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. 2017 Nov 17;49(11):e396.
doi: 10.1038/emm.2017.213.

Integrin Alpha 11 in the Regulation of the Myofibroblast Phenotype: Implications for Fibrotic Diseases

Affiliations
Free PMC article

Integrin Alpha 11 in the Regulation of the Myofibroblast Phenotype: Implications for Fibrotic Diseases

Ruchi Bansal et al. Exp Mol Med. .
Free PMC article

Abstract

Tissue fibrosis, characterized by excessive accumulation of aberrant extracellular matrix (ECM) produced by myofibroblasts, is a growing cause of mortality worldwide. Understanding the factors that induce myofibroblastic differentiation is paramount to prevent or reverse the fibrogenic process. Integrin-mediated interaction between the ECM and cytoskeleton promotes myofibroblast differentiation. In the present study, we explored the significance of integrin alpha 11 (ITGA11), the integrin alpha subunit that selectively binds to type I collagen during tissue fibrosis in the liver, lungs and kidneys. We showed that ITGA11 was co-localized with α-smooth muscle actin-positive myofibroblasts and was correlatively induced with increasing fibrogenesis in mouse models and human fibrotic organs. Furthermore, transcriptome and protein expression analysis revealed that ITGA11 knockdown in hepatic stellate cells (liver-specific myofibroblasts) markedly reduced transforming growth factor β-induced differentiation and fibrotic parameters. Moreover, ITGA11 knockdown dramatically altered the myofibroblast phenotype, as indicated by the loss of protrusions, attenuated adhesion and migration, and impaired contractility of collagen I matrices. Furthermore, we demonstrated that ITGA11 was regulated by the hedgehog signaling pathway, and inhibition of the hedgehog pathway reduced ITGA11 expression and fibrotic parameters in human hepatic stellate cells in vitro, in liver fibrosis mouse model in vivo and in human liver slices ex vivo. Therefore, we speculated that ITGA11 might be involved in fibrogenic signaling and might act downstream of the hedgehog signaling pathway. These findings highlight the significance of the ITGA11 receptor as a highly promising therapeutic target in organ fibrosis.

Conflict of interest statement

JP is the founder and stakeholder of ScarTec Therapeutics BV, Enschede, The Netherlands. The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Upregulation of ITGA11 in CCl4-induced chronic liver fibrosis in mice and fibrotic human livers. (a) Collagen-I, Desmin and ITGA11-stained liver sections from olive-oil-treated (control) and CCl4-treated (8 weeks, fibrotic) mice. n=5 per group. (b) Gene expression of fibrotic parameters (Col1a1, Col3a1, Tgfβ, Timp1, Mmp9 and Mmp13), myofibroblast activation markers (Acta2, Desmin and Pdgfβr) and Itga11 and Itgb1 in the livers of olive-oil-treated non-fibrotic control mice and CCl4-treated fibrotic mice (4 and 8 weeks). n=5 per group. #P<0.05 and ##P<0.01 versus control. (c) Correlative analysis of ITGA11 protein and gene expression with respect to the expression of fibrotic parameters (collagen-I and Desmin). The correlations were assessed using Pearson’s correlative analysis. ‘R2’ denotes Pearson’s correlation coefficient, and ‘P’ denotes statistical significance. (d) α-SMA- and ITGA11-stained human liver sections (n=4). Left, healthy human liver; middle, cirrhotic human liver; right, magnified image. Upper, ITGA11 (red); middle, α-SMA (green); bottom, merged image. Nuclei are stained blue with DAPI. (e) ITGA11 mRNA expression levels from publicly available transcriptome profiling data sets (www.ncbi.nlm.nih.gov/geo). Liver tissues affected with non-alcoholic fatty liver disease (NAFLD) stratified according to fibrosis stage: stage F0 or F1, mild fibrosis (n=40); stage F3 or F4, severe fibrosis (n=32) (GEO accession number: GSE49541).
Figure 2
Figure 2
Increased ITGA11 expression in human hepatic stellate cells following TGFβ treatment. (a) Collagen I-, α-SMA- and ITGA11 (ITGA11, red; DAPI, blue)-stained human HSCs treated with medium (control) or TGFβ (5 ng ml−1). (b) Western blotting and (c) analysis of ITGA11 and β-actin expression in control and TGFβ1-activated human HSCs, human hepatocytes (HepG2) and human monocytes (THP1). ‘nd’ denotes not detected. (d) Gene expression of fibrotic parameters (Col1α1, Acta2, desmin, vimentin, TIMP1 and PDGFβR) and ITGA11 and ITGB1 in control and TGFβ-activated human HSCs, n=4. *P<0.05 and **P<0.01 versus control HSCs.
Figure 3
Figure 3
ITGA11 depletion in human HSCs inhibits HSC activation. (a) Western blotting depicting ITGA11, Collagen-I and β-actin expression in control, TGFβ-treated HSCs and TGFβ-treated ITGA11-KD (ITGA11-knockdown) HSCs. (b) Collagen-I-, α-SMA- and vimentin-stained control and ITGA11-KD HSCs with or without TGFβ (5 ng ml−1). (c) Gene expression of ITGA11, fibrotic parameters (Collagen I, Acta2, vimentin and TIMP1), ECM-adhesion protein (Paxillin), ITGB1 and ITGA5 in control HSCs, TGFβ-treated scrambled HSCs and ITGA11-KD HSCs, n=4. #P<0.05 and ##P<0.01 versus control HSCs. *P<0.05 and **P<0.01 versus TGFβ-treated HSCs. Control cells were transfected with control/scrambled shRNA plasmid. ITGA11-KD cells were transfected with the ITGA11 shRNA plasmid.
Figure 4
Figure 4
ITGA11 depletion in HSCs inhibits phenotypic transformation of HSCs. (a) Percentage of wound closure (24 h) by control HSCs and ITGA11-KD HSCs under different conditions, n=4. #P<0.05, ##P<0.01 versus control or ITGA11-KD HSCs; *P<0.05. Representative images (b) and quantitative analysis (c) of 3D collagen-I gel contraction containing control versus ITGA11-KD HSCs treated with or without TGFβ (5 ng ml−1), n=4. ##P<0.01 versus control HSCs; **P<0.01 versus TGFβ-treated HSCs. (d) Percentage of cell viability of control HSCs and ITGA11-KD HSCs with or without TGFβ (5 ng ml−1) at different time points (0, 24 and 48 h) as assessed using the Alamar Blue assay. n=4. #P<0.05 versus control; *P<0.05 and **P<0.01 versus control or TGFβ-treated HSCs. (e) ITGA11-KD and control HSCs stained with phalloidin (red, first column), ITGA11 (green, second column); third column, merged image; fourth column, magnified merged image (depicting co-localization), and arrows depict the ITGA11/phalloidin co-stained protrusions. Nuclei are stained blue with DAPI. (f) AFM images depicting the adhesion of stained control HSCs and ITGA11-KD HSCs. The upper panel depicts microscopic images, the middle panel depicts AFM images and the bottom panel depicts the magnified edges. The right panel shows the height of cells (average±s.d., n=4) as measured by AFM. (g) Control (first column) and ITGA11-KD (second column) HSCs stained with vinculin (green, upper panel) and phalloidin (red, middle panel); lower panel, merged image. The third column shows the magnified vinculin-stained images. Nuclei are stained blue with DAPI. Control cells were transfected with control/scrambled shRNA plasmid. ITGA11-KD cells were transfected with the ITGA11 shRNA plasmid.
Figure 5
Figure 5
Myofibroblastic localization of Gli1 and the effect of hedgehog inhibition on TGFβ-activated human HSCs in vitro and human liver slices ex vivo. (a) Gene expression of hedgehog pathway-related genes (Shh, Ptch1, Smo, Gli1, Gli2 and Sox9) in ITGA11-KD HSCs versus control HSCs, n=3. **P<0.01 represents significance versus control HSCs. (b) Gene expression of hedgehog pathway-related genes (Sox9 and Gli1) in TGFβ-treated ITGA11-KD HSCs versus TGFβ-treated control HSCs, n=4. #P<0.05 and ##P<0.01 versus control HSCs (dashed line); *P<0.05 and **P<0.01 versus TGFβ-treated HSCs. (c) Gene expression of ITGA11 in control HSCs and ITGA11-KD HSCs with or without Shh (5 μg ml−1), n=3. #P<0.01 versus Shh-treated HSCs, **P<0.01 versus control HSCs. (d) α-SMA and Gli1 co-immuno-stained human liver sections. Gli1 (red, first column), α-SMA (green, second column) and merged image (third column). Fourth column shows the magnified image depicting co-localization. Nuclei are stained blue with DAPI. (e) Collagen-I- and vimentin-stained HSCs treated with or without TGFβ (5 ng ml−1)±10 μM of the hedgehog inhibitor (LDE225). Gene expression of fibrotic parameters collagen-I, α-SMA and vimentin in HSCs treated with medium alone, TGFβ (5 ng ml−1)±10 μM LDE225, n=3. #P<0.05, ##P<0.01 versus the control cells; *P<0.05 versus TGFβ-treated cells. (f) Graph depicts % 3D collagen-I gel contraction after 24, 48 and 72 h of treatment with or without TGFβ (5 ng ml−1)±10 μM LDE225, n=3. #P<0.05, ##P<0.01 versus control cells; *P<0.05 versus TGFβ-treated cells. Gene expression of Acta2, TIMP1, PDGFβR and Collagen-I (g) and ITGA11, Sox9, Gli1 and ITGA5 (h) in the slices obtained from fibrotic livers from human patients incubated with medium (control) or 10 or 15 μM LDE225. n=3 patients (3 slices each). *P<0.05 and **P<0.01 versus control group. Control cells were transfected with control/scrambled shRNA plasmid. ITGA11-KD cells were transfected with the ITGA11 shRNA plasmid.
Figure 6
Figure 6
Effect of hedgehog inhibition in an acute CCl4-induced liver injury mouse model. (a) Representative photomicrographs and quantitative analysis of collagen-I- and desmin-stained liver sections from normal (olive-oil-treated), vehicle-treated CCl4 and LDE225-treated CCl4 mice. (b) Gene expression in the livers of different treated groups, n=5 per group. #P<0.05 and ##P<0.01 versus the olive-oil-treated normal group; *P<0.05 versus CCl4-treated vehicle group. (c) The serum ALT levels of different treated groups. n=5 per group. ##P<0.01 versus olive-oil treated control group; P=0.06 versus CCl4-treated vehicle group.
Figure 7
Figure 7
Integrin alpha 11 (ITGA11) overexpression in the UUO model of kidney fibrosis in mice and in fibrotic human kidneys and fibrotic lung tissues. (a) Collagen-I-, α-SMA- and ITGA11-stained kidney sections obtained from sham- and 7 days UUO-operated mice (n=4 per group). Gene expression of fibrotic parameters (Col1A1, Acta2 and Col3A1) and ITGA11 in the kidneys from contralateral, 3-day and 7-day UUO-operated mice, n=4 per group. *P<0.05 and **P<0.01 versus contralateral kidneys. (b) α-SMA and ITGA11 co-immunostained human fibrotic kidney sections (n=4 per group). Upper, ITGA11 (red); middle, α-SMA (green); bottom, merged image. Nuclei are stained blue using DAPI. The lower panel depicts the magnified image. (c) The ITGA11 mRNA expression levels extracted from publicly available transcriptome profiling data sets (www.ncbi.nlm.nih.gov/geo). Kidney tissues stratified according to the IFTA score: 0, no fibrosis (n=16); 1, mild fibrosis (n=11); 2, moderate fibrosis (n=13); 3, severe fibrosis (n=8) (GSE25902), *P=0.03. (d) α-SMA and ITGA11 co-immunostained human lung sections (n=5 per group). Upper, ITGA11 (red); middle, α-SMA (green); bottom, merged image. Nuclei are stained blue using DAPI. (e) The ITGA11 mRNA expression levels were extracted from publicly available transcriptome profiling data sets (www.ncbi.nlm.nih.gov/geo). Lung tissues with idiopathic pulmonary fibrosis (n=123) compared with normal controls (n=96) (GSE47460). *P<0.001 versus normal lungs. (f) Heat map representation of the gene expression of ITGA11, Col1A1, Vimentin and TIMP1 in human lung slices. N1–N5 and F1–F5 denote normal and fibrotic samples, respectively.

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