Deficiency of biglycan causes cardiac fibroblasts to differentiate into a myofibroblast phenotype

Abstract

Myocardial infarction (MI) is followed by extracellular matrix (ECM) remodeling, which is on the one hand required for the healing response and the formation of stable scar tissue. However, on the other hand, ECM remodeling can lead to fibrosis and decreased ventricular compliance. The small leucine-rich proteoglycan (SLRP), biglycan (bgn), has been shown to be critically involved in these processes. During post-infarct remodeling cardiac fibroblasts differentiate into myofibroblasts which are the main cell type mediating ECM remodeling. The aim of the present study was to characterize the role of bgn in modulating the phenotype of cardiac fibroblasts. Cardiac fibroblasts were isolated from hearts of wild-type (WT) versus bgn(-/0) mice. Phenotypic characterization of the bgn(-/0) fibroblasts revealed increased proliferation. Importantly, this phenotype of bgn(-/0) fibroblasts was abolished to the WT level by reconstitution of biglycan in the ECM. TGF-β receptor II expression and phosphorylation of SMAD2 were increased. Furthermore, indicative of a myofibroblast phenotype bgn(-/0) fibroblasts were characterized by increased α-smooth muscle actin (α-SMA) incorporated into stress fibers, increased formation of focal adhesions, and increased contraction of collagen gels. Administration of neutralizing antibodies to TGF-β reversed the pro-proliferative, myofibroblastic phenotype. In vivo post-MI α-SMA, TGF-β receptor II expression, and SMAD2 phosphorylation were markedly increased in bgn(-/0) mice. Collectively, the data suggest that bgn deficiency promotes myofibroblast differentiation and proliferation in vitro and in vivo likely due to increased responses to TGF-β and SMAD2 signaling.

FIGURE 1.

Lack of bgn causes increased proliferation of cardiac fibroblasts. A–D, morphology of WT (A and B) and bgn^−/0 cardiac fibroblasts (C and D). A and C, 200× and C and D, 400× magnification. E, DNA synthesis as measured by [H³]thymidine incorporation at 24 h in response to 5% FCS was increased in bgn^−/0 fibroblasts; n = 5 (WT), n = 4 (bgn^−/0). F, proliferation as determined by cell counting after 48 and 72 h under standard culture conditions (5% FCS) was increased in bgn^−/0 fibroblasts; n = 4 (48 h), n = 6 (72 h). G and H, effects of cell-free ECM derived from WT versus bgn^−/0 fibroblasts on proliferation of cardiac fibroblasts. Fibroblasts were plated on either plastic or WT fibroblast ECM (G) or bgn^−/0 fibroblasts-derived ECM (H) and grown for 48 h in 5% FCS; n = 4. I, cell proliferation as determined by cell counting in WT and bgn^−/0 fibroblasts on collagen matrix plus and minus purified human bgn, n = 4. J, lentiviral overexpression of bgn in bgn^−/0 fibroblasts inhibited proliferation to WT level; cell counts after 72 h; n = 4. K, stimulation with either PDGF-BB (10 ng/ml) or 10% serum (FCS) failed to stimulate DNA synthesis in bgn^−/0 fibroblasts after serum withdrawal; n = 12 (starved), n = 4 (PDGF), n = 6 (FCS). Data are presented as mean ± S.E.; * and #, p < 0.05; **, p < 0.01.

FIGURE 2.

Cardiac fibroblasts derived from bgn^−/0 mice displayed characteristic features of differentiated myofibroblasts. A and B, immunocytochemical staining of α-SMA in WT (A) and bgn^−/0 fibroblasts (B) at 200 × magnification. C and D, immunostaining of F-actin at 200 × magnification. E, collagen gel contraction by bgn^−/0 fibroblasts was increased after 24 h; n = 4. mRNA expression of α-SMA is elevated in bgn^−/0 fibroblasts, n = xy. G, α-SMA and HSP90 immunoblotting. Quantitative analysis was achieved by the ratio of α-SMA and HSP90 to control for loading, n = 5. H, mRNA expression of fibronectin ED-A fragment was significantly induced in bgn^−/0 fibroblasts; n = 4. Data are presented as mean ± S.E.; *, p < 0.05; **, p < 0.01.

FIGURE 3.

Increased focal adhesions and differential regulation of ECM-related genes in bgn^−/0 myofibroblasts. A and B, immunocytochemical analysis of paxillin revealed increased establishment of focal adhesions (FA) in bgn^−/0 fibroblasts. FA are pointed out by white arrows. C–F, immunoblotting and mRNA expression of paxillin and vinculin 24 h after plating in 5% FCS confirmed the results obtained by immunostaining, n = 3. G–K, mRNA expression of cell surface ECM receptor CD44, the ECM-modifying enzymes plod1 (H), plod2 (I), MMP3, and MMP13 24 h after plating in 5% FCS; n = 3 (PLOD1, 2); n = 4 (MMP3, 13). Data are presented as mean ± S.E.; *, p < 0.05.

FIGURE 4.

Increased expression of TGF-βRII in bgn^−/0 fibroblasts. A, TGF-β ELISA revealed lower levels of secreted TGF-β in medium conditioned by bgn^−/0 fibroblasts; n = 3. B, mRNA expression of TGF-β1 was not different between the two genotypes; n = 6. C, mRNA expression of TGF-βRII was significantly elevated in bgn^−/0 fibroblasts. D, immunoblotting of TGF-βRII; n = 5. E and F, immunocytochemical detection of TGF-βRII in primary cardiac fibroblasts in the first passage, representative images are shown of n = 3. The staining revealed increased TGF-βRII expression in bgn^−/0 fibroblasts. Analysis was performed 24 h after plating in 5% FCS. Data are presented as mean ± S.E.; *, p < 0.05.

FIGURE 5.

Reversion of myofibroblast phenotype by neutralizing endogenous TGF-β. A, cell growth in the presence of either non-immune mouse IgG (mIgG) as isotype control or neutralizing antibody to TGF-β -1, -2, -3 (anti-TGF-β) after 24 h in 5% FCS. Both antibodies were used at 0.5 μg/ml. Anti-TGF-β significantly inhibited proliferation of bgn^−/0 fibroblasts; n = 3. B, OSCR assay in the presence of mIgG or anti-TGF-β. Anti-TGFβ blocked enhanced collagen gel contraction by bgn^−/0 fibroblasts (black bars); n = 3. C–F, immunocytochemical staining of α-α-SMA of WT (C and D) and bgn^−/0 fibroblasts (E and F) treated with either mIgG (C and E) or anti-TGF-β neutralizing antibody (D and F). Anti-TGF-β reduced α-SMA-positive stress fibers in bgn^−/0 fibroblasts; 200× magnification; representative images of n = 3 experiments. G, immunoblotting confirmed the responsiveness of α-SMA to anti-TGFβ antibodies; n = 3. H and I, protein expression as evidenced by immunoblotting of paxillin (H) and TGF-βRII (I) were down-regulated by neutralization of TGF-β in bgn^−/0 fibroblasts as well. HSP90 served as loading control; n = 3. J–L, mRNA expression of α-SMA, paxillin, and the fibronectin fragment ED-A were analyzed by quantitative real-time PCR. α-SMA and fibronectin ED-A mRNA expression were increased in bgn^−/0 fibroblasts and were decreased by neutralizing TGF-β. Paxillin mRNA showed a similar trend; n = 4. Experiments were performed 24 h after plating in 5% FCS Data are presented as mean ± S.E.; *,^# p < 0.05.

FIGURE 6.

Increased phosphorylation of SMAD2 and ERK in bgn^−/0 fibroblasts. Cardiac fibroblasts were grown in 5% serum and Western blot analysis was performed 24 h after plating. A–C, immunoblotting and quantitative analysis of phosphorylated and total SMAD2 (A), SMAD3 (B), and ERK1/2 (C); n = 3. Quantitative data were expressed as the ratio between phosphorylated and total protein. D and E, SMAD7 and β-catenin expression were analyzed by Western blot and HSP90 protein was used as loading control for quantitative analysis; n = 3. F, PARP1 immunoblotting revealed increased PARP cleavage in bgn^−/0 fibroblasts but not in WT. GAPDH was used as loading control; n = 9. Data are presented as mean ± S.E.; *, p < 0.05.

FIGURE 7.

Increased mRNA expression of α-SMA and myofibroblast-associated genes after experimental MI in bgn^−/0 mice. Total RNA was isolated from the apex post-MI and analyzed for α-SMA, TGF-β1, TGF-βRII, fibronectin ED-A, SMAD7, CD44, and paxillin. mRNA expression as determined by quantitative real time RT-PCR and compared with results from sham-operated mice. A, α-SMA mRNA expression 3, 7, and 21 days postexperimental MI; n = 7(3 d), n = 4(7 d), n = 9 (21 d). B–G, mRNA expression of TGF-β1, TGF-βRII, fibronectin ED-A, SMAD7, CD44, and paxillin 3 days post-MI. Data are expressed as mean ± S.E.; *, p < 0.05; n = 5.

FIGURE 8.

TGF-βRII protein expression and SMAD2 phosphorylation were increased post-MI in bgn^−/0 mice. Immunoblotting using left ventricular tissue extracts 3 days post-MI. A, Western blotting of α-SMA in tissue extracts from infarcted ventricles confirmed increased α-SMA expression in bgn^−/0 mice. B, TGF-βRII protein expression. C, increased ratio of phosphorylated SMAD2 to total SMAD2 in bgn^−/0 hearts. D, ratio of phosphorylated ERK to total ERK1/2 revealed only a trend toward increased phosphorylation of ERK1/2; in A–D, n = 5. E and F, immunostaining of α-SMA 7 days after experimental MI. Shown are representative images of the peri-infarct zone confirming up-regulated α-SMA expression in bgn^−/0 mice; n = 4. G, quantification of α-SMA immunohistochemistry as in D and E was performed using ImageJ (NIH). Data are expressed as mean ± S.E.; *, p < 0.05.