Tuesday 16th August 2022

Therapeutic strategies for urethral stricture

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Introduction

Urethral stricture is narrowing of the urethral lumen due to fibrosis and scar formation in the urethral mucosa and surrounding tissues.1–3 Several factors have been implicated in the etiology of this condition, including infection, trauma due to urethral instrumentation, and inflammatory disorders.4 In each case, urethral epithelial injury heals by fibrosis, resulting in a reduction in urethral diameter and impairment of urine flow.3,5 Various surgical procedures, such as urethral dilation, urethroplasty, and internal urethrotomy, are available for the treatment of urethral stricture but they have a high failure rate and stricture often recurs.6–8 Recently introduced treatments include antifibrotic drugs such as mitomycin C, somatostatin analogs, glucocorticoids, bitoxin A, and halofuginone; however, they have shown little benefit.9–12 Therefore, there exists an urgent medical need for new strategies for the treatment of urethral stricture.

The process of wound healing after urethral trauma occurs in three overlapping phases: inflammation, proliferation, and remodeling.13,14 Of these, the inflammation phase is the most appropriate intervention phase to prevent urethral stricture, because hypertrophic repair and scar formation at the wound caused by post-traumatic inflammation are the central processes in urethral stricture.15,16 Among the many cell types involved in this process, fibroblasts and macrophages play the most important roles as mediators and regulators, respectively, of scar formation and/or degradation.17 It is well-established that the phenotype of tissue macrophages evolves during wound healing, and also that the macrophage polarization status influences the activation and biological behavior of fibroblasts.14,18 Pro-inflammatory macrophages, traditionally referred to as M1 macrophages, have positive effects by infiltrating the site of tissue injury or infection and clearing debris, dead cells, and bacteria in the wound. However, excessive inflammation and prolonged activation of fibroblasts can result in scar formation, the ultimate cause of urethral stricture.19 Ueshima et al also reported that macrophage-secreted TGF-β1 would contribute to fibroblast activation and ureteral stricture after ablation injury.20 In contrast, M2 macrophages inhibit excessive inflammation and promote wound healing by remodeling of the extracellular matrix.18 These observations prompted us to consider that enforced macrophage polarization to the M2 subtype could be a potential therapeutic strategy to reduce post-traumatic urethral inflammation and scar formation.

Recent studies have suggested that mesenchymal stem cells (MSCs) can contribute to the resolution of inflammation and facilitate the repair of injured tissues by regulating immune cells in the post-traumatic microenvironment.21,22 For example, Zhao et al reported that the microRNA (miRNA) miR-182 is present in extracellular vesicles, or exosomes, secreted by MSCs and can attenuate myocardial ischemia–reperfusion injury by regulating macrophage polarization.23 Additionally, apoptotic bodies derived from MSCs can promote cutaneous wound healing by regulating macrophage function.24 Inflammatory mediators can induce movement of MSCs to the site of inflammation, where they alter their secretory profile, exert immunomodulatory effects, and promote wound healing.25 These observations suggest that cytokines and/or drugs could be used to augment the immunomodulatory characteristics of MSCs and enhance their roles in tissue repair, thus providing a rationale for the development of such treatments for inflammation-related disorders, including urethral stricture.

Pretreatment of MSCs with IL-1β, a key inflammatory mediator, has been reported to enhance the inhibitory effects of MSCs on sepsis-associated systemic inflammation by inducing macrophage polarization towards the anti-inflammatory M2 phenotype.26 Thus, we hypothesized that IL-1β-induced MSCs may similarly suppress post-traumatic urethral inflammation and scar formation by regulating macrophage polarization. Secretion of exosomes containing various nucleic acids is now recognized as a major mechanism for intercellular communication.27 Therefore, we speculated that MSCs and macrophages may communicate during urethral wound healing via exosomes. In the present study, we investigated the effects of exosomes derived from IL-1β-induced MSCs on macrophage polarization and fibroblast activation in vitro, and additionally on post-traumatic urethral stricture and fibroblast activation in an animal model. We also explored the underlying mechanism driving the direct effects of Exo-MSCsIL−1β on macrophages. The results of our study suggest that miRNA-containing MSC-derived exosomes may be a promising new therapeutic strategy for urethral stricture.

Materials and Methods

Cell Lines and Cell Culture

Specimens of urethral scar tissues were obtained from three patients who underwent urethroplasty for post-traumatic urethral stricture. Primary urethral fibroblasts (UFBs) were obtained and cultured as previously described.2,3 All experiments with primary cells were performed with the consent of the donors and were approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University. Umbilical cord-derived MSCs were purchased from Saliai Stem Cell Science and Technology (Guangzhou, China) and maintained in F12/Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific Life Sciences, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific Life Sciences). The human monocytic cell-line THP-1 was purchased from the American Type Culture Collection (Manassas, VA, USA), and was cultured in RPMI medium containing 10% FBS. To induce differentiation into macrophages, THP-1 monocytes were incubated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma, St. Louis, MO, USA) for 48 h. For polarization, THP-1 cells were incubated with PMA for 48 h in the presence of 20 ng/mL IL-4 (M2 polarization) or 20 ng/mL IFN-γ and 10 pg/mL lipopolysaccharide (M1 polarization). Transforming growth factor-β1 (TGF-β1) was added at 20 ng/mL to activate UFBs as indicated. All cells were cultured at 37°C in a 5% CO2 atmosphere.

Isolation and Characterization of Exosomes

MSCs were incubated with or without IL-1β at 50 ng/mL for 48 h. Exosomes were isolated from the culture supernatants by differential centrifugation as previously described, and then characterized using a range of analytical techniques.28 Exosome morphology was examined by transmission electron microscopy (JEM-200CX, Hitachi, Tokyo, Japan), and size distribution was measured using a NanoSight NS300 system (Malvern Panalytical, Malvern, UK) after dilution of 10 μL isolated exosomes to 1 mL with filtered PBS. The identity of exosomes was confirmed by Western blotting (WB) with antibodies against the exosome marker proteins CD63 and TSG101. Exosomes were quantified by BCA assay of total protein content (Thermo Fisher Scientific Life Sciences). Exosomes from control and IL-1β-primed MSCs were designated Exo-MSCs and Exo-MSCsIL−1β, respectively.

Animal Model and Urethrography

All animal experiments were approved by the Experimental Animal Ethics Committee of Fujian Medical University, which conformed to the principles of animal protection, animal welfare and ethics, and the relevant provisions of national experimental animal welfare ethics. Twenty-four healthy New Zealand white male rabbits (2.5–3.5 kg) were obtained from the Laboratory Animal Center of Fujian Medical University. Animals were housed in a temperature-controlled (22±1°C), humidity-controlled, (40–70%), and light-period controlled (12 h/12 h light/dark cycle) environment. They were fed a standard rabbit pellet diet and had access to tap water ad libitum. Six animals were randomly selected as the negative control group and received an injection of 100 µL saline into the urethral wall. The remaining animals were injected in the spongiosum urethra with 1 mg of TGF-β1 in 100 µL of saline to enhance fibrosis and induce the stricture phenotype. All rabbits underwent a partial incision of 1 cm length in the mucosa on the ventral side urethra of the penis using a surgical blade, with an intravenous anesthesia of sodium pentobarbital (30–40 mg/kg). The penile skin wound was subsequently closed using 5–0 absorbable sutures and the urethral catheter then…

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