The RhoA GTPase controls endothelial cell migration, adhesion, and barrier formation but the role of RhoB is unclear. Kovačević et al. now discover that RhoB is ubiquitinated by the CUL3–Rbx1–KCTD10 complex and that this is a prerequisite for lysosomal degradation of RhoB and the maintenance of endothelial barrier integrity.

Angiogenic homeostasis is maintained by a balance between vascular endothelial growth factor (VEGF) and Notch signaling in endothelial cells (ECs). We screened for molecules that might mediate the coupling of VEGF signal transduction with down-regulation of Notch signaling, and identified B-cell chronic lymphocytic leukemia/lymphoma6-associated zinc finger protein (BAZF). BAZF was induced by VEGF-A in ECs to bind to the Notch signaling factor C-promoter binding factor 1 (CBF1), and to promote the degradation of CBF1 through polyubiquitination in a CBF1-cullin3 (CUL3) E3 ligase complex. BAZF disruption in vivo decreased endothelial tip cell number and filopodia protrusion, and markedly abrogated vascular plexus formation in the mouse retina, overlapping the retinal phenotype seen in response to Notch activation. Further, impaired angiogenesis and capillary remodeling were observed in skin-wounded BAZF−/− mice. We therefore propose that BAZF supports angiogenic sprouting via BAZF-CUL3-based polyubiquitination-dependent degradation of CBF1 to down-regulate Notch signaling.

Perivascular adipose tissue exhibits characteristics of active local inflammation, which contributes to the development of atherosclerotic disease as a complication of obesity/metabolic syndrome. However, the precise role of perivascular adipose tissue in the progression of abdominal aortic aneurysm remains unclear. To test the hypothesis that genetic deletion of angiotensin II type 1a (AT) receptor in perivascular visceral adipose tissue (VAT) can attenuate aortic aneurysm formation in apolipoprotein E-deficient (ApoE) mice, we performed adipose tissue transplantation experiments by using an angiotensin II-induced aneurysm murine model, in which we transplanted VAT from ApoE or ApoE AT donor mice onto the abdominal aorta of ApoE recipient mice. Compared with ApoE VAT transplantation, ApoE AT VAT transplantation markedly attenuated aortic aneurysm formation, macrophage infiltration, and gelatinolytic activity in the abdominal aorta. AT receptor activation led to the polarization of macrophages in perivascular VAT toward the proinflammatory phenotype. Moreover, osteopontin expression and gelatinolytic activity were considerably lower in ApoE AT perivascular VAT than in ApoE perivascular VAT, and angiotensin II-induced osteopontin secretion from adipocytes was eliminated after deletion of AT receptor in adipocytes. Notably, induction of macrophage migration by conditioned medium from angiotensin II-stimulated wild-type adipocytes was suppressed by treatment with an osteopontin-neutralizing antibody, and ApoE OPN VAT transplantation more potently attenuated aortic aneurysm formation than ApoE VAT transplantation. Our findings indicate a previously unrecognized effect of AT receptor in perivascular VAT on the pathogenesis of abdominal aortic aneurysm.

Tumor metastasis is the most common cause of cancer death. Elucidation of the mechanism of tumor metastasis is therefore important in the development of novel, effective anti-cancer therapies to reduce cancer mortality. Interaction between cancer cells and surrounding stromal cells in the tumor microenvironment is a key factor in tumor metastasis. Using a co-culture assay system with human prostate cancer LNCaP cells and primary human prostate stromal cells, we identified epithelial membrane protein 1 (EMP1) as a gene with elevated expression in the cancer cells. The orthotopic injection of LNCaP cells overexpressing EMP1 (EMP1-LNCaP cells) into the prostate of nude mice induced lymph node and lung metastases, while that of control LNCaP cells did not. EMP1-LNCaP cells had higher cell motility and Rac1 activity than control LNCaP cells. These results were also observed in other lines of cancer cells. We newly identified copine-III as an intracellular binding partner of EMP1. Knockdown of copine-III attenuated the increased cell motility and Rac1 activity in EMP1-LNCaP cells. Reduced cell motility and Rac1 activity following knockdown of copine-III in EMP1-LNCaP cells were recovered by re-expression of wild-type copine-III, but not of a copine-III mutant incapable of interacting with EMP1, suggesting the importance of the EMP1–copine-III interaction. Phosphorylated and activated Src and a Rac guanine nucleotide exchange factor Vav2 were found to be involved in the EMP1-induced enhancement of cell motility and Rac1 activation. Moreover, EMP1 was highly expressed in prostate cancer samples obtained from patients with higher Gleason score. These results demonstrate that upregulation of EMP1 significantly increases cancer cell migration that leads to tumor metastasis, suggesting that EMP1 may play an essential role as a positive regulator of tumor metastasis.

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a type I transmembrane glycoprotein, is known as one of the most specific lymphatic vessel markers in the skin. In this study, we found that the ectodomain of LYVE-1 undergoes proteolytic cleavage, and this process produces soluble LYVE-1. We further identified the cleavage site for ectodomain shedding and generated an uncleavable mutant of LYVE-1. In lymphatic endothelial cells, ectodomain shedding of LYVE-1 was induced by vascular endothelial growth factor (VEGF)-A, an important factor for angiogenesis and lymphangiogenesis under pathological conditions. VEGF-A-induced LYVE-1 ectodomain shedding was mediated via the extracellular signal-regulated kinase (ERK) and a disintegrin and metalloproteinase (ADAM) 17. Wild-type LYVE-1, but not uncleavable LYVE-1, promoted migration of lymphatic endothelial cells in response to VEGF-A. Immunostaining analyses in human psoriasis skin lesions and VEGF-A transgenic mouse skin suggested that the ectodomain shedding of LYVE-1 occurred in lymphatic vessels undergoing chronic inflammation. These results indicate that the ectodomain shedding of LYVE-1 might be involved in promoting pathological lymphangiogenesis.Lymphatic vessels play crucial roles in maintaining tissue fluid homeostasis, immune surveillance, and fat absorption. Physiological lymphangiogenesis is regulated by several genes including Prox1 and vascular endothelial growth factor (VEGF)-C that induce the sprouting of lymphatic endothelial cells from embryonic veins during mammalian development (1, 2). Lymphatic endothelial cells express VEGF receptors (VEGFRs) 2 that represent a family of receptor tyrosine kinases. Among them, VEGFR-3 plays an essential role in promoting physiological lymphangiogenesis because VEGFR-3 shows a high affinity toward VEGF-C. In fact, several missense mutations are known among tyrosine kinase domains in Flt4/ VEGFR-3 that lead to the formation of Milroy disease, a congenital lymphedema due to an insufficient development of cutaneous lymphatic vessels. Thus, the VEGF-C/VEGFR-3 pathway plays a crucial role in promoting physiological and pathological lymphangiogenesis.VEGF-A, a specific ligand for VEGFR-1 and VEGFR-2, induces cutaneous angiogenesis in physiological and pathological conditions such as psoriasis (3). Targeted overexpression of mouse VEGF-A164 in the epidermis of transgenic mice leads to the formation of erythematous plaques resembling psoriasis (4), indicating that VEGF-A plays an important role in the pathogenesis of psoriasis. Our previous studies indicated that targeted overexpression of VEGF-A in mouse skin promotes the prominent enlargement of lymphatic vessels during acute inflammation by ultraviolet-B irradiation (5) and induces tumor-associated lymphangiogenesis as well as angiogenesis during multistep chemically induced skin carcinogenesis (6). Importantly, tumor lymphangiogenesis actively promotes enhanced metastasis to draining lymph nodes and beyond in VEGF-A transgenic mice as compared with wild-typ...