Improving erectile function by silencing phosphodiesterase-5.
OBJECTIVE
We investigated whether phosphodiesterase-5 (PDE5) can be down-regulated by a specific small interfering RNA (siRNA) and whether this would improve erectile function.


METHODS
A PDE5 siRNA encoding oligonucleotide was inserted into pSUPER-retro vector, resulting in the siRNA expressing construct, pPDE5-silencer. The construct was packaged into oncoretroviral particles and then transduced into rat cavernous smooth muscle cells (CSMCs). Cells were examined for PDE5 expression by reverse transcriptase-polymerase chain reaction, Western blotting and immunofluorescence microscopy. Cells were then treated with 10 microM sodium nitroprusside (SNP) and examined for cyclic guanosine monophosphate (cGMP) at 0, 10, 30, 60 and 240 minutes. The siRNA expressing cassette was transferred from the oncoretrovirus to lentivirus, which was then injected into rat penises. Three months later erectile function was examined by electrostimulation and PDE5 expression in cavernous smooth muscle was determined by immunohistochemistry.


RESULTS
CSMCs transfected with pPDE5-silencer (CSMC plus siRNA) showed an 88.2% decrease in PDE5 compared with CSMCs transfected with control vector (CSMCs plus vector). Within 10 minutes of SNP treatment cells (CSMCs, CSMCs plus vector and CSMCs plus siRNA) showed similar sharp increases in cGMP. However, while cGMP levels in CSMCs and CSMCs plus vector returned to almost baseline in 1 hour, the cGMP level in CSMCs plus siRNA remained high even 4 hours after SNP treatment. Rats injected with the siRNA expressing lentivirus showed increased electrostimulated erectile function, as measured by peak intracorporeal pressure and the intracorporeal pressure increase, compared with rats injected with control lentivirus. PDE5 expression was decreased in the siRNA treated cavernous smooth muscle.


CONCLUSIONS
PDE5 expression could be decreased by siRNA, resulting in prolonged cGMP accumulation and improved erection.
Lin
Guiting
G
Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California-San Francisco 94143-1695, USA.
Hayashi
Narihiko
N
Carrion
Rafael
R
Chang
Lung-Ji
LJ
Lue
Tom F
TF
Lin
Ching-Shwun
CS
D015106:3',5'-Cyclic-GMP Phosphodiesterases; D000818:Animals; D002478:Cells, Cultured; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D007172:Erectile Dysfunction / Q000473:pathology / Q000503:physiopathology*; D020868:Gene Silencing / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008856:Microscopy, Fluorescence; D009130:Muscle, Smooth / Q000187:drug effects* / Q000473:pathology / Q000503:physiopathology; D009841:Oligonucleotides / Q000235:genetics / Q000494:pharmacology; D010410:Penile Erection / Q000187:drug effects* / Q000502:physiology; D010727:Phosphoric Diester Hydrolases / Q000235:genetics*; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000494:pharmacology*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic
D009841:Oligonucleotides; D034741:RNA, Small Interfering; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; C517384:Pde5a protein, rat