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In vitro systems currently used to investigate angiogenesis include the rat aortic ring assay, the chick aortic arch assay, the mouse metatarsal assay, and human umbilical vein endothelial cell HUVEC bead assay 7 — 13 , and although all of these methods have enhanced our understanding, they also have limitations. Angiogenesis is primarily a microvascular event, so the use of large vessels, such as in the rat aortic ring assay, the chick aortic arch assay and HUVEC assays, is not truly representative of events in small vessels, and variability in the aortic rings used can influence vessel outgrowth.

The ovary is an excellent model in which to study angiogenesis, because it undergoes intense vascular morphogenesis in a cyclical fashion 2 , A culture system that allows the elucidation of the angiogenic process in the corpus luteum is currently in use 15 ; however, to our knowledge, no system exists for the manipulation and assessment of angiogenesis in ovarian follicles in an in vitro setting. A culture method that addresses this problem and that is robust and representative of angiogenesis in vivo would therefore be highly beneficial. In the current study, we have developed a novel culture system, using intact preantral and early antral rat follicles, to provide a new approach to the study of follicular angiogenesis.

Vascular endothelial growth factor VEGF is the key factor involved in promoting angiogenesis, whereas thrombospondin TSP -1 is a putative antiangiogenic factor 4 — 6. The members of the TSP family TSP-1 and TSP-2 are large extracellular matrix glycoproteins, and their effects are mediated through interaction with the cell surface receptors CD36 and integrin-associated protein 16 , TSP-1 has been shown to inhibit angiogenesis both in vitro and in vivo , and studies have shown that treatment with TSP-1 renders endothelial cells unable to respond to many proangiogenic factors 16 , Several studies have used knockout mice to show that the absence of TSP-1 leads to increased vascularization 6 , 19 — 22 , providing the first evidence for the role of TSP-1 in vivo and also suggesting that TSP-1 is an inhibitor of angiogenesis.

However, few studies have investigated the role of TSP-1 in the ovary. In a previous study, we have made the interesting observation that TSP-1 mRNA and protein is up-regulated during follicular atresia in vivo 23 , suggesting that TSP-1 may be involved in the cessation of angiogenesis in follicles undergoing atresia. Studies in rat, bovine, and marmoset models have demonstrated a decrease in both TSP-1 and CD36 expression as follicular development progresses 24 , 25 , leading to the suggestion that it inhibits angiogenesis during early follicular development.

In addition, a correlation between down-regulation of TSP-1 and tumor angiogenesis and invasiveness 26 — 29 has led to the proposed use of TSP-1 as a therapeutic inhibitor of angiogenesis 30 — Because angiogenesis is tightly regulated by both pro and antiangiogenic factors, it is possible that these factors may be able to interact with each other. The direct effect of TSP-1 on granulosa cell apoptosis has not been investigated previously, nor has it been investigated in primary granulosa cells; thus, one aim of the present study was to investigate whether TSP-1 is able to directly induce apoptosis in a more physiological setting.

VEGF is the key factor involved in promoting angiogenesis 1 , 33 — We have previously shown that VEGF has a role in regulating the cyclical angiogenesis that takes place in the developing follicle and corpus luteum, in vivo 36 — Aberrant angiogenesis is associated with various pathological conditions, including tumor growth, coronary artery disease, polycystic ovary syndrome PCOS , endometriosis, and pre-eclampsia 1 , Therefore, Aflibercept presents an ideal molecule to test in our in vitro angiogenesis system, because it is an established angiogenesis inhibitor.

In this study, we describe for the first time a novel culture system for the study of angiogenesis in ovarian follicles. Using this system, we have investigated the role of TSP-1 in regulating in vitro follicular angiogenesis and its effect on follicle development and survival. In addition to providing information on the role of TSP-1 in follicular angiogenesis and development, this system has the potential to be a powerful model for investigating the effects of other putative pro or antiangiogenic compounds in future studies.

Handling and treatment of animals were according to the Animals Scientific Procedures Act, Follicles at these stages were selected as they have undergone recruitment of endothelial cells to the thecal layer and have the highest angiogenic potential.

The Matrigel provides a three-dimensional extracellular matrix support for follicle growth and allows the vascular outgrowths to develop in three-dimensions, as they would in vivo. To test the efficacy of the assay, the known angiogenesis inhibitor Aflibercept Regeneron Pharmaceuticals, Inc. The incorporation of the Fc domain results in homodimerization of the recombinant protein, creating a high-affinity Aflibercept This was done by drawing an area of interest around the outgrowths but excluding the follicle , and the sum of the area of all sprouts was calculated by the software. The mean area of angiogenic sprouting for each treatment was then compared with the control.

Dil-Ac-LDL was added to the culture medium on d 4, and outgrowths were assessed for uptake of the molecule on d 6 via fluorescence microscopy at nm.

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To further characterize the vascular outgrowths, whole-mount fluorescent immunocytochemistry was performed. Follicles were washed in between each treatment step in Tris-buffered saline TBS -Tween, and all incubations were carried out at room temperature unless stated otherwise. After washing, follicles were incubated with donkey antigoat-Alexa Fluor in PBS; Invitrogen, Paisley, UK for 1 h, and then labeling was detected using confocal microscopy at nm red channel to show CDlabeled cells.

Follicles were incubated with goat antimouse peroxidase with Fab fragments in NGS; Dako and kept in the dark before incubating with tyramide signal amplification plus Cy3 system ; PerkinElmer Life Sciences, Beaconsfield, UK for 10 min.

Angiogenesis Assays: A Critical Overview

After washing, labeling was detected using confocal microscopy at nm red channel to show CDlabeled cells. To determine the localization and changes in the number of dying cells, a rabbit antibody to activated caspase-3 Asp; New England Biolabs, Hitchen, UK was used as described previously For each follicle, the section containing the oocyte with a nucleus present was used for quantification of staining.

The cross-sectional area of staining for activated caspase-3 was calculated as a proportion of total cross-sectional area of granulosa cells within each follicle. Follicles were visualized in PBS via fluorescence microscopy using a confocal microscopy at nm green channel to show TUNEL-labeled cells and nm red channel to show propidium iodide stained cells simultaneously.

Two animals were used for each experiment, and experiments were done in triplicate. Animals were killed by asphyxiation with CO 2 and the ovaries removed. Granulosa cells were isolated by puncturing follicles with a gauge needle and gently expelling the cells into culture medium.

Tissue culture grade well plastic plates Corning, Inc. Treatments were carried out in triplicate, and experiments were repeated three times. Because serum can generate a background caspase activity signal, an additional two wells contained cell culture medium and carrier solution without any cells. The well contents were then mixed and incubated at room temperature for up to 3 h. This resulted in cell lysis, followed by caspase cleavage of the substrate and generation of a glow-type luminescent signal, produced by luciferase.

Luminescence was directly proportional to the amount of caspase activity present. The value for the no cell control was subtracted from the experimental values. The experiment was repeated three times to reduce the possible effects of biological variability. To establish whether angiogenesis occurred in our novel in vitro system, follicles were cultured for 6 d in serum-free EBM-2 medium.

Follicles were photographed at the start and end of the culture period, and the d-6 images were compared with the d-0 images. In Fig.

Follicles displayed angiogenic sprouting during the culture period Fig. Establishment of angiogenesis in vitro. In addition, representative images of the outgrowths at higher magnification are shown labeled with CD31 D and CD34 E. We proposed that Aflibercept would inhibit follicular angiogenesis and could be used to establish the efficacy of this culture system, because it is an established angiogenesis inhibitor.

Effect of Aflibercept on angiogenesis. All follicles were late preantral or early antral follicles. White arrows indicate the endothelial outgrowths. Treatment with TSP-1 resulted in a significant inhibition of follicular angiogenesis, in a dose-dependent manner Fig. Effect of TSP-1 on angiogenesis. Representative images show a control follicle Fig. A negative control for the immunohistochemistry is represented in Fig. A dramatic increase in cell death, shown by the green fluorescence, was observed after treatment with TSP Effect of TSP-1 on follicular atresia.

C, A negative control for the immunohistochemistry is shown.

Modeling angiogenesis with micro- and nanotechnology - Lab on a Chip (RSC Publishing)

Black arrows indicate the expression of activated caspase-3, white arrows indicate apoptotic cells, and white arrowheads indicate healthy cells. Effect of TSP-1 on granulosa cell apoptosis. Luminescence is given in arbitrary units au. A novel in vitro system has been developed that uses intact ovarian follicles to investigate follicular angiogenesis.

In this system, tube-like structures with branch points representing angiogenic sprouting are formed and display endothelial cell markers. This system has been used to demonstrate for the first time that TSP-1 is a potent inhibitor of follicular angiogenesis.

Furthermore, we have shown that TSP-1 promotes follicular atresia in vitro by directly inducing apoptosis of granulosa cells. TSP-1 and its receptor CD36 are mainly expressed in the granulosa cells of preantral and early antral follicles within the ovary, with expression declining as follicular development progresses 24 , Primordial and primary follicles are avascular, so angiogenesis must be stimulated during follicle development by proangiogenic factors, including VEGF.

As the antiangiogenic factor TSP-1 is expressed during the follicular stages when vascularization is occurring, it is likely that it acts to limit any overgrowth of vasculature in response to high levels of proangiogenic factors. The localization of TSP-1 in the follicle, together with the studies using TSPdeficient mice 6 , 19 — 22 , strongly suggests that TSP-1 plays a role in inhibiting follicular angiogenesis. In the current study, we have now shown that TSP-1 is able to inhibit follicular angiogenesis in vitro. We have shown previously, in a descriptive study, that TSP-1 expression is up-regulated during follicular atresia in the marmoset ovary We proposed that this phenomenon would also occur in the rat ovary, and in the present study, we have confirmed that TSP-1 is expressed in the granulosa cells of late preantral and antral follicles in the rat ovary and that it is up-regulated in atretic follicles data not shown.

Because CD36 is expressed in granulosa cells, we hypothesized that TSP-1 may be acting directly on these cells in an autocrine fashion to promote follicular atresia via an apoptotic mechanism Analysis of the antiangiogenic mechanisms of TSP-1 in human dermal microvascular endothelial cells has shown that induction of apoptosis by TSP-1 requires the sequential activation of the CD36 receptor, p59 fyn , caspase-like proteases and p38 mitogen-activated protein kinases In the ovary, an extravascular role for TSP-1 has also been suggested 6 , 47 , Using a granulosa cell line, Greenaway et al.

Here, we have shown for the first time that TSP-1 directly induces apoptosis of primary granulosa cells via the activation of caspase This effect on granulosa cell apoptosis occurred both in intact follicles and isolated granulosa cells. Two mechanisms of action of TSP-1 can therefore be proposed. First, TSP-1 is acting on the endothelial cells to inhibit angiogenesis, thus leading to follicular atresia. Second, TSP-1 is acting on the granulosa cells to promote apoptosis directly. Although angiogenesis is essential for the development of follicles in vivo , follicles grown in vitro do not have a requirement for blood vessel growth.

Therefore, at least in vitro , it is unlikely that TSP-1 is promoting granulosa cell apoptosis via the inhibition of follicular angiogenesis. Because CD36 receptors are present on granulosa cells 17 , 25 , and we have shown that TSP-1 promotes apoptosis of isolated granulosa cells, it is likely that the promotion of follicle atresia by TSP-1 is due to a direct effect on the granulosa cells. This suggests that the role of TSP-1 in inhibiting angiogenesis is modulated both through its receptors and through interaction with VEGF, but also that the formation of receptor complexes could play a role in regulating the function of TSP VEGF has also been shown to have a cytoprotective role in the ovary 50 and to promote cell survival.

However, because TSP-1 is capable of directly activating proapoptotic pathways in other cell types 47 , 48 , 51 , a direct mechanism of action of TSP-1 in granulosa cells cannot be ruled out. Further studies are required to fully elucidate the mechanisms of TSP-1 action and the relative roles of pro and antiangiogenic factors during follicular development.

The regulation of TSP-1 in the ovary has been studied in several species 6 , 24 , TSP-1 expression in healthy follicles is highest during the preantral and early antral stages of development and it decreases as the follicles increase in size and progress toward the preovulatory stage.

However, estradiol has been shown to inhibit both TSP-1 mRNA and protein expression in vitro and in vivo , in many different cell types, including endothelial cells, HUVECs, and in an ovariectomized rat model 53 , It has also been demonstrated that estradiol down-regulates the expression of CD36 55 , which may result in reduced TSP-1 action.

Developing follicles produce increasing amounts of estradiol, and these follicles have decreased levels of TSP-1, so it is possible that estradiol plays a role in suppressing the expression of TSP-1 during follicular development. It can therefore be hypothesized that TSP-1 may participate in the process of follicle selection, because follicles that are not selected would have reduced estradiol production, leading to an increase in the levels of TSP-1 in the granulosa cells via autocrine control.

The ability and the extent of the nanoparticles to cross the HUVEC layer to reach the lower hydrogel layer were studied using Z-stack confocal microscopy. The confocal observations were also made in serial intervals of time. Scanning electron microscopy was performed with Ultra-Plus Zeiss microscope equipped with two secondary electron detectors: SE2 and In-Lens to characterize the size and distribution characteristics of the particles. The experiment was repeated at varying magnetic filed strengths and the displacement of the nanoparticles were studied in four different times.

NMR study was performed on the liquid sample with nanoparticles. A problem related to the paramagnetic nature of these particles was encountered. Indeed, interference with the magnetic field does not allow us to obtain an NMR signal, and a broad peak of water that covers the spectrum was seen.

The signal of the nanoparticles was not visible due to the closeness of the super-paramagnetic iron oxide. The scanning electron microscopy picture of the colloidal suspension of magnetic nanoparticles is shown in Additional file 1 : Figure S1. The angiogenesis sprouts were observed predominantly in the basal layer, and the direction of angiogenesis was from above downwards i. Additional file 2: Video S1 showed the effect of angiogenesis when nanoparticles were placed in the basal layer only.

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Additional file 3: Video S2 shows the effect of nanoparticles when placed in the upper layer of hydrogel. The sprouts were seen growing towards the upper nutrient layer. When the nanoparticles were placed in both the layers the sprouts were seen from bottom layer to the top Additional file 4: Video S3. In the 4th scenario where there were no nanoparticles only, insignificant growth was seen Additional file 5: Video S4. The results have proved that the nanoparticles with VEGF are efficient to grow the sprouts and the particles can be manipulated to desired locations in the hydrogel using a magnet.

Additional file 2: Video S1. The effects of magnetic nanoparticles with VEGF when placed in the basal layers. WMV kb. Additional file 3: Video S2. The effects of magnetic nanoparticles with VEGF when placed in the upper layers. Additional file 4: Video S3. The effects of magnetic nanoparticles with VEGF when placed in both upper and lower basal layers.

In the next phase the endothelial barrier crossing of the nanoparticles was analyzed. Z-stack confocal microscopy was used to analyze the effect of the nanoparticles crossing the HUVEC monolayer to lower collagen hydrogel layer. Quantification of the endothelial barrier crossing was not performed, however, by visual assessment a significant number of particles cross the barrier.

Figure 3 a and b envisage the crossing of the nanoparticles fluorescent particles from the nutrient layer located above across the HUVEC monolayer cells to the lower collagen hydrogel layer located underneath. The magnetic nanoparticles-VEGF particles were studied in matrix matrigel. Panels a to d , each has upper and lower panels which correspond to immediate 0 hr , and at 24 hr after the experimental setup.

With electromagnetic interfacing the magnetic nanoparticles were observed to polarize to one end of the drop Fig. This was consistently observed in all 4 different times on a glass slide. The tendency of polarization starts at 0. Access to higher strengths of magnetic field was not available. Hence, the highest limit evaluated was 0.

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Shows polarization of the magnetic nanoparticles under the influence of 0. In another experiment, the droplet with magnetic nanoparticles over a bar magnet retains its shape in all directions and antigravity positions Additional file 1 : Figure S3 , whereas a control droplet without magnetic nanoparticles loses its shape very easily. This study demonstrated the potential possibilities of angiogenesis using magnetic nanoparticles with VEGF.

The results observed a proof-of-concept analysis of these nanoparticles showing the ability of the magnetic nanoparticles with VEGF to form angiogenic sprouts; and also the ability of MN to cross endothelial barrier. The advantage of the method is that the nanoparticles could be manipulated with magnetic force to desired locations, and angiogenesis thereby could be target oriented and therapeutic. This could be of particular use in the setting of coronary artery disease where the nanoparticles could be injected into the coronaries, and the particles could be potentially controlled by magnetic force to desired locations to form collaterals.

The ability of these particles to move towards magnetic area, which was in the opposite direction to the nutrient layer, shows the influence of the magnetic field. Very interestingly it can be used through extravascular method to form arterial connections between left anterior descending artery and left internal mammary artery, which is placed adjacently and also the commonly used connection artery for coronary artery bypass surgery.

To emphasize it has potentials to form percutaneous bypass connections, especially in patients who are not capable to undergo surgery due to comorbidities. The potential advantages could be useful in treatment of peripheral vascular diseases and cerebral ischemic conditions. Also, this study has shown that these MN have potentials to cross the endothelial barrier, which could be of advantage. However, in this setting, it is very difficult to simulate the exact tissue nature as the endothelium has a barrier and tight junctions.

However, due to the nanostructure of the particles, it is expected to cross the endothelium easily, especially, if it is driven by an ischemic stimulus and with further magnetic field augmentation. It is difficult to simulate in-vitro the phenomenon of endothelial barrier crossing.

Matrix Matrigel is a recombinant basement membrane extract.


Though absolute quantification of crossing was not studied this is the first evaluation of such a simulation. Moreover, this endothelial penetration property of the particles could be increased with external magnetic force. The magnetic particles show polarization in this study. The magnetic nanoparticles are small and they could be rapidly eliminated by monocytes. Aihua Fu et al. The particles accumulated in the target areas using the Ni micro-mesh technique. The magnetic nanoparticles could also be injected extravasularly to form collateral connections between left anterior descending artery and left internal mammary or intercostal vessels percutaneous bypass ; and thereafter-magnetic particles can be externally controlled by the external magnetic field.

Targeted nanoparticles are useful to identify tumor cells, modulation of sRNA and cardiosphere derived cells engraftment in myocardial infarction [ 27 , 28 , 29 ]. The magnetic nanoparticles are useful for receptor-mediated gene delivery [ 30 ]. Also, suppression of VEGF has been shown to be associated with regression of the tumor, and also in the control siRNA, and receptor-mediated signal transduction [ 31 ].

Also, it would be quite interesting to study the effect of integrin expression on the cardiomyocyte membranes by these nanoparticles, as VEGF expression can induce integrin expression in cardiomyocyte surface also, which could emerge as a dual advantage [ 32 , 33 ]. These particles need to be studied in-vitro for further information on the magnetic displacement of the particles.

These MN are largely biocompatible, and it could be used for various bioengineering purposes in vitro and in vivo [ 34 ]. Magnetic levitation is a technique of modification of the 3D cell culture with magnetic nanoparticles and studying the modified effect of the tissues used in culture [ 35 ]. The cellular behavior could be controlled in a remote control form [ 36 ]. Magnetic nanoparticles are very useful in bioengineering of the tissues and antibody purification. The magnetic nanoparticles are useful in-vivo gene delivery, antibody purification and photo-acoustic detection of circulating tumor cells.

This study is an initial observation demonstrating the ability of magnetic nanoparticles with VEGF in angiogenesis and endothelial barrier crossing. Further studies need to be preformed to look for the extent, and the magnitude of targeted angiogenesis. Angiogenesis could be induced by the effect of magnetic nanoparticles conjugated with the vascular endothelial growth factor. These nanoparticles could be controlled by magnetic force, and these nanoparticles have possible potentials to cross endothelium. Risau W.

Mechanisms of angiogenesis. Guillemin K. Forsythe JA, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol. Rivard A, et al. Age-dependent defect in vascular endothelial growth factor expression is associated with reduced hypoxia-inducible factor 1 activity. J Biol Chem. Vascular permeability factor vascular endothelial growth factor gene is expressed differentially in normal tissues, macrophages, and tumors. Mol Biol Cell. Enholm B, et al. Ravi R, et al. Regulation of tumor angiogenesis by pinduced degradation of hypoxia-inducible factor 1alpha.

Genes Dev. Couffinhal T, et al. Ferrara N, Kerbel R. Angiogenesis as a therapeutic target. Losordo D. Therapeutic angiogenesis and Vasculogenesis for ischemic disease: part II: cell-based therapies. Grines C. Therapeutic angiogenesis with recombinant fibroblast growth factor-2 improves stress and rest myocardial perfusion abnormalities in patients with severe symptomatic chronic coronary artery disease.

Age-Dependent Impairment of Angiogenesis. Fu A, et al. Fluorescent magnetic Nanoparticles for magnetically enhanced cancer imaging and targeting in living subjects. ACS Nano. Bioengineered blood vessels. Expert Opin Biol Ther. Korff T, Augustin HG. Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation. The Journal of Cell Biology Nov.

J Cell Sci. How does the extracellular matrix direct gene expression? J Theor Biol. Lumen formation by epithelial cell lines in response to collagen overlay: a morphogenetic model in culture. Hypoxia induces capillary network formation in cultured bovine pulmonary micro-vessel endothelial cells. Am J Physiol. Bulick A, et al. Impact of endothelial cells and mechanical conditioning on smooth muscle cell extracellular matrix production and differentiation.

Tissue Eng Part A. Extracellular matrix and growth factors improve the efficacy of intramuscular islet transplantation. PLoS One. The influence of Matrigel or growth factor reduced Matrigel on human intervertebral disc cell growth and proliferation.