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. 2002 May 13;157(4):657-63.
doi: 10.1083/jcb.200201049. Epub 2002 May 6.

The NH2-terminal peptide of alpha-smooth muscle actin inhibits force generation by the myofibroblast in vitro and in vivo

Boris Hinz  1 , Giulio Gabbiani, Christine Chaponnier
Affiliations

The NH2-terminal peptide of alpha-smooth muscle actin inhibits force generation by the myofibroblast in vitro and in vivo

Boris Hinz et al. J Cell Biol. .

Abstract

Myofibroblasts are specialized fibroblasts responsible for granulation tissue contraction and the soft tissue retractions occurring during fibrocontractive diseases. The marker of fibroblast-myofibroblast modulation is the neo expression of alpha-smooth muscle actin (alpha-SMA), the actin isoform typical of vascular smooth muscle cells that has been suggested to play an important role in myofibroblast force generation. Actin isoforms differ slightly in their NH2-terminal sequences; these conserved differences suggest different functions. When the NH2-terminal sequence of alpha-SMA Ac-EEED is delivered to cultured myofibroblast in the form of a fusion peptide (FP) with a cell penetrating sequence, it inhibits their contractile activity; moreover, upon topical administration in vivo it inhibits the contraction of rat wound granulation tissue. The NH2-terminal peptide of alpha-skeletal actin has no effect on myofibroblasts, whereas the NH2-terminal peptide of beta-cytoplasmic actin abolishes the immunofluorescence staining for this isoform without influencing alpha-SMA distribution and cell contraction. The FPs represent a new tool to better understand the specific functions of actin isoforms. Our findings support the crucial role of alpha-SMA in wound contraction. The alpha-SMA-FP will be useful for the understanding of the mechanisms of connective tissue remodeling; moreover, it furnishes the basis for a cytoskeleton-dependent preventive and/or therapeutic strategy for fibrocontractive pathological situations.

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Figures

Figure 1.
Figure 1.
Distribution of FPs in cultured LFs and their action on α-SMA immunostaining. SKA-Rh-FP (A, red) shows diffuse cytoplasmic distribution 30 min after application to the culture medium. At the same time, it does not affect the staining for α-SMA (B, green) and β-CA (C, blue) in stress fibers. Under the same conditions, SMA-Rh-FP localizes along stress fibers (D) and abolishes α-SMA staining (E) without changing the staining for β-CA (F). Bar, 50 μm.
Figure 2.
Figure 2.
Effect of the β-CA–FP on cultured LFs. Control LFs coexpress α-SMA (A, green) and β-CA (B, red) in stress fibers (C, yellow). When added to cultured LFs for 30 min, the β-CA–FP does not affect the staining for α-SMA (D) but abolishes staining for β-CA (E). Moreover, β-CA–FP induces the de novo formation of lamellar protrusions that are free of stress fibers (F, white line indicates putative cell edge before treatment). Bar, 50 μm.
Figure 3.
Figure 3.
SMA-FP inhibits the tension exerted by LFs on silicone substrates. (A) Untreated LFs produce wrinkles on deformable silicone substrates during 60 min recording. (B) Wrinkles decrease in number already 15 min after treatment with SMA-FP and completely disappear after 30 min (C). (D) 10 min after removal of the SMA-FP by repeated washing, LFs contract again followed by gradual wrinkle reformation after 30 (E) and 60 min (F). Bar, 50 μm. Also see the video available at http://www.jcb.org/cgi/content/full/jcb.200201049/DC1.
Figure 4.
Figure 4.
SMA-FP inhibits LF-mediated contraction of collagen lattices. Attached collagen lattices were treated with FPs for 30 min and released; their diameter, measured after another 30 min, was normalized to the diameter before release (equals % contraction). Compared with untreated control lattices, (ct) SKA-FP (SK) has no effect on lattice contraction, whereas SMA-FP (SM) reduces contraction dose dependently; washing out SMA-FP before release (W) reverses this effect. *p ≤ 0.01 and **p ≤ 0.001 compared with control.
Figure 5.
Figure 5.
Long term effect of SMA-FP on LF morphology and α-SMA immunostaining. LFs grown for 5 d in enriched serum-free medium exhibit a highly organized stress fiber apparatus that stains for α-SMA (A, green) and F-actin (B, phalloidin red). Administration of SMA-FP twice a day for 5 d almost completely abolishes staining for α-SMA (C) but does not change cell morphology and F-actin distribution (D). Bar, 50 μm.
Figure 6.
Figure 6.
SMA-FP decreases mRNA expression of collagen I and actin in cultured myofibroblasts. After treating cultured LFs with SMA-FP (3 μg/ml) for 1–5 d, Northern blot analysis shows a time-dependent decrease of collagen I mRNA, detected in two bands at 4.7 and 5.8 kb, and of α-SMA, detected at 1.7 kb. In both cases, the decrease is visible on the third day. Control LFs (ct) do not show changes in collagen I and α-SMA mRNA after similar incubation times.
Figure 7.
Figure 7.
SMA-FP inhibits granulation tissue strip contraction. Strips from 9-d-old granulation tissue were stimulated with ET-1, returned to resting tension by washing for 120 min, then treated with FPs for 60 min or left untreated and stimulated a second time with ET-1. Reversibility was tested by washing 120 min and stimulat-ing with ET-1 a third time. Treatment with SKA-FP (SK) does not change contraction compared with untreated strips (ct). SMA-FP (SM) significantly reduces strip contraction; washing out SMA-FP restores control contraction levels. **p ≤ 0.001 compared with control.
Figure 8.
Figure 8.
SMA-FP reduces in vivo wound contraction. (A) A representative full thickness wound on the rat dorsal region was subjected to mechanical tension by splinting; the frame was left in place for 10 d. The scab was removed 8 d after wounding, and wound tissue was treated with FPs in carrier gel or with carrier gel only. Treatment was repeated on the ninth and tenth day after wounding. (B) 24 h after splint removal, the wound treated with SKA-FP exhibits an important surface reduction comparable to that of untreated controls. (C) The wound treated with SMA-FP exhibits a significantly less important reduction. (D) Wound area was measured 6 and 24 h after splint removal and normalized to the initial wound area. Mean values were calculated using 20 animals per experimental condition. ct, carrier gel only; SK, SKA-FP; SM, SMA-FP. **p ≤ 0.001 compared with control.
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