title: The crucial role of collagen-binding integrins in maintaining the mechanical properties of human scleral fibroblasts-seeded collagen matrix.
abstract: OBJECTIVE
  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.
authors.affiliation: Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, P.R. China.
authors.forename: Shoulong
authors.initials: S
authors.lastname: Hu
authors.forename: Dongmei
authors.initials: D
authors.lastname: Cui
authors.forename: Xiao
authors.initials: X
authors.lastname: Yang
authors.forename: Jianmin
authors.initials: J
authors.lastname: Hu
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Wan
authors.forename: Junwen
authors.initials: J
authors.lastname: Zeng
mesh_terms: D000293:Adolescent; D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D001696:Biomechanical Phenomena; D002448:Cell Adhesion / Q000187:drug effects; D018929:Cell Culture Techniques; D003094:Collagen / Q000378:metabolism*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005782:Gels / Q000378:metabolism; D006801:Humans; D039423:Integrin alpha1 / Q000235:genetics / Q000378:metabolism; D039222:Integrin alpha1beta1 / Q000235:genetics / Q000378:metabolism*; D039421:Integrin alpha2 / Q000235:genetics / Q000378:metabolism; D038982:Integrin alpha2beta1 / Q000235:genetics / Q000378:metabolism*; D019012:Integrin beta1 / Q000235:genetics / Q000378:metabolism; D018583:Pliability / Q000187:drug effects; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012590:Sclera / Q000378:metabolism*; D055815:Young Adult
chemical_list: D000906:Antibodies; D005136:Eye Proteins; D005782:Gels; D039423:Integrin alpha1; D039222:Integrin alpha1beta1; D039421:Integrin alpha2; D038982:Integrin alpha2beta1; D019012:Integrin beta1; D021122:Protein Subunits; D012333:RNA, Messenger; D003094:Collagen