A Comparative Analysis to Dissect the Histological and Molecular Differences among Lipedema, Lipohypertrophy and Secondary Lymphedema

Abstract

Lipedema, lipohypertrophy and secondary lymphedema are three conditions characterized by disproportionate subcutaneous fat accumulation affecting the extremities. Despite the apparent similarities and differences among their phenotypes, a comprehensive histological and molecular comparison does not yet exist, supporting the idea that there is an insufficient understanding of the conditions and particularly of lipohypertrophy. In our study, we performed histological and molecular analysis in anatomically-, BMI- and gender-matched samples of lipedema, lipohypertrophy and secondary lymphedema versus healthy control patients. Hereby, we found a significantly increased epidermal thickness only in patients with lipedema and secondary lymphedema, while significant adipocyte hypertrophy was identified in both lipedema and lipohypertrophy. Interestingly, the assessment of lymphatic vessel morphology showed significantly decreased total area coverage in lipohypertrophy versus the other conditions, while VEGF-D expression was significantly decreased across all conditions. The analysis of junctional genes often associated with permeability indicated a distinct and higher expression only in secondary lymphedema. Finally, the evaluation of the immune cell infiltrate verified the increased CD4+ cell and macrophage infiltration in lymphedema and lipedema respectively, without depicting a distinct immune cell profile in lipohypertrophy. Our study describes the distinct histological and molecular characteristics of lipohypertrophy, clearly distinguishing it from its two most important differential diagnoses.

Keywords: CD4+ cells; VEGFD; adipose tissue disorders; lipedema; lipohypertrophy; lymphatic vessels; macrophages; secondary lymphedema.

Figure 1

Assessment of the skin architecture and fibrosis content in lipedema, secondary lymphedema and lipohypertrophy patients compared to the control group. (A) Representative Hematoxylin/Eosin (H/E) staining of skin from control, lipedema, secondary lymphedema and lipohypertrophy patients. (B) The quantification of epidermal thickness across all four groups, depicted increased epidermal thickness in the skin of lipedema and secondary lymphedema patients compared to the control group. (C) Sirius Red (SR) staining of the skin was used to evaluate the extend of fibrosis in control, lipedema, secondary lymphedema and lipohypertrophy patients. (D) The analysis of the collagen deposition indicated significantly lower levels of fibrosis in lipohypertrophy. Black scale bar represents 100 μm. N = 10 in each group. Statistical significance is signified by asterisks (* p < 0.05, ** p < 0.01).

Figure 2

Adipocyte size was significantly increased in lipedema and lipohypertrophy. (A) Hematoxylin/Eosin (H/E) staining of adipose tissue in all four groups (control, lipedema, secondary lymphedema and lipohypertrophy). (B) Evaluation of adipocyte size comparing all four groups. Significantly larger adipocytes were found in lipedema and lipohypertrophy when compared to the control tissue and secondary lymphedema. (C) Sirius Red (SR) staining of adipose tissue in all four groups (control, lipedema, secondary lymphedema and lipohypertrophy). (D) Fibrosis analyzed by collagen content comparing all four groups showed significant increase in secondary lymphedema. (E) Adipocyte size distribution analysis identified increased number of larger adipocytes in lipedema and lipohypertrophy. Black bar represents 100 μm. N = 10 in each group. Statistical significance is signified by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

Evaluation of the lymphatic vascular milieu. (A) Representative Podoplanin (PDPN) staining of skin sections from all four groups (control, lipedema, lipohypertrophy, secondary lymphedema). (B) Quantification of lymphatic vessels by number of vessels, size of vessels and percentage of surface area covered by vessels. A significant decrease in surface area covered by lymphatic vessels was detected in lipohypertrophy in comparison to control group. (C) Relative gene expression (hereby control group = 1) of VEGF-A, VEGF-C and VEGF-D. The expression of VEGF-D is significantly decreased across all three conditions in comparison to the control group. VEGF-R2 is significantly decreased in secondary lymphedema and lipohypertrophy when compared to control tissue. The expression levels of VEGF-R2 in lipohypertrophy are significantly lower when compared to lipedema as well. VEGF-R3 expression was significantly increased in secondary lymphedema when compared to all groups. (D) Relative gene expression (hereby control group = 1) of GJA1, TJP1 and CLDN5. In GJA1 and CLDN5 expression was significantly increased solely in secondary lymphedema. No change in expression was detected in TJP1. Black bar represents 100 μm. N = 10 in each group (gene expression of CLDN5, TJP1 and GJA1 in LE group = 5). Statistical significance is signified by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

Increased but distinct immune cell presence in lipedema and secondary lymphedema, without signs of increased immune infiltration in lipohypertrophy. (A) CD45+ hematopoietic cell, (B) CD4+ T helper cell and (C) CD68+ macrophage (D) CD163+ macrophage staining of skin in control, lipedema, secondary lymphedema and lipohypertrophy paraffin sections (E–H) Quantification of histological analysis in tissue sections. An increased presence of CD45+ cells was detected in lipedema and secondary lymphedema but not in lipohypertrophy. Secondary lymphedema is characterized by an increased CD4+ infiltration, in comparison to both control and lipedema tissue. In lipedema an increased infiltration of both CD68+ and CD163+ cells in comparison to both the control and lipohypertrophy tissue is detected. (G-I) Relative gene expression (hereby control group = 1) of (I) CD45, (J) CD4, (K) CD68, (L) CD163. The findings here confirm most of the differences detected in the histological analysis. Black scale bar represents 100 μm. N = 10 in each group. Statistical significance is signified by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).