Rho-associated Coiled-coil Kinase Inhibitor Research Articles

Reduction of Phosphorylated Tau in Alzheimer's Disease Induced Pluripotent Stem Cell-Derived Neuro-Spheroids by Rho-Associated Coiled-Coil Kinase Inhibitor Fasudil.

Alzheimer's disease (AD) is the most predominant form of dementia. Rho-associated coiled coil kinase (ROCK) inhibitor, fasudil, is one of the candidate drugs against the AD progression. We aimed to investigate possible changes of AD associated markers in three-dimensional neuro-spheroids (3D neuro-spheroids) generated from induced pluripotent stem cells derived from AD patients or healthy control subjects (HC) and to determine the impact of pharmacological intervention with the ROCK inhibitor fasudil. We treated 3D neuro-spheroids with fasudil and tested the possible effect on AD markers by ELISA, transcriptomic and proteomic analyses. Transcriptomic analysis revealed a reduction in the expression of AKT serine/threonine-protein kinase 1 (AKT1) in AD neuro-spheroids, compared to HC. This decrease was reverted in the presence of fasudil. Proteomic analysis showed up- and down-regulation of proteins related to AKT pathway in fasudil-treated neuro-spheroids. We found an evident increase of phosphorylated tau at four different residues (pTau181, 202, 231, and 396) in AD compared to HC-derived neuro-spheroids. This was accompanied by a decrease of secreted clusterin (clu) and an increase of intracellular clu levels in AD patient-derived neuro-spheroids. Increases of phosphorylated tau in AD patient-derived neuro-spheroids were suppressed in the presence of fasudil. Fasudil modulates clu protein levels and enhances AKT1 that results in the suppression of AD associated tau phosphorylation.

Rho-associated coiled-coil kinase contributes an important role in Xingnaojing injection-induced adverse drug reactions

Xingnaojing injection (XNJI) is widely used for acute cerebral hemorrhage. However, the adverse drug reactions (ADRs) caused by XNJI have attracted broad attention. In this study, mice were intravenously administrated with a single dose of XNJI (10 or 20 mL/kg) to evaluate the vascular permeability. Then, pathological examinations on ears and lungs were performed to further determine the characteristics of ADRs induced by XNJI. In addition, human umbilical vein endothelial cells (HUVECs) were stimulated with XNJI (1/128, 1/64, and 1/32 XNJI pure product dilution) and stained with rhodamine-phalloidin to analyze the alterations on cytoskeleton. In addition, endothelial monolayer was incubated with XNJI and the effect on permeability was tested. Western blot testing was conducted to measure the involvement of Rho-associated coiled-coil kinase (ROCK) in XNJI-induced ADRs and fasudil hydrochloride was applied to additionally confirm the contribution of ROCK. The results indicated that XNJI may lead to dose-associated vascular leakage. None visibly abnormal symptom was noticed in the mice treated with XNJI at 10 mL/kg, while inflammation in ears and lungs was observed in the mice administrated with 20 mL/kg XNJI. In in vitro experiment, stimulation with high concentration of XNJI (1/32 XNJI pure product dilution) could enhance the permeability of confluent endothelial monolayer and induce rearrangement of actin cytoskeleton. Activation of ROCK, which was shown as augmented expressions of p-MLC2 and p-MYPT1, was observed in cells and mice treated with XNJI. ROCK inhibitor could attenuate XNJI-induced dysfunction of endothelial barrier in in vitro experiment and alleviate the vascular leakage and inflammation noticed in mice treated with XNJI. This study indicates that overdose of XNJI can destroy vascular endothelial function and induce increased vascular permeability, which may cause ADRs in clinical practice. Meanwhile, ROCK plays an important role in XNJI-induced ADRs.

Crystal structure of the Rho-associated coiled-coil kinase 2 inhibitor belumosudil bound to CK2α.

The small molecule belumosudil was initially identified as a selective inhibitor of Rho-associated coiled-coil kinase 2 (ROCK2) and has recently been approved for the treatment of graft-versus-host disease. However, recent studies have shown that many of the phenotypes displayed upon treatment with belumosudil were due to CK2α inhibition. CK2α is in itself a very promising therapeutic target for a range of conditions and has recently been put forward as a potential treatment for COVID-19. Belumosudil presents a promising starting point for the development of future CK2α inhibitors as it provides a safe, potent and orally bioavailable scaffold. Therefore, several of the major hurdles in drug development have already been overcome. Here, the crystal structure of belumosudil bound to the ATP site of CK2α is presented. This crystal structure combined with modelling studies further elucidates how belumosudil could be developed into a selective and potent CK2α or ROCK2 inhibitor.

Exploring the therapeutic promise of targeting Rho kinase in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a prevalent systemic autoimmune disease caused by dysregulated inflammatory reactions, T lymphocyte invasion into the joints, and articular thickening. Immune cells, primarily tumor necrosis factor-alpha (TNF-α) and chemokines (interleukin or IL-1), which are predominantly generated by activated macrophages cells, have also been involved with the pathogenesis of rheumatoid arthritis. Rho GTPases are integral factors of biochemical cascades utilized by antigens, and also by cellular receptors, cytokines, and chemokines, to modulate inflammatory reactions, according to growing data. The Rho family is a group of G proteins that govern a variety of biological and physiological activities such as mobility, actin stress fiber production, growth, and polarity. Research suggests that the Rho A and Rho-associated coiled-coil kinase (ROCK) regulatory cascade could be essential in several autoimmune conditions, including RA. ROCK is activated in the synovial of rheumatoid arthritis patients, while the blocking of ROCK with fasudil could also decrease IL-6, TNF-α, and IL-1. This review covers current developments in understanding the overactivation of Rho enzyme activity in RA suppressed by ROCK inhibitors which can be utilized for the treatment of autoimmune disease. We offer an outline of the function of ROCK inhibitors in immune cells and discuss findings which emphasize the rising participation of this category of kinases within the pathological process of autoimmune disorders. Assuming the potential ability of ROCK as a therapeutic, we define approaches that might be used to inhibit Rho kinase activity in rheumatoid disorders.

EpCAM (CD326) Regulates Intestinal Epithelial Integrity and Stem Cells via Rho-Associated Kinase.

Humans with biallelic inactivating mutations in Epithelial Cell Adhesion Molecule (EpCAM) develop congenital tufting enteropathy (CTE). To gain mechanistic insights regarding EpCAM function in this disorder, we prepared intestinal epithelial cell (IEC) organoids and spheroids. IEC organoids and spheroids were generated from ROSA-CreERT2 EpCAMfl/fl mice. Proliferation, tight junctions, cell polarity and epithelial integrity were assessed in tamoxifen-induced EpCAM-deficient organoids via confocal immunofluorescence microscopy and Western blotting. Olfm4-expressing stem cells were assessed in IEC cells in vitro and in vivo via fluorescence in situ hybridization. To determine if existing drugs could ameliorate effects of EpCAM deficiency in IEC cells, a variety of pharmacologic inhibitors were screened. Deletion of EpCAM resulted in increased apoptosis and attenuated growth of organoids and spheroids. Selected claudins were destabilized and epithelial integrity was severely compromised. Epithelial integrity was improved by treatment with Rho-associated coiled-coil kinase (ROCK) inhibitors without restoration of claudin expression. Correspondingly, enhanced phosphorylation of myosin light chain, a serine/threonine ROCK substrate, was observed in EpCAM-deficient organoids. Strikingly, frequencies of Olfm4-expressing stem cells in EpCAM-deficient IEC cells in vitro and in vivo were decreased. Treatment with ROCK inhibitors increased numbers of stem cells in EpCAM-deficient organoids and spheroids. Thus, EpCAM regulates intestinal epithelial homeostasis via a signaling pathway that includes ROCK.

Targeting ROCK2 isoform with its widely used inhibitors for faster post-stroke recovery

Recovery after ischemic stroke is slow and highly variable. Activated ROCK (Rho-associated coiled-coil kinase) pathway hampers recovery of impaired neurons. Though inhibiting ROCK pathway has shown therapeutic effects in vitro, the selectivity of most of the ROCK inhibitors is still not investigated. Present study aims to investigate the binding affinity in silico of nine widely used ROCK inhibitors with brainspecific ROCK2 isoform. Three-dimensional structures of ROCK2 and eight drugs were taken from Protein Data Bank and PubChem Chemical Compound Database, respectively, whereas, FSD-C10 structure was generated based on Xin et al., 2015. In docking, ROCK2 was set to be rigid and drugs were free to rotate. All simulations were carried out using AutoDock 4.2. This study demonstrated strong complexation between all ligands and ROCK2. All ROCK inhibitors, except FSD-C10, were able to bind to ROCK2 more strongly [Binding constant (Ka) between 2.6 – 36.7 × 105 M−1] than fasudil (Ka = 2.5 × 105 M−1). SLx-2119 (KD-025) had the highest binding constant (Ka = 36.7 × 105 M−1) thus succeeding as a better ROCK2 specific inhibitor. Selectivity of ROCK inhibitors (in silico) towards ROCK2 can be an indicative measure to estimate therapeutic benefits or adverse effects prior to in vitro study.

ROCK inhibitors enhance bone healing by promoting osteoclastic and osteoblastic differentiation

Osteoclast and osteoblast are essential for proper bone development and remodeling as well as recovery of bone fracture. In this study, we seek chemical compounds that enhance turnover of bone metabolism for promoting bone healing. First, we screen a chemical library which includes 378 compounds by using murine pre-osteoclastic RAW264.7 cells to identify compounds that promote osteoclastic differentiation. We find that two ROCK (Rho-associated coiled-coil kinase) inhibitors, HA-1077 (Fasudil) and Y-27632, enhance osteoclastogenesis. Subsequently, we identify that these two compounds also increase osteoblastic differentiation of MC3T3-E1 cells. Finally, our in vivo experiment shows that the local administration of ROCK inhibitors accelerate the bone healing of the rat calvarial defect.

P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK

Optimal levels of reactive oxygen species (ROS) play a critical role in cellular physiological function. For production of intracellular superoxide, NADPH oxidase is one of the sources. Rac1/2 and RhoA GTPases are involved in regulation of NADPH oxidase activity and Tyr42 phosphorylation of RhoA (p-Tyr42 RhoA) seems significant in this regard as it was recently shown that hydrogen peroxide was able to increase p-Tyr42 RhoA levels. Phorbol myristate acetate (PMA), a tumor promoter, also induces production of superoxides; PMA activates Src, a tyrosine kinase, and increases p-Tyr42 RhoA levels. In exploring the mechanism of PMA effects, we reduced RhoA levels in test cells with si-RhoA and then restoration of various versions of RhoA for effect in response of the cells to PMA and producing superoxides. Restoration of RhoA Y42F (a dephospho-mimic form) still had reduced superoxide formation in response to PMA, compared with WT and Y42E RhoA. This was similarly seen with assays for cell migration and proliferation with cells responding to PMA. Y27632, a ROCK (Rho associated coiled coil kinase) inhibitor, also inhibited superoxide production, and also reduced p-Y416 Src and p-p47phox levels. A ROCK active fragment was also able to phosphorylate p47phox at Ser345 residue (p-Ser345 p47phox), a component of NADPH oxidase. Overall, we demonstrate that p-Tyr42 RhoA levels increase following PMA treatment and this is through production of superoxide and activation of Src. These in turn amplify superoxide production through ROCK phophorylation of p47phox and maintain a positive feedback loop for superoxide generation, and contribute to tumor progression.

Optimal concentration of Rho-associated coiled-coil kinase (ROCK) inhibitor improved sperm membrane functionality and fertilizing ability of cryopreserved-thawed feline sperm

Sperm cryopreservation induces irreversible loss of viability and fertilizing ability. This study aimed at examining the effects of Rho-associated, coiled-coil kinase (ROCK) inhibitor on quality of frozen-thawed feline sperm. Ejaculated semen from individual cats (n = 6) was examined for the expression of LIMK1 and LIMK2 mediated ROCK cascade. The effects of ROCK inhibitor during cooling and cryopreservation on sperm quality and fertilizing ability were also examined. Feline sperm were treated with different concentrations of ROCK inhibitor (10, 20 and 40 μM) during cooling at 4 °C and cryopreservation. Sperm cooled and conventionally cryopreserved without ROCK inhibitor (0 μM) served as a control group.The ROCK cascade was confirmed in feline sperm as they expressed mRNA of LIMK1 and LIMK2 genes. Cryopreservation significantly reduced sperm quality in terms of viability (91.63 ± 3.96 vs. 60.11 ± 8.93), progressive motility (91.67 ± 3.54 vs. 46.67 ± 8.66) and acrosome integrity (93.49 ± 3.64 vs. 63.81 ± 5.31) for fresh and frozen-thawed sperm, respectively (p < 0.05). The positive effects of ROCK inhibitor on sperm quality were pronounced at 1 and 3 h post-thaw. ROCK inhibitor at 10 μM significantly improved sperm motility and membrane functionality compared to those observed in a control group (0 μM) (p < 0.05). In vitro fertilization revealed that supplement ROCK inhibitor at 10 μM during cryopreservation significantly improved in vitro fertilizing ability of the frozen-thawed sperm (p < 0.05). However, it did not subsequently increase morula and blastocyst rates (p > 0.05). Increased concentrations of ROCK inhibitor to 20 and 40 μM did not further improve the quality of frozen-thawed sperm. In conclusion, an optimal concentration (10 μM) of the ROCK inhibitor added into cooling medium could improve post-thaw sperm quality.

Overexpression of Na+/H+ exchanger 1 specifically induces cell death in human iPS cells via sustained activation of the Rho kinase ROCK

Understanding the specific properties of human induced pluripotent stem cells (iPSCs) is important for quality control of iPSCs. Having incidentally discovered that overexpression of plasma membrane Na+/H+ exchanger 1 (NHE1) induces cell death in iPSCs, we investigated the mechanism of NHE1-induced cell death. Doxycycline-induced NHE1 overexpression arrested cell growth, and nearly all cells were killed by a necrotic process within 72 h. NHE1 overexpression led to sustained activation of Rho-associated coiled-coil kinase (ROCK), accompanied by dramatic changes in cell shape, cell elongation, and swelling of peripheral cells in iPSC colonies, as well as marked stress fiber formation. The ROCK inhibitor Y27632 reduced NHE1-induced cell death. ROCK-dependent phenotypes were suppressed by a loss-of-function mutation of NHE1 and inhibited by an inhibitor of NHE1 activity, indicating that NHE1-mediated transport activity is required. Moreover, ROCK was activated by trimethylamine treatment-mediated cytosolic alkalinization and accumulated in the plasma membrane near NHE1 in peripheral iPSCs of cell colonies. By contrast, cell death did not occur in mesendoderm-like cells that had differentiated from iPSCs, indicating that the NHE1-mediated effects were specific for iPSCs. These results suggest that NHE1 overexpression specifically induces death of iPSCs via sustained ROCK activation, probably caused by an increase in local pH near NHE1. Finally, monensin, a Na+/H+ exchange ionophore, selectively killed iPSCs, suggesting that monensin could help eliminate iPSCs that remain after differentiation, a strategy that might be useful for improving regenerative medicine.

Regenerative potential of mouse embryonic stem cell-derived PDGFRα+ cardiac lineage committed cells in infarcted myocardium.

BACKGROUNDPluripotent stem cell-derived cardiomyocytes (CMs) have become one of the most attractive cellular resources for cell-based therapy to rescue damaged cardiac tissue.AIMWe investigated the regenerative potential of mouse embryonic stem cell (ESC)-derived platelet-derived growth factor receptor-α (DGFRα)+ cardiac lineage-committed cells (CLCs), which have a proliferative capacity but are in a morphologically and functionally immature state compared with differentiated CMs.METHODSWe induced mouse ESCs into PDGFRα+ CLCs and αMHC+ CMs using a combination of the small molecule cyclosporin A, the rho-associated coiled-coil kinase inhibitor Y27632, the antioxidant Trolox, and the ALK5 inhibitor EW7197. We implanted PDGFRα+ CLCs and differentiated αMHC+ CMs into a myocardial infarction (MI) murine model and performed functional analysis using transthoracic echocardiography (TTE) and histologic analysis.RESULTSCompared with the untreated MI hearts, the anterior and septal regional wall motion and systolic functional parameters were notably and similarly improved in the MI hearts implanted with PDGFRα+ CLCs and αMHC+ CMs based on TTE. In histologic analysis, the untreated MI hearts contained a thinner ventricular wall than did the controls, while the ventricular walls of MI hearts implanted with PDGFRα+ CLCs and αMHC+ CMs were similarly thicker compared with that of the untreated MI hearts. Furthermore, implanted PDGFRα+ CLCs aligned and integrated with host CMs and were mostly differentiated into α-actinin+ CMs, and they did not convert into CD31+ endothelial cells or αSMA+ mural cells.CONCLUSIONPDGFRα+ CLCs from mouse ESCs exhibiting proliferative capacity showed a regenerative effect in infarcted myocardium. Therefore, mouse ESC-derived PDGFRα+ CLCs may represent a potential cellular resource for cardiac regeneration.

TRIM59 promotes breast cancer motility by suppressing p62-selective autophagic degradation of PDCD10.

Cancer cells adopt various modes of migration during metastasis. How the ubiquitination machinery contributes to cancer cell motility remains underexplored. Here, we report that tripartite motif (TRIM) 59 is frequently up-regulated in metastatic breast cancer, which is correlated with advanced clinical stages and reduced survival among breast cancer patients. TRIM59 knockdown (KD) promoted apoptosis and inhibited tumor growth, while TRIM59 overexpression led to the opposite effects. Importantly, we uncovered TRIM59 as a key regulator of cell contractility and adhesion to control the plasticity of metastatic tumor cells. At the molecular level, we identified programmed cell death protein 10 (PDCD10) as a target of TRIM59. TRIM59 stabilized PDCD10 by suppressing RING finger and transmembrane domain-containing protein 1 (RNFT1)-induced lysine 63 (K63) ubiquitination and subsequent phosphotyrosine-independent ligand for the Lck SH2 domain of 62 kDa (p62)-selective autophagic degradation. TRIM59 promoted PDCD10-mediated suppression of Ras homolog family member A (RhoA)-Rho-associated coiled-coil kinase (ROCK) 1 signaling to control the transition between amoeboid and mesenchymal invasiveness. PDCD10 overexpression or administration of a ROCK inhibitor reversed TRIM59 loss-induced contractile phenotypes, thereby accelerating cell migration, invasion, and tumor formation. These findings establish the rationale for targeting deregulated TRIM59/PDCD10 to treat breast cancer.

FSD-C10 Shows Therapeutic Effects in Suppressing oxidized low-density lipoprotein (ox-LDL)-Induced Human Brain Microvascular Endothelial Cells Apoptosis via Rho-Associated Coiled-Coil Kinase (ROCK)/Mitogen-Activated Protein Kinase (MAPK) Signaling.

Backgroundox-LDL-induced injury of brain microvascular endothelial cells (BMECs) is strongly associated with cerebral vascular diseases such as cerebral arterial atherosclerosis. ROCK inhibitor was proved to be anti-apoptotic and has been used in treating cerebral vascular diseases. Research on the neuroprotective effects of a novel ROCK inhibitor, FSD-C10, is still limited. The present study investigated the anti-apoptotic effect and underlying molecular mechanism of FSD-C10 in ox-LDL-mediated apoptosis of BMECs.Material/Methodsox-LDL and/or FSD-C10 were used to incubate immortalized human BMECs. MTT assay was used to assess cell viability. Cell apoptosis was evaluated by TUNEL assay. A colorimetric method was used to assess ROCK activity. Western blot analysis was used to examine the expression and phosphorylation levels of proteins.Resultsox-LDL incubation reduced the viability of BMECs by inducing cell apoptosis in a concentration-dependent manner. ROCK activity was also elevated by ox-LDL incubation in BMECs in a concentration-dependent manner. Expression level of Bcl2 was reduced while expression levels of Bax and active caspase3 were increased by ox-LDL treatment in a concentration-dependent manner. ox-LDL also increased the phosphorylation levels of p38, JNK, and ERK1/2 in a concentration-dependent manner. FSD-C10 treatment increased the cell viability by reducing apoptosis of BMECs exposed to ox-LDL. Moreover, FSD-C10 was found to suppress the phosphorylation levels of p38, JNK, and ERK1/2 and the expression levels of Bax and active caspase3 in ox-LDL treated BMECs.ConclusionsFSD-C10 increases cell viability in ox-LDL-treated BMECs by reducing cell apoptosis. ROCK/MAPKs-mediated apoptosis appears to be the underlying molecular mechanism.

Staphylococcus aureus impairs sinonasal epithelial repair: Effects in patients with chronic rhinosinusitis with nasal polyps and control subjects

The effect of Staphylococcus aureus on nasal epithelial repair has never been assessed in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). This study aimed to determine whether (1) nasal epithelial cell cultures from patients with CRSwNP and control subjects repair differently; (2) S aureus exoproducts compromise nasal epithelial repair; (3) S aureus alters lamellipodial dynamics; and (4) deleterious effects could be counteracted by the Rho-associated coiled-coil kinase inhibitor Y-27632. Primary nasal epithelial cells (pNECs) collected during surgeries were cultured and injured under 3 conditions: (1) basal conditions, (2) exposed to S aureus exoproducts, and (3) exposed to S aureus exoproducts and Y-27632. Epithelial repair, lamellipodial dynamics, and cytoskeletal organization were assessed. Under basal conditions, pNEC cultures from patients with CRSwNP presented significantly lower repair rates and reduced lamellipodial protrusion length and velocity than those from control subjects. S aureus exoproducts significantly decreased repair rates and protrusion dynamics in both control subjects and patients with CRSwNP; however, the effect of S aureus on cell protrusions was more sustained over time in patients with CRSwNP. Under basal conditions, immunofluorescence assays showed significantly reduced percentages of cells with lamellipodia at the wound edge in patients with CRSwNP compared with control subjects. S aureus altered cell polarity and decreased the percentage of cells with lamellipodia in both groups. Finally, Y-27632 prevented the deleterious effects of S aureus exoproducts on CRSwNP repair rates, as well as on lamellipodial dynamics and formation. S aureus exoproducts significantly alter epithelial repair and lamellipodial dynamics on pNECs, and this impairment was more pronounced in patients with CRSwNP. Importantly, Y-27632 restored epithelial repair and lamellipodial dynamics in the presence of S aureus exoproducts.

Rho-Associated Coiled-Coil Kinase (ROCK) in Molecular Regulation of Angiogenesis.

Identified as a major downstream effector of the small GTPase RhoA, Rho-associated coiled-coil kinase (ROCK) is a versatile regulator of multiple cellular processes. Angiogenesis, the process of generating new capillaries from the pre-existing ones, is required for the development of various diseases such as cancer, diabetes and rheumatoid arthritis. Recently, ROCK has attracted attention for its crucial role in angiogenesis, making it a promising target for new therapeutic approaches. In this review, we summarize recent advances in understanding the role of ROCK signaling in regulating the permeability, migration, proliferation and tubulogenesis of endothelial cells (ECs), as well as its functions in non-ECs which constitute the pro-angiogenic microenvironment. The therapeutic potential of ROCK inhibitors in angiogenesis-related diseases is also discussed.

Glibenclamide inhibits NLRP3 inflammasome-mediated IL-1β secretion in human trophoblasts

Infection-associated pregnancy complications cause premature delivery. Caspase-1 is involved in the maturation of interleukin (IL)-1β, which is activated by the NLRP3 inflammasome. To characterize the significance of the NLRP3 inflammasome pathway in the placenta, the effects of activators and inhibitors on NLRP3-related molecules were examined using isolated primary trophoblasts. Caspase-1 and IL-1β mRNA expression was markedly increased in response to lipopolysaccharide (LPS), a toll-like receptor (TLR)4 ligand. Treatment with the potassium ionophore nigericin significantly increased the level of activated caspase-1. Treatment with either LPS or nigericin stimulated IL-1β secretion, whereas pretreatment with the ATP-sensitive K+ channel inhibitor glibenclamide, the Rho-associated coiled-coil kinase inhibitor Y-27632, or a caspase-1 inhibitor significantly decreased nigericin-induced IL-1β secretion. In addition, dibutyryl-cAMP, which induces trophoblast differentiation, decreased expression of NLRP3, caspase-1, and IL-1β. These findings suggest that trophoblasts can secrete IL-1β through the NLRP3/caspase-1 pathway, which is suppressed by glibenclamide, and that the TLR4-mediated NLRP3 inflammasome pathway is more likely to be stimulated in undifferentiated than differentiated trophoblasts. Our data support the hypothesis that inhibition of the NLRP3 inflammasome can suppress placental inflammation-associated disorders.

The role of ras homolog family/ras homolog family kinase signaling pathway and its inhibitors in the optic nerve disease

Ras homolog family (Rho)/ Rho-associated coiled-coil kinase (ROCK) signaling pathway widely exists in human and mammal cells, which is closely related to inhibition of repair after optic nerve damage. The expression level of Rho/ROCK signaling pathway-related proteins is up-regulated in glaucoma, and related with the death of retinal ganglionic cell (RGC) and the axon activity. ROCK inhibitors can protect the surviving RGC and promote axon extension with a dose-dependent manner. ROCK inhibitors also can inhibit glial scar formation, lower intraocular pressure and inhibit inflammatory response to some degrees. Rho/ROCK signaling pathway correlates with the optic nerve disease progression, and ROCK inhibitors hope to become a new therapeutic drug. Key words: Optic nerve diseases/etiology; Optic nerve diseases/therapy; G-protein-coupled receptor kinase 1/antagonists & inhibitors; Review

Generation of PDGFR\u03b1+ Cardioblasts from Pluripotent Stem Cells

Isolating actively proliferating cardioblasts is the first crucial step for cardiac regeneration through cell implantation. However, the origin and identity of putative cardioblasts are still unclear. Here, we uncover a novel class of cardiac lineage cells, PDGFRα+Flk1− cardioblasts (PCBs), from mouse and human pluripotent stem cells induced using CsAYTE, a combination of the small molecules Cyclosporin A, the rho-associated coiled-coil kinase inhibitor Y27632, the antioxidant Trolox, and the ALK5 inhibitor EW7197. This novel population of actively proliferating cells is cardiac lineage–committed but in a morphologically and functionally immature state compared to mature cardiomyocytes. Most important, most of CsAYTE-induced PCBs spontaneously differentiated into functional αMHC+ cardiomyocytes (M+CMs) and could be a potential cellular resource for cardiac regeneration.

Single cell time-lapse analysis reveals that podoplanin enhances cell survival and colony formation capacity of squamous cell carcinoma cells

Tumor initiating cells (TICs) are characterized by high clonal expansion capacity. We previously reported that podoplanin is a TIC-specific marker for the human squamous cell carcinoma cell line A431. The aim of this study is to explore the molecular mechanism underlying the high clonal expansion potential of podoplanin-positive A431cells using Fucci imaging. Single podoplanin-positive cells created large colonies at a significantly higher frequency than single podoplanin-negative cells, whereas no difference was observed between the two types of cells with respect to cell cycle status. Conversely, the cell death ratio of progenies derived from podoplanin-positive single cell was significantly lower than that of cells derived from podoplanin-negative cells. Single A431 cells, whose podoplanin expression was suppressed by RNA interference, exhibited increased cell death ratios and decreased frequency of large colony forming. Moreover, the frequency of large colony forming decreased significantly when podoplanin-positive single cells was treated with a ROCK (Rho-associated coiled-coil kinase) inhibitor, whereas no difference was observed in single podoplanin-negative cells. Our current study cleared that high clonal expansion capacity of podoplanin-positive TICs populations was the result of reduced cell death by podoplanin-mediated signaling. Therefore, podoplanin activity may be a therapeutic target in the treatment of squamous cell carcinomas.

Fasudil, an inhibitor of Rho-associated coiled-coil kinase, improves cognitive impairments induced by smoke exposure.

The current study was designed to investigate the pathological changes in brain induced by smoke exposure, and explore whether fasudil could alleviate these impairments.Adult C57BL/6 mice were exposed to tobacco smoking for four months, and fasudil was treated from the third months. To investigate lung injuries, the immunohistochemistry of lung tissue, immune cell infiltrations, cytokine productions in bronchoalveolar lavage (BAL) fluid, and seurm inflammatory cytokines were evaluated. To investigate cognitive impairments, Morris water maze test, hippocampal inflammatory cytokines and Rho associated signaling pathways were evaluated.Our findings showed fasudil administration inhibited the inflitration of inflammatory cells (macrophages, neutrophils, and lymphocytes), suppressed the production of inflammatory cytokines both in the BAL fluid, serum, and hippocampus. Further, fasudil significantly improved the spatial learning and memory impairments and reduced the elevation of hippocampal inflammatory cytokines induced by tobacco smoking. Of note, expressions of RhoA, ROCK1, ROCK2, caspase-3, caspase-9, bax and the phosphorylation of NF-κBp65 were increased accompanying the smoke exposure-induced cognitive impairments, which were significantly inhibited by fasudil treatment as indicted in western blot and immunohistochemistry analysis.Our results showed that fasudil exhibited protective effects on smoke exposure induced cognitive deficits which might involve with the regulation of Rho/ROCK/NF-κB pathways. Further studies are warranted before clinical application of fasudil.