Small GTP-binding Protein Rho Research Articles

Application and Study of ROCK Inhibitors in Pulmonary Fibrosis: Recent Developments and Future Perspectives.

Rho-associated coiled-coil-containing kinases (ROCKs), serine/threonine protein kinases, were initially identified as downstream targets of the small GTP-binding protein Rho. Pulmonary fibrosis (PF) is a lethal disease with limited therapeutic options and a particularly poor prognosis. Interestingly, ROCK activation has been demonstrated in PF patients and in animal PF models, making it a promising target for PF treatment. Many ROCK inhibitors have been discovered, and four of these have been approved for clinical use; however, no ROCK inhibitors are approved for the treatment of PF patients. In this article, we describe ROCK signaling pathways and the structure-activity relationship, potency, selectivity, binding modes, pharmacokinetics (PKs), biological functions, and recently reported inhibitors of ROCKs in the context of PF. We will also focus our attention on the challenges to be addressed when targeting ROCKs and discuss the strategy of ROCK inhibitor use in the treatment of PF.

From outside to inside and back again: the lysophosphatidic acid-CCN axis in signal transduction.

CCN1 and CCN2 are matricellular proteins that are transcriptionally induced by various stimuli, including growth factors. CCN proteins act to facilitate signaling events involving extracellular matrix proteins. Lysophosphatidic acid (LPA) is a lipid that activates G protein-coupled receptors (GPCRs), enhancing proliferation, adhesion, and migration in many types of cancer cells. Our group previously reported that LPA induces production of CCN1 protein in human prostate cancer cell lines within 2-4h. In these cells, the mitogenic activity of LPA is mediated by LPA Receptor 1 (LPAR1), a GPCR. There are multiple examples of the induction of CCN proteins by LPA, and by the related lipid mediator sphingosine-1-phosphate (S1P), in various cellular models. The signaling pathways responsible for LPA/S1P-induced CCN1/2 typically involve activation of the small GTP-binding protein Rho and the transcription factor YAP. Inducible CCNs can potentially play roles in downstream signal transduction events required for LPA and S1P-induced responses. Specifically, CCNs secreted into the extracellular space can facilitate the activation of additional receptors and signal transduction pathways, contributing to the biphasic delayed responses typically seen in response to growth factors acting via GPCRs. In some model systems, CCN1 and CCN2 play key roles in LPA/S1P-induced cell migration and proliferation. In this way, an extracellular signal (LPA or S1P) can activate GPCR-mediated intracellular signaling to induce the production of extracellular modulators (CCN1 and CCN2) that in turn initiate another round of intracellular signaling.

Lysophosphatidylinositol Induced Morphological Changes and Stress Fiber Formation through the GPR55-RhoA-ROCK Pathway.

We previously reported that lysophosphatidylinositol (LPI) functions as an endogenous agonist of GPR55, a novel cannabinoid receptor. However, the physiological roles of LPI-GPR55 have not yet been elucidated in detail. In the present study, we found that LPI induced morphological changes in GPR55-expressing HEK293 cells. LPI induced the cell rounding of GPR55-expressing HEK293 cells but not of empty-vector-transfected cells. LPI also induced the activation of small GTP-binding protein RhoA and increased stress fiber formation in GPR55-expressing HEK293 cells. The inhibition of RhoA and Rho kinase ROCK by the C3 exoenzyme and the ROCK inhibitor reduced LPI-induced cell rounding and stress fiber formation. These results clearly indicated that the LPI-induced morphological changes and the assembly of the cytoskeletons were mediated through the GPR55-RhoA-ROCK pathway.

Effects of the Rho Kinase Inhibitor Fasudil on Adrenergic Contractions of Rat Aorta and Mouse Spleen

G‐protein coupled receptors such as α1‐adrenergic receptors (AR) are linked via the G‐protein Gq/G11 to both Ca2+ entry and release of Ca2+ from stores, but can also activate the small GTP binding protein RhoA and Rho kinase to produce contractions involving Ca2+ sensitization. In rat portal vein, tonic but not phasic components of contractions to the α1‐AR agonist phenylephrine involve predominantly the α1B‐AR subtype, and are at least partly mediated by mechanisms involving Rho kinase sensitive to fasudil (Alsufyani & Docherty, 2021). In this study we examined the involvement of Rho Kinase in contractions involving α1‐AR in two further tissues to investigate the predominant α1‐AR subtype involved. We have examined the Rho kinase inhibitor fasudil in rat aorta and mouse spleen, tissues containing multiple α1‐AR subtypes. Male Wistar rats (250‐300g) and C57 mice were killed by CO2 overdose and cervical dislocation, and rat aortic rings and mouse spleens were mounted in organ baths under 0.5 g tension, and bathed with Krebs‐Henseleit solution at 37oC. Tissues were contracted with cumulative additions of norepinephrine (NE) in 0.5 log unit increments, over the concentration range 10‐8‐10‐3M. Both α2‐ and α1‐ARs mediate contractions of mouse spleen and the major α1‐AR involved is the α1B‐adrenoceptor, and this receptor interacts with an α1D‐AR at low, and an α1A‐AR at high, NE concentrations (Alsufyani et al., 2021). In mouse spleen, fasudil (3 μM) reduced contractions to both low and high concentrations of NE. In rat aorta, contractions to NE are reported to be mediated by two subtypes of α1‐AR: α1B‐ and α1D‐ARs, with responses to low but not high concentrations of NE being sensitive to the antagonist BMY7378. Responses to high concentrations of NE in rat aorta are sensitive to the non‐selective α1‐AR antagonist prazosin and the putative α1B‐AR antagonist cyclazosin. Fasudil (3 and 10 μM) significantly reduced contractions to both low and high concentrations of NE in rat aorta in a dose dependent manner in terms of maximum response. It is concluded that α1‐AR mediated contractions to NE in both rat aorta and mouse spleen are sensitive to non‐competitive block by the Rho kinase inhibitor fasudil in concentrations of 3‐10 μM. Although multiple subtypes of α1‐adrenoceptor are involved in contractions of both rat aorta and mouse spleen, the evidence suggests that, as previously reported in rat portal vein, α1B‐adrenoceptors are most likely linked to the Rho kinase pathwayAlsufyani HA & Docherty, J.R. Can J Physiol Pharmacol. 2021 Jun;99(6):654‐659. Alsufyani HA, et al. Basic Clin Pharmacol Toxicol. 2021 Dec;129(6):416‐426.

Activation of Protein Tyrosine Phosphatase Receptor Type γ Suppresses Mechanisms of Adhesion and Survival in Chronic Lymphocytic Leukemia Cells.

The regulatory role of protein tyrosine kinases in β1- and β2-integrin activation and in the survival of chronic lymphocytic leukemia (CLL) cells is well established. In contrast, the involvement of protein tyrosine phosphatases in CLL biology was less investigated. We show that selective activation of the protein tyrosine phosphatase receptor type γ (PTPRG) strongly suppresses integrin activation and survival in leukemic B cells isolated from patients with CLL. Activation of PTPRG specifically inhibits CXCR4- as well as BCR-induced triggering of LFA-1 and VLA-4 integrins and mediated rapid adhesion. Triggering of LFA-1 affinity is also prevented by PTPRG activity. Analysis of signaling mechanisms shows that activation of PTPRG blocks chemokine-induced triggering of JAK2 and Bruton's tyrosine kinase protein tyrosine kinases and of the small GTP-binding protein RhoA. Furthermore, activated PTPRG triggers rapid and robust caspase-3/7-mediated apoptosis in CLL cells in a manner quantitatively comparable to the Bruton's tyrosine kinase inhibitor ibrutinib. However, in contrast to ibrutinib, PTPRG-triggered apoptosis is insensitive to prosurvival signals generated by CXCR4 and BCR signaling. Importantly, PTPRG activation does not trigger apoptosis in healthy B lymphocytes. The data show that activated PTPRG inhibits, at once, the signaling pathways controlling adhesion and survival of CLL cells, thus emerging as a negative regulator of CLL pathogenesis. These findings suggest that pharmacological potentiation of PTPRG tyrosine-phosphatase enzymatic activity could represent a novel approach to CLL treatment.

Effect of RhoA/Rho‐kinase inhibitor in Human Internal Mammary Artery and Clinical Implications

ObjectivesCoronary artery bypass grafting surgery (CABG) is the treatment of choice for severe coronary artery disease (CAD). Graft failure is a common complication after CABG. Different biological mechanisms contribute to the pathophysiology of vascular graft failure including vasospasm. RhoA/Rho‐kinase (ROCK) pathway plays an important role in cell contraction of cardiovascular disease. ROCK is a downstream effector of small GTP‐binding protein RhoA and has two isoforms, ROCK1 and ROCK2. This study aimed to investigate the inhibitory effect of ROCK inhibitor fasudil on the vasoconstriction in human internal mammary artery (IMA) from patients undergoing CABG and to explore whether the combination of fasudil and nitroglycerin (NTG) is superior than being used alone.MethodsIsolated human IMA rings (n=8 in each experimental group) taken from 58 patients undergoing CABG were studied in myograph. Cumulative concentration‐relaxation curves for fasudil (−9 to −3.5 log M) were established in IMA precontracted with KCl (30 mM) and U46619 (−8 log M). Cumulative concentration‐contraction curves for KCl (5 to 120 mM) and U46619 (−9 to −4.5 log M) were also established in two ways: (1) in three groups pretreated with the plasma concentration (−6.3 log M) and 10‐fold higher concentration of fasudil (−5.3 log M) or vehicle to estimate the depression effect of fasudil; (2) in four groups pretreated with fasudil (−6.3 log M), NTG (0.1 μM), the combination, and vehicle for 40 minutes. Western Blot was used to measure the change of the ROCK2 protein.ResultsFasudil caused similar cumulative relaxation in KCl‐ or U46619‐ contracted (p > 0.05) rings. Pretreatment with −5.3 log M fasudil significantly depressed subsequent contraction by KCl (P < 0.01 vs. control, P < 0.05 vs. −6.3 log M) and U46619 (P < 0.05 vs. control or −6.3 log M). Pretreatment with the combination of NTG and fasudil significantly depressed the cumulative contraction by KCl (P < 0.01 vs. control; P < 0.05 vs. −5.3 log M; P < 0.01 vs. NTG). Similarly, in U46619‐induced contraction, the combination also significantly depressed the contraction (P < 0.001 vs. control; P < 0.05 vs. −5.3 log M fasudil; P < 0.01 vs. NTG). Fasudil also caused a decrease of ROCK2 protein content (p < 0.05).ConclusionsFasudil has a potent inhibitory effect on the vasoconstriction mediated by KCl and U46619 in human conduit arteries. Combination of fasudil and NTG is more effective than fasudil or NTG alone. These findings provide a new antispastic method in CABG and may be beneficial to long‐term patency of the grafts.Support or Funding InformationThis work was supported by the National Natural Science Foundation of China [81870288 & 81870227]; the Non‐profit Central Research Institute Fund of Chinese Academy of Medical Sciences [2019XK310001 & 2018TX31002]; Tianjin Science and Technology Committee[18PTZWHZ00060];Binhai New Area Health Bureau (2016BWKY007 & 2018BWKZ005).

GRAF gene expression in patients with acute and chronic myeloid leukaemia: impact on the prognosis

Objective We aimed to assess the expression level of GRAF gene in patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) and to clarify its prognosticsignificance on the clinical outcome. Background GTPase regulator associated with focal adhesion kinase (GRAF) is a newly identified protein specifically binding to focal adhesion kinase and negatively regulates the small GTP-binding protein RhoA, which is well known for its growth-promoting effect in RAS-mediated malignant transformation. GRAF is recognized as a tumor suppressor gene that binds to focal adhesion kinase. Methods In this study, we investigated the expression of the GRAF transcript by realtime quantitative PCR in 37 AML patients, 35 CML patients and 15 healthy and sex-matched controls, and to clarify its prognostic significance on the clinical outcome. Results We found that the GRAF expression is low in patients with AML and CML. A high GRAF expression is a favorable prognostic marker in patients with AML and CML. In AML, high GRAF transcript. Conclusion High GRAF expression is a favorable prognostic marker in AML patients and a protective factor against CML progression.

SMURF1-mediated ubiquitination of ARHGAP26 promotes ovarian cancer cell invasion and migration

Rho GTPase-activating protein 26 (ARHGAP26) is a negative regulator of the Rho family that converts the small GTP-binding protein RhoA (GTP-RhoA) to its inactive GDP-bound form and is a putative tumor suppressor gene associated with cell growth and migration. Here, the involvement of ARHGAP26 in ovarian cancer cell proliferation and migration was investigated. In this study, low ARHGAP26 expression was observed in ovarian cancer tissues and was associated with a poor overall survival and higher β-catenin expression in patients with ovarian cancer. A2780 and HEY cells with ARHGAP26 upregulation showed decreased cell proliferation, migration, and invasion, along with decreased GTP-RhoA, β-catenin, VEGF, MMP2, and MMP7 expression. ARHGAP26 upregulation in A2780 cells also inhibited lung metastasis in vivo. SKOV3 cells with ARHGAP26 downregulation demonstrated an inverse effect, which was inhibited by ARHGAP26 overexpression or DKK1, an antagonist of the β-catenin pathway. SMURF1, an E3 ubiquitin ligase, interacted with and induced ubiquitination of ARHGAP26. ARHGAP26 upregulation in SKOV3 cells significantly inhibited SMURF1 upregulation-induced cell migration and invasion. Overall, SMURF1-mediated ubiquitination of ARHGAP26 may promote invasion and migration of ovarian cancer cells via the β-catenin pathway.

CXCR4- and BCR-triggered integrin activation in B-cell chronic lymphocytic leukemia cells depends on JAK2-activated Bruton's tyrosine kinase.

Bruton's tyrosine kinase (BTK) regulates the B-cell receptor (BCR) signaling pathway, which, in turn, plays a critical role in B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis. The BTK-specific inhibitor Ibrutinib blocks BCR signaling and is now approved as effective B-CLL therapy. Chemokines, such as the homeostatic chemokine CXCL12, play a central role in B-CLL pathogenesis and progression, by regulating CLL cell interaction with the stromal microenvironment, leading to cells survival and proliferation. In this study, we investigated, in normal versus CLL B-lymphocytes, the role of BTK in signal transduction activated by the CXCL12-CXCR4 signaling axis and its involvement in rapid integrin activation. We show that BTK is rapidly activated by CXCL12 in healthy as well as CLL B-lymphocytes, with a kinetic of tyr-phosphorylation coherent with rapid adhesion triggering. BTK inhibition prevents CXCL12-induced triggering of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. Furthermore, BTK inhibition blocks the activation of the small GTP-binding protein RhoA, controlling integrin affinity. Very importantly, we show that BTK tyr-phosphorylation and activation by CXCL12 depends on upstream activation of JAK2 tyrosine kinase. A comparative analysis of 36 B-CLL patients demonstrates that JAK2-dependent BTK regulatory role on integrin activation by CXCL12 is fully conserved in CLL cells. Finally, we show that the JAK2-BTK axis also regulates signaling to integrin activation by BCR. Thus, BTK and JAK protein tyrosine kinases (PTKs) manifest a hierarchical activity both in chemokine- as well as BCR-mediated integrin activation and dependent adhesion, potentially suggesting the possibility of combined therapeutic approaches to B-CLL treatment.

Long noncoding RNA NORAD, a novel competing endogenous RNA, enhances the hypoxia-induced epithelial-mesenchymal transition to promote metastasis in pancreatic cancer

BackgroundPancreatic cancer, one of the top two most fatal cancers, is characterized by a desmoplastic reaction that creates a dense microenvironment, promoting hypoxia and inducing the epithelial-to-mesenchymal transition (EMT) to facilitate invasion and metastasis. Recent evidence indicates that the long noncoding RNA NORAD may be a potential oncogenic gene and that this lncRNA is significantly upregulated during hypoxia. However, the overall biological role and clinical significance of NORAD remains largely unknown.MethodsNORAD expression was measured in 33 paired cancerous and noncancerous tissue samples by real-time PCR. The effects of NORAD on pancreatic cancer cells were studied by overexpression and knockdown in vitro. Insights into the mechanism of competitive endogenous RNAs (ceRNAs) were gained from bioinformatics analyses and luciferase assays. In vivo, metastatic potential was identified using an orthotopic model of PDAC and quantified using bioluminescent signals. Alterations in RhoA expression and EMT levels were identified and verified by immunohistochemistry and Western blotting.ResultsNORAD is highly expressed in pancreatic cancer tissues and upregulated in hypoxic conditions. NORAD upregulation is correlated with shorter overall survival in pancreatic cancer patients. Furthermore, NORAD overexpression promoted the migration and invasion of pancreatic carcinoma cells, while NORAD depletion inhibited EMT and metastasis in vitro and in vivo. In particular, NORAD may function as a ceRNA to regulate the expression of the small GTP binding protein RhoA through competition for hsa-miR-125a-3p, thereby promoting EMT.ConclusionsElevated expression of NORAD in pancreatic cancer tissues is linked to poor prognosis and may confer a malignant phenotype upon tumor cells. NORAD may function as a ceRNA to regulate the expression of the small GTP binding protein RhoA through competition for hsa-miR-125a-3p. This finding may contribute to a better understanding of the role played by lncRNAs in hypoxia-induced EMT and provide a potential novel diagnostic and therapeutic target for pancreatic cancer.

Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling

Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα12/13-mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gαq/11-mediated or β-arrestin-mediated signaling but rather involves liberation of Gβγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways.

Rho Kinases and Cardiac Remodeling.

Hypertensive cardiac remodeling is characterized by left ventricular hypertrophy and interstitial fibrosis, which can lead to heart failure with preserved ejection fraction. The Rho-associated coiled-coil containing kinases (ROCKs) are members of the serine/threonine protein kinase family, which mediates the downstream effects of the small GTP-binding protein RhoA. There are 2 isoforms: ROCK1 and ROCK2. They have different functions in different types of cells and tissues. There is growing evidence that ROCKs contribute to the development of cardiovascular diseases, including cardiac fibrosis, hypertrophy, and subsequent heart failure. Recent experimental studies using ROCK inhibitors, such as fasudil, have shown the benefits of ROCK inhibition in cardiac remodeling. Mice lacking each ROCK isoform also exhibit reduced myocardial fibrosis in a variety of pathological models of cardiac remodeling. Indeed, clinical studies with fasudil have suggested that ROCKs could be potential novel therapeutic targets for cardiovascular diseases. In this review, we summarize the current understanding of the roles of ROCKs in the development of cardiac fibrosis and hypertrophy and discuss their therapeutic potential for deleterious cardiac remodeling. (Circ J 2016; 80: 1491-1498).

Abstract 17413: Influence of Endothelial Function and Rho-Associated Kinase Activity in Patients With Primary Aldosteronism

Background: Primary aldosteronism (PA) is a most common cause of secondary hypertension and patients are divided into two subtypes in clinically, aldosterone producing adenoma (APA) and idiopathic hyperaldosteronism (IHA). Hypertension is associated with endothelial dysfunction. It is well known that endothelial dysfunction is an independent predictor of cardiovascular events. Rho-associated kinases (ROCKs), one of the first downstream targets of the small GTP-binding protein Rho A, mediate various cellular physiologic functions. It has been reported that an increase in ROCK activity is associated with cardiovascular diseases, including hypertension. However, there is little information on the relationship between subtype of PA and the grade of atherosclerosis. The purpose of this study was to evaluate the vascular function and ROCK activity in patients with PA. Methods: Vascular function, including peripheral arterial tonometry (PAT) and ROCK activity in peripheral leukocytes were evaluated in 18 patients with APA group (49.5±15.5 years, 7 males), 14 patients with IHA group (56.1±11.0 years, 8 males), and 23 age-, gender-, and blood pressure-matched EH group (55.6 ± 9.9 years, 13 males). Results: PAT ratio was significantly lower in the APA and IHA groups than in the EH group (0.62±0.23 vs. 0.62±0.24 and 0.79±0.28, P<0.05, respectively), whereas there was no significant difference in PAT ratio between the APA group and IHA group. ROCK activity was significantly higher in the APA group than in the IHA group and EH group (1.36±0.58 vs. 0.95±0.51 and 0.80±0.39, P<0.05 and P<0.001, respectively), whereas there was no significant difference in ROCK activity between the IHA group and EH group. PAT ratio correlated with plasma aldosterone concentration (PAC) (r=-0.36, P<0.01) and plasma renin activity ratio (ARR) (r=-0.26, P=0.05). ROCK activity correlated with serum potassium (r=-0.27, P=0.05), PAC (r=0.39, P<0.01) and ARR (r=0.49, P<0.01). Conclusions: APA and IHA were associated with endothelial dysfunction and APA increased ROCK activity compared with those in IHA and EH. These findings suggest that APA may have a higher risk of future cardiovascular events.

JAK2 tyrosine kinase mediates integrin activation induced by CXCL12 in B-cell chronic lymphocytic leukemia.

Chemokines participate to B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis by promoting cell adhesion and survival in bone marrow stromal niches and mediating cell dissemination to secondary lymphoid organs. In this study we investigated the role of JAK protein tyrosine kinases (PTK) in adhesion triggering by the CXC chemokine CXCL12 in normal versus CLL B-lymphocytes. We demonstrate that CXCL12 activates JAK2 in normal as well as CLL B-lymphocytes, with kinetics consistent with rapid adhesion triggering. By using complementary methodologies of signal transduction interference, we found that JAK2 mediates CXCL12-triggered activation of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. We also show that JAK2 mediates the activation of the small GTP-binding protein RhoA, in turn controlling LFA-1 affinity triggering by CXCL12. Importantly, comparative analysis of 41 B-CLL patients did not evidence JAK2 functional variability between subjects, thus suggesting that JAK2, differently from other signaling events involved in adhesion regulation in B-CLL, is a signaling molecule downstream to CXCR4 characterized by a conserved regulatory role. Our results reveal JAK2 as critical component of chemokine signaling in CLL B-lymphocytes and indicate JAK inhibition as a potentially useful new pharmacological approach to B-CLL treatment.

Light and Dark of Reactive Oxygen Species for Vascular Function: 2014 ASVB (Asian Society of Vascular Biology).

Vascular-derived hydrogen peroxide (H2O2) serves as an important signaling molecule in the cardiovascular system and contributes to vascular homeostasis. H2O2 is a second messenger, transducing the oxidative signal into biological responses through posttranslational protein modification. The balance between oxidant and antioxidant systems regulates intracellular redox status, and their imbalance causes oxidative or reductive stress, leading to cellular damage in cardiovascular systems. Excessive H2O2 deteriorates vascular functions and promotes vascular disease through multiple pathways. The RhoA/Rho-kinase pathway plays an important role in various fundamental cellular functions, including production of excessive reactive oxygen species, leading to the development of cardiovascular diseases. Rho-kinase (ROCK1 and ROCK2) belongs to the family of serine/threonine kinases and is an important downstream effector of the small GTP-binding protein RhoA. Rho-kinase plays a crucial role in the pathogenesis of vasospasm, arteriosclerosis, ischemia/reperfusion injury, hypertension, pulmonary hypertension, stroke, and heart failure. Thus, Rho-kinase inhibitors may be useful for the treatment of cardiovascular diseases in humans. In this review, we will briefly discuss the roles of vascular-derived H2O2 and review the recent progress in the translational research on the therapeutic importance of the Rho-kinase pathway in cardiovascular medicine.

MEMBRANE TYPE 1-MATRIX METALLOPROTEINASE (MT1-MMP) IDENTIFIED AS A MULTIFUNCTIONAL REGULATOR OF VASCULAR RESPONSES.

Membrane type 1-matrix metalloproteinase (MT1-MMP) functions as a signaling molecules in addition to a transmembrane metalloprotease, which degrades interstitial collagens and extracellular matrix components. This review focuses on the multifunctional roles of MT1-MMP as a signaling molecule in vascular responses to pro-atherosclerotic stimuli in the pathogenesis of cardiovascular diseases. First, the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1)-MT1-MMP signaling axis contributes to endothelial dysfunction, which is mediated via small GTP-binding protein RhoA and Rac1 activation. Second, MT1-MMP plays a crucial role in reactive oxygen species (ROS) generation through the activation of receptor for advanced glycation end products (AGEs) in smooth muscle cells, indicating that MT1-MMP may be a therapeutic target for diabetic vascular complications. Third, MT1-MMP is involved in RhoA/Rac1 activation and Ca(2+) signaling in the mechanism of thrombin-stimulated endothelial dysfunction and oxidant stress. Fourth, the inhibition of the MT1-MMP/Akt signaling pathway may be an attractive strategy for treating endothelial disordered hemostasis in the development of vascular diseases linked to TNF-α-induced inflammation. Fifth, MT1-MMP through RAGE induced RhoA/Rac1 activation and tissue factor protein upregulation through NF-κB phosphorylation in endothelial cells stimulated by high-mobility group box-1, which plays a key role in the systemic inflammation. These findings suggest that the MT1-MMP-mediated signaling axis may be a promising target for treating atherosclerosis and subsequent cardiovascular diseases.

Rho-Kinase Inhibition Ameliorates Metabolic Disorders through Activation of AMPK Pathway in Mice

BackgroundMetabolic disorders, caused by excessive calorie intake and low physical activity, are important cardiovascular risk factors. Rho-kinase, an effector protein of the small GTP-binding protein RhoA, is an important cardiovascular therapeutic target and its activity is increased in patients with metabolic syndrome. We aimed to examine whether Rho-kinase inhibition improves high-fat diet (HFD)-induced metabolic disorders, and if so, to elucidate the involvement of AMP-activated kinase (AMPK), a key molecule of metabolic conditions.Methods and ResultsMice were fed a high-fat diet, which induced metabolic phenotypes, such as obesity, hypercholesterolemia and glucose intolerance. These phenotypes are suppressed by treatment with selective Rho-kinase inhibitor, associated with increased whole body O2 consumption and AMPK activation in the skeletal muscle and liver. Moreover, Rho-kinase inhibition increased mRNA expression of the molecules linked to fatty acid oxidation, mitochondrial energy production and glucose metabolism, all of which are known as targets of AMPK in those tissues. In systemic overexpression of dominant-negative Rho-kinase mice, body weight, serum lipid levels and glucose metabolism were improved compared with littermate control mice. Furthermore, in AMPKα2-deficient mice, the beneficial effects of fasudil, a Rho-kinase inhibitor, on body weight, hypercholesterolemia, mRNA expression of the AMPK targets and increase of whole body O2 consumption were absent, whereas glucose metabolism was restored by fasudil to the level in wild-type mice. In cultured mouse myocytes, pharmacological and genetic inhibition of Rho-kinase increased AMPK activity through liver kinase b1 (LKB1), with up-regulation of its targets, which effects were abolished by an AMPK inhibitor, compound C.ConclusionsThese results indicate that Rho-kinase inhibition ameliorates metabolic disorders through activation of the LKB1/AMPK pathway, suggesting that Rho-kinase is also a novel therapeutic target of metabolic disorders.

Fasudil inhibits ER stress-induced VCAM-1 expression by modulating unfolded protein response in endothelial cells

The process of atherosclerosis is affected by interactions among numerous biological pathways. Accumulating evidence shows that endoplasmic reticulum (ER) stress plays a crucial role in the development of atherosclerosis. Rho-kinase is an effector of small GTP-binding protein Rho, and has been implicated as an atherogenic factor. Previous studies demonstrated that fasudil, a specific Rho-kinase inhibitor, exerts a cardioprotective effect by downregulating ER stress signaling. However, the molecular link between ER stress and Rho-kinase in endothelial cells has not been elucidated. In this study, we investigated the mechanisms by which fasudil regulates endothelial inflammation during ER stress. Tunicamycin, an established ER stress inducer, increased vascular cellular adhesion molecule (VCAM)-1 expression in endothelial cells. Intriguingly, fasudil inhibited VCAM-1 induction. From a mechanistic stand point, fasudil inhibited expression of activating transcription factor (ATF)4 and subsequent C/EBP homologous protein (CHOP) induction by tunicamycin. Furthermore, fasudil attenuated tunicamycin-induced phophorylation of p38MAPK that is crucial for the atherogenic response during ER stress. These findings indicate that Rho-kinase regulates ER stress-mediated VCAM-1 induction by ATF4- and p38MAPK-dependent signaling pathways. Rho-kinase inhibition by fasudil would be an important therapeutic approach against atherosclerosis, in particular, under conditions of ER stress.

Abstract 1793: ADAR1 regulates ARHGAP26 expression by RNA editing through destruction of the miR-30b-3p and miR-573 target sites.

Abstract Rho GTPase activating protein 26 (ARHGAP26) is a putative tumor suppressor gene. It was observed to negatively regulate the small GTP-binding protein RhoA and be down-regulated in myeloid malignancies and metastatic brain cancers. Herein, we demonstrated that ARHGAP26 transcript undergoes extensive A-to-I RNA editing catalyzed by ADAR1 in its 3’-UTR. Either knocking down of ADAR1 by silencing or overexpression by ADAR1 expression vector decreased or increased the expression level of ARHGAP26 gene. Furthermore, luciferase assay showed that miR-30b-3p and miR-573 targeted the ARHGAP26, but the RNA editing abolished the effect of miR-30b-3p or miR-573. In addition, we observed that the level of the A-to-I RNA editing on ARHGAP26 transcript decreased significantly in human brain cancer tissues comparing to their controls, which contributes to the down-regulation of the ARHGAP26 expression in human cancer tissues. These data suggested that ADAR1 regulated the expression of ARHGAP26 gene by A-to-I RNA editing through destruction of the existing miR-30b-3p and miR-573 target sites. It implied that down-regulation of tumor suppressors through RNA editing is one crucial way to explain how dyregulation of RNA editing involves in tumorigenesis. Citation Format: Qiong Wang, Zhendong Guo, Weina Zhang, Li Wang. ADAR1 regulates ARHGAP26 expression by RNA editing through destruction of the miR-30b-3p and miR-573 target sites. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1793. doi:10.1158/1538-7445.AM2013-1793 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation

Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.