI2and PDE-5i are well known potent smooth muscle relaxant and vasodilators. Benefiting by PGI2-IP-cAMP-PKT signal pathway, PGI2therapy may directly dilate the artery and sinusoid trabeculae, and overcome the cavernous nerve injury barrier. However, it’s short time half-life (<2min) limits its clinical application. In2006, Ruan et al reported an innovative engineering of a recombinant protein COX2-10aa-PGIS with triple catalytic activities directly converting AA into PGI2. This special protein structure could induce PGI2high express. It looks COX2-10aa-PGIS could be the promising candidate for RPED treatment. We tried to use adenovirus with COX1-10aa-PGIS gene (Ad-COX2-10aa-PGIS) to BCNC ED. The effect was observed and the underlying mechanisms were explored. Recently, Ruan et al optimized the structure of the recombinant protein named as COX1-10aa-PGIS. Adenovirus showed some limitation to clinical use. ADSCs are arising as a new therapy for ED, The aim of our second study was to applying the Adipose Derived Stem Cells (ADSCs) with COX1-10aa-PGIS gene to BCNC Rat ED model and observing the effect, then exploring the underlying mechanisms. The main body of this paper is divided into two parts and narrated as following. Chapter Two:COX2-10aa-PGIS Gene Therapy Improving Erectile Function in Rats after Cavernous Nerve InjuryObjectiveThe purpose of this study was to explore the effect and mechanism of COX2-10aa-PGIS gene therapy in penile rehabilitation.MethodsBilateral cavernous nerve crush (BCNC) in adult Sprague-Dawley(SD) rats was used to mimic radical prostatectomy induced ED. SD rats were randomly assigned into four groups:1. sham surgery;2. BCNC;3. BCNC+null control recombinant adenovirus(NCRA) intracavernous injection; and4. BCNC+Ad-COX2-10aa-PGIS intracavernous injection.28days later, intracavernosal pressure (ICP) was recorded under cavernous nerve stimulation; in the meantime the blood pressure was monitored. At the end of the measurement, the penis was harvested and processed for (1) immunohistochemistry analysis for:endothelial nitric oxide synthase(eNOS), a-smooth muscle actin (ASMA) and transforming growth factor beta-1(TGFβ1);(2) Western Blot for COX2-10aa-PGI;(3) Masson’s Trichrome stain for smooth muscle/collagen ratios; and (4) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis.Results1. COX2-10aa-PGIS gene therapy preserved erectile function in the BCNC rat model;2. COX2-10aa-PGIS gene therapy increased eNOS and ASMA expression and decreased TGF(β1; 3. COX2-10aa-PGIS gene therapy reduced cell apoptosis after cavernous nerve injury;4. COX2-10aa-PGIS gene therapy preserved smooth muscle/collagen ratios.ConclusionOur data demonstrated that COX2-10aa-PGIS protected erectile function after cavernous nerve injury through anti-fibrosis and anti-apoptotic mechanism. Chapter Three:Adipose Derived Stem Cells with Coxl-10aa-PGIS Gene Improve Erectile Function in Rats with Bilateral Cavernous Nerve CrushObjective:Use Adipose Derived Stem Cells as the carrier of COX1-10aa-PGIS gene to improve the penile rehabilitation after bilateral cavernous nerve crush (BCNC) in the rat ED model.MethodsAdipose Derived Stem Cells (ADSCs) were isolated from perigonadal fat of Sprague-Dawley (SD) rats and cultured in DMEM/F12medium. The COX1-10aa-PGIS gene was transferred into ADSCs. ADSCs with COX1-10aa-PGIS stable cell line was filtered by G418and identified by Western Blot and LC-MSMS. BCNC in adult SD rats was used to replicate radical prostatectomy induced ED. SD rats were randomly assigned into four groups:1. sham surgery;2. BCNC;3. BCNC+ADSCs intracavernous injection;4、BCNC+ADSCs with COX1-10aa-PGIS gene intracavernous injection;28days later, intracavernous pressure (ICP) was recorded under cavernous nerve stimulation; meanwhile the blood pressure was monitored. At the end of the measurement, the penis and urine was harvested and processed as required for1、Immunohistochemistry for Neural nitric oxide synthase(nNOS),a-smooth muscle actin (ASMA) and Vascular endothelial growth factor (VEGF) expression level;2、Western blot for ASMA, Transf forming growth factor β1(TGFβ1) and Hypoxia-inducible factor-1a(HIF-1a);3、Liquid Chromatography-Mass Spectrometry (LC-MS/MS) for6-keto-PGF1a;4、TUNEL for apoptosis.Results1. The ADSCs was successfully isolated and passaged. 2. ADSCs with COX1-10aa-PGIS gene stable cell line were successfully established.3. The ADSCs stable cell line with COX1-10aa-PGIS gene which can high express PGI2was identified by LC-MS/MS, G418and Western blot.4. ADSCs with COX1-10aa-PGIS gene therapy improved erectile function in rats after BCNC injury.5. ADSCs with COX1-10aa-PGIS up-regulated nNOS, ASMA and VEGF expression level in Corpus cavernosum6. ADSCs with COX1-10aa-PGIS down-regulated TGFβ1and HIF-1a expression level in Corpus cavernosum7. ADSCs with COX1-10aa-PGIS inhibit cell apoptosis after cavernous nerve injuryConclusionOur data also suggested that COX1-10aa-PGIS gene can be successfully transferred into ADSCs for gene therapy for the treatment of ED after cavernous nerve injury in an animal model. ADSC with COX1-10aa-PGIS gene might be a potential candidate in post radical prostatectomy penile rehabilitation. The underlying mechanisms of ADSCs with COX1-10aa-PGIS protected erectile function after cavernous nerve injury may through anti-hypoxia, anti-fibrosis and anti-apoptotic, and paracrine secretion and wound healing by ADSCs. Conclusion of the whole paper1. Our data demonstrated that COX2-10AA-PGIS protected erectile function after cavernous nerve injury through anti-fibrosis and anti-apoptotic mechanism.2. Our data also suggested that COX1-10aa-PGIS gene can be successfully transferred into ADSCs for gene therapy for the treatment of ED after cavernous nerve injury in an animal model. ADSC with COX1-10aa-PGIS gene might be a potential candidate in post radical prostatectomy penile rehabilitation. The underlying mechanisms of ADSCs with COX1-10aa-PGIS protected erectile function after cavernous nerve injury may through anti-hypoxia, anti-fibrosis and anti-apoptotic, and paracrine secretion and wound healing by ADSCs.