Target-seeking antifibrotic compound enhances wound healing and suppresses scar formation in mice

Construction of Recombinant Decorins.

We produced a wound-directed recombinant decorin fusion protein, CAR–decorin, by adding a wound homing peptide, CAR (sequence CARSKNKDC), which recognizes angiogenic blood vessels in skin wounds and in other regenerating tissues, to an extended C terminus of human decorin (Fig. 1) (19). We also generated native decorin, human serum albumin with the CAR peptide (CAR–HSA), and decorin with an inactive CAR peptide variant (mCAR–DCN). We have previously shown that replacing two basic amino acids in the CAR sequence with neutral ones (CARSKNKDC mutated to CAQSNNKDC in mCAR) eliminates the wound homing activity (19). His-tagged fusion proteins were produced in a mammalian cell expression system. The yields of purified fusion proteins were 5–15 mg/L of cell culture medium, providing ample material for characterization and in vivo treatment studies.

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Fig. 1.

Cloning and production of recombinant fusion proteins. (A) Schematic representation of the CAR–decorin fusion protein showing the insertion of the CAR peptide sequence and a polyhistidine-tag C-terminal of full-length decorin. (B) Gel electrophoretic analysis of recombinant decorins and CAR–HSA. The recombinant proteins were expressed in mammalian cells, purified on a Ni-column, separated on gradient SDS/PAGE gels, and detected with a monoclonal anti–6-histidine tag antibody. The decorins migrate as sharp bands at 45 kDa with a smear above the sharp band. (C) Digestion of the recombinant decorins with chondroitinase ABC before electrophoresis removed the smear. Thus, the sharp bands correspond to the core proteins and the smear is caused by the chondroitin sulfate chain attached to most of the decorin molecules (7, 8, 22, 23).

In SDS gel electrophoresis, decorin and the decorin fusion proteins migrated as sharp bands at 45 kDa with a smear above the band (Fig. 1). The sharp bands correspond to the core proteins, and the smear is caused by heterogeneity in the chondroitin sulfate chain attached to most of the decorin molecules (23). Mass spectrometry confirmed protein identities as decorin (or HSA), and differential scanning calorimetry produced a sharp peak with a melting temperature of Tm = 49.3 °C, indicating native protein folding.

Activities of Decorin and CAR Peptide Are Retained in the Fusion Protein.

We tested the activity of the CAR homing motif in the decorin fusion protein in cultured Chinese hamster ovary cells (CHO-K). In line with the strong binding of the synthetic CAR peptide to CHO-K cells (ref. 19; Fig. S1), CAR–decorin bound to these cells more avidly than decorin (Fig. 2A, 4 h). Fig. 2A shows that the increased binding of CAR–decorin to the CHO cells was not seen when we used mutant CHO cells (pgsA-745) that lack the receptor for the CAR (19). Decorin inhibits CHO-K cell proliferation, which is dependent on autologous TGF-β production (7, 23), but CAR–decorin was active at 10-fold lower concentrations than decorin (Fig. S1B). Decorin also inhibits cell spreading (24), and CAR–decorin was more potent in this regard than decorin (Fig. 2A, 72 h). Thus, the CAR peptide imparts its binding specificity and other activities to the CAR–decorin fusion protein that make it more potent than decorin itself.

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Fig. 2.

Cell binding and inhibition of cell spreading and TGF-β–dependent cell proliferation by decorins. (A) The culture media of CHO-K and pgsA-745 cells were supplemented with 0.3 μg/mL of decorins daily for 4 h or 3 d. After washing, the decorins were detected with anti–his-tag antibody and FITC-conjugated secondary antibody (green). The CAR-targeted decorin bound to CHO-K cells more (4 h) and inhibited cell spreading more strongly (3 d) than unmodified decorin, whereas the two decorins show identical low binding to mutant pgsA-745 cells that lack the receptor for the CAR peptide (19). Cell nuclei were stained by DAPI (blue). (Scale bar, 50 μm.) (B) CHO-K cells were grown in the presence of TGF-β1, (C) TGF-β2, or (D) TGF-β3 (all at 7.5 ng/mL) and 0.3 μg/mL of decorins. Half of the medium was changed daily for 4 d (7). Error bars represent mean ± SD. Two to four separate experiments were performed in triplicate. CAR-targeted decorin was particularly potent in inhibiting TGF-β1– and -β2–driven cell proliferation (B and C, ***P ≤ 0.001 both compared with control and decorin; ANOVA).

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Fig. 3.

Homing of CAR–decorin in wound tissue. Mice with full thickness skin wounds received an i.v. injection of His-tagged fusion proteins on day 5 after wounding. After 4 h, the location of decorin was determined with an anti–His-tag antibody (brown). (A) The wounds of mice injected with nontargeted decorin (DCN) or mCAR–DCN were weakly positive, whereas strong staining was observed in CAR–DCN wounds. The accumulation of CAR–HSA in the wounds confirmed the ability of the CAR peptide to enhance targeting to the wounds. No staining was observed in normal skeletal muscle underlying the skin wounds of mice treated with any of the decorins (shown for CAR–DCN; CAR–DCN/muscle). No staining was seen in wound tissue when class-matched mouse IgG was substituted for the anti–6-histidine tag antibody (no ab). (Scale bar, 200 μm.) (B) Quantitative analysis of homing in skin wounds. The statistical significance was examined with ANOVA; *P < 0.05, **P ≤ 0.01, ***P < 0.001, n = 16 per group. The results are expressed as mean ± SD.

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Fig. 4.

Reduced scar formation during wound healing in mice treated with CAR–decorin. Mice with full thickness skin wounds received daily i.v. injections as indicated from day 3 after the wounding until day 14. Scars were harvested on day 21, and the cross-sectional area (A) and the width (diameter) (B) of the scars were quantified by examining two microscopic sections from each wound. The results are expressed as the average of the two values. There were seven animals, each with four wounds, in every treatment group. *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA. The results are expressed as mean ± SEM, n = 28.

Enhanced TGF-β1 Neutralizing Activity of CAR-Modified Decorin.

To determine whether CAR–decorin can inhibit proliferation induced by exogenously added TGF-β, we tested CHO-K cells cultured in the presence of the three isoforms of TGF-β. Decorin partially inhibited cell proliferation induced by the major scar-inducing isoform, TGF-β1, whereas CAR–decorin completely suppressed this TGF-β1 activity (Fig. 2B; P < 0.001). CAR–decorin was significantly more active than decorin used at 100-fold higher concentrations (Fig. S1C; P < 0.001). Neither protein affected TGF-β1–stimulated pgsA-745 cells (Fig. S1D). CAR–decorin also almost completely inhibited TGF-β2–induced CHO cell proliferation at a concentration at which decorin was inactive (Fig. 2C; P < 0.001). Remarkably, CAR–decorin had no effect on cell proliferation induced by TGF-β3 (Fig. 2D).

Systemic Delivery of Decorin to Wounds.

Decorin has been reported to home to angiogenic vasculature through a core protein-dependent interaction (25, 26), an effect possibly further enhanced by the binding of its glycosaminoglycan side chain to integrins (27). We found that adding the CAR peptide to decorin greatly enhanced the accumulation of i.v.-injected decorin in skin wounds in mice as detected by an anti–His-tag antibody (the recombinant proteins contained a His tag). We observed an almost fivefold stronger signal in the wound granulation tissue in mice that received CAR–decorin compared with nonmodified decorin (Fig. 3 and Fig. S2). The difference persisted even when a 2.5-fold higher dose of decorin was used (Fig. 3; P < 0.001), and after multiple daily injections (days 3–5) of the recombinant decorins had been given (Fig. S2). CAR–decorin also penetrated into granulation tissue more than nonmodified decorin (Fig. S2). The mCAR–decorin control protein accumulated in wounds to approximately the same extent as nonmodified decorin (Fig. 3). The normal tissues (skin and underlying muscle) around the skin wound were almost completely negative for decorin immunoreactivity regardless of which decorin protein was used. The accumulation of the CAR–decorin in wound tissue parallels the accumulation we have obtained in using the CAR peptide to deliver bacteriophage and fluorescein into wounds (19). The results show that homing peptide-enhanced delivery can markedly increase the concentration of decorin in wounds.

Wound Treatment with CAR-Modified Decorin.

Next, we examined the effect of the peptide-modified decorins on wound healing. I.v. administration of decorins was started on day 3 after wounding, when granulation tissue first starts to form in wounds (5). The treatment was continued for 7 or 11 d in three independent treatment experiments with 20 mice in each treatment group (n = 140). The daily dose of 40 μg/d (by tail vein injection) was chosen on the basis of previous decorin studies (8, 25, 26). The dose was doubled on days 4–6, coincident with the peak in TGF-β expression in wounds (5).

The 3-d delay in starting the treatment was designed to rule out the possibility that decorin would accumulate into the wounds by extravasation during the inflammatory phase and to preserve a window for the recruitment of inflammatory cells to the wounds (5). The influx of inflammatory cells into a wound contributes to subsequent scar formation (1–3, 28, 29), but inflammatory cells are also important in combating infection, particularly in humans (28). Furthermore, macrophages, especially the early influx, are crucial for all aspects of skin wound healing in mice (29–31), not just for the removal of necrotic material (28). Indeed, we found that the accumulation of macrophages and neutrophils in wounds treated with CAR–decorin did not differ from controls (Fig. S3). By limiting the duration of the therapy to the period when granulation tissue is formed, we left the early, TGF-β–induced accumulation of inflammatory cells untouched and preserved the beneficial effects of macrophages on subsequent tissue regeneration (29–31).

Daily inspection of the wounds (n = 560) showed no wound rupture during the treatment trials. Histological analysis of wounds at day 10 showed that the amount of granulation tissue was strikingly smaller in the groups that received CAR–decorin; the reduction was ≈50% relative to control groups (Figs. S4 and S5) at the 10-d timepoint. Nonmodified decorin, synthetic CAR peptide alone, CAR–HSA, or mCAR–decorin produced no statistically significant change in the amount of the granulation tissue compared with the vehicle control (Figs. S4 and S5). We also tested nonmodified decorin at a 2.5-fold higher dose (100/200 μg/d; DCN × 2.5) and saw no statistically significant change in wound size compared with the control wounds.

After the granulation tissue has formed, myofibroblasts cause contraction of the wound into scar tissue. A treatment trial ending at day 14 showed that the difference seen in the size of the granulation tissue by CAR–decorin treatment persisted through the wound contraction process (Figs. S4 and S5). Wound length (the longitudinal diameter of the wound) was also significantly decreased in groups receiving CAR–decorin (Fig. S4). Wound closure measurements indicated acceleration of closure in the animals that received CAR–decorin (Fig S6). The small differences to the controls became highly significant for CAR–decorin when combined results from three experiments were analyzed, and there may have been a slight effect by the nontargeted decorins as well in this assay (Table S1). A significantly shorter distance between the tips of the epithelial tongues was measured for the CAR–DCN treated wounds compared with control wounds in the histological analysis (Fig. S6B).

Reduction of Scar Formation by Wound-Targeted Decorin.

To determine whether the reduction seen in the granulation tissue and scar formation by CAR–decorin treatment persisted in fully healed wounds, we treated mice between days 3 and 14 after wounding and collected the scar tissue on day 21. The scars of CAR–decorin-treated mice were nearly 50% smaller than the scars in the other groups (P < 0.001, Fig. 4). The length of the scars (hyperproliferative epidermis) was also significantly decreased in the group that received CAR–decorin (Fig. 4). These results show that homing peptide-targeted decorin administered during wound healing can significantly reduce the size of the resultant scar.

Pharmacokinetics of Recombinant Decorins in Vivo.

The enhanced effect of CAR–decorin appears to be based on active targeting rather than other mechanisms, such as pharmacokinetic factors. The results described above show that, whereas CAR–HSA homes to granulation tissue (Fig. 3), it has no effect on the wounds (Fig. 4 and Fig. S4). This result excludes the possibility that prolonging the half-life of the CAR peptide in the circulation by its coupling to decorin would have rendered the peptide itself active in wound healing. Moreover, the various recombinant decorins used in this study had identical half-lives in the circulation of normal mice without wounds (Fig. S7), excluding the possibility that the activity of CAR–decorin would be due to unusual pharmacokinetics (other than the homing to wounds).

CAR–Decorin Inhibits the Expression of TGF-β–Induced Genes in Wounds.

TGF-β1 is likely to be an important target of decorin in wound healing, because TGF-β1 plays a key role in scar formation (1–5), and decorin inhibits TGF-β–dependent responses (4, 6–18). CAR–decorin inhibited gene expression of several TGF-β–induced genes that play a role in scar formation (32–35) by about 50% at day 5 after wounding when TGF-β activity peaks in wounds (5) (Fig. S8). The transformation of fibroblasts to α-SMA–positive myofibroblasts, which is a TGF-β–dependent process and responsible for converting granulation tissue to permanent scar tissue (36), was significantly reduced (Fig. 5A). This result is in agreement with previous data showing that decorin can block the TGF-β–induced transdifferentiation of myogenic cells to myofibroblasts (9). Connective tissue growth factor (CTGF/CCN2) is required for TGF-β–induced transdifferentiation of fibroblasts to myofibroblasts (32–34). CAR–decorin also reduced the expression of CCN2 mRNA and protein at various timepoints during granulation tissue formation (Fig. 5 and Fig. S8). As myofibroblasts are responsible for converting the granulation tissue into permanent scar (36), these data provide an explanation for the reduced scar formation we observe in the mice treated with the targeted decorin.

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Fig. 5.

α-SMA–positive fibroblasts (myofibroblasts) and connective tissue growth factor (CCN2/CTGF) in wounds treated with targeted decorin. Representative sections from wounds collected on day 10 after wounding are shown for α-SMA–positive myofibroblasts (A) and for CCN2/CTGF (B). A wound from a decorin-treated mouse was stained with class-matched mouse IgG as a specificity control (no ab). The wounds of mice treated with CAR–DCN showed diminished myofibroblast reaction and CCN2/CTGF protein expression in their skin wounds. The arrows indicate α-SMA–positive smooth muscle cells in the walls of blood vessels in CAR–DCN treated wound. Quantitative analysis of α-SMA–positive myofibroblasts (C) and CCN2/CTGF protein expression (D) in skin wounds at day 10. The statistical significance was examined with ANOVA, *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001. The results are expressed as mean ± SEM, n = 16. (Scale bars, 150 μm.) (C) PBS/BSA control treatment; CAR refers to the CAR peptide.