The chick embryo chorioallantoic membrane as a model system for the study of tumor angiogenesis, invasion and development of anti-angiogenic agents.

Angiogenesis, the formation of new blood vessels, is essential for tumor growth, progression and metastasis. The development of agents that target tumor vasculature is ultimately dependent on the availability of appropriate preclinical screening assays. The chorioallantoic membrane (CAM) assay is well established and widely used as a model to examine angiogenesis, and anti-angiogenesis. This review 1) summarizes the currently used angiogenesis assays and the importance of CAM model among them; 2) summarizes the current knowledge about the development and structure of the CAM's capillary bed; 3) reports findings regarding the role played by molecular signaling pathways in angiogenesis process; 4) discusses the use, advantages and limitations of the CAM as a model for studying tumor angiogenesis and invasiveness, as well as development of angiogenic and/or anti-angiogenic agents; 5) discusses the importance of standardization of the major methodologies for all aspects of the use of the CAM in angiogenesis-related studies; 6) and finally, summarizes major findings regarding the agents developed by the use of CAM model in the study of tumor angiogenesis, invasion and development of anti-angiogenic agents

Amelioration of ethanol-induced growth retardation by all-trans-retinoic acid and alpha-tocopherol in shell-less culture of the chick embryo.

The mechanisms of teratogenic action of ethanol (EtOH) were investigated by testing the hypothesis that all-trans-retinoic acid and/or alpha-tocopherol ameliorates ethanol-induced embryonic growth retardation. Chicken embryos were explanted in shell-less cultures and a single dose of EtOH (15, 30, or 50%) or 50% EtOH with either all-trans-retinoic acid (10(-8)M) or alpha-tocopherol (0.05 M) or a mix of all-trans-retinoic acid (10(-8)M) and alpha-tocopherol (0.05 M) was applied to the center of the blastodisc. EtOH significantly increased the mortality rate and induced growth retardation in a dose-dependent manner. In addition, EtOH increased malondialdehyde (MDA) levels, an indicator of oxidative stress and cell damage, in a dose dependent manner. All-trans-retinoic acid, the active form of Vitamin A, and/or alpha-tocopherol, an antioxidant, co-treatment with EtOH significantly diminished both the EtOH-induced mortality and growth retardation. However, only alpha-tocopherol co-treatment reduced the MDA levels. Thus, the mechanisms of teratogenic action of EtOH appear to involve initiation of oxidative stress as well as perturbation of retinoic acid (RA) signaling. It also appears likely that these mechanisms work independently of each other

the chick area vasculosa

The effect of ethanol (EtOH) exposure on extraembryonic vascular development was examined using the chick embryo area vasculosa (AV) in shell-less culture. Embryos were placed in cultures at Hamburger Hamilton (HH) stage 11/12 and a single dose of EtOH (10, 30 or 50%) was applied to the center of the blastodisc. Untreated/sodium-chloride-treated controls showed normal embryonic growth and well-developed extraembryonic vessels at 24/48 h of treatment. At doses of 30 and 50%, the mortality rate was significantly increased, and survivors demonstrated significant growth retardation and inhibition of normal vascular development in a dose-dependent manner. Immunostaining for vascular endothelial growth factor ( VEGF) showed that mesenchymal cells continued to differentiate into angioblasts to form blood islands, but their assembly into primitive vessels was perturbed in a dose-dependent manner. Northern blot analyses of basic fibroblast growth factor, VEGF, Flt-1 and Flk-1 mRNA expression supported these findings and showed a dose-dependent decrease in EtOH-treated cultures compared to controls. Co-treatment with alpha-tocopherol (0.05 M) or all-trans-retinoic acid (10(-8) M) significantly decreased the mortality rate and improved both embryonic growth and extraembryonic vascular development in the cultures. On the other hand, almost all embryos treated with 10% EtOH survived the first 48 h after treatment. However, the complexity of the vascular tree measured as the relative vasculogenesis index, the surface area of the AV and the mRNA expression of vasculogenic molecules were increased during the first 24 h. This acute effect disappeared 48 h after treatment and the vascular tree continued to develop parallel to the controls. No significant growth retardation was observed in this group. These results suggest that, in terms of extraembryonic vascular development, an early, single, low-dose EtOH exposure may have an acute, short-term positive effect, whereas moderate- or high-dose EtOH exposure may severely perturb this process disabling the necessary absorption of the nutrients for the embryo to develop properly. The mechanisms of action of EtOH on extraembryonic vascular development may involve the establishment of reactive oxygen species, resulting in the initiation of oxidative stress and perturbation of retinoic acid signaling and alterations in the expression of growth-regulatory vasculogenic factors and their receptors

agents

Angiogenesis, the formation of new blood vessels, is essential for tumor growth, progression and metastasis. The development of agents that target tumor vasculature is ultimately dependent on the availability of appropriate preclinical screening assays. The chorioallantoic membrane (CAM) assay is well established and widely used as a model to examine angiogenesis, and anti-angiogenesis. This review 1) summarizes the currently used angiogenesis assays and the importance of CAM model among them; 2) summarizes the current knowledge about the development and structure of the CAM's capillary bed; 3) reports findings regarding the role played by molecular signaling pathways in angiogenesis process; 4) discusses the use, advantages and limitations of the CAM as a model for studying tumor angiogenesis and invasiveness, as well as development of angiogenic and/or anti-angiogenic agents; 5) discusses the importance of standardization of the major methodologies for all aspects of the use of the CAM in angiogenesis-related studies; 6) and finally, summarizes major findings regarding the agents developed by the use of CAM model in the study of tumor angiogenesis, invasion and development of anti-angiogenic agents

acid and alpha-tocopherol in shell-less culture of the chick embryo

The mechanisms of teratogenic action of ethanol (EtOH) were investigated by testing the hypothesis that all-trans-retinoic acid and/or alpha-tocopherol ameliorates ethanol-induced embryonic growth retardation. Chicken embryos were explanted in shell-less cultures and a single dose of EtOH (15, 30, or 50%) or 50% EtOH with either all-trans-retinoic acid (10(-8) M) or alpha-tocopherol (0.05 M) or a mix of all-trans-retinoic acid (10(-8) M) and alpha-tocopherol (0.05 M) was applied to the center of the blastodisc. EtOH significantly increased the mortality rate and induced growth retardation in a dose-dependent manner. In addition, EtOH increased malondialdehyde (MDA) levels, an indicator of oxidative stress and cell damage, in a dose dependent manner. All-trans-retinoic acid, the active form of Vitamin A, and/or alpha-tocopherol, an antioxidant, co-treatment with EtOH significantly diminished both the EtOH-induced mortality and growth retardation. However, only alpha-tocopherol co-treatment reduced the MDA levels. Thus, the mechanisms of teratogenic action of EtOH appear to involve initiation of oxidative stress as well as perturbation of retinoic acid (RA) signaling. It also appears likely that these mechanisms work independently of each other. (C) 2004 Elsevier Inc. All rights reserved

The effect of ethanol exposure on extraembryonic vascular development in the chick area vasculosa.

The effect of ethanol (EtOH) exposure on extraembryonic vascular development was examined using the chick embryo area vasculosa (AV) in shell-less culture. Embryos were placed in cultures at Hamburger Hamilton (HH) stage 11/12 and a single dose of EtOH (10, 30 or 50%) was applied to the center of the blastodisc. Untreated/sodium-chloride-treated controls showed normal embryonic growth and well-developed extraembryonic vessels at 24/48 h of treatment. At doses of 30 and 50%, the mortality rate was significantly increased, and survivors demonstrated significant growth retardation and inhibition of normal vascular development in a dose-dependent manner. Immunostaining for vascular endothelial growth factor (VEGF) showed that mesenchymal cells continued to differentiate into angioblasts to form blood islands, but their assembly into primitive vessels was perturbed in a dose-dependent manner. Northern blot analyses of basic fibroblast growth factor, VEGF, Flt-1 and Flk-1 mRNA expression supported these findings and showed a dose-dependent decrease in EtOH-treated cultures compared to controls. Co-treatment with alpha-tocopherol (0.05 M) or all-trans-retinoic acid (10(-8) M) significantly decreased the mortality rate and improved both embryonic growth and extraembryonic vascular development in the cultures. On the other hand, almost all embryos treated with 10% EtOH survived the first 48 h after treatment. However, the complexity of the vascular tree measured as the relative vasculogenesis index, the surface area of the AV and the mRNA expression of vasculogenic molecules were increased during the first 24 h. This acute effect disappeared 48 h after treatment and the vascular tree continued to develop parallel to the controls. No significant growth retardation was observed in this group. These results suggest that, in terms of extraembryonic vascular development, an early, single, low-dose EtOH exposure may have an acute, short-term positive effect, whereas moderate- or high-dose EtOH exposure may severely perturb this process disabling the necessary absorption of the nutrients for the embryo to develop properly. The mechanisms of action of EtOH on extraembryonic vascular development may involve the establishment of reactive oxygen species, resulting in the initiation of oxidative stress and perturbation of retinoic acid signaling and alterations in the expression of growth-regulatory vasculogenic factors and their receptors

Accurate diagnosis of a homozygous G1138A mutation in the fibroblast growth factor receptor 3 gene responsible for achondroplasia.

Achondroplasia is the most common genetic form of dwarfism inherited as an autosomal dominant disorder. Individuals affected with achondroplasia have impaired ability to form bone from cartilage (endochondral bone formation). Homozygous achondroplasia is a neonatal lethal condition. The vast majority of patients with achondroplasia have a G-to-A transition at position 1138 of the fibroblast growth factor receptor 3 (FGFR3) cDNA sequence, resulting in the Gly-to-Arg substitution at position 380 of the FGFR3 protein. This mutation has been diagnosed by SfcI digestion of amplified genomic DNA. However, it has also been demonstrated that the SfcI digestion protocol does not consistently distinguish between DNA samples heterozygous and homozygous for the G1138A substitution. This study was designed to improve the molecular diagnosis based on the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques for the FGFR3 G1138A mutation. The newly designed forward primer contains one mismatch (G at position 1136) from the FGFR3 cDNA sequence (A at position 1136), thereby creating a PstI site (CTGCAG at positions 1134 to 1139) in the amplified DNA from alleles containing the G1138A mutation. The PCR-RFLP technique based on the PstI digestion of amplified genomic DNA with a novel forward primer shows 100% accuracy in diagnosis of the G1138A mutation in heterozygous and homozygous individuals

growth factor receptor 3 gene responsible for achondroplasia

Achondroplasia is the most common genetic form of dwarfism inherited as an autosomal dominant disorder. Individuals affected with achondroplasia have impaired ability to form bone from cartilage (endochondral bone formation). Homozygous achondroplasia is a neonatal lethal condition. The vast majority of patients with achondroplasia have a G-to-A transition at position 1138 of the fibroblast growth factor receptor 3 (FGFR3) cDNA sequence, resulting in the Gly-to-Arg substitution at position 380 of the FGFR3 protein. This mutation has been diagnosed by SfcI digestion of amplified genomic DNA. However, it has also been demonstrated that the SfeI digestion protocol does not consistently distinguish between DNA samples heterozygous and homozygous for the G1138A substitution. This study was designed to improve the molecular diagnosis based on the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques for the FGFR3 G1138A mutation. The newly designed forward primer contains one mismatch (G at position 1136) from the FGFR3 cDNA sequence (A at position 1136), thereby creating a PstI site (CTGCAG at positions 1134 to 1139) in the amplified DNA from alleles containing the G1138A mutation. The PCR-RFLP technique based on the PstI digestion of amplified genomic DNA with a novel forward primer shows 100% accuracy in diagnosis of the G1138A mutation in heterozygous and homozygous individuals