Kunie Eguchi

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Abstract 012: Involvement of Drp1, A Mitochondrial Fission Inducer, in Angiotensin Ii-induced Hypertensive Vascular Remodeling in vitro and in vivo

Hypertension (2016)

Tatsuo Kawai Steven J. Forrester Katherine J Elliot Kunie Eguchi Victor Rizzo

Mitochondrial dysfunction has been implicated in various types of cardiovascular diseases which may involve overload and de-compensation in mitochondrial quality/quantity control. However, limited mechanistic insight is available regarding the contribution and mechanism of mitochondrial quality control in hypertension. In the present study, we tested our hypothesis that enhancement of mitochondrial fission via Drp1 activation in vascular smooth muscle cells (VSMCs) is involved in hypertensive vascular remodeling. Rat aortic VSMCs pretreated with adenovirus encoding Drp1 siRNA (Ad-siDrp1) or control non-silencing RNA (100 moi) were stimulated with 100 nM angiotensin II (AngII) up to 72 h. 8 week old male C57/BL6 mice were infused with (1000 ng/kg/min) for 2 weeks with or without treatment of Drp1 inhibitor mdivi1 (25 mg/kg ip every other day). In VSMCs, AngII induced transient mitochondrial fission (max at 2-4 h assessed by mito-tracker staining) associated with Drp1 phosphorylation at Ser616 (10-30 min). Pretreatment of ad-siDrp1 (100 moi) or mdivi1 (5 μM) attenuated AngII-induced mitochondrial fission. Ad-siDrp1 or mdivi1 also attenuated AngII-induced enhancements of mitochondrial reactive oxygen species (ROS) generation, total cell protein, cell volume and extracellular collagen content. In mice, mdivi1 significantly suppressed vascular hypertrophy and perivascular fibrosis induced by AngII in aorta, heart and kidney. mdivi1 also inhibited AngII-induced left ventricular hypertrophy assessed by heart weight body weight ratio (mg/g: 7.8±0.9 vs 6.3±0.2 p<0.01) as well as by echocardiogram. However, mdivi1 did not affect hypertension induced by AngII assessed by telemetry (mean arterial pressure: sham 150±8 vs mdivi1 155±7 mmHg). KDEL and nitro-tyrosine staining of the heart and kidney suggest attenuation of vascular ER stress and oxidative stress, respectively. In conclusion, this data suggests that Drp1-dependent mitochondrial fission contributes to AngII-induced cardiovascular remodeling independently of hypertension via enhancement of mitochondrial ROS and ER stress in target organs.

10.1161/hyp.68.suppl_1.012

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Distinct G proteins mediate the EGFR transactivation pathway and the Rho/Rho‐kinase (ROCK) pathway activated by angiotensin II in vascular smooth muscle cells.

The FASEB Journal (2007)

Haruhiko Ohtsu Sadaharu Higuchi Kunie Eguchi Hiroyuki Suzuki Gerald D. Frank

Angiotensin II (AngII) and its G protein-coupled AT1 receptor, play critical roles in mediating cardiovascular diseases, in part through the induction of hypertrophy and hyperplasia of vascular smooth muscle cells (VSMCs). The AT1 receptor appears to have two major growth-promoting pathways, the EGFR/ERK and Rho/ROCK pathways in VSMCs. This study examines the requirement of select G protein(s) on these pathways that lead to vascular remodeling. Intracellular Ca2+ elevation induced by AngII but not by PDGF was completely inhibited by a selective Gq inhibitor, YM-254890. In addition, EGFR and ERK activation induced by AngII, but not Ca2+ ionophore, was inhibited by YM. Also, YM partially inhibited AngII-induced phosphorylation of MYPT, a substrate of ROCK. Thus, AngII activates G12/13 as selectively measured with the GST-TPR pull-down assay. Stimulation of quiescent VSMCs with AngII for 72 h resulted in an increase of cellular protein and cell volume, but not in proliferation. YM completely inhibited these hypertrophic effects. However, a G protein-independent AT1 receptor agonist did not stimulate the EGFR or the Rho/ROCK pathway in VSMCs. Furthermore, adenovirus encoding a Gq inhibitor completely inhibited the EGFR/ERK pathway and partially inhibited the Rho/ROCK pathway activated by AngII. These results suggest that Gq plays a major role in the EGFR/ERK pathway leading to vascular hypertrophy induced by AngII, whereas both Gq and G12/13 partially participates in the Rho/ROCK pathway.

Gq alpha subunit

10.1096/fasebj.21.6.a1244-d

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Central Role of G_qin the Hypertrophic Signal Transduction of Angiotensin II in Vascular Smooth Muscle Cells

Endocrinology (2008)

Haruhiko Ohtsu Sadaharu Higuchi Heigoro Shirai Kunie Eguchi Hiroyuki Suzuki

The angiotensin II (AngII) type 1 receptor (AT1) plays a critical role in hypertrophy of vascular smooth muscle cells (VSMCs). Although it is well known that Gq is the major G protein activated by the AT1 receptor, the requirement of Gq for AngII-induced VSMC hypertrophy remains unclear. By using cultured VSMCs, this study examined the requirement of Gq for the epidermal growth factor receptor (EGFR) pathway, the Rho-kinase (ROCK) pathway, and subsequent hypertrophy. AngII-induced intracellular Ca2+ elevation was completely inhibited by a pharmacological Gq inhibitor as well as by adenovirus encoding a Gq inhibitory minigene. AngII (100nm)-induced EGFR transactivation was almost completely inhibited by these inhibitors, whereas these inhibitors only partially inhibited AngII (100nm)-induced phosphorylation of a ROCK substrate, myosin phosphatase target subunit-1. Stimulation of VSMCs with AngII resulted in an increase of cellular protein and cell volume but not in cell number. The Gq inhibitors completely blocked these hypertrophic responses, whereas a G protein-independent AT1 agonist did not stimulate these hypertrophic responses. In conclusion, Gq appears to play a major role in the EGFR pathway, leading to vascular hypertrophy induced by AngII. Vascular Gq seems to be a critical target of intervention against cardiovascular diseases associated with the enhanced renin-angiotensin system.

Myosin-light-chain phosphatase

Gq alpha subunit

10.1210/en.2007-1694

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Abstract 12454: Mitochondrial Transcription Factor 2B as a Mediator of Mitochondrial Retrograde Signaling and Endothelial Cell Shape

Circulation (2015)

Steven J. Forrester Tatsuo Kawai Katherine J Elliot Kunie Eguchi Joon‐Young Park

Mitochondrial dysfunction, such as observed in endothelial cells, has been implicated in various cardiovascular diseases including, hypertension and atherosclerosis. Mitochondrial transcription factor 2B (TFB2M) is an essential component to maintain proper transcriptional and functional control of mitochondrial DNA. As well, elongation of endothelial cells is a characteristic of atheroprotective regions within the vasculature, and the relationship between the mitochondria and EC shape is currently unknown. Additionally, recent interest has been focused on mechanisms by which the mitochondria may signal to the nucleus to affect cell function. The aim of our study is to investigate the hypothesis that TFB2M has a novel role in enhancing endothelial function. Human umbilical vein endothelial cells (HUVECs) were harvested 72 hours after adenoviral transduction with TFB2M (100 moi). HUVECs transduced with TFB2M showed an elongated cell morphology when compared to GFP control. To further investigate the effect of TFB2M on regulating mitochondrial function and cell shape, immunoblotting was carried out for markers involved in mitochondrial function/dynamics and markers indicative of cytoskeleton reorganization. TFB2M transduction resulted in increased expression of mitochondrial biogenesis marker VDAC (2.6 fold increase), mitochondrial fusion protein MFN2 (2.1 fold increase), and phosphorylated myosin phosphatase targeting protein MYPT1 at Thr850 (2.2 fold increase, p < 0.05 for all proteins). Additionally, fluorescence microscopy showed enhanced mitochondrial fluorescence in TFB2M transduced cells using mitotracker red staining (3.5 fold increase, p < 0.001). These data indicate that TFB2M has a previously undiscovered function contributing to altered EC function and shape, potentially through a novel mitochondrial retrograde signaling mechanism. Further research will focus on distinguishing the exact mechanisms culminating in a protective EC phenotype and the beneficial role of endothelial TFB2M-mediated enhanced mitochondrial function in the treatment of EC dysfunction associated with various cardiovascular diseases.

Mediator

10.1161/circ.132.suppl_3.12454

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Abstract 087: Mitochondrial Fission Inducer, Dynamin-Related Protein 1 (DRP1), is Required for Endothelial Inflammatory Responses

Hypertension (2016)

Steven J. Forrester Tatsuo Kawai Katherine J. Elliott Kunie Eguchi Victor Rizzo

Among various cardiovascular diseases, hypertension (HTN) is considered to be a disease plagued by chronic low-grade inflammation associated with endothelial dysfunction. Interestingly, recent studies have identified mitochondrial adaptation and/or dysfunction as components to hypertensive vascular dysfunction. While mitochondria are indispensable to maintain cellular metabolism, they also participate in adaptive and maladaptive cell/tissue responses via several retro grade signaling pathways. DRP1 plays a major role in mitochondrial quality control. However, whether DRP1 is involved in mitochondrial dysfunction and endothelial inflammation during development of HTN remains unknown. In the present study, we tested the hypothesis that inflammatory stimuli, through DRP1-dependent mitochondrial alteration, enhance endothelial inflammation. In cultured rat aortic endothelial cells (RAECs), TNFα (10 μg/mL) transiently induced mitochondrial fission maximally at 3h which was inhibited using a mitochondrial fission inhibitor, Mdivi1 (10 μM) (0.16±0.04 vs 0.10±0.02 mitochondria fragmentation count with MitoTracker, p<.01 ). TNFα and FCCP (a fission agonist, 10 μM) increased THP-1 monocyte adhesion to RAECs, which was also inhibited with Mdivi1 (256±17 vs 139±16 for TNFα, 238±30 vs 156±14 for FCCP, attached cells per field scanned, p<.01 ). Likewise, mdivi1 and adenoviruses encoding siRNA for DRP1 or dominant-negative K38A DRP1 (50 moi) attenuated TNFα-induced VCAM-1 induction in RAECs. TNFα increased aerobic respiration, which was prevented by mdivi1 or ER stress inhibitor PBA (10 mM). Inhibition of ER stress, glycolysis or mitochondrial respiration using PBA, 2-DG (1 mg/mL) or oligomycin (1 μM) prevented VCAM-1 induction. However, suppression of TNFα-induced mitochondrial ROS production by mito-Tempo (25 nM) was unable to prevent VCAM-1 induction. In C57BL6 mice receiving AngII (1000 ng/kg/min, 2 weeks) infusion, treatment with Mdivi-1 (25 mg/kg ip every other day) or PBA (1g/kg/day) prevented vascular VCAM-1 induction. In conclusion, our data suggests a critical role for ER stress and subsequent functional and structural remodeling of mitochondria induced by DRP1 in mediating endothelial inflammatory activation in HTN.

DNM1L

MFN2

Endothelial Dysfunction

MFN1

Oligomycin

Cellular respiration

10.1161/hyp.68.suppl_1.087

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Transduction Efficiency of Adenovirus Vectors in Endothelial Cells and Vascular Smooth Muscle Cells

Journal of Cardiovascular Pharmacology (2020)

Nikita Mokhashi Robert Y. Choi Stephanie Cicalese Kunie Eguchi Michael Boyer

Adenoviral vectors are useful tools in manipulating a gene of interest in vitro and in vivo, including in the vascular system. The transduction efficiencies of adenoviral vectors in vascular cells such as endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are known to be lower than those in epithelial cell types. The effective entry for adenoviral vectors is primarily mediated through the coxsackievirus and adenovirus receptor (CAR), which has been shown to be expressed in both cell types. Cationic liposomes have been used to enhance adenovirus transduction efficiency in nonepithelial cells. Accordingly, the aim of this study is to obtain new information regarding differences in transduction efficiencies, cationic liposome sensitivity, and CAR expression between ECs and VSMCs. Using cultured rat aortic ECs and VSMCs, here, we have compared transduction efficiency of adenoviruses with or without inclusion of liposomes and CAR expression. A significant increase in basal transduction efficiency was observed in ECs compared with VSMCs. Cationic liposome polybrene enhanced transduction efficiency in VSMCs, whereas decreased efficiency was observed in ECs. Western blotting demonstrated expression of the CAR in ECs but not in VSMCs. Proteomic analysis and mouse aorta immunostaining further suggests significant expression of the CAR in ECs but not in VSMCs. In conclusion, adenoviruses can effectively transduce the gene of interest in aortic ECs likely because of abundant expression of the CAR, whereas cationic liposomes such as polybrene enhance the transduction efficiency in VSMCs lacking CAR expression.

Transduction (biophysics)

10.1097/fjc.0000000000000821

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Insulin-Induced Akt Activation Is Inhibited by Angiotensin II in the Vasculature Through Protein Kinase C-α

Hypertension (2003)

Evangeline D. Motley Kunie Eguchi Carla D. Gardner Adrienne L. Hicks Cherilynn M. Reynolds

Insulin resistance is an important risk factor in the development of cardiovascular diseases such as hypertension and atherosclerosis. However, the specific role of insulin resistance in the etiology of these diseases is poorly understood. Angiotensin (Ang) II is a potent vasculotrophic and vasoconstricting factor. We hypothesize that in vascular smooth muscle cells (VSMCs), Ang II interferes with insulin action by inhibiting Akt, a major signaling molecule implicated in the biological actions of insulin. By immunoblotting with a phospho-specific antibody for Akt, we found that Ang II inhibits insulin-induced Akt phosphorylation in a time- and concentration-dependent manner. The inhibitory effect of Ang II was blocked by a Ang II type 1 receptor antagonist, RNH6270. A protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, also inhibited insulin-induced Akt phosphorylation. PKC inhibitors, including Go6976 (specific for alpha- and beta-isoforms), blocked the Ang II- and PMA-induced inhibition of Akt phosphorylation by insulin. Moreover, overexpression of PKC-alpha but not PKC-beta isoform by adenovirus inhibited insulin-induced Akt phosphorylation. By contrast, an epidermal growth factor receptor inhibitor (AG1478), a p42/44 mitogen-activated protein kinase (MAPK) kinase inhibitor (PD 598,059), and a p38 MAPK inhibitor (SB 203,580) did not block the Ang II-induced inhibition of Akt phosphorylation. From these data, we conclude that Ang II negatively regulates the insulin signal, Akt, in the vasculature specifically through PKC-alpha activation, providing an alternative molecular mechanism that may explain the association of hyperinsulinemia with cardiovascular diseases.

10.1161/01.hyp.0000051891.90321.12

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Distinct Roles of Protease-Activated Receptors in Signal Transduction Regulation of Endothelial Nitric Oxide Synthase

Hypertension (2008)

Hiroyuki Suzuki Evangeline D. Motley Kunie Eguchi Akinari Hinoki Heigoro Shirai

Protease-activated receptors (PARs), such as PAR1 and PAR2, have been implicated in the regulation of endothelial NO production. We hypothesized that PAR1 and PAR2 distinctly regulate the activity of endothelial NO synthase through the selective phosphorylation of a positive regulatory site, Ser(1179), and a negative regulatory site, Thr(497), in bovine aortic endothelial cells. A selective PAR1 ligand, TFLLR, stimulated the phosphorylation of endothelial NO synthase at Thr(497). It had a minimal effect on Ser(1179) phosphorylation. In contrast, a selective PAR2 ligand, SLIGRL, stimulated the phosphorylation of Ser(1179) with no noticeable effect on Thr(497). Thrombin has been shown to transactivate PAR2 through PAR1. Thus, thrombin, as well as a peptide mimicking the PAR1 tethered ligand, TRAP, stimulated phosphorylation of both sites. Also, thrombin and SLIGRL, but not TFLLR, stimulated cGMP production. A G(q) inhibitor blocked thrombin- and SLIGRL-induced Ser(1179) phosphorylation, whereas it enhanced thrombin-induced Thr(497) phosphorylation. In contrast, a G(12/13) inhibitor blocked thrombin- and TFLLR-induced Thr(497) phosphorylation, whereas it enhanced the Ser(1179) phosphorylation. Although a Rho-kinase inhibitor, Y27632, blocked the Thr(497) phosphorylation, other inhibitors that targeted Rho-kinase failed to block TFLLR-induced Thr(497) phosphorylation. These data suggest that PAR1 and PAR2 distinctly regulate endothelial NO synthase phosphorylation and activity through G(12/13) and G(q), respectively, delineating the novel signaling pathways by which the proteases act on protease-activated receptors to potentially modulate endothelial functions.

Protease-activated receptor

10.1161/hypertensionaha.108.125229

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Caveolin-1 negatively regulates a metalloprotease-dependent epidermal growth factor receptor transactivation by angiotensin II

Journal of Molecular and Cellular Cardiology (2010)

Akira Takaguri Heigoro Shirai Keita Kimura Akinari Hinoki Kunie Eguchi

Lipid raft

Caveolin 1

10.1016/j.yjmcc.2010.12.009

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Citations (44)

p21-Activated Kinase 1 Participates in Vascular Remodeling In Vitro and In Vivo

Hypertension (2009)

Akinari Hinoki Keita Kimura Sadaharu Higuchi Kunie Eguchi Akira Takaguri

Vascular smooth muscle cell hypertrophy, proliferation, or migration occurs in hypertension, atherosclerosis, and restenosis after angioplasty, leading to pathophysiological vascular remodeling. Angiotensin II and platelet-derived growth factor are well-known participants of vascular remodeling and activate a myriad of downstream protein kinases, including p21-activated protein kinase (PAK1). PAK1, an effector kinase of small GTPases, phosphorylates several substrates to regulate cytoskeletal reorganization. However, the exact role of PAK1 activation in vascular remodeling remains to be elucidated. Here, we have hypothesized that PAK1 is a critical target of intervention for the prevention of vascular remodeling. Adenoviral expression of dominant-negative PAK1 inhibited angiotensin II-stimulated vascular smooth muscle cell migration. It also inhibited vascular smooth muscle cell proliferation induced by platelet-derived growth factor. PAK1 was activated in neointima of the carotid artery after balloon injury in the rat. Moreover, marked inhibition of the neointima hyperplasia was observed in a dominant-negative PAK1 adenovirus-treated carotid artery after the balloon injury. Taken together, these results suggest that PAK1 is involved in both angiotensin II and platelet-derived growth factor-mediated vascular smooth muscle cell remodeling, and inactivation of PAK1 in vivo could be effective in preventing pathophysiological vascular remodeling.

Neointima

PAK1

10.1161/hypertensionaha.109.143057

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Citations (26)