Hypoxia regulates the expression of extracellular matrix associated proteins in equine dermal fibroblasts via HIF1

Proliferation of EDF in normoxia and hypoxia. Cell proliferation measured using Trypan blue exclusion and counting. (A) Graph showing cell division (log base 2 of cell count) as a function of time (hypoxic chamber calibrated at 1% O₂). N=3 for each group. Symbol: ♢, limb EDF in normoxia; □, body EDF in normoxia; ▵, limb EDF in hypoxia; and ×, body EDF in hypoxia. (B) Table compiling proliferation rate (linear regression of cell division as a function of time) and coefficient of determination (R²) for the four groups (mean±SEM; **statistically significant difference between normoxia and hypoxia, limb p<0.0001 and body p=0.0002).

Cleaved caspase-3 (CASP3) protein expression in EDF cultured in CoCl₂ hypoxia. Western blot analysis of CASP3 in cell lysate total protein extraction. α-Tubulin was used as a loading control protein. (A) Histogram compiling results (mean±SEM; *tendency p<0.05; non significant after Bonferroni correction). Results are compared to normoxia values (time 0) and data are presented as fold-increase. Grey columns represent body EDF values and black columns represent limb EDF values. N=4 for each group at each time. (B) Western blot bands of CASP3 and α-tubulin for limb EDF.

Vascular endothelial growth factor A (VEGF A) and solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1) mRNA expression in EDF exposed to 6h treatment of CoCl₂ hypoxia, with or without echinomycin. qRT-PCR analysis of (A) VEGF A and (B) SLC2A1 in cell lysate RNA extraction. b-Actine (ACTB) used as a housekeeping gene. Histogram compiling results (mean±SEM). N=2 for each group.

Precursor collagen α1 type I (COL1A1) and matrix metalloproteinase 2 (MMP2) protein expression in EDF cultured in CoCl₂ hypoxia. Western blot analysis of (A) COL1A1 or (B) MMP2 in cell lysate total protein extraction. α-Tubulin used as a loading control protein. Histogram compiling results (mean±SEM; **statistically significant difference between normoxia and hypoxia p<0.005, *tendency p<0.05; non significant after Bonferroni correction). Results are compared to normoxia values (time 0) and data are presented as fold-increase. Grey columns represent body EDF values and black columns represent limb EDF values. N=4 for each group at each time. Western blot bands of (C) COL1A1 or (D) MMP2 and α-tubulin for limb EDF.

Precursor collagen α1 type I (COL1A1) and matrix metalloproteinase 2 (MMP2) protein expression in EDF exposed to 6h treatment of CoCl₂ hypoxia, with or without echinomycin. Western blot analysis of (A) COL1A1 or (B) MMP2 in cell lysate total protein extractions. α-Tubulin used as a loading control protein. Histogram compiling results (mean±SEM; **statistically significant difference between treatments: COL1A1 p<0.01; MMP2 p<0.03). Results are normalized to normoxic values and data are presented as fold-increase. N=7 for normoxia and hypoxia; N=3 for hypoxia+echinomycin.

Col1A1 and MMP2 under HIF1A regulation. Results presented show Col1A1 and MMP2 regulated by HIF1A in equine dermal fibroblasts.