Ultra high frequency ultrasound enables real-time visualization of blood supply from chorioallantoic membrane to human autosomal dominant polycystic kidney tissue.

Ultra high frequency (UHF) ultrasound enables the visualization of very small structures that cannot be detected by conventional ultrasound. The utilization of UHF imaging as a new imaging technique for the 3D-in-vivo chorioallantoic membrane (CAM) model can facilitate new insights into tissue perfusion and survival. Therefore, human renal cystic tissue was grafted onto the CAM and examined using UHF ultrasound imaging. Due to the unprecedented resolution of UHF ultrasound, it was possible to visualize microvessels, their development, and the formation of anastomoses. This enabled the observation of anastomoses between human and chicken vessels only 12 h after transplantation. These observations were validated by 3D reconstructions from a light sheet microscopy image stack, indocyanine green angiography, and histological analysis. Contrary to the assumption that the nutrient supply of the human cystic tissue and the gas exchange happens through diffusion from CAM vessels, this study shows that the vasculature of the human cystic tissue is directly connected to the blood vessels of the CAM and perfusion is established within a short period. Therefore, this in-vivo model combined with UHF imaging appears to be the ideal platform for studying the effects of intravenously applied therapeutics to inhibit renal cyst growth.

Phytochemical profiling, cytotoxic, anti-migration, and anti-angiogenic potential of phenolic-rich fraction from Peganum harmala: in vitro and in ovo studies.

Peganum harmala has been extensively employed in Algerian traditional medicine practices. This study aimed to explore the impact of n-butanol (n-BuOH) extract sourced from Peganum harmala seeds on cell proliferation, cell migration, and angiogenesis inhibition. Cytotoxic potential of n-BuOH extract was evaluated using MTT (3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide) assay against human breast adenocarcinoma MCF-7 cells, cell migration was determined using scratch assay, and anti-angiogenic effect was evaluated through macroscopic and histological examinations conducted on chick embryo chorioallantoic membrane. Additionally, this research estimated the phytochemical profile of n-BuOH extract. Fifteen phenolic compounds were identified using Ultra-performance liquid chromatography UPLC-ESI-MS-MS analysis. In addition, the n-BuOH extract of P. harmala exhibited potent antioxidant and free radical scavenging properties. The n-BuOH extract showed potent cytotoxicity against MCF-7 cell with an IC50 value of 8.68 ± 1.58 µg/mL. Furthermore, n-BuOH extract significantly reduced migration. A strong anti-angiogenic activity was observed in the groups treated with n-BuOH extract in comparison to the negative control. Histological analysis confirmed the anti-angiogenic effect of the n-BuOH extract. This activity is probably a result of the synergistic effects produced by different polyphenolic classes.

Immunometric and functional measurement of endogenous vasoinhibin in human sera.

Introduction: Circulating levels of the antiangiogenic protein vasoinhibin, a fragment of prolactin, are of interest in vasoproliferative retinopathies, preeclampsia, and peripartum cardiomyopathy; however, it is difficult to determine the circulating levels of vasoinhibin due to the lack of quantitative assays. Methods: This study used human serum samples to assess the concentration and bioactivity of vasoinhibin using a novel enzyme-linked immunosorbent assay (ELISA) for human vasoinhibin, which employs an anti-vasoinhibin monoclonal antibody, a human umbilical vein endothelial cell (HUVEC) proliferation assay, and a chick chorioallantoic membrane (CAM) angiogenesis assay. Results: Serum samples from 17 pregnant women without (one group) and with preeclampsia and pregnancy induced hypertension (another group) demonstrated endogenous vasoinhibin concentrations in the range of 5-340 ng/ml. Immunoactive vasoinhibin levels were significantly higher in preeclampsia serum compared to healthy pregnancy serum (mean 63.09 ± 22.15 SD vs. 19.67 ± 13.34 ng/ml, p = 0.0003), as was the bioactive vasoinhibin level as determined by the HUVEC proliferation assay (56.12 ± 19.83 vs. 13.38 ± 4.88 ng/ml, p < 0.0001). There was a correlation between the concentration of vasoinhibin measured by ELISA and the HUVEC proliferation assay (Pearson r = 0.95, p < 0.0001). Healthy serum demonstrated a proangiogenic effect in the CAM assay (p < 0.05, compared to control), while serum from preeclamptic patients demonstrated an antiangiogenic effect (p < 0.05 vs. control), as did recombinant human vasoinhibin and a synthetic circular retro-inverse vasoinhibin analogue (CRIVi45-51). The antiangiogenic effects in the CAM assay and the inhibition of HUVEC proliferation were abolished by addition of the ELISA anti-vasoinhibin monoclonal antibody, but not by mouse IgG. Discussion: These results demonstrate the first quantitation of endogenous vasoinhibin in human sera and the elevation of it levels and antiangiogenic activity in sera from women with preeclampsia. The development and implementation of a quantitative assay for vasoinhibin overcomes a long-standing barrier and suggests the thorough clinical verification of vasoinhibin as a relevant biomarker.

Chemical composition and anti-angiogenic activity of the essential oil from Blumea eriantha D.C.

Blumea eriantha D.C is a weed from Asteraceae family and is reported to have anticancer activity. The essential oil from the aerial parts was extracted by steam distillation method with the yield of 0.36%. Through GC-MS analysis of the oil, seventeen compounds could be identified by comparing with linear retention indices with the library. Out of the seventeen compounds ß-Caryophylline oxide was isolated by column chromatography with gradient elution and the structure was determined through FT-IR, MS, 1HNMR, 13 C NMR and DEPT. The oil was evaluated for its effect on angiogenesis using Chorioallantoic Membrane Assay (CAM Assay). The concentration dependent antiangiogenic effect was observed with IC 50 value of 19.28 ppm.

Anti-angiogenic and anti-tumour activities of Lignosus rhinocerus (Cooke) Ryvarden water extracts on HCT116 human colorectal carcinoma cells implanted in chick embryos.

ETHNOPHARMACOLOGICAL RELEVANCE: The sclerotium of Lignosus rhinocerus (Cooke) Ryvarden is used by the local communities in Southeast Asia and China to treat cancer, asthma, fever, and other ailments based on traditional knowledge. The sclerotial water extracts were previously reported to exhibit cytotoxic, apoptotic, and immunomodulatory activities - providing a scientific basis for its use in treating cancer; however, there is still a lack of evidence on its potential anti-angiogenic activity. AIM OF THE STUDY: This study aimed to investigate the toxicity, anti-angiogenic, and anti-tumour activities of the hot-water and cold-water extracts of L. rhinocerus using HCT116 human colorectal carcinoma cells implanted in the chick chorioallantoic membrane (CAM) model. MATERIALS AND METHODS: The toxicity of L. rhinocerus extracts towards the chick embryos was determined 24 h post-treatment. The anti-angiogenic activity of the extracts was then investigated at 0.1-10 µg/embryo (6.7-670 µg/mL) at targeted blood vessels. The anti-tumour effect of selected extracts against the HCT116 human colorectal carcinoma cells xenografted onto the chick embryos was also studied. RESULTS: The cold-water extracts of L. rhinocerus displayed strong in ovo toxicity (LC50: 1.2-37.7 µg/mL) while the hot-water extracts are non-toxic up to 670 µg/mL. Among the extracts, the hot-water extracts demonstrated the highest anti-angiogenic activity with 44.0 ± 17.7% reduction of capillary diameter (relative to the saline-treated control). Moreover, treatment of the HCT116 cells xenografted onto the chick embryos with the hot-water extracts resulted in smaller tumour size and lower number of blood vessels compared to the saline-treated control. CONCLUSIONS: The hot-water extracts of L. rhinocerus sclerotium demonstrated anti-angiogenic and anti-tumour activities but most of the cold-water extracts at similar concentrations were devoid of that. Our findings provide further scientific validation of the medicinal use of the sclerotium in treating cancer and thus, expanding our knowledge on the possible mechanism of its anti-cancer effect apart from direct cytotoxicity, induction of apoptosis and immunomodulation that have been studied thus far.

Enhancing bone tissue engineering using iron nanoparticles and magnetic fields: A focus on cytomechanics and angiogenesis in the chicken egg chorioallantoic membrane model.

AIM: To evaluate the potential of iron nanoparticles (FeNPs) in conjunction with magnetic fields (MFs) to enhance osteoblast cytomechanics, promote cell homing, bone development activity, and antibacterial capabilities, and to assess their in vivo angiogenic viability using the chicken egg chorioallantoic membrane (CAM) model. SETTINGS AND DESIGN: Experimental study conducted in a laboratory setting to investigate the effects of FeNPs and MFs on osteoblast cells and angiogenesis using a custom titanium (Ti) substrate coated with FeNPs. MATERIALS AND METHODS: A custom titanium (Ti) was coated with FeNPs. Evaluations were conducted to analyze the antibacterial properties, cell adhesion, durability, physical characteristics, and nanoparticle absorption associated with FeNPs. Cell physical characteristics were assessed using protein markers, and microscopy, CAM model, was used to quantify blood vessel formation and morphology to assess the FeNP-coated Ti's angiogenic potential. This in vivo study provided critical insights into tissue response and regenerative properties for biomedical applications. STATISTICAL ANALYSIS: Statistical analysis was performed using appropriate tests to compare experimental groups and controls. Significance was determined at P < 0.05. RESULTS: FeNPs and MFs notably improved osteoblast cell mechanical properties facilitated the growth and formation of new blood vessels and bone tissue and promoted cell migration to targeted sites. In the group treated with FeNPs and exposed to MFs, there was a significant increase in vessel percentage area (76.03%) compared to control groups (58.11%), along with enhanced mineralization and robust antibacterial effects (P < 0.05). CONCLUSION: The study highlights the promising potential of FeNPs in fostering the growth of new blood vessels, promoting the formation of bone tissue, and facilitating targeted cell migration. These findings underscore the importance of further investigating the mechanical traits of FeNPs, as they could significantly advance the development of effective bone tissue engineering techniques, ultimately enhancing clinical outcomes in the field.

Effects of neratinib on angiogenesis and the early stage of the embryo using chicken embryo as a model.

Angiogenesis is the process of forming new blood capillaries from pre-existing vessels. Even though it is essential during normal development, it plays a major role in cancer progression. Neratinib is a pan-human epidermal growth factor receptor (HER) inhibitor that has recently been approved for the treatment of HER2-positive breast cancer. However, its effects on angiogenesis and embryogenesis remain unknown. This study examined the antiangiogenic effects of neratinib using the chorioallantoic membrane (CAM) of chicken embryos. We also evaluated neratinib's toxicity during the early stages of normal development using the chicken embryos, primary embryonic fibroblasts (EFBs), and human umbilical vein endothelial cells (HUVEC). Our findings revealed that neratinib significantly inhibited the CAM angiogenesis compared to controls by reducing vessel percentage area and the average vessel length. Furthermore, neratinib downregulated vascular endothelial growth factor (VEGF), a key mediator of angiogenesis. At lower concentrations, neratinib was well-tolerated during early stages of normal development. Additionally, EFBs treated with neratinib showed no morphological or viability changes when compared to controls. However, at the highest concentration tested, neratinib treatment reduced HUVEC cell viability. This effect may be associated with the dysregulation of key apoptotic genes, including caspase-3, caspase-8, caspase-9, and the B-cell lymphoma 2 (Bcl2) gene. Our findings indicate a novel potential application of neratinib as an antiangiogenic agent, exhibiting tolerable toxicity in the early stages of embryogenesis.

Vascularization of the adult mouse lung grafted onto the chick chorioallantoic membrane.

In the later stages of angiogenesis, the vascular sprout transitions into a functional vessel by fusing with a target vessel. Although this process appears to routinely occur in embryonic tissue, the biologic rules for sprout fusion and lumenization in adult regenerating tissue are unknown. To investigate this process, we grafted portions of the regenerating post-pneumonectomy lung onto the chick chorioallantoic membrane (CAM). Grafts from all 4 lobes of the post-pneumonectomy right lung demonstrated peri-graft angiogenesis as reflected by fluorescent plasma markers; however, fluorescent microsphere perfusion primarily occurred in the lobe of the lung that is the dominant site of post-pneumonectomy angiogenesis-namely, the cardiac lobe. Vascularization of the cardiac lobe grafts was confirmed by active tissue growth (p < .05). Functional vascular connections between the cardiac lobe and the CAM vascular network were demonstrated by confocal fluorescence microscopy as well as corrosion casting and scanning electron microscopy (SEM). Bulk transcriptional profiling of the cardiac lobe demonstrated the enhanced expression of many genes relative to alveolar epithelial cell (CD11b-/CD31-) control cells, but only the upregulation of Ereg and Fgf6 compared to the less well-vascularized right upper lobe. The growth of actively regenerating non-neoplastic adult tissue on the CAM demonstrates that functional lumenization can occur between species (mouse and chick) and across the developmental spectrum (adult and embryo).

Pedunculagin and tellimagrandin-I stimulate inflammation and angiogenesis and upregulate vascular endothelial growth factor and tumor necrosis factor-alpha in vivo.

Pedunculagin (PD) and tellimagrandin-I (TL), isolated from Myrciaria cauliflora seeds and Eucaliptus microcorys leaves, respectively, have attracted great attention owing to their relevant biological activities, such as antitumor, antioxidant, and hepatoprotective activities. This study investigated the angiogenic potential of PD and TL using a chick embryo chorioallantoic membrane (CAM) assay. Using the CAM assay, our results showed that both PD and TL promoted a significant increase in the number and caliber of blood vessels, the thickness of the CAM, and the presence of fibroblasts and inflammatory cells. Moreover, an increase of tumor necrosis factor-α and vascular endothelial growth factor was observed in the CAM treated with PD and TL, indicating the induction of angiogenic factors. Thus, the remarkable profile of PD and TL in inducing angiogenesis opens up new perspectives for their potential utilization in different therapeutic approaches involving neovascularization.

Exploration of Chick Embryo and Chorioallantoic Membrane on Imaging Navigated Platforms for Anticancer Pharmaceutical Evaluations.

Conforming to the current replace-reduce-refine 3Rs' guidelines in animal experiments, a series of explorative efforts have been made to set up operable biomedical imaging-guided platforms for qualitative and quantitative evaluations on pharmacological effects of tumor vascular-disrupting agents (VDAs), based on the chick embryos (CEs) with its chorioallantoic membrane (CAM), in this overview. The techniques and platforms have been hierarchically elaborated, from macroscopic to microscopic and from overall to specific aspects. A protocol of LED lamplight associated with a new deep-learning algorithm was consolidated to screen out weak CEs by using the CAM vasculature imaging. 3D magnetic resonance imaging (MRI) and laser speckle contrast imaging (LSCI) to monitor the evolution of CE and vascular changes in CAM are introduced. A LSCI-CAM platform for studying the effects of VDAs on normal and cancerous vasculature of CAM and possible molecular mechanisms has been demonstrated. Finally, practical challenges and future perspectives are highlighted. The aim of this article is to help peers in biomedical research to familiarize with the CAM platform and to optimize imaging protocols for the evaluation of vasoactive pharmaceuticals, especially anticancer vascular targeted therapy.

Methods of In Ovo and Ex Ovo Ostrich Embryo Culture with Observations on the Development and Maturation of the Chorioallantoic Membrane.

Culture of shell-free and windowed eggs for drug testing and other experiments has been perfected for smaller eggs such as those of chickens, where the developing blood vessels of the chorioallantoic membrane (CAM) become accessible for manipulative studies. However, due to the thickness and hardness of the ostrich egg shell, such techniques are not applicable. Using a tork craft mini rotary and a drill bit, we established windowed egg, in-shell-membrane windowed egg, and in-shell-membrane shell-free methods in the ostrich egg, depending on whether the shell membranes were retained or not. Concomitant study of the developing CAM revealed that at embryonic day 16 (E16), the three layers of the CAM were clearly delineated and at E25, the chorionic capillaries had fused with the epithelium while the CAM at E37 had reached maturity and the chorion and the allantois were both 3-4 times thicker and villous cavity (VC) and capillary-covering cells were well delineated. Both intussusceptive and sprouting angiogenesis were found to be the predominant modes of vascular growth in the ostrich CAM. Development and maturation of the ostrich CAM are similar to those of the well-studied chicken egg, albeit its incubation time being twice in duration.

The Chick Embryo and Its Structures as a Model System for Experimental Ophthalmology.

The possibilities of using the chick embryo and its individual structures as a model system in experimental ophthalmology are considered. Cultures of the retina and spinal ganglia from chick embryos are used in the development of new methods for the treatment of glaucomatous optic neuropathy and ischemic optic neuropathy. The chorioallantoic membrane is used for modelling vascular pathologies of the eye, screening of anti-VEGF drugs, and assessing biocompatibility of implants. Co-culturing of chick embryo nervous tissue and human corneal cells makes it possible to study the processes of corneal reinnervation. The use of chick embryo cells and tissues in the "organ-on-a-chip" system opens up wide opportunities for fundamental and applied ophthalmological studies.

Use of Chicken Embryos as an Angiogenesis Model for Central Nervous System Malignant Tumor Research.

AIM: To demonstrate the usability of chicken chorioallantoic membrane (CAM) as an angiogenesis model for the development and treatment of malignant tumors of the central nervous system. MATERIAL AND METHODS: A fresh tumor tissue piece taken from Glioblastoma patients, a malignant tumor of the central nervous system, was transferred to the CAM of chicken embryos and left to incubate in the incubator and their development was monitored. After examining the results of the study macroscopically, CAM tissue samples were evaluated both histochemically and immunohistochemically in terms of angiogenic factors VEGF (Vascular Endothelial Growth Factor), bFGF (basic Fibroblast Growth Factor) and PDGF (Platelet Derived Growth Factor). RESULTS: According to histochemical findings obtained from our study when compared with control embryos, blood vessels, fibroblast count and inflammatory infiltration were observed more in the tumor transplanted groups, especially in the tumordeveloping CAM region. There was also intense pleomorphism and marked hypercellularity in the cells. In our immunohistochemical findings, it was determined that bFGF, PDGF, VEGF staining intensities were higher in tumor transplanted groups compared to control groups, and this elevation was more pronounced in the tumor-developing region. CONCLUSION: As a result, it has been shown that the chicken embryo CAM model may be a suitable in vivo model for cancer angiogenesis studies. The protocol we created in this study will be a source for projects related to the use of therapeutic agents in cancer angiogenesis.

Application of Laser Speckle Contrast Imaging (LSCI) for the Angiogenesis Measurement of Tumors in the Chorioallantoic Membrane (CAM) Model.

Tumor angiogenesis is one essential aspect for the growth and metastasis of cancer cells, which means that adequate in vivo angiogenesis models are of utmost importance for the investigation of such diseases. The chick chorioallantoic membrane (CAM) model is one established method for this purpose and has already been used for research on multiple cancer types. One important part of the evaluation of tumors grafted onto the CAM is the measurement of tumor-induced angiogenesis. In order to address this central aspect, we utilized the novel PeriCam perfusion speckle imager (PSI) system high resolution (HR) model (Perimed AB, Järfälla, Sweden), which is based on laser speckle contrast imaging (LSCI) for the semiquantitative measurement of blood flow in the CAM model. This method enables a fast and accurate analysis of the angiogenesis of cell line tumors and primary tumors that are grafted onto the CAM. The proposed model can be regarded as a precursor model for personalized cancer therapy.

Angiogenesis Assays for the Analysis of CCN Proteins.

Angiogenesis, the process of generating new blood vessels from an existing vasculature, is essential in normal developmental processes such as endochondral ossification and in numerous kinds of pathogenesis including tumor growth. A part from the actin of angiogenic factor or antiangiogenic factor, it is still unknown at which stage of the angiogenic cascade these agents affect angiogenesis. Here, we describe methods for the use of cellular communication network factor/connective tissue growth factor (CTGF/CCN2) and CCN2-neutralizing antibody in the currently used principal angiogenesis assays, including those in vitro ones for the proliferation, migration, adhesion, and tube formation of endothelial cells and in vivo assays such as those utilizing type I collagen implantation and the chick chorioallantoic membrane (CAM). In addition, we introduce an autofluorescence imaging of blood vessels in the subcutaneous tumor xenograft mouse model. These assays can be applied to studies on roles of CCN proteins in tumor metastasis and development of treatment strategies targeting CCN proteins.

Chick chorioallantoic membrane: a valuable 3D in vivo model for screening nanoformulations for tumor antiangiogenic therapeutics.

Drug discovery is an extensive process. From identifying lead compounds to approval for clinical application, it goes through a sequence of labor-intensive in vitro, in vivo preclinical screening and clinical trials. Among thousands of drugs screened only a few get approval for clinical trials. Furthermore, these approved drugs are often discontinued due to systemic toxicity and comorbidity at clinically administered dosages. To overcome these limitations, nanoformulations have emerged as the most sought-after strategy to safely and effectively deliver drugs within tumors at therapeutic concentrations. Most importantly, the employment of suitably variable preclinical models is considered highly critical for the therapeutic evaluation of candidate drugs or their formulations. A review of literature from the past 10 years on antiangiogenic nanoformulations shows the employment of limited types of preclinical models mainly the 2-dimensional (2D) monolayer cell culture and murine models as the mainstay for drug uptake, toxicity and efficiency studies. To top it all, murine models are highly expensive, time-consuming and require expertise in handling them. The current review highlights the utilization of the age-old chicken chorioallantoic membrane (CAM), a well-defined angiogenic model in the investigation of antiangiogenic compounds and nanoformulations in an economic framework. For practical applicability, we have evaluated the CAM model to demonstrate the screening of antiangiogenic compounds and that tumor cells can be implanted onto developing CAM for growing xenografts by recruiting host endothelial and other cellular components. In addition, the exploitation of CAM tumor xenograft models for the evaluation of nanoparticle distribution has also been reinforced by demonstrating that intravenously administered iron oxide nanoparticles (IONPs) passively accumulate and exhibit intracellular as well as extracellular compartment accumulation in highly vascular xenografts. Finally, the ethical considerations, benefits, and drawbacks, of using CAM as an experimental model for testing potential therapeutics are also highlighted.

The Effects of Abscisic Acid on Angiogenesis in Both ex vivo and in vivo Assays.

BACKGROUND: Angiogenesis is a complex biological process wherein novel capillary blood vessels mature from pre-existing vasculature for delivering tissues with oxygen and nutrients. Natural molecules that have anti-angiogenic activity and toxicity can raise the focus on using plant sources as essential therapeutic agent. OBJECTIVE: The current research was intended to estimate the probable anti-angiogenic activity of abscisic acid alone and in combination with prednisolone, a well-known angiostatic glucocorticoid. METHODS: two months old albino rats were used in this study. ABA and prednisolone stock solutions were prepared and added after embedding aortic rings in growth media. The ex vivo rat aorta ring assay (RAR) was applied to explore the anti-angiogenic effect of ABA. The in vivo chorioallantoic membrane assay (CAM) was applied to quantify the blood vessels inhibition zone by ABA effect. That zone was calculated as the mean inhibition region on eggs in mm ± SD. RESULTS: Abscisic acid screened byex vivo and in vivo assays, revealed a significant dose-dependent blood vessels inhibition in comparison to the negative control with IC50= 7.5µg/ml and a synergism effect when combined with prednisolone. CONCLUSION: The synergism activity of ABA with prednisolone can significantly inhibit blood vessels growth in RAR and CAM assays. These results shed the light on the potential clinic values of ABA, and prednisolone combination in angiogenesis-dependent tumors.

The Chicken Embryo Chorioallantoic Membrane as an In Vivo Model for Photodynamic Therapy.

For many decades the chicken embryo chorioallantoic membrane (CAM) has been used for research as an in vivo model in a large number of different fields, including toxicology, bioengineering, and cancer research. More specifically, the CAM is also a suitable and convenient model system in the field of photodynamic therapy (PDT), mainly due to the easy access of its membrane and the possibility of grafting or growing tumors on the membrane and, interestingly, to study the PDT effects on its dense vascular network. In addition, the CAM is simple to handle and cheap. Since the CAM is not innervated until later stages of the embryo development, its use in research is simplified compared to other in vivo models as far as ethical and regulatory issues are concerned. In this review different incubation and drug administration protocols of relevance for PDT are presented. Moreover, data regarding the propagation of light at different wavelengths and CAM development stages are provided. Finally, the effects induced by photobiomodulation on the CAM angiogenesis and its impact on PDT treatment outcome are discussed.

Development and characterization of a chick embryo chorioallantoic membrane (CAM) based platform for evaluation of vasoactive medications.

Among various anti-cancer therapies, tumor vascular disrupting agents (VDAs) play a crucial role, for which their off-targeting effects on normal vessels need also to be investigated. The purpose of this study was to set up an in-ovo platform that combines a laser speckle contrast imaging (LSCI) modality with chick embryo chorioallantoic membrane (CAM) to real-time monitor vascular diameters and perfusion without and with intravascular injection. Two eggshell windows for both observation or measurement and injection were opened. Dynamic blood perfusion images and corresponding statistic graphs were acquired by using a LSCI unit on CAMs from embryo date (ED) 9 to ED15. A dedicated fine needle catheter was made for slow intravascular administration over 30 min with simultaneous LSCI acquisition. To verify the connectivity between CAM vessels and the embryonic circulations in the egg, contrast-enhanced 3D micro computed tomography (µCT), 2D angiography and histology were executed. This platform was successfully established to acquire, quantify and demonstrate vascular and hemodynamic information from the CAM. Chick embryos even with air cell opened remained alive from ED9 to ED15. Through collecting LSCI derived CAM vascular diameter and perfusion parameters, ED12 was determined as the best time window for vasoactive drug studies. A reverse correlation between CAM vessel diameter and blood perfusion rate was found (p < 0.002). Intravascular infusion and simultaneous LSCI acquisition for 30 min in ovo proved feasible. Contrast-enhanced angiography and histomorphology could characterize the connectivity between CAM vasculature and embryonic circulation. This LSCI-CAM platform was proved effective for investigating the in-ovo hemodynamics, which paves the road for further preclinical research on vasoactive medications including VDAs.

Investigations on the Wound Healing Potential of Tilapia Piscidin (TP)2-5 and TP2-6.

Wound healing is a highly orchestrated process involving many cell types, such as keratinocytes, fibroblasts and endothelial cells. This study aimed to evaluate the potential application of synthetic peptides derived from tilapia piscidin (TP)2, TP2-5 and TP2-6 in skin wound healing. The treatment of HaCaT keratinocytes with TP2-5 and TP2-6 did not cause cytotoxicity, but did enhance cell proliferation and migration, which could be attributed to the activation of epidermal growth factor receptor signaling. In CCD-966SK fibroblasts, although TP2-5 (31.25 µg/mL) and TP2-6 (125 µg/mL) showed cytotoxic effects, we observed the significant promotion of cell proliferation and migration at low concentrations. In addition, collagen I, collagen III, and keratinocyte growth factor were upregulated by the peptides. We further found that TP2-5 and TP2-6 showed pro-angiogenic properties, including the enhancement of human umbilical vein endothelial cell (HUVEC) migration and the promotion of neovascularization. In a murine model, wounds treated topically with TP2-5 and TP2-6 were reduced by day 2 post-injury and healed significantly faster than untreated wounds. Taken together, these findings demonstrate that both TP2-5 and TP2-6 have multifaceted effects when used as topical agents for accelerating wound healing.