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In this study an analysis of identified ICG lymphography features of the superficial lymphatics of the lower extremity was undertaken [Table 2.2]. The absence or presence and extent of the superficial lymphatics in the limb with LLLE was assessed to identify if any of these features or combination of features were significantly associated with particular diagnostic groups of primary or secondary cancer related or secondary non-cancer related LLLE. ICG lymphography studies undertaken by the researcher (submitted for publication) had identified two compensatory lymph drainage regions, the contralateral inguinal and axillo-inguinal nodal regions, associated with secondary cancer related LLLE which were not seen or very rarely seen in clinically diagnosed primary LLLE and less so in secondary non-cancer related LLLE. Conversely, retrograde flow in lymph vessels demonstrated during the ICG lymphography procedure was not seen in secondary cancer related LLLE. These observations provided a background rationale to exploring the diagnostic capacity of ICG lymphography in LLLE. One purpose of this study was to explore the feasibility of using ICG lymphography features as a diagnostic tool. It was hoped that identified ICG lymphographic features would allow the individual presenting with persistent leg swelling of unknown causation to be able to be advised that their condition is most likely a secondary non cancer related LLLE rather than an adult onset primary LE and therefore be possibly less likely to progress or deteriorate. This may potentially provide better prognostic information and improve individualised therapeutic plans of management and support compliance (Pigott, 2021). The aim of Part A of this study was to identify the prevalence and characteristics of ICG lymphography features of the lymph vessels, position and extent of DBF and drainage patterns. In addition, Part B of this study was to explore whether using the ALERT standardised ICG lymphography technique and a structured analysis of ICG lymphography features could be translated into a simple score to distinguish the ICG pattern of adult onset primary LLLE from secondary non-cancer related LLLE.

Angiogenesis, the growth of blood vessels from pre-existing vasculature, is primarily regulated by vascular endothelial growth factor receptors (VEGFRs). Dysregulated angiogenesis is associated with cancers, obesity, and over 70 vascular diseases. Upregulated VEGFR protein expressions in diseased vasculature are promising biomarkers for predicting clinical outcomes, as indicated by non-quantitative immunohistochemical studies in patients with impaired vascularization or tumor angiogenesis. While the quantitative characterization of VEGFRs is critical in identifying biomarkers for anti-angiogenic therapies, VEGFR biomarker development presents two particular challenges: (1) The invasive tissue biopsy needed limits the amount of VEGFR data that can be collected from both normal and diseased vasculatures, and (2) we poorly understand the significance of endothelial and various non-endothelial VEGFR-expressing cells in angiogenic therapies. To address these challenges, here I pioneer a blood biopsy-based proteomic approach that allows non-invasive VEGFR quantification. More significantly, I identify and establish age- and sex-specific basal levels of VEGFRs on endothelial cells and bone marrow-derived progenitor cells (Chapter 2). In recent years, blood biopsies have expanded our knowledge of vascular pathology. In particular, circulating angiogenic cells, such as circulating endothelial cells (cECs) and circulating progenitor cells (cPCs), are isolated and counted, and their elevated abundances are often correlated with vascular disease progression and cancer prognosis. However, cECs and cPCs have been overlooked as accessible proxies for profiling vascular biomarker expressions by activated or damaged vasculatures. For the first time, I show that cPCs and cECs exhibit heterogeneous plasma membrane expression of VEGFRs, which are correlated with donor sexes and ages, particularly pre- vs. post-menopausal status. Menopause is known to reduce regenerative and angiogenic capacities, as manifested by decreased capillary growth in skeletal muscle and increased risks for cardiovascular diseases. Here I provide baseline VEGFR expression ranges for these cells, showing that ~50% of cECs in premenopausal females exhibit intermediate-to-high plasma membrane expression (138,000 VEGFR1 and 39,000-236,000 VEGFR2/cell) and ~25% of cECs in males exhibit high VEGFR plasma membrane expression (206,000 VEGFR1 and 155,000 VEGFR2/cell). In marked contrast, nearly all cECs in postmenopausal females are VEGFR-low (2,900 VEGFR1 and 3,400 VEGFR2/cell), agreeing with the reduced angiogenic capacities after menopause. Additionally, VEGFR1 signaling is critical for cPC localization to activated or damged blood vessels. My data show that VEGFR1 plasma membrane localization in cPCs occurs only in postmenopausal females, suggesting menopause activates VEGFR1 signaling pathways in cPCs. Therefore, my data offer quantitative insights into how VEGFR-regulated regenerative and angiogenic capacities are altered due to menopause. Overall, these findings provide the first insights into how sex and age interactions, particularly menopause, influence VEGFR plasma membrane localization in circulating angiogenic cells. More importantly, the findings help establish age- and sex-specific VEGFR baselines for predicting vascular disease progression and therapeutic outcomes. The second challenge is quantitatively characterize how endothelial and non-endothelial VEGFR-expressing cells contribute to angiogenic regulation. Here, I quantitatively elucidate the changes in VEGFR expressions by endothelial cells and non-enodthelial cells in adipose tissues, and identify biomarkable adipose tissue cells that show altered VEGFR membrane expressions in normal versus high-adiposity states (Chapter 3). Obesity is a major risk factor for vascular disorders, including peripheral artery disease, critical limb ischemia, and several cancers. I hypothesize that VEGFR membrane expression by adipose tissue cells is altered as body fat accumulates (increased adiposity). The VEGFR quantification data presented here indicate that ~ 20% of activated lymphocytes upregulate their membrane expressions of VEGFR1 and VEGFR3 by tenfold in response to increased subcutaneous adiposity induced by lipedema, which is very commonly accompanied by impaired vascularization and chronic inflammation. On the other hand, in murine visceral adipose tissue, myeloid progenitor cells exhibit the highest VEGFR membrane expressions (16,000 ± 4,700 VEGFR1, 50,000 ± 6,200 VEGFR2, and 2,100 ± 460 VEGFR3/cell). Compared to myeloid progenitor cells, visceral endothelial cells exhibit an order of magnitude lower VEGFR1 and VEGFR2 levels (2,400 ± 710 VEGFR1/cell, 1,100 ± 190 VEGFR2/cell, and 1,200 ± 220 VEGFR3/cell, respectively). My approach and findings are foundational to a systematic understanding of how VEGFR-expressing adipose cells regulate adipose angiogenesis and adipogenesis. Future studies are warranted to compare how VEGFR membrane expressions differ in chow-fed and high fat-fed mice, and the quantitative proteomic findings will guide therapies for visceral obesity-associated vascular disorders. Last but not least, unlike VEGFRs, many receptors of clinical interest, particularly the oxytocin receptor (OXTR) and its genetic variants, do not have specific antibodies that enable quantitative characterization. To overcome this issue, I have designed a transfected cell model that is engineered to express HA-OXTR-GFP protein complexes, in which an N-terminal HA acts as a proxy for membrane OXTR detection and a C-terminal GFP acts as an indicator in selecting transfected cells from untransfected cells (Chapter 4). This transfected cell model is applied to characterize the varied dose-response profiles of OXTR wild-type and variant cells to oxytocin, a common labor induction drug. My OXTR quantification data show clear correlations to oxytocin-induced functional outcomes, including calcium release and cell desensitization, suggesting that the quantities of different OXTR variants are predictive of cell responses to administered oxytocin and should be considered when making personalized oxytocin dosing decisions. Overall, my results demonstrate that membrane expression of VEGFRs is significantly associated with physiological factors such as sex, age, and menopause, and with pathological adipose tissue expansion. Although VEGFR protein expression is a promising biomarker for many vascular diseases and cancers, quantitative and baseline VEGFR data are still needed for VEGFR-driven pathology. My work on both VEGFRs and other biomarkable receptors, such as OXTR, provides much-needed standardized approaches and quantitative data, a first step towards proteomic biomarker-driven precision medicine.

In recent years stem cell research has become increasingly important for regenerativemedicine and tissue engineering. The isolation of stem cells from adipose tissue evades ethicalconcerns with which embryonic stem cells and induces pluripotent stem cells (iPS) are afflicted,because of its declaration as clinical waste material. Tumescent liposuction is a minimallyinvasive procedure providing high amounts of adipose tissue rich in therapeutically relevantcells within a short time. The isolated stromal vascular fraction (SVF) and the adipose derivedstromal/stem cells (ASC) contained therein show a high regenerative potential and have beensuccessfully used in many clinical studies. Maintaining SVF cells in their natural environmentand therefore providing the maximum possible regenerative potential of adipose tissue-derivedcells is a prerequisite for successful autologous clinical application. With an improved gentleand fast isolation process by minor manipulation it is possible to obtain a therapeuticallyrelevant cell population. A physical stimulus already used in clinics is the extracorporealshockwave therapy (ESWT), shockwaves are characterized by their high rise in pressurewithin a very short time followed by cavitation wave with a negative amplitude. By applyinglow-energy ESWT on freshly obtained human liposuction material and isolated SVF cells (invitro) we aimed to equalize and enhance stem cell properties and their functionality. We wereable to show an increased adenosine tri-phosphate (ATP) concentration after applying ESWTon adipose tissue as well as a significantly increased expression of single mesenchymal andvascular surface markers in comparison with the untreated group. Additionally, the proteinsecretion of insulin-like growth factor 1 (IGF-1) and placental growth factor (PLGF) wassignificantly enhanced. Further it was investigated if there is the same beneficial effect whenapplying ESWT on the adipose tissue harvest site before liposuction to improve cell propertiesin situ. We showed a significantly enhanced viability, ATP concentration and populationdoublings after 3 weeks in culture for cells isolated from ESW treated adipose tissue harvestsite. Further the expression of mesenchymal and endothelial/pericytic markers was elevatedcollaborating with the increased angiogenic differentiation potential as well as the increasedsecretion of certain angiogenic proteins after ESWT in situ. Besides ESWT the effect of anotherphysical stimulus on SVF/ASC cells was tested - Low level laser therapy (LLLT) has alreadyshown beneficial effects. Therefore, we investigated effects of pulsed blue (475nm), green(516nm) and red (635nm) light from light-emitting diodes (LEDs) applied on freshly isolatedSVF cells. Cells had a stronger capacity to vascular tube formation after exposure to greenand red light concomitant with an increased concentration of vascular endothelial growth factor(VEGF) in the secretome. In a side project during the PhD program the hormone-relatedwomens disease lipedema was investigated. The SVF cell properties of healthy and lipedemapatients were investigated and a significant enhancement in cell yield as well as a reduction inadipogenic differentiation capacity of lipedema SVF cells was revealed. Within this workdifferent physical forces applied on adipose tissue and adipose tissue-derived cells werepresented as well as an improved isolation method and characteristics of degenerated adiposetissue. This are promising applications for the clinical use in the field of regenerative medicineand tissue regeneration.

The lymphatic system regulates tissue fluid homeostasis, intestinal fat absorption, and immune cell trafficing. Lymphedema is soft tissue swelling secondary to lymphatic dysfunction, which results in the accumulation of tissue fluid in the interstitial space. This might occur as a primary disorder of the developing lymphatic system, or alternatively lymphedema might be an acquired disorder secondary to lymphatic injury. For example, secondary lymphedema is a common problem following cancer and cancer treatments such as lymph node surgery and radiotherapy, resulting in significant morbidity. Radiotherapy is an established risk factor for lymphedema, and in addition to causing direct injury to the lymphatic vessel, it is possible that alternative mechanisms might also contribute to radiation-induced lymphatic dysfunction, such as localized ischemia of the lymphatic wall. It is also likely that predisposing genetic risk factors are at play, as not all individuals exposed to the same risk factors will develop secondary lymphedema. Lipoedema is a different form of soft tissue swelling due to the abnormal accumulation of adipose tissue. Lipoedema and lymphatic dysfunction appear to be linked, as individuals frequently develop a degree of lymphedema, particularly as the condition progresses in severity, where it may be decribed as lipo-lymphedema. The cause of lipoedema and the genetic basis of the condition are currently unknown. This thesis aims to discover and define alternative mechanisms for lymphtic dysfunction in the context of secondary lymphedema, particularly focussing on the supply of oxygenated blood to the lymphatic vessel wall. We also aim to describe inheritance patterns and the genetic factors involved in lipoedema and lipo-lymphedema. Such knowledge might uncover therapeutic targets and facilitate the development of treatments for lymphedema and lipoedema, including gene therapy.