ORIGINAL RESEARCH
Sildenafil Promotes Smooth Muscle Preservation and Ameliorates Fibrosis Through Modulation of Extracellular Matrix and Tissue Growth Factor Gene Expression After Bilateral Cavernosal Nerve Resection in the Rat

https://doi.org/10.1111/j.1743-6109.2010.02195.xGet rights and content

ABSTRACT

Introduction

It has been shown that phosphodiesterase type 5 (PDE5) inhibitors preserve smooth muscle (SM) content and ameliorate the fibrotic degeneration normally seen in the corpora cavernosa after bilateral cavernosal nerve resection (BCNR). However, the downstream mechanisms by which these drugs protect the corpora cavernosa remain poorly understood.

Aim

To provide insight into the mechanism, we aimed to determine the gene expression profile of angiogenesis‐related pathways within the penile tissue after BCNR with or without continuous sildenafil (SIL) treatment.

Methods

Five‐month‐old Fisher rats were subjected to BCNR or sham operation and treated with or without SIL (20 mg/kg/BW drinking water) for 3 days or 45 days (N = 8 rats per group). Total RNAs isolated from the denuded penile shaft and prostate were subjected to reverse transcription and to angiogenesis real‐time‐polymerase chain reaction arrays (84 genes). Changes in protein expression of selected genes such as epiregulin (EREG) and connective tissue growth factor (CTGF) were corroborated by Western blot and immunohistochemistry.

Main Outcomes Measures

Genes modulated by BCNR and SIL treatment.

Results

A decreased expression of genes related to SM growth factors such as EREG, platelet‐derived growth factor (PDGF), extracellular matrix regulators such as metalloproteinases 3 and 9, endothelial growth factors, together with an upregulation of pro‐fibrotic genes such as CTGF and transforming growth factor beta 2 were found at both time points after BCNR. SIL treatment reversed this process by upregulating endothelial and SM growth factors and downregulating pro‐fibrotic factors. SIL did not affect the expression of EREG, VEGF, and PDGF in the ventral prostate of BCNR animals

Conclusions

SIL treatment after BCNR activates genes related to SM preservation and downregulates genes related to fibrosis in the corpora cavernosa. These results provide a mechanistic justification for the use of SIL and other PDE5 inhibitors as protective therapy against corporal SM loss and fibrosis after radical prostatectomy. Sirad F, Hlaing S, Kovanecz I, Artaza JN, Garcia LA, Rajfer J, and Ferrini MG. Sildenafil promotes smooth muscle preservation and ameliorates fibrosis through modulation of extracellular matrix and tissue growth factor gene expression after bilateral cavernosal nerve resection in the rat.

Introduction

Erectile dysfunction (ED) associated with radical prostatectomy (RP) is a common complication after surgery due to intra‐operative injury to the cavernosal nerves 1, 2. Although nerve‐sparing RP (NSRP) has significantly decreased the rates of postoperative ED 2, 3, 4, novel approaches for preserving both the normal architecture of the corporal tissue as well as its function continue to undergo investigation because of the still unacceptable high rates of ED with NSRP.

Cavernosal nerve damage ultimately leads to apoptosis of the corporal smooth muscle (SM) cells as well as fibrosis of the corporal tissue 5, 6. This condition leads to the development of cavernosal veno‐occlusive dysfunction (CVOD) [6] which is sometimes referred to as venous leakage. In this condition, blood entering the corporal tissue during an erectile event fails to remain within the sinusoids mainly because the remaining SM within the cavernosa is unable to relax sufficiently enough to achieve an intracorporeal pressure high enough to occlude the veins egressing the corpora. This alteration in the corporal histology is initiated immediately after the nerve injury occurs and it is assumed that a certain threshold of SM impairment/apoptosis has to be reached before a functional impairment, i.e., ED due to CVOD become manifest [7]. Therefore, in this setting, therapies to protect corporal SM viability either by reducing its apoptosis and/or sustaining its proliferation should be initiated as early as possible after the injury.

It has been previously demonstrated in rats that long‐term treatment with the phosphodiesterase type 5 (PDE5) inhibitors such as vardenafil [6], sildenafil (SIL) [8], and tadalafil [9] preserves corporal SM and ameliorates fibrotic degeneration normally seen after bilateral cavernosal nerve resection (BCNR). Anecdotally, acute SIL treatment is able to downregulate penile hypoxia markers [10]. There is still ongoing debate as to the exact role that PDE5 inhibitors play in the post‐RP setting [11]. One school of thought states that these PDE5 inhibitors increase “oxygenation” to the penile tissues 10, 12, 13, 14, while the other school of thought adheres to the concept that these are simply anti‐fibrotic compounds which primarily inhibit the oxidative stress to the cavernosal tissue induced by the neurotomy 6, 7, 8, 9, 15, 16, 17, 18.

Although animal studies support the concept of so‐called corporal preservation post‐prostatectomy, in the clinical setting, there is no consensus regarding its benefit, mainly because all the studies performed so far have marked differences in their design that comparisons between studies cannot be made. For example, the study of Schwartz et al. [19] reported on 40 patients who had undergone RP and were treated with one of two doses of SIL started when the catheter was removed and given every other day for 6 months and who had a cavernosal biopsy both prior to and at the end of the study period. The results demonstrated not only preservation of corporal histology and absence of fibrosis, but in the higher‐dose group, there was a substantial increased content of corporal SM cells. Padma Nathan and et al. [20] found in a randomized, double‐blind, placebo‐controlled study of postoperative nightly SIL started 4 weeks after the NSRP and administered nightly for 36 weeks a fourfold increase in the return of normal spontaneous erections when compared to the control group, although in the control group, only 8% had normal erections at the end of the study period. More recently, Montorsi et al. [21], in the triple arm REINVENT study where drug treatment was initiated 14 days after the NSRP and given for 9 months, reported that vardenafil was more efficacious in preserving erectile function when given as an on‐demand rather than as a nightly dose, although in the final open label phase of the study, there was no difference in terms of recovery of erections as determined by an on‐demand vardenafil trial. In fact, both the daily and on‐demand treatment for 9 months was no different than the placebo group

From these data, it is not surprising that penile rehabilitation or corporal preservation after RP remains controversial and more research is required in order to establish, at least with PDE5 inhibitors, when to start treatment post‐RP, as well as the dose and the appropriate outcome metric to determine efficacy 22, 23.

To date, little is known about the mechanism of action by which long‐term PDE5 inhibitors would have a beneficial effect on the corporal tissue. While it is known that elevated levels of cyclic guanosine monophosphate may inhibit SM apoptosis and/or inhibit collagen deposition, the downstream pathways involved in this process are poorly understood. Angiogenesis, the formation of thin‐walled endothelium‐lined structures with muscular SM wall, plays an important role during the adult life span as “repair mechanism” of damaged tissues [24]. Emerging evidence has demonstrated that SIL can induce angiogenesis after pulmonary hypertension [25] and stroke [26]. Moreover, SIL has also shown promise in the management of ischemia‐reperfusion injury in the heart [27]. However, there is no molecular evidence regarding the mechanisms by which SIL therapy may confer protection on the corporal SM after nerve injury or a neuropathy, although Musicki et al. has suggested that SIL may activate pathways that are involved in the repair of the corporal SM 28, 29.

Expression of angiogenic factors such as vascular endothelial growth factor (VEGF) and its respective receptors are present in human corpus cavernosum, indicating the possibility of mechanisms associated with angiogenesis that could be involved in repairing the penile tissue structure after injuries or diseases 30, 31.

Section snippets

Aims

The specific aim of this study was to determine (i) whether BCNR can cause changes in the gene expression profile involved in fibrosis and SM biology in the penis and (ii) which molecular mechanisms that induce preservation of the SM content and ameliorates fibrosis in rat penile tissue following BCNR are modulated by SIL.

Animal Surgery and Treatments

Five‐month‐old male Fisher 344 rats (Harlan Sprague–Dawley, San Diego, CA, USA) were randomly divided into sham‐operated, BCNR and BCNR + SIL groups. Treatment with SIL was initiated immediately after surgery, dissolving the drug in drinking water in a dose of 20 mg/kg/BW.

Animals were sacrificed at 3 days (short‐term treatment) and at 45 days (long‐term treatment) after surgery (N = 8 each group). The selection of these two time points was based on our previous results where we determined that

Effect of BCNR and SIL Treatment on the Expression of α‐SMA

In order to verify whether the expression of α‐SMA in penile tissue after BCNR plus SIL treatment can be observed under these experimental settings, Western blot analysis was carried out in animals that were used for gene expression analyses. Figure 1 shows that by 45 days after BCNR, α‐SMA expression was decreased by 57% compared to the sham group, while the addition of SIL to the BCNR group was able to preserve the SM content levels even 45 days after BCNR.

Short‐Term Treatment with SIL Modulates the Expression of Genes Involved in Both the Preservation of the SM Content and the Amelioration of Fibrosis

The impact of BCNR and SIL treatment

Discussion

To the best of our knowledge, this is the first report to examine the gene expression profile after BCNR and its subsequent treatment with a PDE5 inhibitor. Our initial objective was to look for changes in inflammation, ECM remodeling, and angiogenesis‐related pathways after BCNR and to understand the mechanism by which SIL preserves the SM and ameliorates fibrosis after BCNR. Our findings demonstrate that both short‐term and long‐term BCNR promotes downregulation of the gene expression profile

Conclusion

In conclusion, cavernosal nerve damage appears within the corporal tissue to downregulate genes related to SM preservation and activates genes related to fibrosis, while both short‐term and long‐term continuous SIL treatment revert this impaired gene expression profile. Taken together, the data from this present study suggest that PDE5 inhibitors could be used as an alternative treatment in disease states where amelioration of fibrosis and neo‐vessel growth are desired as seen, e.g., in

Category 1

  • (a)

    Conception and Design

    Monica G. Ferrini

  • (b)

    Acquisition of Data

    Fara Sirad; Su Hlaing; Istvan Kovanecz; Leah A. Garcia

  • (c)

    Analysis and Interpretation of Data

    Monica G. Ferrini; Jorge N. Artaza; Istvan Kovanecz; Fara Sirad; Su Hlaing; Leah A. Garcia

Category 2

  • (a)

    Drafting the Article

    Monica G. Ferrini; Jorge N. Artaza; Jacob Rajfer; Istvan Kovanecz

  • (b)

    Revising It for Intellectual Content

    Monica G. Ferrini; Jorge N. Artaza; Jacob Rajfer

Category 3

  • (a)

    Final Approval of the Completed Article

    Monica G. Ferrini

Acknowledgments

This work was supported by National Institute of Neurological Disorders and Stroke and the National Institute of General Medicine NINDS/NIGMS SC1NS064611 (MGF), National Center for Research Resources (NCRR) 1U54RR026138‐01 (JA), Research Centers in Minority Institutions (RTRN) U54RR022762, and the National Center on Minority Health & Disparities (NCMHD) UCLA/Drew Project EXPORT 2P20MD000182 (JA).

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