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Thrombin-activated platelets induce proliferation of human skin fibroblasts by stimulating autocrine production of insulin-like growth factor-1

Ferdinando Giacco^*, Giuseppe Perruolo^*, Elio D’Agostino^*, Giorgio Fratellanza^*, Enzo Perna^*, Saverio Misso^*, Gennaro Saldalamacchia, Francesco Oriente^*, Francesca Fiory^*, Claudia Miele^*, Salvatore Formisano^*, Francesco Beguinot^* and Pietro Formisano^*,1

^* Dipartimento di Biologia e Patologia Cellulare e Molecolare and Istituto di Endocrinologia ed Oncologia Sperimentale del CNR; and

Dipartimento di Medicina Clinica e Sperimentale, Università di Napoli "Federico II", Napoli, Italy

¹Correspondence: Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano" Università di Napoli "Federico II" Via Pansini 5, 80131 Napoli, Italy. E-mail: fpietro{at}unina.it

SPECIFIC AIM

This work aims to elucidate the molecular mechanisms elicited by factors released by activated platelets in wound healing processes in vivo and in cultured cell systems. In particular, we have investigated the molecular pathways regulating growth-related events induced by thrombin-activated platelets in the healing of ulcerative lesions of diabetic patients and in cultured human fibroblasts.

PRINCIPAL FINDINGS

1. Application of thrombin-activated platelets to ulcerative lesions in diabetic patients induce activation of Akt/PKB and ERK1/2
Thrombin-activated platelets (TAPs) are commonly used in the treatment of ulcerative skin lesions and in other processes requiring wound healing and tissue regeneration. We have found that, when applied to diabetic foot lesions, TAPs induce the activation of Akt/PKB and ERK1/2. This observation derives from Western blot analysis with phospho-specific Akt/PKB and ERK1/2 antibodies of bioptical specimens obtained by seven consecutive patients with II/III grade ulcers, treated with TAPs for al least 1 wk.

2. Thrombin-activated platelets induce proliferation of cultured human fibroblasts
To analyze the molecular details of TAPs-induced cell growth, cultured human fibroblasts have been treated with TAPs as a serum substitute (Fig. 1 ). Application of TAPs to the fibroblasts increased the cell number in a manner comparable to that obtained in the presence of 10% FBS. This increase in cell growth was dependent on platelet number and accompanied by increased DNA synthesis, as assessed by thymidine incorporation experiments.

3. Thrombin-activated platelets activate Akt/PKB and ERK1/2 with different kinetics
Treatment of human fibroblasts with TAPs rapidly increased phosphorylation and activity of both Akt/PKB and ERK1/2. However, while TAPs-induced Akt/PKB activation reached an early plateau, ERK activity progressively increased up to 48 h in the cultured fibroblasts. Interestingly, a raising profile of ERK activation, but not of Akt/PKB, was also displayed in vivo, in the protein extracts of the sequential peri-lesional biopsies obtained by TAPs-treated diabetic patients. Moreover, both Akt/PKB and ERK1/2 activity are needed to propagate the growth effect, since treatment of fibroblasts with LY294002 and PD98059, which inhibit PI3K/PKB and MEK/ERK pathways, respectively, drastically reduced thymidine incorporation in response to TAPs.

4. TAPs-induced proliferation of human fibroblasts requires platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1 signaling
Two lines of evidence indicate that PDGF and IGF-1 largely mediate the TAPs effect on cell growth. First, detectable amounts of both PDGF and IGF-1 have been found in the platelet releasates and in media of TAPs-treated fibroblasts and are sufficient to induce the activation of their own receptors. Second, selective inhibition of PDGF receptor tyrosine kinase activity by AG1296 and of IGF-1 receptor by AG1024, reduced TAPs-stimulated thymidine incorporation.

5. Autocrine production of IGF-1 by human fibroblasts is required for sustained ERK1/2 activation and proliferation
PDGF levels were well detectable in media incubated with TAPs alone. However, PDGF concentration was slightly lower in conditioned media following coincubation with TAPs and fibroblasts. This finding is consistent with PDGF consumption due to the utilization by the targeted fibroblasts. At variance, IGF-1 concentration was relatively low in TAPs alone and progressively increased in TAPs + fibroblasts conditioned media. Treatment of the recipient fibroblasts with the PDGF-R blocker AG1296 slightly inhibited ERK1/2 and strongly reduced Akt/PKB, while AG1024 selectively reduced ERK1/2 activity induced by the TAPs + fibroblast-conditioned media. Moreover, TAPs application onto NIH-3T3 cells overexpressing a dominant-negative mutant of the IGF-1R failed to induce sustained ERK1/2 activation. This was accompanied by reduced TAPs stimulation of cell growth (Fig. 2 ).

CONCLUSIONS AND SIGNIFICANCE

Platelet components have been successfully used in orthopedic and maxillo-facial surgery for bone reconstruction and soft tissue regeneration. According to recent recommendations and our own previous investigation, treatment with autologous platelets also represents an important therapeutic tool for diabetic patients with ulcers at the lower extremities (diabetic foot). We sought therefore to investigate whether activated platelets could induce a cellular growth response when applied to ulcerative skin lesions of diabetic individuals. We found that the growth-related molecules Akt/PKB and ERK1/2 were activated in the peri-lesional skin following TAPs applications. We have then explored the molecular mechanisms responsible for the beneficial effect of platelets in wound healing processes. To this end, a method was devised to analyze the effect of TAPs in cultured cells. Aliquots of TAPs were applied to monolayers of cultured cells as serum substitute. Interestingly, the addition of TAPs to human fibroblasts induced cell growth in a fashion comparable with 10% FBS. This novel observation supports the possible utilization of human platelet factors as a substitute for animal serum for cell-based therapeutic applications. The effect of TAPs, however, was cell-specific, as we failed to observe a similar growth induction in human umbilical vein endothelial cells.

We have also shown that, in the human fibroblasts, the activities of Akt/PKB and ERK1/2 were required to elicit TAPs-stimulated growth effect. However, while the platelet released PDGF preferentially regulates Akt/PKB activity, IGF-1 signaling is necessary for the sustained activation of ERK1/2. Indeed, inhibition of IGF-1 signaling by Ag1024 and expression of a dominant negative IGF-1R mutant selectively reduced the stimulation of ERK1/2 by TAPs and fibroblast-released factors, with minor changes of Akt/PKB activity. Moreover, IGF-1 is detected at levels higher then expected in media coincubated with TAPs and fibroblasts, compared to those incubated with TAPs or fibroblasts alone. These observations are consistent with the hypothesis that platelet factors, including PDGF, can activate Akt/PKB and, at lesser extent, ERK1/2 and induce the release of IGF-1 by the fibroblasts. IGF-1, in turn may participate in an autocrine loop to sustain the activation of ERK1/2, and to elicit DNA synthesis and fibroblast proliferation (Fig. 3) . The combined action of PDGF and IGF-1 may be therefore responsible for a large fraction of the growth effect of TAPs on human fibroblasts and implicated in the repair mechanisms involving connective tissue.

Thus, to our knowledge, this is the first report investigating the molecular mechanisms by which the local application of activated platelets can induce growth effects in wound healing processes. Further elucidation of such mechanisms may lead to the identification of novel and more effective therapeutic strategies for the treatment of the diabetic foot as well as of other degenerative disorders requiring tissue reconstruction.

View larger version (13K): [in this window] [in a new window]   Figure 1. Effect of TAPs on cell growth in cultured human fibroblasts. A) Cells (105) were plated and supplied with complete medium (10% BS) or serum-free media or were serum-free (–). Where indicated, a 1 cm2 aliquot of TAPs (TAPs 1=1.0x109; TAPs 2=0.75x109; TAPs 3=0.5x109; TAPs 4=0.2x109), obtained as described in Materials and Methods, has been added to the serum-free medium. At the indicated times, the cells were trypsinized and counted. Data represent mean ± SD of six independent experiments in duplicate. B) Alternatively, equal amounts of cells were placed in serum-free media (–) and were supplied with thrombin-treated platelet-poor plasma (T-PPP), thrombin-activated platelets (TAPs; 1.0x109), 10% human serum and thrombin (T; 0.5 NIH U), as indicated. At the indicated times, the cells were trypsinized and counted. Data represent means ± SD of three independent experiments in duplicate. C) Six-well plates were seeded with 105 cells/plate in 1 ml complete medium. After incubation for 24 h at 37°C, the medium was removed and replaced with Dulbecco’s modified Eagle medium (DMEM) containing 0.25% BSA and no serum. After an additional 24 h, the medium was removed again and replaced with complete DMEM, or DMEM 0.25% albumin with or without TAPs. Incubation was prolonged for additional 16 h, and the incubation media replaced with the same media supplemented with [3H]thymidine (500 nCi/ml). After 1 h, media were removed and thymidine incorporation was measured as described in Materials and Methods. The bar graph represents means ± SD of three independent experiments in triplicate.

View larger version (13K): [in this window] [in a new window]   Figure 2. Effect of TAPs on IGF-1R defective cells. Thrombin-activated platelets have been applied to parental NIH-3T3 cells and to clones stably overexpressing wt IGF-1R or IGF-1R-DN. A) Cells have been detached and counted. B, C) Alternatively, at the indicated times, cells have been solubilized and the activity of Akt/PKB and ERK1/2 determined. The graphs represent the means ± SD of three independent experiments in triplicate.

View larger version (16K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6104fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6104fjev1 20/13/2402    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Giacco, F. Articles by Formisano, P. Search for Related Content PubMed PubMed Citation Articles by Giacco, F. Articles by Formisano, P.