Figure 4.

Obesity-associated extracellular matrix (ECM) regulates macrophage morphology and proliferation in vitro. A: Representative photomicrographs illustrating the distribution of human adipose tissue interstitial ECM in between adipocytes via hematoxylin and eosin (H&E; top panel) and visualizing fibronectin (Fn) and collagen, two key components of interstitial fibrosis, by immunohistochemistry (IHC; middle panel) and second harmonic generation (SHG) imaging (bottom panel), respectively. B: Schematic illustrating protocol for adipose stromal cell (ASC) isolation from the inguinal fat pad of lean wild-type and obese (ob/ob) age-matched mice and their use for preparation of decellularized ECMs. C: Representative confocal microscopy images of decellularized ECM assembled by lean and obese ASCs after immunostaining for Fn. D–F: Bone marrow–derived macrophages (BMDMs) cultured on lean and obese decellularized matrices (dashed lines highlight the long axis of the macrophages; D) are more aligned with the matrix (0 on x axis represents perfect alignment; E) and exhibit increased elongation (F), as determined by confocal microscopy and image analysis (top 25% of cells plotted) after stimulation with IL-4 and IL-13. F:U-test was performed. G: BMDMs cultured on obese decellularized ECM are more proliferative, as determined by bromodeoxyuridine (BrdU) incorporation with immunofluorescence image analysis. A t-test analysis was performed. Data are expressed as median and interquartile range (F) or means ± SD (G). n = 3 independent experiments with 10 images per condition per experiment (D and E); n = 3 independent experiments with 30 representative images per condition per experiment (G). ***P < 0.001. Scale bars: 50 μm (A and C); 25 μm (D and G).