Platelets stimulate fibroblast-mediated contraction of collagen gels

Abstract

Background: Platelets are thought to play a role in a variety of inflammatory conditions in the lung, some of which may lead to fibrosis. In the current study we tested the hypothesis that whole platelets and platelet lysate can mediate remodelling of extracellular matrix in vitro by affecting fibroblast-mediated contraction of a collagen gel. We also sought to determine to what extent platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) contribute to this effect.

Methods: Washed platelets, isolated from healthy blood donors, and platelet lysate (freezing and thawing), were cast together with human lung fibroblasts in three-dimensional collagen gels. The gels were then released and cultured for four days. PDGF and TGF-beta1 concentrations were measured in culture supernatants by ELISA.

Results: Both platelets and platelet lysate augmented fibroblast-mediated gel contraction in a time and concentration dependent manner (19.9% +/- 0.1 (mean +/- SEM) of initial area vs. 48.0% +/- 0.4 at 48 hours; P < 0.001 and 41.5% +/- 0.6 vs. 60.6% +/- 0.3 at 48 hours; P < 0.001, respectively). Fixed platelets had no effect in the system. Both TGF-beta1 and PDGF-AA/AB were released in co-culture. PDGF-AA/AB had a maximum release at 24 hours whereas TGF-beta1 release increased with longer culture periods. Neutralising antibodies to these mediators partially inhibited platelet-induced gel contraction.

Conclusion: We conclude that platelets may promote remodelling of extracellular matrix in vitro and that PDGF and TGF-beta partially mediate this effect, also indicating a role for other mediators. The findings may be an important mechanism in regulating repair processes after injury.

Figure 1

Time dependent augmentation of fibroblast-mediated collagen gel contraction induced by platelets (Plt). Fibroblasts (HFL1: 3 × 10⁵/mL gel) and Plt (10⁸/mL gel) or the two cell types together (HFL1/Plt) were cast into a 24-well tissue culture plate. The assay was performed by releasing the gels into medium immediately after gelation. The area of each gel was measured by an image analyser on four consecutive days. Vertical axis: gel area expressed as percentage of original gel size. Horizontal axis: time (days). Data are presented as mean of triplicate. Standard errors of mean (± SEM) were less than 1%. P < 0.001: HFL1 vs. HFL1/Plt on day 2.

Figure 2

Concentration dependent augmentation of fibroblast-mediated collagen gel contraction. Increasing numbers of platelets were mixed together with fibroblasts in a three-dimensional collagen gel. After gelation, the gels were released and the area of the floating gels was measured after 48 hours of culture. Vertical axis: gel area expressed as percentage of original gel size. Horizontal axis: number of platelets added to gels. Data shown as mean of triplicate ± SEM. P < 0.001: 0 (HFL1) vs. 10⁷ at all data points.

Figure 3

Fixed platelets do not augment fibroblast-mediated collagen gel contraction. Fibroblasts, platelets, fixed platelets (PFA 10 min) or combinations of cell types were cast into collagen gels. The gels were released into medium and the area was measured on four consecutive days. Vertical axis: gel area expressed as percentage of original gel size. Horizontal axis: time (days). Data shown as mean of triplicate. ± SEM were less than 1%. P < 0.001: HFL1/Plt vs. HFL1/fix Plt at all data points.

Figure 4

Time and concentration dependent effect of platelet lysate on fibroblast mediated collagen gel contraction. Various dilutions of platelet lysate were mixed together with fibroblasts in collagen gels. After gelation, the gels were released and the area of the floating gels was measured after 2 (solid line) and 4 (dotted line) days. Vertical axis: gel area expressed as percentage of original gel size. Horizontal axis: dilutions of platelet lysate, control contain no lysate. Data presented as mean of triplicate. ± SEM were less than 1%. P < 0.001: HFL1 vs. platelet lysate at all data points.

Figure 5

Cell numbers in collagen gels estimated by DNA content. Floating collagen gels containing 300 000 fibroblasts/mL gel (HFL1), HFL1 and 10⁸ platelets/mL gel (HFL1/Plt) or fibroblasts and undiluted platelet lysate, (HFL1/Lys) were cultured for two days. The gels were then dissolved in collagenase and DNA contents in pellets were determined by a fluorometric assay. Control is collagen gels without cells or lysate. Vertical axis: Optical Density (OD) measured at wavelength 355/460 nm. Data shown as means ± SD (n = 6). ** P < 0.01, *** P < 0.001.

Figure 6

Fibroblast-mediated collagen gel contraction is dependent of cell concentration. Increasing number of fibroblasts (HFL1) were added to three-dimensional collagen gels. After gelation, the gels were released and the area of the floating gels was measured after 48 h of culture. Vertical axis: gel area expressed as percentage of original gel size. Horizontal axis: number of fibroblasts added to gels. Data shown as mean of gel triplicate and ± SEM.

Figure 7

Release of PDGF-AB and TGF-β₁ in co-culture. Fibroblasts (HFL1) or fibroblasts together with platelets (Plt), fixed platelets (fix Plt) or platelet lysate (Lys) were cast into collagen gels. After two days of culture, the media surrounding the gels were collected and analysed for a) PDGF-AB and b) TGF-β₁ (ELISA). Vertical axis: total concentration of PDGF-AB respectively TGF-β₁ (pg/mL). Data shown as mean of duplicates ± SD. *** P < 0.001.

Figure 8

Time-dependent release of PDGF-AA and -AB in fibroblast/platelet co-culture. Fibroblasts (HFL1) together with platelets (Plt) were cast into collagen gels. After 2, 12, 24 or 48 hours of culture, the medium surrounding the gels were collected and analysed for PDGF-AA and PDGF-AB by ELISA. Vertical axis: concentration of PDGF (pg/mL) in medium. Data shown as mean of duplicates ± SD.

Figure 9

Time-dependent release of TGF-β₁ in fibroblast/platelet co-culture. Fibroblasts (HFL1) or fibroblasts together with platelets (Plt) were cast into collagen gels. After 2, 12, 24 or 48 hours of culture, the medium surrounding the gels were collected and analysed for TGF-β₁ by ELISA. Vertical axis: concentration of TGF-β₁ (pg/mL) in medium. Data shown as mean of duplicates ± SD.

Figure 10

Antibodies to PDGF (αPDGF) and TGF-β (αTGF) partially block platelet-induced stimulation of fibroblast-mediated collagen gel contraction. Collagen gels containing fibroblasts (HFL1) or fibroblasts and platelets (Plt) with or without antibodies were prepared. After gelation, the gels were released into media containing either antibodies to PDGF, TGF-β or the antibodies together. After 24 hours of culture the gel area was measured by an image analyser. Vertical axis: gel area expressed as percentage of initial gel area. Data presented as mean of triplicates ± SEM. ** P <0.01, *** P < 0.001.