A Human Cellular Model for Colorectal Anastomotic Repair: The Effect of Localization and Transforming Growth Factor-β1 Treatment on Collagen Deposition and Biomarkers

Abstract

Anastomotic leakage (AL) is a devastating complication after colorectal surgery, possibly due to the loss of stabilizing collagen fibers in the submucosa. Our aim was to assess the formation of collagen in the colon versus the rectum with or without transforming growth factor (TGF)-β1 exposure in a human cellular model of colorectal repair. Primary fibroblasts were isolated by an explant procedure from clinically resected tissue rings during anastomosis construction in 19 consecutive colorectal patients who underwent laparoscopy. The cells, identified as fibroblasts by morphologic characteristics and flow cytometry analysis (CD90+), were cultured for 8 days and in 12 patients in the presence of 1 ng/mL TGF-β1. Total collagen deposition was measured colorimetrically after Sirius red staining of fixed cell layers, and type I, III, and VI collagen biosynthesis and degradation were specifically determined by the biomarkers PINP, PRO-C3, PRO-C6, and C3M in conditioned media by competitive enzyme-linked immunosorbent assays. Total collagen deposition by fibroblasts from the colon and rectum did not significantly differ. TGF-β1 treatment increased PINP, PRO-C6, and total collagen deposition. Mechanistically, TGF-β1 treatment increased COL1A1 and ACTA2 (encoding α-smooth muscle actin), and decreased COL6A1 and MMP2 mRNA levels in colorectal fibroblasts. In conclusion, we found no effect of anatomic localization on collagen production by fibroblasts derived from the large intestine. TGF-β1 represents a potential therapeutic agent for the prevention of AL by increasing type I collagen synthesis and collagen deposition.

Gene Expression Linked to Reepithelialization of Human Skin Wounds

“…Previous studies in NHDFs indicated that IL-6 is anti-proliferative at ≥10 ng/mL while IL-1β (0.1 ng/mL) is proliferative [ 22 ]. In our studies of NHDFs using the BrdU incorporation assay [ 23 , 24 ], IL-6 and IL-1β did not significantly influence DNA synthesis at 30 ng/mL and 1 ng/mL, while IL-6 at 100 ng/mL tended to reduce ( p = 0.052) BrdU incorporation into NHDFs ( Supplementary Figure S2 ).…”

“…NHDFs were cultured in DMEM with GlutaMAX™, glucose (4.5 g/L) and pyruvate (Gibco, Life Technologies, Grand Island, NY, USA) with 10% fetal bovine serum (FBS; Gibco heat-inactivated qualified FBS, 10500064, Thermo Fisher Scientific, Waltham, MA USA), penicillin (100 IU/mL) and streptomycin (100 μg/mL). Cells were incubated in a humidified atmosphere of 5% CO 2 /air at 37 °C and were passaged using 0.05% trypsin-0.02% EDTA (Biological Industries, Kibbutz Bet-Haemek, Israel) [ 24 ].…”

Healing of colonic anastomosis in rats under obstructive ileus conditions

“…Promoter agents include FGF, TGF-α and VEGF. Turlu et al showed that TGF-β1 represents a potential therapeutic agent for the prevention of anastomotic leakage by increasing collagen synthesis and collagen deposition 104 . Positive effects also have local factors, such as hypoxia, low pH and high levels of lactic acid 89 .…”

Understanding the functional inflammatory factors involved in therapeutic response to immune checkpoint inhibitors for pan-cancer

“…In bladder cancer patients, PD-L1 expression is discordant at metastatic sites and the primary tumor, suggesting the dynamic nature of the ECM ( Mukherji et al, 2016 ). TGF-β has been reported to increase the synthesis and deposition of type I collagen and downregulate MMP-2 ( Türlü et al, 2021 ). Activation of the RhoA/ROCK signaling pathway and transcription of SNAIL1 and TWIST1 genes are both triggered by TGF-β ( Gaggioli et al, 2007 ; García-Palmero et al, 2016 ; Chung et al, 2021 ).…”