Validation and testing of an in vitro model to study medical treatments for anterior urethral stricture disease: assessing the potential efficacy of phosphodiesterase-4 (PDE4) inhibition and testosterone

Objective

To 1) determine the expression and distribution of all PDE4 isozymes (A-D) along the length of the anterior urethra, 2) culture fibroblasts and epithelial cells from healthy and strictured urethras, 3) investigate an in vitro model of anterior urethral stricture disease (aUSD), and 4) assess the therapeutic potential of phosphodiesterase-4 (PDE4) inhibitors and testosterone compared to paclitaxel.

Methods

The presence and relative abundance of PDE4 isozymes (A-D) was confirmed using immunohistochemistry on 5 male cadaveric urethras. Human urethral fibroblasts (FBs) were cultured from healthy control urethras of patients undergoing vaginoplasty (n=3) and from idiopathic bulbar urethral strictures (L2S1E2) of patients undergoing urethroplasty (n=3). Epithelial cells (ECs) were cultured from a healthy control urethra and two urethral strictures. To investigate a model of aUSD, Control FBs were stimulated with TGFβ1 and compared to Stricture FBs on assays of cell proliferation and expression of genes relevant to aUSD pathophysiology. To test therapeutics, Stricture FBs were treated with the PDE4 inhibitor, roflumilast, testosterone (T), or paclitaxel and compared to Control FBs on the previously mentioned assays and cell viability.

Results

PDE4- A, B, and D were detected along the length of the urethra. Expression levels did not differ between urethral regions. TGFβ1 altered proliferation and gene expression in a dose-dependent manner. Roflumilast and T preserved cell viability and proliferation and decreased expression of genes positively associated with auSD.

Conclusion

Urethral FBs and ECs can be cultured from healthy and strictured surgical specimens, enabling in vitro research. PDE4 inhibitors and T may be non-cytotoxic alternatives or additions to paclitaxel for aUSD.

Highlights

• PDE4 isozymes A, B, and D are expressed in adult anterior urethras

• PDE4 is expressed equally from proximal bulbar to meatal urethra

• Epithelial cells and fibroblasts can be cultured from healthy and stricture urethra

• TGFβ1 may not be an optimal method to model aUSD in vitro

• Unlike paclitaxel, roflumilast and testosterone are not toxic to urethral cells