August 8, 2021
doi: 10.1126/technology.1079562. -14 as well as lower manifestation of TIMP-2 in liver endothelial cells. In addition, we report improved manifestation of MMP-13 in scar-associated macrophages as well as improved liver regeneration upon ablation of VHL in myeloid cells. Finally, restorative infusion of macrophages nulli-zygous for VHL or treated with the pharmacologic hydroxylase inhibitor and HIF-inducer Dimethyloxalylglycine (DMOG) accelerates resolution of fibrosis. Hence, improving the HIF-VEGF signaling axis in macrophages represents a encouraging restorative avenue for the treatment of liver fibrosis. = 5) and VHL fl/fl- LysMCre+ mice (= 6). (B) Time-schedule for CCl4-treatment, bone-marrow transplantation and subsequent fibrosis resolution. (C) Representative histological images of Sirius Red-stained liver sections from mice after reconstitution with bone marrow (BM) from VHL LysMCre- (remaining panel) and VHL LysMCre+ mice (ideal panel) after 12 weeks of treatment with CCl4 and 4 weeks of recovery. (D) Quantification of Sirius Red-positive area on murine livers sections (= 5 for VHL LysMCre- and = 7 for VHL Rabbit Polyclonal to KR1_HHV11 LysMCre+). (E) Dedication of free hydroxyproline in liver tissue samples (= 5 for VHL LysMCre- and = 7 for VHL LysMCre+). (F) Representative histological images of -SMA-stained liver sections from the Carbenoxolone Sodium two groups of mice and quantification of -SMA-positive area (= 5 for VHL LysMCre- and = 7 for VHL LysMCre+). Error bars symbolize SEM. Scale bars equivalent 100 m. Interestingly, reconstitution with BM from VHLfl/fl-LysMCre+ mice results in reduced liver collagen content compared to mice reconstituted with wildtype (VHLfl/fl-LysMCre -) bone marrow (Number 1C, 1D and 1E) as assessed by quantitative analysis of the Sirius red-positive area on liver sections (Number ?(Figure1D)1D) as well as by dedication of the total liver hydroxy-proline content (Figure ?(Figure1E).1E). Consistent with the notion that ECM Carbenoxolone Sodium degradation itself can contribute to myofibroblast contraction upon fibrosis regression [15, 16], we observe reduced numbers of -SMA-expressing myofibroblasts after reconstitution with VHLfl/fl-LysMCre+ BM (Number ?(Figure1F).1F). Taken together, this indicates that improving the hypoxic response in myeloid cells by deleting VHL accelerates the resolution of fibrosis. Deletion of VHL in myeloid cells upon resolution enhances ECM degradation activity and endothelial manifestation of matrix degrading enzymes The resolution of fibrosis requires the breakdown of the ECM network. Hence, we performed an zymography by incubating liver sections with fluorescein-labeled gelatin (DQ-gelatin?). This fluorogenic substrate yields a bright fluorescent transmission upon proteolytic digestion and allows the detection of ECM degradation (Number ?(Figure2A).2A). Quantitative analysis of the fluorescent transmission revealed improved zymographic activity in mice with BM from VHLfl/fl-LysMCre+ mice compared to mice after reconstitution with wildtype (VHLfl/fl-LysMCre-) bone marrow (Number ?(Figure2B).2B). This further suggests that mice reconstituted with BM from VHLfl/fl-LysMCre+ mice are more efficient in breaking down ECM and resolving liver fibrosis. We have previously demonstrated that, despite an overall increase in vascular denseness, the fibrotic scar is mostly devoid of sinusoids, suggesting sinusoidal rarefication in this area . Upon regression of the fibrotic scar, though, the fibrotic areas become revascularized inside a VEGF-dependent manner, resulting in a more homogenous distribution of sinusoidal vessels and a decrease in vascular denseness [9, 17]. This was linked to a proresolution phenotype of the liver endothelium, involving improved manifestation of MMP-2 and -14 as well as reduced manifestation of TIMP-1 and -2 in response to myeloid cell-derived VEGF. In order to determine whether focusing on of VHL in myeloid cells translates into vascular changes, we performed simultaneous detection of sinusoidal vessels and the fibrotic scar by means of double immunofluorescence for VEGFR2 and SMA on liver sections from both genotypes. As demonstrated in Number ?Number2C,2C, accelerated resolution of the fibrotic scar in VHLfl/fl-LysMCre+ BM-reconstituted mice was indeed associated with a more homogenous pattern of sinusoids and a reduction of vascular density (Number ?(Figure2D).2D). Strikingly, this was associated with enhanced manifestation of MMP-2 and -14 and a decrease in TIMP-2 manifestation in sorted liver endothelial cells (Number ?(Number2E),2E), therefore further substantiating the part of VEGF like a driver of fibrolysis. Open in a separate window Number 2 Transplantation Carbenoxolone Sodium of bone marrow from VHLfl/fl-LysMcre+ mice into C57Bl6/J mice after CCl4-challenge accelerates matrix degradation activity and the manifestation of matrix degrading enzymes in liver endothelial cells(A) DQ?-gelatin images illustrating the gelatinolytic activity in liver sections from the two experimental groups. (B) Quantification of DQ?-gelatin-positive areas about liver sections. (C) Representative images of murine liver sections co-immunolabeled for VEGFR2 and -SMA. (D) Quantitative analysis of the VEGFR2-positive area. (E) Quantitative actual time-analysis of MMP2, MMP14, and TIMP2-manifestation, respectively, in.