Dilated Blood and Lymphatic Microvessels, Angiogenesis, Increased Macrophages, and Adipocyte Hypertrophy in Lipedema Thigh Skin and Fat Tissue

Abstract

Background and aim: Lipedema is a common painful SAT disorder characterized by enlargement of fat primarily in the legs of women. Case reports of lipedema tissue samples demonstrate fluid and fibrosis in the interstitial matrix, increased macrophages, and adipocyte hypertrophy. The aims of this project are to investigate blood vasculature, immune cells, and structure of lipedema tissue in a cohort of women.

Methods: Forty-nine participants, 19 controls and 30 with lipedema, were divided into groups based on body mass index (BMI): Non-Obese (BMI 20 to <30 kg/m²) and Obese (BMI 30 to <40 kg/m²). Histological sections from thigh skin and fat were stained with H&E. Adipocyte area and blood vessel size and number were quantified using ImageJ software. Markers for macrophages (CD68), mast cells (CD117), T cells (CD3), endothelial cells (CD31), blood (SMA), and lymphatic (D2-40 and Lyve-1) vessels were investigated by IHC and IF.

Results: Non-Obese Lipedema adipocyte area was larger than Non-Obese Controls (p=0.005) and similar to Obese Lipedema and Obese Controls. Macrophage numbers were significantly increased in Non-Obese (p < 0.005) and Obese (p < 0.05) Lipedema skin and fat compared to Control groups. No differences in T lymphocytes or mast cells were observed when comparing Lipedema to Control in both groups. SMA staining revealed increased dermal vessels in Non-Obese Lipedema patients (p < 0.001) compared to Non-Obese Controls. Lyve-1 and D2-40 staining showed a significant increase in lymphatic vessel area but not in number or perimeter in Obese Lipedema participants (p < 0.05) compared to Controls (Obese and Non-Obese). Areas of angiogenesis were found in the fat in 30% of lipedema participants but not controls.

Conclusion: Hypertrophic adipocytes, increased numbers of macrophages and blood vessels, and dilation of capillaries in thigh tissue of non-obese women with lipedema suggest inflammation, and angiogenesis occurs independent of obesity and demonstrates a role of altered vasculature in the manifestation of the disease.

Figure 1

Adipocyte area measured in Non-Obese and Obese participants with and without lipedema (controls). (a) Representative H&E images showing heterogeneity and hypertrophy in cell size in adipocytes in leg fat of Non-Obese participants (scale bar = 200 µm). (b) Box and whisker plot representing the average adipocyte area in Non-Obese (Control n=10; Lipedema n=10) and Obese groups (Control n=9; Lipedema n=11) (^∗ p < 0.05; ^∗∗ p < 0.01).

Figure 2

Immunohistochemistry of (a) CD68-labeled macrophages, (c) CD3-labeled T-lymphocytes, and (e) CD117-labeled mast cells in leg fat of lipedema participants (arrows). Images captured with 20x objective lens. Box and whisker plots of (b) CD68, (d) CD3, and (f) CD117 cell counts in skin and fat of control and lipedema participants. Non-Obese group (Control n=10; Lipedema n=10); Obese group (Control n=7; Lipedema n=12) (^∗∗ p < 0.01).

Figure 3

Number of blood vessels counted in the thigh skin of Non-Obese and Obese participants with and without lipedema (controls). (A, B) Immunohistochemistry of smooth muscle actin (SMA) positive vessels in the papillary dermal layer of lipedema leg skin (arrows). Note large lymphatic vessels in B (arrowheads) (scale bar 100 µm). (C) Box and whisker plots representing the blood vessel count in groups. Non-Obese group (Control n=10; Lipedema n=13); Obese group (Control n=8, Lipedema n=15). (^∗ p < 0.05, ^∗∗∗∗ p < 0.001).

Figure 4

Quantification of capillaries in the leg fat of Non-Obese and Obese participants with and without lipedema (controls). Box and whisker plots of the (a) number of capillaries per adipocyte and (b) capillary diameter measured in Obese and Non-Obese Lipedema participants compared to Controls. Non-Obese groups (Control n=10; Lipedema n=13); Obese groups (Control n=8; Lipedema n=14).

Figure 5

Angiogenesis in Non-Obese Lipedema leg fat. (a) H&E stain. Arrows indicate blood vessel lumens around adipocytes. (b, c) Immunohistochemistry of CD31 and SMA confirms the blood vessel origin of the tissue structures. (d) Trichrome stain demonstrating fibrosis in the areas of increased blood vessels. (e, f) Immunohistochemistry of CD68-labeled macrophages and CD117-labeled mast cells (arrows). Images captured at 40x objective lens (scale bar: 100 μm).

Figure 6

Measurement of the lymphatic vessel area and perimeter in the dermal skin of Non-Obese and Obese participants with and without lipedema (controls). (a, b) Box and whisker plots representing the quantification of lymphatic vessel area and area/perimeter ratio measurements. Non-Obese groups (Control n=7; Lipedema n=13); Obese groups (Control n=6; Lipedema n=14) (^∗ p < 0.05; ^∗∗ p < 0.01).