The crucial role of collagen-binding integrins in maintaining the mechanical properties of human scleral fibroblasts-seeded collagen matrix

Abstract

Purpose: The aim of this study was to identify the presence of collagen-binding integrin subunits in human scleral fibroblasts (HSFs) and investigate their actual functions in maintaining the mechanical creep properties of the HSFs-seeded collagen matrix.

Methods: Primary HSFs were cultured in vitro. Reverse- transcription PCR was used to detect mRNA expression of integrin α1, α2, and β1 subunits in HSFs. In addition, western blot analysis and immunofluorescence were used to detect their protein in HSFs. Monoclonal antibodies were applied directly against the extracellular domains of integrin subunits in HSFs cultured in the three-dimensional collagen gels to block the interaction between HSFs and the extracellular collagen matrix. The effects of anti-integrin antibodies on HSFs morphology in collagen gel were observed. The effects of the added antibodies on fibroblast-mediated collagen gels' contraction were evaluated. Furthermore, the changes in mechanical creep properties of collagen gel were measured by a biomechanics test instrument.

Results: The mRNA and protein expressions of collagen-binding integrin α1, α2, and β1 subunits were present in HSFs. The elongated bipolar cells converted to spherical shapes after 6 h after the addition of integrin α1β1 and α2β1 antibody. The blocking of integrin α1β1 and α2β1 subunits noticeably decreased the contraction in the collagen gels. In addition, all samples were subjected to a constantly applied load of 0.03 N for 600 s. The blocking of integrin α1β1 and α2β1 subunits also induced increases in the values of final extension, creep extension, and creep rate, compared to those of the controls (p<0.01). Furthermore, the creep elements were significantly increased with the augmentation of the integrin antibody dose (p<0.01). The final extension of the integrin α2β1 antibody (1 μg/ml or 4 μg/ml) group was significantly higher compared to that of the integrin α1β1 antibody (1 μg/ml or 4 μg/ml) group (p<0.01). However, the creep extension and creep rate of the integrin α2β1 antibody (1 μg/ml or 4 μg/ml) group were not significantly different from those in the integrin α1β1 antibody (1 μg/ml or 4 μg/ml) group (p>0.05).

Conclusions: Our findings suggested that HSF integrin α1β1 and α2β1 participated in maintaining the mechanical creep properties of the HSFs-seeded collagen matrix. Furthermore, integrin α2β1 might play a more crucial role in maintaining the mechanical creep properties of the collagen matrix than does integrin α1β1.

Figure 1

Identification of the collagen-binding integrins subtypes expressed in human scleral fibroblasts (HSFs). A: Amplification products representing the integrin α1 (280 bp), integrin α2 (226 bp) and integrin β1 (180 bp) subunits were detected in HSFs using reverse-transcription PCR. Molecular markers were included for product size comparison. B: The products representing the integrin α1 (130 kDa), integrin α2 (129 kDa), and integrin β1 (88 kDa) subunits were detected in HSFs using western blot analysis. C-K: Distribution of integrin α1, α2 and β1 in HSFs were observed by indirect immunofluorescence. FITC marked the secondary antibody (green; 1) and Hoechst33358 dyed the nucleus (blue; 2). The first (1) and second images (2) combined to form the third image (3). Integrin α1 (C-E), α2 (F-H), and β1 (I-K) were localized in the plasmalemma of HSFs.

Figure 2

Morphological changes caused by the addition of anti-human integrin antibodies to human scleral fibroblasts (HSFs) in the collagen gel lattice. Anti-human integrin antibody (at the concentration 4µg/ml, which was diluted with serum-free DMEM) was added dropping it onto an HSF-populated collagen gel. Morphological changes of fibroblasts were monitored after 6 h addition using light microscopy (original magnification 200×). A: mouse anti-human IgG (0.1 mg/ml) was used as a control; B: A mixture of anti-human integrin a1 (4 µg/ml) and anti-human integrin β1 (4 µg/ml) antibodies were added. C: A mixture of anti-human integrin a2 (4 µg/ml), and anti-human integrin β1 (4 µg/ml) antibodies were added.

Figure 3

Gel contraction was dose-dependent inhibited by anti-integrin α1β1 and α2β1 antibody. Human fibroblasts were incubated in the absence (mouse anti-human IgG as control) or presence of a combination of anti-human integrin α and anti-human integrin β1 antibodies at final concentrations of 1 µg/ml and 4 µg/ml for 3 days. A: Representative photomicrographs show collagen gel changes. B: Contraction was indicated as percentage of the initial gel surface area.

Figure 4

Anti-integrin a2β1 and anti-integrin a1β1 affect on mechanical creep properties of collagen matrix. A: Typical extension-versus-time behavior of the samples was shown, respectively. A constantly applied load of 0.03 N for 600 s was subjected to each sample. B: Anti-integrin a2β1 and anti-integrin a1β1 acted on the final extension of the HSF-populated collagen gel. C: Anti-integrin a2β1 and anti-integrin a1β1 acted on creep extension of the HSF-populated collagen gel. D: Anti-integrin a2β1 and anti-integrin a1β1 acted on the creep rate of the HSF-populated collagen gel. Results were expressed as mean±SEM *p<0.01 versus CON; ^#p<0.01 versus α2β1 (1 μg/ml) or α1β1 (1 μg/ml); †p<0.01 versus α1β1 (1 μg/ml) or α1β1 (4 μg/ml).