TGF-β2 Promotes Oxidative Stress in Human Trabecular Meshwork Cells by Selectively Enhancing NADPH Oxidase 4 Expression.
TGF-β2 Promotes Oxidative Stress in Human Trabecular Meshwork Cells by Selectively Enhancing NADPH Oxidase 4 Expression.
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DOI：
10.1167/iovs.62.4.4
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发表时间：
2021-04-01
影响因子：
4.4
通讯作者：
Stubbs EB Jr
中科院分区：
文献类型：
--
作者：
Rao VR;Stubbs EB Jr
多功能促纤维化细胞因子TGF-β2参与原发性开角型青光眼（POAG）的病理生理学。虽然POAG的根本原因尚不清楚，但小梁网（TM）微环境内细胞外基质（ECM）的TGF-β2依赖性重塑被认为是与房水（AH）流出受损和IOP升高相关的早期病理后果。我们的研究小组最近发现，线粒体靶向抗氧化剂可显著减弱TGF-β2促纤维化反应，强烈暗示氧化应激是人TM细胞中TGF-β2信号传导的关键促进剂。在这项研究中，我们确定了氧化应激促进TGF-β2促纤维化反应在培养的原代人TM细胞的机制。将原代或转化的人TM细胞的半流体培养物在无血清培养基中调节过夜，随后在无或有TGF-β2（5 ng/mL）的情况下激发。在不存在或存在GKT 137831（一种Nox 1-Nox 4双酶抑制剂）的情况下，测定了烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶（Nox）亚型、结缔组织生长因子（CTGF）、胶原1α1和4α1亚型mRNA含量的相对变化或Nox 4、磷酸化和总-Smad 2和-Smad 3、胶原I和IV蛋白含量的相对变化，并分别通过实时qPCR或通过免疫印迹进行定量。通过免疫细胞化学半定量I型和IV型胶原以及α平滑肌肌动蛋白（αSMA）的相对原位变化，而通过鬼笔环肽染色半定量丝状肌动蛋白应力纤维形成的相对变化。在TGF-β2存在下培养的静止原代人TM细胞显示内源性Nox 4 mRNA和Nox 4蛋白表达的显著选择性增加。放线菌素D阻止TGF-β2介导的Nox 4 mRNA表达的增加。用针对Smad 3的siRNA反向转染的TM细胞阻止了TGF-β2介导的Nox 4 mRNA表达的增加。用GKT 137831预孵育TM细胞可减弱TGF-β2介导的细胞内活性氧（ROS）、COL 1A 1、COL 4A 1和CTGF mRNA表达、Smad 3蛋白磷酸化、胶原蛋白I、胶原蛋白IV和αSMA蛋白表达以及丝状肌动蛋白应力纤维形成的增加。TGF-β2通过选择性增加NADPH氧化酶4的表达促进原代人TM细胞中的氧化应激。通过诱导NADPH氧化酶4表达导致的氧化还原平衡失调似乎是TGF-β2经典信号传导引发的病理性促纤维化反应（包括ECM重塑、丝状肌动蛋白应力纤维形成和αSMA表达）中的关键早期事件。选择性抑制Nox 4的表达/活化，与神经靶向抗氧化剂组合，代表了一种新的策略，通过该策略来减缓TGF-β2在TM内引起的促纤维化反应的进展。
The multifunctional profibrotic cytokine TGF-β2 is implicated in the pathophysiology of primary open angle glaucoma (POAG). While the underlying cause of POAG remains unclear, TGF-β2 dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment is considered an early pathologic consequence associated with impaired aqueous humor (AH) outflow and elevated IOP. Mitochondrial-targeted antioxidants have been recently shown by our group to markedly attenuate TGF-β2 profibrotic responses, strongly implicating oxidative stress as a key facilitator of TGF-β2 signaling in human TM cells. In this study, we determined the mechanism by which oxidative stress facilitates TGF-β2 profibrotic responses in cultured primary human TM cells. Semiconfluent cultures of primary or transformed human TM cells were conditioned overnight in serum-free media and subsequently challenged without or with TGF-β2 (5 ng/mL). Relative changes in the mRNA content of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) isoforms, connective tissue growth factor (CTGF), collagen 1α1 and 4α1 isoforms or relative changes in the protein content of Nox4, phospho- and total-Smad2 and -Smad3, collagens I and IV were determined in the absence or presence of GKT137831, a Nox1-Nox4 dual enzyme inhibitor, and quantified by real-time qPCR or by immunoblot, respectively. Relative in situ changes in collagens I and IV and in alpha smooth muscle actin (αSMA) were semiquantified by immunocytochemistry, whereas relative changes in filamentous actin stress fiber formation was semiquantified by phalloidin staining. Quiescent primary human TM cells cultured in the presence of TGF-β2 exhibited a marked selective increase in endogenous Nox4 mRNA and Nox4 protein expression. Actinomycin D prevented TGF-β2 mediated increases in Nox4 mRNA expression. TM cells reverse transfected with siRNA against Smad3 prevented TGF-β2 mediated increases in Nox4 mRNA expression. Pre-incubating TM cells with GKT137831 attenuated TGF-β2 mediated increases in intracellular reactive oxygen species (ROS), in COL1A1, COL4A1, and CTGF mRNA expression, in Smad3 protein phosphorylation, in collagens I, collagens IV, and αSMA protein expression, and in filamentous actin stress fiber formation. TGF-β2 promotes oxidative stress in primary human TM cells by selectively increasing expression of NADPH oxidase 4. Dysregulation of redox equilibrium by induction of NADPH oxidase 4 expression appears to be a key early event involved in the pathologic profibrotic responses elicited by TGF-β2 canonical signaling, including ECM remodeling, filamentous actin stress fiber formation, and αSMA expression. Selective inhibition of Nox4 expression/activation, in combination with mitochondrial-targeted antioxidants, represents a novel strategy by which to slow the progression of TGF-β2 elicited profibrotic responses within the TM.
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影响因子：
8
作者：
Altenhofer, Sebastian;Kleikers, Pamela W. M.;Radermacher, Kim A.;Scheurer, Peter;Hermans, J. J. Rob;Schiffers, Paul;Ho, Heidi;Wingler, Kirstin;Schmidt, Harald H. H. W.
通讯作者：
Schmidt, Harald H. H. W.
DOI：
10.1159/000458482
发表时间：
2017-01-01
期刊：
GLAUCOMA SURGERY, 2ND REVISED AND EXTENDED EDITION
影响因子：
--
作者：
Kalouda, Pelagia;Keskini, Christina;Topouzis, Fotis
通讯作者：
Topouzis, Fotis
影响因子：
4.8
作者：
Jain, Manu;Rivera, Stephanie;Chandel, Navdeep S.
通讯作者：
Chandel, Navdeep S.
影响因子：
4.4
作者：
Junn, E;Lee, KN;Choi, I
通讯作者：
Choi, I
DOI：
10.2147/opth.s32933
发表时间：
2012
期刊：
Clinical ophthalmology (Auckland, N.Z.)
影响因子：
--
作者：
Beidoe G;Mousa SA
通讯作者：
Mousa SA