Pentoxifylline attenuates transforming growth factor-β1-stimulated collagen deposition and elastogenesis in human tunica albuginea-derived fibroblasts part 1: impact on extracellular matrix

Abstract

Introduction: Transforming growth factor-β1 (TGF-β1) has been implicated in the pathogenesis of Peyronie's disease (PD) and also plays a role in collagen and elastin metabolism. Pentoxifylline (PTX) antagonizes the effects of TGF-β1 and has been utilized in our clinic for the management of PD.

Aim: We studied the effects of TGF-β1 and PTX on collagen metabolism and elastogenesis in tunica albuginea-derived fibroblasts (TADFs).

Methods: TADFs from men with and without PD were cultured and treated with TGF-β1 and PTX as monotherapy at differing concentrations and time points. Combination treatment (TGF-β1 followed by PTX and vice versa) was also investigated.

Main outcome measures: Cell proliferation assay, enzyme-linked immunosorbent assay, and immunohistochemistry were utilized to assess the impact of TGF-β1 and PTX on TADF with respect to elastin and collagen I metabolism.

Results: PTX inhibited fibroblast proliferation at doses of 100 µM. TGF-β1 stimulated elastogenesis and collagen I fiber deposition in TADF in a dose- and time-dependent fashion. Pretreatment with PTX dramatically attenuated TGF-β1-mediated elastogenesis and collagen fiber deposition in TADF from men with and without PD. Interestingly, production of collagen I was higher in untreated Peyronie's tunica (PT) cells relative to normal tunica (NT) cells; furthermore, PTX attenuated collagen production to levels similar to untreated control TADF in PT cells but not in NT cells, suggesting important intrinsic differences between PT and NT cells.

Conclusion: Both elastin and collagen are upregulated by TGF-β1 in TADF. This likely contributes to the PD phenotype. Pretreatment with PTX attenuates both collagen fiber deposition and elastogenesis in TADF exposed to TGF-β1; these effects suggest a useful role for PTX in the management of PD.

Figure 1

Tunica albuginea-derived fibroblast cells were treated with vehicle or transforming growth factor-β1 (TGF-β1; 1 ng/mL) in Dulbecco’s modified Eagle medium with 5% fetal bovine serum for 14 days. In the native status (vehicle treated; A, B), there were no mature elastic fibers formed although small quantities of tropoelastin were identified (arrowheads). After treatment with TGF-β1, elastogenesis was activated. Many matured elastic fibers (arrows) were formed in the extracellular matrix (green) (C, D). Elastin fiber formation was more robust in PT cells.

Figure 2

Pentoxifylline (PTX) attenuates elastogenesis activated by transforming growth factor-β1 (TGF-β1) in tunica albuginea-derived fibroblast. PT TADF cells were pretreated with 0, 0.01, 10, and 100 uM PTX for 6 hours followed by 1 ng/mL TGF-β1 for 14 days in Dulbecco’s modified Eagle medium with 5% fetal bovine serum. Inhibition of elastogenesis showed a dose–response increase with progressively higher levels of PTX in PT cells; a PTX dose–response relationship was not clearly evident in normal tunica cells (arrows indicate elastic fibers, arrowhead indicates tropoelastin. ×200).

Figure 3

Pentoxifylline (PTX) attenuates elastogenesis activated by transforming growth factor-β1 (TGF-β1) in normal tunica (NT) and PT cells. (A) PT tunica albuginea-derived fibroblast cells were treated with TGF-β1 (1 ng/mL) or combination TGF-β1 (1 ng/mL) and PTX (100 uM). PTX attenuated elastogenesis in both PT and NT cells.

Figure 4

Pentoxifylline (PTX) decreases collagen I in tunica albuginea-derived fibroblast. Normal tunica (NT) and PT were treated with transforming growth factor-β1 (TGF-β1), TGF-β followed by PTX, and PTX followed by TGF-β1. Expression of collagen I was higher in PT relative to NT in the untreated control group (*P < 0.05). TGF-β1 significantly increased collagen I in NT and PT cells from 0.169 3 0.02 and 0.703 3 0.1 ug/mL to 1.342 3 0.01 and 1.8525 3 0.05 ug/mL, respectively (#P < 0.05 vs. controls). This effect was not observed in cells pretreated with PTX prior to TGF-β1 (0.569 3 0.02 and 0.75 3 0.05 ug/mL in NT and PT, respectively, P < 0.05 vs. TGF-β1 treated groups) although collagen I production in these NT cells remained significantly higher than what was observed in NT controls (#P < 0.05 vs. control cells). PTX following TGF-β1 treatment did not affect collagen I increase relative to TGF-β1 alone (P > 0.05; *P < 0.05 vs. NT cells, #P < 0.05 vs. control cells [PT and NT]).