Lymphatic vasculature requires estrogen receptor alpha signaling to protect from lymphedema

Rationale: Estrogen play a crucial role on the cardiovascular system and, particularly, on the vascular endothelium. However, the effect of estrogen on the lymphatic system has been poorly investigated. Objective: We aimed to demonstrate the protective effect of the 17 estradiol (E2), the most potent endogenous estrogen in lymphedema, a lymphatic dysfunction, which results in a massive fluid and fat accumulation. Methods and Results: Immunohistochemistry revealed that high doses of E2 modified skin microenvironment and stimulated lymphangiogenesis. E2 activates lymphatic endothelium through a transcriptional activation induced by its receptor ER. Using an original model of secondary lymphedema, we found a protective effect of E2 that prevents limb swelling. Loss of ER function using selective estrogen modulator (SERM) and in Tie2 Cre(+); ER-/- mice lead to a disruption of the lymphatic network promoting lymphedema. This effect was associated with a remodeling of LEC, an inhibition of gene expression, and ERK, but not AKT E2-induced phosphorylation. Conclusion: These findings reveal a new facet of the influence of estrogens in the management of the lymphatic system in pathological condition and provides more evidences that secondary lymphedema is not only a side effect of surgery, but is worsen by hormone therapy

Hypoxia Induces VEGF-C Expression in Metastatic Tumor Cells via a HIF-1α-Independent Translation-Mediated Mechanism

SummaryVarious tumors metastasize via lymph vessels and lymph nodes to distant organs. Even though tumors are hypoxic, the mechanisms of how hypoxia regulates lymphangiogenesis remain poorly characterized. Here, we show that hypoxia reduced vascular endothelial growth factor C (VEGF-C) transcription and cap-dependent translation via the upregulation of hypophosphorylated 4E-binding protein 1 (4E-BP1). However, initiation of VEGF-C translation was induced by hypoxia through an internal ribosome entry site (IRES)-dependent mechanism. IRES-dependent VEGF-C translation was independent of hypoxia-inducible factor 1α (HIF-1α) signaling. Notably, the VEGF-C IRES activity was higher in metastasizing tumor cells in lymph nodes than in primary tumors, most likely because lymph vessels in these lymph nodes were severely hypoxic. Overall, this transcription-independent but translation-dependent upregulation of VEGF-C in hypoxia stimulates lymphangiogenesis in tumors and lymph nodes and may contribute to lymphatic metastasis

Single and combined impacts of irradiation and surgery on lymphatic vasculature and fibrosis associated to secondary lymphedema

peer reviewedLymphedema (LD) refers to a condition of lymphatic dysfunction associated with excessive fluid accumulation, fibroadipose tissue deposition and swelling. In industrialized countries, LD development mainly results from a local disruption of the lymphatic network by an infection or cancer-related surgery (secondary LD). In the absence of efficient therapy, animal models are needed to decipher the cellular and molecular mechanisms underlying LD and test putative drugs. In this study, we optimized and characterized a murine model of LD that combines an irradiation of the mice hind limb and a radical surgery (lymph node resection associated to lymphatic vessel ligation). We investigated the respective roles of irradiation and surgery in LD formation by comparing their impacts, alone or in combination (with different intervention sequences), on eight different features of the pathology: swelling (paw thickness), indocyanine green (ICG) clearance, lymphatic vasculature remodeling, epidermal and dermal thickening, adipocyte accumulation, inflammatory cell infiltration and collagen deposition. This study supports the importance of radiation prior to surgery to experimentally induce a rapid, severe and sustained tissue remodeling harboring the different hallmarks of LD. We provide the first experimental evidence for an excessive deposition of periostin (POSTN) and tenascin-C (TNC) in LD. Through a computerized method of digital image quantification, we established the spatial map of lymphatic expansion, as well as collagen, POSTN and TNC deposition in papillary and reticular dermis of lymphedematous skins. This mouse model is available to study the patho-physiology of LD and test potential therapeutic targets

Development and characterization of a microfluidic model of the tumour microenvironment

The physical microenvironment of tumours is characterized by heterotypic cell interactions and physiological gradients of nutrients, waste products and oxygen. This tumour microenvironment has a major impact on the biology of cancer cells and their response to chemotherapeutic agents. Despite this, most in vitro cancer research still relies primarily on cells grown in 2D and in isolation in nutrient- and oxygen-rich conditions. Here, a microfluidic device is presented that is easy to use and enables modelling and study of the tumour microenvironment in real-time. The versatility of this microfluidic platform allows for different aspects of the microenvironment to be monitored and dissected. This is exemplified here by real-time profiling of oxygen and glucose concentrations inside the device as well as effects on cell proliferation and growth, ROS generation and apoptosis. Heterotypic cell interactions were also studied. The device provides a live ‘window’ into the microenvironment and could be used to study cancer cells for which it is difficult to generate tumour spheroids. Another major application of the device is the study of effects of the microenvironment on cellular drug responses. Some data is presented for this indicating the device’s potential to enable more physiological in vitro drug screening

Hypoxia Induces VEGF-C Expression in Metastatic Tumor Cells via a HIF-1 α-Independent Translation-Mediated Mechanism.

Various tumors metastasize via lymph vessels and lymph nodes to distant organs. Even though tumors are hypoxic, the mechanisms of how hypoxia regulates lymphangiogenesis remain poorly characterized. Here, we show that hypoxia reduced vascular endothelial growth factor C (VEGF-C) transcription and cap-dependent translation via the upregulation of hypophosphorylated 4E-binding protein 1 (4E-BP1). However, initiation of VEGF-C translation was induced by hypoxia through an internal ribosome entry site (IRES)-dependent mechanism. IRES-dependent VEGF-C translation was independent of hypoxia-inducible factor 1α (HIF-1α) signaling. Notably, the VEGF-C IRES activity was higher in metastasizing tumor cells in lymph nodes than in primary tumors, most likely because lymph vessels in these lymph nodes were severely hypoxic. Overall, this transcription-independent but translation-dependent upregulation of VEGF-C in hypoxia stimulates lymphangiogenesis in tumors and lymph nodes and may contribute to lymphatic metastasis

Induction of WNT11 by hypoxia and hypoxia-inducible factor-1α regulates cell proliferation, migration and invasion

Changes in cellular oxygen tension play important roles in physiological processes including development and pathological processes such as tumor promotion. The cellular adaptations to sustained hypoxia are mediated by hypoxia-inducible factors (HIFs) to regulate downstream target gene expression. With hypoxia, the stabilized HIF-α and aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIF-β) heterodimer bind to hypoxia response elements (HREs) and regulate expression of target genes. Here, we report that WNT11 is induced by hypoxia in many cell types, and that transcription of WNT11 is regulated primarily by HIF-1α. We observed induced WNT11 expression in the hypoxic area of allograft tumors. In addition, in mice bearing orthotopic malignant gliomas, inhibition with bevacizumab of vascular endothelial growth factor, which is an important stimulus for angiogenesis, increased nuclear HIF-1α and HIF-2α, and expression of WNT11. Gain- and loss-of-function approaches revealed that WNT11 stimulates proliferation, migration and invasion of cancer-derived cells, and increases activity of matrix metalloproteinase (MMP)-2 and 9. Since tumor hypoxia has been proposed to increase tumor aggressiveness, these data suggest WNT11 as a possible target for cancer therapies, especially for tumors treated with antiangiogenic therapy

Molecular regulations of lymphangiogenic growth factors in vascular pathologies

Le système lymphatique a pour rôle principal de drainer les fluides tissulaires et de participer à la surveillance immune. La lymphangiogenèse est principalement activée par deux facteurs : les vascular endothelial growth factors-C et -D (VEGF-C et VEGF-D). Ces facteurs participent à la progression métastatique à la fois en stimulant la prolifération des vaisseaux lymphatiques dans la tumeur et en périphérie et en provoquant la dilatation des vaisseaux collecteurs facilitant ainsi le passage des cellules tumorales de la tumeur primaire aux ganglions lymphatiques. Lors de son développement, une tumeur est soumise à de nombreux stress cellulaires tels que l'hypoxie, l'inflammation ou la déprivation en nutriments. Ma thèse a donc été consacrée à l'analyse des régulations moléculaires permettant une surexpression des facteurs lymphangiogéniques en condition de stress cellulaire. Lors d'un premier projet de recherche j'ai démontré que l'hypoxie réduit la transcription de VEGF-C ainsi que sa traduction coiffe-dépendante mais activait une traduction médiée par un site interne d'entrée du ribosome (IRES). De plus, cette activation de la synthèse de VEGF-C en hypoxie est indépendante de HIF-1a et stimule la lymphangiogenèse dans les tumeurs ainsi que dans les ganglions lymphatiques participant ainsi à la dissémination tumorale. Dans un second temps, j'ai démontré que le VEGF-D possédait lui aussi un IRES activé par un choc thermique. L'activité de l'IRES du VEGF-D est régulée par la localisation subcellulaire d'un facteur trans-activateur de l'IRES (ITAF) : la nucléoline. Cette protéine est exportée du noyau vers le cytoplasme lors d'un choc thermique. Ce processus est inhibé par un traitement avec un anti-inflammatoire non stéroïdien ce qui supprime l'activation de la traduction IRES dépendante du VEGF-D. Enfin lors d'un troisième projet, j'ai travaillé sur le lymphœdème secondaire, une pathologie caractérisée par une destruction du système lymphatique où la lymphangiogenèse doit être stimulée. J'ai évalué l'impact de l'hormonothérapie, principal traitement du cancer du sein, sur l'expression du VEGF-C et -D. J'ai démontré que l'estradiol stimulait l'expression de ces facteurs alors que l'hormonothérapie diminue leur synthèse et provoque une destruction de l'endothélium lymphatique. En conclusion, mes trois projets de thèse m'ont permis d'étudier les régulations moléculaires des deux facteurs lymphangiogéniques principaux dans des pathologies provoquées par une lymphangiogenèse excessive ou insuffisante.Tumor lymphangiogenesis promotes lymph node metastasis using two mechanisms consisting of lymphatic vessels proliferation and dilatation to facilitate tumor spread. Lymphangiogenesis is mainly promoted by two growth factors: the vascular endothelial growth factor C and D. My PhD was focused on the molecular regulations inducing a tumoral over-expression of these two factors during stress. I demonstrated that hypoxia reduced VEGF-C transcription and cap-dependent translation while activating an Internal Ribosome Entry Site (IRES)-dependent mechanism of translation. This upregulation of VEGF-C in hypoxia is independent of HIF-1a; and stimulates lymphangiogenesis in tumors and lymph nodes and contribute to lymphatic metastasis. Then, I have discovered that VEGF-D had an IRES selectively activated by heat shock. VEGF-D IRES is regulated by a subcellular relocalization of an ITAF, the nucleolin. This molecular process is reversed by non-steroidal anti-inflammatory drugs (NSAID) that decrease VEGF-D IRES translation initiation by targeting the nucleolin. Finally, I worked on secondary lymphedema, a pathology that is characterized by a disruption of the lymphatic network where lymphangiogenesis thus need to be restored. In this condition, I have evaluated the impact of hormone therapy, the main treatment for breast cancer, on the regulation of lymphangiogenic factors. I found that estradiol stimulates the expression of VEGF-C and-D contrary to the hormone therapy that inhibits VEGF-C and -D and thus mediates a disruption of the lymphatic endothelium. Taken together, my PhD projects have allowed me to study the regulations of VEGF-C and -D in pathologies mediated either by an excessive or an insufficient lymphangiogenesis