Measuring and mapping flow in lipedema: impact of adipose tissue dysfunction on interstitial fatty acid and fluid transport

Introduction:: Adipose tissue (AT) is the master regulator of whole body metabolism. During pathological states, such as obesity, AT expands rapidly without adequate vascularization, leading to hypoxia. Hypoxia can then drive fibrosis – the pathological deposition of extracellular matrix (ECM), which is known to alter interstitial transport. Lipedema, a disorder characterized by excess AT deposition, most frequently in the lower extremities, primarily affects women, and is both underdiagnosed and understudied. We aim to develop engineered lipedemic AT models, benchmarked to ex vivo lipedema tissue, to understand how lipedema may impact fluid and lipid transport in this key tissue.


Materials and Methods:: Tissue engineered models: Human adipose derived stem cells (ADSCs) are isolated from patients undergoing either abdominoplasty or thigh plasty. ADSCs are differentiated into adipocytes via DMEM/F-12 with 3% Fetal Bovine Serum, 1% Penicillin-Streptomycin, rosiglitazone (2 μM), isobutylmethylxanthine (500 μM), dexamethasone (1 μM), D-calcium pantothenate (17 μM), biotin (33 μM) and insulin (20 nM). Adipocytes are encapsulated at 1 million/mL in 2 mg/mL Type I collagen gels. After 24 hours, hypoxia is initiated with 1 μM Dimethyloxalylglycine (DMOG). Rhodamine Palmitic Acid (PA) is conjugated to bovine serum albumin and added above the AT models after one week of normoxia or hypoxia. Flow through media is collected and gels are homogenized to determine PA transport and uptake. For hormone treatment, 17 beta estradiol is added to the constructs at 0.5, 5, 25, and 50 ng/mL for one week. For all experiments, AT models are fixed in 4% paraformaldehyde and stained with Bodipy and DAPI.


Ex vivo MRI: Thigh AT from a male lymphedema patient was cut to approximately 0.4 grams, secured in a tissue culture insert with transglutaminase, and placed inside of a Bruker 9.4T ClinScan MRI. Gado-DTPA (BioPal) was added to the upper well above the tissue at a 1:100 dilution. T1-weighted images were then taken every 3 minutes for 30 minutes. Lymph4D in Matlab is used to create maps of diffusivity, magnitude of flow, and direction of flow.

In silico modeling: COMSOL Multiphysics was used to model flow velocity around varying sizes of adipocytes, governed by Darcy's Law.


Results, Conclusions, and Discussions:: Overall, female adipocytes are smaller than male (p< 0.05), regardless of depot, but have larger lipid droplets (Fig 1A-C). Between depots, adipocytes from the thigh have a smaller area range than from the abdomen (data not shown). Hypoxia has varying impacts on adipocyte maturity and morphology in male and female cells. Adipocyte area trends downwards in female adipocytes under hypoxia, and in male adipocytes, is significantly reduced under hypoxia (p< 0.05) (Fig 1B). Lipid droplet area trends downward in female cells under hypoxia and upward in male (Fig 1C). In male adipocytes, number of lipid droplets per cell trends downward under hypoxia (Fig 1D). PA transport is also altered by hypoxia. Interestingly, male adipocytes trend toward higher PA transport under hypoxia, whereas female adipocytes demonstrate the opposite trend (Fig 1E). Preliminary data suggests estradiol does not change adipocyte and lipid droplet area, but does decrease lipid droplets per adipocyte. Additionally, preliminary staining suggests less fibronectin deposition by female cells than male.



In ex vivo AT (Fig 2A), flow has been characterized using dynamic contrast enhanced MRI, with an average diffusivity of 35.6 µm2/s (Fig 2B) and an average flow magnitude of 0.6 µm/s (Fig 2C). Flow maps and histograms (Fig 2B-E) demonstrate that transport in AT is heterogeneous and correlates with structures visualized in T2 images (Fig 2A). Computational modeling demonstrates that adipocyte size alters flow paths and velocities (Fig 3).



These results suggest sex differences among adipocytes which may advance our understanding of why lipedema primarily affects female patients. Adipocyte maturity and size could alter transport biochemically, in terms of lipid uptake and storage, as well as physically, by altering the microenvironment. The enhanced interstitial transport of PA by hypoxic female adipocytes may translate to interstitial accumulation, which could contribute to AT dysfunction. Understanding changes to transport in healthy and dysfunctional AT and how flow impacts adipocyte function will provide essential information toward understanding AT-related disorders. Next steps include examining how estradiol impacts lipid transport and acquiring lipedema tissue samples to study transport on the MRI and to build lipedemic engineered AT.


Acknowledgements (Optional): : This work was supported by the Lipedema Foundation.

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