Dysregulated Vaginal Fibrotic Response to Mesh Under Diabetic Conditions

Objectives: Urogynecologic mesh implantation is used to augment surgeries with native tissue, whereas diabetes has been identified as an independent risk for mesh exposure. Successful mesh-tissue integration requires a balanced fibrotic response in tissue regeneration. However, diabetes may interfere with this process, resulting in impaired would healing. Here we aimed to define the impact of diabetes on the fibrotic response in the vagina following mesh implantation. As macrophages, a dominant immune cell type at mesh-tissue interface, regulate tissue fibrotic response, we further examined their alterations in the process.

Methods: A diabetic rat model (Wistar, 9-12 mo) was established using streptozotocin for 2 weeks. Diabetic vs. nondiabetic rats underwent sham surgeries or mesh implantation upon the vaginas via a modified sacrocolpopexy following hysterectomy and ovariectomy for 3-, 7- and 42-days (n5 per time point per group). Tissue collagen architecture was characterized by picrosirius red staining of tissue sections (7μm) with polarized microscopic imaging. Green (G), orange and red (R) fibers, representing thin, medium and thick collagen fibers, and collagen content (% of tissue area) were quantified. Macrophage (CD68, CD163) and myofibroblast (aSMA) markers, were co-labeled with immunofluorescence. Macrophages were isolated from the enzyme-digested vaginal tissue with flow sorting, followed by RNA sequencing to examine macrophage transcriptomics. Profibrotic factor TGF-β1 in tissue protein extract was quantified by ELISA. Data were analyzed with CLC Genomics Workbench (fold change>2, FDR < .1) and Kruskal-Wallis tests (p < .05).

Results: Under normoglycemic conditions, mesh implantation resulted in an increased R/G ratio across all tissue layers at both 7- and 42-days relative to Sham (p=.014, .002), demonstrating a long-lasting overall increase of vaginal fibrotic response. In contrast, this response was not significant at 42 days under diabetic conditions, despite a similarly increased R/G ratio at 7 days when compared to Sham. At 42- vs. 7- days, the non-diabetic group with mesh implantation showed increased collagen content (p=.008) with no change in the R/G ratio (Figure1). In addition, the tissue TGF-β1 level was decreased over time (p=.007). In diabetic groups, however, such longitudinal fibrotic response in the vagina was not observed, as evidenced by no change in collagen content and TGF-β1 levels. Instead, a decrease in the R/G ratio (p=.014) was noted at 42- vs. 7- days. Interestingly, mRNA levels of aSMA and major collagens including I, III, VI and VI, which are normally expressed in myofibroblasts (acitvated fibroblasts producing extracellular matrix), were all increased by ~4-fold in macrophages in the diabetic vs. non-diabetic group at 42 days (all p< .001, FDR < .05), indicating an increased macrophage phenotypic transition toward myofibroblasts (Figure 2). CD68 and aSMA co-labeling was observed at mesh-tissue interface.

Conclusions: Long-term diabetes induces dysregulated fibrotic response to mesh in the vagina, which is associated with a likely increased macrophage-myofibroblast trandifferentiation.