Preferential secretion of collagen type 3 versus type 1 from adventitial fibroblasts stimulated by TGF-β/Smad3-treated medial smooth muscle cells

Abstract

Restenosis, or arterial lumen re-narrowing, occurs in 30-50% of the patients undergoing angioplasty. Adaptive remodeling is the compensatory enlargement of the vessel size, and has been reported to prevent the deleterious effects of restenosis. Our previous studies have shown that elevated transforming growth factor (TGF-β) and its signaling protein Smad3 in the media layer induce adaptive remodeling of angioplastied rat carotid artery accompanying an increase of total collagen in the adventitia. In order to gain insights into a possible role of collagen in Smad3-induced adaptive remodeling, here we have investigated a mechanism of cell-cell communication between medial smooth muscle cells (SMCs) and adventitial fibroblasts in regulating the secretion of two major collagen subtypes. We have identified a preferential collagen-3 versus collagen-1 secretion by adventitial fibroblasts following stimulation by the conditioned medium from the TGF-β1-treated/Smad3-expressing medial smooth muscle cells (SMCs), which contained higher levels of CTGF and IGF2 as compared to control medium. Treating the TGF-β/Smad3-stimulated SMCs with an siRNA to either CTGF or IGF2 reversed the effect of conditioned media on preferential collagen-3 secretion from fibroblasts. Moreover, recombinant CTGF and IGF2 together stimulated adventitial fibroblasts to preferentially secrete collagen-3 versus collagen-1. This is the first study to identify a preferential secretion of collagen-3 versus collagen-1 from adventitial fibroblasts as a result of TGF-β/Smad3 stimulation of medial SMCs, and that CTGF and IGF2 function together to mediate this signaling communication between the two cell types.

Fig. 1

Diagram of the in vitro experimental procedures to assess fibroblast collagen secretion in response to the stimulation of conditioned media from TGF-β1/Smad3-treated SMCs. TGF-β1 (5 ng) was added to Smad3-expressing medial SMCs that were previously starved for 24 h in the culture medium containing 0.5% serum. GFP-expressing SMCs served as control. After treatment for 48 h, conditioned media which contained factors produced by the TGF-β1/Smad3-treated SMCs, were collected, concentrated, and then added into the starved (for 24 h) rat aortic adventitial fibroblasts. Following a 72 h incubation, the quantity of CN-3 and CN-1 secreted from the fibroblasts (media)was assessed by either immunoblotting or ELISA. (CN, collagen).

Fig. 2. Measurement of differential secretion of CN-3 versus CN-1 from adventitial fibroblasts following stimulation with the conditioned media from TGF-β1/Smad3-treated SMCs

A. CN-3 and CN-1 secretion from adventitial fibroblasts. These experiments were conducted as described in Fig. 1. After stimulation with the SMC-conditioned media for 72 h, samples were collected from fibroblast culture media and analyzed for CN-3 (black) or CN-1 (white) by Western blotting, as shown in lanes 2, 4, 6, and 8. The control samples of SMC conditioned media were loaded in lanes 1, 3, 5, and 7, respectively, indicating absence of detectable collagen signal. Lanes 2 and 4 represent stimulation with the conditioned media from the GFP-expressing SMCs without or with TGF-β1, respectively. Lanes 6 and 8 represent stimulation with conditioned media from the Smad3-expressing SMCs without or with TGF-β1, respectively. The quantitated data normalized by β-actin loading control (see B) are presented in the bar graph. Each bar represents a mean±SEM (at least 3 independent experiments). *p>0.05, as compared to GFP control. B. Western blotting of β-actin as loading control. Shown are the cell lysate samples taken from the fibroblast cultures from which the media samples were analyzed for CN-3 and CN-1 secretion in A in lanes 2, 4, 6, and 8, respectively. C. Western blotting of Smad3. The representative blot shows overexpression of Smad3 in Smad3-expressing SMCs as compared to the control of GFP-expressing SMCs.

Fig. 3

Measurement of CTGF and IGF2 in the conditioned media of the Smad3-expressing and TGF-β1-treated SMCs. Conditioned media from the GFP-expressing SMCs without (Bar 1) or with (Bar 2) TGF-β1 treatment, or from the Smad3-expressing SMCs without (Bar 3) or with (Bar 4) TGF-β1 treatment, were analyzed for secretion of CTGF (A) or IGF2 (B) by ELISA. Each bar represents a mean±SEM (at least 3 independent experiments). *p>0.05, as compared to GFP control.

Fig. 4

Measurement of CN-3 and CN-1 secretion from adventitial fibroblasts stimulated with recombinant CTGF and/or IGF2. Recombinant CTGF (100 ng/ml) or recombinant IGF2 (300 ng/ml) were added either alone (lanes 2 and 3, respectively) or together (lane 4) into starved (for 24 h) fibroblasts, and incubated for 72 h. A solvent equivalent was added to the control (lane 1). Fibroblast culture media were then analyzed by Western blotting for secretion of CN-3 and CN-1. The data presented in A are normalized using β-actin loading control (see B). Each bar represents a mean±SEM (at least 3 independent experiments). *p>0.05, as compared to GFP control.

Fig. 5. Effect of CTGF or IGF2 siRNA applied to TGF-β/Smad3-treated SMCs on CN-3 versus CN-1 secretion from adventitial fibroblasts

A. Measurement of CN-3 and CN-1 by ELISA. Experiments were performed as depicted in Fig. 1, except that respective siRNAs (100 pmol, 6 h) were used to knock down IGF2 or CTGF expression in the Smad3-expressing SMCs. Scramble siRNA (100 pmol) was used as negative control. At 72 h following the addition of the SMC-conditioned media, samples were taken from the fibroblast cultures and analyzed for CN-3 (black) and CN-1 (white) by ELISA. Each bar represents a mean±SEM (at least 3 independent experiments). Different conditions are labeled by numbers (1–6) under the respective bars. **p>0.05, as compared to no TGF-β/Smad3. *p>0.05, as compared to respective maximum CN-3 or CN-1 change (TGF-β/Smad3+scramble siRNA). B. Western blotting of collagen. In addition to the ELISA measurements in A, CN-3 and CN-1 in the fibroblast media were also detected by immunobotting. Lanes 4, 5, and 6 correspond to the same conditions in A (4, 5, and 6), respectively. C. Knockdown of IGF2 and CTGF by their respective siRNA, as measured by ELISA. The conditions of scramble siRNA and IGF2 siRNA correspond to those in 4 and 5 in A, respectively; the conditions of scramble siRNA and CTGF siRNA correspond to those in 4 and 6 in A, respectively.

Fig. 6. Measurement of CN-3 and CN-1 in the injured rat carotid artery following periadventitial application of CTGF. Rat common carotid arteries were injured using a balloon catheter, and pluronic gel (200 μl) containing recombinant CTGF (CTGF, 80 ng) or PBS (Control) of equal volume was applied to the outside of arteries, as described in Methods [5]. Cross-sections of the arteries, harvested at 14 days post injury, were immunostained for either CN-1 or CN-3

A. Representative CN-1 and CN-3 immunostaining of cross-sections from control or CTGF-treated arteries. B. Quantification of adventitial CN-1 or CN-3 immunostaining. The effect of CTGF on adventitial CN-1 or CN-3 content is expressed as fold induction versus the control without CTGF treatment (n=6 rats, *p>0.05).