Buxuhuayu decoction accelerates angiogenesis by activating the PI3K-Akt-eNOS signalling pathway in a streptozotocin-induced diabetic ulcer rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: Buxuhuayu decoction (BXHYD) has been frequently used to treat patients with diabetic ulcers (DUs), without notable adverse reactions. However, the related molecular mechanism remains unelucidated. AIM OF THE STUDY: This study assessed the potential mechanism of BXHYD against DUs by using network pharmacology and animal experiments. MATERIALS AND METHODS: First, high-performance liquid chromatography (HPLC) was used for quality control of BXHYD. Further, the hub compounds and targets were screened from the Active Compound-Targets (ACT) network and the protein and protein interaction (PPI) network. Enrichment analysis was performed using DAVID, and molecular docking technology was used to identify active compounds that may play a key role in pub targets. Finally, a DUs animal model was established and used to elucidate the effect of BXHYD on the PI3K/Akt/eNOS signalling pathway. RESULTS: (1) Calycosin-7-glucoside, amygdalin, and tanshinone iiA were detected in the freeze-dried powder of BXHYD. (2) Twelve hub compounds and eight hub targets were screened using the ACT and PPI networks. Through molecular docking, it was found that the four hub targets (TP53, IL6, VEGFA, and AKT1) binds luteolin and quercetin more tightly. (3) BXHYD is most likely to promote angiogenesis and wound healing by activating the PI3K/Akt/eNOS signalling pathway. CONCLUSIONS: This research revealed that BXHYD might activate the PI3K/Akt/eNOS signalling pathway to promote DUs healing. These findings support the clinical use of BXHYD and provide the foundation for its subsequent studies.

Lab-On-A-Chip for the Development of Pro-/Anti-Angiogenic Nanomedicines to Treat Brain Diseases.

There is a huge demand for pro-/anti-angiogenic nanomedicines to treat conditions such as ischemic strokes, brain tumors, and neurodegenerative diseases such as Alzheimer's and Parkinson's. Nanomedicines are therapeutic particles in the size range of 10-1000 nm, where the drug is encapsulated into nano-capsules or adsorbed onto nano-scaffolds. They have good blood-brain barrier permeability, stability and shelf life, and able to rapidly target different sites in the brain. However, the relationship between the nanomedicines' physical and chemical properties and its ability to travel across the brain remains incompletely understood. The main challenge is the lack of a reliable drug testing model for brain angiogenesis. Recently, microfluidic platforms (known as "lab-on-a-chip" or LOCs) have been developed to mimic the brain micro-vasculature related events, such as vasculogenesis, angiogenesis, inflammation, etc. The LOCs are able to closely replicate the dynamic conditions of the human brain and could be reliable platforms for drug screening applications. There are still many technical difficulties in establishing uniform and reproducible conditions, mainly due to the extreme complexity of the human brain. In this paper, we review the prospective of LOCs in the development of nanomedicines for brain angiogenesis-related conditions.

Design, Synthesis and Biological Evaluation of Diosgenin-Amino Acid Derivatives with Dual Functions of Neuroprotection and Angiogenesis.

Diosgenin, a natural product with steroidal structure, has a wide range of clinical applications in China. It also shows great potential in the treatment of blood clots and nerve damage. To enhance the bioavailability as well as efficacy of diosgenin, eighteen diosgenin-amino acid derivatives were designed and synthesized. The neuroprotective effects of these compounds were evaluated by SH-SY5Y cell line and the biosafety was evaluated by H9c2 cell line. The results displayed that part of the derivatives' activities (EC50 < 20 µM) were higher than positive control edaravone (EC50 = 21.60 ± 3.04 µM), among which, DG-15 (EC50 = 6.86 ± 0.69 µM) exhibited the best neuroprotection. Meanwhile, biosafety evaluation showed that DG-15 had no cytotoxicity on H9c2 cell lines. Interestingly, combined neuroprotective and cytotoxic results, part of the derivatives without their protecting group were superior to compounds with protecting group. Subsequently, Giemsa staining and DAPI (4',6-diamidino-2-phenylindole) staining indicated that DG-15 had a protective effect on damaged SH-SY5Y cells by reducing apoptosis. Moreover, DG-15 showed a higher role in promoting angiogenesis at high concentrations (4 mg/mL) on the chorioallantoic membrane model. This finding displayed that DG-15 had dual functions of neuroprotection and angiogenesis, which provided further insight into designing agent for the application in treatment of ischemic stroke.

Pro-angiogenesis effect and transcriptome profile of Shuxinyin formula in zebrafish.

BACKGROUND: Angiogenesis plays a critical role in ischemia disease like coronary heart disease. Shunxinyin formula has been developed for treating coronary heart disease according to the principle of traditional Chinese medicine while its underlying mechanism is not fully elucidated. PURPOSE: Here, we hypothesize Shuxinyin formula could promote angiogenesis and microcirculation, and the underlying mechanism is also investigated. METHODS: We established the chemical profile of Shuxinyin (SXY) extract utilizing a UHPLC-Q/Exactive analysis system and evaluated its pro-angiogenesis effect in zebrafish model. The underlying mechanisms were investigated by combination of pharmacological experiments with transcriptome analysis in zebrafish. Zebrafish treated with VEGF was served as the positive control in present study. RESULTS: We found SXY significantly enhanced the sub-intestinal vessel plexus (SIVs) growth in zebrafish. Co-treatment and post-treatment SXY attenuated VEGF receptor tyrosine kinase inhibitor II (VRI)-induced deficiency of intersegmental vessels (ISVs) in a concentration dependent manner. Post-treatment VEGF, which is a well-known angiogenesis driver, also partially ameliorated VRI-induced ISVs deficiency. In addition, SXY inhibited the down-regulation of VEGF receptors, including kdr, flt1 and kdrl, induced by VRI in zebrafish. The pro-angiogenesis effect of SXY on VRI-induced ISVs deficiency was suppressed by PI3K and JNK inhibitors, and Akt inhibitor abolished the pro-angiogenesis effect of SXY. The transcriptome profile of SXY preventing from VRI-induced vascular growth deficiency revealed that the underlying mechanisms were also co-related to cell junction, apoptosis and autophagy. CONCLUSION: We could conclude that SXY presented pro-angiogenesis effect and the action mechanisms were involved in VEGF/PI3K/Akt/MAPK signaling pathways, cell junction, apoptosis and autophagy.

Synthesis of Novel Baicalein Amino Acid Derivatives and Biological Evaluation as Neuroprotective Agents.

Baicalein, a famously effective component of the traditional Chinese medicine Rhizoma Huang Qin (Scutellaria altissima L.), has been proved to have potent neuroprotection and anti-platelet aggregation effects with few side effects. Meanwhile, recent studies have revealed that the introduction of amino acid to baicalein could improve its neuroprotective activity. In the present study, a series of novel baicalein amino acid derivatives were designed, synthesized, and screened for their neuroprotective effect against tert-butyl, hydroperoxide-induced, SH-SY5Y neurotoxicity cells and toxicity on the normal H9C2 cell line by standard methylthiazol tetrazolium (MTT) assay. In addition, all of the newly synthesized compounds were characterized by 1H-NMR, 13C-NMR, and high resolution mass spectrometry (HR-MS). The results showed that most of the compounds provided more potent neuroprotection than baicalein, and were equivalent to the positive drug edaravin. They showed no obvious cytotoxicity on normal H9C2 cells. Notably, the most active compound 8 displayed the highest protective effect (50% effective concentration (EC50) = 4.31 µM) against tert-butyl, hydroperoxide-induced, SH-SY5Y neurotoxicity cells, which was much better than the baicalein (EC50 = 24.77 µM) and edaravin (EC50 = 5.62 µM). Further research on the chick chorioallantoic membrane (CAM) model indicated that compound 8 could significantly increase angiogenesis, which might promote neurovascular proliferation. The detection of apoptosis analysis showed that compound 8 could dramatically alleviate morphological manifestations of cell damage. Moreover, the benzyloxycarbonyl (cbz)-protected baicalein amino acid derivatives showed better neuroprotective activity than the t-Butyloxy carbonyl (boc)-protected derivatives.

Angiosuppressive effects of bio-fabricated silver nanoparticles synthesis using Clitoria ternatea flower: an in vitro and in vivo approach.

Biosynthesis of silver nanoparticles (CTNP's) by Clitoria ternatea flower in the aqueous extract was investigated. Synthesized nanoparticles were characterized by using UV-Visible spectroscopy, followed by DLS, Zeta potential, XRD, FTIR, SEM, and AFM. The biocompatibility nature of CTNP's was determined using erythrocytes model system. Cytotoxicity of CTNP's against MCF-7 and EAC cells were determined by using MTT and Trypan blue exclusion method and their IC50 was found to be 19.37 µg/mL and 24 µg/mL. Cytotoxic potential of CTNP's was further confirmed by clonogenic assay. Further in vivo studies using EAC mice model supports the anti-cancer potential of silver nanoparticles. Results found that the CTNP's effectively control the proliferation rate by inhibiting the ascites secretion and cellular density. Further quantification of VEGF, microvessel density counts and CAM assays show the anti-angiogenic potential of the CTNP's. The apoptotic inducing activity of CTNP's was confirmed by DNA fragmentation, fluorescent staining studies. More interestingly, EAC treated mice exhibit significant increase in lifespan (~ 2.25 fold) compared to control EAC mice. Interestingly CTNP's did not exhibit any secondary complications against normal mice. The present findings give an experimental proof that the CTNP's could serve as a promising candidate to overcome limitations of existing conventional cancer therapeutics.

Novel Insights into the Effect of Hyperforin and Photodynamic Therapy with Hypericin on Chosen Angiogenic Factors in Colorectal Micro-Tumors Created on Chorioallantoic Membrane.

Photodynamic therapy with hypericin (HY-PDT) and hyperforin (HP) could be treatment modalities for colorectal cancer (CRC), but evidence of their effect on angiogenic factors in CRC is missing. Convenient experimental model utilization is essential for angiogenesis research. Therefore, not only 2D cell models, but also 3D cell models and micro-tumors were used and compared. The micro-tumor extent and interconnection with the chorioallantoic membrane (CAM) was determined by histological analyses. The presence of proliferating cells and HY penetration into the tumor mass were detected by fluorescence microscopy. The metabolic activity status was assessed by an colorimetric assay for assessing cell metabolic activity (MTT assay) and HY accumulation was determined by flow cytometry. Pro-angiogenic factor expression was determined by Western blot and quantitative real-time polymerase chain reaction (RT-qPCR). We confirmed the cytotoxic effect of HY-PDT and HP and showed that their effect is influenced by structural characteristics of the experimental model. We have pioneered a method for analyzing the effect of HP and cellular targeted HY-PDT on pro-angiogenic factor expression in CRC micro-tumors. Despite the inhibitory effect of HY-PDT and HP on CRC, the increased expression of some pro-angiogenic factors was observed. We also showed that CRC experimental micro-tumors created on quail CAM could be utilized for analyses of gene and protein expression.

Ameliorating effects of Cuscuta chinensis Lamak extract on hind­limb ischemia, and angiogenic­ or inflammatory associated factors in ovariectomized mice.

Cuscuta chinensis Lamak (CCL) has traditionally been used in Korea to treat sexual disorders and skin problems. The aim of the present study was to investigate the effects of CCL extract on surgical injury­induced ischemia in the hind limbs of mice. Specifically, female C57BL/6 mice were ovariectomized, and their hind­limb vessels were ligated with surgical silk (6­0) and excised. CCL (150 or 450 mg/kg/BW) was then administered to the mice for 3 weeks, and the blood flow rate was evaluated using a laser Doppler system at ­7, 0, 7, 14 and 21 days following hind­limb ischemia. The serum expression profiles of angiogenic and inflammatory mediators were measured using an antibody array, and the transcript levels were reverse transcription­quantitative polymerase chain reaction. The rate of hind limb blood flow was normalized to non­ischemic lesions and revealed to be markedly elevated at 14 and 21 days following ischemia when compared with the vehicle group. The density of capillaries in the hind limbs was also significantly increased following treatment with CCL in a dose­dependent manner. In addition, the transcriptional expression of angiogenetic factors were upregulated, whereas that of inflammatory cytokines were downregulated. Finally, vascular endothelial cell migration and tube formation were evaluated in vitro using human umbilical vein endothelial cells (HUVECs) and identified to be significantly increased following treatment with CCL. Overall, the results of the present study indicate that CCL extract exhibits therapeutic potential for the treatment of hind­limb ischemia as it promotes peripheral angiogenic and anti­inflammatory effects in mice.

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.

Apratyramide, a Marine-Derived Peptidic Stimulator of VEGF-A and Other Growth Factors with Potential Application in Wound Healing.

A novel linear depsipeptide enriched with tyrosine-derived moieties, termed apratyramide, was isolated from an apratoxin-producing cyanobacterium. The structure was determined using a combination of NMR spectroscopy, mass spectrometry, and chiral analysis of the acid hydrolyzate and confirmed by total synthesis. Apratyramide up-regulated multiple growth factors at the transcript level in human keratinocyte (HaCaT) cells and induced the secretion of vascular endothelial growth factor A (VEGF-A) from HaCaT cells, suggesting the compound's potential wound-healing properties through growth factor induction. Transcriptome analysis and sequential validation supported the hypothesis and indicated its mode of action (MOA) through the unfolded protein response (UPR) pathway, which is functionally related to wound healing and angiogenesis. The conditioned medium of HaCaT cells treated with apratyramide induced angiogenesis in vitro. An ex vivo rabbit corneal epithelial model was applied to confirm the VEGF-A induction in this wound-healing model.

Angiogenic peptide nanofibers repair cardiac tissue defect after myocardial infarction.

Myocardial infarction remains one of the top leading causes of death in the world and the damage sustained in the heart eventually develops into heart failure. Limited conventional treatment options due to the inability of the myocardium to regenerate after injury and shortage of organ donors require the development of alternative therapies to repair the damaged myocardium. Current efforts in repairing damage after myocardial infarction concentrates on using biologically derived molecules such as growth factors or stem cells, which carry risks of serious side effects including the formation of teratomas. Here, we demonstrate that synthetic glycosaminoglycan (GAG) mimetic peptide nanofiber scaffolds induce neovascularization in cardiovascular tissue after myocardial infarction, without the addition of any biologically derived factors or stem cells. When the GAG mimetic nanofiber gels were injected in the infarct site of rodent myocardial infarct model, increased VEGF-A expression and recruitment of vascular cells was observed. This was accompanied with significant degree of neovascularization and better cardiac performance when compared to the control saline group. The results demonstrate the potential of future clinical applications of these bioactive peptide nanofibers as a promising strategy for cardiovascular repair. STATEMENT OF SIGNIFICANCE: We present a synthetic bioactive peptide nanofiber system can enhance cardiac function and enhance cardiovascular regeneration after myocardial infarction (MI) without the addition of growth factors, stem cells or other biologically derived molecules. Current state of the art in cardiac repair after MI utilize at least one of the above mentioned biologically derived molecules, thus our approach is ground-breaking for cardiovascular therapy after MI. In this work, we showed that synthetic glycosaminoglycan (GAG) mimetic peptide nanofiber scaffolds induce neovascularization and cardiomyocyte differentiation for the regeneration of cardiovascular tissue after myocardial infarction in a rat infarct model. When the peptide nanofiber gels were injected in infarct site at rodent myocardial infarct model, recruitment of vascular cells was observed, neovascularization was significantly induced and cardiac performance was improved. These results demonstrate the potential of future clinical applications of these bioactive peptide nanofibers as a promising strategy for cardiovascular repair.

Physicochemical/photophysical characterization and angiogenic properties of Curcuma longa essential oil.

This study analyzed the physicochemical and photophysical properties of essential oil of Curcuma longa and its angiogenic potential. The results showed that curcumin is the main fluorescent component present in the oil, although the amount is relatively small. The experimental chorioallantoic membrane model was used to evaluate angiogenic activity, showing a significant increase in the vascular network of Curcuma longa and positive control groups when compared to the neutral and inhibitor controls (P <0.05), but no significant difference was found between Curcuma longa essential oil and the positive control (P >0.05). Histological analysis showed extensive neovascularization, hyperemia and inflammation in the positive control group and Curcuma longa when compared to other controls (P <0.05), characteristic factors of the angiogenesis process. In conclusion, Curcuma longa oil showed considerable proangiogenic activity and could be a potential compound in medical applications.

Treatment with a herbal formula B401 enhances neuroprotection and angiogenesis in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disease characterized by motor dysfunction and early death. Despite years of research, the mechanisms responsible for chronic neurodegeneration of HD remain elusive. Chinese traditional medicines might provide new insights or new therapy for HD. The Chinese herbal formula B401 is a well-known Taiwan-US patent formula and a health supplement for promoting blood circulation and enhancing brain function. This study aimed to elucidate the neuroprotective effects of the Chinese herbal formula B401 on the syndrome of HD. Then, we compared the life span and body weight of R6/2 HD mice with and without oral B401 treatment. The ameliorative effects of B401 on the symptom of HD mice were investigated through behavior tests. Expressions of proteins for neuroprotection, angiogenesis, and inflammation in the brain tissue of R6/2 HD mice were compared by using immunostaining and Western blotting techniques. Our study in vitro showed that viabilities of glutamate-treated SH-SY5Y cells were significantly increased under B401 treatment. Our results in vivo showed that duration of survival was increased, body weight loss was reduced, and motor ability was improved in R6/2 HD mice under oral B401 treatment. Subcutaneous microcirculation was enhanced in 3-month R6/2 HD mice under intraperitoneal B401 injections as observed by using moorFLPI laser Doppler imager. Atrophy of cerebrum, midbrain, and cerebellum in 3-month R6/2 HD mice under oral B401 treatment was alleviated as observed by utilizing magnetic resonance imaging. Evidence from immunostaining and Western blotting analysis showed that expressions of mutant huntingtin and tumor necrosis factor-alpha were reduced, while expressions of brain-derived neurotrophic factor and vascular endothelial growth factor were enhanced in the brain tissue of 2-month R6/2 HD mice under oral B401 treatment. We suggest that the herbal formula B401 can be developed as a medical supplement for ameliorating neurodegenerative diseases of HD via reducing mutant huntingtin aggregation and excitotoxicity, enhancing neuroprotection and angiogenesis, and alleviating inflammation in the brain.

[Synthesis and characterization of protocatechuic acid derivants].

To explore the effects of protocatechuic acid (PCA) and its derivants on angiogenesis of the chick embryo chorioallantoic membrane (CAM) and scavenging DPPH radical in vitro. The protection of benzyl and alkaline hydrolysis of benzyl ester were employed. The structures of PCA-1, PCA-2 and PCA-3, the derivates of PCA, were elucidated by 1H, 13C-NMR and MS data The bioactivity of PCA and its derivants was evaluated on the models of DPPH radical and chick embryo chorioallantoic membrane (CAM), respectively. PCA and PCA-1 showed the best activity of scavenging DPPH radical among all the compounds. In contrast to PCA-2, PCA and PCA-3 displayed inhibition to angiogenesis (P < 0.001). Pyrocatechol hydroxyl is the active site of PCA on scavenging DPPH radical in vitro. PCA with carboxyl and without pyrocatechol hydroxyl seems to show promotion to angiogenesis, but it needs more evidences.

New alkaloids and cytotoxic principles from Sinomenium acutum.

Two new alkaloids, 2-demethyl-oxypalmatine (1) and 5-ethoxycarbonylsinoracutine (2), were isolated from the rhizomes of Sinomenium acutum, along with thirty-four known compounds. Cytotoxicity of the isolated compounds was examined for the MCF-7, H460, HT-29, and CEM human cancer cell lines. Dauriporphine (16), 6-O-demethylmenisporphine (17), bianfugecine (18), menisporphine (19), and 6-O-demethyldauriporphine (20) showed differential effects in their cytotoxic activity on the target cancer cell lines. Significant angiogenesis inhibitions of 16 and 19 were also observed.

Bioassay-guided isolation of norviburtinal from the root of Rehmannia glutinosa, exhibited angiogenesis effect in zebrafish embryo model.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Rehmannia glutinosa (RR) is commonly used as a wound-healing agent in various traditional Chinese herbal formulae; while angiogenesis is one of the crucial aspects in wound-healing. AIM OF THE STUDY: The objective of the present study was to investigate the angiogenesis effects of RR aqueous crude extract and its active component(s) using zebrafish model. MATERIALS AND METHODS: The in vivo angiogenesis effect was studied using transgenic TG(fli1:EGFP)(y1)/+(AB) zebrafish embryos by observing the capillary sprouts formation in sub-intestinal vessel (SIV) of zebrafish embryos after 72 h post-fertilization under fluorescence microscopy. RESULTS: Our results indicated that RR aqueous crude extract (250 µg/ml) exhibited significant angiogenesis effect, with an increase in capillary sprouts formation in SIV. Following sequential solvent partition of the RR aqueous crude extract with dichloromethane, ethyl acetate and n-butanol successively, the dichloromethane fraction (DCM) was found to have the most sprouts formation in the SIV region. Subjected to column chromatography, DCM fraction was further fractionated into six sub-fractions and among these tested, the sub-fraction C2 exhibited the most potent angiogenesis effect. The major component, C2A, was isolated and identified as norviburtinal using nuclear magnetic resonance (NMR) and mass spectrometry (MS). The compound norviburtinal (at 50 µg/ml) was shown to possess significant angiogenesis effect in zebrafish model (p < 0.001). CONCLUSIONS: Norviburtinal was, for the first time, found in the extract of RR and possessed novel angiogenesis effect. Bioassay-guided fractionation suggested that norviburtinal was not the only active component responsible for the angiogenesis effect of RR.

Role of microRNA-214 in ginsenoside-Rg1-induced angiogenesis.

MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional gene modulators. Ginsenoside-Rg1, one of the active components of ginseng, has been confirmed as an angiogenesis inducer. Using miRNA microarray analysis, a total of 17 (including miR-214) and 5 miRNAs were found to be down- or up-regulated by Rg1 in human umbilical vein endothelial cells (HUVECs), respectively. Since miR-214 is closely related to endothelial nitric oxide synthase (eNOS) and hence angiogenesis, its expression was further validated by qRT-PCR. We also investigated the role of miR-214 on eNOS expression and in tubulogenesis and motility of HUVEC by transfection of specific miRNA inhibitor or precursor. Our results suggested that Rg1 can down-regulate miR-214 expression in HUVEC, leading to an increase in eNOS expression, and in vitro cell migration and tube formation which can possibly promote angiogenesis. These results signify a new understanding towards how a simple natural compound can affect physiological changes through modulation of miRNA expression.

Stability of angiogenic agents, ginsenoside Rg1 and Re, isolated from Panax ginseng: in vitro and in vivo studies.

The study was designed to investigate the stability of ginsenoside Rg(1) (Rg(1)) and Re (Re), two natural herbal compounds isolated from Panax ginseng, based on their activity to promote angiogenesis in vitro and in vivo. After being treated at different temperatures, pHs, and solvent species for distinct durations, the remaining activities of Rg(1) and Re on human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation were examined in vitro. Additionally, the remaining activity of each treated test agent, mixed in a growth factor-reduced Matrigel, in stimulating angiogenesis was evaluated subcutaneously in a mouse model. Basic fibroblast growth factor (bFGF) was used as a control. It was found in vitro that HUVEC proliferation, migration in a Transwell plate, and tube formation on Matrigel were all significantly enhanced in the presence of bFGF, Rg(1), or Re. However, after being treated at different temperatures, pHs, or solvent species, the remaining activity of bFGF on HUVEC behaviors reduced significantly. This observation was more significant with increasing the duration of treatment. In contrast, the activities of Rg(1) and Re remained unchanged throughout the entire course of the study. The in vivo results observed on day 7 after implantation showed that the blank control (Matrigel alone) was slightly vascularized. In contrast, the density of neo-vessels in the Matrigel plug mixed with bFGF, Rg(1), or Re was significantly enhanced. However, after being treated, the density of neo-vessels was significantly reduced in the Matrigel plug mixed with bFGF, while those of Rg(1) and Re remained unchanged. The aforementioned results suggested that Rg(1) and Re could be a novel group of nonpeptide angiogenic agents with a superior stability and may be used for the management of tissue regeneration.

Identification of HIV-1 Tat peptides for future therapeutic angiogenesis.

Therapeutic angiogenesis represents a novel approach to treat critical limb ischemia when revascularization is no more an option. The clinical use of the vascular endothelial growth factor is questioned, because of its side effects. This study was designed to identify and characterize human immunodeficiency virus type 1 (HIV-1) Tat-derived peptides based on their pro-angiogenic properties. A series of Tat-derived peptides were synthesized containing mutations in the basic domain. To minimize side effects Tat peptides were selected exerting no effects on the proteasome and on the viability of human umbilical vein endothelial cells (HUVEC). Tatpep5, 15, and 16 increased the endogenous levels of the pro-angiogenic transcription factors c-Jun and SP-1 as well as the production of the plasminogen activator inhibitor-1 (PAI-1) by HUVEC. A significant induction of endothelial cell invasion was observed upon treatment of HUVEC with Tat peptides. In addition, selected Tat peptides induced tube formation by HUVEC as visualized and quantified in a Matrigel matrix. Our data demonstrate that the selected Tat peptides fulfill essential criteria for pro-angiogenic substances. They represent the basis for the development of novel pro-angiogenic drugs for future therapeutic angiogenesis, which might be applied for treatment of unreconstructible critical limb ischemia.

Uncaria rhynchophylla induces angiogenesis in vitro and in vivo.

Angiogenesis consists of the proliferation, migration, and differentiation of endothelial cells, and angiogenic factors and matrix protein interactions modulate this process. The aim of this study was to determine the angiogenic properties of Uncaria rhynchophylla. Uncaria rhynchophylla significantly enhanced human umbilical vein endothelial cells (HUVECs) proliferation in a dose-dependent manner. Neutralization of vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) by monoclonal antibody suppressed the Uncaria rhynchophylla stimulatory effect on proliferation. In addition, Uncaria rhynchophylla significantly increased chemotactic-migration on gelatin and tubular structures on Matrigel of HUVECs in a dose-dependent manner. Interestingly, Uncaria rhynchophylla dose-dependently increased VEGF, and bFGF gene expression and protein secretion of HUVEC. The angiogenic activity of Uncaria rhynchophylla was confirmed using an in vivo Matrigel angiogenesis model, showing promotion of blood vessel formation. These results suggest that Uncaria rhynchophylla could potentially used to accelerate vascular wound healing or to promote the growth of collateral blood vessel in ischemic tissues.