(091) RNAseq and Pathway Analysis of Rat Penis with Cavernous Nerve Injury and Sonic Hedgehog Treatment by Peptide Amphiphile
Authors: Searl T , Martin S , Harrington D , Stupp S , Ohlander S , Mcvary K , Podlasek C 
Published: May 2023

Abstract

Introduction
Erectile dysfunction (ED) treatments are minimally effective in prostatectomy and diabetic patients due to injury to the cavernous nerve (CN). When the CN is injured, loss of innervation initiates penile remodeling, which includes abundant apoptosis in corpora cavernosal smooth muscle, followed by fibrosis. Decreased smooth muscle makes the erectile tissue less able to respond to normal neurotransmitter signaling, and ED results. When developing novel therapies, the goal is to prevent the apoptotic response and penile remodeling, to preserve erectile function. Our previous studies identify Sonic hedgehog (SHH) as a key mediator of penile remodeling. We examine what signaling mechanisms are altered with CN injury, and how SHH impacts changes in signaling to prevent penile remodeling and ED.

Objective
We performed RNA seq analysis and subsequent pathway analysis, to identify changes in gene expression and signaling pathways that are altered in response to cavernous nerve injury and with SHH treatment by peptide amphiphile (PA), in a rat prostatectomy model of ED.

Methods
Corpora cavernosa were obtained from Sprague Dawley rats (n=36) that underwent bilateral CN crush, CN crush plus SHH treatment of the penis by PA, or sham surgery (controls). Rats were sacrificed 1, 2 and 4 days after surgery. RNA was extracted using TRIzol, DNase treated, and purified by Qiagen mini kit. RNAseq analysis was performed, and subsequent directional pathway enrichment analysis was examined using Ingenuity software. A two-fold change with a q-value &lt;0.05 was used as a threshold for differential expression. Z value measures were used to predict altered activity for the identified enrichments.

Results
mRNA representing 523 genes were significantly differentially expressed (&gt;2 fold; Q&lt;0.05) in CN crushed corpora cavernosal tissue relative to sham controls. Ingenuity analysis showed altered activity in canonical signaling and disease-function enrichments. These changes include increased necrosis and netrin, and decreased Integrin linked kinase, integrin, actin cytoskeleton, acute phase response signaling, cell movement, angiogenesis, and myeloid cell movement activity. With SHH PA treatment at the time of CN injury, these changes were reversed or significantly diminished. In addition, SHH PA impacted known mediators of ED by reducing apoptosis, and increasing cell survival, cell viability, muscle cell proliferation, VEGF, p38 MAPK, and PI3K/AKT pathways.

Conclusions
RNAseq and pathway analysis identified changes in gene expression and pathway signaling that is associated with CN injury in a rat prostatectomy model. Pathway analysis showed increases in processes associated with cell death and a loss of functions necessary for maintenance of corpora cavernosal architecture. Most of the CN injury associated changes were reversed or greatly diminished with SHH treatment of the penis at the time of CN injury by PA. These results support the significant potential of SHH PA to be developed as a preventative treatment for ED in prostatectomy patients. The miriad of altered signaling pathways that were identified with CN injury offer multiple, novel avenues for intervention independently and in combination with SHH PA treatment.

Disclosure
No