Fig 2: Combined blockade of TNFa, CCL2, and CXCL2 reduces atherogenesis after visceral fat transplantation from aged donors to Ldlr -/- recipients. (a) Representative images of H&E or Mac2 stained right carotid artery, aortic root and BCA sections of Ldlr -/- mice that received sham surgery or visceral fat transplants from IgG control (Donor-IgG) or TNFa, CCL2, and CXCL2 blockade (Donor-B) treated aged donor mice. (b–d) The quantifications of lesion area, necrotic area, Mac2 positive area and the percentage of Mac2 positive area in each section of (b) right carotid artery, (c) aortic root and (d) BCA are shown (n = 7-8/group). Note: no necrosis was observed in carotid artery. Results are presented as means ± SEM. Two-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. p values indicate the main effect between groups of Donor-IgG vs. Donor-B.

Fig 3: Macrophages are major contributors to the inflammatory profile in visceral adipose tissue with aging. Aged mice were treated with control liposome or liposome-encapsulated clodronate by intraperitoneal injection, and then the macrophage depletion efficiency in visceral fat (GWAT) was determined, as well as the secretion of inflammatory cytokines and chemokines. (a) Representative FACS contour plots of macrophages (F4/80+CD11b+) in visceral fat, and the numbers of macrophages per milligram tissue are shown (n = 9-10/group). (b) Representative images of Mac2-stained visceral fat sections are shown, and the percentage of Mac2 positive areas was quantified and is shown on the right (n = 8/group). (c) Levels of TNFa, IL-6, IL-1ß, CCL2, CCL3, and CXCL2 in the tissue culture medium (CM) of visceral fat were determined by multiplex assay (n = 9-10/group). Results are presented as means ± SEM. Unpaired two-tailed Student's t-test was used for statistical analysis.

Fig 4: Macrophages within the aged fat transplant contribute to atherogenesis. Aged WT donor mice were treated with clodronate liposomaes according to the experimental schema described in Figure 5a. (a) Representative images of H&E or Mac2 stained right carotid artery, aortic root and BCA sections of Ldlr -/- mice that received sham surgery or visceral fat transplants from control liposome treated (Donor-L) or clodronate-liposome treated (Donor-C) aged donor mice. (b–d) The quantifications of lesion area, necrotic area, Mac2 positive area, and the percentage of Mac2 positive area in each section of (b) right carotid artery, (c) aortic root, and (d) BCA are shown (n = 8/group). Note: no necrosis was observed in carotid artery. Results are presented as means ± SEM. Two-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. p values indicate the main effect between groups of Donor-L and Donor-C.

Fig 5: Visceral fat transplants from aged mice increase macrophage numbers within athersclerotic lesions and fat transplants after transplantation. (a–c) Representative images of Mac2-stained (a) right carotid artery, (b) aortic root and (c) brachiocephalic artery (BCA) sections of Ldlr -/- mice that received sham surgery or visceral fat transplants from young (Donor-Y) or aged (Donor-A) mice. The quantification of Mac2 positive area in each section is shown on the right (n = 7-10/group). (d, f) Representative images of (d) Mac2 or (f) F4/80 stained visceral fat (GWAT) sections of young (Y) or aged (A) donors, before (bef) and after (aft) fat transplant (FT) surgery, are shown. The quantification of Mac2 (n = 14-17/group) or F4/80 (n = 7-9/group) positive area is shown on the right. (e, g) The change in the percentage of (e) Mac2 or (g) F4/80 positive area after FT, compared to before FT, in young (Y) or aged (A) visceral fat is shown (n = 7 or 14 / group). Results are presented as means ± SEM. Two-way ANOVA followed by Tukey's post hoc test was used for statistical analysis in (a–c). p values indicate the main effect between Donor-Y vs. Donor-A groups. Unpaired two-tailed Student's t-test was used for statistical analysis in (d–g).