Antisense and short hairpin RNA (shRNA) constructs targeting PIN (Protein Inhibitor of NOS) ameliorate aging-related erectile dysfunction in the rat.

Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA.
Journal of Sexual Medicine (Impact Factor: 3.51). 05/2007; 4(3):633-43. DOI: 10.1111/j.1743-6109.2007.00459.x
Source: PubMed

ABSTRACT Over-expression of penile neuronal nitric oxide synthase (PnNOS) from a plasmid ameliorates aging-related erectile dysfunction (ED), whereas over-expression of the protein inhibitor of NOS (PIN), that binds to nNOS, increases ED.
To improve this form of gene therapy for ED by comparing the electrical field response of short hairpin RNA (shRNA) for PIN with that of antisense PIN RNA.
Both shRNA and antisense RNA gene therapy vectors increased intracavernosal pressure in aged rats.
PIN small interfering RNA (siRNA), and plasmid constructs for cytomegalovirus promoter plasmid vector (pCMV-PIN), pCMV-PIN antisense RNA, pSilencer2.1-U6-PIN-shRNA; and pSilencer2.1-U6-randomer-shRNA were prepared and validated by transfection into HEK293 cells, determining the effects on PIN expression by Western blot. Plasmid constructs were then injected, followed by electroporation, into the penile corpora cavernosa of aged (20-month-old) Fisher 344 rats and, 1 month later, the erectile response was measured by intracavernosal pressure increase following electrical field stimulation (EFS) of the cavernosal nerve. PIN was estimated in penile tissue by Western blot and real-time reverse transcriptase-polymerase chain reaction. Cyclic guanosine monophosphate (cGMP) measurements were conducted by competitive enzyme immunoassay (EIA). Immunohistofluorescence detected PIN in corporal tissue sections.
In cell culture, PIN siRNA and plasmid-expressed pU6-PIN-shRNA effectively reduced PIN expression from pCMV-PIN. pSilencer2.1-U6-PIN-shRNA corrected the impaired erectile response to EFS in aged rats and raised it above the value for young rats, more efficiently than pCMV-PIN antisense RNA. PIN mRNA expression in the penis was decreased by >70% by the shRNA but remained unaffected by the antisense RNA, whereas PIN protein expression was reduced in both cases, particularly in the dorsal nerve. PIN antisense increased cGMP concentration in treated tissue by twofold.
pSilencer2.1-U6-PIN-shRNA gene therapy was more effective than the antisense PIN mRNA in ameliorating ED in the aged rat, thereby suggesting that PIN is indeed a physiological inhibitor of nNOS and nitrergic neurotransmission in the penis.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Bisphenol A (BPA), a suspected reproductive biohazard and endocrine disruptor, released from plastics is associated with ED in occupationally exposed workers. However, in rats, despite the induction of hypogonadism, apoptosis of the penile corporal smooth muscle (SM), fat infiltration into the cavernosal tissue and changes in global gene expression with the intraperitoneal administration of high dose BPA, ED was not observed. We investigated whether BPA administered orally rather than intraperitoneally to rats for longer periods and lower doses will lead to ED. Main outcome measures are ED, histological, and biochemical markers in rat penile tissues. In all, 2.5-month-old rats were given drinking water daily without and with BPA at 1 and 0.1 mg kg(-1) per day. Two months later, erectile function was determined by cavernosometry and electrical field stimulation (EFS) and serum levels of testosterone (T), estradiol (E2) and BPA were measured. Penile tissue sections were assayed by Masson (SM/collagen), Oil Red O (fat), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (apoptosis), immunohistochemistry for Oct4 (stem cells), and α-SM actin/calponin (SM and myofibroblasts), applying quantitative image analysis. Other markers were assayed by western blotting. DNA microarrays/microRNA (miR) assays defined transcription profiles. Orally administered BPA did not affect body weight, but (1) decreased serum T and E2; (2) reduced the EFS response and increased the drop rate; (3) increased within the corporal tissue the presence of fat, myofibroblasts and apoptosis; (4) lowered the contents of SM and stem cells, but not nerve terminals; and (5) caused alterations in the transcriptional profiles for both mRNA and miRs within the penile shaft. Long-term exposure of rats to oral BPA caused a moderate corporal veno-occlusive dysfunction (CVOD), possibly due to alterations within the corporal tissue that pose gene transcriptional changes related to inflammation, fibrosis and epithelial/mesenchymal transition (EMT).International Journal of Impotence Research advance online publication, 5 December 2013; doi:10.1038/ijir.2013.37.
    International journal of impotence research 12/2013; · 2.73 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The aim of this study is to investigate whether injection of short hairpin RNA (shRNA) constructs targeting IGFBP-3 in penis in old rat would improve erectile function. The most validated IGFBP-3 shRNA plasmid vector (pGPU6/GFP/Neo-sh IGFBP-3) was prepared and injected into the penile corpus cavernosum tissue. 30 old (24 months) Sprague-Dawley male rats were randomized divided into 3 groups: PBS-only (100ul), pGPU6/GFP/Neo-shNC (100ug) and the most validated plasmid constructs pGPU6/GFP/Neo-shIGFBP-3 (100ug) treatment group (n=10/group). The erectile response was measured by intracavernosal pressure (ICP) after 4 weeks. The percent of smooth muscle in corpus cavernosum tissue was evaluated. The activity of nitric oxide synthase (NOS) and concentration of guanosine 3', 5'-cyclic-monophosphates (cGMP) in penile tissue were also analyzed. IGFBP-3 was estimated in penile tissue by Western blot, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. pGPU6/GFP/Neo-shIGFBP-3 corrected the impaired erectile response in aged rats, compared with rats injected with PBS and pGPU6/GFP/Neo-shNC (p<0.01, respectively). The percentage of cavernosal smooth muscle was increased in pGPU6/GFP/Neo-shIGFBP-3 treatment group. NOS activities and cGMP concentrations were also significantly increased in the pGPU6/GFP/Neo-shIGFBP-3 treatment rats. IGFBP-3 shRNA effectively reduced the IGFBP-3 mRNA and protein expression in the penile corpus cavernosum tissue. Decreasing the IGFBP-3 expression by plasmid-expressed shRNA can improve the erectile function in aged rats, which may modulate the integrity of smooth muscle and accumulate the concentration of the cGMP. It may be the new direction for the treatment of ED in the clinical practice.
    The Journal of urology 02/2014; · 4.02 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The ability to get and keep an erection is important to men for several reasons and the inability is called as erectile dysfunction (ED). ED started to be accepted as an early indicator for systemic endothelial dysfunction and subsequent of cardiovascular diseases. The role of nitric oxide (NO) in endothelial relaxation and erectile function is well accepted. The discovery of NO as small signaling gasotransmitter led to the investigation of the role of other endogenously derived gases, carbon monoxide (CO) and hydrogen sulfide (H2 S) in physiological and pathophysiological conditions. The role of NO and CO in sexual function and dysfunction has been investigated more extensively and recently the involvement of H2 S in erectile function has also been confirmed. In this review, we focused on the role of these 3 sister gasotransmitters in the physiology, pharmacology and pathophysiology of sexual function in man, specifically erectile function. We also reviewed the role of soluble guanylyl cyclase /cyclic GMP pathway as a common target of these gasotransmitters. Several studies proposed alternative therapies targeting different mechanisms in addition to phosphodiesterase-5 inhibition for ED treatment, since some patients do not respond to these drugs. This review highlights complementary and possible coordinated roles for these mediators and treatments targeting these gasotransmitters in erectile function/ED.
    British Journal of Pharmacology 03/2014; · 5.07 Impact Factor

Full-text (2 Sources)

Available from
May 16, 2014