Fig. 2.
Collagen XVIII variants contribute to the composition and development of WAT. (A) Percentages of the Lin−:CD29+:CD34+: Sca1+:CD24+ cell subpopulation, enriched in early adipocyte progenitors, in eWAT of 22- to 26-d-old male mice fed standard chow: WT (n = 11), Col18a1−/− (n = 6), Col18a1P1/P1 (n = 6), and Col18a1P2/P2 (n = 10). (B) Immunostaining of extracellular matrix of adipocytes and vasculature with anti–medium/long-18 (red) and anti-CD31 (green) antibodies, respectively. The yellow fluorescence indicates colocalization of medium/long collagen XVIII isoforms with CD31+ blood vessels. Samples from 12-wk-old mice fed a Western diet for 5 wk. (Scale bar, 100 µm.) (C) Western blots of eWAT cell homogenates from specific mice without (w/o) and with heparitinase I treatment. The distinct immunoreactive products in heparitinase I-treated samples indicate that medium/long collagen XVIII contains HS chains. Estimated sizes of medium and long collagen XVIII isoforms are respectively 156 and 182 kDa, but collagens run anomalously on SDS/PAGE. (D) Representative images of H&E-stained eWAT from specified male mice on standard chow at specified ages. (Scale bars, 100 µm.) (E) Mean adipocyte sizes of eWAT from age-matched WT and mutant mice reared on standard chow. All cells in two to five microscopic fields per sample were counted at each weekly time point. Error bars show SD calculated from ≥3 mice. *P < 0.05, **P < 0.01, ***P < 0.001.