HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

10.3791/58591 ◽  
2019 ◽  
Author(s):  
Maria Teresa Gentile ◽  
Olga Pastorino ◽  
Maurizio Bifulco ◽  
Luca Colucci-D'Amato
Keyword(s):  
Natural Products ◽  
Tube Formation ◽  
Tube Formation Assay ◽  
The Impact

Endothelial k-RasV12 Expression Induces Capillary Deficiency Attributable to Marked Tube Network Expansion Coupled to Reduced Pericytes and Basement Membranes

2021 ◽  
Author(s):  
Zheying Sun ◽  
Scott S. Kemp ◽  
Prisca K. Lin ◽  
Kalia N. Aguera ◽  
George E. Davis
Keyword(s):  
Protein Kinase ◽  
Basement Membrane ◽  
Arteriovenous Malformations ◽  
Network Formation ◽  
Tube Formation ◽  
Small Gtpase ◽  
Basement Membranes ◽  
Formation Mechanisms ◽  
Lumen Formation ◽  
The Impact

Objective: We sought to determine how endothelial cell (EC) expression of the activating k-Ras mutation, k-RasV12, affects their ability to form lumens and tubes and interact with pericytes during capillary assembly Approach and Results: Using defined bioassays where human ECs undergo observable tubulogenesis, sprouting behavior, pericyte recruitment to EC-lined tubes, and pericyte-induced EC basement membrane deposition, we assessed the impact of EC k-RasV12 expression on these critical processes that are necessary for proper capillary network formation. This mutation, which is frequently seen in human ECs within brain arteriovenous malformations, was found to markedly accentuate EC lumen formation mechanisms, with strongly accelerated intracellular vacuole formation, vacuole fusion, and lumen expansion and with reduced sprouting behavior, leading to excessively widened tube networks compared with control ECs. These abnormal tubes demonstrate strong reductions in pericyte recruitment and pericyte-induced EC basement membranes compared with controls, with deficiencies in fibronectin, collagen type IV, and perlecan deposition. Analyses of signaling during tube formation from these k-RasV12 ECs reveals strong enhancement of Src, Pak2 (P21 [RAC1 (Rac family small GTPase 1)] activated kinase 2), b-Raf (v-raf murine sarcoma viral oncogene homolog B1), Erk (extracellular signal–related kinase), and Akt activation and increased expression of PKCε (protein kinase C epsilon), MT1-MMP (membrane-type 1 matrix metalloproteinase), acetylated tubulin and CDCP1 (CUB domain-containing protein 1; most are known EC lumen regulators). Pharmacological blockade of MT1-MMP, Src, Pak, Raf, Mek (mitogen-activated protein kinase) kinases, Cdc42 (cell division cycle 42)/Rac1, and Notch markedly interferes with lumen and tube formation from these ECs. Conclusions: Overall, this novel work demonstrates that EC expression of k-RasV12 disrupts capillary assembly due to markedly excessive lumen formation coupled with strongly reduced pericyte recruitment and basement membrane deposition, which are critical pathogenic features predisposing the vasculature to develop arteriovenous malformations.

scholarly journals Regiospecific Methylation of a Dietary Flavonoid Scaffold Selectively Enhances IL-1β Production following Toll-like Receptor 2 Stimulation in THP-1 Monocytes

2013 ◽  
Vol 288 (29) ◽  
pp. 21126-21135 ◽  
Author(s):  
Eng-Kiat Lim ◽  
Paul J. Mitchell ◽  
Najmeeyah Brown ◽  
Rebecca A. Drummond ◽  
Gordon D. Brown ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Natural Products ◽  
Intestinal Microflora ◽  
Rank Order ◽  
Inflammasome Activation ◽  
Toll Like Receptor ◽  
Immune Health ◽  
Toll Like Receptor 2 ◽  
Dietary Flavonoid ◽  
The Impact

It is now recognized that innate immunity to intestinal microflora plays a significant role in mediating immune health, and modulation of microbial sensing may underpin the impact of plant natural products in the diet or when used as nutraceuticals. In this context, we have examined five classes of plant-derived flavonoids (flavonols, flavones, flavanones, catechins, and cyanidin) for their ability to regulate cytokine release induced by the Toll-like receptor 2 (TLR2) agonist Pam3CSK4. We found that the flavonols selectively co-stimulated IL-1β secretion but had no impact on the secretion of IL-6. Importantly, this costimulation of TLR2-induced cytokine secretion was dependent on regiospecific methylation of the flavonol scaffold with a rank order of quercetin-3,4′-dimethylether > quercetin-3-methylether > casticin. The mechanism underpinning this costimulation did not involve enhanced inflammasome activation. In contrast, the methylated flavonols enhanced IL-1β gene expression through transcriptional regulation, involving mechanisms that operate downstream of the initial NF-κB and STAT1 activation events. These studies demonstrate an exquisite level of control of scaffold bioactivity by regiospecific methylation, with important implications for understanding how natural products affect innate immunity and for their development as novel immunomodulators for clinical use.

scholarly journals Influence of tillage methods on the yield of winter rye variety Sudarushka

2022 ◽  
Vol 51 (6) ◽  
pp. 5-10
Author(s):  
P. N. Brazhnikov ◽  
A. B. Sainakova ◽  
O. V. Litvinchuk
Keyword(s):  
Sandy Loam ◽  
Humus Content ◽  
Tube Formation ◽  
Winter Rye ◽  
Taiga Zone ◽  
Average Yield ◽  
New Variety ◽  
Soil Cultivation ◽  
In The Beginning ◽  
The Impact

The results of studying the elements of the technology of cultivation of a new variety of winter rye Sudarushka are presented. The research took place in 2019, 2020 in the sub-taiga zone of the Tomsk region. The impact of soil cultivation methods as well as the impact of humic fertiliser from peat Gumostim on the yield of winter rye Sudarushka is evaluated. The soils of the experimental plot are acidic (pH 4.3) soddy-podzolic loamy sandy loam. The arable horizon has a low humus content (1.5%), a low (0.2 mg/100g) content of nitrate nitrogen, a medium content of mobile phosphorus (19.2) and exchangeable potassium (7.1) and a high (11.0 mg/100g) content of mobile aluminium. The scheme of the experiment included four variants of cultivation: ploughing of fallow lands with PlN-4-35 + cultivation KPN-4,2, sowing without packing and with packing; disking with BDMK-2,8 + cultivation KPN-4,2, sowing without packing and with packing. Treatment with humic fertilizer from peat Gumostim at a concentration of 0.001% was carried out during the growing season in the beginning phase of the leaf-tube formation. The average yield of winter rye in the variety Sudarushka during the years of research was 4.66 t/ha in the variant with disking, subsequent cultivation before sowing and further sowing with packing, which is 0.34 t/ha higher than with the traditional method of soil cultivation. The application of humic fertilizer from peat Gumostim on cold sod-podzolic soils enabled the average yield of winter rye to be 4.31 t/ha y, which is 0.39 t/ha higher than without the fertilizer.

Abstract 466: Vascular Claudin-11 Plays a Protective Role for Human Atherosclerosis Progression

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Eun Hee Kim ◽  
Moon Hee Jung ◽  
Man Seok Park ◽  
Kang Ho Choi ◽  
Su Jin Lee ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Coronary Atherosclerosis ◽  
Tube Formation ◽  
Total N ◽  
Tube Formation Assay ◽  
Junction Protein

Background and Aims: The expression and the role of tight junction protein, Claudin-11 (CLDN11) in vasculat smooth muscle cell (vSMC) is unknown. Methods: To understand the role of CLDN11 in the vSMC, we transfected siRNA-CLDN11 into human coronary vascular smooth muscle cell line (hCSMC). cDNA microarray using Agilent Human mRNA arrays, immunocytochemistry, tube formation assay, FACS, and cell cycle analysis were performed 7 days after CLDN11 knock-down. To know the role of CLDN11 in the human cardiovascular system, we obtained vascular smooth muscle layer from autopsied left anterior descending artery and CLDN11 mRNA expression was evaluated following modified AHA Consensus Classification Based on Morphologic Descriptions (three groups, total n=45). Results: CLDN11 was well expressed in vascular smooth mucscle layer in immunohistochemisty and western blot analysis. We observed the angiogenesis (CXCL8, SOX17, HEY1), cell proliferation (EGR3, ITGB2), and extracellular matrix (BMPER, WNT1) associated gene expression. Following CLDN11-siRNA transfection, the tube formation assay and proliferating cellular phase was markedly increased in siRNA treatment group (p<0.01, respectively). In the human sample, CLDN11 expression was inversely correlated with the progression of coronary atherosclerosis (p=0.0026), and the sudden cardiac death with complicated coronary atherosclerosis (p<0.05). Conclusions: These results indicate that CLDN11 could tightly regulate the vascular smooth muscle physiology. And CLDN11 might play a certain role for atherosclerosis propagation to regulate the vSMCs plasticity and possible new etiology for cardiovascular events.

scholarly journals The quest for supernatural products: the impact of total synthesis in complex natural products medicinal chemistry

2020 ◽  
Vol 37 (11) ◽  
pp. 1511-1531
Author(s):  
Zhi-Chen Wu ◽  
Dale L. Boger
Keyword(s):  
Natural Products ◽  
Total Synthesis ◽  
Medicinal Chemistry ◽  
Recent Advances ◽  
The Impact

This review summarizes and highlights recent advances in medicinal chemistry of natural products enabled by total synthesis that provide “supernatural products” with improved properties superseding the natural products themselves.

scholarly journals Redox Modulations, Antioxidants, and Neuropsychiatric Disorders

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Erik A. Fraunberger ◽  
Gustavo Scola ◽  
Victoria L. M. Laliberté ◽  
Angela Duong ◽  
Ana C. Andreazza
Keyword(s):  
Natural Products ◽  
Current Literature ◽  
Biological Significance ◽  
Neuropsychiatric Disorders ◽  
Redox Status ◽  
Primary Objective ◽  
The Body ◽  
Brain Health ◽  
Current Role ◽  
The Impact

Although antioxidants, redox modulations, and neuropsychiatric disorders have been widely studied for many years, the field would benefit from an integrative and corroborative review. Our primary objective is to delineate the biological significance of compounds that modulate our redox status (i.e., reactive species and antioxidants) as well as outline their current role in brain health and the impact of redox modulations on the severity of illnesses. Therefore, this review will not enter into the debate regarding the perceived medical legitimacy of antioxidants but rather seek to clarify their abilities and limitations. With this in mind, antioxidants may be interpreted as natural products with significant pharmacological actions in the body. A renewed understanding of these often overlooked compounds will allow us to critically appraise the current literature and provide an informed, novel perspective on an important healthcare issue. In this review, we will introduce the complex topics of redox modulations and their role in the development of select neuropsychiatric disorders.

scholarly journals The BET protein inhibitor apabetalone (RVX-208) restores angiogenic response in type 1 and type 2 diabetes by transcriptional regulation of thrombospondin-1

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
S Mohammed ◽  
M A Mattia ◽  
G K Gergely ◽  
G S Gaia ◽  
S A Ambrosini ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Blood Flow ◽  
Gastrocnemius Muscle ◽  
Tube Formation ◽  
Public Institution ◽  
Diabetic Patients ◽  
Angiogenic Response ◽  
Bet Protein ◽  
The Impact

Abstract Background Peripheral artery disease (PAD) is highly prevalent in people with type 2 diabetes and associates with chronic limb ischemia and poor prognosis. Understanding the mechanisms of impaired blood vessel growth in diabetic patients is of paramount importance to develop new angiogenic therapies in this setting. Dysregulation of epigenetic mechanisms of gene transcription in vascular cells contributes to cardiovascular disease development but is currently not targeted by therapies. Apabetalone (RVX-208) – an FDA approved small molecule inhibitor of the epigenetic readers bromodomain and extra-terminal (BET) proteins – has recently shown to modulate transcriptional programs implicated in vascular inflammation and atherosclerosis. Purpose To investigate RVX-208 effects in modulating angiogenic response and post-ischemic vascularization in diabetes. Methods Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 20 mM) for 48 hours in presence of RVX-208 (20μM) or vehicle (DMSO). Scratch and tube formation assays were performed to investigate the impact of RVX-208 on angiogenic properties of HAECs. T1D mice (streptozotocin-induced diabetes) and T2D mice (Lepdb/db) were orally treated with apabetalone or vehicle for 5 days. Hindlimb ischemia was induced in T1D mice & blood flow recovery analysed at 30 minutes, 7 and 14 days by laser Doppler imaging. Sprouting and matrigel plug assays were performed in Lepdb/db mice. Gastrocnemius muscle samples from patients with and without T2D were employed to translate our experimental findings. Results HG impaired HAECs migration and tube formation as compared to NG, whereas treatment with RVX-208 rescued HG-induced impairment of angiogenic properties. Real time PCR arrays in HG-treated HAECs showed that RVX-208 treatment prevents the dysregulation of genes implicated in endothelial migration, sprouting and inflammation, namely the anti-angiogenic molecule thrombospondin (THBS1), VEGF-A, IL-1β, IL-6, VCAM-1, and CXCL1. Of interest, both gene silencing of BET protein (BRD4) or its pharmacological inhibition by RVX-208 reduced THBS1 expression while restoring VEGFA levels in HG-treated HAECs. ChIP assays showed the enrichment of both BRD4 and the active chromatin mark H3K27Ac on THBS1 promoter. Mechanistic experiments uncovered the inhibitory role of THBS1 on VEGFA signalling, as also confirmed by STRING analysis. Treatment of T1D mice with RVX-208 improved blood flow reperfusion and vascular density at 14 days as compared to vehicle-treated animals. Moreover, RVX-208 restored endothelial sprouting in T2D-Lepdb/db mice. Of clinical relevance, THBS1 was upregulated while VEGFA expression was reduced in gastrocnemius muscle specimens from T2D patients with PAD as compared to non-diabetic controls. Conclusion In vivo targeting of BET-proteins by RVX-208 may represents a novel therapeutic approach to boost post-ischemic neovascularization in diabetes. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Zurich

scholarly journals The effect of cycling hypoxia on MCF-7 cancer stem cells and the impact of their microenvironment on angiogenesis using human umbilical vein endothelial cells (HUVECs) as a model

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e5990 ◽  
Author(s):  
Fuad M. Alhawarat ◽  
Hana M. Hammad ◽  
Majd S. Hijjawi ◽  
Ahmad S. Sharab ◽  
Duaa A. Abuarqoub ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wound Healing ◽  
Endothelial Cells ◽  
Tumor Microenvironment ◽  
Tube Formation ◽  
Tumor Vascularization ◽  
And Migration ◽  
The Impact ◽  
Mcf 7

Background Breast cancer is the most common type of cancer among females. Hypoxia mediates cancer hallmarks and results from reduced oxygen level due to irregularities in tumor vascularization or when the tumor size prevents oxygen diffusion and triggers angiogenesis to compensate for low oxygen. Cancer stem cells (CSCs) are a rare subpopulation, able to self-renew and to give rise to tumor-initiating cells. It is proposed that CSCs’ secretions help to recruit endothelial cells via angiogenic factors to establish tumor vascularization. In the tumor microenvironment, the effect of hypoxia on CSCs and the impact of their secretions on triggering angiogenesis and tumor vascularization remain questionable. In this study, three-dimensional (3D) CSCs derived from MCF-7 were directly exposed to repetitive long-term cycles of hypoxia to assess its effect on CSCs and then to evaluate the role of the hypoxic CSCs’ (CSCsHYP) secretions in angiogenesis using (HUVECs) as a model for tumor neovascularization response. Methods CSCs derived from MCF-7 cell-line were expanded under repetitive, strictly optimized, long-term/continuous and intermittent hypoxic shots for almost four months to assess hypoxic effect on CSCs, sorted based on CD44+/CD24− biomarkers. Hypoxic phenotype of CSCsHYP was evaluated by assessing the acquired chemoresistance using MTT assay and elevated stemness properties were assessed by flow cytometry. To evaluate the effect of the secretions from CSCsHYP on angiogenesis, HUVECs were exposed to CSCsHYP conditioned-medium (CdM)—in which CSCs had been previously grown—to mimic the tumor microenvironment and to assess the effect of the secretions from CSCsHYP on the HUVECs’ capability of tube formation, migration and wound healing. Additionally, co-culture of CSCsHYP with HUVECs was performed. Results CSCsHYP acquired higher chemoresistance, increased stemness properties and obtained greater propagation, migration, and wound healing capacities, when compared to CSCs in normoxic condition (CSCsNOR). HUVECs’ tube formation and migration abilities were mediated by hypoxic (CSCs) conditioned media (CdM). Discussion This study demonstrates that chemoresistant and migrational properties of CSCs are enhanced under hypoxia to a certain extent. The microenvironment of CSCsHYP contributes to tumor angiogenesis and migration. Hypoxia is a key player in tumor angiogenesis mediated by CSCs.

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