PPARγ agonist treatment reduces fibroadipose tissue in secondary lymphedema by exhausting fibroadipogenic PDGFRα+ mesenchymal cells

Abstract

Secondary lymphedema occurs in up to 20% of patients after lymphadenectomy performed for the surgical management of tumors involving the breast, prostate, uterus, and skin. Patients develop progressive edema of the affected extremity due to retention of protein-rich lymphatic fluid. Despite compression therapy, patients progress to chronic lymphedema in which noncompressible fibrosis and adipose tissue are deposited within the extremity. The presence of fibrosis led to our hypothesis that rosiglitazone, a PPARγ agonist that inhibits fibrosis, would reduce fibrosis in a mouse model of secondary lymphedema after hind limb lymphadenectomy. In vivo, rosiglitazone reduced fibrosis in the hind limb after lymphadenectomy. Our findings verified that rosiglitazone reestablished the adipogenic features of TGF-β1-treated mesenchymal cells in vitro. Despite this, rosiglitazone led to a reduction in adipose tissue deposition. Single-cell RNA-Seq data obtained from human tissues and flow cytometric and histological evaluation of mouse tissues demonstrated increased presence of PDGFRα+ cells in lymphedema; human tissue analysis verified these cells have the capacity for adipogenic and fibrogenic differentiation. Upon treatment with rosiglitazone, we noted a reduction in the overall quantity of PDGFRα+ cells and LipidTOX+ cells. Our findings provide a framework for treating secondary lymphedema as a condition of fibrosis and adipose tissue deposition, both of which, paradoxically, can be prevented with a pro-adipogenic agent.

Keywords: Adipose tissue; Cell Biology; Fibrosis.

Figure 1. A mouse model of hind limb lymphedema recapitulates augmented swelling and adipose tissue deposition observed in patients with lymphedema.

(A) Surgical procedure for hind limb lymphadenectomy in mice. (B) Normalized circumference of hind limbs with/without lymphadenectomy and rosiglitazone treatment (n = 8); comparison between No injury + Vehicle and LN + Vehicle is indicated by *; comparison between LN + Vehicle and LN + Rosiglitazone is indicated by ^#. (C) Normalized edema volume of hind limbs with/without lymphadenectomy and rosiglitazone treatment (n = 8); comparison between No injury + Vehicle and LN + Vehicle is indicated by *; comparison between LN + Vehicle and LN + Rosiglitazone is indicated by ^#. (D) H&E staining of No injury + Vehicle and LN + Vehicle day 7 and day 21, as well as LN + Rosiglitazone day 7 and day 21 hind limbs, original magnification 4×, scale bar: 500 μm. (E) Quantification of overall hind limb skin thickness (n = 8). (F) Quantification of hind limb fibroadipose tissue thickness (n = 8). (G) Quantification of hind limb dermis thickness (n = 8). (H) Quantification of hind limb epidermis thickness (n = 8). Statistical significance is established using 1-way ANOVA and Tukey’s post hoc tests, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. LN, lymphadenectomy; E, epidermis; D, dermis; F, fibroadipose tissue; M, muscle.

Figure 2. Treatment effect of rosiglitazone on fibrosis in hind limb lymphedema.

(A) Immunostaining of No injury + Vehicle and LN + Vehicle day 7 and LN + Rosiglitazone day 7 sections for phosphorylated (p-) SMAD 2/3; original magnification 20×, scale bar: 100 μm. (B) Quantification of p-SMAD2/3⁺ cells in fibroadipose tissue layer (n = 3). (C) Expression levels of adipogenic genes among adipose-derived mesenchymal cells treated with TGF-β1 with or without rosiglitazone (n = 3). (D) Expression levels of fibrogenic genes among adipose-derived mesenchymal cells treated with TGF-β1 with or without rosiglitazone (n = 3). (E) Picrosirius red staining of No injury + Vehicle and LN + Vehicle day 7 and day 21, as well as LN + rosiglitazone day 7 and day 21 hind limbs; original magnification 4×, scale bar: 500 μm. (F) Normalized quantification of hind limb fibrosis (n = 8). Statistical significance is established using 1-way ANOVA and Tukey’s post hoc tests, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. LN, lymphadenectomy; E, epidermis; D, dermis; F, fibroadipose tissue; M, muscle.

Figure 3. Lymphedema modifies the presence of fate of PDGFRα⁺ cells.

(A) Left: Uniform manifold approximation and projection (UMAP) of PDGFRA⁺ cells subclustered from total lymphedema scRNA-Seq data set (Supplemental Figure 6). Right: PDGFRA⁺ UMAP split by lymphedema and healthy SVF (LSVF and NSVF, respectively). (B) Heatmap of top 10 differentially expressed transcripts identifying each cluster in the PDGFRA⁺ UMAP space. (C) Proportion of cells in each cluster for LSVF and NSVF samples, respectively. Box plots show the interquartile range (box), median (line), and minimum and maximum (whiskers). Statistical significance is established using Mann-Whitney nonparametric t test, *P < 0.05, **P < 0.01. (D) Volcano plots of top differentially expressed genes between lymphedema and healthy samples for cluster 2 and cluster 6; fold-change cutoff 0.5, P-adjusted cutoff 0.001. Labeled transcripts colored in red meet both fold-change and significance cutoff; transcripts to the right are increased in lymphedema; and transcripts to the left of plot are increased in healthy.

Figure 4. Rosiglitazone reduces presence of PDGFRα⁺ adipocytes after lymphadenectomy.

(A) Representative flow cytometry plots showing detection of PDGFRα⁺ and LipidTOX⁺ cells in hind limb tissue from No injury + Vehicle, LN + Vehicle, and LN + Rosiglitazone mice. (B) Quantification of PDGFRα⁺ cells in 1 cm × 1 cm area of skin from No injury + Vehicle, LN + Vehicle, and LN + Rosiglitazone hind limbs (n = 3). (C) Quantification of LipidTOX⁺ cells in 1 cm × 1 cm area of skin from No injury + Vehicle, LN + Vehicle, and LN + Rosiglitazone hind limbs (n = 3). (D) Immunostaining for PDGFRα and Ki67 in No injury + Vehicle and LN + Vehicle day 7 and LN + Rosiglitazone day 7 sections; original magnification 20×, scale bar: 100 μm. (E) Quantification of PDGFRα⁺ cells in fibroadipose tissue layer (n = 3). (F) Quantification of Ki67⁺ cells in fibroadipose tissue layer (n = 3). Statistical significance is established using 1-way ANOVA and Tukey’s post hoc tests, *P < 0.05, **P < 0.01, ****P < 0.0001. LN, lymphadenectomy; D, dermis; F, fibroadipose tissue; M, muscle.