Peroxiredoxin II is an essential antioxidant enzyme that prevents the oxidative inactivation of VEGF receptor-2 in vascular endothelial cells.

Cellular antioxidant enzymes play crucial roles in aerobic organisms by eliminating detrimental oxidants and maintaining the intracellular redox homeostasis. Therefore, the function of antioxidant enzymes is inextricably linked to the redox-dependent activities of multiple proteins and signaling pathways. Here, we report that the VEGFR2 RTK has an oxidation-sensitive cysteine residue whose reduced state is preserved specifically by peroxiredoxin II (PrxII) in vascular endothelial cells. In the absence of PrxII, the cellular H(2)O(2) level is markedly increased and the VEGFR2 becomes inactive, no longer responding to VEGF stimulation. Such VEGFR2 inactivation is due to the formation of intramolecular disulfide linkage between Cys1199 and Cys1206 in the C-terminal tail. Interestingly, the PrxII-mediated VEGFR2 protection is achieved by association of two proteins in the caveolae. Furthermore, PrxII deficiency suppresses tumor angiogenesis in vivo. This study thus demonstrates a physiological function of PrxII as the residential antioxidant safeguard specific to the redox-sensitive VEGFR2.

Propranolol increases vascular permeability through pericyte apoptosis and exacerbates oxygen-induced retinopathy.

Retinopathy of prematurity (ROP) is an eye disease that causes blindness due to delayed vascular growth, retinal ischemia, and resulting abnormal angiogenesis. Nonselective ß-antagonist propranolol is in clinical trials for the treatment of ROP due to its effect of reducing VEGF expression and inhibiting retinal angiogenesis in oxygen-induced ROP models (OIR), but the mechanism by which propranolol acts on ROP vessels is still unclear. In the present study, we have focused on the effect of propranolol on pericyte survival and vascular permeability. We demonstrated that propranolol increases pericyte apoptosis more sensitively than endothelial cells (ECs), thereby weakening EC tight junctions to increase endothelial permeability in co-cultures of pericytes and ECs. Mechanistically, pericyte apoptosis by propranolol was due to the inhibition of Akt signaling pathway. We also demonstrated that propranolol increases pericyte loss and vascular permeability of retinal vessels in a mouse model of OIR. These results suggest that propranolol may be negative for blood vessels in retinas of OIR, and that the efficacy of propranolol for the treatment of ROP needs to be more thoroughly verified.

Conditional ablation of LYVE-1+ cells unveils defensive roles of lymphatic vessels in intestine and lymph nodes.

To unveil the organotypic role and vulnerability of lymphatic vessels, we generated a lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)-Cre/iDTR double-transgenic mouse and ablated LYVE-1-expressing lymphatic vessels in adult mice in a diphtheria toxin (DT)-inducible manner based on selective expression of LYVE-1 in most lymphatic vessels. Strikingly, lymphatic vessels in the small intestine and lymph nodes were rapidly ablated, but lymphatic vessels in the other organs were relatively intact at 24 hours after DT administration. Unexpectedly, LYVE-1-Cre/iDTR mice died of sepsis without visible edema at 24 and 60 hours after DT administration. The cause of death appeared to be related to acute failure of immune surveillance systems in the small intestine and draining lymph nodes. Of note, acute loss of lymphatic lacteals in intestinal villi appeared to trigger distortion of blood capillaries and the whole architecture of the villi, whereas acute loss of lymphatic vessels in lymph nodes caused dysfunction of lymph drainage and abnormal distribution of dendritic cells and macrophages. Thus, intact lymphatic vessels are required for structural and functional maintenance of surrounding tissues in an organotypic manner, at least in the intestine and lymph nodes.

Endothelial deletion of phospholipase D2 reduces hypoxic response and pathological angiogenesis.

OBJECTIVE: Aberrant regulation of the proliferation, survival, and migration of endothelial cells (ECs) is closely related to the abnormal angiogenesis that occurs in hypoxia-induced pathological situations, such as cancer and vascular retinopathy. Hypoxic conditions and the subsequent upregulation of hypoxia-inducible factor-1α and target genes are important for the angiogenic functions of ECs. Phospholipase D2 (PLD2) is a crucial signaling mediator that stimulates the production of the second messenger phosphatidic acid. PLD2 is involved in various cellular functions; however, its specific roles in ECs under hypoxia and in vivo angiogenesis remain unclear. In the present study, we investigated the potential roles of PLD2 in ECs under hypoxia and in hypoxia-induced pathological angiogenesis in vivo. APPROACH AND RESULTS: Pld2 knockout ECs exhibited decreased hypoxia-induced cellular responses in survival, migration, and thus vessel sprouting. Analysis of hypoxia-induced gene expression revealed that PLD2 deficiency disrupted the upregulation of hypoxia-inducible factor-1α target genes, including VEGF, PFKFB3, HMOX-1, and NTRK2. Consistent with this, PLD2 contributed to hypoxia-induced hypoxia-inducible factor-1α expression at the translational level. The roles of PLD2 in hypoxia-induced in vivo pathological angiogenesis were assessed using oxygen-induced retinopathy and tumor implantation models in endothelial-specific Pld2 knockout mice. Pld2 endothelial-specific knockout retinae showed decreased neovascular tuft formation, despite a larger avascular region. Tumor growth and tumor blood vessel formation were also reduced in Pld2 endothelial-specific knockout mice. CONCLUSIONS: Our findings demonstrate a novel role for endothelial PLD2 in the survival and migration of ECs under hypoxia via the expression of hypoxia-inducible factor-1α and in pathological retinal angiogenesis and tumor angiogenesis in vivo.

Vascular endothelial growth factor-angiopoietin chimera with improved properties for therapeutic angiogenesis.

BACKGROUND: There is an unmet need for proangiogenic therapeutic molecules for the treatment of tissue ischemia in cardiovascular diseases. However, major inducers of angiogenesis such as vascular endothelial growth factor (VEGF/VEGF-A) have side effects that limit their therapeutic utility in vivo, especially at high concentrations. Angiopoietin-1 has been considered to be a blood vessel stabilization factor that can inhibit the intrinsic property of VEGF to promote vessel leakiness. In this study, we have designed and tested the angiogenic properties of chimeric molecules consisting of receptor-binding parts of VEGF and angiopoietin-1. We aimed at combining the activities of both factors into 1 molecule for easy delivery and expression in target tissues. METHODS AND RESULTS: The VEGF-angiopoietin-1 (VA1) chimeric protein bound to both VEGF receptor-2 and Tie2 and induced the activation of both receptors. Detailed analysis of VA1 versus VEGF revealed differences in the kinetics of VEGF receptor-2 activation and endocytosis, downstream kinase activation, and VE-cadherin internalization. The delivery of a VA1 transgene into mouse skeletal muscle led to increased blood flow and enhanced angiogenesis. VA1 was also very efficient in rescuing ischemic limb perfusion. However, VA1 induced less plasma protein leakage and myeloid inflammatory cell recruitment than VEGF. Furthermore, angioma-like structures associated with VEGF expression were not observed with VA1. CONCLUSIONS: The VEGF-angiopoietin-1 chimera is a potent angiogenic factor that triggers a novel mode of VEGF receptor-2 activation, promoting less vessel leakiness, less tissue inflammation, and better perfusion in ischemic muscle than VEGF. These properties of VA1 make it an attractive therapeutic tool.

Adipose tissue is an extramedullary reservoir for functional hematopoietic stem and progenitor cells.

The stromal vascular fraction (SVF) in adipose tissue contains a pool of various stem and progenitor cells, but the existence of hematopoietic stem and progenitor cells (HSPCs) in the SVF has not been seriously considered. We detected the presence of HSPCs in the SVF by phenotypically probing with Lin(-)Sca-1(+)c-kit(+) (LSK) and functionally confirming the presence using colony-forming cell assay and assessing the long-term multilineage reconstitution ability after SVF transplantation. The LSK population in the SVF was 0.004% plus or minus 0.001%, and 5 x 10(5) freshly isolated SVF cells gave rise to 13 plus or minus 4 multilineage colonies. In addition, 0.15% plus or minus 0.03% of SVF cells was home to bone marrow (BM), especially near vascular and endosteal regions, 24 hours after blood transplantation. SVF transplantation was capable of generating a long-term (> 16 weeks), but variable extent (2.1%-32.1%) multilineage reconstitution in primary recipients, which was subsequently transferred to the secondary recipients by BM transplantation. All HSPCs within the SVF originated from the BM. Furthermore, the granulocyte-colony-stimulating factor (G-CSF) mobilization of HSPCs from BM markedly elevated the number of phenotypic and functional HSPCs in the SVF, which induced a high efficiency long-term reconstitution in multilineage hematopoiesis in vivo. Our results provide compelling evidence that adipose tissue is a novel extramedullary tissue possessing phenotypic and functional HSPCs.

Intracavernous delivery of freshly isolated stromal vascular fraction rescues erectile function by enhancing endothelial regeneration in the streptozotocin-induced diabetic mouse.

INTRODUCTION: Men with diabetic erectile dysfunction (ED) often have severe endothelial dysfunction and respond poorly to oral phosphodiesterase-5 inhibitors. AIM: To examine whether and how freshly isolated stromal vascular fraction (SVF) promotes cavernous endothelial regeneration and restores erectile function in diabetic animals. METHODS: Eight-week-old C57BL/6J mice were used. Diabetes was induced by intraperitoneal injection of streptozotocin. SVF was isolated from epididymal adipose tissues of green fluorescence protein transgenic mice. At 8 weeks after the induction of diabetes, the animals were divided into six groups: controls, diabetic mice, and diabetic mice treated with a single intracavernous injection of phosphate-buffered saline (PBS) or SVF (1 × 10(4) cells, 1 × 10(5) cells, or 2 × 10(5) cells/20 µL, respectively). MAIN OUTCOME MEASURES: Two weeks later, erectile function was measured by cavernous nerve stimulation. The penis was stained with antibodies to CD31, CD34, phosphohistone H3, phospho-endothelial nitric oxide synthase (eNOS), and vascular endothelial growth factor-A (VEGF-A). We also performed Western blot for phospho-eNOS and eNOS, and determined cyclic guanosine monophosphate (cGMP) concentration in the corpus cavernosum tissue. RESULTS: Significant improvement in erectile function was noted in diabetic mice treated with SVF at concentrations of 1 × 10(5) and 2 × 10(5) cells, which reached up to 82% of the control values. Local delivery of SVF significantly increased cavernous endothelial cell proliferation, eNOS phosphorylation, and cGMP expression compared with that in the untreated group and the PBS-treated diabetic group. Intracavernous injection of SVF increased cavernous VEGF-A expression and induced recruitment of CD34(+)CD31(-) progenitor cells. Some SVF underwent differentiation into cavernous endothelial cells. SVF-induced promotion of cavernous angiogenesis and erectile function was abolished in the presence of VEGF-Trap, a soluble VEGF-A neutralizing antibody. CONCLUSION: The results support the concept of cavernous endothelial regeneration by use of SVF as a curative therapy for diabetic ED.

Stromal vascular fraction from adipose tissue forms profound vascular network through the dynamic reassembly of blood endothelial cells.

OBJECTIVE: Tremendous efforts have been made to establish effective therapeutic neovascularization using adipose tissue-derived stromal vascular fraction (SVF), but the efficiency is low, and underlying mechanisms and their interaction with the host in a new microenvironment are poorly understood. METHODS AND RESULTS: Here we demonstrate that direct implantation of SVF derived from donor adipose tissue can create a profound vascular network through the disassembly and reassembly of blood endothelial cells at the site of implantation. This neovasculature successfully established connection with recipient blood vessels to form a functionally perfused circuit. Addition of vascular growth factors to the SVF implant improved the efficiency of functional neovasculature formation. In contrast, spheroid culture of SVF before implantation reduced the capacity of vasculature formation, possibly because of cellular alteration. Implanting SVF into the mouse ischemic hindlimb induced the robust formation of a local neovascular network and salvaged the limb. Moreover, the coimplantation of SVF prevented fat absorption in the subcutaneous adipose tissue graft model. CONCLUSIONS: Freshly isolated SVF can effectively induce new vessel formation through the dynamic reassembly of blood endothelial cells and could be applied to achieve therapeutic neovascularization for relieving ischemia and preventing fat absorption in an autologous manner.

Adipocytokine orosomucoid integrates inflammatory and metabolic signals to preserve energy homeostasis by resolving immoderate inflammation.

Orosomucoid (ORM), also called alpha-1 acid glycoprotein, is an abundant plasma protein that is an immunomodulator induced by stressful conditions such as infections. In this study, we reveal that Orm is induced selectively in the adipose tissue of obese mice to suppress excess inflammation that otherwise disturbs energy homeostasis. Adipose Orm levels were elevated by metabolic signals, including insulin, high glucose, and free fatty acid, as well as by the proinflammatory cytokine tumor necrosis factor-alpha, which is found in increased levels in the adipose tissue of morbid obese subjects. In both adipocytes and macrophages, ORM suppressed proinflammatory gene expression and pathways such as NF-kappaB and mitogen-activated protein kinase signalings and reactive oxygen species generation. Concomitantly, ORM relieved hyperglycemia-induced insulin resistance as well as tumor necrosis factor-alpha-mediated lipolysis in adipocytes. Accordingly, ORM improved glucose and insulin tolerance in obese and diabetic db/db mice. Taken together, our results suggest that ORM integrates inflammatory and metabolic signals to modulate immune responses to protect adipose tissue from excessive inflammation and thereby from metabolic dysfunction.

Anti-Angiogenic Property of Free Human Oligosaccharides.

Angiogenesis, a fundamental process in human physiology and pathology, has attracted considerable attention owing to its potential as a therapeutic strategy. Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) are deemed major mediators of angiogenesis. To date, inhibition of the VEGF-A/VEGFR-2 axis has been an effective strategy employed in the development of anticancer drugs. However, some limitations, such as low efficacy and side effects, need to be addressed. Several drug candidates have been discovered, including small molecule compounds, recombinant proteins, and oligosaccharides. In this review, we focus on human oligosaccharides as modulators of angiogenesis. In particular, sialylated human milk oligosaccharides (HMOs) play a significant role in the inhibition of VEGFR-2-mediated angiogenesis. We discuss the structural features concerning the interaction between sialylated HMOs and VEGFR-2 as a molecular mechanism of anti-angiogenesis modulation and its effectiveness in vivo experiments. In the current state, extensive clinical trials are required to develop a novel VEGFR-2 inhibitor from sialylated HMOs.

Anti-Neuroinflammatory Effects and Mechanism of Action of Fructus ligustri lucidi Extract in BV2 Microglia.

For centuries, Fructus ligustri lucidi (FLL; the fruit of Ligustrum lucidum Aiton or Ligustrum japonicum Thunb.) has been commonly used in traditional Chinese medicine for treating hepatitis and aging-related symptoms and in traditional Korean medicine to detoxify kidneys and the liver. Pharmacological research has shown FLL has antioxidant, anti-inflammatory, anticancer, anti-osteoporosis, and hepatoprotective activities. This study was undertaken to investigate the effects of FLL extract (FLLE) on neuroinflammation. After setting a non-toxic concentration using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay data, we investigated the effects of FLLE using Western blotting, cell migration, enzyme-linked immunosorbent assay, a nitric oxide (NO) assay, and immunofluorescence staining in lipopolysaccharide (LPS)-stimulated murine BV2 microglial cells. FLLE was non-toxic to BV2 cells up to a concentration of 500 µg/mL and concentration-dependently inhibited the production of NO and prostaglandin E2 and the protein levels of inducible nitric oxide synthase and cyclooxygenase-2 under LPS-induced inflammatory conditions. It also inhibited the secretion of the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Furthermore, FLLE pretreatment attenuated LPS-induced increases of CD68 (a marker of microglia activation) and suppressed the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) signaling pathways in LPS-stimulated BV2 cells, and significantly increased heme oxygenase (HO)-1 levels. FLLE also reduced the LPS-induced increase in the migratory ability of BV2 cells and the phosphorylation of vascular endothelial growth factor receptor 1. Collectively, FLLE effectively inhibited inflammatory response by suppressing the MAPK and NF-κB signaling pathways and inducing HO-1 in LPS-stimulated BV2 microglial cells. Our findings provide a scientific basis for further study of FLL as a candidate for preventing or alleviating neuroinflammation.

Wrist-wearable bioelectrical impedance analyzer with miniature electrodes for daily obesity management.

Bioelectrical impedance analysis (BIA) is used to analyze human body composition by applying a small alternating current through the body and measuring the impedance. The smaller the electrode of a BIA device, the larger the impedance measurement error due to the contact resistance between the electrode and human skin. Therefore, most commercial BIA devices utilize electrodes that are large enough (i.e., 4 × 1400 mm2) to counteract the contact resistance effect. We propose a novel method of compensating for contact resistance by performing 4-point and 2-point measurements alternately such that body impedance can be accurately estimated even with considerably smaller electrodes (outer electrodes: 68 mm2; inner electrodes: 128 mm2). Additionally, we report the use of a wrist-wearable BIA device with single-finger contact measurement and clinical test results from 203 participants at Seoul St. Mary's Hospital. The correlation coefficient and standard error of estimate of percentage body fat were 0.899 and 3.76%, respectively, in comparison with dual-energy X-ray absorptiometry. This result exceeds the performance level of the commercial upper-body portable body fat analyzer (Omron HBF-306). With a measurement time of 7 s, this sensor technology is expected to provide a new possibility of a wearable bioelectrical impedance analyzer, toward obesity management.

Effects of Ginsenoside Rg3 on Inhibiting Differentiation, Adipogenesis, and ER Stress-Mediated Cell Death in Brown Adipocytes.

OBJECTIVES: Ginsenoside Rg3 (Rg3), a main active component of Panax ginseng, has various therapeutic properties in literatures, and it has been studied for its potential use in obesity control due to its antiadipogenic effects in white adipocytes. However, little is known about its effects on brown adipocytes. METHODS: The mechanisms through which Rg3 inhibits differentiation, adipogenesis, and ER stress-mediated cell death in mouse primary brown adipocytes (MPBAs) are explored. RESULTS: Rg3 significantly induced cytotoxicity in differentiated MPBAs but not in undifferentiated MPBAs. Rg3 treatment downregulated the expression of differentiation and adipogenesis markers and the level of perilipin in MPBAs while upregulating the expression of lipolytic Kruppel-like factor genes. Rg3 also induced lipolysis and efflux of triglycerides from MPBAs and subsequently increased proinflammatory cytokine levels. Notably, Rg3 treatment resulted in elevation of ER stress and proapoptotic markers in MPBAs. CONCLUSIONS: Our results demonstrate that Rg3 is able to selectively exert cytotoxicity in differentiated MPBAs while leaving undifferentiated MPBAs intact, resulting in the induction of ER stress and subsequent cell death in MPBAs via regulation of various genes related to adipocyte differentiation, adipogenesis, lipolysis, and inflammation. These results indicate that further studies on the potential therapeutic applications of Rg3 are warranted.

Bone marrow-derived circulating progenitor cells fail to transdifferentiate into adipocytes in adult adipose tissues in mice.

Little is known about whether bone marrow-derived circulating progenitor cells (BMDCPCs) can transdifferentiate into adipocytes in adipose tissues or play a role in expanding adipocyte number during adipose tissue growth. Using a mouse bone marrow transplantation model, we addressed whether BMDCPCs can transdifferentiate into adipocytes under standard conditions as well as in the settings of diet-induced obesity, rosiglitazone treatment, and exposure to G-CSF. We also addressed the possibility of transdifferentiation to adipocytes in a murine parabiosis model. In each of these settings, our findings indicated that BMDCPCs did not transdifferentiate into either unilocular or multilocular adipocytes in adipose tissues. Most BMDCPCs became resident and phagocytic macrophages in adipose tissues--which resembled transdifferentiated multilocular adipocytes by appearance, but displayed cell surface markers characteristic for macrophages - in the absence of adipocyte marker expression. When exposed to adipogenic medium in vitro, bone marrow cells differentiated into multilocular, but not unilocular, adipocytes, but transdifferentiation was not observed in vivo, even in the contexts of adipose tissue regrowth or dermal wound healing. Our results suggest that BMDCPCs do not transdifferentiate into adipocytes in vivo and play little, if any, role in expanding the number of adipocytes during the growth of adipose tissues.

Role of CD11b+ macrophages in intraperitoneal lipopolysaccharide-induced aberrant lymphangiogenesis and lymphatic function in the diaphragm.

Lymphatic vessels in the diaphragm are essential for draining peritoneal fluid, but little is known about their pathological changes during inflammation. Here we characterized diaphragmatic lymphatic vessels in a peritonitis model generated by daily i.p. administration of lipopolysaccharide (LPS) in mice. Intraperitoneal LPS increased lymphatic density, branching, sprouts, connections, and network formation in the diaphragm in time- and dose-dependent manners. These changes were reversible on discontinuation of LPS administration. The LPS-induced lymphatic density and remodeling occur mainly through proliferation of lymphatic endothelial cells. CD11b+ macrophages were massively accumulated and closely associated with the lymphatic vessels changed by i.p. LPS. Both RT-PCR assays and experiments with vascular endothelial growth factor-C/D blockade and macrophage-depletion indicated that the CD11b+ macrophage-derived lymphangiogenic factors vascular endothelial growth factor-C/D could be major mediators of LPS-induced lymphangiogenesis and lymphatic remodeling through paracrine activity. Functional assays with India ink and fluorescein isothiocyanate-microspheres indicated that impaired peritoneal fluid drainage in diaphragm of LPS-induced peritonitis mice was due to inflammatory fibrosis and massive attachment of CD11b+ macrophages on the peritoneal side of the diaphragmatic lymphatic vessels. These findings reveal that CD11b+ macrophages play an important role in i.p. LPS-induced aberrant lymphangiogenesis and lymphatic dysfunction in the diaphragm.

Angiopoietin-2 exocytosis is stimulated by sphingosine-1-phosphate in human blood and lymphatic endothelial cells.

OBJECTIVE: Although diverse functions of angiopoietin-2 (Ang2) have been revealed, little is known about upstream signaling molecules regulating Ang2 exocytosis. We therefore investigated the mechanism of Ang2 exocytosis in human blood and lymphatic endothelial cells (BECs and LECs) by stimulation with sphingosine-1-phosphate (S1P). METHODS AND RESULTS: By immunostaining and ELISA analyses using our newly developed human Ang2-specific antibodies, Ang2 exocytosis from human endothelial cells was examined. Both exogenous and endogenous S1P trigger rapid Ang2 exocytosis in time- and dose-dependent manners. Intriguingly, S1P-induced Ang2 exocytosis is higher in LECs than BECs. These effects of S1P are mainly mediated by the endothelial differentiation gene receptor 1, which subsequently activates its downstream phospholipase C and intracellular calcium mobilization to trigger Ang2 exocytosis. Consistently, S1P also dramatically stimulates Ang2 exocytosis from the ECs of ex vivo-incubated blood vessels. CONCLUSION: These results imply that the rapid secretion of Ang2 by exocytosis from endothelial cells is another possible mechanism underlying S1P-induced angiogenesis and inflammation.

Effects of exercise training and detraining on atheromatous matrix metalloproteinase activity in mice.

BACKGROUND AND AIMS: Exercise training (ET) helps treat atherosclerosis. However, many patients stop regular ET for various reasons. The effect of detraining on atherosclerosis is not well studied. We examined the effects of ET vs. short-term detraining on atheromatous matrix-metalloproteinase (MMP) activity in preexisting plaque and circulating cytokines/lipids. METHODS AND RESULTS: Eighteen-week-old apolipoprotein-E-/- mice (n = 56) on a Western diet underwent: 1) ET for 6-weeks (ET5+1), 2) ET for 5-weeks and detraining for 1-week (ET5+0), 3) ET for the last 1-week (ET0+1), or 4) no treadmill ET at all for 6-weeks (ET0+0). Atheromatous MMP-activity was visualized using molecular imaging with an MMP-2/9-activatable near-infrared-fluorescent probe. Compared with no ET (ET0+0), regular ET (ET5+1) decreased carotid atheromatous MMP activity, but this protective effect was significantly blunted by short-term detraining (ET5+0). Short-term detraining after longer-term ET showed a reduction in MMP-activity similar to short-term ET (ET0+1). Blood levels of lipids and cytokines paralleled the molecular imaging results: exercise caused higher levels of high-density lipoprotein, adiponectin, and interleukin-10 and lower levels of vascular cell adhesion molecule, monocyte chemoattractant protein-1, interleukin-1ß, and low-density lipoprotein. However, this beneficial effect was short-lived, with the ET5+0 group being similar to the ET0+0 group, and the ET0+1 group being similar to the ET5+1 group. The effect of exercise can be modeled with an exponential-decay of the protective factor of about 15%/day. CONCLUSIONS: Even short-term detraining reduces atheroprotective effects, and tips the balance towards atherosclerosis. This suggests that ET, to be effective, needs to be prolonged and regular, and that detraining should be avoided.

Angiogenic role of LYVE-1-positive macrophages in adipose tissue.

Here we report the discovery of a characteristic dense vascular network (DVN) in the tip portion of epididymal adipose tissue in adult mice. The DVN is formed by angiogenesis rather than by vasculogenesis, and has functional blood circulation. This DVN and its subsequent branching may provide a new functional route for adipogenesis. The recruitment, infiltration, and accumulation of bone marrow-derived LYVE-1(+) macrophages in the tip region are crucial for the formation of the DVN. Matrix metalloproteinases (MMPs) and the VEGF-VEGFR2 system are responsible not only for the formation of the DVN, but also for the recruitment and infiltration of LYVE-1(+) macrophages into the epididymal adipose tissue tip region. SDF-1, but not the MCP-1-CCR2 system, is a critical factor in recruitment and ongoing retention of macrophages in this area. We also demonstrate that the tip region of epididymal adipose tissue is highly hypoxic, and thus provides a microenvironment conducive to the high expression and enhanced activities of VEGF, VEGFR2, MMPs, and SDF-1 in autocrine and paracrine manners, to create an ideal niche for the recruitment, retention, and angiogenic action of macrophages. These findings shed light on the complex interplay between macrophage infiltration, angiogenesis, and adipogenesis in the tip region of adult epididymal adipose tissue, and provide novel insight into the regulation of alternative outgrowth of adipose tissue.

Aqueous extract of Dipsacus asperoides suppresses lipopolysaccharide-stimulated inflammatory responses by inhibiting the ERK1/2 signaling pathway in RAW 264.7 macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Dipsaci Radix, which is the dried root of Dipsacus asperoides C. Y. Cheng and T. M. Ai (Dipsacaceae), is used to treat back pain and blood stasis syndrome in Korean traditional medicine. AIM OF THE STUDY: To understand the mechanisms responsible for the pharmacological activities of D. asperoides, we investigated the inhibitory effect of D. asperoides on lipopolysaccharide (LPS)-induced inflammation in mouse macrophages RAW 264.7 cells. MATERIALS AND METHODS: Aqueous extract of D. asperoides (AEDA) was prepared by boiling D. asperoides in water and then administered to LPS treated RAW 264.7 cells. Cell viabilities were measured using an MTT assay, and protein levels were determined by western blotting. The ROS scavenging activity of AEDA was measured using a DCFH-DA assay and levels of nitric oxide (NO) were determined using a NO assay. The nuclear translocations of NF-κB and Nrf2 were investigated immunocytochemically, and pro-inflammatory cytokines in supernatant were evaluated by ELISA. RESULTS: Treatment with AEDA suppressed the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 macrophages. AEDA also reduced ROS, pro-inflammatory cytokine (IL-6 and IL-1ß) levels, and iNOS-derived NO and COX-2-derived prostaglandin E2 release to medium, and suppressed the phosphorylation and degradation of IκB and the activation of NF-κB in macrophages. Furthermore, treatment with AEDA inhibited the ERK1/2 pathway but not the JNK or p38 MAPK pathways. In addition, AEDA significantly promoted Nrf2 translocation from cytoplasm to nucleus and up-regulated the expression of HO-1. CONCLUSION: These results suggest that AEDA has anti-inflammatory and anti-oxidative effects through the inhibition of NF-κB and ERK1/2 and the activation of Nrf2/HO-1 in macrophages.

6'-Sialylgalactose inhibits vascular endothelial growth factor receptor 2-mediated angiogenesis.

Angiogenesis should be precisely regulated because disordered neovascularization is involved in the aggravation of multiple diseases. The vascular endothelial growth factor (VEGF)-A/VEGF receptor 2 (VEGFR-2) axis is crucial for controlling angiogenic responses in vascular endothelial cells (ECs). Therefore, inactivating VEGFR-2 signaling may effectively suppress aberrant angiogenesis and alleviate related symptoms. In this study, we performed virtual screening, identified the synthetic disaccharide 6'-sialylgalactose (6SG) as a potent VEGFR-2-binding compound and verified its high binding affinity by Biacore assay. 6SG effectively suppressed VEGF-A-induced VEGFR-2 phosphorylation and subsequent in vitro angiogenesis in HUVECs without inducing cytotoxicity. 6SG also inhibited VEGF-A-induced extracellular-regulated kinase (ERK)/Akt activation and actin stress fiber formation in HUVECs. We demonstrated that 6SG inhibited retinal angiogenesis in a mouse model of retinopathy of prematurity and tumor angiogenesis in a xenograft mouse model. Our results suggest a potential therapeutic benefit of 6SG in inhibiting angiogenesis in proangiogenic diseases, such as retinopathy and cancer.