Objective To develop an experimental method for routine isolation and short‐term culture of primary lymphatic endothelial cells from specific collecting vessels. Methods Lymphatic endothelial cell tubes (LECTs) were isolated from micro‐dissected collecting vessels. LECTs were allowed to attach and grow for ~3 weeks before being passaged. Non‐purified cultures were partially characterized by immunofluorescence and RT‐PCR at passages 1–2. Results The method was validated in cultures of primary lymphatic endothelial cells (LECs) from male and female mice. After 1 or 2 passages, >60% of the LECs maintained expression of Prox1. Expression of 22 different genes was assessed using RT‐PCR. Prox1, Vegfr3, eNos, Cdh5, Pecam1, Cx43, Cx37, and Cx47, among others, were expressed in these short‐term cultured LECs, while Myh11, Cnn1, Desmin, and Cd11b were not detected. Prox1 expression, as determined by western blotting, was similar in cultured LECs from age‐matched male and female mice. Confocal imaging of intracellular calcium in cultures of primary LECs from Cdh5‐GCaMP8 mice demonstrated that a functional phenotype was maintained, similar to lymphatic endothelial cells in freshly isolated vessels. Conclusions This method provides an innovative tool for routine isolation and study of primary LECs from specific collecting lymphatic vessels from any mouse, and in fact, from other species.

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A two‐way connection between obesity and lymphatic dysfunction has now been established. Clinical studies have demonstrated that obesity significantly increases the risk for developing secondary lymphedema. Using animal‐models, obesity has been linked to different aspects of lymphatic dysfunction including impaired contractility, flow‐mediated responses, and fluid transport, as well as increased permeability, and impaired dendritic cell migration among others. Dysfunction of lymphatic valves is a main form of lymphatic dysfunction, known to result in severe edematous phenotypes; however, the extent of lymphatic valve deficiency in secondary lymphedema, including obesity‐induced lymphedema, remains unknown. Therefore, the aim of the present study was to determine whether obesity resulted in lymphatic valve dysfunction. We quantitatively assessed and compared valve function in isolated popliteal and mesenteric collecting lymphatic vessels from control and diet‐induced obese mice. Feeding a western diet (N=7) for 14 weeks induced obesity, resulting in significant gain of body weight, i.e., 18.8±1.4 g, versus 9.7±0.7 g for mice fed a control diet (N=6). Obese mice displayed significantly higher non‐fasted blood glucose concentration when compared to controls. The function of lymphatic valves in popliteal lymphatics was not affected by diet‐induced obesity; however, obesity resulted in significant back‐leak of pressure in mesenteric lymphatic valves (0.1±0.1 versus 0.7±0.1 cmH2O for control (n=11) and obese (n=16) groups respectively). Dysfunctional, leaky valves from obese animals required significantly higher adverse pressure to trigger valve closure (i.e., 5.5±1.6 cmH2O, versus 0.9±0.3 cmH2O for controls, when upstream pressure was maintained at 0.5 cmH2O). In obese animals, valve dysfunction in mesenteric lymphatics was associated with structural and mechanical modifications including shortening of valve leaflets, enlargement of the lymphatic vasculature, and increased cross‐sectional distensibility. This is the first study that reports on quantitative assessment of lymphatic valve function in an animal model of obesity, and the first to link obesity with lymphatic valve dysfunction. Finally, utilizing a newly developed method for the isolation and culturing of primary lymphatic endothelial cells (LECs) from collecting vessels, our current and future experiments seek to assess and understand functional modifications to LECs induced by obesity in‐vitro. Specifically, using high‐speed confocal microscopy, mouse lines carrying genetically encoded Ca2+ indicators (i.e., Cdh5‐GCaMP8 and acta2‐RCaMP), and lymphatic cellular co‐cultures, we aim to determine whether obesity results in intracellular calcium dysregulation in LECs, and/or abnormal crosstalk between LECs and lymphatic muscle cells.

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 diabet...