Neuroprotection in early stages of Alzheimer’s disease is promoted by transthyretin angiogenic properties

Abstract Background While still controversial, it has been demonstrated that vascular defects can precede the onset of other AD hallmarks features, making it an important therapeutic target. Given that the protein transthyretin (TTR) has been established as neuroprotective in AD, here we investigated the influence of TTR in the vasculature. Methods We evaluated the thickness of the basement membrane and the length of brain microvessels, by immunohistochemistry, in AβPPswe/PS1A246E (AD) transgenic mice and non-transgenic mice (NT) bearing one (TTR+/−) or two (TTR+/+) copies of the TTR gene. The angiogenic potential of TTR was evaluated in vitro using the tube formation assay, and in vivo using the chick chorioallantoic membrane (CAM) assay. Results AD transgenic mice with TTR genetic reduction, AD/TTR+/−, exhibited a thicker BM in brain microvessels and decreased vessel length than animals with normal TTR levels, AD/TTR+/+. Further in vivo investigation, using the CAM assay, revealed that TTR is a pro-angiogenic molecule, and the neovessels formed are functional. Also, TTR increased the expression of key angiogenic molecules such as proteins interleukins 6 and 8, angiopoietin 2, and vascular endothelial growth factor, by endothelial cells, in vitro, under tube formation conditions. We showed that while TTR reduction also leads to a thicker BM in NT mice, this effect is more pronounced in AD mice than in NT animals, strengthening the idea that TTR is a neuroprotective protein. We also studied the effect of TTR tetrameric stabilization on BM thickness, showing that AD mice treated with the TTR tetrameric stabilizer iododiflunisal (IDIF) displayed a significant reduction of BM thickness and increased vessel length, when compared to non-treated littermates. Conclusion Our in vivo results demonstrate the involvement of TTR in angiogenesis, particularly as a modulator of vascular alterations occurring in AD. Since TTR is decreased early in AD, its tetrameric stabilization can represent a therapeutic avenue for the early treatment of AD through the maintenance of the vascular structure.

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Neuroprotection in early stages of Alzheimers Disease is promoted by Transthyretin angiogenic properties

10.1101/2021.04.16.440131 ◽

2021 ◽

Author(s):

Tiago Gião ◽

Joana Saavedra ◽

José Ricardo Vieira ◽

Marta Teixeira Pinto ◽

Gemma Arsequell ◽

...

Keyword(s):

Chorioallantoic Membrane ◽

Tube Formation ◽

Cam Assay ◽

Vessel Length ◽

Chick Chorioallantoic Membrane ◽

Brain Microvessels ◽

Formation Conditions ◽

Therapeutic Avenue ◽

Important Therapeutic Target

While still controversial, it has been demonstrated that vascular defects can precede the onset of the other AD hallmarks features, making it an important therapeutic target. Given that the protein transthyretin (TTR) has been established as neuroprotective in AD, here we investigated the influence of TTR in the vasculature. AD transgenic mice with TTR genetic reduction, AD/TTR+/-, exhibited a thicker BM in brain microvessels and decreased vessel length than animals with normal TTR levels, AD/TTR+/+. Further in vivo investigation, using the chick chorioallantoic membrane (CAM) assay, revealed that TTR is a pro-angiogenic molecule. Also, TTR increased the expression of key angiogenic molecules, by endothelial cells under tube formation conditions. We showed that TTR reduction leads to a thicker BM in AD mice than in NT animals, strengthening the idea that TTR is a neuroprotective protein. We also studied the effect of TTR tetrameric stabilization on BM thickness, showing that AD mice treated with iododiflunisal (IDIF) displayed a significant reduction of BM thickness and increased vessel length when compared to non-treated littermates. Our in vivo results show the involvement of TTR in angiogenesis, particularly as a modulator of vascular alterations occurring in AD. Since TTR is decreased early in AD, its tetrameric stabilization can represent a therapeutic avenue for the early treatment of AD through the maintenance of the vascular structure.

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Nymphaeol-A Isolated from Okinawan Propolis Suppresses Angiogenesis and Induces Caspase-Dependent Apoptosis via Inactivation of Survival Signals

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/826245 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 11

Author(s):

Ikumi Tsuchiya ◽

Takahiro Hosoya ◽

Motoko Ushida ◽

Kazuhiro Kunimasa ◽

Toshiro Ohta ◽

...

Keyword(s):

Screening Program ◽

Biological Activities ◽

Umbilical Vein ◽

Chorioallantoic Membrane ◽

Tube Formation ◽

Mitogen Activated Protein Kinase ◽

Cam Assay ◽

Prenylated Flavonoids

Propolis, a resinous substance that honeybees collect to protect their beehive from enemies, is reported to have various biological activities. In our screening program to search for antiangiogenic compounds from propolis, the ethanol extracts of Okinawan propolis (EEOP) showed significant antiangiogenic activities in a tube formation assay with human umbilical vein endothelial cells (HUVECs)in vitroat 3.13 μg/mL and chorioallantoic membrane (CAM) assayin vivoat 25 μg/egg. To elucidate the active compounds of EEOP and their mode of action, we isolated some prenylated flavonoids from EEOP and found that nymphaeol-A had the strongest antiangiogenic activity among them. Nymphaeol-A significantly reducedin vivoneovessel formation in the CAM assay at 25 μg/egg. At the molecular level, nymphaeol-A markedly inactivated mitogen-activated protein kinase/ERK kinase 1/2 (MEK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2), whose molecular activations signal new vessel formation in HUVECs. In addition, nymphaeol-A dose- and time-dependently induced caspase-dependent apoptosis in tube-forming HUVECs. Taken together, nymphaeol-A was shown to inhibit angiogenesis at least in part via inactivation of MEK1/2–ERK1/2 signaling and induction of caspase-dependent apoptosis. Okinawan propolis and its major component, nymphaeol-A, may be useful agents for preventing tumor-induced angiogenesis.

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N6-Methyladenosine Methyltransferase METTL3 Promotes Angiogenesis and Atherosclerosis by Upregulating the JAK2/STAT3 Pathway via m6A Reader IGF2BP1

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.731810 ◽

2021 ◽

Vol 9 ◽

Author(s):

Guo Dong ◽

Jiangbo Yu ◽

Gaojun Shan ◽

Lide Su ◽

Nannan Yu ◽

...

Keyword(s):

Molecular Mechanisms ◽

Chorioallantoic Membrane ◽

Tube Formation ◽

Luciferase Reporter ◽

Dependent Manner ◽

Stat3 Pathway ◽

Protein Levels ◽

Chick Chorioallantoic Membrane

Atherosclerosis (AS) is a life-threatening vascular disease. RNA N6-methyladenosine (m6A) modification level is dysregulated in multiple pathophysiologic processes including AS. In this text, the roles and molecular mechanisms of m6A writer METTL3 in AS progression were explored in vitro and in vivo. In the present study, cell proliferative, migratory, and tube formation capacities were assessed through CCK-8, Transwell migration, and tube formation assays, respectively. RNA m6A level was examined through a commercial kit. RNA and protein levels of genes were measured through RT-qPCR and western blot assays, respectively. VEGF secretion level was tested through ELISA assay. JAK2 mRNA stability was detected through actinomycin D assay. The relationship of METTL3, IGF2BP1, and JAK2 was investigated through bioinformatics analysis, MeRIP, RIP, RNA pull-down, and luciferase reporter assays. An AS mouse model was established to examine the effect of METTL3 knockdown on AS development in vivo. The angiogenetic activity was examined through chick chorioallantoic membrane assay in vivo. The results showed that METTL3 was highly expressed in ox-LDL-induced dysregulated HUVECs. METTL3 knockdown inhibited cell proliferation, migration, tube formation, and VEGF expression/secretion in ox-LDL-treated HUVECs, hampered AS process in vivo, and prevented in vivo angiogenesis of developing embryos. METTL3 positively regulated JAK2 expression and JAK2/STAT3 pathway in an m6A dependent manner in HUVECs. IGF2BP1 positively regulated JAK2 expression through directly binding to an m6A site within JAK2 mRNA in HUVECs. METTL3 knockdown weakened the interaction of JAK2 and IGF2BP1. METTL3 exerted its functions through JAK2/STAT3 pathway. In conclusion, METTL3 knockdown prevented AS progression by inhibiting JAK2/STAT3 pathway via IGF2BP1.

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In VitroEvaluation of Scaffolds for the Delivery of Mesenchymal Stem Cells to Wounds

BioMed Research International ◽

10.1155/2015/108571 ◽

2015 ◽

Vol 2015 ◽

pp. 1-14 ◽

Cited By ~ 25

Author(s):

Elizabeth A. Wahl ◽

Fernando A. Fierro ◽

Thomas R. Peavy ◽

Ursula Hopfner ◽

Julian F. Dye ◽

...

Keyword(s):

Stem Cells ◽

Wound Healing ◽

Mesenchymal Stem Cells ◽

Three Dimensional ◽

Chorioallantoic Membrane ◽

Angiogenic Potential ◽

Chick Chorioallantoic Membrane ◽

Bovine Collagen

Mesenchymal stem cells (MSCs) have been shown to improve tissue regeneration in several preclinical and clinical trials. These cells have been used in combination with three-dimensional scaffolds as a promising approach in the field of regenerative medicine. We compare the behavior of human adipose-derived MSCs (AdMSCs) on four different biomaterials that are awaiting or have already received FDA approval to determine a suitable regenerative scaffold for delivering these cells to dermal wounds and increasing healing potential. AdMSCs were isolated, characterized, and seeded onto scaffolds based on chitosan, fibrin, bovine collagen, and decellularized porcine dermis.In vitroresults demonstrated that the scaffolds strongly influence key parameters, such as seeding efficiency, cellular distribution, attachment, survival, metabolic activity, and paracrine release. Chick chorioallantoic membrane assays revealed that the scaffold composition similarly influences the angiogenic potential of AdMSCsin vivo. The wound healing potential of scaffolds increases by means of a synergistic relationship between AdMSCs and biomaterial resulting in the release of proangiogenic and cytokine factors, which is currently lacking when a scaffold alone is utilized. Furthermore, the methods used herein can be utilized to test other scaffold materials to increase their wound healing potential with AdMSCs.

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Microvascular Experimentation in the Chick Chorioallantoic Membrane as a Model for Screening Angiogenic Agents including from Gene-Modified Cells

International Journal of Molecular Sciences ◽

10.3390/ijms23010452 ◽

2021 ◽

Vol 23 (1) ◽

pp. 452

Author(s):

Donna C. Kennedy ◽

Barbara Coen ◽

Antony M. Wheatley ◽

Karl J. A. McCullagh

Keyword(s):

Low Cost ◽

Chorioallantoic Membrane ◽

Visual Assessment ◽

Cam Assay ◽

Chick Chorioallantoic Membrane ◽

Assay Procedure ◽

Angiogenic Effect ◽

Cultivation Techniques

The chick chorioallantoic membrane (CAM) assay model of angiogenesis has been highlighted as a relatively quick, low cost and effective model for the study of pro-angiogenic and anti-angiogenic factors. The chick CAM is a highly vascularised extraembryonic membrane which functions for gas exchange, nutrient exchange and waste removal for the growing chick embryo. It is beneficial as it can function as a treatment screening tool, which bridges the gap between cell based in vitro studies and in vivo animal experimentation. In this review, we explore the benefits and drawbacks of the CAM assay to study microcirculation, by the investigation of each distinct stage of the CAM assay procedure, including cultivation techniques, treatment applications and methods of determining an angiogenic response using this assay. We detail the angiogenic effect of treatments, including drugs, metabolites, genes and cells used in conjunction with the CAM assay, while also highlighting the testing of genetically modified cells. We also present a detailed exploration of the advantages and limitations of different CAM analysis techniques, including visual assessment, histological and molecular analysis along with vascular casting methods and live blood flow observations.

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Novel Nargenicin A1 Analog Inhibits Angiogenesis by Downregulating the Endothelial VEGF/VEGFR2 Signaling and Tumoral HIF-1α/VEGF Pathway

Biomedicines ◽

10.3390/biomedicines8080252 ◽

2020 ◽

Vol 8 (8) ◽

pp. 252

Author(s):

Jang Mi Han ◽

Ye Seul Choi ◽

Dipesh Dhakal ◽

Jae Kyung Sohng ◽

Hye Jin Jung

Keyword(s):

Capillary Tube ◽

Umbilical Vein ◽

Hypoxia Inducible Factor ◽

Chorioallantoic Membrane ◽

Tube Formation ◽

Ags Cells ◽

Vegf Pathway ◽

Vegfr2 Signaling

Targeting angiogenesis is an attractive strategy for the treatment of angiogenesis-related diseases, including cancer. We previously identified 23-demethyl 8,13-deoxynargenicin (compound 9) as a novel nargenicin A1 analog with potential anticancer activity. In this study, we investigated the antiangiogenic activity and mode of action of compound 9. This compound was found to effectively inhibit in vitro angiogenic characteristics, including the proliferation, invasion, capillary tube formation, and adhesion of human umbilical vein endothelial cells (HUVECs) stimulated by vascular endothelial growth factor (VEGF). Furthermore, compound 9 suppressed the neovascularization of the chorioallantoic membrane of growing chick embryos in vivo. Notably, the antiangiogenic properties of compound 9 were related to the downregulation of VEGF/VEGFR2-mediated downstream signaling pathways, as well as matrix metalloproteinase (MMP)-2 and MMP-9 expression in HUVECs. In addition, compound 9 was found to decrease the in vitro AGS gastric cancer cell-induced angiogenesis of HUVECs by blocking hypoxia-inducible factor-1α (HIF-1α) and VEGF expression in AGS cells. Collectively, our findings demonstrate for the first time that compound 9 is a promising antiangiogenic agent targeting both VEGF/VEGFR2 signaling in ECs and HIF-1α/VEGF pathway in tumor cells.

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Apigenin Suppresses Angiogenesis by Inhibiting Tube Formation and Inducing Apoptosis

Natural Product Communications ◽

10.1177/1934578x1601101005 ◽

2016 ◽

Vol 11 (10) ◽

pp. 1934578X1601101

Author(s):

Hyun Ju Kim ◽

Mok-Ryeon Ahn

Keyword(s):

Umbilical Vein ◽

Chorioallantoic Membrane ◽

Tube Formation ◽

Anticancer Activities ◽

Apoptotic Pathway ◽

Inhibition Of Angiogenesis ◽

Induced Apoptosis ◽

Umbilical Vein Endothelial Cells

Apigenin has been reported to exert angiogenic and anticancer activities in vitro. The mechanism of inhibition of angiogenesis by apigenin, however, has not been well-established. In this study, we investigated whether apigenin not only inhibited tube formation but also induced apoptosis in human umbilical vein endothelial cells (HUVECs). Furthermore, strong antiangiogenic activity of apigenin was observed in the in vivo assay using chick embryo chorioallantoic membrane (CAM). We also analyzed changes in survival signals and the apoptotic pathway through Western blotting. The results indicate that apigenin exerts its antiangiogenic effects through induction of endothelial apoptosis.

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Bactericidal/permeability-increasing protein (BPI) inhibits angiogenesis via induction of apoptosis in vascular endothelial cells

Blood ◽

10.1182/blood.v96.1.176 ◽

2000 ◽

Vol 96 (1) ◽

pp. 176-181 ◽

Cited By ~ 42

Author(s):

Daisy W. J. van der Schaft ◽

Elly A. H. Toebes ◽

Judith R. Haseman ◽

Kevin H. Mayo ◽

Arjan W. Griffioen

Keyword(s):

Vascular Endothelial Cells ◽

Flow Cytometric Analysis ◽

Chorioallantoic Membrane ◽

Collagen Gel ◽

Tube Formation ◽

Therapeutic Modality ◽

Angiogenesis Assay ◽

Induction Of Apoptosis

Abstract Bactericidal/permeability-increasing protein (BPI) has been known for some time to function in killing bacteria and in neutralizing the effects of bacterial endotoxin lipopolysaccharide. In the present study, BPI is found to be a novel endogenous inhibitor of angiogenesis. Within the sub-μM range, BPI shows a concentration-dependent inhibition of endothelial cell (EC) proliferation that is mediated by cell detachment and subsequent induction of apoptosis. As measured by flow cytometric analysis of the percentage of subdiploid cells, apoptosis induction was half-maximal at about 250 nmol/L BPI. Apoptosis was confirmed by quantification of cells with nuclear fragmentation. Apoptosis was found to be EC specific. In an in vitro collagen gel-based angiogenesis assay, BPI at 1.8 μmol/L inhibited tube formation by 81% after only 24 hours. Evidence for in vivo inhibition of angiogenesis was obtained, using the chorioallantoic membrane assay in which BPI was seen to be significantly effective at concentrations as low as 180 nmol/L. This newly discovered function of BPI might provide a possible therapeutic modality for the treatment of various pathologic disorders that depend on angiogenesis.

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Evaluation of the Anti-angiogenic Activity of Saponins from Maesa lanceolata by Different Assays

Natural Product Communications ◽

10.1177/1934578x1200700910 ◽

2012 ◽

Vol 7 (9) ◽

pp. 1934578X1200700

Author(s):

Kenn Foubert ◽

Annelies Breynaert ◽

Mart Theunis ◽

Rita Van Den Bossche ◽

Guido R.Y. De Meyer ◽

...

Keyword(s):

Ex Vivo ◽

Chorioallantoic Membrane ◽

Rat Aorta ◽

Cam Assay ◽

Plant Metabolites ◽

Angiogenic Activity ◽

Test Systems ◽

Aorta Ring

Angiogenesis, in which a vascular network is established from pre-existing vessels, is a complex multistep process. Mechanisms underlying angiogenesis can be investigated using a variety of in vitro, ex vivo and in vivo approaches. Evaluation of several promising plants and plant metabolites, including terpenoids, revealed promising anti-angiogenic activity. Since the maesasaponins displayed anti-angiogenic activity in the chick chorioallantoic membrane (CAM) assay, their activity was further investigated in several test systems. The rat aorta ring assay was compared with the placental vein assay and then selected for the ex vivo investigation of the saponins. Besides their effect on the viability of HUVEC, the anti-angiogenic capacity of the compounds was also investigated in an in vivo zebrafish assay. The activity of the saponins in the viability assay was more pronounced than in the rat aorta ring assay and similar to the effect observed in the CAM assay. The use of different test systems, however, implies different results in the case of saponins.

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Bactericidal/permeability-increasing protein (BPI) inhibits angiogenesis via induction of apoptosis in vascular endothelial cells

Blood ◽

10.1182/blood.v96.1.176.013k32_176_181 ◽

2000 ◽

Vol 96 (1) ◽

pp. 176-181

Author(s):

Daisy W. J. van der Schaft ◽

Elly A. H. Toebes ◽

Judith R. Haseman ◽

Kevin H. Mayo ◽

Arjan W. Griffioen

Keyword(s):

Vascular Endothelial Cells ◽

Flow Cytometric Analysis ◽

Chorioallantoic Membrane ◽

Collagen Gel ◽

Tube Formation ◽

Therapeutic Modality ◽

Angiogenesis Assay ◽

Induction Of Apoptosis

Bactericidal/permeability-increasing protein (BPI) has been known for some time to function in killing bacteria and in neutralizing the effects of bacterial endotoxin lipopolysaccharide. In the present study, BPI is found to be a novel endogenous inhibitor of angiogenesis. Within the sub-μM range, BPI shows a concentration-dependent inhibition of endothelial cell (EC) proliferation that is mediated by cell detachment and subsequent induction of apoptosis. As measured by flow cytometric analysis of the percentage of subdiploid cells, apoptosis induction was half-maximal at about 250 nmol/L BPI. Apoptosis was confirmed by quantification of cells with nuclear fragmentation. Apoptosis was found to be EC specific. In an in vitro collagen gel-based angiogenesis assay, BPI at 1.8 μmol/L inhibited tube formation by 81% after only 24 hours. Evidence for in vivo inhibition of angiogenesis was obtained, using the chorioallantoic membrane assay in which BPI was seen to be significantly effective at concentrations as low as 180 nmol/L. This newly discovered function of BPI might provide a possible therapeutic modality for the treatment of various pathologic disorders that depend on angiogenesis.

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