Combined pharmacological targeting of CD9+ progenitors alleviates obesity-induced adipose tissue fibrosis and metabolic impairment

Abstract

Fibrosis in visceral white adipose tissue (vWAT) is closely associated with tissue dysfunction and systemic metabolic disturbances in obesity. Identifying pathways amenable to drug intervention to prevent fibrotic changes in vWAT is a critical step in addressing the array of metabolic complications associated with obesity. CD9⁺ adipose progenitors (Progs) are key drivers of vWAT fibrosis. Here, we explore pharmacological strategies to target these cells and improve metabolic health. Profiling of CD9⁺ Progs reveals pro-fibrotic pathways that can be targeted by the Food and Drug Administration (FDA)-approved drugs nintedanib and celecoxib. Treatment with this combination blocks the progression of vWAT fibrosis and improves systemic metabolism in obese mice. Within the CD9⁺ Prog population, both Ly-6C⁺ Progs and mesothelial cells adopt a pro-fibrotic phenotype during obesity, a shift markedly reduced by the drug treatment. Our data highlight the importance of targeting adipose progenitors to counteract fibrosis and preserve adipose tissue function.

Keywords: adipose tissue fibrosis; mesothelium; obesity; progenitors; type 2 diabetes.

Graphical abstract

Figure 1

CD9⁺ progenitor frequency in visceral WAT is associated with loss of glucose control and bariatric surgery outcomes (A) Heatmap of multivariable linear regressions between bioclinical variables and CD9⁺ progenitor percentage in omental white adipose tissue (oWAT), with or without adjustment (n = 88). Standardized adjusted β coefficient is presented; ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p < 0.0005. (B) Pearson correlation between hydroxyproline content and CD9⁺ Progs in oWAT from individuals with obesity (n = 60). R and p values are indicated. (C–K) Analysis of T2D remission 1 year after bariatric surgery (BS) in patients with obesity and T2D (OBD), stratified by low or high CD9⁺ Progs in oWAT at the time of BS (n = 23). (C) Comparison of full, partial, or no T2D remission rates (chi-squared test, n = 19). The ratio of patients undergoing full, partial, and no T2D remission are presented. (D–K) Clinical parameters at baseline and 1 year post-BS are represented as mean ± SEM: (D) HbA1c (n = 18–23), (E) glycemia (n = 18–23), (F) insulinemia (n = 16–22), (G) HOMA-IR (n = 13–18), (H) BMI (n = 19–23), (I) fat mass (n = 18–22), (J) fat-free mass (n = 18–22), and (K) leptinemia (n = 18–22). Group comparisons used Student’s t test and generalized linear models adjusted for baseline values. p_adj: post hoc contrasts; plots show unadjusted data. ns, not significant.

Figure 2

The fibrogenic phenotype of the progenitors is curbed with nintedanib and celecoxib (A) Gene set enrichment analysis of differentially expressed genes in CD9⁺ progenitors isolated from lean and obese fibrotic EpiWAT of C3H mice. (B) Heatmap of selected significantly up- or downregulated genes in CD9⁺ progenitors from lean and obese EpiWAT (n = 4; fold change (FC) > 1.5; adjusted p < 0.05). (C) EpiWAT mass in mice fed chow or HFD for 7 days (n = 5). (D) Representative bright-field and polarized images of picrosirius red-stained EpiWAT sections and adipocyte size distribution (scale bar, 100 μm; n = 3). (E) Flow cytometry plots of Ki-67 expression in CD9⁺ progenitors and quantification of Ki-67⁺ cells (n = 4–5). (F) CD9⁺ progenitor count per gram of EpiWAT in chow- and HFD-fed mice (n = 4–5). (G) Relative mRNA expression of fibrosis markers in whole EpiWAT (normalized to chow; n = 5). (H) mRNA expression in CD9⁺ progenitors from obese fibrotic EpiWAT treated with vehicle (Veh), nintedanib (Nin), and/or celecoxib (Cel) (n = 4). (I) mRNA expression in CD9⁺ progenitors from human oWAT treated with Veh, Nin, and/or Cel (n = 4). (A and B) Linear models for microarray data (LIMMA) analysis with multiple-testing correction. (C–H) Data are represented as mean ± SEM. (C–G) Student’s t test. (H and I) One-way ANOVA with Newman-Keuls post hoc test.

Figure 3

The combination of nintedanib and celecoxib treatment improved glucose and lipid metabolism in vivo (A) Experimental design: 1 week after initiating high-fat diet (HFD), C3H male mice were treated for 4 weeks with vehicle (Veh), nintedanib (Nin), celecoxib (Cel), or Nin+Cel. (B–D) Body weight, fat mass, and lean mass measured pre- and post-treatment (n = 8–10); dashed lines indicate mean values from age-matched chow-fed controls. (E–H) Mass of EpiWAT, IngWAT, liver, and kidney post-treatment (n = 8–10). Dashed lines indicate mean values from age-matched chow-fed controls. (I) Glucose tolerance test (GTT) and area under the curve (AUC) (n = 8–10). (J) Fasting glycemia (n = 8–10) and insulinemia (n = 8–9). (K) Liver histology (H&E; scale, 50 μm) and steatosis quantification (n = 4–5); dashed lines represent chow-fed controls. (L–N) VO₂ normalized to lean mass, food intake, and respiratory exchange ratio (RER) in Veh vs. Nin+Cel groups (n = 5). (O) Plasma glycerol levels pre- and post-CL 316,243 injection (n = 5–6). (B–O) Data are represented as mean ± SEM. (B–J) One-way ANOVA with Newman-Keuls post hoc test; (K, M, and O) Student’s t test; (L and N) two-way repeated-measures ANOVA.

Figure 4

Combination treatment with nintedanib and celecoxib exerts anti-fibrotic effects in adipose tissue (A) Hydroxyproline content in EpiWAT of HFD-fed C3H mice treated with Veh, Nin, Cel, or Nin+Cel (n = 8–10). (B) Representative bright-field and polarized light images of picrosirius red-stained EpiWAT (scale, 100 μm). (C–E) (C) Mean adipocyte size (n = 6); (D) fibrosis per adipocyte (arbitrary units) (n = 5–6); (E) mRNA expression in EpiWAT (n = 7–8). (F) Percentage of Ki-67⁺ macrophages and macrophage density (CD45⁺CD64⁺CD31⁻) in EpiWAT (n = 6–8). (G–H and J) mRNA expression in EpiWAT (n = 7–8). (I) Insulin-stimulated p(S473)AKT and pan-AKT levels in EpiWAT; quantification relative to basal (n = 5). (K–M) Flow cytometry of EpiWAT progenitors: (K) left, representative fluorescence-activated cell sorting (FACS) plot showing percentage of CD9⁺PDGFRα⁺Ki-67⁺; right, the percentages of CD9⁺PDGFRα⁺Ki-67⁺ are represented as mean ± SEM. (L) CD9⁺ progenitor density and (M) CD9⁻ preadipocyte density (n = 6–8). (N) Hydroxyproline content in IngWAT (n = 8–10). (O–Q) mRNA expression in IngWAT (n = 5–8). (R–T) IngWAT progenitor analysis (n = 6–8): (R) percentage of PDGFRα⁺Ki-67⁺, (S) PDGFRα⁺ cell density, and (T) percentage of CD36⁺ progenitors. (A–T) Data are represented as mean ± SEM. (A, D–H, and J–T) One-way ANOVA with Newman-Keuls post hoc test; (C and I) Student’s t test.

Figure 5

The combination of nintedanib and celecoxib blocks the progression of established EpiWAT fibrosis (A) Experimental design: C3H male mice fed HFD for 5 weeks and then treated with Veh or Nin+Cel for 3 weeks. (B) Hydroxyproline content in EpiWAT from chow- or HFD-fed mice (n = 4–5). (C and D) Body weight and liver mass after treatment (n = 10); dashed lines: age-matched chow-fed mice. (E and F) Liver H&E staining (scale, 50 μm) and steatosis quantification (n = 5). Dashed lines: age-matched chow-fed mice. (G and H) EpiWAT mass and hydroxyproline content (n = 10); dashed line in (H): baseline HFD-fed mice. Dashed lines: age-matched chow-fed mice. (I) Representative picrosirius red-stained EpiWAT images (scale, 100 μm). (J and K) Adipocyte size and fibrosis per adipocyte in EpiWAT (n = 7–8). (L and M) GTT, AUC, fasted glycemia, and insulinemia (n = 4–10). (N) Insulin tolerance test (ITT) in Veh-vs. Nin+Cel-treated mice (n = 5–10). (O–Q) EpiWAT progenitor analysis: (O) left, representative FACS plot showing the percentage of CD9⁺PDGFRα⁺Ki-67⁺; right, the percentages of CD9⁺PDGFRα⁺Ki-67⁺ are represented as mean ± SEM. (P) CD9⁺ progenitor density and (Q) PDGFRα⁺CD9⁻ preadipocyte density (n = 8). (R and S) Macrophage counts and Ki-67⁺ percentage among macrophages in EpiWAT (n = 8). (T) IngWAT mass (n = 10). (U and V) IngWAT histology (picrosirius red; scale, 100 μm), adipocyte size, and fibrosis per adipocyte (n = 4–6). (W–Y) IngWAT progenitor analysis: (W) PDGFRα⁺ cell density, (X) percentage of PDGFRα⁺Ki-67⁺, and (Y) percentage of CD36⁺ progenitors (n = 8). (Z) Spearman correlation between CD36⁺ progenitors and IngWAT mass (n = 42). R and p values are indicated. (B–Y) Data are represented as mean ± SEM. (B–J and O–Y) Student’s t test; (K–N) one-way ANOVA with Newman-Keuls post hoc test; (Z) Spearman correlation.

Figure 6

Mesothelial cells and fibro-inflammatory progenitors undergo a fibrotic transformation in EpiWAT of obese C3H mice (A) Left: gating strategy for FIPs (GP38⁺CD9⁺Ly-6C⁺CD45⁻CD31⁻) vs. mesothelial cells (MCs, GP38⁺CD9⁺Ly-6C⁻CD45⁻CD31⁻). Right: heatmap of MC marker expression (Msln, Krt19, Upk3b, and Lrrn4) in sorted FIPs and MCs (n = 5). (B) GO term enrichment analysis of genes differentially expressed between FIPs and MCs from EpiWAT of HFD-fed mice. (C) Heatmap of ECM-related gene expression upregulated in FIPs vs. MCs (n = 5, FC > 1.5, adjusted p < 0.05). (D) mRNA expression of fibrosis marker genes in FIPs and MCs; Msln in MCs (n = 5). (E) Flow cytometric quantification of MSLN⁺ cells per gram of EpiWAT in chow- and HFD-fed mice (n = 6). (F) Representative picrosirius red staining of EpiWAT perilobular area (scale, 50 μm) and quantification of collagen deposition (n = 4–5). (G and H) Two-photon microscopy of EpiWAT from Krt19-CreERT; tdTomato mice fed HFD. (G) Top view: Tomato⁺ MCs (red) and second harmonic generation (SHG)-collagen (green) at tissue surface. (H) Side view: mesothelial layer closely associated with collagen fibers (scale, 20 μm). (I) Tomato⁺ cell density in EpiWAT from control (Krt19-CreERT; tdTomato) and Krt19-αK (Krt19-CreERT; tdTomato; PDGFRαK) mice (n = 5–7). (J) mRNA expression of fibrosis genes in sorted Tomato⁺ MCs from controls and Krt19-αK (n = 3–4). (K and L) Human WAT histology from patients with obesity (n = 3). (K) Representative oWAT images showing COL1 (green), MSLN (red), DAPI (blue), and bright field (gray); scale, 20 μm. (L) MSLN expression on mesWAT and oWAT but not on scWAT surfaces; autofluorescence (green), DAPI (blue), and bright field (gray); scale, 130 μm. (D–F, I, and J) Data are represented as mean ± SEM. (A–C) LIMMA analysis with adjusted p values. (D) One-way ANOVA with Newman-Keuls test. (D–J) Student’s t test.

Figure 7

FIPs and MCs lowered their fibrotic potential in mice treated with nintedanib and celecoxib (A) Density of CD9⁺Ly-6c⁺ FIPs and CD9⁺Ly-6c⁻ MCs in EpiWAT of HFD-fed C3H mice treated with vehicle or Nin+Cel (treatment start after 5 weeks of HFD, n = 8). (B) Percentage of Ki-67⁺ FIPs and MCs (n = 8). (C) Msln mRNA expression in MCs sorted from EpiWAT (n = 6). (D) Venn diagram of genes downregulated in FIPs and MCs following Nin+Cel treatment. (E) Gene set enrichment analysis (GSEA) plots showing enrichment of CD9⁺ Prog fibrosis-associated signatures among genes downregulated in FIPs/MCs by the combined treatment (Phantasus). (F) Pathway enrichment analysis of downregulated genes in FIPs and MCs (Phantasus). (G) Heatmap of ECM-associated genes downregulated in sorted FIPs and MCs (fold change > 1.5, adjusted p < 0.05; n = 5). (H) Representative images (bright-field and polarized light) and quantification of perilobular collagen in EpiWAT from HFD-fed mice treated with Veh or Nin+Cel (n = 5–7; scale bar, 50 μm). (A–C and H) Data are represented as mean ± SEM. (A and B) One-way ANOVA with Newman-Keuls test; (C and H) Student’s t test; (D–G) LIMMA analysis with adjusted p values.