18 results on '"Michelle Surma"'
Search Results
2. Dissecting the Mechanisms of Doxorubicin and Oxidative Stress-Induced Cytotoxicity: The Involvement of Actin Cytoskeleton and ROCK1.
- Author
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Lei Wei, Michelle Surma, Gina Gough, Stephanie Shi, Nathan Lambert-Cheatham, Jiang Chang, and Jianjian Shi
- Subjects
Medicine ,Science - Abstract
We have recently reported that ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has superior anti-apoptotic and pro-survival effects than antioxidants against doxorubicin, a chemotherapeutic drug. Although oxidative stress is the most widely accepted mechanism, our studies suggest that ROCK1-dependent actin cytoskeleton remodeling plays a more important role in mediating doxorubicin cytotoxicity on MEFs. To further explore the contributions of ROCK1-dependent actin cytoskeleton remodeling in response to stress, this study investigates the mechanistic differences between the cytotoxic effects of doxorubicin versus hydrogen peroxide (H2O2), with a focus on cytoskeleton alterations, apoptosis and necrosis induction. We found that both types of stress induce caspase activation but with different temporal patterns and magnitudes in MEFs: H2O2 induces the maximal levels (2 to 4-fold) of activation of caspases 3, 8, and 9 within 4 h, while doxorubicin induces much higher maximal levels (15 to 25-fold) of caspases activation at later time points (16-24 h). In addition, necrosis induced by H2O2 reaches maximal levels within 4 h while doxorubicin-induced necrosis largely occurs at 16-24 h secondary to apoptosis. Moreover, both types of stress induce actin cytoskeleton remodeling but with different characteristics: H2O2 induces disruption of stress fibers associated with cytosolic translocation of phosphorylated myosin light chain (p-MLC) from stress fibers, while doxorubicin induces cortical F-actin formation associated with cortical translocation of p-MLC from central stress fibers. Furthermore, N-acetylcysteine (an antioxidant) is a potent suppressor for H2O2-induced cytotoxic effects including caspase activation, necrosis, and cell detachment, but shows a much reduced inhibition on doxorubicin-induced changes. On the other hand, ROCK1 deficiency is a more potent suppressor for the cytotoxic effects induced by doxorubicin than by H2O2. These results support the notion that doxorubicin induces caspase activation, necrosis, and actin cytoskeleton alterations largely through ROCK1-dependent and oxidative stress-independent pathways.
- Published
- 2015
- Full Text
- View/download PDF
3. ROCK1 deficiency enhances protective effects of antioxidants against apoptosis and cell detachment.
- Author
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Michelle Surma, Caitlin Handy, Jiang Chang, Reuben Kapur, Lei Wei, and Jianjian Shi
- Subjects
Medicine ,Science - Abstract
We have recently reported that the homologous Rho kinases, ROCK1 and ROCK2, play different roles in regulating stress-induced stress fiber disassembly and cell detachment, and the ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has remarkable anti-apoptotic, anti-detachment and pro-survival effects against doxorubicin, a chemotherapeutic drug. This study investigated the roles of ROCK isoforms in doxorubicin-induced reactive oxygen species (ROS) generation which is believed to be the major mechanism underlying its cytotoxicity to normal cells, and especially to cardiomyocytes. Different antioxidants have been shown to provide a protective role reported in numerous experimental studies, but clinical trials of antioxidant therapy showed insufficient benefit against the cardiac side effect. We found that both ROCK1-/- and ROCK2-/- MEFs exhibited reduced ROS production in response to doxorubicin treatment. Interestingly, only ROCK1 deficiency, but not ROCK2 deficiency, significantly enhanced the protective effects of antioxidants against doxorubicin-induced cytotoxicity. First, ROCK1 deficiency and N-acetylcysteine (an anti-oxidant) treatment synergistically reduced ROS levels, caspase activation and cell detachment. In addition, the reduction of ROS generation in ROCK1-/- MEFs in response to doxorubicin treatment was in part through inhibiting NADPH oxidase activity. Furthermore, ROCK1 deficiency enhanced the inhibitory effects of diphenyleneiodonium (an inhibitor of NADPH oxidase) on ROS generation and caspase 3 activation induced by doxorubicin. Finally, ROCK1 deficiency had greater protective effects than antioxidant treatment, especially on reducing actin cytoskeleton remodeling. ROCK1 deficiency not only reduced actomyosin contraction but also preserved central stress fiber stability, whereas antioxidant treatment only reduced actomyosin contraction without preserving central stress fibers. These results reveal a novel strategy to enhance the protective effect of antioxidant therapy by targeting the ROCK1 pathway to stabilize the actin cytoskeleton and boost the inhibitory effects on ROS production, apoptosis and cell detachment.
- Published
- 2014
- Full Text
- View/download PDF
4. Glaucomatous Optineurin (E50K) Mutation Disrupts Mitochondrial Homeostasis in Human Stem Cell Derived RGCs
- Author
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Leonardo Olivera Perez, Michelle Surma, Sayanta Dutta, and Arupratan Das
- Subjects
Ocean Engineering - Abstract
Background:Retinal ganglion cells (RGCs) are highly energy dependent due to their continuous action potential firing requirements and long unmyelinated axons hence highly susceptible to mitochondrial dysfunctions, observed in glaucoma. Dr. Das’s lab recently had identified Tank-binding kinase 1 (TBK1) inhibition by BX795 drug activates mitochondrial biogenesis and promotes RGC protection with glaucomatous Optineurin (OPTN-E50K) mutation. OPTN is a critical player for mitophagy. It is still not clear if activation of mito-biogenesis improved mitochondrial homeostasis which I investigated in this project.MethodsTo investigate mitochondrial homeostasis in human RGCs, I have used a robust well-characterized human stem cell differentiated RGC (hRGC) model with wild-type (WT) and E50K mutation background which Dr. Das’s lab routinely uses. To investigate mitochondrial homeostasis, I used JC1 live cell mitochondrial dye which fluoresces red when bound to healthy mitochondria and green when bound to damaged mitochondria. I used this assay on hRGCs treated with BX795 and mitochondrial stressor CCCP and measured red to green mitochondria ratio on confocal z-stacks using ImageJ.ResultsUnder basal level, we found hRGCsWT had a significantly increased healthy (red:green) mitochondria compared to hRGCsE50K. This suggests E50K mutation disrupts mitochondrial homeostasis. To gain mitochondrial homeostasis, it is possible that hRGCsE50K will produce more mitochondria over time than the WT. Indeed, we observed significant increase in healthy mitochondria for hRGCsE50K at 3h and 24h of DMSO and BX795 treatment, but not for hRGCsWT. We also observed under CCCP damage for 3h, hRGCsE50K had significantly higher amount of damaged mitochondria (p=0.051) while hRGCsWT maintained homeostasis.Conclusion and Potential ImpactMy study suggests glaucomatous OPTN-E50K mutation disrupts mitochondrial homeostasis and activation of mito-biogenesis by BX enriches healthy mitochondria leading to hRGCE50K protection. This study has high impact as further avenues for promoting mito-biogenesis could lead to glaucoma neuroprotection therapy.
- Published
- 2023
5. Enhanced mitochondrial biogenesis promotes neuroprotection in human stem cell derived retinal ganglion cells of the central nervous system
- Author
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Michelle Surma, Kavitha Anbarasu, Sayanta Dutta, Leonardo J. Olivera Perez, Kang-Chieh Huang, Jason S. Meyer, and Arupratan Das
- Abstract
Mitochondrial dysfunctions are widely afflicted in central nervous system (CNS) disorders with minimal understanding on how to improve mitochondrial homeostasis to promote neuroprotection. Here we used human stem cell differentiated retinal ganglion cells (hRGCs) of the CNS, which are highly sensitive towards mitochondrial dysfunctions due to their unique structure and function, to identify mechanisms for improving mitochondrial quality control (MQC). We found that hRGCs are efficient in maintaining mitochondrial homeostasis through rapid degradation and biogenesis of mitochondria under acute damage. Using a glaucomatous Optineurin mutant (E50K) stem cell lines, we saw that at basal level mutant hRGCs possess less mitochondrial mass and suffer mitochondrial swelling due to excess ATP production load. Activation of mitochondrial biogenesis through pharmacological inhibition of the Tank binding kinase 1 (TBK1) restored energy homeostasis, mitigated mitochondrial swelling with neuroprotection against acute mitochondrial damage for glaucomatousE50KhRGCs, revealing a novel neuroprotection mechanism.
- Published
- 2022
6. Disruption of both ROCK1 and ROCK2 genes in cardiomyocytes promotes autophagy and reduces cardiac fibrosis during aging
- Author
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Yang Yang, Lei Wei, Michelle Surma, and Jianjian Shi
- Subjects
Male ,0301 basic medicine ,Aging ,Myosin light-chain kinase ,Cardiac fibrosis ,Mice, Transgenic ,Biochemistry ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Autophagy ,Genetics ,medicine ,Animals ,Myocytes, Cardiac ,ROCK1 ,Protein kinase A ,Molecular Biology ,Protein kinase B ,Rho-associated protein kinase ,Crosses, Genetic ,PI3K/AKT/mTOR pathway ,Mice, Knockout ,rho-Associated Kinases ,Chemistry ,TOR Serine-Threonine Kinases ,Research ,medicine.disease ,Fibrosis ,Recombinant Proteins ,Cell biology ,Tamoxifen ,030104 developmental biology ,Gene Expression Regulation ,Enzyme Induction ,Female ,030217 neurology & neurosurgery ,Biotechnology - Abstract
In this study, we investigated the pathophysiological impact of Rho-associated coiled-coil–containing protein kinase (ROCK)1 and ROCK2 double deletion vs. single deletion on cardiac remodeling. Utilizing a cardiomyocyte-specific and tamoxifen-inducible MerCreMer recombinase (MCM), 3 mouse lines (MCM/ROCK1(fl/fl)/ROCK2(fl/fl), MCM/ROCK1(fl/fl), and MCM/ROCK2(fl/fl)) were generated. As early as 5 d after inducible deletion, the double ROCK knockout hearts exhibited reduced phosphorylation of myosin light chain (MLC) and focal adhesion kinase (FAK), supporting a role for ROCK activity in regulating the nonsarcomeric cytoskeleton. Moreover, the autophagy marker microtubule-associated proteins 1A-1B light chain 3B was increased in the double ROCK knockout, and these early molecular features persisted throughout aging. Mechanistically, the double ROCK knockout promoted age-associated or starvation-induced autophagy concomitant with reduced protein kinase B (AKT), mammalian target of rapamycin (mTOR), Unc-51–like kinase signaling, and cardiac fibrosis. In contrast, ROCK2 knockout hearts showed increased phosphorylated (p)-MLC and p-FAK levels, which were mostly attributable to a compensatory ROCK1 overactivation. Autophagy was inhibited at the baseline accompanying increased mTOR activity, leading to increased cardiac fibrosis in the ROCK2 knockout hearts. Finally, the loss of ROCK1 had no significant effect on p-MLC and p-FAK levels, mTOR signaling, or autophagy at baseline. In summary, deletions of ROCK isoforms in cardiomyocytes have different, even opposite, effects on endogenous ROCK activity and the MLC/FAK/AKT/mTOR signaling pathway, which is involved in autophagy and fibrosis of the heart.—Shi, J., Surma, M., Yang, Y., Wei, L. Disruption of both ROCK1 and ROCK2 genes in cardiomyocytes promotes autophagy and reduces cardiac fibrosis during aging.
- Published
- 2019
7. Selection-Free Non-Viral Method Revealed Highly Efficient Crispr-Cas9 Genome Editing of Human Pluripotent Stem Cells Guided by Cellular Autophagy
- Author
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Arupratan Das, Michelle Surma, and Kavitha Anbarasu
- Subjects
Genome editing ,Lipofectamine ,Cas9 ,CRISPR ,Transfection ,Guide RNA ,Gene delivery ,Biology ,Induced pluripotent stem cell ,Cell biology - Abstract
SUMMARYCRISPR-Cas9 mediated genome editing of human pluripotent stem cells (hPSCs) provides strong avenues for human disease modeling, drug discovery and cell replacement therapy. Genome editing of hPSCs is an extremely inefficient process and requires complex gene delivery and selection methods to improve edit efficiency which are not ideal for clinical applications. Here, we have shown a selection free simple lipofectamine based transfection method where a single plasmid encoding guide RNA (gRNA) and Cas9 selectively transfected hPSCs at the colony edges. Upon dissection and sequencing, the edge cells showed more than 30% edit frequency compared to the reported 3% rate under no selections. Increased cellular health of the edge cells as revealed by reduced autophagy gene-expressions is critical for such transfection pattern. Edge specific transfection was inhibited by blocking lysosomal activity which is essential for autophagy. Hence, our method provides robust scarless genome-editing of hPSCs which is ideal for translational research.
- Published
- 2021
8. Disruption of ROCK1 gene restores autophagic flux and mitigates doxorubicin-induced cardiotoxicity
- Author
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Michelle Surma, Jianjian Shi, and Lei Wei
- Subjects
0301 basic medicine ,Cardiotoxicity ,autophagy ,Chemistry ,Autophagy ,cardiotoxicity ,apoptosis ,Pharmacology ,medicine.disease ,doxorubicin ,3. Good health ,03 medical and health sciences ,030104 developmental biology ,Oncology ,Apoptosis ,Fibrosis ,Knockout mouse ,medicine ,ROCK1 ,Doxorubicin ,Rho kinase ,Rho-associated protein kinase ,medicine.drug ,Research Paper - Abstract
Doxorubicin is among the essential medicines with a wide antitumor spectrum, but its clinical application is limited by its cardiotoxicity. We recently discovered that ROCK1 is a key molecule in mediating cardiac remodeling in response to various stresses. To determine the roles of ROCK1 in doxorubicin cardiotoxicity, we gave three doses of doxorubicin injections to wild type (WT) and ROCK1-/- mice with one week intervals between treatments, the cumulative dose being 24 mg/kg. ROCK1-/- mice exhibited preserved cardiac function, reduced apoptosis, autophagy and fibrosis compared to the WT mice. To further determine the cellular mechanisms, we have examined the role of ROCK1 in cardiomyocytes using cardiomyocyte-specific knockout mice, MHC-Cre/ROCK1fl/fl, which partially reproduced the cardioprotective characteristics of ROCK1-/- mice, indicating that ROCK1 in both cardiomyocytes and non-cardiomyocytes mediates doxorubicin cardiotoxicity. To elucidate the molecular mechanisms, a detailed time course study after a single doxorubicin injection at 10 mg/kg was performed in ROCK1-/- and MHC-Cre/ROCK1fl/fl mice. The molecular analysis revealed that both ROCK1-/- and MHC-Cre/ROCK1fl/fl hearts exhibited significant reduction of doxorubicin-induced early responses including increased apoptotic (Bax) and autophagic (p62/SQSTM1 and LC3-II) markers, associated with reduced Beclin 1 phosphorylation on Thr119, supporting reduced Beclin 1-mediated autophagy initiation due to increased association of Beclin 1 with Bcl 2 or Bcl-XL in these hearts compared to the WT or ROCK1fl/fl mice. These results support that ROCK1 deficiency is cardioprotective against doxorubicin-induced cardiotoxicity at least in part through reducing Beclin 1-mediated autophagy initiation in cardiomyocytes and restoring autophagic flux to ameliorate doxorubicin cardiotoxicity.
- Published
- 2018
9. ROCK2 inhibition enhances the thermogenic program in white and brown fat tissue in mice
- Author
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Michelle Surma, Yang Yang, Sarah A. Tersey, Jianjian Shi, and Lei Wei
- Subjects
0301 basic medicine ,medicine.medical_specialty ,RHOA ,Adipose Tissue, White ,Adipose tissue ,White adipose tissue ,Diet, High-Fat ,Biochemistry ,Article ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Insulin resistance ,Adipose Tissue, Brown ,In vivo ,Internal medicine ,Genetics ,medicine ,Animals ,ROCK1 ,Obesity ,Molecular Biology ,Mice, Knockout ,rho-Associated Kinases ,Adipogenesis ,biology ,Chemistry ,Cell Differentiation ,Thermogenesis ,medicine.disease ,Actin cytoskeleton ,030104 developmental biology ,Endocrinology ,biology.protein ,Insulin Resistance ,Energy Metabolism ,030217 neurology & neurosurgery ,Biotechnology ,Signal Transduction - Abstract
The RhoA/ROCK-mediated actin cytoskeleton dynamics have been implicated in adipogenesis. The two ROCK isoforms, ROCK1 and ROCK2, are highly homologous. The contribution of ROCK2 to adipogenesis in vivo has not been elucidated. The present study aimed at the in vivo and in vitro roles of ROCK2 in the regulation of adipogenesis and the development of obesity. We performed molecular, histological and metabolic analyses in ROCK2(+/−) and ROCK2(+/KD) mouse models, the latter harboring an allele with a kinase-dead (KD) mutation. Both ROCK2(+/−) and ROCK2(+/KD) mouse models showed a lean body mass phenotype during aging, associated with increased amounts of beige cells in subcutaneous white adipose tissue (sWAT) and increased thermogenic gene expression in all fat depots. ROCK2(+/−) mice on a high-fat diet showed increased energy expenditure accompanying by reduced obesity, and improved insulin sensitivity. In vitro differentiated ROCK2(+/−) stromal-vascular (SV) cells revealed increased beige adipogenesis associated with increased thermogenic gene expressions. Treatment with a selective ROCK2 inhibitor, KD025, to inhibit ROCK2 activity in differentiated SV cells reproduced the pro-beige phenotype of ROCK2(+/−) SV cells. In conclusion, ROCK2 activity-mediated actin cytoskeleton dynamics contribute to the inhibition of beige adipogenesis in WAT, and also promotes age-related and diet-induced fat mass gain and insulin resistance.
- Published
- 2019
10. Dissecting the roles of ROCK isoforms in stress-induced cell detachment
- Author
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Lumin Zhang, Jianjian Shi, Michelle Surma, and Lei Wei
- Subjects
Cofilin 1 ,actin cytoskeleton ,Stress fiber ,Cell Survival ,Biology ,serum starvation ,doxorubicin ,Cell Line ,Stress fiber assembly ,Focal adhesion ,Adherens junction ,stress fibers ,Mice ,Cell Adhesion ,Animals ,Rho kinase ,ROCK2 ,Phosphorylation ,Cell adhesion ,Egtazic Acid ,Molecular Biology ,Rho-associated protein kinase ,Mice, Knockout ,Extra Views ,Focal Adhesions ,rho-Associated Kinases ,Antibiotics, Antineoplastic ,Membrane Proteins ,isoform ,Adherens Junctions ,Cell Biology ,Actin cytoskeleton ,Cell biology ,detachment ,cell-matrix and cell-cell adhesion ,Developmental Biology - Abstract
The homologous Rho kinases, ROCK1 and ROCK2, are involved in stress fiber assembly and cell adhesion and are assumed to be functionally redundant. Using mouse embryonic fibroblasts (MEFs) derived from ROCK1(-/-) and ROCK2(-/-) mice, we have recently reported that they play different roles in regulating doxorubicin-induced stress fiber disassembly and cell detachment: ROCK1 is involved in destabilizing the actin cytoskeleton and cell detachment, whereas ROCK2 is required for stabilizing the actin cytoskeleton and cell adhesion. Here, we present additional insights into the roles of ROCK1 and ROCK2 in regulating stress-induced impairment of cell-matrix and cell-cell adhesion. In response to doxorubicin, ROCK1(-/-) MEFs showed significant preservation of both focal adhesions and adherens junctions, while ROCK2(-/-) MEFs exhibited impaired focal adhesions but preserved adherens junctions compared with the wild-type MEFs. Additionally, inhibition of focal adhesion or adherens junction formations by chemical inhibitors abolished the anti-detachment effects of ROCK1 deletion. Finally, ROCK1(-/-) MEFs, but not ROCK2(-/-) MEFs, also exhibited preserved central stress fibers and reduced cell detachment in response to serum starvation. These results add new insights into a novel mechanism underlying the anti-detachment effects of ROCK1 deletion mediated by reduced peripheral actomyosin contraction and increased actin stabilization to promote cell-cell and cell-matrix adhesion. Our studies further support the differential roles of ROCK isoforms in regulating stress-induced loss of central stress fibers and focal adhesions as well as cell detachment.
- Published
- 2013
11. Downregulation of doxorubicin-induced myocardial apoptosis accompanies postnatal heart maturation
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Yi Wei Zhang, Lei Wei, R. Mark Payne, Michelle Surma, Lumin Zhang, and Jianjian Shi
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Genetically modified mouse ,medicine.medical_specialty ,Anthracycline ,Physiology ,Down-Regulation ,Apoptosis ,Mice, Transgenic ,Biology ,Mitochondria, Heart ,Mice ,Downregulation and upregulation ,Physiology (medical) ,Internal medicine ,In Situ Nick-End Labeling ,medicine ,Animals ,Myocytes, Cardiac ,Doxorubicin ,Cardiotoxicity ,Antibiotics, Antineoplastic ,Myocardium ,Heart ,Immunohistochemistry ,Enzyme Activation ,Endocrinology ,Animals, Newborn ,Lac Operon ,Terminal deoxynucleotidyl transferase ,Caspases ,Cancer research ,Signaling and Stress Response ,Signal transduction ,Apoptosis Regulatory Proteins ,Cardiology and Cardiovascular Medicine ,Signal Transduction ,Subcellular Fractions ,medicine.drug - Abstract
Doxorubicin is a highly effective chemotherapeutic agent used for treating a wide spectrum of tumors, but its usage is limited because of its dose-dependent cardiotoxicity, especially in pediatric patients. Accumulating evidence indicates that caspase-dependent apoptosis contributes to the cardiotoxicity of doxorubicin. However, less attention has been paid to the effects of age on doxorubicin-induced apoptosis signaling in myocardium. This study focused on investigating differential apoptotic sensitivity between neonatal and adult myocardium, in particular, between neonatal and adult cardiomyocytes in vivo. Our results show that caspase-3 activity in normal mouse hearts decreased by ≥20-fold within the first 3 wk after birth, associated with a rapid downregulation in the expression of key proapoptotic proteins in intrinsic and extrinsic pathways. This rapid downregulation of caspase-3 activity was confirmed by immunostaining for cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP-mediated nick-end label staining. Doxorubicin treatment induced a dose-dependent increase in caspase-3 activity and apoptosis in neonatal mouse hearts, and both caspase-8 and caspase-9 activations were involved. Using transgenic mice with a nuclear localized LacZ reporter gene to label cardiomyocytes in vivo, we observed a fourfold higher level of doxorubicin-induced cardiomyocyte apoptosis in 1-wk-old mice compared with that in 3-wk-old mice. This study points to a major difference in apoptotic signaling in doxorubicin cardiotoxicity between neonatal and adult mouse hearts and reveals a critical transition from high to low susceptibility to doxorubicin-induced apoptosis during postnatal heart maturation.
- Published
- 2012
12. Rho kinase as a therapeutic target in cardiovascular disease
- Author
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Michelle Surma, Lei Wei, and Jianjian Shi
- Subjects
Male ,RHOA ,Pyridines ,Apoptosis ,Coronary Artery Disease ,Biology ,Gene Expression Regulation, Enzymologic ,Article ,MAP2K7 ,1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ,Humans ,Protein Isoforms ,Myocytes, Cardiac ,ROCK1 ,ASK1 ,ROCK2 ,Enzyme Inhibitors ,Protein Kinase Inhibitors ,Rho-associated protein kinase ,Heart Failure ,rho-Associated Kinases ,MAP kinase kinase kinase ,Amides ,Cell biology ,Vasodilation ,Cardiovascular Diseases ,biology.protein ,Molecular Medicine ,Female ,Cyclin-dependent kinase 9 ,Cardiology and Cardiovascular Medicine - Abstract
Rho kinase (ROCK) belongs to the AGC (PKA/PKG/PKC) family of serine/threonine kinases and is a major downstream effector of the small GTPase RhoA. ROCK plays central roles in the organization of the actin cytoskeleton and is involved in a wide range of fundamental cellular functions such as contraction, adhesion, migration, proliferation and gene expression. Two ROCK isoforms, ROCK1 a n d ROCK2, are assumed to be functionally redundant, based largely on the major common activators, the high degree of homology within the kinase domain and studies from overexpression with kinase constructs a n d chemical inhibitors (e.g., Y27632 a n d fasudil), which inhibit both ROCK1 and ROCK2. Extensive experimental a n d clinical studies support a critical role for the RhoA/ROCK pathway in the vascular bed in the pathogenesis of cardiovascular diseases, in which increased ROCK activity mediates vascular smooth muscle cell hypercontraction, endothelial dysfunction, inflammatory cell recruitment and vascular remodeling. Recent experimental studies, using ROCK inhibitors or genetic mouse models, indicate that the RhoA/ROCK pathway in myocardium contributes to cardiac remodeling induced by ischemic injury or persistent hypertrophic stress, thereby leading to cardiac decompensation and heart failure. This article, based on recent molecular, cellular and animal studies, focuses on the current understanding of ROCK signaling in cardiovascular diseases and in the pathogenesis of heart failure.
- Published
- 2011
13. Dissecting the Mechanisms of Doxorubicin and Oxidative Stress-Induced Cytotoxicity: The Involvement of Actin Cytoskeleton and ROCK1
- Author
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Michelle Surma, Lei Wei, Jiang Chang, Jianjian Shi, Gina Gough, Nathan Lambert-Cheatham, and Stephanie Shi
- Subjects
Cell Survival ,lcsh:Medicine ,Caspase 3 ,Apoptosis ,Caspase 8 ,Antioxidants ,03 medical and health sciences ,Mice ,Necrosis ,0302 clinical medicine ,Stress Fibers ,Animals ,ROCK1 ,Phosphorylation ,Cytoskeleton ,lcsh:Science ,Caspase ,Actin ,030304 developmental biology ,0303 health sciences ,rho-Associated Kinases ,Multidisciplinary ,Antibiotics, Antineoplastic ,biology ,lcsh:R ,Hydrogen Peroxide ,Fibroblasts ,Actin cytoskeleton ,Actins ,Caspase 9 ,Cell biology ,Enzyme Activation ,Actin Cytoskeleton ,Oxidative Stress ,Microscopy, Fluorescence ,Doxorubicin ,030220 oncology & carcinogenesis ,biology.protein ,lcsh:Q ,Research Article - Abstract
We have recently reported that ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has superior anti-apoptotic and pro-survival effects than antioxidants against doxorubicin, a chemotherapeutic drug. Although oxidative stress is the most widely accepted mechanism, our studies suggest that ROCK1-dependent actin cytoskeleton remodeling plays a more important role in mediating doxorubicin cytotoxicity on MEFs. To further explore the contributions of ROCK1-dependent actin cytoskeleton remodeling in response to stress, this study investigates the mechanistic differences between the cytotoxic effects of doxorubicin versus hydrogen peroxide (H2O2), with a focus on cytoskeleton alterations, apoptosis and necrosis induction. We found that both types of stress induce caspase activation but with different temporal patterns and magnitudes in MEFs: H2O2 induces the maximal levels (2 to 4-fold) of activation of caspases 3, 8, and 9 within 4 h, while doxorubicin induces much higher maximal levels (15 to 25-fold) of caspases activation at later time points (16–24 h). In addition, necrosis induced by H2O2 reaches maximal levels within 4 h while doxorubicin-induced necrosis largely occurs at 16–24 h secondary to apoptosis. Moreover, both types of stress induce actin cytoskeleton remodeling but with different characteristics: H2O2 induces disruption of stress fibers associated with cytosolic translocation of phosphorylated myosin light chain (p-MLC) from stress fibers, while doxorubicin induces cortical F-actin formation associated with cortical translocation of p-MLC from central stress fibers. Furthermore, N-acetylcysteine (an antioxidant) is a potent suppressor for H2O2-induced cytotoxic effects including caspase activation, necrosis, and cell detachment, but shows a much reduced inhibition on doxorubicin-induced changes. On the other hand, ROCK1 deficiency is a more potent suppressor for the cytotoxic effects induced by doxorubicin than by H2O2. These results support the notion that doxorubicin induces caspase activation, necrosis, and actin cytoskeleton alterations largely through ROCK1-dependent and oxidative stress-independent pathways.
- Published
- 2015
14. Disruption of both ROCK1 and ROCK2 genes in cardiomyocytes promotes autophagy and reduces cardiac fibrosis during aging.
- Author
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Jianjian Shi, Michelle Surma, Yang Yang, and Lei Wei
- Abstract
In this study, we investigated the pathophysiological impact of Rho-associated coiled-coil-containing protein kinase (ROCK)1 and ROCK2 double deletion vs. single deletion on cardiac remodeling. Utilizing a cardiomyocyte-specific and tamoxifen-inducible MerCreMer recombinase (MCM), 3 mouse lines (MCM/ROCK1fl/fl/ROCK2fl/fl, MCM/ROCK1fl/fl, and MCM/ROCK2fl/fl) were generated. As early as 5 d after inducible deletion, the double ROCK knockout hearts exhibited reduced phosphorylation of myosin light chain (MLC) and focal adhesion kinase (FAK), supporting a role for ROCK activity in regulating the nonsarcomeric cytoskeleton. Moreover, the autophagy marker microtubule-associated proteins 1A-1B light chain 3B was increased in the double ROCK knockout, and these early molecular features persisted throughout aging. Mechanistically, the double ROCK knockout promoted age-associated or starvation-induced autophagy concomitant with reduced protein kinase B (AKT), mammalian target of rapamycin (mTOR), Unc-51-like kinase signaling, and cardiac fibrosis. In contrast, ROCK2 knockout hearts showed increased phosphorylated (p)-MLC and p-FAK levels, which were mostly attributable to a compensatory ROCK1 overactivation. Autophagy was inhibited at the baseline accompanying increased mTOR activity, leading to increased cardiac fibrosis in the ROCK2 knockout hearts. Finally, the loss of ROCK1 had no significant effect on p-MLC and p-FAK levels, mTOR signaling, or autophagy at baseline. In summary, deletions of ROCK isoforms in cardiomyocytes have different, even opposite, effects on endogenous ROCK activity and the MLC/FAK/AKT/mTOR signaling pathway, which is involved in autophagy and fibrosis of the heart. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
15. ROCK1 deficiency enhances protective effects of antioxidants against apoptosis and cell detachment
- Author
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Lei Wei, Michelle Surma, Reuben Kapur, Jiang Chang, Caitlin Handy, and Jianjian Shi
- Subjects
Cancer Treatment ,lcsh:Medicine ,Apoptosis ,Antioxidants ,Mice ,Oxidative Damage ,0302 clinical medicine ,Molecular Cell Biology ,Basic Cancer Research ,Signaling in Cellular Processes ,ROCK1 ,ROCK2 ,lcsh:Science ,Cells, Cultured ,Cytoskeleton ,Cellular Stress Responses ,chemistry.chemical_classification ,0303 health sciences ,rho-Associated Kinases ,Multidisciplinary ,NADPH oxidase ,Antibiotics, Antineoplastic ,Cell Death ,Antiapoptotic Signaling ,Cellular Structures ,Cell biology ,Oncology ,030220 oncology & carcinogenesis ,Medicine ,Public Health ,Research Article ,Signal Transduction ,Stress fiber ,Caspase 3 ,Biology ,03 medical and health sciences ,Cell Adhesion ,Animals ,030304 developmental biology ,Reactive oxygen species ,lcsh:R ,NADPH Oxidases ,Fibroblasts ,Chemotherapy and Drug Treatment ,Actin cytoskeleton ,Enzyme Activation ,chemistry ,Doxorubicin ,biology.protein ,lcsh:Q ,Preventive Medicine ,Reactive Oxygen Species ,Gene Deletion - Abstract
We have recently reported that the homologous Rho kinases, ROCK1 and ROCK2, play different roles in regulating stress-induced stress fiber disassembly and cell detachment, and the ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has remarkable anti-apoptotic, anti-detachment and pro-survival effects against doxorubicin, a chemotherapeutic drug. This study investigated the roles of ROCK isoforms in doxorubicin-induced reactive oxygen species (ROS) generation which is believed to be the major mechanism underlying its cytotoxicity to normal cells, and especially to cardiomyocytes. Different antioxidants have been shown to provide a protective role reported in numerous experimental studies, but clinical trials of antioxidant therapy showed insufficient benefit against the cardiac side effect. We found that both ROCK1−/− and ROCK2−/− MEFs exhibited reduced ROS production in response to doxorubicin treatment. Interestingly, only ROCK1 deficiency, but not ROCK2 deficiency, significantly enhanced the protective effects of antioxidants against doxorubicin-induced cytotoxicity. First, ROCK1 deficiency and N-acetylcysteine (an anti-oxidant) treatment synergistically reduced ROS levels, caspase activation and cell detachment. In addition, the reduction of ROS generation in ROCK1−/− MEFs in response to doxorubicin treatment was in part through inhibiting NADPH oxidase activity. Furthermore, ROCK1 deficiency enhanced the inhibitory effects of diphenyleneiodonium (an inhibitor of NADPH oxidase) on ROS generation and caspase 3 activation induced by doxorubicin. Finally, ROCK1 deficiency had greater protective effects than antioxidant treatment, especially on reducing actin cytoskeleton remodeling. ROCK1 deficiency not only reduced actomyosin contraction but also preserved central stress fiber stability, whereas antioxidant treatment only reduced actomyosin contraction without preserving central stress fibers. These results reveal a novel strategy to enhance the protective effect of antioxidant therapy by targeting the ROCK1 pathway to stabilize the actin cytoskeleton and boost the inhibitory effects on ROS production, apoptosis and cell detachment.
- Published
- 2013
16. ROCK1 Plays an Essential Role in Doxorubicin-Induced Cardiotoxicity
- Author
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Michelle Surma, Jianjian Shi, and Lei Wei
- Subjects
Cardiotoxicity ,business.industry ,Medicine ,ROCK1 ,Doxorubicin ,Pharmacology ,Cardiology and Cardiovascular Medicine ,business ,medicine.drug - Published
- 2014
17. Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment
- Author
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Jianjian Shi, Yu Yang, Xiangbing Wu, Michelle Surma, Reuben Kapur, Lumin Zhang, Sasidhar Vemula, and Lei Wei
- Subjects
Cofilin 1 ,Cancer Research ,Stress fiber ,isoform function ,Cytochalasin D ,Myosin Light Chains ,Cell Survival ,Pyridines ,Immunology ,actin cytoskeleton remodeling ,Arp2/3 complex ,macromolecular substances ,Biology ,Microfilament ,Cell Line ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Mice ,0302 clinical medicine ,Animals ,Rho-kinase ,Phosphorylation ,Cell Shape ,030304 developmental biology ,Mice, Knockout ,0303 health sciences ,rho-Associated Kinases ,Antibiotics, Antineoplastic ,Actin cytoskeleton reorganization ,apoptosis ,Actin remodeling ,Cell Biology ,Cofilin ,Actin cytoskeleton ,Amides ,Actins ,Cell biology ,Actin Cytoskeleton ,Doxorubicin ,030220 oncology & carcinogenesis ,Caspases ,detachment ,biology.protein ,Original Article ,Cardiac Myosins - Abstract
This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1(-/-) and ROCK2(-/-) mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1(-/-) MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2(-/-) MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.
- Published
- 2013
18. Novel Insights into the Roles of Rho Kinase in Cancer
- Author
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Lei Wei, Michelle Surma, Jianjian Shi, Nathan Lambert-Cheatham, and Stephanie Shi
- Subjects
0301 basic medicine ,ROCK isoform-specific inhibitor ,RHOA ,Cancer therapy ,Cell Survival ,Immunology ,Apoptosis ,Review ,Biology ,Metastasis ,03 medical and health sciences ,Cell Movement ,Neoplasms ,Cell Adhesion ,medicine ,Humans ,Protein Isoforms ,Immunology and Allergy ,Rho kinase ,ROCK1 ,Rho kinase pan-inhibitor ,ROCK2 ,Neoplasm Metastasis ,Cell adhesion ,Rho-associated protein kinase ,Cell Proliferation ,rho-Associated Kinases ,Polymorphism, Genetic ,Neovascularization, Pathologic ,Kinase ,General Medicine ,medicine.disease ,Actin cytoskeleton ,3. Good health ,Cell biology ,MicroRNAs ,030104 developmental biology ,Drug Resistance, Neoplasm ,Mutation ,Disease Progression ,Neoplastic Stem Cells ,biology.protein ,Signal Transduction - Abstract
Rho-associated coiled-coil kinase (ROCK) is a major downstream effector of the small GTPase RhoA. The ROCK family, consisting of ROCK1 and ROCK2, plays a central role in the organization of the actin cytoskeleton, and is involved in a wide range of fundamental cellular functions such as contraction, adhesion, migration, proliferation, and apoptosis. Since the discovery of effective inhibitors such as fasudil and Y27632, the biological roles of ROCK have been extensively explored in numerous diseases, including cancer. Accumulating evidence supports the concept that ROCK plays important roles in tumor development and progression through regulating many key cellular functions associated with malignancy, including tumorigenicity, tumor growth, metastasis, angiogenesis, tumor cell apoptosis/survival and chemoresistance as well. This review focuses on the new advances of the most recent 5 years from the studies on the roles of ROCK in cancer development and progression; the discussion is mainly focused on the potential value of ROCK inhibitors in cancer therapy.
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