ROCK Pathway Research Articles

Spiromesifen contributes vascular developmental toxicity via disrupting endothelial cell proliferation and migration in zebrafish embryos

Spiromesifen (SPF) is a specific contact pesticide, which has been widely used to control the growth of sucking insects like mites and whiteflies on crops. Although its residues in crops and effects on organisms has been extensively reported, its impact on the vasculature is still not being reported. In the present study, using human umbilical vein endothelial cells (HUVECs) and zebrafish embryos, we investigated the effects of SPF on blood vessel development and its mechanism of action. SPF exposure triggered abnormal blood vessel development, including vascular deletions and malformations, inhibition of CCV remodeling, and decrease of SIV areas. SPF exposure also obstructed the migration of endothelial cell from caudal hematopoietic tissue in zebrafish embryos. SPF damaged cytoskeleton, caused cell cycle arrest, inhibited the viability and migration of HUVECs. In addition, SPF also inhibited the expression of the VEGF/VEGFR pathway-related genes (hif1a, vegfa, flt1, and kdrl), cell cycle-related genes (ccnd1, ccne1, cdk2, and pcna), and Rho/ROCK pathway-related genes (itgb1, rho, rock, mlc-1, and vim-1). Taken together, SPF may inhibit the proliferation and migration of vascular endothelial cells through disturbing cytoskeleton via the Rho/ ROCK pathway, resulting in vascular malformation. Our study contributes to potential insight into the mechanism of SPF toxicity in angiocardiopathy.

Tuning of Liver Sieve: The Interplay between Actin and Myosin Regulatory Light Chain Regulates Fenestration Size and Number in Murine Liver Sinusoidal Endothelial Cells.

Liver sinusoidal endothelial cells (LSECs) facilitate the efficient transport of macromolecules and solutes between the blood and hepatocytes. The efficiency of this transport is realized via transcellular nanopores, called fenestrations. The mean fenestration size is 140 ± 20 nm, with the range from 50 nm to 350 nm being mostly below the limits of diffraction of visible light. The cellular mechanisms controlling fenestrations are still poorly understood. In this study, we tested a hypothesis that both Rho kinase (ROCK) and myosin light chain (MLC) kinase (MLCK)-dependent phosphorylation of MLC regulates fenestrations. We verified the hypothesis using a combination of several molecular inhibitors and by applying two high-resolution microscopy modalities: structured illumination microscopy (SIM) and scanning electron microscopy (SEM). We demonstrated precise, dose-dependent, and reversible regulation of the mean fenestration diameter within a wide range from 120 nm to 220 nm and the fine-tuning of the porosity in a range from ~0% up to 12% using the ROCK pathway. Moreover, our findings indicate that MLCK is involved in the formation of new fenestrations—after inhibiting MLCK, closed fenestrations cannot be reopened with other agents. We, therefore, conclude that the Rho-ROCK pathway is responsible for the control of the fenestration diameter, while the inhibition of MLCK prevents the formation of new fenestrations.

Identification of lysophosphatidic acid in serum as a factor that promotes epithelial apical junctional complex organization

The apical junctional complex (AJC) consists of adherens junctions (AJs) and tight junctions and regulates epithelial integrity and remodeling. However, it is unclear how AJC organization is regulated based on environmental cues. We found here using cultured EpH4 mouse mammary epithelial cells that fetal bovine serum (FBS) in a culture medium showed an activity to promote AJC organization and that FBS showed an activity to promote tight junction formation even in the absence of AJ proteins, such as E-cadherin, αE-catenin, and afadin. Furthermore, we purified the individual factor responsible for these functions from FBS and identified this molecule as lysophosphatidic acid (LPA). In validation experiments, purified LPA elicited the same activity as FBS. In addition, we found that the AJC organization–promoting activity of LPA was mediated through the LPA receptor 1/5 via diacylglycerol–novel PKC and Rho–ROCK pathway activation in a mutually independent, but complementary, manner. We demonstrated that the Rho–ROCK pathway activation–mediated AJC organization was independent of myosin II-induced actomyosin contraction, although this signaling pathway was previously shown to induce myosin II activation. These findings are in contrast to the literature, as previous results suggested an AJC organization–disrupting activity of LPA. The present results indicate that LPA in serum has an AJC organization–promoting activity in a manner dependent on or independent of AJ proteins.

Identification of a Potent, Selective, and Brain-Penetrant Rho Kinase Inhibitor and its Activity in a Mouse Model of Huntington's Disease.

The Rho kinase (ROCK) pathway is implicated in the pathogenesis of several conditions, including neurological diseases. In Huntington's disease (HD), ROCK is implicated in mutant huntingtin (HTT) aggregation and neurotoxicity, and members of the ROCK pathway are increased in HD mouse models and patients. To validate this mode of action as a potential treatment for HD, we sought a potent, selective, central nervous system (CNS)-penetrant ROCK inhibitor. Identifying a compound that could be dosed orally in mice with selectivity against other AGC kinases, including protein kinase G (PKG), whose inhibition could potentially activate the ROCK pathway, was paramount for the program. We describe the optimization of published ligands to identify a novel series of ROCK inhibitors based on a piperazine core. Morphing of the early series developed in-house by scaffold hopping enabled the identification of a compound exhibiting high potency and desired selectivity and demonstrating a robust pharmacodynamic (PD) effect by the inhibition of ROCK-mediated substrate (MYPT1) phosphorylation after oral dosing.

RHOA/ROCK PATHWAY PARTICIPATES IN THE STUDY OF RELATED MECHANISMS OF APATINIB IN THE TREATMENT OF BREAST CANCER-INDUCED HYPERTENSION

Objective: To explore activation of the RhoA/ROCK pathway whether involved in the mechanism of apatinib in the treatment of breast cancer-induced hypertension and whether ROCK pathway inhibitors can prevent and reverse the increase in blood pressure and target organ damage caused by apatinib in the treatment of breast cancer, thus confirming its therapeutic effect on this type of hypertension. Design and method: Thirty female Balb/c nude mice aged 4–6 weeks and the human breast cancer cell MDA-MB-231 were used for breast cancer tumorigenesis. Mice were randomly divided into 5 groups, each with 6 mice; 1): no tumor model, 2): just tumor model; 3): high-salt diet (giving customized 8% high-salt feed); 4) apatinib 50 mg/kg.d; 5): apatinib 50 mg/kg. d + Y27632 10 mg/kg. d. After the tumor grew to the target volume of 100–150mm3, intervention was performed for 3 weeks, blood pressure was measured weekly, and body weight was measured every day. Results: 1. The blood pressure of the fourth group was higher than other groups. 2. The ventricular thickness of the mice in the third and fourth groups was thicker than the first and second groups, and the fifth group was less than the fourth group. 3. Masson staining of mice aorta from strong to weak is: the fourth group, the third group, the fifth group, the second group, the first group. 4. The results of mice aortic mRNA and Western Blot showed that RhOA and ROCK1 in the third, fourth, and fifth groups were higher than those in the first and second groups, and the fifth group was lower than the fourth group. 5.The mice aortic immunohistochemistry show that the third and fourth groups of COLI and COLIII are higher than the first and the second group, the fifth group has a lower than the fourth group. Conclusions: The RhoA/ROCK pathway is involved in the apatinib-induced hypertension. After applying apatinib to breast cancer, the blood pressure, the vascular stiffness, the Endothelin-1, RhoA/ROCK pathway protein and mRNA are increased, the endothelial function decreased, and RhoA/ROCK pathway inhibitors can prevent and reverse the changes by apatinib.

Protective effects of adipose-derived biogenic nanoparticles on the pulmonary microvascular endothelial barrier in mice with ventilator-induced lung injury via the TRPV4/ROCK1 signalling pathway

PurposeThe "obesity paradox" phenomenon occurs in critically ill patients who receive mechanical ventilation. Our previous studies found that the adipose-derived exosomes secreted by obese mice have a protective effect on the pulmonary microvascular endothelial barrier. However, the extraction of exosomes is cumbersome, their yield is low, and their storage is difficult. After further research, we discovered a new type of adipose-derived bioactive material called: lipoaspirate nanoparticles (Lipo-NPs). MethodsLipo-NPs were extracted and identified using a tangential flow filtration system. The Lipo-NPs were used as an intervention in ventilator-induced lung injury (VILI) models in vivo and in vitro to investigate whether they have a protective effect on lung tissue damage (haematoxylin and eosin staining), lung barrier function (lung wet/dry [W/D] weight ratio, protein concentration in bronchoalveolar lavage fluid (BALF), and Vascular endothelial (VE)-expression), as well as their related mechanisms. ResultsIn both in vivo and in vitro studies, Lipo-NPs can attenuate lung injury, reduce lung W/D ratio and protein concentration in BALF, and augment the expression of the adhesion link-protein VE-cadherin, thus playing a protective role in lung barrier function. This protective effect involves the activation of the transient receptor potential vanilloid 4 (TRPV4)/Rho-associated kinase1 (ROCK1) signalling pathway. We further verified the role of this signalling pathway via activation and inhibition of TRPV4 and ROCK1. Moreover, phosphorylation of myosin light chain 2 (MLC2) regulates F-actin and is a target of the ROCK pathway. ConclusionLipo-NPs can enhance the expression of VE-cadherin by inhibiting the TRPV4/ROCK1/pMLC2 signalling pathway in the mechanical ventilation model, thereby exerting a protective effect on the VILI pulmonary microvascular endothelial barrier.

Cyclocarya paliurus triterpenoids attenuate glomerular endothelial injury in the diabetic rats via ROCK pathway

Ethnopharmacological relevanceCyclocarya paliurus (Batal.) Iljinskaja. (C. paliurus) is a distinctive traditional Chinese herb, with remarkable hypoglycemic capacity. Emerging evidence suggested that glomerular endothelial injury is a crucial pathological process of diabetic kidney disease (DKD). Our previous research found that C. paliurus triterpenoids fraction (CPT) has ameliorative effects on DKD. However, whether C. paliurus could counteract the glomerular endothelial injury of DKD is still undefined. Aim of the studyWe aimed to investigate the effects of CPT on glomerular endothelial function and explore its underlying mechanisms with in vivo and in vitro experiments. Materials and methodsThe effects and possible mechanisms of CPT on glomerular endothelial injury in streptozotocin (STZ)-induced diabetic rats and H2O2-challenged primary rat glomerular endothelial cells were successively investigated. ResultsIn vivo, we found that CPT treatment obviously decreased the levels of blood glucose, microalbumin, BUN and mesangial expansion. Additionally, CPT could ameliorate renal endothelium function by reducing the content of VCAM-1 and ICAM-1, and blocking the loss of glycocalyx. In vitro, CPT could also alleviate H2O2-induced endothelial injury. Mechanistically, CPT remarkably increased the phosphorylation levels of Akt and eNOS, decreased the expression of ROCK and Arg2in vivo and in vitro. Noticeably, the favorable effects mediated by CPT were abolished following ROCK overexpression with plasmid transfection. ConclusionThese findings suggested that CPT could be sufficient to protect against glomerular endothelial injury in DKD through regulating ROCK pathway.

Influence of ROCK Pathway Manipulation on the Actin Cytoskeleton Height.

The actin cytoskeleton with its dynamic properties serves as the driving force for the movement and division of cells and gives the cell shape and structure. Disorders in the actin cytoskeleton occur in many diseases. Deeper understanding of its regulation is essential in order to better understand these biochemical processes. In our study, we use metal-induced energy transfer (MIET) as a tool to quantitatively examine the rarely considered third dimension of the actin cytoskeleton with nanometer accuracy. In particular, we investigate the influence of different drugs acting on the ROCK pathway on the three-dimensional actin organization. We find that cells treated with inhibitors have a lower actin height to the substrate while treatment with a stimulator for the ROCK pathway increases the actin height to the substrate, while the height of the membrane remains unchanged. This reveals the precise tuning of adhesion and cytoskeleton tension, which leads to a rich three-dimensional structural behaviour of the actin cytoskeleton. This finetuning is differentially affected by either inhibition or stimulation. The high axial resolution shows the importance of the precise finetuning of the actin cytoskeleton and the disturbed regulation of the ROCK pathway has a significant impact on the actin behavior in the z dimension.

DL0805-1, a novel Rho-kinase inhibitor, attenuates lung injury and vasculopathy in a rat model of monocrotaline-induced pulmonary hypertension

Pulmonary hypertension (PH) is a severe chronic cardiopulmonary dysfunction characterized by impaired of pulmonary circulation. Current therapeutic drugs mainly act as vasodilators, leading to an unsatisfactory prognosis. The Rho/ROCK pathway plays an important role in the cardiovascular system. DL0805-1, a novel Rho kinase inhibitor, synthesized by our institute and showed a protective effect on lung tissues and reduced right ventricular systolic pressure in a hypertensive crisis rat model in our previous study. The present study aims to explore the efficacy of DL0805-1 on PH. The classical PH rat model induced by a single subcutaneous injection of monocrotaline was used to investigate the therapeutic effect of DL0805-1 on PH and the underlying mechanisms. The results showed that the high dose of DL0805-1 had a better effect on the survival rate and controlled right ventricular systolic pressure (RVSP) of PH rats than fasudil. DL0805-1 also exhibited a superior lung protective effect and significantly improved pulmonary vascular function compared with bosentan. Regarding molecular mechanisms, DL0805-1 inhibited the ROCK pathway in both pulmonary arteries and lung tissues. Taken together, DL0805-1 alleviated lung injury and vasculopathy in experimental PH rats. DL0805-1 has the potential to be developed as a candidate drug for the treatment of PH.

Stretch increases alveolar type 1 cell number in fetal lungs through ROCK-Yap/Taz pathway.

Accurate fluid pressure in the fetal lung is critical for its development, especially at the beginning of the saccular stage when alveolar epithelial type 1 (AT1) and type 2 (AT2) cells differentiate from the epithelial progenitors. Despite our growing understanding of the role of physical forces in lung development, the molecular mechanisms that regulate the transduction of mechanical stretch to alveolar differentiation remain elusive. To simulate lung distension, we optimized both an ex vivo model with precision cut lung slices and an in vivo model of fetal tracheal occlusion. Increased mechanical tension showed to improve alveolar maturation and differentiation toward AT1. By manipulating ROCK pathway, we demonstrate that stretch-induced Yap/Taz activation promotes alveolar differentiation toward AT1 phenotype via ROCK activity. Our findings show that balanced ROCK-Yap/Taz signaling is essential to regulate AT1 differentiation in response to mechanical stretching of the fetal lung, which might be helpful in improving lung development and regeneration.

3D printed porous microgel for lung cancer cells culture in vitro

Currently, in vitro cancer cell culture technology is very important for cancer research. Existing studies have shown that two-dimensional (2D) cell environment may cause significant differences from tumor cells in vivo, resulting in high failure rates when the therapies developed in 2D tumor models translated to the clinic. And compared with 2D culture, the isotropic three-dimensional (3D) culture can construct an environment more similar to that in the body. Here, we utilized 3D printed porous microgel to culture for lung cancer cell and also explored possible mechanism of how 3D culture environment regulates actin cytoskeleton. Compared with 2D cultured lung cancer cells, cells in porous microgel are more similar to those extracted from the existing gold standard: mouse transplanted tumors, in terms of the ROCK-actin pathway. In addition, this study also revealed that ROCK pathway altered by environment played an important role in regulating the drug sensitivity of lung cancer cells. Hence, this 3D printed porous microgel is a promising lung cancer cell culture method, which shows potentional application in future cancer research and drug development.

Small-molecule modulators of INAVA cytosolic condensate and cell-cell junction assemblies.

Epithelial cells lining mucosal surfaces distinctively express the inflammatory bowel disease risk gene INAVA. We previously found that INAVA has dual and competing functions: one at lateral membranes where it affects mucosal barrier function and the other in the cytosol where INAVA enhances IL-1β signal transduction and protein ubiquitination and forms puncta. We now find that IL-1β-induced INAVA puncta are biomolecular condensates that rapidly assemble and physiologically resolve. The condensates contain ubiquitin and the E3 ligase βTrCP2, and their formation correlates with amplified ubiquitination, suggesting function in regulation of cellular proteostasis. Accordingly, a small-molecule screen identified ROS inducers, proteasome inhibitors, and inhibitors of the protein folding chaperone HSP90 as potent agonists for INAVA condensate formation. Notably, inhibitors of the p38α and mTOR pathways enhanced resolution of the condensates, and inhibitors of the Rho-ROCK pathway induced INAVA's competing function by recruiting INAVA to newly assembled intercellular junctions in cells where none existed before.

RELATIONSHIP BETWEEN ATRIAL FIBRILLATION, RHO KINASE ACTIVITY AND CONNEXIN40. STUDY IN END-STAGE RENAL DISEASE PATIENTS ON HAEMODIALYSIS

Objective: Evidence on cellular/molecular mechanisms leading to atrial fibrillation (AF) are scanty. Recently, it has been proposed a role for Rho Kinase (ROCK) pathway in terms of increased expression of ROCK, and myosin-phosphatase-target-subunit-1 (MYPT-1), downstream target of ROCK pathway, which correlated with the increased expression of Connexin40 (Cx40), an integral membrane protein of heart gap junctions, fundamental for the rapid cell-cell transfer of action potential. AF is the most frequent arrhythmia in CKD and dialysis patients in which we observed increased MYPT-1 phosphorylation which correlated with their LV mass. Given the ROCK established role in cardiovascular-renal remodelling, induction of impairment of cell-to-cell coupling/potential conduction leading to AF, we evaluated in dialysis patients with AF, the phosphorylation state of MYPT-1, expression of Cx40 and their relationship to support their involvement in AF. Design and method: 11 AF-dialysis patients (DPAF) under chronic bicarbonate dialysis 3-times/week (8 males, 3 females, 49–91yo), 11 sinus rhythm-dialysis patients (DP) (7 males, 4 females, 53–91yo) and 11 healthy subjects (C) (7 males, 4 females, 26–38yo) were enrolled. Mononuclear cells MYPT-1 phosphorylation state, Cx40 expression and ROCK inhibitor fasudil's effect were determined by Western blot. LV mass and left atrial systolic volume were measured by M-mode echocardiography. Results: DPAF's phospho-MYPT-1 was increased vs DP and C (1.57 ± 0.17 du vs 0.69 ± 0.04 vs 0.51 ± 0.05, p < 0.0001). MYPT-1 phosphorylation in DP was also higher vs C, p = 0.009. Cx40 was higher in DPAF (1.23 ± 0.12 vs 0.74 ± 0.03 vs 0.69 ± 0.03, p < 0.0001). DPAF's phospho-MYPT-1 correlated with Cx40 (r = 0.84, p = 0.0014), with cardiac left atrial systolic volume (r = 0.72, p = 0.013) and with LV mass (p = 0.014). Cardiac left atrial systolic volume was increased in DPAF compared with DP (44.45 ± 1,38 vs 35.09 ± 2,62, p = 0.005) and also significantly correlated with LV mass (r = 0.60, p = 0.049). In DPAF Fasudil reduced MYPT-1 phosphorylation (p < 0.01) and Cx40 expression (p = 0.03). Conclusions: These data confirm at mechanistic level a significant role of ROCK pathway, MYPT-1 and Cx40 in the mechanisms leading to AF in dialysis patients. The clarification on a mechanistic basis of this relationship/association should contribute to provide mechanistic explanations for AF generation and identify potential pharmacological targets for translations into treatment to AF.

Piezo1 regulates intestinal epithelial function by affecting the tight junction protein claudin-1 via the ROCK pathway

AimsDefective tight junctions (TJs) can induce intestinal epithelial dysfunction, which participates in various diseases such as irritable bowel syndrome. However, the mechanisms of TJ defects remain unclear. Our study revealed the role of Piezo1 in regulating intestinal epithelial function and TJs. Materials and methodsThe human colonic adenocarcinoma cell line Caco-2 were cultured on Transwell plate to form an epithelial barrier in vitro, and Piezo1 expression was manipulated using a lentivirus vector. Epithelial function was evaluated by measuring transepithelial electronic resistance (TEER) and 4-kDa FITC-dextran (FD4) transmission. TJ proteins (claudin-1, occludin, ZO-1) were evaluated by RT-PCR, western blot, and immunostaining analysis. Potential signal pathways, including the ROCK and Erk pathways, were detected. Moreover, to explore the regulatory effect of Piezo1 activity on epithelial function, inhibitors (ruthenium red, GsMTx4) and an agonist (Yoda1) were introduced both ex vivo and in vitro. Key findingsAlteration of Piezo1 expression altered epithelial function and the expression of the tight junction protein claudin-1. Piezo1 expression regulated phosphorylated ROCK1/2 expression, whereas interference on ROCK1/2 prevented the regulation of claudin-1 by Piezo1. In both Caco-2 monolayer and mouse colon epithelium, Piezo1 activity directly modulated epithelial function and permeability. SignificancePiezo1 negatively regulates epithelial barrier function by affecting the expression of claudin-1. Such regulation may be achieved partially via the ROCK1/2 pathway. Moreover, activating Piezo1 can induce epithelial dysfunction.

Abstract PD15-05: Sipa1 effects rock pathway in human breast cancer linking to HGF mediated changes in tight junction functions controlling metastasis

Abstract Background: SIPA1 (Signal-induced proliferation-associated gene 1) is a mitogen-inducible gene encoding a GTPase-activating protein for Rap1 and Rap2 and has been suggested to be involved in metastatic progression. We have previously shown SIPA1 may regulate barrier function in breast cancer cells as part of hepatocyte growth factor-determined changes and that this may be via a number of regulatory pathways, most likely via the ROCK pathway. Our aim with this current work was to definitively identify which pathway is involved. Methods: After knockdown of SIPA1 in MDA-MB-231 cells, they were treated to a number of inhibitors to proteins involved in pathways involved in TJ barrier function (ROCK, N-WASP, ARPs, WAVEs, MAPK, ERK, PLC-gamma). Following the identification of suitable inhibitor a large scale in vivo study was carried out using a tumour model of SIPA1 knockdown versus WT control was treated with/without inhibitors and immunohistochemistry (IHC) used to assess changes in tumour growth, architecture and protein expression. We also assessed the expression of SIPA-1 in a cohort of human breast cancer tissues using IHC. Results: After knockdown of SIPA1 in MDA-MB-231 cells, we subjected cells to a number of inhibitors to proteins involved in pathways involved barrier function (ROCK, N-WASP, ARPs, WAVEs, MAPK, ERK, PLC-gamma). Analysis of behaviour showed that knockdown cells no longer responded to the ROCK inhibitor (ROCKi Y-27632, p<0.05, n=10). In vivo tumour samples were assessed for expression of ROCKI, expression of phospho-ROCK, and any differences when treated with ROCKi. Control tissues showed good levels of all three proteins (SIPA1, ROCKI and phosphor-ROCK). Knockdown tissues exhibited significantly lower levels of ROCK and phosphor-ROCK (p<0.01, n=12). In control tumours, phosphor-ROCK was increased after treatment with ROCKi; conversely, in SIPA1 knockdown tumours, phosphor-ROCK was decreased after treatment with ROCKi (p<0.05). In SIPA1 knockdown tissues, ROCK and phosphor-ROCK was diffuse in staining pattern when compared to the control where the staining was located at the cell membrane. Conclusion: Our results confirm our hypothesis that SIPA1 is involved in the control of TJ in breast cancer and that this is through the ROCK pathway. Moreover, these results demonstrate that this may be due to the effect that SIPA1 has on cellular ratios of ROCK to phosphor-ROCK in breast cancer. Citation Format: Chang Liu, Fiona Ruge, Lin Ye, Gregory M. Harrison, Wen G. Jiang, Tracey A. Martin. Sipa1 effects rock pathway in human breast cancer linking to HGF mediated changes in tight junction functions controlling metastasis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-05.

Constraint-Induced Movement Therapy Promotes Neural Remodeling and Functional Reorganization by Overcoming Nogo-A/NgR/RhoA/ROCK Signals in Hemiplegic Cerebral Palsy Mice

Background. Little is known about the induction of functional and brain structural reorganization in hemiplegic cerebral palsy (HCP) by constraint-induced movement therapy (CIMT). Objective. We aimed to explore the specific molecular mechanism of functional and structural plasticity related to CIMT in HCP. Methods. The mice were divided into a control group and HCP groups with different interventions (unconstraint-induced movement therapy [UNCIMT], CIMT or siRNA-Nogo-A [SN] treatment): the HCP, HCP+UNCIMT, HCP+CIMT, HCP+SN, and HCP+SN+CIMT groups. Rotarod and front-limb suspension tests, immunohistochemistry, Golgi-Cox staining, transmission electron microscopy, and Western blot analyses were applied to measure motor function, neurons and neurofilament density, dendrites/axon areas, myelin integrity, and Nogo-A/NgR/RhoA/ROCK expression in the motor cortex. Results. The mice in the HCP+CIMT group had better motor function, greater neurons and neurofilament density, dendrites/axon areas, myelin integrity, and lower Nogo-A/NgR/RhoA/ROCK expression in the motor cortex than the HCP and HCP+UNCIMT groups (P < .05). Moreover, the expression of Nogo-A/NgR/RhoA/ROCK, the improvement of neural remodeling and motor function of mice in the HCP+SN group were similar to those in the HCP+CIMT group (P > .05). The neural remodeling and motor function of the HCP+SN+CIMT group were significantly greater than those in the HCP+SN and HCP+CIMT groups (P < .05). Motor function were positively correlated with the density of neurons (r = 0.450 and 0.309, respectively; P < .05) and neurofilament (r = 0.717 and 0.567, respectively; P < .05). Conclusions. CIMT might promote the remodeling of neurons, neurofilament, dendrites/axon areas, and myelin in the motor cortex by partially inhibiting the Nogo-A/NgR/RhoA/ROCK pathway, thereby promoting the improvement of motor function in HCP mice.

Crocetin Exerts a Cardio-protective Effect on Mice with Coxsackievirus B3-induced Acute Viral Myocarditis.

Previous research has proven that coxsackievirus B3 (CVB3) is broadly considered virus used in the experimental model of animals, which causes myocarditis in humans. To investigate whether there exists a cardio-protective effect of crocetin in an experimental murine model of acute viral myocarditis (AVM). Male BALB/c mice were randomly assigned to three groups: control, myocarditis treated with placebo and myocarditis treated with crocetin (n = 40 animals per group). Myocarditis was established by intraperitoneal injection with CVB3. Twenty-four hours after infection, crocetin was intraperitoneally administered for 14 consecutive days. Twenty mice were randomly selected from each group to monitor a 14-day survival rate. On day 7 and day 14, eight surviving mice from each group were sacrificed and their hearts and blood were obtained to perform serological and histological examinations. Expression of ROCKs, interleukin-17 (IL-17), interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), RORγt, and Foxp3 was quantified by RT-PCR. Plasma levels of TNFα, IL-1β and IL-17 were measured by ELISA. In addition, protein levels of IL-17 and ROCK2 in cardiac tissues were analyzed by Western blot. Crocetin treatment significantly increased survival, attenuated myocardial necrotic lesions, reduced CVB3 replication and expression of ROCK2 and IL-17 in the infected hearts. ROCK pathway inhibition was cardio-protective in viral myocarditis with increased survival, decreased viral replication, and inflammatory response. These findings suggest that crocetin is a potential therapeutic agent for patients with viral myocarditis.

Non-Canonical WNT5A Signaling Through RYK Contributes to Aggressive Phenotype of the Rheumatoid Fibroblast-Like Synoviocytes

We hypothesized that WNT5A could contribute to the enhanced migration and invasiveness of rheumatoid arthritis fibroblast-like synoviocytes (RA FLS), which is one of the incompletely understood aspects of the RA FLS aggressive phenotype. This hypothesis is based on the previous evidence of a WNT5A role in both, RA and cell migration. Migration and invasion of RA FLS were assessed after incubation with recombinant Wnt5a (rWnt5a) or silencing of the endogenous WNT5A expression. The expression of WNT5A, WNT receptors, cytokines, chemokines, and metalloproteinases was quantified with RT-PCR. The WNT pathway was explored with gene silencing, antibody and pharmacological inhibition followed by migration assays and phosphoprotein western blots. Here, we reported that rWnt5a promoted migration and invasion of RA FLS, whereas knockdown of the endogenous WNT5A reduced them. These effects were specific to the RA FLS since they were not observed in FLS from osteoarthritis (OA) patients. Also, rWnt5a induced the expression of IL6, IL8, CCL2, CXCL5, MMP1, MMP3, MMP9, and MMP13 from baseline or potentiating the TNF induction, WNT5A signaling required the RYK receptor and was mediated through the WNT/Ca2+ and the ROCK pathway. These pathways involved the RYK and ROCK dependent activation of the p38, ERK, AKT, and GSK3β kinases, but not the activation of JNK. Together these findings indicate that WNT5A contributes to the enhanced migration and invasiveness of RA FLS through RYK and the specific activation of ROCK and downstream kinases.

A novel mechanism of inhibiting in-stent restenosis with arsenic trioxide drug-eluting stent: Enhancing contractile phenotype of vascular smooth muscle cells via YAP pathway

ObjectiveArsenic trioxide (ATO or As2O3) has beneficial effects on suppressing neointimal hyperplasia and restenosis, but the mechanism is still unclear. The goal of this study is to further understand the mechanism of ATO's inhibitory effect on vascular smooth muscle cells (VSMCs). Methods and resultsThrough in vitro cell culture and in vivo stent implanting into the carotid arteries of rabbit, a synthetic-to-contractile phenotypic transition was induced and the proliferation of VSMCs was inhibited by ATO. F-actin filaments were clustered and the elasticity modulus was increased within the phenotypic modulation of VSMCs induced by ATO in vitro. Meanwhile, Yes-associated protein (YAP) nuclear translocation was inhibited by ATO both in vivo and in vitro. It was found that ROCK inhibitor or YAP inactivator could partially mask the phenotype modulation of ATO on VSMCs. ConclusionsThe interaction of YAP with the ROCK pathway through ATO seems to mediate the contractile phenotype of VSMCs. This provides an indication of the clinical therapeutic mechanism for the beneficial bioactive effect of ATO-drug eluting stent (AES) on in-stent restenosis (ISR).

Abstract 3530: Investigating aneuploidy, CIN and mechanisms of DNA content reduction in cancer

Abstract Chromosomal instability (CIN) is a form of genomic instability that refers to a dynamic state in which the cells are constantly changing the number and/or the structure of their chromosomes. CIN leads to aneuploidy, a state in which a cell's set of chromosomes deviates from the euploid set. Causes of aneuploidy include errors during DNA replication, mitosis and cell fusion events. Furthermore, aneuploidy has been shown to promote tumorigenesis and tumor progression, but also to reduce cell fitness or induce cell death, especially when high levels of aneuploidy are present. Nevertheless, antimitotic drugs that induce high levels of aneuploidy, such as Paclitaxel, are not always effective in the clinic. In this study, we aim to investigate the mechanisms by which cancer cells tolerate aneuploidy, to elucidate the mechanisms by which cell fusion can lead to aneuploidy and resistance to paclitaxel and to identify new targets in aneuploidy and CIN. Towards this direction, aneuploid clones of pseudo-diploid cancer cells were generated after treatment with anti-mitotic drugs and single cell sorting and were characterized by cell cycle profile, metaphase spreads and CGH or SNP profile. Furthermore, CIN was induced by inhibition of CENP-E or ATM or impairment of the SWI/ SNF complex and quantified by induction of chromosome missegregation and micronuclei formation. Interestingly, several near tetraploid clones that were isolated after anti-mitotic drug treatment, reduced their DNA content to near diploid during propagation in culture. Thus, we hypothesize that cancer cells may utilize specific mechanisms to change their DNA content and maintain tolerable levels of aneuploidy. By time-lapse microscopy, we observed that cells treated with Paclitaxel, can increase their DNA content by abortive mitosis, cytokinesis failure or by undergoing cell fusion. Interestingly, these multinucleated cells could reduce their DNA content by undergoing independent mitosis, independent nucleus catastrophe or independent nucleus separation (cell exodus). We identify cell exodus, as a potential mechanism by which cells can recover after treatment with Paclitaxel. Inhibition of ROCK pathway, that stabilizes actin filaments and prevents cellular contractility, reduced cell fusion and exodus events and delayed cell recovery after paclitaxel treatment. Finally, in order to identify new targets in CIN and aneuploidy, we setup sgRNA loss of function screening conditions in the generated CIN and aneuploid cell models and we aim to identify genes that are essential for the survival of aneuploid and/or CIN cancer cells. Citation Format: Mara Mandelia, Kostas Drosopoulos, Spiros Linardopoulos. Investigating aneuploidy, CIN and mechanisms of DNA content reduction in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3530.