Fibronectin extra domain-A promotes hepatic stellate cell motility but not differentiation into myofibroblasts

Abstract

Background & aims: Myofibroblasts are the primary cell type involved in physiologic wound healing and its pathologic counterpart, fibrosis. Cellular fibronectin that contains the alternatively spliced extra domain A (EIIIA) is up-regulated during these processes and is believed to promote myofibroblast differentiation. We sought to determine the requirement for EIIIA in fibrosis and differentiation of myofibroblasts in rodent livers.

Methods: We used a mechanically tunable hydrogel cell culture system to study differentiation of primary hepatic stellate cells and portal fibroblasts from rats into myofibroblasts. Liver fibrosis was induced in mice by bile duct ligation or administration of thioacetamide.

Results: EIIIA was not required for differentiation of rat hepatic stellate cells or portal fibroblasts into fibrogenic myofibroblasts. Instead, hepatic stellate cells cultured on EIIIA-containing cellular fibronectin formed increased numbers of lamellipodia; their random motility and chemotaxis also increased. These increases required the receptor for EIIIA, the integrin α(9)β(1). In contrast, the motility of portal fibroblasts did not increase on EIIIA, and these cells expressed little α(9)β(1). Male EIIIA(-/-) mice were modestly protected from thioacetamide-induced fibrosis, which requires motile hepatic stellate cells, but not from bile duct ligation-induced fibrosis, in which portal fibroblasts are more important. Notably, myofibroblasts developed during induction of fibrosis with either thioacetamide or bile duct ligation in EIIIA(-/-) mice.

Conclusions: EIIIA is dispensable for differentiation of hepatic stellate cells and portal fibroblasts to myofibroblasts, both in culture and in mouse models of fibrosis. Our findings, however, indicate a role for EIIIA in promoting stellate cell motility and parenchymal liver fibrosis.

Figure 1. EIIIA is not required for myofibroblast differentiation of hepatic stellate cells or portal fibroblasts

A. Stellate cells were cultured on pFN-coated or cFN-coated gels for 7 days. qRT-PCR for αSMA (Acta2) on 0.4–12 kPa gels. Error bars represent SD from 2 experiments. B. qRT-PCR on 12 kPa gels. Col1a2 = collagen I, Col1a3 = collagen III, Col4a1 = collagen IV, Tnc = tenascin C, and EIIIB/FN1 = ratio of EIIIB+ cFN to total fibronectin. Error bars represent SEM from 4 experiments. p values from a paired t-test are indicated. C. Stellate cells were cultured on pFN-coated or cFN-coated gels for 7 days then stained for αSMA (red) and DAPI (blue). Scale is the same as in (D). D. Portal fibroblasts were cultured on 12 kPa hydrogels coated with pFN or cFN in the presence or absence of TGF-β1 and a TβR1 inhibitor. Cells were stained for αSMA and DAPI (blue). Scale bar = 50 μm.

Figure 2. Hepatic stellate cells cultured on cFN have a motile morphology

Stellate cells were cultured on pFN-coated or cFN-coated gels for 7 days. A. Cells on 12 kPa gels stained for αSMA. Scale bar = 20 μm. B. Cell circularity was quantified (circularity = 4πA/P², where A = cell area and P = perimeter). n = 18–23 cells per condition from 2 experiments. C. Stellate cells on 12 kPa gels stained for vinculin. Scale is the same as in (A). D. Focal adhesion length was calculated using NIH ImageJ. N = 125–150 focal adhesions per cell from 3 representative cells per matrix.

Figure 3. Hepatic stellate cells cultured on cFN are more motile

Cells were cultured on pFN-coated or cFN-coated 12 kPA gels for 7 days, followed by time-lapse microscopy performed over 6 hours. A–C. Cells were outlined on still frames taken at 40 minute intervals. Area, perimeter, and circularity were calculated, and the average percent change in each parameter from one frame to the next is graphed. N = 15 cells per matrix. D–E. Nuclear coordinates were traced. Each line represents the path of an individual cell. N = 15 cells per matrix. F. The average total path length per cell was calculated.

Figure 4. Hepatic stellate cells but not portal fibroblasts cultured on cFN have increased chemotaxis

A. Stellate cells were plated on transwell inserts coated with pFN or cFN. After 19 hours, cells were stained with calcein a.m. and visualized (10× magnification). B. Mean fluorescence intensity was measured. p-values were calculated with a paired t-test from 8 experiments. C. Inserts were coated with pFN or cFN, then incubated 1 hour with antibodies before plating cells. Results represent 5 experiments. D. Portal fibroblasts were plated on pFN or cFN-coated inserts. After 19 hours, cells were stained, and mean fluorescence intensity was measured. Results represent 3 experiments. E. Inserts were coated with pFN or cFN, then incubated 1 hour with antibodies before plating cells. Results represent 3 experiments.

Figure 5. α₉β₁ is required for increased stellate cell chemotaxis on cFN

A–C. Stellate cells were cultured for 4 days then transduced with an adenovirus expressing either an integrin-targeting shRNA or a scrambled (scr) control. Transwell chemotaxis assay was performed 72 hours post-transduction. D–F. Cells were pre-incubated with blocking antibodies against α₉β₁, α₅β₁, or α_vβ₃ for 1 hour prior to beginning the assay. Error bars represent SEM from 3–4 experiments.

Figure 6. Male EIIIA^−/− mice are protected from thioacetamide-induced fibrosis

Mice were administered 200 mg/L thioacetamide for 12 weeks. n = 13 H₂O control mice (7 male, 6 female), 11 EIIIA^−/− mice (6 male, 5 female), and 12 wild type mice (6 male, 6 female). A. αSMA (Act2a) was measured by qRT-PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and beta 2 microglobulin (B2M). B. Liver sections were stained for αSMA (brown) and with hematoxylin (blue). Scale bar = 100 μm. C. Sections were stained with sirius red. Fibrosis was scored by a blinded observer on a scale of 0 (normal) to 4 (cirrhosis). Mean fibrosis score is indicated in the bottom right corner of each photo. D–E. Hydroxyproline content was assayed and reported as relative (D) or total hydroxyproline (E). F. ECM transcripts were measured by qRT-PCR. Col1a2 = collagen I, Col3a1 = collagen III.

Figure 7. EIIIA^−/− and wild type mice develop equivalent fibrosis after bile duct ligation

Mice were euthanized 14 days post-bile duct ligation. Depending on the experiment, n = 6–16 sham-operated animals (up to 10 male, 6 female), 12 EIIIA^−/− mice (7 male, 5 female), and 13 wild type mice (7 male, 6 female). A. Liver sections were stained for αSMA (brown) and with hematoxylin (blue). Scale bar = 200 μm. B. αSMA transcript (Act2a) was measured by qRT-PCR and normalized to expression of 18s ribosomal rRNA and glucuronidase B (Gusb). C. Sections were stained with sirius red. Mean fibrosis score is indicated in the bottom right corner of each photo. D–E. Hydroxyproline content was assayed and reported as relative (D) or total hydroxyproline (E). F. Transcripts were measured by qRT-PCR. Col1a2 = collagen I and Col3a1 = collagen III. *, ** = significant over sham control.