Blood vessels sense dermal stiffness via a novel mechanotransducer, APJ.

Microvascular dysfunction accompanied by a dramatic alteration of stable capillary structure is a major hallmark of numerous age-related diseases. In skin, although the role of angiogenesis during dermal reconstitution is well documented, the functional relevance of the extracellular matrix (ECM) stiffness to vascular remodeling and its molecular mechanisms was poorly understood. Here, we developed an ex vivo 3-dimensional angiogenic model using human fat, revealing that "appropriate" stiffness induces vascular maturation associated with upregulated APJ expression, whereas the overexpression of APJ promotes the formation of large vessels even in the absence of the "appropriate" stiffness. Taken together, APJ could be a novel mechanotransducer that accelerates the maturation of cutaneous blood vessels, leading to the prevention of human skin aging.

Antiedema effects of Siberian ginseng in humans and its molecular mechanism of lymphatic vascular function in vitro.

The lymphatic system in the skin plays a major role in tissue fluid homeostasis, in the afferent phase of the immune response, and in tumor metastasis. Although lymphangiogenic factors involved in embryonic development and the metastatic spread of tumor cells have been well studied, little is known about small-molecule compounds that activate lymphatic function, especially under physiological conditions. We hypothesized that the identification of a lymphatic-activating compound could provide a method for improving edema. Here, we show that Siberian ginseng (Eleutherococcus senticosus) and its component eleutheroside E induce phosphorylation of the endothelial-specific receptor Tie2 in vitro. The activation of Tie2 on lymphatic endothelial cells (LECs) is known to stabilize lymphatic vessels, so we examined the effects of Siberian ginseng on LECs. We found that Siberian ginseng induces the migration and cord formation of LECs. Permeability assays demonstrated that it stabilizes LECs by promoting the intercellular localization of vascular endothelial cadherin, which is an endothelium-specific cell-cell adhesion molecule involved in endothelial barrier function, and it induces the phosphorylation of endothelial nitric oxide synthase by LECs. These effects appear to be mediated by the activation of Tie2 in LECs. Finally, we investigated whether the consumption of Siberian ginseng powder improves edema in a 2-way, randomized, crossover study in 50 healthy female volunteers. Edema of the lower limbs was significantly attenuated at 2 and 4hours after ingestion as compared with the control group. Thus, we demonstrate that Siberian ginseng exerts its potent antiedema activity mainly by promoting lymphatic function.

Electric current-induced lymphatic activation.

The lymphatic system in skin plays important roles in drainage of wastes and in the afferent phase of immune response. We previously showed that activation of vascular endothelial growth factor receptor (VEGFR), specifically the VEGFC/VEGFR-3 pathway, attenuates oedema and inflammation by promoting lymphangiogenesis, suggesting a protective role of lymphatic vessels against skin inflammation. However, it remains unknown how physical stimuli promote lymphatic function. Here, we show that lymphatic endothelial cells (LECs) are activated by direct-current (DC) electrical stimulation, which induced extension of actin filaments of LECs, increased calcium influx into LECs, and increased phosphorylation of p38 mitogen-activated protein kinase (MAPK). An inhibitor of focal adhesion kinase, which plays a role in cellular adhesion and motility, diminished the DC-induced extension of F-actin and abrogated p38 phosphorylation. Time-lapse imaging revealed that pulsed-DC stimulation promoted proliferation and migration of LECs. Overall, these results indicate that electro-stimulation activates lymphatic function by activating p38 MAPK.

The effect of podoplanin inhibition on lymphangiogenesis under pathological conditions.

PURPOSE: Podoplanin has been shown to be a reliable marker of lymphatic endothelium, but its role in the lymphatic system has not been well investigated. The purpose of this study is to investigate the role of podoplanin in lymphangiogenesis and macrophage functions under inflammatory conditions. METHODS: Mouse corneal suture and ear section models were used to induce lymphangiogenesis and macrophage infiltration. Antilymphatic vessel endothelial hyaluronan-1 (Anti-LYVE-1) antibody was used to visualize lymphatic vessels. Thioglycollate-induced macrophages (mps) were collected and cultured with lipopolysaccharide (LPS), IFN-γ, and anti-mouse podoplanin antibody (PMab-1). Podoplanin, NF-κB, and mitogen-activated protein kinase (MAPK) pathway expression were detected by Western blot analysis. The TNF-α secretion was measured by ELISA. RESULTS: Administration of PMab-1, reduced lymphangiogenesis in the corneal suture and ear wound healing models. Also, PMab-1 suppressed mps infiltration at the site of wound healing. Moreover, administration of PMab-1 led to a significant suppression of the rejection reaction in the corneal transplantation model. Our in vitro experiments showed that PMab-1 suppressed TNF-α secretion from mps under inflamed conditions, especially secretion caused by LPS stimulation. We confirmed the effect of PMab-1 on mps under inflamed conditions with a Western blot experiment, which clearly showed that the phosphorylation signal of the MAPK and NF-κB pathways was suppressed by PMab-1. CONCLUSIONS: Podoplanin neutralization resulted in inhibition of lymphatic growth associated with corneal and ear wound healing as well as mps inflammation. These data suggest that podoplanin is a novel therapeutic target for suppressing lymphangiogenesis and inflammation.

Apelin inhibits diet-induced obesity by enhancing lymphatic and blood vessel integrity.

Angiogenesis is tightly associated with the outgrowth of adipose tissue, leading to obesity, which is a risk factor for type 2 diabetes and hypertension, mainly because expanding adipose tissue requires an increased nutrient supply from blood vessels. Therefore, induction of vessel abnormality by adipokines has been well-studied, whereas how altered vascular function promotes obesity is relatively unexplored. Also, surviving Prox1 heterozygous mice have shown abnormal lymphatic patterning and adult-onset obesity, indicating that accumulation of adipocytes could be closely linked with lymphatic function. Here, we propose a new antiobesity strategy based on enhancement of lymphatic and blood vessel integrity with apelin. Apelin knockout (KO) mice fed a high-fat diet (HFD) showed an obese phenotype associated with abnormal lymphatic and blood vessel enlargement. Fatty acids present in the HFD induced hyperpermeability of endothelial cells, causing adipocyte differentiation, whereas apelin promoted vascular stabilization. Moreover, treatment of apelin KO mice with a selective cyclooxygenase-2 inhibitor, celecoxib, that were fed an HFD improved vascular function and also attenuated obesity. Finally, apelin transgenic mice showed decreased subcutaneous adipose tissue attributable to inhibition of HFD-induced hyperpermeability of vessels. These results indicate that apelin inhibits HFD-induced obesity by enhancing vessel integrity. Apelin could serve as a therapeutic target for treating obesity and related diseases.

Ultraviolet light-induced changes of lymphatic and blood vasculature in skin and their molecular mechanisms.

Ultraviolet light in the 290- to 320-nm wavelength range (UVB) induces angiogenesis and lymphatic dysfunction in skin. This review deals with UVB-induced alterations to the blood and lymphatic systems in skin and the molecular mechanisms involved. We also discuss potential strategies to block photoageing of skin by inhibiting angiogenesis and/or promoting lymphatic vascular function.

Promotion of lymphatic integrity by angiopoietin-1/Tie2 signaling during inflammation.

The cutaneous lymphatic system plays a major role in tissue fluid homeostasis and inflammation of the skin. Although several lymphangiogenic factors are known to be involved in the formation of lymphatic vessels, the molecular mechanisms that maintain lymphatic integrity and control the functional drainage of interstitial fluid and resolution of inflammation remain unknown. Here we show that angiopoietin-1 (Ang1) enhances lymphatic integrity and function during inflammation. Ang1 transgenic mice under the control of keratin-14 (K14-Ang1) showed attenuated edema formation and inflammation after UV B (UVB) exposure. After UVB irradiation, blood vascular permeability was inhibited in K14-Ang1 mice compared with wild-type (WT) mice. Moreover, lymphatic vessels of WT mice were markedly enlarged and leaky in inflamed skin, whereas K14-Ang1 mice showed relatively contracted lymphatic vessels together with enhanced lymphatic vascularization. Expression of endothelial-specific tight junction molecules claudin-5 and zonula occludens protein 1 (ZO-1) was strongly down-regulated in the inflamed lymphatic vessels of UVB-exposed WT mice, whereas down-regulation of both claudin-5 and ZO-1 was blocked in UVB-exposed K14-Ang1 mice. In vitro studies revealed that the stability of lymphatic endothelial cells was enhanced in the presence of Ang1, presumably via up-regulation of claudin-5, as well as ZO-1. Claudin-5 knockdown markedly increased the permeability of lymphatic endothelial cells. Overall, our data strongly support the idea that Ang1/Tie2 signaling promotes lymphatic integrity by modulating tight junction molecule expression during inflammation.

Apelin attenuates UVB-induced edema and inflammation by promoting vessel function.

Apelin, the ligand of the G protein-coupled receptor APJ, is involved in the regulation of cardiovascular functions, fluid homeostasis, and vessel formation. Recent reports indicate that apelin secreted from endothelial cells mediates APJ regulation of blood vessel caliber size; however, the function of apelin in lymphatic vessels is unclear. Here we report that APJ was expressed by human lymphatic endothelial cells and that apelin induced migration and cord formation of lymphatic endothelial cells dose-dependently in vitro. Furthermore, permeability assays demonstrated that apelin stabilizes lymphatic endothelial cells. In vivo, transgenic mice harboring apelin under the control of keratin 14 (K14-apelin) exhibited attenuated UVB-induced edema and a decreased number of CD11b-positive macrophages. Moreover, activation of apelin/APJ signaling inhibited UVB-induced enlargement of lymphatic and blood vessels. Finally, K14-apelin mice blocked the hyperpermeability of lymphatic vessels in inflamed skin. These results indicate that apelin plays a functional role in the stabilization of lymphatic vessels in inflamed tissues and that apelin might be a suitable target for prevention of UVB-induced inflammation.

Activation of the VEGFR-3 pathway by VEGF-C attenuates UVB-induced edema formation and skin inflammation by promoting lymphangiogenesis.

We have previously demonstrated that UVB irradiation resulted in impaired function of cutaneous lymphatic vessels, suggesting a crucial role of lymphatic function in the mediation of UVB-induced inflammation. Nonetheless, the molecular mechanisms of lymphatic involvement in inflammation have remained unclear. Here, we show that vascular endothelial growth factor (VEGF)-C expression is downregulated after UVB irradiation, associated with enlargement of lymphatic vessels and with an increase of macrophage infiltration in the dermis. To determine whether activation of VEGF-C/VEGFR-3 signaling might reduce UVB-induced inflammation, mice were exposed to a single dose of UVB irradiation together with intradermal injection of mutant VEGF-C (Cys156Ser), which specifically binds to VEGFR-3 on lymphatic endothelium. We found that the activation of VEGFR-3 attenuated UVB-induced edema formation, associated with a decreased number of CD11b-positive macrophages. Moreover, mutant VEGF-C injection inhibited UVB-induced enlargement of lymphatic vessels and also induced the proliferation of lymphatic endothelial cells. In contrast, treatment with mutant VEGF-C had no effect on blood vessel size or number. These results demonstrate that UVB-induced lymphatic impairment is mediated by downregulation of VEGF-C expression and that the activation of the VEGF-C/VEGFR-3 pathway might represent a feasible target for the prevention of UVB-induced inflammation by promoting lymphangiogenesis.