An In Vitro Model of Diabetic Retinal Vascular Endothelial Dysfunction and Neuroretinal Degeneration

Background. Diabetic retinopathy (DR) is a leading cause of blindness in working-age populations. Proper in vitro DR models are crucial for exploring pathophysiology and identifying novel therapeutic targets. This study establishes a rational in vitro diabetic retinal neuronal-endothelial dysfunction model and a comprehensive downstream validation system. Methods. Human retinal vascular endothelial cells (HRMECs) and retinal ganglion cells (RGCs) were treated with different glucose concentrations with mannitol as matched osmotic controls. Cell proliferation and viability were evaluated by the Cell Counting Kit-8. Cell migration was measured using a transwell migration assay. Cell sprouting was assessed by a tube formation assay. The VEGF expression was assessed by ELISA. RGCs were labeled by neurons and RGC markers TUJ1 and BRN3A for quantitative and morphological analysis. Apoptosis was detected using PI/Hoechst staining and TUNEL assay and quantified by ImageJ. Results. Cell proliferation and migration in HRMECs were significantly higher in the 25 mM glucose-treated group ( p < 0.001 ) but lower in the 50 mM and 100 mM groups ( p < 0.001 ). The permeability and the apoptotic index in HRMECs were statistically higher in the 25 mM, 50 mM, and 100 mM groups ( p < 0.05 ). The tube formation assay found that all the parameters were significantly higher in the 25 mM and 50 mM groups ( p < 0.001 ) concomitant with the elevated VEGFA expression in HRMECs ( p = 0.016 ). Cell viability was significantly lower in the 50 mM, 100 mM, and 150 mM groups in RGCs ( p 50 mM = 0.013 , p 100 mM = 0.019 , and p 150 mM = 0.002 ). Apoptosis was significantly elevated, but the proportion of RGCs with neurite extension was significantly lower in the 50 mM, 100 mM, and 150 mM groups ( p 50 mM < 0.001 , p 100 m M < 0.001 , and p 150 mM < 0.001 ). Conclusions. We have optimized glucose concentrations to model diabetic retinal endothelial (25-50 mM) or neuronal (50-100 mM) dysfunction in vitro, which have a wide range of downstream applications.

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Total flavonoids from Astragalus alleviate endothelial dysfunction by activating the Akt/eNOS pathway

Journal of International Medical Research ◽

10.1177/0300060517717358 ◽

2017 ◽

Vol 46 (6) ◽

pp. 2096-2103 ◽

Cited By ~ 4

Author(s):

Qian Liu ◽

Ling Zhang ◽

Qiyuan Shan ◽

Yuxia Ding ◽

Zhaocai Zhang ◽

...

Keyword(s):

Cell Proliferation ◽

Vascular Endothelial Cells ◽

Ex Vivo ◽

Umbilical Vein ◽

Cell Counting ◽

Total Flavonoids ◽

Vascular Endothelial ◽

Protective Effects ◽

Hypoxic Conditions ◽

Underlying Mechanisms

Objective To investigate the vasodilative and endothelial-protective effects and the underlying mechanisms of total flavonoids from Astragalus (TFA). Methods The vasodilative activities of TFA were measured with a myograph ex vivo using rat superior mesenteric arterial rings. The primary human umbilical vein endothelial cell (HUVEC) viabilities were assayed using the cell counting kit-8 after hypoxia or normoxia treatment with or without TFA. Akt, P-Akt, eNOS, P-eNOS, Erk, P-Erk, Bcl-2 and Bax expression were analyzed using western blotting. Results TFA showed concentration-dependent vasodilative effects on rat superior mesenteric arterial rings, but had no effects on normal or potassium chloride precontracted arterial rings. TFA did not affect HUVEC viabilities in normoxia, but dramatically promoted cell proliferation in the concentration range of 1 to 30 µg/mL under hypoxia. Moreover, TFA significantly increased the ratios of P-Akt/Akt and P-eNOS/eNOS in vascular endothelial cells under hypoxic conditions, but did not change the P-Erk/Erk or Bcl-2/Bax ratios. Conclusions TFA might exhibit vasorelaxant and endothelial-protective effects via the Akt/eNOS signaling pathway.

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Translocation of protein tyrosine phosphatase Pez/PTPD2/PTP36 to the nucleus is associated with induction of cell proliferation

Journal of Cell Science ◽

10.1242/jcs.113.17.3117 ◽

2000 ◽

Vol 113 (17) ◽

pp. 3117-3123 ◽

Cited By ~ 2

Author(s):

C. Wadham ◽

J.R. Gamble ◽

M.A. Vadas ◽

Y. Khew-Goodall

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Nuclear Protein ◽

Vascular Endothelial Cells ◽

Tyrosine Phosphatase ◽

Subcellular Localisation ◽

Vascular Endothelial ◽

Proliferating Cells ◽

Wound Edge

Pez is a non-transmembrane tyrosine phosphatase with homology to the FERM (4.1, ezrin, radixin, moesin) family of proteins. The subcellular localisation of Pez in endothelial cells was found to be regulated by cell density and serum concentration. In confluent monolayers Pez was cytoplasmic, but in cells cultured at low density Pez was nuclear, suggesting that it is a nuclear protein in proliferating cells. This notion is supported by the loss of nuclear Pez when cells are serum-starved to induce quiescence, and the rapid return of Pez to the nucleus upon refeeding with serum to induce proliferation. Vascular endothelial cells normally exist as a quiescent confluent monolayer but become proliferative during angiogenesis or upon vascular injury. Using a ‘wound’ assay to mimic these events in vitro, Pez was found to be nuclear in the cells that had migrated and were proliferative at the ‘wound’ edge. TGFbeta, which inhibits cell proliferation but not migration, inhibited the translocation of Pez to the nucleus in the cells at the ‘wound’ edge, further strengthening the argument that Pez plays a role in the nucleus during cell proliferation. Together, the data presented indicate that Pez is a nuclear tyrosine phosphatase that may play a role in cell proliferation.

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CYLD regulates angiogenesis by mediating vascular endothelial cell migration

Blood ◽

10.1182/blood-2009-10-248526 ◽

2010 ◽

Vol 115 (20) ◽

pp. 4130-4137 ◽

Cited By ~ 52

Author(s):

Jinmin Gao ◽

Lei Sun ◽

Lihong Huo ◽

Min Liu ◽

Dengwen Li ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Endothelial Cell ◽

Vascular Endothelial Cells ◽

Vascular Endothelial Cell ◽

Tube Formation ◽

Endothelial Cell Migration ◽

Vascular Endothelial

Cylindromatosis (CYLD) is a deubiquitinase that was initially identified as a tumor suppressor and has recently been implicated in diverse normal physiologic processes. In this study, we have investigated the involvement of CYLD in angiogenesis, the formation of new blood vessels from preexisting ones. We find that knockdown of CYLD expression significantly impairs angiogenesis in vitro in both matrigel-based tube formation assay and collagen-based 3-dimensional capillary sprouting assay. Disruption of CYLD also remarkably inhibits angiogenic response in vivo, as evidenced by diminished blood vessel growth into the angioreactors implanted in mice. Mechanistic studies show that CYLD regulates angiogenesis by mediating the spreading and migration of vascular endothelial cells. Silencing of CYLD dramatically decreases microtubule dynamics in endothelial cells and inhibits endothelial cell migration by blocking the polarization process. Furthermore, we identify Rac1 activation as an important factor contributing to the action of CYLD in regulating endothelial cell migration and angiogenesis. Our findings thus uncover a previously unrecognized role for CYLD in the angiogenic process and provide a novel mechanism for Rac1 activation during endothelial cell migration and angiogenesis.

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17β-Estradiol Enhances Vascular Endothelial Ets-1/miR-126-3p Expression: The Possible Mechanism for Attenuation of Atherosclerosis

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2016-2974 ◽

2016 ◽

Vol 102 (2) ◽

pp. 594-603 ◽

Cited By ~ 14

Author(s):

Ping Li ◽

Jinzhi Wei ◽

Xiaosa Li ◽

Yang Cheng ◽

Weiyu Chen ◽

...

Keyword(s):

Cell Proliferation ◽

Vascular Endothelial Cells ◽

Cell Model ◽

Tube Formation ◽

Luciferase Assay ◽

Endothelial Cell Proliferation ◽

Vascular Endothelial ◽

Atherosclerotic Lesions ◽

17Β Estradiol ◽

Cell Adhesion Molecule 1

Abstract Context: Endothelial microRNA 126 (miR-126) attenuates the development of atherosclerosis (AS). However, there is no evidence showing the role of miR-126 in estrogen’s antiatherogenic effects. Objective: We hypothesized that 17β-estradiol (E2) modulates miR-126 expression and thus may improve endothelial function and retard AS development. Design/Setting/Participants: This was a prospective cohort study of 12 healthy regularly menstruating female volunteers. ApoE−/− mice were used as the atherosclerosis model and human umbilical vascular endothelial cells (HUVECs) were cultured as the cell model. Main Outcome Measures: Serum hormones and miR-126-3p levels were measured up to 3 times for 1 cycle. Real-time polymerase chain reaction, histology for atherosclerotic lesions, immunofluorescence, luciferase assay, transfection experiments, cell proliferation, migration and tube formation assay, and western blot were performed. Results: Serum concentrations of miR-126-3p in cycling women were higher at the ovulatory and luteal phases than in the follicular phase, and they were positively correlated with E2 values. Administration of miR-126-3p mimics to ApoE−/− mice-attenuated atherogenesis, and antagomir-126-3p partially reversed the protective effect of E2 on atherogenesis. In HUVECs, E2 increased miR-126-3p expression via upregulation of Ets-1 (a transcription factor for miR-126). c-Src/Akt signaling was important for E2-mediated expression of Ets-1/miR-126. E2 decreased expression of miR-126-3p target Spred1 (a protein that inhibits mitogenic signaling). Overexpression of Spred1 partially blocked enhancement of endothelial cell proliferation, migration, and tube formation by E2. Additionally, E2 regulates miR-126-3p–mediated expression of vascular cell adhesion molecule-1 to inhibit monocyte adhesion into HUVECs. Conclusions: E2 protection against atherogenesis is possibly mediated by Ets-1/miR-126.

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Effects of nicardipine on tube formation of bovine vascular endothelial cells in vitro.

Stroke ◽

10.1161/01.str.23.11.1637 ◽

1992 ◽

Vol 23 (11) ◽

pp. 1637-1642 ◽

Cited By ~ 10

Author(s):

T Kaneko ◽

I Nagata ◽

S Miyamoto ◽

H Kubo ◽

H Kikuchi ◽

...

Keyword(s):

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Tube Formation ◽

Vascular Endothelial

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RBM23 Drives Hepatocellular Carcinoma by Activating NF-κB Signaling Pathway

BioMed Research International ◽

10.1155/2021/6697476 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Hexu Han ◽

Ting Lin ◽

Ziyi Fang ◽

Guoxiong Zhou

Keyword(s):

Hepatocellular Carcinoma ◽

Signaling Pathway ◽

Rna Binding ◽

Vascular Endothelial Cells ◽

Tube Formation ◽

Nuclear Accumulation ◽

Binding Motif ◽

Strong Positive Correlation ◽

Vascular Endothelial

Purpose. Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, and angiogenesis has been proven to be significantly involved in its progression. However, the molecular mechanism underlying HCC angiogenesis has not been well researched. In this study, RNA Binding Motif Protein 23 (RBM23) was identified as a novel proangiogenic factor in HCC cell lines and tissues. Materials and Methods. Firstly, we analyzed the correlation of clinical specimens. In HCC tissues, the levels of RBM23 and microvessel density (MVD) showed a strong positive correlation. Furthermore, data from related cytology experiments showed that the knockdown of RBM23 expression in HCC cells significantly inhibited the tube formation by the human vascular endothelial cells in vitro. The mechanism of this phenomenon was found to be through increasing the mRNA of p65 and enhanced the nuclear accumulation of p65. Consequently, RBM23 activated the NF-κB signaling pathway and promoted expression of the proangiogenic cytokines selectively. Results and Conclusion. In summary, this study revealed that RBM23 promotes the angiogenesis properties of HCC via the NF-κB signaling pathway. It may, therefore, be a potential therapeutic target for the treatment of hepatocellular carcinoma.

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NDRG1 Targets TAF15 to Promote Endothelial Dysfunction and Hypoxia-Induced Pulmonary Hypertension

10.21203/rs.3.rs-794105/v1 ◽

2021 ◽

Author(s):

Chengwei Li ◽

Liang Dong ◽

Ning Zhu ◽

Xiujuan Zhang ◽

Ruzetuoheti Yiminniyaze ◽

...

Keyword(s):

Endothelial Cells ◽

Pulmonary Hypertension ◽

Endothelial Dysfunction ◽

Vascular Remodeling ◽

Tube Formation ◽

Cell Counting ◽

Dependent Manner ◽

Rat Models ◽

Chain Reactions

Abstract Background: The mechanism underlying vascular remodeling of hypoxia-induced pulmonary hypertension (HPH) is not fully elucidated. We hypothesized that hypoxia promotes expression of N-myc downstream regulated gene-1 (NDRG1) in human pulmonary arterial endothelial cells (HPAECs), which in turn leads to endothelial dysfunction and contributing to HPH. Methods: Lung samples were obtained from qualified patients and HPH rat models. Quantitative polymerase chain reactions, western blotting and immunohistochemistry were used to measure the expression of NDRG1. EdU incorporation assays, cell counting kit-8 (CCK-8) assays, transwell migration assays, and matrigel assays were conducted to detect the role of NDRG1 in HPACE function in vitro. HPH models were established in SD rats and were treated with plasmids expressing short hairpin RNAs (shRNAs) to silence NDRG1. The candidate binding partner(s) of NDRG1 was screened and validated via co-immunoprecipitation and immunofluorescence staining. Results: NDRG1 is up-regulated by hypoxia in a time-dependent manner in HPAECs. Expression of NDRG1 was increased in lung tissues of HPH patient and rat model. In vitro, silencing NDRG1 attenuated proliferation, migration and tube formation of HPAECs under hypoxia, while NDRG1 over-expression promoted these behaviors of HPAECs in normoxia. NDRG1 knock-down alleviated vascular remodeling and right ventricular hypertrophy in rat models of HPH. NDRG1 can directly interact with TATA-box binding protein associated factor 15 (TAF15) and promote its nuclear localization. Bioinformatics study found that Notch1 signaling was downstream of TAF15 in endothelial cells. TAF15 can promote HPAECs dysfunction via binding to Notch1 promoter region and subsequently increasing Notch1 expression. Conclusions: Taken together, hypoxia-induced up-regulation of NDRG1 contributes to endothelial dysfunction and HPH development through TAF15 upregulation of Notch1, suggesting the applicability of targeting NDRG1 in clinical treatment of HPH.

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Abstract W MP77: The Role of Ischemia/Reperfusion Generated Nitrative Stress on VEGF Angiogenic Signaling in Diabetic rats.

Stroke ◽

10.1161/str.45.suppl_1.wmp77 ◽

2014 ◽

Vol 45 (suppl_1) ◽

Author(s):

Mohammed A Abdelsaid ◽

Roshini Prakash ◽

Weiguo Li ◽

Maha Coucha ◽

Susan Fagan ◽

...

Keyword(s):

Caspase 3 ◽

Vascular Endothelial Cells ◽

Ischemia Reperfusion ◽

Artery Occlusion ◽

Tube Formation ◽

Dependent Manner ◽

Vascular Endothelial ◽

Migration Assay ◽

Gk Rats ◽

Nitrative Stress

Diabetes worsens the outcome and impairs recovery after stroke. We have previously shown that diabetes caused a nitration-dependent dysfunctional cerebral neovascularization via increased vascular endothelial growth factor (VEGF) signalling. Our recent study showed that ischemia/reperfusion (I/R) impaired vascular repair in the post-stroke period in diabetes. Yet, the molecular mechanism is unclear. Our present hypothesis is that I/R-mediated excess peroxynitrite formation impairs VEGF survival and angiogenic signal in a nitration-dependent manner. Methods: Acute ischemic stroke was induced via 90 min middle cerebral artery occlusion in Wistar (Wis) and diabetic Goto-Kakizaki rats (GK). 14 days later brain sections were examined for peroxynitrite generation (nitrotyrosine), survival markers (Akt and P38 activation), and apoptosis markers (caspase-3 and cleaved PARP). Brain micro-vascular endothelial cells (BMVEC) were isolated from Wis and GK rats and subjected to 6 hr hypoxia (0.2% oxygen) followed by 18 h normoxia. Nitrotyrosine, P85 nitration, Akt and P38 activity and caspase-3 cleavage were determined. VEGF signal was assessed via cell proliferation, tube formation and cell migration assay. Results: There was increased basal tyrosine nitration in diabetes. I/R increased nitrotyrosine in control (*2-fold) and diabetic (*3-fold) rats (*p<0.05). This increase was associated with decreased survival signal (20%, *60%) and increased apoptic signal (1.5-fold and *6-fold in Wis and GK, respectively, *p<0.05). In parallel, BMVEC isolated from GK rats showed increased nitrative stress compared to controls (*p<0.05). A second hit by hypoxia/reoxygenation dramatically increased the nitration of P85 subunit of PI3kinase* and activation of p38 MAPK* while decreased Akt activation* (*p<0.05). These effects were associated with impairment of VEGF-induced migration* and restored by treatments with peroxynitrite scavenger FeTPPS* (5 uM) or nitration inhibitor Epicatechin* (200uM) (*p<0.05). Conclusion: Our results demonstrate that I/R diverts prosurvial/angiogenic effects of VEGF to a proapoptotic pathway and suggest peroxynitrite as a novel therapeutic target for improvement of reparative angiogenesis after stroke in diabetes.

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