Endothelial‐Cell Modifications in Murine Collecting Lymphatic Vessels in Obesity

Abstract

The presence of edema within tissues and cavities presented by patients afflicted with obesity suggests that lymphatic dysfunction may be a contributing factor during the onset and/or development of the various ailments associated with this disease. Importantly, obesity has been shown to significantly increase the chances of developing type-2 diabetes, cardiovascular diseases, and lymphedema in breast cancer patients undergoing surgical intervention and radiotherapy. In obesity, vascular endothelial dysfunction is known to result as a consequence of the chronic systemic inflammation. Relevant to this study, the extent of lymphatic endothelial dysfunction in obesity and type-2 diabetes remains to be fully assessed. In order to 1) elucidate the root cause of lymphatic dysfunction and subsequent impaired lymph transport in obesity, and 2) determine if this dysfunction involves an endothelial component; it is essential to isolate the various cell types in the lymphatic wall and compare the specific genetic and proteomic makeup in health and disease. Here we focus on the study and understanding of the disease-driven modifications to lymphatic endothelial cells (LECs). We hypothesize that LECs of collecting lymphatic vessels from obese mice will exhibit differential expression of transcription factors commonly associated with endothelial dysfunction (i.e. LEC specification, valve dysfunction, and barrier disruption). We utilized a diet-induced obesity mouse model. Obesity was induced by feeding C57BL/6J male mice a western diet (ResearchDiets Inc. Cat.No. D12079Bi) for 14 weeks starting at 5 weeks of age. Consistent with previous studies, diet-induced obese animals gained twice as much weight when compared to aged-matched animals fed a control diet (weight gain 18.8 g vs 9.7 g). Non-fasted blood glucose levels were 41.6±10.6% higher in obese animals, which also displayed increased skin moisture content, 45.1±4.7% (obese) vs 32.6±4.8% (control), as determined by a wet-to-dry weight ratio. Our preliminary qRT-PCR results on fluorescence-activated cell sorting (FACS) purified LECs show that diet-induced obesity results in decreased expression of the transcription factor, and LEC marker, Prox1. We have developed a novel protocol for establishing cell cultures of primary LECs from collecting lymphatic vessels isolated from different species. Notably, the same protocol with minor modifications can be used to establish cultures of primary endothelial cells from blood vessels. Preliminary characterization of murine primary LECs using RT-PCR, immunofluorescence, and confocal microscopy suggests that these cultures could be a useful novel tool for lymphatic research. Currently, we are using these cultured primary LECs from db/db mice and a proteomics approach to further assess the effects of obesity and type-2 diabetes in the lymphatic endothelium of collecting lymphatics. We anticipate that these studies will provide critical information to identify novel mechanisms of lymphatic endothelial dysfunction in obesity, type-2 diabetes, and lymphedema, and propose novel therapeutic targets to ameliorate lymphatic dysfunction in these diseases.