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Wang L, Zhang C, Pang L, Wang Y. Integrated network pharmacology and experimental validation to explore the potential pharmacological mechanism of Qihuang Granule and its main ingredients in regulating ferroptosis in AMD. BMC Complement Med Ther 2023; 23:420. [PMID: 37990310 PMCID: PMC10664676 DOI: 10.1186/s12906-023-04205-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2023] [Accepted: 10/07/2023] [Indexed: 11/23/2023] Open
Abstract
BACKGROUND Qihuang Granule (QHG) is a traditional prescription that has exhibited potential in safeguarding against age-related maculopathy (AMD). Salvia miltiorrhiza (SM) and Fructus lycii (FL) are the main components of QHG. Ferroptosis, a newly discovered, iron-dependent, regulated cell death pathway, have been implicated in the pathogenesis of AMD. This study delves into the intricate mechanism by which SM/FL and QHG confer protection against AMD by modulating the ferroptosis pathway, employing a combination of network pharmacology and experimental validation. METHODS Bioactive compounds and potential targets of SM and FL were gathered from databases such as TCMSP, GeneCard, OMIM, and FerrDb, along with AMD-related genes and key genes responsible for ferroptosis regulation. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction (PPI) network were performed to discover the potential mechanism. The construction of an interaction network involving AMD, ferroptosis, SM/FL potential target genes was facilitated by the STRING database and realized using Cytoscape software. Subsequent validation was accomplished through molecular docking and in vitro cell experiments. RESULTS Noteworthy active compounds including quercetin, tanshinone IIA, luteolin, cryptotanshinone, and hub targets such as HIF-1α, EGFR, IL6, and VEGFA were identified. KEGG enrichment unveiled the HIF-1 signalling pathway as profoundly enriched, and IL6 and VEGF were involved. The molecular docking revealed the significant active compounds with hub genes and quercetin showed good binding to HIF-1α, which is involved in inflammation and angiogenesis. Experimental results verified that both herbs and QHG could regulate key ferroptosis-related targets in the retinal pigment epithelium and inhibit the expression of HIF-1α, VEGFA, and IL-6, subsequently increase cell viability and decrease the ROS content induced by H2O2. CONCLUSION This study demonstrates the molecular mechanism through which SM/FL and QHG protect against AMD and emerges as a plausible mechanism underlying this protection.
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Affiliation(s)
- Lu Wang
- Department of Ophthalmology, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, 111DaDe Road, Guangzhou, Guangdong, 510120, China
| | - Canyang Zhang
- Department of Ophthalmology, The Second Clinical College of Guangzhou University of Chinese Medicine, Guangzhou, 510006, China
| | - Long Pang
- Department of Ophthalmology, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, 111DaDe Road, Guangzhou, Guangdong, 510120, China.
| | - Yan Wang
- Department of Ophthalmology, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, 111DaDe Road, Guangzhou, Guangdong, 510120, China.
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2
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Li J, Han J, Shi Y, Liu M. Rapamycin inhibits corneal inflammatory response and neovascularization in a mouse model of corneal alkali burn. Exp Eye Res 2023:109539. [PMID: 37315833 DOI: 10.1016/j.exer.2023.109539] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2022] [Revised: 05/26/2023] [Accepted: 06/11/2023] [Indexed: 06/16/2023]
Abstract
Alkali burn-induced corneal injury often causes inflammation and neovascularization and leads to compromised vision. We previously reported that rapamycin ameliorated corneal injury after alkali burns by methylation modification. In this study, we aimed to investigate the rapamycin-medicated mechanism against corneal inflammation and neovascularization. Our data showed that alkali burn could induce a range of different inflammatory response, including a stark upregulation of pro-inflammatory factor expression and an increase in the infiltration of myeloperoxidase- and F4/80-positive cells from the corneal limbus to the central stroma. Rapamycin effectively downregulated the mRNA expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), toll-like receptor 4 (TLR4), nucleotide binding oligomerization domain-like receptors (NLR) family pyrin domain-containing 3 (NLRP3), and Caspase-1, and suppressed the infiltration of neutrophils and macrophages. Inflammation-related angiogenesis mediated by matrix metalloproteinase-2 (MMP-2) and rapamycin restrained this process by inhibiting the TNF-α upregulation in burned corneas of mice. Rapamycin also restrained corneal alkali burn-induced inflammation by regulating HIF-1α/VEGF-mediated angiogenesis and the serum cytokines TNF-α, IL-6, Interferon-gamma (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The findings of this study indicated rapamycin may reduce inflammation-associated infiltration of inflammatory cells, shape the expression of cytokines, and balance the regulation of MMP-2 and HIF-1α-mediated inflammation and angiogenesis by suppressing mTOR activation in corneal wound healing induced by an alkali injury. It offered novel insights relevant for a potent drug for treating corneal alkali burn.
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Affiliation(s)
- Jiande Li
- College of Life Sciences, Northwest Normal University, Lanzhou, 730070, Gansu, China.
| | - Jiangyuan Han
- College of Life Sciences and Technology, Gansu Agricultural University, Lanzhou, 730070, Gansu, China.
| | - Yongpeng Shi
- School of Life Sciences, Lanzhou University, Lanzhou, 730000, Gansu, China.
| | - Minrui Liu
- College of Life Sciences and Technology, Gansu Agricultural University, Lanzhou, 730070, Gansu, China.
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Qiu F, Tong HJ. Inhibitory effect of maspinon neovascularization in diabetic retinopathy. World J Diabetes 2021; 12:2050-2057. [PMID: 35047119 PMCID: PMC8696638 DOI: 10.4239/wjd.v12.i12.2050] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/07/2021] [Revised: 08/09/2021] [Accepted: 12/08/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Diabetic retinopathy (DR) is a serious and potentially blinding complication of diabetes mellitus. Retinal neovascularization is one of the main pathological features of proliferative DR, and inhibiting retinal neovascularization is a research focus.
AIM The aim was to evaluate the effect of intravitreal injection of recombinant human maspin on neovascularization in DR.
METHODS An oxygen-induced retinopathy (OIR) mouse model was used to simulate neovascularization in DR. New born C57BL/6J mice were randomly divided to a normal control group, a maspin injection OIR group, and an OIR group. The mice in the maspin injection OIR group were injected with recombinant human maspin in the bilateral vitreous cavity on postnatal day P12, and those in the OIR group were injected with sterile phosphate buffered saline. The protein expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1-alpha (HIF-1α) in the retina was measured by western blotting, and the mRNA expression of VEGF and HIF-1α was measured by real-time polymerase chain reaction. The vascular cell nuclei that broke through the inner limiting membrane (ILM) were counted in haematoxylin-eosin stained retinal sections.
RESULTS It was found that the number of vascular cell nuclei breaking through the ILM was 31.8 ± 8.75 in the OIR group, which was significantly more than that in the normal control group (P < 0.001). The number of vascular cell nuclei breaking through the ILM was 6.19 ± 2.91 in the maspin injection OIR group, which was significantly less than that in OIR group (P < 0.01). The relative protein and mRNA expression of VEGF and HIF-1α was significantly lower in the retinas in the maspin injection OIR group than in those in the OIR group (P < 0.01).
CONCLUSION Maspin inhibited neovascularization in DR by modulating the HIF-1α/VEGF pathway, which provides a potential and effective strategy for the treatment of DR.
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Affiliation(s)
- Feng Qiu
- Department of Ophthalmology, Shenyang Fourth People’s Hospital, Shenyang 110031, Liaoning Province, China
| | - Hui-Juan Tong
- Department of Nursing, Shenyang Medical College, Shenyang 110034, Liaoning Province, China
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4
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Plastino F, Pesce NA, André H. MicroRNAs and the HIF/VEGF axis in ocular neovascular diseases. Acta Ophthalmol 2021; 99:e1255-e1262. [PMID: 33729690 DOI: 10.1111/aos.14845] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2020] [Revised: 02/18/2021] [Accepted: 02/23/2021] [Indexed: 12/28/2022]
Abstract
Ocular neovascular diseases, such as proliferative diabetic retinopathy, retinopathy of prematurity and neovascular age-related macular degeneration, are the leading causes of visual impairment worldwide. The hypoxia-inducible factors and vascular endothelial growth factors are key molecular promoters of ocular neovascularization. Moreover, the role of microRNAs as regulators of angiogenesis has been expanding, particularly hypoxia-associated microRNA; hypoxamiRs. This review provides a summary of hypoxamiRs that directly and specifically target HIF1A and VEGF mRNAs, thus critically involved in the regulation of ocular neovascular pathologies. The discussed microRNAs highlight putative diagnostic markers and therapeutic agents in choroidal and retinal angiogenic diseases, including proliferative diabetic retinopathy, retinopathy of prematurity and neovascular age-related macular degeneration.
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Affiliation(s)
- Flavia Plastino
- Department of Clinical Neurosciences Division of Eye and Vision St. Erik Eye Hospital Karolinska Institutet Stockholm Sweden
| | - Noemi Anna Pesce
- Department of Clinical Neurosciences Division of Eye and Vision St. Erik Eye Hospital Karolinska Institutet Stockholm Sweden
| | - Helder André
- Department of Clinical Neurosciences Division of Eye and Vision St. Erik Eye Hospital Karolinska Institutet Stockholm Sweden
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Wong HL, Hung LT, Kwok SS, Bu Y, Lin Y, Shum HC, Wang H, Lo ACY, Yam GHF, Jhanji V, Shih KC, Chan YK. The anti-scarring role of Lycium barbarum polysaccharide on cornea epithelial-stromal injury. Exp Eye Res 2021; 211:108747. [PMID: 34450184 DOI: 10.1016/j.exer.2021.108747] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2021] [Revised: 08/07/2021] [Accepted: 08/22/2021] [Indexed: 12/16/2022]
Abstract
PURPOSE Cornea epithelial-stromal scarring is related to the differentiation of fibroblasts into opaque myofibroblasts. Our study aims to assess the effectiveness of Lycium barbarum polysaccharide (LBP) solution as a pre-treatment in minimizing corneal scarring. METHODS Human corneal fibroblasts were cultured in a three-dimensional collagen type I-based hydrogel in an eye-on-a-chip model. Fibroblasts were pre-treated with 2 mg/mL LBP for 24 h, followed by another 24-h incubation with 10 ng/mL transforming growth factor-beta 1 (TGF-β1) to induce relevant physiological events after stromal injury. Intracellular pro-fibrotic proteins, extracellular matrix proteins, and pro-inflammatory cytokines that involved in fibrosis, were assessed using immunocytochemistry and enzyme-linked immunosorbent assays. RESULTS Compared to the positive control TGF-β1 group, LBP pre-treated cells had a significantly lower expression of alpha-smooth muscle actin, marker of myofibroblasts, vimentin (p < 0.05), and also extracellular matrix proteins both collagen type II and type III (p < 0.05) that can be found in scar tissues. Moreover, LBP pre-treated cells had a significantly lower secretion of pro-inflammatory cytokines interleukin-6 and interleukin-8 (p < 0.05). The cell-laden hydrogel contraction and stiffness showed no significant difference between LBP pre-treatment and control groups. Fibroblasts pretreated with LBP as well had reduced angiogenic factors expression and suppression of undesired proliferation (p < 0.05). CONCLUSION Our results showed that LBP reduced both pro-fibrotic proteins and pro-inflammatory cytokines on corneal injury in vitro. We suggest that LBP, as a natural Traditional Chinese Medicine, may potentially be a novel topical pre-treatment option prior to corneal refractive surgeries with an improved prognosis.
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Affiliation(s)
- Ho Lam Wong
- Department of Ophthalmology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region
| | - Lap Tak Hung
- Department of Mechanical Engineering, Faculty of Engineering, The University of Hong Kong, Hong Kong Special Administrative Region
| | - Sum Sum Kwok
- Department of Ophthalmology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region
| | - Yashan Bu
- Department of Ophthalmology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region
| | - Yuan Lin
- Department of Mechanical Engineering, Faculty of Engineering, The University of Hong Kong, Hong Kong Special Administrative Region
| | - Ho Cheung Shum
- Department of Mechanical Engineering, Faculty of Engineering, The University of Hong Kong, Hong Kong Special Administrative Region
| | - Hua Wang
- Eye Center of Xiangya Hospital, Central South University, China; Hunan Key Laboratory of Ophthalmology, China
| | - Amy Cheuk Yin Lo
- Department of Ophthalmology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region
| | - Gary Hin Fai Yam
- Department of Ophthalmology, University of Pittsburgh Medical Centre, USA
| | - Vishal Jhanji
- Department of Ophthalmology, University of Pittsburgh Medical Centre, USA
| | - Kendrick Co Shih
- Department of Ophthalmology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region.
| | - Yau Kei Chan
- Department of Ophthalmology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region.
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Vinekar A, Nair AP, Sinha S, Vaidya T, Chakrabarty K, Shetty R, Ghosh A, Sethu S. Tear Fluid Angiogenic Factors: Potential Noninvasive Biomarkers for Retinopathy of Prematurity Screening in Preterm Infants. Invest Ophthalmol Vis Sci 2021; 62:2. [PMID: 33646290 PMCID: PMC7938022 DOI: 10.1167/iovs.62.3.2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Purpose To determine the status of proangiogenic factors in the tear fluid of preterm infants with and without retinopathy of prematurity (ROP). Methods Preterm infants (n = 36) undergoing routine ROP screening included in the prospective study were categorized as No-ROP (n = 13, no ROP at any visits), ROP (if ROP was present at first visit; n = 18), or No-ROP to ROP (no disease at first visit, but developed ROP subsequently; n = 5). Infants with ROP were also grouped as progressing (n = 7) and regressing (n = 16) based on ROP evolution between the first and subsequent visits. Schirmer's strips were used to collect tear fluid and proangiogenic factors (VEGF, angiogenin, soluble vascular cell adhesion molecule, and fractalkine) levels (in picograms per milliliter) in tear fluid were measured by multiplex ELISA. Results Lower levels of VEGF (135 ± 69; mean ± standard deviation) and higher levels of angiogenin (6568 ± 4975) were observed in infants with ROP compared with infants without ROP (172.5 ± 54.0; 4139 ± 3909) at the first visit. Significantly lower levels of VEGF were observed in the No-ROP to ROP group compared with the No-ROP and ROP groups. The VEGF and angiogenin levels at the first visit were significantly lower in infants with ROP with progressing disease. Angiogenin levels negatively correlated with birth weight and gestational age in ROP. The area under the curve (AUC) and odds ratio (OR) analysis demonstrated that angiogenin/birth weight (AUC = 0.776; OR, 8.6); angiogenin/gestational age (AUC = 0.706; OR, 7.3) and Angiogenin/VEGF (AUC = 0.806; OR, 14.3) ratios were able to differentiated preterm infants with and without ROP. Conclusions The association between angiogenin and ROP suggests its possible role in ROP. The ratio of angiogenin level with birth weight, gestational age, and/or VEGF could serve as a potential noninvasive screening biomarker for ROP.
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Affiliation(s)
- Anand Vinekar
- Department of Pediatric Retina, Narayana Nethralaya, Bangalore, India
| | - Archana Padmanabhan Nair
- GROW Research Lab, Narayana Nethralaya Foundation, Bangalore, India.,Manipal Academy of Higher Education, Manipal, India
| | - Shivani Sinha
- Department of Pediatric Retina, Narayana Nethralaya, Bangalore, India
| | - Tanuja Vaidya
- GROW Research Lab, Narayana Nethralaya Foundation, Bangalore, India.,Manipal Academy of Higher Education, Manipal, India
| | | | - Rohit Shetty
- Department of Cornea and Refractive Surgery, Narayana Nethralaya, Bangalore, India
| | - Arkasubhra Ghosh
- GROW Research Lab, Narayana Nethralaya Foundation, Bangalore, India.,Singapore Eye Research Institute, Singapore
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Echinomycin mitigates ocular angiogenesis by transcriptional inhibition of the hypoxia-inducible factor-1. Exp Eye Res 2021; 206:108518. [PMID: 33639134 DOI: 10.1016/j.exer.2021.108518] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2020] [Revised: 01/29/2021] [Accepted: 02/15/2021] [Indexed: 12/11/2022]
Abstract
BACKGROUND Echinomycin (EKN), an inhibitor of hypoxia-inducible factor (HIF)-1 DNA-binding activity, has been implied as a possible therapeutic agent in ischemic diseases. Here, we assess EKN in hypoxia-driven responses in vitro using human primary adult retinal pigment epithelium cells (aRPE) and retinal endothelial cells (hREC), and in vivo using the laser-induced mouse choroidal neovascularization (CNV) model. METHODS Effects of EKN on hypoxia-mediated pathways in aRPE were analyzed by Western blotting for HIF-1α protein, quantitative PCR of HIF-target genes, and proteome array for soluble angiogenic factors. In vitro inhibition of angiogenesis by EKN was determined in hREC. In vivo inhibition of angiogenesis by EKN was determined in the mouse laser-induced CNV, as a model of HIF-associated ocular neovascularization. CNV lesion area was determined by fundus fluorescein angiography. RESULTS aRPE treated with EKN showed hypoxia-dependent significantly decreased cell recovery in the wound healing assay. These results were supported by lower levels of HIF-mediated transcripts detected in hypoxic aRPE cells treated with EKN compared with non-treated controls, and confirmed by proteome profiler for angiogenic factors. hREC exposed to aRPE EKN-conditioned medium displayed reduced sprouting angiogenesis. Mice with laser-induced CNV treated with intravitreally injected EKN showed significantly decreased vascular lesion area when compared with a mouse equivalent of aflibercept, or vehicle-treated controls. CONCLUSIONS Our data proposes EKN as a potent inhibitor of HIF-mediated angiogenesis in retinal cells and in the mouse model of CNV, which could have future implications in the treatment of patients with neovascular age-related macular degeneration.
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8
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Zhang J, Jiang C, Ruan L, Huang X. Associations of cytokine concentrations in aqueous humour with retinal vascular abnormalities and exudation in Coats' disease. Acta Ophthalmol 2019; 97:319-324. [PMID: 30414256 DOI: 10.1111/aos.13971] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2018] [Accepted: 10/11/2018] [Indexed: 01/15/2023]
Abstract
PURPOSE To investigate the associations of cytokine concentrations in aqueous humour with the severity of retinal vascular abnormalities, exudation and fibrosis in patients with Coats' disease. METHODS Aqueous humour samples were collected in 23 paediatric patients (23 eyes) with Coats' disease and six age-matched control patients (six eyes) with congenital cataract in this cross-sectional, case-control study. Through Cytometric Bead Array technology, six angiogenic, inflammatory and fibrotic cytokines were measured for their concentrations in aqueous humour. Ophthalmologic characteristics including retinal vessel abnormalities, exudation and fibrosis of Coats' disease were also clinically evaluated for analysis. RESULTS The aqueous levels of vascular endothelial growth factor (VEGF) (p = 0.006) and monocyte chemoattractant protein-1 (MCP-1) (p < 0.001) were significantly higher in the Coats' disease group than in the control group. The concentrations of angiogenin were peaked in eyes with first-grade vessels tortuosity (p < 0.001), and also positively correlated with the severity of retinal capillary abnormalities (r = 0.910, p < 0.001). The concentrations of MCP-1 (r = 0.966, p < 0.001) and VEGF (r = 0. 765, p = 0.002) were significantly correlated with the extent of retinal exudation. The aqueous humour transforming growth factor-β (TGFβ) concentrations were higher in eyes with retinal fibrosis than in non-fibrotic eyes with Coats' disease (p = 0.004). CONCLUSION In Coats' disease, angiogenin may act as a potential biomarker for retinal vascular abnormalities. The concentrations of VEGF and MCP-1 may positively correlate with the severity of retinal exudation.
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Affiliation(s)
- Juan Zhang
- Department of Ophthalmology at Eye and ENT Hospital Shanghai Medical College Fudan University Shanghai China
- Shanghai Key Laboratory of Visual Impairment and Restoration Fudan University Shanghai China
| | - Chen Jiang
- Department of Ophthalmology at Eye and ENT Hospital Shanghai Medical College Fudan University Shanghai China
- Shanghai Key Laboratory of Visual Impairment and Restoration Fudan University Shanghai China
| | - Lu Ruan
- Department of Ophthalmology at Eye and ENT Hospital Shanghai Medical College Fudan University Shanghai China
| | - Xin Huang
- Department of Ophthalmology at Eye and ENT Hospital Shanghai Medical College Fudan University Shanghai China
- Shanghai Key Laboratory of Visual Impairment and Restoration Fudan University Shanghai China
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Yamamoto K, Yano I. Genetic polymorphisms associated with adverse reactions of molecular-targeted therapies in renal cell carcinoma. Med Oncol 2018; 35:16. [PMID: 29302760 DOI: 10.1007/s12032-017-1077-0] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2017] [Accepted: 12/27/2017] [Indexed: 12/28/2022]
Abstract
The prognosis of patients with metastatic renal cell carcinoma has drastically improved due to the development of molecular-targeted drugs and their use in clinical practice. However, these drugs cause some diverse adverse reactions in patients and sometimes affect clinical outcomes of cancer therapy. Therefore, predictive markers are necessary to avoid severe adverse reactions, to establish novel and effective prevention methods, and to improve treatment outcomes. Some genetic factors involved in these adverse reactions have been reported; however, perspectives on each adverse response have not been integrated yet. In this review, genetic polymorphisms relating to molecular-targeted therapy-induced adverse reactions in patients with renal cell carcinoma are summarized in the points of pharmacokinetic and pharmacodynamic mechanisms. We also discuss about the relationship between systemic drug exposure and adverse drug reactions.
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Affiliation(s)
- Kazuhiro Yamamoto
- Department of Pharmacy, Kobe University Hospital, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
| | - Ikuko Yano
- Department of Pharmacy, Kobe University Hospital, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan
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10
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A Critical Analysis of the Available In Vitro and Ex Vivo Methods to Study Retinal Angiogenesis. J Ophthalmol 2017; 2017:3034953. [PMID: 28848677 PMCID: PMC5564124 DOI: 10.1155/2017/3034953] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2017] [Revised: 06/20/2017] [Accepted: 06/28/2017] [Indexed: 12/15/2022] Open
Abstract
Angiogenesis is a biological process with a central role in retinal diseases. The choice of the ideal method to study angiogenesis, particularly in the retina, remains a problem. Angiogenesis can be assessed through in vitro and in vivo studies. In spite of inherent limitations, in vitro studies are faster, easier to perform and quantify, and typically less expensive and allow the study of isolated angiogenesis steps. We performed a systematic review of PubMed searching for original articles that applied in vitro or ex vivo angiogenic retinal assays until May 2017, presenting the available assays and discussing their applicability, advantages, and disadvantages. Most of the studies evaluated migration, proliferation, and tube formation of endothelial cells in response to inhibitory or stimulatory compounds. Other aspects of angiogenesis were studied by assessing cell permeability, adhesion, or apoptosis, as well as by implementing organotypic models of the retina. Emphasis is placed on how the methods are applied and how they can contribute to retinal angiogenesis comprehension. We also discuss how to choose the best cell culture to implement these methods. When applied together, in vitro and ex vivo studies constitute a powerful tool to improve retinal angiogenesis knowledge. This review provides support for researchers to better select the most suitable protocols in this field.
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11
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Beaujean O, Locri F, Aronsson M, Kvanta A, André H. A novel in vivo model of puncture-induced iris neovascularization. PLoS One 2017; 12:e0180235. [PMID: 28658313 PMCID: PMC5489193 DOI: 10.1371/journal.pone.0180235] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2017] [Accepted: 06/12/2017] [Indexed: 01/08/2023] Open
Abstract
PURPOSE To assess iris neovascularization by uveal puncture of the mouse eye and determine the role of angiogenic factors during iris neovascularization. METHODS Uveal punctures were performed on BalbC mouse eyes to induce iris angiogenesis. VEGF-blockage was used as an anti-angiogenic treatment, while normoxia- and hypoxia-conditioned media from retinal pigment epithelium (RPE) cells was used as an angiogenic-inducer in this model. Iris vasculature was determined in vivo by noninvasive methods. Iris blood vessels were stained for platelet endothelial cell adhesion molecule-1 and vascular sprouts were counted as markers of angiogenesis. Expression of angiogenic and inflammatory factors in the puncture-induced model were determined by qPCR and western blot. RESULTS Punctures led to increased neovascularization and sprouting of the iris. qPCR and protein analysis showed an increase of angiogenic factors, particularly in the plasminogen-activating receptor and inflammatory systems. VEGF-blockage partly reduced iris neovascularization, and treatment with hypoxia-conditioned RPE medium led to a statistically significant increase in iris neovascularization. CONCLUSIONS This study presents the first evidence of a puncture-induced iris angiogenesis model in the mouse. In a broader context, this novel in vivo model of neovascularization has the potential for noninvasive evaluation of angiogenesis modulating substances.
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Affiliation(s)
- Ophélie Beaujean
- Department of Clinical Neuroscience, Section of Eye and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden
| | - Filippo Locri
- Department of Clinical Neuroscience, Section of Eye and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden
| | - Monica Aronsson
- Department of Clinical Neuroscience, Section of Eye and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden
| | - Anders Kvanta
- Department of Clinical Neuroscience, Section of Eye and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden
| | - Helder André
- Department of Clinical Neuroscience, Section of Eye and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden
- * E-mail:
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12
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Affiliation(s)
- Hassan Ghasemi
- Department of Ophthalmology, Shahed University, Tehran, Iran
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13
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D'Amico AG, Maugeri G, Bucolo C, Saccone S, Federico C, Cavallaro S, D'Agata V. Nap Interferes with Hypoxia-Inducible Factors and VEGF Expression in Retina of Diabetic Rats. J Mol Neurosci 2016; 61:256-266. [PMID: 27909871 DOI: 10.1007/s12031-016-0869-6] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2016] [Accepted: 11/22/2016] [Indexed: 02/14/2023]
Abstract
The retinal microvascular damage is a complication of diabetic retinopathy (DR). Hyperglycemia and hypoxia are responsible of aberrant vessel's proliferation. The cellular response to hypoxia is mediated through activation of hypoxia-inducible factors (HIFs). Among these, HIF-1α modulates expression of its target gene, VEGF, whose upregulation controls the angiogenic event during DR development. In a previous study, we have demonstrated that a small peptide, NAP, is able to protect retina from hyperglycemic insult. Here, we have demonstrated that its intraocular administration in a rat model of diabetic retinopathy has reduced expression of HIF-1α, HIF-2α, and VEGF by increasing HIF-3α levels. These data have been also confirmed by immunolocalization study by confocal microscopy. Although these evidences need to be further deepened to understand the molecular mechanism involved in the protective NAP action, the present data suggest that this small peptide may be effective to prevent the development of this ocular pathology.
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Affiliation(s)
- Agata Grazia D'Amico
- San Raffaele Open University of Rome, Rome, Italy.,Department of Biomedical and Biotechnological Sciences, Section of Human Anatomy and Histology, University of Catania, Via S. Sofia, 87, 95123, Catania, Italy
| | - Grazia Maugeri
- Department of Biomedical and Biotechnological Sciences, Section of Human Anatomy and Histology, University of Catania, Via S. Sofia, 87, 95123, Catania, Italy
| | - Claudio Bucolo
- Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy
| | - Salvatore Saccone
- Department of Biological, Geological and Environmental Sciences, University of Catania, Catania, Italy
| | - Concetta Federico
- Department of Biological, Geological and Environmental Sciences, University of Catania, Catania, Italy
| | - Sebastiano Cavallaro
- Institute of Neurological Sciences, Italian National Research Council, Catania, Italy
| | - Velia D'Agata
- Department of Biomedical and Biotechnological Sciences, Section of Human Anatomy and Histology, University of Catania, Via S. Sofia, 87, 95123, Catania, Italy.
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Jafari Sani M, Yazdi F, Masoomi Karimi M, Alizadeh J, Rahmati M, Zarei Mahmudabadi A. The siRNA-Mediated Down-Regulation of Vascular Endothelial Growth Factor Receptor1. IRANIAN RED CRESCENT MEDICAL JOURNAL 2016; 18:e23418. [PMID: 27275397 PMCID: PMC4893410 DOI: 10.5812/ircmj.23418] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/10/2014] [Revised: 04/16/2015] [Accepted: 06/08/2015] [Indexed: 11/24/2022]
Abstract
Background Angiogenesis is an important biological process involved in the proliferation of endothelial cells, tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is considered as a prominent regulator of angiogenesis which exerts the aforementioned effect(s) through its respective receptors (VEGFR1 and VEGFR2). VEGF receptors are targeted as a therapeutic candidate for cancer growth inhibition. RNAi as a new and promising strategy has provided a useful means to specifically suppress gene expression in cancer cells. Objectives The current study aimed to down-regulate expression of the VEGFR1 using siRNA. Materials and Methods This experimental study designed specific siRNAs against VEGFR1. Total RNA was extracted from human umbilical vain endothelial cell (HUVEC) and subsequently cDNA was synthetized. PCR was performed using specific primers to amplify the target gene. After double digestion and purification, the gene was cloned into pEFGP-N1 expression vector. Then, AGS cells were transfected with recombinant pEGFP-N1 using lipofectamin. The gene expression and down-regulation were evaluated by fluorescence scanning, reverse transcription PCR (RT-PCR) and Western blot techniques. Results Fluorescent scanning, RT-PCR (27.68%) and western blot analysis (31.06%) showed that the expression of VEGFR1 was suppressed effectively. Conclusions The results of the current study showed that specifically designed siRNA can be considered as an appropriate strategy to suppress gene expression and might be a promising tool to prevent angiogenesis.
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Affiliation(s)
- Moslem Jafari Sani
- Biochemistry Department, School of Medicine, Shahroud University of Medical Sciences, Shahroud, IR Iran
| | - Foad Yazdi
- Biotechnology Department, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, IR Iran
| | - Masoomeh Masoomi Karimi
- Immunology Department, School of Medicine, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, IR Iran
| | - Javad Alizadeh
- Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, IR Iran
| | - Majid Rahmati
- Biotechnology Department, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, IR Iran
| | - Ali Zarei Mahmudabadi
- Biochemical Department, Chemical Injuries Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran
- Corresponding Author: Ali Zarei Mahmudabadi, Biochemical Department, Chemical Injuries Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran. Tel: +98-9123277532, Fax: +98-2122830262, E-mail:
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Yang C, Xu Y, Zhou H, Yang L, Yu S, Gao Y, Huang Y, Lu L, Liang X. Tetramethylpyrazine protects CoCl2-induced apoptosis in human umbilical vein endothelial cells by regulating the PHD2/HIF/1α-VEGF pathway. Mol Med Rep 2015; 13:1287-96. [PMID: 26676934 DOI: 10.3892/mmr.2015.4679] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2015] [Accepted: 11/17/2015] [Indexed: 11/06/2022] Open
Abstract
Tetramethylpyrazine (TMP), one of the active ingredients isolated from a Chinese herbal prescription, possesses protective effects against apoptosis in endothelial cells. However, the underlying mechanism of its protective effects in endothelial cells remains to be elucidated. Using human umbilical vein endothelial cells (HUVECs), the present study assessed the protective effects of TMP on CoCl2-induced apoptosis. Following pre-incubation with CoCl2 (150 µM/ml) for 4 h, the HUVECs were treated with TMP at different concentrations (50, 100 and 200 µM/ml) for 8 h. TMP upregulated the expression of prolyl hydroxylase (PHD)2, reduced the protein and mRNA expression levels of vascular endothelial growth factor (VEGF), and reduced the expression of HIF-1α only at the protein level, not at the mRNA level in HUVECs, in a concentration-dependent manner. Furthermore, silencing of the PHD2 gene with small interfering (si)RNAs abolished the reduction in the expression of hypoxia-inducible factor (HIF)-1α and VEGF by TMP. In addition, TMP protected CoCl2-induced HUVEC injury via an apoptosis pathway, as characterized by the increased ratio of cell viability and the reduced percentage of apoptotic and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive HUVECs, activation of caspase-3, -8 and -9, B-cell lymphoma (Bcl)-2/Bcl-2-activated X protein expression, as well as the release of cytochrome c. The protective properties of TMP were partially attributed to the mRNA and protein expression levels of PHD, since silencing of the PHD2 gene with siRNAs abolished these effects. The present study demonstrated that the antiapoptotic effect of TMP in CoCl2-induced HUVECs was, at least in part, via the regulation of the PHD2/HIF-1α signaling pathway.
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Affiliation(s)
- Cheng Yang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‑sen University, Guangzhou, Guangdong 510000, P.R. China
| | - Yue Xu
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‑sen University, Guangzhou, Guangdong 510000, P.R. China
| | - Huanjiao Zhou
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‑sen University, Guangzhou, Guangdong 510000, P.R. China
| | - Lu Yang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‑sen University, Guangzhou, Guangdong 510000, P.R. China
| | - Shanshan Yu
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‑sen University, Guangzhou, Guangdong 510000, P.R. China
| | - Yi Gao
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‑sen University, Guangzhou, Guangdong 510000, P.R. China
| | - Yongsheng Huang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‑sen University, Guangzhou, Guangdong 510000, P.R. China
| | - Lin Lu
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‑sen University, Guangzhou, Guangdong 510000, P.R. China
| | - Xiaoling Liang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‑sen University, Guangzhou, Guangdong 510000, P.R. China
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Gutierrez LS, Ling J, Nye D, Papathomas K, Dickinson C. Thrombospondin peptide ABT-898 inhibits inflammation and angiogenesis in a colitis model. World J Gastroenterol 2015; 21:6157-66. [PMID: 26034351 PMCID: PMC4445093 DOI: 10.3748/wjg.v21.i20.6157] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/25/2014] [Revised: 11/10/2014] [Accepted: 12/20/2014] [Indexed: 02/06/2023] Open
Abstract
AIM To evaluate the efficacy of the improved thrombospondin mimetic peptide ABT-898 in a murine model of ulcerative colitis. METHODS The dextran sodium sulfate (DSS) was used for the induction of colitis in both TSP-1 deficient (TSP-1(-/-)) and wild type (WT) mice during 7 d. While mice were receiving the DSS dissolved in the drinking water, the ABT-898 peptide was dissolved in sterile 5% glucose solution and delivered using mini pumps subcutaneously implanted. Plasma samples were analyzed for interleukin (IL)-6 by ELISA assay and colonic tissues were harvested, fixed and processed for histological evaluation. Immunohistochemistry using antibodies for the detection of CD31 and MECA in endothelial cells was performed. Inflammation was graded in colonic sections and the number of microvessels in each lesion was assessed. Activation of signal transducer and activator of transcription 3 (STAT3) in colonic samples was quantified by immunohistochemistry and Western blotting using antibodies against total STAT3 and phosphorylated STAT3 (pSTAT3) (Ser727). RESULTS Treatment with ABT-898 considerably diminished the inflammatory response in WT and TSP-1(-/-) mice (P < 0.0001 in both groups vs control). Identification of blood vessels highlighted by CD31/MECA immunohistochemistry, showed significantly reduced vessel counts in colitic lesions of WT and TSP-1(-/-) mice treated with ABT898 (TSP-1(-/-) controls/TSP-1(-/-) treated, P = 0.0002; WT controls/WT treated, P = 0.0005). Consistently, IL-6 was significantly diminished in plasma samples of TSP-1(-/-) and WT treated with the peptide when compared to the control mice (P = 0.0002 and P = 0.0148, respectively). pSTAT3 positive cells were quantified in WT and TSP-1(-/-) treated with ABT-898. A significant decrease in positive cells for pSTAT3 was observed in treated mice (TSP-1(-/-) controls/TSP-1(-/-) treated, P = 0.0089; WT/WT treated, P = 0.0110). These results were confirmed by Western blotting analyses showing lower levels of pSTAT3 in colitic lesions from mice treated with the peptide ABT-898. CONCLUSION These findings indicate that the new peptide ABT-898 ameliorates inflammation and angiogenesis and might be a therapeutic alternative in IBD and inflammatory diseases.
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HIF-α/MIF and NF-κB/IL-6 axes contribute to the recruitment of CD11b+Gr-1+ myeloid cells in hypoxic microenvironment of HNSCC. Neoplasia 2014; 16:168-79. [PMID: 24709424 DOI: 10.1593/neo.132034] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2013] [Revised: 01/14/2014] [Accepted: 01/27/2014] [Indexed: 02/05/2023]
Abstract
CD11b+Gr-1+ myeloid cells have gained much attention due to their roles in tumor immunity suppression as well as promotion of angiogenesis, invasion, and metastases. However, the mechanisms by which CD11b+Gr-1+ myeloid cells recruit to the tumor site have not been well clarified. In the present study, we showed that hypoxia could stimulate the migration of CD11b+Gr-1+ myeloid cells through increased production of macrophage migration inhibitory factor (MIF) and interleukin-6 (IL-6) by head and neck squamous cell carcinoma (HNSCC) cells. Hypoxia-inducible factor-1α (HIF-1α)- and HIF-2α-dependent MIF regulated chemotaxis, differentiation, and pro-angiogenic function of CD11b+Gr-1+ myeloid cells through binding to CD74/CXCR2, and CD74/CXCR4 complexes, and then activating p38/mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinases (PI3K)/AKT signaling pathways. Knockdown (KD) of HIF-1α and HIF-2α in HNSCC cells decreased MIF level but failed to inhibit the CD11b+Gr-1+ myeloid cell migration, because HIF-1α/2α KD enhanced nuclear factor κB (NF-κB) activity that increased IL-6 secretion. Simultaneously blocking NF-κB and HIF-1α/HIF-2α had better inhibitory effect on CD11b+Gr-1+ myeloid cell recruitment in the hypoxic zone than individually silencing HIF-1α/2α or NF-κB. In conclusion, the interaction between HIF-α/MIF and NF-κB/IL-6 axes plays an important role in the hypoxia-induced accumulation of CD11b+Gr-1+ myeloid cells and tumor growth in HNSCC.
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Gene profiling of human VEGF signaling pathways in human endothelial and retinal pigment epithelial cells after anti VEGF treatment. BMC Res Notes 2014; 7:617. [PMID: 25201034 PMCID: PMC4167513 DOI: 10.1186/1756-0500-7-617] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2013] [Accepted: 08/27/2014] [Indexed: 11/23/2022] Open
Abstract
Background Ranibizumab (Lucentis®) is a Fab-antibody fragment developed from Bevacizumab, a full-length anti-VEGF antibody. Both compounds are used for treating age-related macular degeneration (AMD). The influence of bevacizumab and ranibizumab on genes involved in signal transduction and cell signaling downstream of VEGF were compared in order to detect possible differences in their mode of action, which are not related to their Fab-antibody fragments. Methods Human umbilical vein cell lines (EA.hy926) and retinal pigment epithelial cells (ARP-19) were exposed to oxidative stress. The cells were treated with therapeutic concentrations of bevacizumab (0.25 mg/mL) and ranibizumab (125 mg/mL) for 24 hours prior to all experiments, and their effects on gene expressions were determined by RT- PCR. Results After exposure to bevacizumab, more genes in the endothelial cells were up-regulated (KDR, NFATc2) and down-regulated (Pla2g12a, Rac2, HgdC, PRKCG) compared to non-treated controls. After exposure to ranibizumab, fewer genes were up-regulated (PTGS2) and down-regulated (NOS3) compared to controls. In comparison between drugs, more genes were up-regulated (NFATc2 and KDR) and more were down-regulated (Pla2g12a, Pla2g1b, Ppp3r2, Rac2) by bevacizumab than by ranibizumab. In RPE cells, NOS3 and PGF were up-regulated and Pla2g12b was down-regulated after exposure to ranibizumab, while PIK3CG was up-regulated and FIGF was down-regulated after exposure to bevacizumab, but the differences in gene expression were minor between drugs (PIK3CGand PGF were down-regulated more by ranibizumab than by bevacizumab). Conclusions The different gene expressions after exposure to ranibizumab and bevacizumab in endothelial and RPE cells may indicate a somewhat different biological activity of the two compounds.
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Luo DJ, Wu JH. Roles of HIF-1 in hepatocellular carcinoma. Shijie Huaren Xiaohua Zazhi 2014; 22:1-8. [DOI: 10.11569/wcjd.v22.i1.1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Hypoxia inducible factor-1 (HIF-1) is a key regulator of the cellular response to hypoxia. Since cell growth is out of control in hepatocellular carcinoma (HCC), HIF-1 activity is significantly enhanced in HCC to help cells adapt to the hypoxic microenvironment. HIF-1 plays a critical role in the occurrence and development of HCC through activating the target genes that participate in the regulation of cell proliferation and apoptosis, energy metabolism, angiogenesis, invasion and metastasis, resistance to chemotherapy and radiotherapy. Given the specific expression and regulation of HIF-1 in HCC growth, HIF-1 may become a new target for drug therapy and gene therapy, which provides a new avenue for neoadjuvant therapy of HCC in the future.
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Ling S, Birnbaum Y, Nanhwan MK, Thomas B, Bajaj M, Ye Y. MicroRNA-dependent cross-talk between VEGF and HIF1α in the diabetic retina. Cell Signal 2013; 25:2840-7. [PMID: 24018047 DOI: 10.1016/j.cellsig.2013.08.039] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2013] [Accepted: 08/30/2013] [Indexed: 12/13/2022]
Abstract
Both HIF1α (hypoxia-inducible factor alpha) and VEGF (vascular endothelial growth factor) are implicated in the pathogenesis of diabetic retinopathy (DR). Competitive endogenous RNAs (ceRNAs) are messenger RNA (mRNA) molecules that affect each other expression through competition for their shared microRNAs (miRNA). However, little is known about the role of ceRNAs in DR. We assess whether the expression of HIF1α and VEGF in DR is interdependent through sequestration of common miRNAs. We used bioinformatics to identify potential miRNAs that affect both genes and validated the interdependence of the genes by silencing or overexpression of the genes and assessed the luciferase-HIF1α 3'UTR activity. We found that HIF1α and VEGF are targeted by 12 common miRNAs. Silencing either HIF1α or VEGF increased the availabilities of the shared miRNAs, therefore suppressed the luciferase-HIF1α 3'UTR activity, whereas over-expressing HIF1α or VEGF increased the luciferase activity. HIF1α was co-expressed with VEGF in-vivo and in-vitro in DR models. Silencing HIF1α transcripts resulted in a significant reduction in VEGF protein levels and vice versa. This interdependence was miRNA- and 3'UTR-dependent, as silencing Dicer abolished the interdependence. Over-expression of a common miRNA (miR-106a) significantly reduced the expression of HIF1α and VEGF and prevented high glucose-induced increased permeability. There is a cross-talk between HIF1α and VEGF through interactions with their common miRNAs. miRNA based therapy can affect the expression of both HIF1α and VEGF and may represent a therapeutic potential for the treatment of DR.
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Affiliation(s)
- Shukuan Ling
- The Department of Biochemistry and Molecular Biology, University of Texas Medical Branch; Galveston, TX, USA; School of Life Science and Technology, Harbin Institute of Technology, Harbin, China; State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center, Beijing, China
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Kapadia S, Hapani S, Choueiri TK, Wu S. Risk of liver toxicity with the angiogenesis inhibitor pazopanib in cancer patients. Acta Oncol 2013; 52:1202-12. [PMID: 23594201 DOI: 10.3109/0284186x.2013.782103] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
PURPOSE The angiogenesis inhibitor pazopanib has been approved for the treatment of advanced renal cell cancer (RCC) and soft tissue sarcoma. Severe and fatal hepatotoxicity has been observed in its clinical studies. This analysis was conducted to determine the risk of liver toxicity by a systematic review and meta-analysis of clinical trials. PATIENTS AND METHODS Databases from PubMed, Web of Science and abstracts presented at ASCO meetings up to January, 2012 were searched to identify relevant studies. Eligible studies included prospective trials of cancer patients treated with pazopanib starting at 800 mg daily. Summary incidence rates, relative risks, and 95% confidence intervals (CIs) were calculated using a fixed- or random-effects model. RESULTS A total of 1478 patients from 10 clinical trials were included. The incidences of all-grade aspartate aminotransferase (AST), alanine transaminase (ALT), and bilirubin elevation were 39.6% (95% CI 31.2-48.6%), 41.4% (95% CI 34.1-49.0%), and 24.8% (95% CI 16.3-35.3%), respectively. The incidences of high-grade (Grade 3 and 4) AST, ALT and bilirubin elevation were 6.9% (95% CI 5.5-8.6%), 9.4% (95% CI 7.8-11.4%), and 3.4% (2.4-5.0%), respectively. In comparison with placebo, pazopanib significantly increased the risk of high-grade AST elevation (RR 6.56, 95% CI 2.04-21.07, p = 0.002) and ALT elevation (RR 4.33, 95% CI 1.88-10.0, p = 0.001). However, the risks of high-grade bilirubin elevation (RR 1.31, 95% CI 0.47-3.64) and fatal hepatotoxicity (RR 2.51, 95% CI 0.12-51.91, p = 0.55) were not significantly elevated. CONCLUSION The use of pazopanib was associated with a significantly increased risk of severe non-fatal hepatotoxicity in cancer patients.
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Affiliation(s)
- Samir Kapadia
- Division of Hematology and Oncology, Department of Medicine, Stony Brook University School of Medicine, Stony Brook, New York, USA
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Nawaz MI, Abouammoh M, Khan HA, Alhomida AS, Alfaran MF, Ola MS. Novel drugs and their targets in the potential treatment of diabetic retinopathy. Med Sci Monit 2013; 19:300-308. [PMID: 23619778 PMCID: PMC3659065 DOI: 10.12659/msm.883895] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2012] [Accepted: 01/25/2013] [Indexed: 02/05/2023] Open
Abstract
Diabetic retinopathy (DR) is the most common complication of diabetes. It causes vision loss, and the incidence is increasing with the growth of the diabetes epidemic worldwide. Over the past few decades a number of clinical trials have confirmed that careful control of glycemia and blood pressure can reduce the risk of developing DR and control its progression. In recent years, many treatment options have been developed for clinical management of the complications of DR (e.g., proliferative DR and macular edema) using laser-based therapies, intravitreal corticosteroids and anti-vascular endothelial growth factors, and vitrectomy to remove scarring and hemorrhage, but all these have limited benefits. In this review, we highlight and discuss potential molecular targets and new approaches that have shown great promise for the treatment of DR. New drugs and strategies are based on targeting a number of hyperglycemia-induced metabolic stress pathways, oxidative stress and inflammatory pathways, the renin-angiotensin system, and neurodegeneration, in addition to the use of stem cells and ribonucleic acid interference (RNAi) technologies. At present, clinical trials of some of these newer drugs in humans are yet to begin or are in early stages. Together, the new therapeutic drugs and approaches discussed may control the incidence and progression of DR with greater efficacy and safety.
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Affiliation(s)
- Mohd Imtiaz Nawaz
- Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia
| | - Marwan Abouammoh
- Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia
| | - Haseeb A. Khan
- Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia
| | - Abdullah S. Alhomida
- Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia
| | - Mubarak F. Alfaran
- Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia
| | - Mohammad Shamsul Ola
- Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia
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Endler A, Chen L, Li Q, Uchida K, Hashimoto T, Lu L, Xu GT, Shibasaki F. Int6/eIF3e silenced HIF2α stabilization enhances migration and tube formation of HUVECs via IL-6 and IL-8 signaling. Cytokine 2013; 62:115-22. [DOI: 10.1016/j.cyto.2013.01.021] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2012] [Revised: 08/04/2012] [Accepted: 01/25/2013] [Indexed: 11/28/2022]
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Spraggs CF, Xu CF, Hunt CM. Genetic characterization to improve interpretation and clinical management of hepatotoxicity caused by tyrosine kinase inhibitors. Pharmacogenomics 2013; 14:541-54. [DOI: 10.2217/pgs.13.24] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Tyrosine kinase inhibitors (TKIs) represent important therapeutic alternatives to, or combinations with, traditional cytotoxic chemotherapy. Despite their selective molecular targeting and demonstrated clinical benefit, TKIs produce a range of serious adverse events, including drug-induced liver injury, that require careful patient management to maintain treatment benefit without harm. Genetic characterization of serious adverse events can identify mechanisms of injury and improve safety risk management. This review presents pharmacogenetic comparisons of two approved TKIs, lapatinib and pazopanib, which reveal different mechanisms of injury and inform the characteristics and risk of serious liver injury in treated patients. The data presented demonstrate the utility of genetic studies to investigate drug-induced liver injury and potentially support its management in patients.
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Affiliation(s)
- Colin F Spraggs
- Genetics, Quantitative Sciences, GlaxoSmithKline Research & Development, Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK.
| | - Chun-Fang Xu
- Genetics, Quantitative Sciences, GlaxoSmithKline Research & Development, Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK
| | - Christine M Hunt
- Clinical Safety Systems, GlaxoSmithKline Research & Development, Research Triangle Park, NC, USA
- Duke University, Durham, NC, USA
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Loboda A, Jozkowicz A, Dulak J. HIF-1 versus HIF-2--is one more important than the other? Vascul Pharmacol 2012; 56:245-51. [PMID: 22366374 DOI: 10.1016/j.vph.2012.02.006] [Citation(s) in RCA: 110] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2012] [Revised: 02/10/2012] [Accepted: 02/11/2012] [Indexed: 01/16/2023]
Abstract
Hypoxia is a state where oxygen availability/delivery is below the level necessary to maintain physiological oxygen tension for a particular tissue. It is well-established that when tissue demand exceeds its oxygen supply, a cascade of intracellular events is activated, with the elevation of the expression of hypoxia-inducible factors (HIFs). As a consequence, the extensive transcriptional response regulating angiogenesis, glucose metabolism, cell growth, metastasis and others processes is induced. The discovery of differences between HIF isoforms has provided new insights into HIFs biology. Importantly, the opposite effects can be exerted by HIF-1 and HIF-2 on the regulation of angiogenic response. Although both isoforms may upregulate the expression of pro-angiogenic vascular endothelial growth factor (VEGF), HIF-1 diminished the expression of interleukin-8 (IL-8) by inhibition of the Nrf2 transcription factor whereas HIF-2 augmented expression of IL-8 in an Nrf2-independent way but via upregulation of SP-1 activity. Moreover, the opposite regulation of c-Myc transcription factor by both HIF isoforms may influence IL-8 regulation. Complexity of effects exerted by both HIF isoforms resulting from the cooperation with other transcription factors should be subjected to intensive investigation especially in the context of pro-and anti-angiogenic therapies.
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Affiliation(s)
- Agnieszka Loboda
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
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Ghasemi H, Ghazanfari T, Yaraee R, Faghihzadeh S, Hassan ZM. Roles of IL-8 in Ocular Inflammations: A Review. Ocul Immunol Inflamm 2011; 19:401-12. [DOI: 10.3109/09273948.2011.618902] [Citation(s) in RCA: 90] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
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Expression Characteristics of Hypoxia-Inducible Factor-1α and Its Clinical Values in Diagnosis and Prognosis of Hepatocellular Carcinoma. HEPATITIS MONTHLY 2011. [DOI: 10.5812/kowsar.1735143x.1305] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
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Zhao G, Guo LL, Xu JY, Yang H, Huang MX, Xiao G. Integrin-linked kinase in gastric cancer cell attachment, invasion and tumor growth. World J Gastroenterol 2011; 17:3487-96. [PMID: 21941415 PMCID: PMC3163246 DOI: 10.3748/wjg.v17.i30.3487] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/05/2010] [Revised: 02/15/2011] [Accepted: 02/22/2011] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the effects of integrin-linked kinase (ILK) on gastric cancer cells both in vitro and in vivo.
METHODS: ILK small interfering RNA (siRNA) was transfected into human gastric cancer BGC-823 cells and ILK expression was monitored by real-time quantitative polymerase chain reaction, Western blotting analysis and immunocytochemistry. Cell attachment, proliferation, invasion, microfilament dynamics and the secretion of vascular endothelial growth factor (VEGF) were also measured. Gastric cancer cells treated with ILK siRNA were subcutaneously transplanted into nude mice and tumor growth was assessed.
RESULTS: Both ILK mRNA and protein levels were significantly down-regulated by ILK siRNA in human gastric cancer cells. This significantly inhibited cell attachment, proliferation and invasion. The knockdown of ILK also disturbed F-actin assembly and reduced VEGF secretion in conditioned medium by 40% (P < 0.05). Four weeks after injection of ILK siRNA-transfected gastric cancer cells into nude mice, tumor volume and weight were significantly reduced compared with that of tumors induced by cells treated with non-silencing siRNA or by untreated cells (P < 0.05).
CONCLUSION: Targeting ILK with siRNA suppresses the growth of gastric cancer cells both in vitro and in vivo. ILK plays an important role in gastric cancer progression.
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Xu CF, Reck BH, Goodman VL, Xue Z, Huang L, Barnes MR, Koshy B, Spraggs CF, Mooser VE, Cardon LR, Pandite LN. Association of the hemochromatosis gene with pazopanib-induced transaminase elevation in renal cell carcinoma. J Hepatol 2011; 54:1237-43. [PMID: 21145803 DOI: 10.1016/j.jhep.2010.09.028] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/11/2010] [Revised: 09/09/2010] [Accepted: 09/20/2010] [Indexed: 02/01/2023]
Abstract
BACKGROUND & AIMS Pazopanib has demonstrated clinical benefit in patients with advanced renal cell carcinoma (RCC) and is generally well tolerated. However, transaminase elevations have commonly been observed. This 2-stage study sought to identify genetic determinants of alanine transaminase (ALT) elevations in pazopanib-treated white patients with RCC. METHODS Data from two separate clinical studies were used to examine the association of genetic polymorphisms with maximum on-treatment ALT levels. RESULTS Of 6852 polymorphisms in 282 candidate genes examined in an exploratory dataset of 115 patients, 92 polymorphisms in 40 genes were significantly associated with ALT elevation (p<0.01). Two markers (rs2858996 and rs707889) in the HFE gene, which are not yet known to be associated with hemochromatosis, showed evidence for replication. Because of multiple comparisons, there was a 12% likelihood the replication occurred by chance. These two markers demonstrated strong linkage disequilibrium (r(2)=0.99). In the combined dataset, median (25-75th percentile) maximum ALT values were 1.2 (0.7-1.9), 1.1 (0.8-2.5), and 5.4 (1.9-7.6)×ULN for rs2858996 GG (n=148), GT (n=82), and TT (n=1 2) genotypes, respectively. All 12 TT patients had a maximum ALT>ULN, and 8 (67%) had ALT≥3×ULN. The odds ratio (95% CI) for ALT≥3×ULN for TT genotype was 39.7 (2.2-703.7) compared with other genotypes. As a predictor of ALT≥3×ULN, the TT genotype had a negative predictive value of 0.83 and positive predictive value of 0.67. No TT patients developed liver failure. CONCLUSIONS The rs2858996/rs707889 polymorphisms in the HFE gene may be associated with reversible ALT elevation in pazo-panib-treated patients with RCC.
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Affiliation(s)
- Chun-Fang Xu
- GlaxoSmithKline Research and Development, Genetics Division, Stevenage, UK.
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Liu Y, Han ZP, Zhang SS, Jing YY, Bu XX, Wang CY, Sun K, Jiang GC, Zhao X, Li R, Gao L, Zhao QD, Wu MC, Wei LX. Effects of inflammatory factors on mesenchymal stem cells and their role in the promotion of tumor angiogenesis in colon cancer. J Biol Chem 2011; 286:25007-15. [PMID: 21592963 DOI: 10.1074/jbc.m110.213108] [Citation(s) in RCA: 144] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Mesenchymal stem cells (MSCs), which are modulated by cytokines present in the tumor microenvironment, play an important role in tumor progression. It is well documented that inflammation is an important part of the tumor microenvironment, so we investigated whether stimulation of MSCs by inflammatory cytokines would contribute to their ability to promote tumor growth. We first showed that MSCs could increase C26 colon cancer growth in mice. This growth-promoting effect was further accelerated when the MSCs were pre-stimulated by inflammatory factors IFN-γ and TNF-α. At the same time, we demonstrated that MSCs pre-stimulated by both inflammatory factors could promote tumor angiogenesis in vivo to a greater degree than untreated MSCs or MSCs pre-stimulated by either IFN-γ or TNF-α alone. A hen egg test-chorioallantoic membrane (HET-CAM) assay showed that treatment of MSC-conditioned medium can promote chorioallantoic membrane angiogenesis in vitro, especially treatment with conditioned medium of MSCs pretreated with IFN-γ and TNF-α together. This mechanism of promoting angiogenesis appears to take place via an increase in the expression of vascular endothelial growth factor (VEGF), which itself takes place through an increase in signaling in the hypoxia-inducible factor 1α (HIF-1α)-dependent pathway. Inhibition of HIF-1α in MSCs by siRNA was found to effectively reduce the ability of MSC to affect the growth of colon cancer in vivo in the inflammatory microenviroment. These results indicate that MSCs stimulated by inflammatory cytokines such as IFN-γ and TNF-α in the tumor microenvironment express higher levels of VEGF via the HIF-1α signaling pathway and that these MSCs then enhance tumor angiogenesis, finally leading to colon cancer growth in mice.
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Affiliation(s)
- Yan Liu
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai 200438, China
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Marek N, Raczyńska K, Siebert J, Myśliwiec M, Zorena K, Myśliwska J, Reiwer-Gostomska M, Trzonkowski P. Decreased angiogenin concentration in vitreous and serum in proliferative diabetic retinopathy. Microvasc Res 2011; 82:1-5. [PMID: 21539846 DOI: 10.1016/j.mvr.2011.04.006] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2011] [Revised: 03/07/2011] [Accepted: 04/18/2011] [Indexed: 01/07/2023]
Abstract
Diabetic retinopathy is the most common cause of vision loss in young adults in developed countries. The disease therapy with anti-vascular endothelial growth factor (VEGF) agents gives some positive results, but is associated with retinal ischemia and vasoconstriction. Therefore, determination of factors involved in the physiological and pathological angiogenesis in the diabetic eye is of great importance for understanding of the pathogenesis of diabetic retinopathy and its effective treatment. Previously, we found that diabetic patients were characterized by increased serum concentration of VEGF, but decreased levels of other proangiogenic factor-angiogenin. The involvement of VEGF in pathogenesis of diabetic retinopathy is well established, but there is lack of data regarding angiogenin in retinopathy. Therefore, in the present study we measured angiogenin concentration in vitreous and serum samples of the patients with type 1 diabetes to determine its role in diabetic retinopathy. In addition, in each time, we compared the level of angiogenin with level of VEGF as a known factor involved in the pathogenesis of the disease. Angiogenin was found to be significantly more abundant in serum than in vitreous in both diabetic groups. In addition, patients with retinopathy had twofold lower vitreous angiogenin levels than diabetic individuals without complications. On the contrary, vitreous concentration of VEGF was dramatically increased only in participants with retinopathy. Patients without diabetic complications had significantly lower VEGF levels in vitreous than in serum and were characterized by high local and systemic concentration of angiogenin. These data suggest a local imbalance between two proangiogenic factors-VEGF and angiogenin in retinopathy. Low vitreous concentration of angiogenin in diabetic patients suggests that this factor is not responsible for pathological neovascularization in diabetic eye. Further studies will elucidate if angiogenin can be used to improve the insufficient angiogenesis in diabetes and prevent retinal ischemia after retinopathy treatment with anti-VEGF agents.
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Affiliation(s)
- Natalia Marek
- Department of Clinical Immunology and Transplantology, Medical University of Gdańsk, ul. Dębinki 7, 80-211 Gdańsk, Poland.
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Mousa SA, Mousa SS. Current status of vascular endothelial growth factor inhibition in age-related macular degeneration. BioDrugs 2010; 24:183-94. [PMID: 20210371 DOI: 10.2165/11318550-000000000-00000] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Angiogenesis, the process by which new vessels are created from pre-existing vasculature, has become the subject of intense research in recent years. Increased rates of angiogenesis are associated with several disease states, including cancer, age-related macular degeneration (AMD), psoriasis, rheumatoid arthritis, and diabetic retinopathy. Vascular endothelial growth factor (VEGF) is an important modulator of angiogenesis, and has been implicated in the pathology of a number of conditions, including AMD, diabetic retinopathy, and cancer. AMD is a progressive disease of the macula and the third major cause of blindness worldwide. If not treated appropriately, AMD can progress to involve both eyes. Until recently, the treatment options for AMD have been limited, with photodynamic therapy (PDT) the mainstay of treatment. Although PDT is effective at slowing disease progression, it rarely results in improved vision. Several therapies have been or are now being developed for neovascular AMD, with the goal of inhibiting VEGF. These VEGF inhibitors include the RNA aptamer pegaptanib, partial and full-length antibodies ranibizumab and bevacizumab, the VEGF receptor decoy aflibercept, small interfering RNA-based therapies bevasiranib and AGN 211745, sirolimus, and tyrosine kinase inhibitors, including vatalanib, pazopanib, TG 100801, TG 101095, AG 013958, and AL 39324. At present, established therapies have met with great success in reducing the vision loss associated with neovascular AMD, whereas those still under investigation offer the potential for further advances. In AMD patients, these therapies slow the rate of vision loss and in some cases increase visual acuity. Although VEGF-inhibitor therapies are a milestone in the treatment of these disease states, several concerns need to be addressed before their impact can be fully realized.
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Affiliation(s)
- Shaker A Mousa
- The Pharmaceutical Research Institute at Albany College of Pharmacy and Health Sciences, Rensselaer, New York 12144, USA.
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Li SS, Yao DF, Dong ZZ. Advances in understanding the relationship between hepatic hypoxia-inducible factor-1 alpha and hepatocellular carcinoma. Shijie Huaren Xiaohua Zazhi 2010; 18:361-367. [DOI: 10.11569/wcjd.v18.i4.361] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Hepatocellular carcinoma (HCC) is characterized by hypoxia due to robust cell proliferation. Hypoxia can promote tumor cell proliferation, metastasis and neovasculogenesis, inhibit differentiation and apoptosis, and decrease chemosensitivity and radiosensitivity. Hypoxia-inducible factor-1α (HIF-1α) is a key mediator of physiological and pathological hypoxia response and controls the transcription of numerous genes that are of pivotal importance for angiogenesis and cellular metabolism. Therefore, HIF-1α is closely related with the proliferation, metastasis and apoptosis of HCC cells. Recently, HIF-1α-based gene therapy has become a novel adjunctive strategy for the management of HCC. This review focuses on the relationship between HIF-1α and the progression and therapy of HCC.
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Sheridan CM, Pate S, Hiscott P, Wong D, Pattwell DM, Kent D. Expression of hypoxia-inducible factor-1alpha and -2alpha in human choroidal neovascular membranes. Graefes Arch Clin Exp Ophthalmol 2009; 247:1361-7. [PMID: 19590888 DOI: 10.1007/s00417-009-1133-3] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2008] [Accepted: 06/15/2009] [Indexed: 10/20/2022] Open
Abstract
PURPOSE Up-regulation of pro-angiogenic cytokine expression occurring secondary to hypoxia in physiologic and pathophysiologic conditions is mediated by the family of transcription regulators know as hypoxia inducible factors (HIF). The present study was undertaken to investigate the expression of HIF occurring in human choroidal neovascularization (CNV) and the posterior segment of young and old eyes. METHODS Surgically excised CNV from patients with either age-related macular degeneration (AMD; n = 9), punctuate inner choroidopathy (PIC; n = 3) and young normal eyes were immunohistochemically probed with monoclonal antibodies against HIF-1alpha and -2alpha and compared to that for cell markers specific for vascular endothelial cells (CD34), macrophages (CD68), retinal pigment epithelial cells (RPE; panel cytokeratins/CK18) and VEGF. Following secondary antibody amplification, reactions were visualized with fast red chromogen. RESULTS Cellular immunoreactivity of membranes for HIF-2alpha was strong in eight out of nine AMD specimens but it was only weakly positive for HIF-1alpha in five specimens. In contrast, two out of three PIC specimens were weakly positive for HIF-1alpha but demonstrated no staining for HIF-2alpha. Immunohistochemical analysis revealed areas within the CNV membranes that were predominantly immunopositive for CD68 and cytokeratin indicating the presence of RPE and/or macrophages and that these cells strongly co-localized with the presence of HIF and VEGF. No immunochemical co-localization was observed with HIF and the endothelial cell marker CD34 in any membranes studied. Normal globes also demonstrated HIF-2 positivity to be predominantly localized to the central RPE rather than peripheral RPE irrespective of age of donor. CONCLUSIONS The localization of HIF expression supports the concept that hypoxia is a major stimulus for the development of submacular wound healing and within this context CNV is but one component of this process.
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Affiliation(s)
- Carl M Sheridan
- Unit of Ophthalmology, School of Clinical Sciences, University of Liverpool, Daulby Street, Liverpool, UK.
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Loboda A, Stachurska A, Florczyk U, Rudnicka D, Jazwa A, Wegrzyn J, Kozakowska M, Stalinska K, Poellinger L, Levonen AL, Yla-Herttuala S, Jozkowicz A, Dulak J. HIF-1 induction attenuates Nrf2-dependent IL-8 expression in human endothelial cells. Antioxid Redox Signal 2009; 11:1501-17. [PMID: 19254160 DOI: 10.1089/ars.2008.2211] [Citation(s) in RCA: 83] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Through hypoxia-inducible factor 1 (HIF-1), hypoxia regulates the expression of numerous genes and is a potent inducer of angiogenesis. However, interleukin-8 (IL-8), an important angiogenic mediator, has been reported to be downregulated by HIF-1, although the mechanisms have not been elucidated. HIF-1 was induced in human endothelial cells by hypoxia and dimethyloxaloylglycine (DMOG). Interestingly, both hypoxia and DMOG attenuated IL-8 expression, and a similar effect has been obtained by adenoviral overexpression of the stable form of HIF-1alpha. Heme oxygenase-1 (HO-1) expression was also downregulated by HIF-1 induction. This suggests similar mechanisms of regulation of IL-8 and HO-1, indicating the involvement of Nrf2, a transcription factor previously linked to hypoxia-mediated inhibition of HO-1. Indeed, HIF-1-mediated downregulation of both IL-8 and HO-1 was associated with both lowered Nrf2 expression and induction of Bach1, a repressor of Nrf2 transcriptional activity. Accordingly, overexpression of Nrf2 reversed the inhibitory effect of HIF-1 on IL-8 and HO-1 expression. However, neither overexpression of HO-1 nor HO-1 inhibition affected IL-8 synthesis. The data indicate that HIF-1-dependent inhibition of IL-8 expression is caused by downregulation of Nrf2. However, expression of IL-8 is independent of HO-1. Cross-talk between HIF-1 and Nrf2 may influence the outcome of anti-angiogenic therapies aimed at targeting HIF-1. Antioxid.
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Affiliation(s)
- Agnieszka Loboda
- Department of Medical Biotechnology, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
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Saidi A, Hagedorn M, Allain N, Verpelli C, Sala C, Bello L, Bikfalvi A, Javerzat S. Combined targeting of interleukin-6 and vascular endothelial growth factor potently inhibits glioma growth and invasiveness. Int J Cancer 2009; 125:1054-64. [DOI: 10.1002/ijc.24380] [Citation(s) in RCA: 79] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
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Kaposi's sarcoma-associated herpesvirus upregulates angiogenin during infection of human dermal microvascular endothelial cells, which induces 45S rRNA synthesis, antiapoptosis, cell proliferation, migration, and angiogenesis. J Virol 2009; 83:3342-64. [PMID: 19158252 DOI: 10.1128/jvi.02052-08] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is associated with the angioproliferative KS lesions characterized by spindle-shaped endothelial cells, inflammatory cells, cytokines, growth factors, and angiogenic factors. De novo KSHV infection of human microvascular dermal endothelial cells results in increased secretion of several growth factors, cytokines, chemokines, and angiogenic factors, and the multifunctional angiogenic protein angiogenin is one of them. KS tissue sections were positive for angiogenin, highlighting the importance of angiogenin in KS pathogenesis. Examination of KSHV-mediated angiogenin upregulation and secretion and potential outcomes revealed that during infection of primary endothelial cells, KSHV induced a time- and dose-dependent increase in angiogenin gene expression and protein secretion beginning as early as 8 h postinfection and lasting until the fifth day of our observation period. TIVE latently transformed cells (TIVE-LTC) latently infected with KSHV secreted high levels of angiogenin. Angiogenin was also detected in BCBL-1 cells (human B cells) carrying KSHV in a latent state. Significant induction of angiogenin was observed in cells expressing KSHV ORF73 (LANA-1; latent) and ORF74 (lytic) genes alone, and moderate induction was seen with the lytic KSHV ORF50 gene. Angiogenin bound to surface actin, internalized in a microtubule-independent manner, and translocated into the nucleus and nucleolus of infected cells. In addition, it increased 45S rRNA gene transcription, antiapoptosis, and proliferation of infected cells, thus demonstrating the multifunctional nature of KSHV-induced angiogenin. These activities were dependent on angiogenin nuclear translocation, which was inhibited by neomycin. Upregulation of angiogenin led to increased activation of urokinase plasminogen activator and generation of active plasmin, which facilitated the migration of endothelial cells toward chemoattractants, including angiogenin, and chemotaxis was prevented by the inhibition of angiogenin nuclear translocation. Treatment of KSHV-infected cell supernatants with antiangiogenin antibodies significantly inhibited endothelial tube formation, and inhibition of nuclear translocation of angiogenin also blocked the expression of KSHV-induced vascular endothelial growth factor C. Collectively, these results strongly suggest that by increasing infected endothelial cell 45S rRNA synthesis, proliferation, migration, and angiogenesis, KSHV-induced angiogenin could be playing a pivotal role in the pathogenesis of KSHV infection, including a contribution to the angioproliferative nature of KS lesions. Our studies suggested that LANA-1 and vGPCR play roles in KSHV-induced angiogenesis and that the angiogenic potential of vGPCR might also be due to its ability to induce angiogenin.
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Slevin M, Kumar P, Wang Q, Kumar S, Gaffney J, Grau-Olivares M, Krupinski J. New VEGF antagonists as possible therapeutic agents in vascular disease. Expert Opin Investig Drugs 2008; 17:1301-14. [DOI: 10.1517/13543784.17.9.1301] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
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Inhibition of proliferation, migration and tube formation of choroidal microvascular endothelial cells by targeting HIF-1alpha with short hairpin RNA-expressing plasmid DNA in human RPE cells in a coculture system. Graefes Arch Clin Exp Ophthalmol 2008; 246:1413-22. [PMID: 18523795 DOI: 10.1007/s00417-008-0858-8] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2008] [Revised: 04/23/2008] [Accepted: 04/28/2008] [Indexed: 10/22/2022] Open
Abstract
BACKGROUND Retinal pigment epithelial (RPE) cells and choroidal microvascular endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), and hypoxia plays an important role in CNV formation via hypoxia inducible factor 1 (HIF-1). Our aim was to evaluate the role of HIF-1 in human RPE cells with regard to proliferation, migration and tube formation of CECs under hypoxia. METHODS RPE cells were cultured under chemical hypoxia induced by 200 muM CoCl(2), and RNA interference (RNAi) technique was used to knock down HIF-1alpha gene in RPE cells. mRNA and protein expression of HIF-1alpha and VEGF in RPE cells were investigated by real-time RT-PCR and Western blot. Three kinds of coculture models were used to observe the effects of RPE cells transfected by short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) (pshHIF-1alpha) on the proliferation, migration and tube formation of CECs respectively. RESULTS Transfection of shRNA-expressing pDNA targeting HIF-1alpha to RPE cells resulted in the knock down of HIF-1alpha gene and reduction of the corresponding mRNA and protein of HIF-1alpha and VEGF under hypoxia. Consequently, the proliferation, migration and tube formation of CECs were significantly inhibited by the knocked-down RPE cells compared with the control in the coculture system. The proliferation rates of CECs decreased by 40.2%, 36.6% and 36.8% on days 3, 4 and 5 respectively. Migration reduced by 49.6% at 5 h, and tube formation decreased by 40.4% at 48 h. CONCLUSION RNAi of HIF-1alpha in RPE cells can inhibit angiogenesis in vitro and provide a possible strategy for treatment of choroidal neovascularization diseases by targeting HIF-1alpha.
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