Saponins from Nigella glandulifera seeds attenuate collagen-induced rheumatoid arthritis in rats via the OPG/RANKL/NF-κB and Ang/Tie-2 pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Nigella glandulifera Freyn et Sint. (N. glandulifera) seeds are widely used in traditional Uyghur medicine for a variety of immuno-inflammatory diseases. The total saponins from N. glandulifera seeds (TSNGS) have been shown to have analgesic, antioxidant, and anti-inflammatory effects that can alleviate joint pain and swelling. AIM OF THE STUDY: Rheumatoid arthritis (RA) is a chronic and progressive, debilitating autoimmune disease for which current treatments are not sufficiently effective and result in unsatisfactory side effects. This study aimed to mechanistically investigate the therapeutic effects of TSNGS on RA. MATERIALS AND METHODS: Qualitative analysis of TSNGS was performed using ultra-high-performance liquid chromatography-Q-Orbitrap-high-resolution mass spectrometry. Rats with collagen-induced arthritis (CIA), IL-1ß-induced HFLS-RAs, and VEGF-induced HUVECs were analyzed to determine the efficacy and mechanism of TSNGS on RA. RESULTS: Twenty-one compounds were identified in TSNGS. TSNGS (10, 50, or 250 mg/kg) reduced the severity of arthritis, indicated by a lower arthritis score, reduced paw swelling, and body weight in rats with CIA. TSNGS ameliorated histopathological changes involving inflammatory infiltration, bone degeneration, and angiogenesis in knee and ankle joints. TSNGS improved the immuno-inflammatory response by restoring the levels of the cytokines IFN-γ, TNF-α, IL-1ß, IL-6, IL-17A, IL-4, and IL-10, and increasing the number of CD4+CD25+ Tregs in the peripheral circulation and Foxp3 levels in knee joints in rats with CIA. Furthermore, TSNGS increased the OPG/RANKL ratio and downregulated p-p65 in serum and joint synovia. Inhibition of angiogenesis by TSNGS was associated with recovery of the angiogenesis-related Ang/Tie-2 signaling pathway. CONCLUSIONS: It was established that TSNGS provides a therapeutic effect on RA by alleviating synovitis, bone degeneration, and angiogenesis via the OPG/RANKL/NF-κB and Ang/Tie-2 pathways and may be used for the treatment of RA.

Notoginsenoside R1 activates the Ang2/Tie2 pathway to promote angiogenesis.

BACKGROUND: Therapeutic angiogenesis is a novel strategy for the treatment of ischemic diseases that involves promotion of angiogenesis in ischemic tissues via the use of proangiogenic agents. However, effective proangiogenic drugs that activate the Ang2/Tie2 signaling pathway remain scarce. PURPOSE: We aimed to investigate the proangiogenic activity of notoginsenoside R1 (NR1) isolated from total saponins of Panax notoginseng with regard to activation of the Ang2/Tie2 signaling pathway. METHODS: We examined the proangiogenic effects of NR1 by assessing the effects of NR1 on the proliferation, migration, invasion and tube formation of human umbilical vein endothelial cells (HUVECs). The aortic ring assay and vascular endothelial growth factor receptor inhibitor (VRI)-induced vascular regression in the zebrafish model were used to confirm the proangiogenic effects of NR1 ex vivo and in vivo. Furthermore, the molecular mechanism was investigated by Western blot analysis. RESULTS: We found that NR1 promoted the proliferation, mobility and tube formation of HUVECs in vitro. NR1 also increased the number of sprouting vessels in rat aortic rings and rescued VRI-induced vascular regression in zebrafish. NR1-induced angiogenesis was dependent on Tie2 receptor activation mediated by increased autocrine Ang2 in HUVECs, and inhibition of the Ang2/Tie2 pathway abrogated the proangiogenic effects of NR1. CONCLUSIONS: Our results suggest that NR1 promotes angiogenesis by activating the Ang2/Tie2 signaling pathway. Thus, NR1-induced activation of the Ang2/Tie2 pathway is an effective proangiogenic approach. NR1 may be useful agent for the treatment of ischemic diseases.

Angiopoietin-1 protects 3T3-L1 preadipocytes from saturated fatty acid-induced cell death.

Cross talk between endothelial cells and adipocytes is vital to adipocyte functions, but little is known about the mechanisms or factors controlling the process. Angiogenesis is a critical component linking the endothelium to healthy adipogenesis, yet it is not known if or how it is involved in adipocyte physiology. Therefore, the purpose of this study was to determine the effect of angiopoietin-1 (Ang-1) and -2 (Ang-2) as well as their receptor, Tie-2, on adipocyte physiology. 3T3-L1 pre- and mature adipocytes were found to express Ang-1, Ang-2, and Tie-2, which decrease upon polyunsaturated fatty acid treatment. Furthermore, 3T3-L1 cells treated with recombinant Ang-1 or Ang-2 increased expression of the antiapoptotic gene Bcl-x and decreased expression of the proapoptotic gene Casp-8. Next, preadipocytes were treated with saturated fatty acids (SFAs) to induce cell stress. SFA-mediated splicing of X-box-binding protein-1 was reduced by co-treatment with Ang-1, and cell viability was improved in the presence of SFAs + Ang-1. Taken together, these results indicate that Ang-1 may protect preadipocytes from SFA-induced apoptosis and endoplasmic reticulum stress.

3-O-Acetyloleanolic acid inhibits angiopoietin-1-induced angiogenesis and lymphangiogenesis via suppression of angiopoietin-1/Tie-2 signaling.

Tumor angiogenesis and lymphangiogenesis are important processes in tumor progression and metastasis. The inhibitory effects of 3-O-acetyloleanolic acid (3AOA), a pentacyclic triterpenoid compound isolated from Vigna sinensis K., on tumor-induced angiogenesis and lymphangiogenesis in vitro and in vivo were studied. Angiopoietin-1 is an important angiogenic and lymphangiogenic factor secreted from colon carcinoma CT-26 cells under hypoxia conditions. 3AOA inhibited proliferation, migration, and tube formation of angiopoietin-1-treated human umbilical vein endothelial cells (HUVEC) and human lymphatic microvascular endothelial cells (HLMEC). 3AOA reduced angiogenesis and lymphangiogenesis in angiopoietin-1-stimulated Matrigel plugs. Also, 3AOA inhibited tumor growth and tumor-induced angiogenesis and lymphangiogenesis in an angiopoietin-1-induced CT-26 allograft colon carcinoma animal model. 3AOA inhibited activation of the angiopoietin-1 receptor Tie-2 and activation of the downstream signaling factors FAK, AKT, and ERK1/2 that are involved in the angiopoietin-1/Tie-2-signaling pathway. Thus, 3AOA has an inhibitory effect on angiogenesis and lymphangiogenesis induced by angiopoietin-1 both in vitro and in vivo, and the inhibitory effect of 3AOA is probably due to suppression of angiopoietin-1/Tie-2 signaling in HUVEC and HLMEC.

Gamma-Tocotrienol Induces Apoptosis in Prostate Cancer Cells by Targeting the Ang-1/Tie-2 Signalling Pathway.

Emerging evidence suggests that gamma-tocotrienol (γ-T3), a vitamin E isomer, has potent anti-cancer properties against a wide-range of cancers. γ-T3 not only inhibited the growth and survival of cancer cells in vitro, but also suppressed angiogenesis and tumour metastasis under in vivo conditions. Recently, γ-T3 was found to target cancer stem cells (CSCs), leading to suppression of tumour formation and chemosensitisation. Despite its promising anti-cancer potential, the exact mechanisms responsible for the effects of γ-T3 are still largely unknown. Here, we report the identification of Ang-1 (Angiopoietin-1)/Tie-2 as a novel γ-T3 downstream target. In prostate cancer cells, γ-T3 treatment leads to the suppression of Ang-1 at both the mRNA transcript and protein levels. Supplementing the cells with Ang-1 was found to protect them against the anti-CSC effect of γ-T3. Intriguingly, inactivation of Tie-2, a member receptor that mediates the effect of Ang-1, was found to significantly enhance the cytotoxic effect of γ-T3 through activation of AMP-activated protein kinase (AMPK) and subsequent interruption of autophagy. Our results highlighted the therapeutic potential of using γ-T3 in combination with a Tie-2 inhibitor to treat advanced prostate cancer.

1-Methoxycarbony-ß-carboline from Picrasma quassioides exerts anti-angiogenic properties in HUVECs in vitro and zebrafish embryos in vivo.

Angiogenesis is a crucial process in the development of inflammatory diseases, including cancer, psoriasis and rheumatoid arthritis. Recently, several alkaloids from Picrasma quassioides had been screened for angiogenic activity in the zebrafish model, and the results indicated that 1-methoxycarbony-ß-carboline (MCC) could effectively inhibit blood vessel formation. In this study, we further confirmed that MCC can inhibit, in a concentration-dependent manner, the viability, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro, as well as the regenerative vascular outgrowth of zebrafish caudal fin in vivo. In the zebrafish xenograft assay, MCC inhibited the growth of tumor masses and the metastatic transplanted DU145 tumor cells. The proteome profile array of the MCC-treated HUVECs showed that MCC could down-regulate several angiogenesis-related self-secreted proteins, including ANG, EGF, bFGF, GRO, IGF-1, PLG and MMP-1. In addition, the expression of two key membrane receptor proteins in angiogenesis, TIE-2 and uPAR, were also down-regulated after MCC treatment. Taken together, these results shed light on the potential therapeutic application of MCC as a potent natural angiogenesis inhibitor via multiple molecular targets.

Biogenic gold nanoparticles synthesis mediated by Mangifera indica seed aqueous extracts exhibits antibacterial, anticancer and anti-angiogenic properties.

During the last few decades, gold nanoparticles (AuNP's) have gained considerable attention in nanomedicine and expanded its application in clinical diagnosis and as therapeutics. Employing plant extract for synthesising gold nanoparticles proves to be an eco-friendly technology for large scale production. It is highly economical and suitable for biological applications by negating the use of chemicals involved in conventional route. In this study, AuNP's was prepared by a simple one step method of employing aqueous Mangifera indica seed extract as a reducing agent. Scanning electron microscopy and transmission electron microscopy revealed spherical shaped nanoparticles and dynamic light scattering analysis indicated the AuNP's to be approximately 46.8 nm in size. AuNP's efficiently inhibited the growth of E. coli and S. aureus by its inherent ability to generate reactive oxygen species (ROS) and exhibited detrimental effects towards the tested bacterial species. Biocompatibility assessment indicated the non-toxic nature of AuNP's towards mesenchymal stem cells at 25 µg/ml and interestingly, suppressed the growth of human gastric cancer cells under in vitro culture conditions. AuNP's significantly exhibited anti-angiogenic property in chick chorioallantoic membrane model (CAM) by downregulating Ang-1/Tie2 pathway. Overall, the synthesized AuNP's exhibited antibacterial and anti-angiogenic properties with high biocompatibility thereby supporting its candidature for various biomedical applications. It can be employed in suppressing tumor growth, combat inflammatory diseases that necessitate the involvement of angiogenesis suppression, and antibacterial activity is suitable for its clinical translation to negate surgery associated infections.

Complementary actions of melatonin on angiogenic factors, the angiopoietin/Tie2 axis and VEGF, in co­cultures of human endothelial and breast cancer cells.

Melatonin exerts oncostatic activity in breast cancer through antiangiogenic actions. There, the aim of the present study was to ascertain whether melatonin modulates, in a coordinated action, angiopoietin-1 (ANG-1), ANG-2, their cognate Tie2 receptor and VEGF in co-cultures of human endothelial cells (HUVECs) and breast cancer (MCF-7) cells. To accomplish this we used co-cultures of human breast cancer cells (MCF-7) or non-malignant human mammary epithelial cells (MCF­10A) with endothelial cells (HUVECs). The presence of breast cancer cells increased HUVEC proliferation and 1 mM melatonin prevented this effect. ANG-1, ANG-2 and VEGF levels in co-culture media and mRNA expression were upregulated and Tie2 mRNA expression was downregulated in the HUVECs and MCF-7. Melatonin (1 mM) downregulated ANG-1, ANG-2 and VEGF levels in the co-culture media and mRNA expression in both types of cells and upregulated Tie2 mRNA expression in HUVECs. ANG-1, ANG-2, Tie2 and VEGF mRNA expression were not modified during HUVEC/MCF-10A co-culture. Estradiol (10 nM) increased ANG-1, ANG-2 and VEGF mRNA expression in HUVECs and melatonin (1 mM) counteracted this effect. We conclude that melatonin simultaneously coordinates downregulation of angiopoietins with a reduction in VEGF, which could be an effective therapeutic strategy for blocking tumor angiogenesis.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-Glucoside Attenuates Ischemia/Reperfusion-Induced Brain Injury in Rats by Promoting Angiogenesis.

Cerebral ischemia can cause brain infarcts, which are difficult to recover due to poor angiogenesis. 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-glucoside is a natural polyphenol, has antioxidant and anti-inflammatory activity, and can protect from ischemic neuronal injury. However, little is known about the effect of 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside on brain microcirculation after stroke. This study aimed at investigating the influence of 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside on brain lesions and angiogenesis after stroke. Sprague-Dawley rats were subjected to right middle cerebral artery occlusion and treated with vehicle, nimodipine, or different doses of 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside daily beginning at 6 h post-middle cerebral artery occlusion for 14 days. The volume of cerebral infarcts, degree of neurological dysfunction, and level of microvessel density were determined longitudinally. The levels of vascular endothelial growth factor, angiopoietin 1, and angiopoietin receptor-2 expression in the brain lesions were characterized by immunohistochemistry and Western blot assays at 14 days post-middle cerebral artery occlusion. We found that 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside significantly promoted postoperative recovery in rats by minimizing the volume of cerebral infarcts and improving neurological dysfunction in a dose- and time-dependent manner. Additionally, 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside significantly increased the microvessel density in the brain and upregulated CD31 expression in ischemic penumbra, relative to that in the control. Finally, treatment with 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside significantly upregulated the relative levels of vascular endothelial growth factor, angiopoietin 1, and angiopoietin receptor-2 expression in the brain lesions of rats. Therefore, these data indicated that 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside treatment promoted angiogenesis and recovery from ischemia/reperfusion-induced brain injury in rats.

Novel TIE-2 inhibitor BAY-826 displays in vivo efficacy in experimental syngeneic murine glioma models.

Targeting the vascular endothelial growth factor signaling axis in glioblastoma inevitably leads to tumor recurrence and a more aggressive phenotype. Therefore, other angiogenic pathways, like the angiopoietin/tunica interna endothelial cell kinase (TIE) signaling axis, have become additional targets for therapeutic intervention. Here, we explored whether targeting the receptor tyrosine kinase TIE-2 using a novel, highly potent, orally available small molecule TIE-2 inhibitor (BAY-826) improves tumor control in syngeneic mouse glioma models. BAY-826 inhibits TIE-2 phosphorylation in vitro and in vivo as demonstrated by suppression of Angiopoietin-1- or Na3 VO4 -induced TIE-2 phosphorylation in glioma cells or extracts of lungs from BAY-826-treated mice. There was a trend toward prolonged survival upon single-agent treatment in two of four models (SMA-497 and SMA-540) and there was a significant survival benefit in one model (SMA-560). Co-treatment with BAY-826 and irradiation was ineffective in one model (SMA-497), but provided synergistic prolongation of survival in another (SMA-560). Decreased vessel densities and increased leukocyte infiltration were observed, but might be independent processes as the effect was also observed in single treatment modalities. These data demonstrate that TIE-2 inhibition may improve tumor response to treatment in highly vascularized tumors such as glioblastoma.

Hydroxysafflor Yellow A Promotes Angiogenesis via the Angiopoietin 1/ Tie-2 Signaling Pathway.

BACKGROUND: The flowers of Carthamus tinctorius L. are widely used in traditional Chinese medicine to treat cerebrovascular and cardiovascular diseases. Hydroxysafflor yellow A (HSYA), the main constituent of C. tinctorius L. flowers, is known for its multiple biological activities. The present study investigated the effects of HSYA on angiogenesis in vitro and in a mouse hindlimb ischemia model. METHODS: Using human umbilical vein endothelial cells (HUVEC) in vitro and a mouse hindlimb ischemia model in vivo, the angiogenic role of HSYA was evaluated. RESULTS: HSYA significantly increased the capillary-like tube formation and migration of HUVEC. HSYA not only induced a rise in the expression of angiopoietin 1 and Tie-2 but it also increased phosphorylation of Tie-2, Akt, and extracellular signal-regulated kinase 1/2. Furthermore, an anti-Tie-2 neutralizing antibody significantly inhibited HSYA-induced HUVEC tube formation and migration. In vivo, the recovery of perfusion of ischemic hindlimb tissue after femoral artery interruption was significantly increased in HSYA-treated mice compared to vehicle controls. Consistent with these results, the arteriole and capillary densities in ischemic gastrocnemius muscles were significantly increased in HSYA-treated mice. CONCLUSIONS: These results indicate the potential utility of HSYA for the treatment of ischemic diseases.

In Silico Exploration for Novel Type-I Inhibitors of Tie-2/TEK: The Performance of Different Selection Strategy in Selecting Virtual Screening Candidates.

The receptor tyrosine kinase Tie-2 is involved in vessel remodeling and maturation, and has been regarded as a potential target for the treatment of various solid tumors. The absence of novel, potent and selective inhibitors severely hampers the understanding of the therapeutic potential of Tie-2. In the present work, we describe the discovery of novel type-I inhibitors of Tie-2 by structure-based virtual screening. Preliminary SAR was also performed based on one active compound, and several novel inhibitors with low micro-molar affinity were discovered. To directly compare the efficiency between different filtering strategies in selecting VS candidates, two methods were separately carried out to screen the same chemical library, and the selected VS candidates were then experimentally assessed by in vitro enzymatic assays. The results demonstrate that the hit rate is improved when stricter drug-likeness criteria and less number of molecules for clustering analysis are used, and meanwhile, the molecular diversity of the compounds still maintains. As a case study of TIE-2, the information presented in this work underscores the importance of selecting an appropriate selection strategy in VS campaign, and the novel inhibitors identified and the detailed binding modes of action provide a starting point for further hit-to-lead optimization process.

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.

Angiopoietin-1 receptor Tie2 distinguishes multipotent differentiation capability in bovine coccygeal nucleus pulposus cells.

BACKGROUND: The intervertebral disc (IVD) has limited self-healing potential and disc repair strategies require an appropriate cell source such as progenitor cells that could regenerate the damaged cells and tissues. The objective of this study was to identify nucleus pulposus-derived progenitor cells (NPPC) and examine their potential in regenerative medicine in vitro. METHODS: Nucleus pulposus cells (NPC) were obtained from 1-year-old bovine coccygeal discs by enzymatic digestion and were sorted for the angiopoietin-1 receptor Tie2. The obtained Tie2- and Tie2+ fractions of cells were differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro. Colony-forming units were prepared from both cell populations and the colonies formed were analyzed and quantified after 8 days of culture. In order to improve the preservation of the Tie2+ phenotype of NPPC in monolayer cultures, we tested a selection of growth factors known to have stimulating effects, cocultured NPPC with IVD tissue, and exposed them to hypoxic conditions (2 % O2). RESULTS: After 3 weeks of differentiation culture, only the NPC that were positive for Tie2 were able to differentiate into osteocytes, adipocytes, and chondrocytes as characterized by calcium deposition (p < 0.0001), fat droplet formation (p < 0.0001), and glycosaminoglycan content (p = 0.0095 vs. Tie2- NPC), respectively. Sorted Tie2- and Tie2+ subpopulations of cells both formed colonies; however, the colonies formed from Tie2+ cells were spheroid in shape, whereas those from Tie2- cells were spread and fibroblastic. In addition, Tie2+ cells formed more colonies in 3D culture (p = 0.011) than Tie2- cells. During expansion, a fast decline in the fraction of Tie2+ cells was observed (p < 0.0001), which was partially reversed by low oxygen concentration (p = 0.0068) and supplementation of the culture with fibroblast growth factor 2 (FGF2) (p < 0.0001). CONCLUSIONS: Our results showed that the bovine nucleus pulposus contains NPPC that are Tie2+. These cells fulfilled formally progenitor criteria that were maintained in subsequent monolayer culture for up to 7 days by addition of FGF2 or hypoxic conditions. We propose that the nucleus pulposus represents a niche of precursor cells for regeneration of the IVD.

Cornin increases angiogenesis and improves functional recovery after stroke via the Ang1/Tie2 axis and the Wnt/ß-catenin pathway.

We investigated whether cornin, an iridoid glycoside isolated from fruits of Verbena officinalis L., regulated angiogenesis and thereby improved functional outcomes after stroke and discovered a potential mechanism. The effects of cornin on proliferation of rat artery smooth muscle cell (RASMC) and signalling was investigated in vitro. Adult male rats were subjected to 1 h of middle cerebral artery occlusion (MCAO) and reperfusion and treated with or without 25 mg/kg of cornin, starting 24 h after ischemia and reperfusion, by continuous intravenous injection daily for 14 days. Neurological functional tests were performed and cerebral Evans blue extravasation was measured. Angiogenesis and angiogenic factor expressions were measured by immunohistochemistry and Western blotting, respectively. Cornin increased the proliferation of RASMC and enhanced the expression of Wnt5a, ß-catenin, cyclin D1 and angiopoietin-1 (Ang1). Cornin treatment promoted angiogenesis in the ischemic brain core and improved functional outcomes after stroke. Cornin-treated MCAO rats showed significant increase in vascularization and expression of vascular endothelial growth factor and Ang1 and phosphorylation of Tie2 and Akt compared with vehicle-treated MCAO rats. The Ang1/Tie2 axis and Wnt/ß-catenin pathways appear to mediate cornin-induced angiogenesis.

The effect of Chinese medicine Pu-Ren-Dan on pancreatic angiogenesis in high fat diet/streptozotocin-induced diabetic rats.

OBJECTIVES: The islet vascular system is critical for ß-cell function. This study investigated the antidiabetic effect of the Chinese Pu-Ren-Dan (PRD) recipe by regulating the pancreatic angiogenic factors in T2DM rats. MATERIALS METHODS: High fat diet/streptozotocin-induced obese type-2 diabetes mellitus rats were developed and treated with PRD for 4 weeks. Then glucolipid metabolism, insulin secretion, pancreatic blood flow, ultrastructure of islet ß-cell, histological changes of islet and protein expressions of pancreatic angiogenic factors were investigated. RESULTS: PRD-reduced T2DM rats' body weight and blood glucose level resisted the lipid metabolism disturbance, and ameliorated the insulin resistance and ß-cell function. In addition, the histological and morphological studies proved that PRD could maintain the normal distribution of endocrine cell in islet and normal ultrastructure of ß cell. An increased pancreatic blood flow was observed after the PRD treatment. In the investigation of pancreatic angiogenic factors, PRD inhibited the decreased expression of VEGF and Ang-1, and reversed the reduction of VEGFR2 and Tie2 phosphorylation in T2DM rats; the Ang-2 and TGFß expression were up-regulated by PRD while PKC was activated; endostatin and angiostatin were down-regulated by PRD. CONCLUSIONS: The results suggest that increasing VEGF expression, regulating VEGF/VEGFR2 signaling, stimulating Ang-1/Tie-2 signaling pathway, and inhibiting PKC-TGFß signaling and antiangiogenic factors might be the underlying mechanism of PRD's antidiabetic effect.

Isoxanthohumol modulates angiogenesis and inflammation via vascular endothelial growth factor receptor, tumor necrosis factor alpha and nuclear factor kappa B pathways.

Angiogenesis and inflammation are becoming distinguished players in the pathogenesis of many heterogeneous diseases, such as diabetes, cardiovascular disease, and cancer. Therefore, it is crucial to study new compounds that are able to modulate these events. Isoxanthohumol (IXN) is a polyphenol with antioxidant, anti-inflammatory, and antiangiogenic properties. The aim of this study was to evaluate the effects of IXN on blood vessel proliferation and maturation and describe underlying molecular mechanisms in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). Angiogenic profile of IXN was analyzed by retinal angiogenesis at different time points. IXN modulation of angiogenic and inflammatory signaling pathways was evaluated by Western blotting on EC and VSMC cultures. IXN inhibited by 20% sprouting angiogenesis and decreased vascular coverage by mural cells up to 39%. IXN of 10 µM also decreased inflammatory signals, namely tumor necrosis factor alpha (TNF-α) (26 and 40%) and factor nuclear kappa B (24 and 42%) in human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVECs). Angiogenic regulators, including vascular endothelial growth factor receptor 2 (HUVEC, 55%), angiopoietins 1 (HUVEC, 39%; HASMC, 35%), angiopoietin 2 (HUVEC, 38%), and Tie2 (HUVEC, 56%) were also inhibited by 10 µM of IXN treatments. Akt activation was reduced by 47% in HUVEC-treated cells and Erk activation was also reduced by 52 and 69% upon IXN treatment of HUVEC and HASMC. IXN seems to regulate in vivo vascular proliferation and stabilization and the EC-VSMC-inflammatory crosstalk, leaving this molecule as an interesting nutritional player for angiogenesis and inflammation-related diseases.

Inhibitory effect of glyceollins on vasculogenesis through suppression of endothelial progenitor cell function.

SCOPE: Endothelial progenitor cells (EPCs) are derived from hematopoietic stem cells, and have the ability to differentiate into mature endothelial cells and contribute to neovascularization. Glyceollins are a type of phytoalexin produced in soybeans under stress conditions. The aim of this study is to determine the effect of glyceollin treatment on EPCs during early tumor vasculogenesis. METHODS AND RESULTS: We found that glyceollin treatment significantly decreased the number of EPC colony-forming units in human cord blood-derived AC133⁺ cells and mouse bone-marrow-derived c-Kit⁺/Sca-1⁺/Lin⁻ cells. Glyceollin treatment diminished the number of lineage-committed EPC cells in a dose-dependent manner (1-20 µM). Glyceollin treatment inhibited EPC migration, tube formation and the mRNA expression of angiopoietin-1 (Ang-1), Tie-2, stromal-derived factor-1 (SDF-1), C-X-C-chemokine receptor-4 (CXCR4), and endothelial nitric oxide synthase (eNOS) in cultured EPCs. Glyceollin treatment suppressed activation of Akt, Erk, and eNOS induced by SDF-1α or vascular endothelial growth factor (VEGF). Treatment with 10 mg/kg glyceollins significantly reduced the number of tumor-induced circulating EPCs and the incorporation of EPCs into neovessels in bone marrow transplanted mice. CONCLUSION: These results suggest that glyceollins inhibit the function of EPCs in tumor neovascularization. Glyceollins from soybean elicitation could be beneficial in prevention of cancer development via vasculogenesis.

[Effect of Taohong Siwu decoction on angiogenesis of medicine-induced incomplete-abortion in early pregnancy rats and expressions of Ang-1, Ang-2 and Tie-2].

OBJECTIVE: To observe the effect of Taohong Siwu decoction (THSWD) on micro-vascular density (MVD) in rat uterus, the content of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in serum, and the expression of tyrosine kinasa receptor (Tie-2) in uterus. METHOD: Early pregnancy rats were intragastrically administrated with misoprostol (100 microg x kg(-1)) and mifepristong (8.3 mg x kg(-1)) to established the incomplete-abortion model. The incomplete-abortion rats were randomly divided into the model group (the same volume of distilled water), the positive control group (at the daily dose of 4.3 g x kg(-1) Motherwort Particles), and THSWD-treated groups (at the daily dose of 18.0, 9.0 and 4.5 g x kg(-1)). Pregnant rats were taken as the control group (the same volume of distilled water). After the successive oral administration for 7 days, blood was collected from aorta abdominalis, and rat uterine tissues were collected. The content of serum Ang-1 and Ang-2 were detected by ELISA; And the levels of Tie-2 and MVD in uterine tissues were detected by SP immunohistochemistry. RESULT: THSWD remarkably increased the levels of MVD in uterus of medicine-induced abortion rats, the content of Ang-1 and Ang-2 in serum, and the expression of Tie-2 in uterine tissues. CONCLUSION: THSWD has the effect in markedly promoting angiogenesis in incomplete-abortion rats. Its mechanism may be related to the regulation of concentrations of Ang-1 and Ang-2 in serum and Tie-2 in uterine tissues.

Anti-angiogenic effect and mechanism of rhein from Rhizoma Rhei.

PURPOSE: Rhein is a major bioactive component in rhubarb (Dahuang), a famous traditional Chinese medicine derived from the rhizome of Rheum palmatum and related species. It was reported to have antitumor and anti-inflammatory properties. Our previous studies found rhein displaying potent anti-angiogenic activities in a zebrafish embryo model. Its action mechanisms need further elucidation. METHODS: The inhibition effect of vessel formation was checked by microscopic imaging on Tg(fli1a:EGFP)y1 zebrafish embryos. Then the action mechanism of rhein was investigated by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) on wild type zebrafish embryos and further tested on human umbilical vein endothelial cells. RESULTS: At 20µM, rhein could almost completely block intersegmental blood vessels formation at both 48 and 72hpf, and completely inhibit subintestinal vessel plexus formation at 72hpf. Rhein affected multiple molecular targets related to angiogenesis, particularly angpt2 and tie2, and also inhibited endothelial cell migration. CONCLUSION: Rhein could inhibit angiogenesis, which may play a role in antitumor and anti-inflammatory actions.