Chorioallantoic Membrane Angiogenesis Model Research Articles

Disruption of Angiogenesis by Anthocyanin-Rich Extracts of Hibiscus sabdariffa.

Abnormal vessel formations contribute to the progression of specific angiogenic diseases including age-related macular degeneration. Adequate vessel growth and maintenance represent the coordinated process of endothelial cell proliferation, matrix remodeling, and differentiation. However, the molecular mechanism of the proper balance between angiogenic activators and inhibitors remains elusive. In addition, quantitative analysis of vessel formation has been challenging due to complex angiogenic morphology. We hypothesized that conjugated double bond containing-natural products, including anthocyanin extracts from Hibiscus sabdariffa, may control the proper angiogenesis. The current study was designed to determine whether natural molecules from African plant library modulate angiogenesis. Further, we questioned how the proper balance of anti- or pro-angiogenic signaling can be obtained in the vascular microenvironment by treating anthocyanin or fatty acids using chick chorioallantoic membrane angiogenesis model in ovo. The angiogenic morphology was analyzed systematically by measuring twenty one angiogenic indexes using Angiogenic Analyzer software. Chick chorioallantoic model demonstrated that anthocyanin-rich extracts inhibited angiogenesis in time- and concentration-dependent manner. Molecular modeling analysis proposed that hibiscetin as a component in Hibiscus may bind to the active site of vascular endothelial growth factor receptor 2 (VEGFR2) with ΔG= -8.42 kcal/mol of binding energy. Our results provided the evidence that anthocyanin is an angiogenic modulator that can be used to treat uncontrolled neovascular-related diseases, including age-related macular degeneration.

Development of collateral vessels: A new paradigm in CAM angiogenesis model

The chorioallantoic membrane (CAM) assay is one of the most widely used models to study angiogenesis. In this study, collateral vessel development is reported in CAM assay useful in analysis of angiogenesis. Four days old white Leghorn fertilized chicken eggs were inoculated with vehicle, standard or test angiogenesis inhibitor using standard protocol. Central vessel growth was seen tapering down and collateral vessels were developed from the lower side of the chorioallantoic membrane moving upward in 12days old standard or test treated CAMs. In the absence of the central vessel, collateral blood supply helped in survival of embryos. Hence, development of collateral vessels was used for ranking of blood vessels and angiogenesis in addition to well-known standard parameters related to central vessel. The finding could differentiate molecules inhibiting angiogenesis with or without collateralization which is crucial in anti-angiogenic therapy used for cardiovascular diseases and cancer.This study proposes a new avenue to distinguish pro-angiogenic molecules from anti-angiogenic ones as well as anti-angiogenic molecules which may or may not support alternative vascularization pathway that would have great impact on future angiogenic and anti-angiogenic therapy.

Inhibition of endogenous hydrogen sulfide production in clear-cell renal cell carcinoma cell lines and xenografts restricts their growth, survival and angiogenic potential

Clear cell renal cell carcinoma (ccRCC) is characterized by Von Hippel–Lindau (VHL)-deficiency, resulting in pseudohypoxic, angiogenic and glycolytic tumours. Hydrogen sulfide (H2S) is an endogenously-produced gasotransmitter that accumulates under hypoxia and has been shown to be pro-angiogenic and cytoprotective in cancer. It was hypothesized that H2S levels are elevated in VHL-deficient ccRCC, contributing to survival, metabolism and angiogenesis. Using the H2S-specific probe MeRhoAz, it was found that H2S levels were higher in VHL-deficient ccRCC cell lines compared to cells with wild-type VHL. Inhibition of H2S-producing enzymes could reduce the proliferation, metabolism and survival of ccRCC cell lines, as determined by live-cell imaging, XTT/ATP assay, and flow cytometry respectively. Using the chorioallantoic membrane angiogenesis model, it was found that systemic inhibition of endogenous H2S production was able to decrease vascularization of VHL-deficient ccRCC xenografts. Endogenous H2S production is an attractive new target in ccRCC due to its involvement in multiple aspects of disease.

Establishment of experimental angiogenic models with applications of quantitative digital image analysis

To establish experimental models for tumor neovascularization and to apply quantitative digital imaging analysis in the study. An endothelial tube formation model was established by human umbilical vein endothelial cells (HUVECs). A vasculogenic mimicry model was established by SGC-7901 gastric cancer cell line. Fertilized eggs were used to establish a chorioallantoic membrane angiogenesis model. Using gene transfection experiment, IRX1 tumor suppressor gene was chosen as a therapeutic target. Image Pro Plus (IPP) analysis software was used for digital vascular images analysis with parameters including points, lines, angles and integral absorbance (IA) for the tubular formation or vasculogenic mimicry. Digital image analysis by IPP showed that HUVEC tubular formation was significantly inhibited in IRX1 transfectant, compared with controls. The tubular numbers in three groups were 12.80 +/- 3.83, 29.00 +/- 5.34 and 28.20 +/- 4.32 (P<0.01). The connection points of tubules in three groups were 13.20 +/- 2.59, 25.00 +/- 2.24 and 24.60 +/- 3.21 (P<0.01). The tubular lengths of three groups were (821.5 +/- 12.5), (930.9 +/- 13.5) and (948.4 +/- 18.1) microm (P=0.022). The IA values of PAS stain in three groups were 3606 +/- 363, 14 200 +/- 1251 and 15 043 +/- 1220 (P<0.01). In chick chorioallantoic membrane model, the angular numbers of tubules in three groups were 6.41 +/- 2.60, 10.27 +/- 2.65 and 9.18 +/- 1.99 (P<0.01). The endothelial tube formation model, vasculogenic mimicry model and chorioallantoic membrane angiogenesis model are useful for gene therapy and drug screening with targeting neoplastic vascularization. Professional image analysis software may greatly facilitate the quantitative analysis of tumor neovascularization.

The effect of topiramate on tumor-related angiogenesis and on the serum proteome of mice bearing Lewis lung carcinoma

Topiramate has been used in patients with brain tumors who develop epilepsy. In our previous research we found topiramate could inhibit tumor metastases of Lewis lung carcinoma in C57BL/6 mice. In this study we aimed to assess the antimetastatic activity of topiramate and determine its mechanism of action. After confirming the effects of topiramate on Lewis lung carcinoma in C57BL/6 mice, we assessed the mRNA expression of carbonic anhydrases II and IX, and the vascular endothelial growth factor (VEGF) distribution in tumor tissue. We studied the role of topiramate on primary angiogenesis using a chicken embryo chorioallantoic membrane angiogenesis model, and analyzed the protein profile of serum from mice treated with or without topiramate by two-dimensional electrophoresis. We found that topiramate significantly reduced the primary tumor growth ( P < 0.05) and the degree of damage to the lung alveoli caused by metastatic tumor deposits. The two-dimensional electrophoresis revealed changes that occurred with topiramate treatment and four down-regulated protein spots were clearly identified as tropomyosin, osteopontin, transthyretin, and serum amyloid A-1. The mRNA and protein expression of serum amyloid A-1, osteopontin and its receptor, integrin α vβ 3 in tumor tissue were reconfirmed. The results suggest that topiramate has antitumor and antimetastatic effects on Lewis lung carcinoma. Its mechanism of action may be related to its inhibition of angiogenesis by down-regulation of osteopontin, VEGF and carbonic anhydrase II.

Enhanced angiogenesis of modified porcine small intestinal submucosa with hyaluronic acid‐poly(lactide‐co‐glycolide) nanoparticles: From fabrication to preclinical validation

Hyaluronic acid-poly(de-co-glycolide) nanoparticles (HA-PLGA NPs) were synthesized to stabilize the porous structure of porcine small intestinal submucosa (SIS), to improve surface biocompatibility and to enhance performance in tissue regeneration. HA-PLGA NPs were characterized for size, zeta potential, surface morphology, and HA loading. Human microvascular endothelial cells responded to HA-PLGA NPs and HA-PLGA modified SIS (HA-PLGA-SIS) with elevated cell proliferation. HA-PLGA-SIS significantly enhanced neo-vascularization in an in ovo chorioallantoic membrane angiogenesis model. The angiogenic capability of the newly fabricated HA-PLGA-SIS was tested in a canine bladder augmentation model. Urinary bladder augmentation was performed in beagle dogs following hemi-cystectomy using HA-PLGA-SIS. The regenerated bladder was harvested at 10 weeks post augmentation and vascularization was evaluated using CD31 immunohistochemical staining. Bladder regenerated with HA-PLGA-SIS had significantly higher vascular ingrowth compared to unmodified SIS. This study shows that HA-PLGA NPs may represent a new approach for modifying naturally derived SIS biomaterials in regenerative medicine.

Bilberry inhibits angiogenesis in chick chorioallontoic membrane

Angiogenesis is the process of generating new blood vessels from preexisting vessels and is considered essential in many pathological conditions. Bilberry has been traditionally used as a folk medicine for some countries. The purpose of the present study was to evaluate the effect of bilberry in chick chorioallantoic membrane angiogenesis model in vivo. In this well characterized model, bilberry inhibited angiogenesis in a concentration-dependent manner. Compared with the normal group, bilberry group has significant decreased vessels proliferation. These results provide evidence that bilberry inhibits angiogenesis and may be useful for treating angiogenesis-dependent human diseases.

Methylene blue inhibits angiogenesis in chick chorioallontic membrane through a nitric oxide-independent mechanism

Angiogenesis is the process of generating new blood vessels from preexisting vessels and is considered essential in many pathological conditions. The purpose of the present study was to evaluate the effect of methylene blue in chick chorioallantoic membrane angiogenesis model in vivo. In this well characterized model, methylene blue inhibited angiogenesis in a concentration-dependent manner. In addition, when methylene blue was combined with sodium nitroprusside, a spontaneous generator of nitric oxide, an inhibition of angiogenesis was evident which was comparable with that observed by the application of methylene blue alone. Sodium nitroprusside, alone, caused a significant inhibition in basal angiogenesis. These results provide evidence that methylene blue inhibits angiogenesis independently of nitric oxide pathway and suggest that methylene blue may be useful for treating angiogenesis-dependent human diseases.