| Over the past decade, molecular details of lymphatic vessels (lymphatics) have been rapidly acquired due to the identification of lymphatic endothelial-specific markers. Separate from, but like the blood vascular system, the lymphatic system is also an elaborate network of vessels that are important in normal physiology. Lymphatic vessels function to regulate fluid homeostasis, assist in immune surveillance, and transport dietary lipids from the gut. Dysfunctional lymphatic vessels can cause pathology, and even normal lymphatics can exacerbate pathology, in some cases. Therefore, lymphatic vessels have generated interest as targets in certain diseases. However, not much is known about lymphatic signaling during adult lymphangiogenesis. The Min Laboratory is interested in signaling mechanisms that mediate vasculogenesis in pathological settings. The goal of this study was to investigate the novel hypothesis that bone marrow kinase in the X chromosome (Bmx), an established inflammatory mediator of pathological angiogenesis, promotes lymphangiogenesis. Here, I show that in wild-type mice, Bmx is upregulated in lymphatic vessels in response to Vascular Endothelial Growth Factor-A (VEGF-A) and lymph node remodeling mediated by oxazolone. In comparison to wild-type mice, Bmx-deficient mice mount weaker lymphangiogenic responses to VEGF-A, VEGF-C, and lymphatic remodeling induced by oxazolone. In vitro, Bmx is expressed in cultured human dermal microvascular lymphatic endothelial cells. Furthermore, pharmacological inhibition and short interfering RNA-mediated silencing of Bmx reduces VEGF-A and VEGF-C-induced signaling and lymphatic endothelial cell tube formation in vitro. Mechanistically, I demonstrate that Bmx differentially regulates VEGFR-2 and VEGFR-3 receptor signaling pathways: Bmx associates with and directly regulates VEGFR-2 activation, whereas Bmx associates with VEGFR-3 and regulates downstream signaling without an effect on the receptor autophosphorylation. Downstream of VEGFR-3-Bmx signaling, I identified a microRNA that fine-tunes VEGFR-3 through a negative feedback mechanism. Specifically, I present evidence that hsa-miR-1236 (miR-1236) is induced by binding of VEGF-C to VEGFR-3. VEGFR-3 can be negatively regulated by miR1236, which is basally expressed in primary human lymphatic endothelial cells. In addition to being responsive to VEGF-C, miR-1236 is also upregulated in response to IL-1beta, a negative regulator of VEGFR-3. MiR-1236 binds the 3' untranslated region of VEGFR-3, resulting in translational inhibition. Overexpression of miR-1236 significantly decreased expression of VEGFR-3, but not VEGFR-2, in human lymphatic endothelial cells. Compared to a control miR, overexpression of miR-1236 also led to decreased VEGFR-3 signaling, while VEGFR-2-specific signaling was not affected. MiR-1236 can attenuate human lymphatic endothelial cell migration and tube formation in vitro, as well as in vivo lymphangiogenesis. In summary, Bmx and MiR-1236 may represent a novel therapeutic target and tool, respectively, to inhibit lymphangiogenesis during inflammation. |
