Wound healing potential of licorice extract in rat model: Antioxidants, histopathological, immunohistochemical and gene expression evidences.

Wound healing is a public health concern. Licorice gained a great attention for its antioxidant and anti-inflammatory properties which expand its valuable effects as a herbal medicine. In this study, we pointed out to the wound healing potential and the mechanism by which licorice alcoholic extract can modulate cutaneous wound healing through immune, antioxidant, histopathological, immunohistochemical (IHC) and molecular studies. 24 Wister rats were assigned into 3 groups (n = 8 each); control group, topical and oral supplied groups. Licorice extract administration significantly increased total and differential leucocyte counts, phagocytic activity of neutrophils, antioxidant biomarkers as superoxide dismutase (SOD), glutathione peroxidase activities (GPx) and reduced glutathione (GSH) content with a notable reduction in oxidative stress marker malondialdehyde (MDA). Moreover, histopathological findings detected complete re-epithelialization with increasing collagen synthesis while IHC results revealed a significant enhancement in the expression of α-SMA, PDGFR-α, FGFR1 and Cytokeratin 14 in licorice treated groups compared with the control group. Licorice extract supplementation accelerated wound healing by increasing angiogenesis and collagen deposition through up-regulation of bFGF, VEGF and TGF-ß gene expression levels compared with the control group. UPLC-PDA-MS/MS aided to authenticate the studied Glycyrrihza species and recognized 101 potential constituents that may be responsible for licorice-exhibited potentials. Based on our observations we concluded that licorice enhanced cutaneous wound healing via its free radical-scavenging potential, potent antioxidant activities, and anti-inflammatory actions. Therefore, licorice could be used as a potential alternative therapy for wound injury which could overcome the associated limitations of modern therapeutic products.

Characterisation of Novel Angiogenic and Potent Anti-Inflammatory Effects of Micro-Fragmented Adipose Tissue.

Adipose tissue and more specifically micro-fragmented adipose tissue (MFAT) obtained from liposuction has recently been shown to possess interesting medicinal properties whereby its application supports pain reduction and may enhance tissue regeneration particularly in osteoarthritis. Here we have characterised samples of MFAT produced using the Lipogems® International Spa system from eight volunteer individuals in order to understand the critical biological mechanisms through which they act. A variation was found in the MFAT cluster size between individual samples and this translated into a similar variation in the ability of purified mesenchymal stem cells (MSCs) to form colony-forming units. Almost all of the isolated cells were CD105/CD90/CD45+ indicating stemness. An analysis of the secretions of cytokines from MFAT samples in a culture using targeted arrays and an enzyme-linked immunosorbent assay (ELISA) showed a long-term specific and significant expression of proteins associated with anti-inflammation (e.g., interleukin-1 receptor alpha (Il-1Rα) antagonist), pro-regeneration (e.g., hepatocyte growth factor), anti-scarring and pro-angiogenesis (e.g., transforming growth factor beta 1 and 2 (TGFß1/2) and anti-bacterial (e.g., chemokine C-X-C motif ligand-9 (CXCL-9). Angiogenesis and angiogenic signalling were notably increased in primary bovine aortic endothelial cells (BAEC) to a different extent in each individual sample of the conditioned medium whilst a direct capacity of the conditioned medium to block inflammation induced by lipopolysaccharides was shown. This work characterises the biological mechanisms through which a strong, long-lasting, and potentially beneficial effect can be observed regarding pain reduction, protection and regeneration in osteoarthritic joints treated with MFAT.

Separation, identification and cardiovascular activities of phospholipid classes from the head of Penaeus vannamei by lipidomics and zebrafish models.

Phospholipids not only have high nutritional value, but also have a positive effect on cardiovascular disease, cancer and nervous system diseases. However, the activity of individual phospholipid classes of shrimp phospholipids is rarely studied. This paper researched phospholipids in the by-products of Penaeus vannamei processing. The phospholipid classes of the head from P. vannamei (PV) were separated by column chromatography, analyzed with UHPLC-Q-Exactive HF/MS, and quantified using ammonium ferrothiocyarate spectrophometry. In addition, their cardiovascular activities in zebrafish models were evaluated. A total of 5 phospholipid classes were obtained, including PV-PC, PV-PE, PV-PI, PV-PS and PV-SM, and identified as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and sphingomyelin (SM), respectively. In the phospholipid profiling analysis, PV-PC (308 molecules) had the highest proportion with 85.24%, followed by PV-PE (139 types) with 9.32%, PV-SM (41 structures) with 4.75%, PV-PS (24 types) with 0.16%, and PV-PI (6 molecules) with 0.03%. In the quantitative analysis, the content of PV was 45.7%, and the purity of phospholipid classes was 75.5-88.1%. In the cardiovascular activity assays, the effects of different phospholipid classes were different. For example, PV-PC groups had strong angiogenesis activity, but PV-PE groups showed the opposite property. Our comprehensive profiling analysis and in vivo bioactivity evaluation of phospholipids from the head of P. vannamei can provide evidence for their targeted applications in the future.

Evaluation of VEGF mediated pro-angiogenic and hemostatic effects and chemical marker investigation for Typhae Pollen and its processed product.

ETHNOPHARMACOLOGICAL RELEVANCE: Typhae Pollen (TP) is a well-known Traditional Chinese Medicine (TCM) to remove blood stasis. Carbonized Typhae Pollen (CTP), a processed product of TP after being stir-fried, has been widely applied to clinical practice with its capability of hemostasis. However, the underlying mechanism of TP and CTP are still not fully elucidated and discrimination against TP and CTP remains a challenge. AIM OF STUDY: The aim of this study is to investigate whether TP could remove blood stasis by promoting angiogenesis and the process of carbonizing it could enhance hemostatic effect. Meanwhile, some chemical markers for quality control of CTP had better to be found. MATERIAL AND METHODS: The changes of constituents between TP and CTP were analyzed by UPLC-QTOF-MS/MS. We investigated pro-angiogenic and hemostatic effects of TP and CTP in two zebrafish models: VRI-induced ISV insufficiency model and Ator-induced cerebral hemorrhage model. Subsequently, quantitative real-time PCR (qRT-PCR) was applied to investigate the mechanism of pharmacological effects. Finally, chemometric method was applied to find chemical markers. RESULTS: A total of 19 compounds were identified in qualitative analysis. The loss rate of each compound was calculated and compared. Two compounds (huaicarbon A/B) could only be detected in CTP and the content of flavonoid glycosides in CTP was significantly decreased compared with TP. The average content of the three identified flavonoid aglycones (quercetin, isorhamnetin and kaempferol) was increased about 30 percent in CTP. TP promoted pro-angiogenesis by up-regulating the expression of VEGFA, flt1 and kdr. After heating process, the pro-angiogenic activity was reduced and hemostatic activity was enhanced in CTP. Then qRT-PCR analysis found that CTP could significantly up-regulate the expression of VEGFA and vWF. In the discovery of markers, 6 chemical markers for discrimination of TP and CTP were obtained by chemometric method. CONCLUSION: Our research indicated that the pro-angiogenic activity of TP was involved in VEGF signaling pathway. After processing, hemostatic activity of CTP has been enhanced by up-regulating the expression of VEGFA and vWF. A chemical marker database was established to provide a scientific evidence for quality control, mechanism and the clinical application of TP and CTP.

A non-anticoagulant heparin-like snail glycosaminoglycan promotes healing of diabetic wound.

Diabetic foot ulcer (DFU) is a common high-risk complication in patients with diabetes mellitus, but current drugs and therapies in management of this disease cannot meet the urgent clinical needs. In this study, a snail glycosaminoglycan (SGAG) from the cultured China white jade snail was purified and structurally clarified. This snail glycosaminoglycan is a regular sulfated polysaccharide, composed of iduronic acid (IdoA) and N-acetyl-glucosamine (GlcNAc) with the repeating sequence of →4)-α-GlcNAc (1→4)-α-IdoA2S (1→. The biological assays showed that SGAG had no anticoagulant activity for lacking specific heparin pentasaccharide sequence. The pharmacological experiments suggested that SGAG markedly accelerated the healing of full-thickness wounds in diabetic mice skin. Histologic and immunohistochemical analysis revealed that SGAG treatment alleviated the inflammation and dermal edema, and promoted angiogenesis. This is the first report applying the snail glycosaminoglycan to favor diabetic wound healing.

Hancornia speciosa serum fraction latex stimulates the angiogenesis and extracellular matrix remodeling processes.

The Hancornia speciosa latex reveals angiogenic, osteogenic, and anti-inflammatory properties, which present its potential for developing of wound healing drugs; however, the latex compounds responsible for angiogenesis remain unknown. One strategy to screen these active compounds is evaluation of latex fractions. This study aimed to obtain different fractions of latex and evaluate its angiogenic activity separately using the chick chorioallantoic membrane (CAM) assay. The serum (SE) fraction was responsible for angiogenesis, which was subject to biochemical characterization and computational simulations in order to understand the contribution of H. speciosa latex in wound healing process. Our results revealed weak antioxidant potential and absence of antimicrobial activity in the SE fraction. Phytochemical analysis identified chlorogenic acids (CGA) as the main compound of SE fraction. CGA bioactivity predictions identify different molecules associated with extracellular matrix (ECM) remodeling, such as metalloproteinases, which also are overexpressed in our CAM assay experiment. Docking simulations revealed the interactions between CGA and matrix metalloproteinase 2. In conclusion, SE latex fraction stimulates angiogenesis and may influence ECM remodeling. These properties may contribute to the wound healing process, and also confirm the widespread use of this plant.

Clavukoellians G-K, New Nardosinane and Aristolane Sesquiterpenoids with Angiogenesis Promoting Activity from the Marine Soft Coral Lemnalia sp.

The chemical examination of the marine soft coral Lemnalia sp., collected at the Xisha islands in the South China Sea, resulted in the isolation of four new nardosinane-type sesquiterpenoids, namely clavukoellians G-J (1-4), and one new aristolane sesquiterpene, namely clavukoellian K (5), together with five known compounds, 6-10. The structure elucidation of the isolated natural products was based on various spectroscopic techniques including HRESIMS and NMR, while their absolute configurations were resolved on the basis of comparisons of the ECD spectra with the calculated ECD data. The isolated new compounds 1-5 were evaluated for their anti- and pro- angiogenesis activities in a transgenic fluorescent zebrafish (Tg(vegfr2:GFP)) model. Quantitative analysis revealed that compound 5 displayed pro-angiogenesis activity in a PTK787-induced vascular injury zebrafish model at 2.5 µM. Data showed that compound 5 significantly promoted the angiogenesis in a dose-dependent manner.

Síndrome de HELLP posparto tras cribado de preeclampsia positivo y óbito fetal: sFlt-1/PIGF / Postpartum HELLP syndrome after positive preeclampsia screening and fetal death: sFlt-1/PIGF

Presentamos el caso de una gestante que desarrolló un síndrome de HELLP a las 48 horas posparto tras la inducción del mismo en la semana 27 por óbito fetal. La paciente se encontraba en seguimiento estricto ante un cribado contingente de preeclampsia positivo iniciado en la semana 21 con doppler positivo de las arterias uterinas y un marcador angiogénico (ratio sFlt-1/PIGF) elevado desde la semana 26

We expose the case of a pregnant woman who developed a HELLP syndrome at 48 hours post-delivery, after labour induction in 27 week due to stillbirth. The patient was being rigorously monitorized because of a positive contingent screening of preeclampsia. The study started at 21 week with positive uterine arteries doppler and an angiogenic marker (ratio sFlt-1/PIGF) elevated from 26 week

Inulin with a low degree of polymerization protects human umbilical vein endothelial cells from hypoxia/reoxygenation-induced injury.

Here, we identified inulin-type oligosaccharides with 3-13 degrees of polymerization from Morinda officinalis. Radical-scavenging assays showed that Inulins 4-7 had modest anti-oxidative effects. Inulins 4 and 5 dose-dependently increased human umbilical vein endothelial cell survival during hypoxia/re-oxygenation (H/R)-induced injury, and Inulin 5 promoted angiogenesis. Triplicate assays with the Affymetrix Human Transcriptome Array 2.0 showed that Inulin 5 exposure up-regulated genes associated with cell cycle progression, apoptosis, DNA replication and repair, ubiquitin-mediated proteolysis, the mitogen-activated protein kinase pathway, and the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-signaling pathway. Flow cytometry, reverse transcription-quantitative polymerase chain reaction, and western blot experiments verified the microarray results and demonstrated that Inulin 5 influenced cell cycle progression and the PI3K-protein kinase B (PKB)-endothelial nitric oxide synthase (eNOS) pathway. Thus, inulin-type oligosaccharides from M. officinalis roots may protect against H/R-induced injury, primarily through an anti-oxidative effect, and promote angiogenesis by activating the PI3K-PKB-eNOS-signaling pathway.

Promotion of mammalian angiogenesis by neolignans derived from soybean extracellular fluids.

Excessive or insufficient angiogenesis is associated with major classes of chronic disease. Although less studied, small molecules which can promote angiogenesis are being sought as potential therapeutics for cardiovascular and peripheral arterial disease and stroke. Here we describe a bioassay-directed discovery approach utilising size exclusion and liquid chromatography to purify components of soybean xylem sap that have pro-angiogenic activity. Using high resolution accurate mass spectrometry and nuclear magnetic resonance spectroscopy, the structure of two pro-angiogenic molecules (FK1 and FK2) were identified as erythro-guaiacylglycerol-8-O-4'-(coniferyl alcohol) ether (eGGCE), and threo-guaiacylglycerol-8-O-4'-(coniferyl alcohol) ether (tGGCE). These two molecules, which are coniferyl neolignan stereoisomers, promoted in vitro angiogenesis in the µM to nM range. Independently sourced samples of eGGCE and tGGCE exhibited comparable pro-angiogenic activity to the soybean derived molecules. The cellular mode of action of these molecules was investigated by studying their effect on endothelial cell proliferation, migration, tube formation and adhesion to the extracellular matrix (ECM) components, fibronectin and vitronectin. They were found to enhance endothelial cell proliferation and endothelial cell tube formation on Matrigel, but did not affect endothelial cell migration or adhesion to fibronectin and vitronectin. Thus, this study has identified two coniferyl neolignan stereoisomers, eGGCE and tGGCE, as pro-angiogenic molecules, with eGGCE being less active than tGGCE.

N-Terminomics identifies HtrA1 cleavage of thrombospondin-1 with generation of a proangiogenic fragment in the polarized retinal pigment epithelial cell model of age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population. Variants in the HTRA1-ARMS2 locus have been linked to increased AMD risk. In the present study we investigated the impact of elevated HtrA1 levels on the retina pigment epithelial (RPE) secretome using a polarized culture system. Upregulation of HtrA1 alters the abundance of key proteins involved in angiogenesis and extracellular matrix remodeling. Thrombospondin-1, an angiogenesis modulator, was identified as a substrate for HtrA1 using terminal amine isotope labeling of substrates in conjunction with HtrA1 specificity profiling. HtrA1 cleavage of thrombospondin-1 was further corroborated by in vitro cleavage assays and targeted proteomics together with small molecule inhibition of HtrA1. While thrombospondin-1 is anti-angiogenic, the proteolytically released N-terminal fragment promotes the formation of tube-like structure by endothelial cells. Taken together, our findings suggest a mechanism by which increased levels of HtrA1 may contribute to AMD pathogenesis. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier. For quantitative secretome analysis, project accession: PXD007691, username: reviewer45093@ebi.ac.uk, password: 1FUpS6Yq. For TAILS analysis, project accession: PXD007139, username: reviewer76731@ebi.ac.uk, password: sNbMp7xK.

Biospecific isolation and characterization of angiogenesis-promoting ingredients in Buyang Huanwu decoction using affinity chromatography on rat brain microvascular endothelial cells combined with solid-phase extraction, and HPLC-MS/MS.

Buyang Huanwu decoction (BHD) was reported to exert angiogenesis-promoting effects, but its active ingredients remain unknown. In this study, we developed a method to screen potential angiogenesis-promoting compounds in BHD, which involved biospecific isolation using live rat brain microvascular endothelial cells (rBMECs) and characterization using solid-phase extraction (SPE) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Six compounds showed binding affinity to rBMECs and were further identified as 6-hydroxykaempferol-di-O-glucoside, paeoniflorin, calycosin-7-O-ß-D-glucoside, galloylpaeoniflorin, formononetin-7-O-ß-D-glucoside, and (3R)-7,2'-hydroxy-3',4'-dimethoxy-isoflavan. The results indicated that five of them except 6-hydroxykaempferol-di-O-glucoside showed a protective effect against oxygen glucose deprivation/reperfusion injury in rBMECs and upregulated the secretion of vascular endothelial growth factor and basic fibroblast growth factor, suggesting a mechanism underlying their angiogenic activity. Our findings suggest that biospecific live cell-based isolation combined with SPE and HPLC-MS/MS is an effective method for screening potential bioactive components in traditional Chinese medicines.

Anti-thrombotic and pro-angiogenic effects of Rubia cordifolia extract in zebrafish.

ETHNOPHARMACOLOGICAL RELEVANCE: Rubia cordifolia is a common traditional Chinese medicine that promotes blood circulation and eliminates blood stasis, and has been used to cure diseases related to blood stasis syndrome (BSS) clinically for many years. It has been previously demonstrated that anti-thrombosis and pro-angiogenesis can improve BSS. However, the anti-thrombotic and pro-angiogenic activities of Rubia cordifolia have not been well investigated. AIM OF STUDY: To determine the potential anti-thrombotic and pro-angiogenic activities of Rubia cordifolia and to elucidate the underlying mechanisms. In addition, the major chemical constituents of Rubia cordifolia extract (QC) were qualitatively analysed by UPLC-Q-TOF/MS to explore the association between pharmacological activity and chemical constituents. MATERIAL AND METHODS: The QC samples were composed of a 95% ethanol extract and an aqueous extract following extraction using 95% ethanol. UPLC-Q-TOF/MS was used to analyse the major chemical constituents of QC. For the anti-thrombotic experiment of QC, a phenylhydrazine (PHZ)-induced AB strain zebrafish thrombosis model was used. The zebrafish larvae were stained using O-dianisidine, and the heart and caudal vein of the zebrafish were observed and imaged with a fluorescence microscope. The staining intensity of erythrocytes in the heart (SI) of each group and the morphology of thrombus in the caudal vein were used to assess the anti-thrombotic effect of QC. For the pro-angiogenic assay of QC, the intersegmental blood vessel (ISV) insufficiency model of Tg(fli-1: EGFP)y1 transgenic zebrafish (Flik zebrafish), which was induced by the VEGF receptor tyrosine kinase inhibitor II (VRI), was used. The morphology of the intact ISVs and defective ISVs was observed to evaluate the pro-angiogenic activity of QC. The mechanism involved in promoting angiogenesis was studied with real-time PCR. RESULTS: A total of 12 components in QC were identified based on standard compounds and references, including nine anthraquinones and three naphthoquinones. After treatment with QC, the PHZ-induced thrombosis in AB strain zebrafish larvae decreased to a certain degree, which we believe was related to its dosages, and the therapeutic effect within the 50-200 µg/mL QC treatment groups was especially prominent (P < 0.01, P < 0.001) compared to that in the PHZ model group. Similarly, QC also recovered the loss of the ISVs, which was induced by VRI in Flik zebrafish larvae, which have a certain dose-effect relationship. The pro-angiogenic activity of QC was also conspicuous (P < 0.01, P < 0.001) compared to that of the VRI model group. The following real-time PCR assay proved that QC significantly restored the VRI-induced downregulation of vWF, VEGF-A, kdrl, and flt-1 in Flik zebrafish (P < 0.05, P < 0.01, P < 0.001). CONCLUSIONS: A total of 12 compounds from QC were analysed by UPLC-Q-TOF/MS. The data of the pharmacological experiments demonstrated that QC presented anti-thrombotic and pro-angiogenic activities in zebrafish, and the principal active components were likely anthraquinones and naphthoquinones. Thus, the current study provided a theoretical basis for the clinical use of Rubia cordifolia as a traditional Chinese medicine in promoting blood circulation and eliminating stasis.

Picrotoxane sesquiterpenoids from the stems of Dendrobium nobile and their absolute configurations and angiogenesis effect.

Five picrotoxane sesquiterpenoids belonging to the unusual dendrobine-type (1 and 4) and the picrotoxinin-type (2, 3, and 5) were isolated from the stems of Dendrobium nobile Lindl. Their structures were established by spectroscopic analyses and physical properties. Compound 1 was a new dendrobine analogue. Although the planar structure of 2 and 3 had been reported, their absolute configurations were first determined by single-crystal X-ray diffraction and circular dichroism. Compound 2 exhibited angiogenesis effect against sunitinib-induced damage on intersegmental blood vessels in Tg (flk1: EGFP) and Tg (fli1: nEGFP) transgenic zebrafish at concentrations of 3.13, 6.25, 12.50, and 25.00µM.

Isolation and screening of proangiogenic and antiangiogenic metabolites producing rare actinobacteria from soil.

AIMS: Angiogenesis is a physiological process that has important impacts on the pathology and healing of various diseases, and its induction or inhibition by bioactive actinobacterial metabolites can help the treatment of some diseases. In this study, the effects of actinobacterial extract in the process of angiogenesis have been explored. METHODS AND RESULTS: In this research, proangiogenic and antiangiogenic metabolites producing actinobacteria were isolated from soil samples and their fermentation broth were extracted and after evaluation of their toxicity by MTT assay, antiangiogenic and proangiogenic activities were screened against human umbilical vein endothelial cells (HUVECs) by in vitro tube formation and migration assay. Isolated strains were identified through molecular techniques. The results showed that Nocardiopsis arvandica UTMC 103 and Nonomuraea sp. UTMC 2180 extracts had a high potential of anti-angiogenic activity on HUVECs. CONCLUSIONS: For the first time proangiogenic potency of a rare actinobacterium, Kribbella sp. UTMC 522, was reported, and N. arvandica UTMC 103 and Nonomuraea sp. UTMC 2180 extracts inhibits the proliferation, migration and angiogenesis activity of HUVECs with reasonable potency. SIGNIFICANCE AND IMPACT OF THE STUDY: Metabolites of the introduced rare actinobacteria are potent proangiogenic and angiogenic inhibitors. Identification of angiogenic-antiangiogenic mechanisms and purification of the extracts would be useful in therapeutic angiogenesis.

Effects of Nigella sativa Extract on Markers of Cerebral Angiogenesis after Global Ischemia of Brain in Rats.

BACKGROUND: Reduction of permanent or transient cerebral blood flow may lead to some structural and functional changes of the brain, causing high mortality and morbidity. The aim of this experimental study was to investigate the effects of hydroalcoholic extract of Nigella sativa (NS) on markers of cerebral angiogenesis in rats induced by global brain ischemia. METHODS: Thirty-two male Wistar rats (250 ± 20 g) were randomly divided into 4 groups: group 1, control group receiving only normal saline; group 2, sham group undergoing surgery and stroke induction without treatment; and groups 3 and 4 treated with 10 and 20 mg/kg NS, respectively, after induction of stroke. Global ischemia was induced by ligation of the right carotid artery for 20 minutes. RESULTS: According to the results of this study, brain edema and infarct volume were significantly decreased in the group treated with 20 mg/kg NS compared with the group treated with 10 mg/kg NS (P < .05). Global ischemia caused a significant reduction in gene expression of vasoactive endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF) in the sham group compared with the control group (P < .05), but NS groups, in led to a significant increase in gene expression of VEGF and HIF compared with the sham group (P < .05). In addition, the activity level of matrix metallopeptidase-9 was decreased among NS groups compared with the control group (P < .05). CONCLUSIONS: Application of NS extract among rats with brain ischemia is associated with increase of VEGF and HIF as angiogenic markers and inhibition of matrix metallopeptidase-9 activities.

Spatholobi Caulis extracts promote angiogenesis in HUVECs in vitro and in zebrafish embryos in vivo via up-regulation of VEGFRs.

ETHNOPHARMACOLOGICAL RELEVANCE: Spatholobi Caulis is a traditional blood-activating and stasis-dispelling herb medicine, which has been used to treat diseases related to blood stasis syndrome (BSS) by inhibiting platelet aggregation, stimulate hematopoiesis, etc. It has been demonstrated that pro-angiogenesis could improve BSS. However, the pro-angiogenic activity of Spatholobi Caulis was not well elucidated AIM OF STUDY: To determine the potential pro-angiogenic activity of Spatholobi Caulis and elucidate its underlying mechanism. The active fractions of Spatholobi Caulis were further screened. MATERIAL AND METHODS: Gelatin precipitation and reversed-phase liquid chromatography (RPLC) were used to purify the methanol extracts of Spatholobi Caulis, respectively. The RPLC was also used to prepare fractions. Total flavonoids of purified methanol extracts of Spatholobi Caulis (PSC) were determined using ultraviolet spectrophotometry. The morphological observation of subintestinal vessel plexus (SIVs) and tyrosine kinase inhibitor II (VRI)-induced intersegmental blood vessels (ISVs) loss in transgenic zebrafish Tg(fli-1a: EGFP)y1 were selected to evaluate the pro-angiogenic activity of PSC in vivo. Cell proliferation by MTT assay and cell migration assay were used to evaluate the pro-angiogenesis effect of PSC in human umbilical vein endothelial cells (HUVECs) in vitro. Both zebrafish and HUVECs were used in screening active fractions of PSC. The mechanism of PSC promoting angiogenesis were studied by real-time PCR in zebrafish and western blotting in HUVECs. RESULTS: Co-treatment PSC dramatically rescued VRI-induced ISVs loss in zebrafish embryos in a dose-dependent manner and 80% of the defective vascular recovered at the concentration of 30µg/ml compared with VRI-only group. PSC also concentration-dependently increased average sprouting number and diameter of SIVs in zebrafish embryo. Real-time PCR assay proved that PSC significantly restored the down regulation of VEGFRs including Flt-1, Kdr and Kdrl induced by VRI in zebrafish (P<0.001). Furthermore, PSC not only promoted proliferation and migration of normal HUVECs but also ameliorated VRI-induced HUVECs cytotoxicity. Western blotting assay showed that co-treatment of PSC increased the expression of VEGFRs and phosphorylation of MAPKs which decreased by VRI treatment. In addition, quality evaluation experiments showed that the content of total flavonoids of PSC reached 56.36% and the main pro-angiogenic fractions of PSC were F3, F4 and F5 both in zebrafish and HUVECs. CONCLUSIONS: Our data demonstrated that PSC presented pro-angiogenic activity both in zebrafish and HUVECs, and principal pro-angiogenic active components were likely flavonoids. Thus, the current study provided evidence for the clinical usage of Spatholobi Caulis in promoting blood circulation and removing stasis in traditional Chinese medicine (TCM).

A Novel Natural Product-Derived Compound, Vestaine A1, Exerts both Pro-Angiogenic and Anti-Permeability Activity via a Different Pathway from VEGF.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) is a key molecule in the regulation of both angiogenesis and vascular permeability. However, it is known that overproduction of VEGF induces abnormal blood vessel formation and these vessels cause several disease pathologies, such as diabetic retinopathy. The purpose of this study was to find novel vasoactive compounds which have different properties from VEGF. METHODS/RESULTS: We screened a natural product library using a co-culture angiogenic assay of endothelial cells and fibroblasts. By focusing on morphological changes of endothelial cells, we isolated the novel compounds vestaine A1 and vestaine B1 from the cultured broth of an actinomycete strain, Streptomyces sp. SANK 63697. Vestaine A1 enhanced tube formation of endothelial cells in Matrigel and suppressed cell death induced by serum deprivation. Vestaine A1 activated both MEK1/2 and PI-3 kinase pathways independently of the VEGF pathway in a dose- and time-dependent fashion. Finally, vestaine A1 potently suppressed VEGF-induced vascular permeability both in vitro and in vivo. CONCLUSION: Vestaine A1 has the potential to exhibit both pro-angiogenic and anti-permeability properties, and would therefore be useful for therapeutic treatment for abnormal vascular permeability-related diseases.

Anti-inflammatory and angiogenic activity of polysaccharide extract obtained from Tibetan kefir.

The search for new bioactive molecules is a driving force for research pharmaceutical industries, especially those molecules obtained from fermentation. The molecules possessing angiogenic and anti-inflammatory attributes have attracted attention and are the focus of this study. Angiogenic activity from kefir polysaccharide extract, via chorioallantoic membrane assay, exhibited a pro-angiogenic effect compared with vascular endothelial factor (pro-angiogenic) and hydrocortisone (anti-angiogenic) activity as standards with an EC50 of 192ng/mL. In terms of anti-inflammatory activity determined via hyaluronidase enzyme assay, kefir polysaccharide extract inhibited the enzyme with a minimal activity of 2.08mg/mL and a maximum activity of 2.57mg/mL. For pharmaceutical purposes, kefir polysaccharide extract is considered to be safe because it does not inhibit VERO cells in cytotoxicity assays.

Evaluation of the angiogenic potency of a novel exopolysaccharide produced by the MK1 bacterial strain.

Angiogenesis is an essential physiological step in wound healing and other regenerative processes. Here, we evaluated the angiogenic properties of an exopolysaccharide (EPS) secreted by MK1 (MK1-EPS), a novel bacterial strain isolated from Neungee mushrooms. MK1-EPS significantly increased human umbilical vein endothelial cell (HUVEC) proliferation, migration, and vascular tube formation. MK1-EPS enhanced the phosphorylation of extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, which are mitogen-activated protein kinases. In addition, the expression of p21 and intercellular adhesion molecule 1 (ICAM1), and phosphorylation of signal transducer and activator of transcription 3 (STAT3), but not of protein kinase B (AKT), were increased. Specific inhibitors of p38 (SB203580), ERK (PD98059), and JNK (SP600125) inhibited MK1-EPS-induced HUVEC proliferation, tube formation, and cell migration, and partially attenuated MKI-EPS-induced expression of p21 and ICAM1, and STAT3 phosphorylation. After surgical implantation into rabbit calvarial bone defects, new blood vessel formation was significantly higher with MK1-EPS composite bone granules than with granules alone, and new bone formation increased significantly. Therefore, MK1-EPS induces angiogenesis and may have potential for use as a bone regeneration agent in bone tissue engineering applications.