RhoA regulates Drp1 mediated mitochondrial fission through ROCK to protect cardiomyocytes

Cardiac ischemia/reperfusion, loss of blood flow and its subsequent restoration, causes damage to the heart. Oxidative stress from ischemia/reperfusion leads to dysfunction and death of cardiomyocytes, increasing the risk of progression to heart failure. Alterations in mitochondrial dynamics, in particular mitochondrial fission, have been suggested to play a role in cardioprotection from oxidative stress. We tested the hypothesis that activation of RhoA regulates mitochondrial fission in cardiomyocytes. Our studies show that expression of constitutively active RhoA in cardiomyocytes increases phosphorylation of Dynamin-related protein 1 (Drp1) at serine-616, and leads to localization of Drp1 at mitochondria. Both responses are blocked by inhibition of Rho-associated Protein Kinase (ROCK). Endogenous RhoA activation by the GPCR agonist sphingosine-1-phosphate (S1P) also increases Drp1 phosphorylation and its mitochondrial translocation in a RhoA and ROCK dependent manner. Consistent with the role of mitochondrial Drp1 in fission, RhoA activation in cardiomyocytes leads to formation of smaller mitochondria and this is attenuated by inhibition of ROCK, by siRNA knockdown of Drp1 or by expression of a phosphorylation-deficient Drp1 S616A mutant. In addition, activation of RhoA prevents cell death in cardiomyocytes challenged by oxidative stress and this protection is blocked by siRNA knockdown of Drp1 or by Drp1 S616A expression. Taken together our findings demonstrate that RhoA activation can regulate Drp1 to induce mitochondrial fission and subsequent cellular protection, implicating regulation of fission as a novel mechanism contributing to RhoA-mediated cardioprotection.     

MALAT1/miR-185-5p mediated high glucose-induced oxidative stress,mitochondrial injury and cardiomyocyte apoptosis via the RhoA/ROCK pathway

To explore the underlying mechanism of lncRNA MALAT1 in the pathogenesis of diabetic cardiomyopathy (DCM). DCM models were confirmed in db/db mice. MiRNAs in myocardium were detected by miRNA sequencing. The interactions of miR-185-5p with MALAT1 and RhoA were validated by dual-luciferase reporter assays. Primary neonatal cardiomyocytes were cultured with 5.5 or 30 mmol/L D-glucose (HG) in the presence or absence of MALAT1-shRNA and fasudil, a ROCK inhibitor. MALAT1 and miR-185-5p expression were determined by real-time quantitative PCR. The apoptotic cardiomyocytes were evaluated using flow cytometry and TUNEL staining. SOD activity and MDA contents were measured. The ROCK activity, phosphorylation of Drp1^S616, mitofusin 2 and apoptosis-related proteins were analysed by Western blotting. Mitochondrial membrane potential was examined by JC-1. MALAT1 was significantly up-regulated while miR-185-5p was down-regulated in myocardium of db/db mice and HG-induced cardiomyocytes. MALAT1 regulated RhoA/ROCK pathway via sponging miR-185-5p in cardiomyocytes in HG. Knockdown of MALAT1 and fasudil all inhibited HG-induced oxidative stress, and alleviated imbalance of mitochondrial dynamics and mitochondrial dysfunction, accompanied by reduced cardiomyocyte apoptosis. MALAT1 activated the RhoA/ROCK pathway via sponging miR-185-5p and mediated HG-induced oxidative stress, mitochondrial damage and apoptosis of cardiomyocytes in mice.     

Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

The diterpene triepoxide triptolide is a major active component of Tripterygium wilfordii Hook F, a popular Chinese herbal medicine with the potential to treat hematologic malignancies. In this study, we investigated the roles of triptolide in apoptosis and cell signaling events in human leukemia cell lines and primary human leukemia blasts. Triptolide selectively induced caspase-dependent cell death that was accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. Furthermore, we found that triptolide dramatically induced ROCK1 cleavage/activation and MLC and MYPT phosphorylation. ROCK1 was cleaved and activated by caspase-3, rather than RhoA. Inhibiting MLC phosphorylation by ML-7 significantly attenuated triptolide-mediated apoptosis, caspase activation, and cytochrome c release. In addition, ROCK1 inhibition also abrogated MLC and MYPT phosphorylation. Our in vivo study showed that both ROCK1 activation and MLC phosphorylation were associated with the tumor growth inhibition caused by triptolide in mouse leukemia xenograft models. Collectively, these findings suggest that triptolide-mediated ROCK1 activation and MLC phosphorylation may be a novel therapeutic strategy for treating hematological malignancies.     

RhoA/ROCK-dependent moesin phosphorylation regulates AGE-induced endothelial cellular response

Background

The role of advanced glycation end products (AGEs) in the development of diabetes, especially diabetic complications, has been emphasized in many reports. Accumulation of AGEs in the vasculature triggers a series of morphological and functional changes in endothelial cells (ECs) and induces an increase of endothelial permeability. This study was to investigate the involvement of RhoA/ROCK-dependent moesin phosphorylation in endothelial abnormalities induced by AGEs.

Methods

Using human dermal microvascular endothelial cells (HMVECs), the effects of human serum albumin modified-AGEs (AGE-HSA) on the endothelium were assessed by measuring monolayer permeability and staining of F-actin in HMVECs. Activations of RhoA and ROCK were determined by a luminescence-based assay and immunoblotting. Transfection of recombinant adenovirus that was dominant negative for RhoA (RhoA N19) was done to down-regulate RhoA expression, while adenovirus with constitutively activated RhoA (RhoA L63) was transfected to cause overexpression of RhoA in HMVECs. H-1152 was employed to specifically block activation of ROCK. Co-immunoprecipitation was used to further confirm the interaction of ROCK and its downstream target moesin. To identify AGE/ROCK-induced phosphorylation site in moesin, two mutants pcDNA3/HA-moesinT^558A and pcDNA3/HA-moesinT^558D were applied in endothelial cells.

Results

The results showed that AGE-HSA increased the permeability of HMVEC monolayer and triggered the formation of F-actin-positive stress fibers. AGE-HSA enhanced RhoA activity as well as phosphorylation of ROCK in a time- and dose-dependent manner. Down-regulation of RhoA expression with RhoA N19 transfection abolished these AGE-induced changes, while transfection of RhoA L63 reproduced the AGE-evoked changes. H-1152 attenuated the AGE-induced alteration in monolayer permeability and cytoskeleton. The results also confirmed the AGE-induced direct interaction of ROCK and moesin. Thr558 was further identified as the phosphorylating site of moesin in AGE-evoked endothelial responses.

Conclusion

These results confirm the involvement of RhoA/ROCK pathway and subsequent moesin Thr558 phosphorylation in AGE-mediated endothelial dysfunction.     

Mitochondrial Remodeling in Endothelial Cells under Cyclic Stretch is Independent of Drp1 Activation

Mitochondria in endothelial cells remodel morphologically when supraphysiological cyclic stretch is exerted on the cells. During remodeling, mitochondria become shorter, but how they do so remains elusive. Drp1 is a regulator of mitochondrial morphologies. It shortens mitochondria by shifting the balance from mitochondrial fusion to fission. In this study, we hypothesized that Drp1 activation is involved in mitochondrial remodeling under supraphysiological cyclic stretch. To verify the involvement of Drp1, its activation was first quantified with Western blotting, but Drp1 was not significantly activated in endothelial cells under supraphysiological cyclic stretch. Next, Drp1 activation was inhibited with Mdivi-1, but this did not inhibit mitochondrial remodeling. Intracellular Ca²⁺ increase activates Drp1 through calcineurin. First, we inhibited the intracellular Ca²⁺ increase with Gd³⁺ and thapsigargin, but this did not inhibit mitochondrial remodeling. Next, we inhibited calcineurin with cyclosporin A, but this also did not inhibit mitochondrial remodeling. These results indicate that mitochondrial remodeling under supraphysiological cyclic stretch is independent of Drp1 activation. In endothelial cells under supraphysiological cyclic stretch, reactive oxygen species (ROS) are generated. Mitochondrial morphologies are remodeled by ROS generation. When ROS was eliminated with N-acetyl-L-cysteine, mitochondrial remodeling was inhibited. Furthermore, when the polymerization of the actin cytoskeleton was inhibited with cytochalasin D, mitochondrial remodeling was also inhibited. These results suggest that ROS and actin cytoskeleton are rather involved in mitochondrial remodeling. In conclusion, the present results suggest that mitochondrial remodeling in endothelial cells under supraphysiological cyclic stretch is induced by ROS in association with actin cytoskeleton rather than through Drp1 activation.     

Simvastatin Alleviates Hyperpermeability of Glomerular Endothelial Cells in Early-Stage Diabetic Nephropathy by Inhibition of RhoA/ROCK1

Background

Endothelial dysfunction is an early sign of diabetic cardiovascular disease and may contribute to progressive diabetic nephropathy (DN). There is increasing evidence that dysfunction of the endothelial tight junction is a crucial step in the development of endothelial hyperpermeability, but it is unknown whether this occurs in glomerular endothelial cells (GEnCs) during the progression of DN. We examined tight junction dysfunction of GEnCs during early-stage DN and the potential underlying mechanisms. We also examined the effect of simvastatin (3-Hydroxy-3-methylglutaryl CoA reductase inhibitor) on dysfunction of the tight junctions of cultured GEnCs and in db/db mice with early-stage DN.

Methods

We assessed the expression of occludin and ZO-1, two major components of the tight junction complex, in cultured rat GEnCs treated with high glucose and in 12 week-old db/db mice with early-stage DN. We also investigated activation of RhoA/ROCK1 signaling, GEnC permeability, and renal function of the mice.

Results

High glucose suppresses occludin expression and disrupts occludin/ZO-1 translocation in GEnCs. These changes were associated with increased permeability to albumin and activation of RhoA/ROCK1 signaling. Occludin and ZO-1 dysregulation also occurred in the glomeruli of mice with early-stage DN, and these abnormalities were accompanied by albuminuria and activation of RhoA/ROCK1 in isolated glomeruli. Simvastatin prevented high glucose or hyperglycemia-induced dysregulation of occludin and ZO-1 by inhibition of RhoA/ROCK1 signaling in cultured GEnCs and in db/db mice with early-stage DN.

Conclusion

Our results indicate that activation of RhoA/ROCK1 by high glucose disrupts the expression and translocation of occludin/ZO-1 and that simvastatin alleviates occludin/ZO-1 dysregulation and albuminuria by suppressing RhoA/ROCK1 signaling during early-stage DN. These results suggest a potential therapeutic strategy for preventing the onset of albuminuria in early-stage DN.     

RhoA modulates functional and physical interaction between ROCK1 and Erk1/2 in selenite-induced apoptosis of leukaemia cells

RhoA GTPase dysregulation is frequently reported in various tumours and haematologic malignancies. RhoA, regulating Rho-associated coiled-coil-forming kinase 1 (ROCK1), modulates multiple cell functions, including malignant transformation, metastasis and cell death. Therefore, RhoA/ROCK1 could be an ideal candidate target in cancer treatment. However, the roles of RhoA/ROCK1 axis in apoptosis of leukaemia cells remain elusive. In this study, we explored the effects of RhoA/ROCK1 cascade on selenite-induced apoptosis of leukaemia cells and the underlying mechanism. We found selenite deactivated RhoA/ROCK1 and decreased the association between RhoA and ROCK1 in leukaemia NB4 and Jurkat cells. The inhibited RhoA/ROCK1 signalling enhanced the phosphorylation of Erk1/2 in a Mek1/2-independent manner. Erk1/2 promoted apoptosis of leukaemia cells after it was activated. Intriguingly, it was shown that both RhoA and ROCK1 were present in the multimolecular complex containing Erk1/2. GST pull-down analysis showed ROCK1 had a direct interaction with GST-Erk2. In addition, selenite-induced apoptosis in an NB4 xenograft model was also found to be associated with the RhoA/ROCK1/Erk1/2 pathway. Our data demonstrate that the RhoA/ROCK1 signalling pathway has important roles in the determination of cell fates and the modulation of Erk1/2 activity at the Mek–Erk interplay level.     

RhoA/ROCK/PTEN signaling is involved in AT-101-mediated apoptosis in human leukemia cells in vitro and in vivo

R-(−)-gossypol acetic acid (AT-101) is a natural cottonseed product that exhibits anticancer activity. However, the molecular mechanism behind the antileukemic activity of AT-101 has not been well characterized. In this study, we investigated how AT-101 induces apoptosis in human leukemia cells. Exposure to AT-101 significantly increased apoptosis in both human leukemia cell lines and primary human leukemia cells. This increase was accompanied by the activation of caspases, cytochrome c release, Bcl2-associated X protein (Bax) translocation, myeloid cell leukemia-1 (Mcl-1) downregulation, Bcl-2-associated death promoter (Bad) dephosphorylation, Akt inactivation, and RhoA/Rho-associated coiled-coil containing protein kinase 1/phosphatase and tensin homolog (RhoA/ROCK1/PTEN) activation. RhoA, rather than caspase-3 cleavage, mediated the cleavage/activation of ROCK1 that AT-101 induced. Inhibiting RhoA and ROCK1 activation by C3 exoenzyme (C3) and Y27632, respectively, attenuated the ROCK1 cleavage/activation, PTEN activity, Akt inactivation, Mcl-1 downregulation, Bad dephosphorylation, and apoptosis mediated by AT-101. Knocking down ROCK1 expression using a ROCK1-specific siRNA also significantly abrogated AT-101-mediated apoptosis. Constitutively active Akt prevented the AT-101-induced Mcl-1 downregulation, Bad dephosphorylation, and apoptosis. Conversely, AT-101 lethality was potentiated by the phosphatidylinositol 3-kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. In vivo, the tumor growth inhibition caused by AT-101 was also associated with RhoA/ROCK1/PTEN activation and Akt inactivation in a mouse leukemia xenograft model. Collectively, these findings suggest that AT-101 may preferentially induce apoptosis in leukemia cells by interrupting the RhoA/ROCK1/PTEN pathway, leading to Akt inactivation, Mcl-1 downregulation, Bad dephosphorylation, and Bax translocation, which culminate in mitochondrial injury and apoptosis.     

Vildagliptin improves high glucose‐induced endothelial mitochondrial dysfunction via inhibiting mitochondrial fission

The dipeptidyl peptidase 4 inhibitor vildagliptin (VLD), a widely used anti‐diabetic drug, exerts favourable effects on vascular endothelium in diabetes. We determined for the first time the improving effects of VLD on mitochondrial dysfunction in diabetic mice and human umbilical vein endothelial cells (HUVECs) cultured under hyperglycaemic conditions, and further explored the mechanism behind the anti‐diabetic activity. Mitochondrial ROS (mtROS) production was detected by fluorescent microscope and flow cytometry. Mitochondrial DNA damage and ATP synthesis were analysed by real time PCR and ATPlite assay, respectively. Mitochondrial network stained with MitoTracker Red to identify mitochondrial fragmentation was visualized under confocal microscopy. The expression levels of dynamin‐related proteins (Drp1 and Fis1) were determined by immunoblotting. We found that VLD significantly reduced mtROS production and mitochondrial DNA damage, but enhanced ATP synthesis in endothelium under diabetic conditions. Moreover, VLD reduced the expression of Drp1 and Fis1, blocked Drp1 translocation into mitochondria, and blunted mitochondrial fragmentation induced by hyperglycaemia. As a result, mitochondrial dysfunction was alleviated and mitochondrial morphology was restored by VLD. Additionally, VLD promoted the phosphorylation of AMPK and its target acetyl‐CoA carboxylase in the setting of high glucose, and AMPK activation led to a decreased expression and activation of Drp1. In conclusion, VLD improves endothelial mitochondrial dysfunction in diabetes, possibly through inhibiting Drp1‐mediated mitochondrial fission in an AMPK‐dependent manner.     

High glucose induces Drp1-mediated mitochondrial fission via the Orai1 calcium channel to participate in diabetic cardiomyocyte hypertrophy

Mitochondrial dysfunction and impaired Ca²⁺ handling are involved in the development of diabetic cardiomyopathy (DCM). Dynamic relative protein 1 (Drp1) regulates mitochondrial fission by changing its level of phosphorylation, and the Orai1 (Ca²⁺ release-activated calcium channel protein 1) calcium channel is important for the increase in Ca²⁺ entry into cardiomyocytes. We aimed to explore the mechanism of Drp1 and Orai1 in cardiomyocyte hypertrophy caused by high glucose (HG). We found that Zucker diabetic fat rats induced by administration of a high-fat diet develop cardiac hypertrophy and impaired cardiac function, accompanied by the activation of mitochondrial dynamics and calcium handling pathway-related proteins. Moreover, HG induces cardiomyocyte hypertrophy, accompanied by abnormal mitochondrial morphology and function, and increased Orai1-mediated Ca²⁺ influx. Mechanistically, the Drp1 inhibitor mitochondrial division inhibitor 1 (Mdivi-1) prevents cardiomyocyte hypertrophy induced by HG by reducing phosphorylation of Drp1 at serine 616 (S616) and increasing phosphorylation at S637. Inhibition of Orai1 with single guide RNA (sgOrai1) or an inhibitor (BTP2) not only suppressed Drp1 activity and calmodulin-binding catalytic subunit A (CnA) and phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) expression but also alleviated mitochondrial dysfunction and cardiomyocyte hypertrophy caused by HG. In addition, the CnA inhibitor cyclosporin A and p-ERK1/2 inhibitor U0126 improved HG-induced cardiomyocyte hypertrophy by promoting and inhibiting phosphorylation of Drp1 at S637 and S616, respectively. In summary, we identified Drp1 as a downstream target of Orai1-mediated Ca²⁺ entry, via activation by p-ERK1/2-mediated phosphorylation at S616 or CnA-mediated dephosphorylation at S637 in DCM. Thus, the Orai1–Drp1 axis is a novel target for treating DCM.Subject terms: Molecular biology, Cardiac hypertrophy, Pathogenesis     

Chemerin induced by Treponema pallidum predicted membrane protein Tp0965 mediates the activation of endothelial cell via MAPK signaling pathway

Chemerin, a chemoattractant protein, is involved in endothelial dysfunction and vascular inflammation in pathological conditions. In a recent study, we observed the upregulation of chemerin in endothelial cells following in vitro treatment with Treponema pallidum. Here, we investigated the role of chemerin in endothelial cells activation induced by the T. pallidum predicted membrane protein Tp0965. Following stimulation of human umbilical vein endothelial cells (HUVECs) with Tp0965, chemerin and its receptor chemerin receptor 23 (ChemR23) were upregulated, companied with elevated expression of Toll-like receptor 2. Furthermore, chemerin from HUVECs activated endothelial cells via chemerin/ChemR23 signaling in an autocrine/paracrine manner, characterized by upregulated expression of intercellular adhesion molecule 1, E-selectin, and matrix metalloproteinase-2. Activation of endothelial cells depended on the mitogen-activated protein kinase signaling pathway. In addition, Tp0965-induced chemerin promoted THP-1-derived macrophages migration to endothelial cells, also via the chemerin/ChemR23 pathway. The RhoA/ROCK signaling pathway was also involved in THP-1-derived macrophages migration in response to chemerin/ChemR23. Our results highlight the role of Tp0965-induced chemerin in endothelial cells dysfunction, which contributes to the immunopathogenesis of vascular inflammation of syphilis.     

MiRNA‐30a inhibits AECs‐II apoptosis by blocking mitochondrial fission dependent on Drp‐1

Apoptosis of type II alveolar epithelial cells (AECs‐II) is a key determinant of initiation and progression of lung fibrosis. However, the mechanism of miR‐30a participation in the regulation of AECs‐II apoptosis is ambiguous. In this study, we investigated whether miR‐30a could block AECs‐II apoptosis by repressing mitochondrial fission dependent on dynamin‐related protein‐1 (Drp‐1). The levels of miR‐30a in vivo and in vitro were determined through quantitative real‐time PCR (qRT‐PCR). The inhibition of miR‐30a in AECs‐II apoptosis, mitochondrial fission and its dependence on Drp‐1, and Drp‐1 expression and translocation were detected using miR‐30a mimic, inhibitor‐transfection method (gain‐ and loss‐of‐function), or Drp‐1 siRNA technology. Results showed that miR‐30a decreased in lung fibrosis. Gain‐ and loss‐of‐function studies revealed that the up‐regulation of miR‐30a could decrease AECs‐II apoptosis, inhibit mitochondrial fission, and reduce Drp‐1 expression and translocation. MiR‐30a mimic/inhibitor and Drp‐1 siRNA co‐transfection showed that miR‐30a could inhibit the mitochondrial fission dependent on Drp‐1. This study demonstrated that miR‐30a inhibited AECs‐II apoptosis by repressing the mitochondrial fission dependent on Drp‐1, and could function as a novel therapeutic target for lung fibrosis.     

Annexin A1 restores Aβ1‐42‐induced blood–brain barrier disruption through the inhibition of RhoA‐ROCK signaling pathway

The blood–brain barrier (BBB) is composed of brain capillary endothelial cells and has an important role in maintaining homeostasis of the brain separating the blood from the parenchyma of the central nervous system (CNS). It is widely known that disruption of the BBB occurs in various neurodegenerative diseases, including Alzheimer's disease (AD). Annexin A1 (ANXA1), an anti‐inflammatory messenger, is expressed in brain endothelial cells and regulates the BBB integrity. However, its role and mechanism for protecting BBB in AD have not been identified. We found that β‐Amyloid 1‐42 (Aβ42)‐induced BBB disruption was rescued by human recombinant ANXA1 (hrANXA1) in the murine brain endothelial cell line bEnd.3. Also, ANXA1 was decreased in the bEnd.3 cells, the capillaries of 5XFAD mice, and the human serum of patients with AD. To find out the mechanism by which ANXA1 recovers the BBB integrity in AD, the RhoA‐ROCK signaling pathway was examined in both Aβ42‐treated bEnd.3 cells and the capillaries of 5XFAD mice as RhoA was activated in both cases. RhoA inhibitors alleviated Aβ42‐induced BBB disruption and constitutively overexpressed RhoA‐GTP (active form of RhoA) attenuated the protective effect of ANXA1. When pericytes were cocultured with bEnd.3 cells, Aβ42‐induced RhoA activation of bEnd.3 cells was inhibited by the secretion of ANXA1 from pericytes. Taken together, our results suggest that ANXA1 restores Aβ42‐induced BBB disruption through inhibition of RhoA‐ROCK signaling pathway and we propose ANXA1 as a therapeutic reagent, protecting against the breakdown of the BBB in AD.     

Mitochondrial fission triggered by hyperglycemia is mediated by ROCK1 activation in podocytes and endothelial cells

Several lines of evidence suggest that mitochondrial dysfunction plays a critical role in the pathogenesis of microvascular complications of diabetes, including diabetic nephropathy. However, the signaling pathways by which hyperglycemia leads to mitochondrial dysfunction are not fully understood. Here we examined the role of Rho-associated coiled coil-containing protein kinase 1 (ROCK1) on mitochondrial dynamics by generating two diabetic mouse models with targeted deletions of ROCK1 and an inducible podocyte-specific knockin mouse expressing a constitutively active (cA) mutant of ROCK1. Our findings suggest that ROCK1 mediates hyperglycemia-induced mitochondrial fission by promoting dynamin-related protein-1 (Drp1) recruitment to the mitochondria. Deletion of ROCK1 in diabetic mice prevented mitochondrial fission, whereas podocyte-specific cA-ROCK1 mice exhibited increased mitochondrial fission. Importantly, we found that ROCK1 triggers mitochondrial fission by phosphorylating Drp1 at serine 600 residue. These findings provide insights into the unexpected role of ROCK1 in a signaling cascade that regulates mitochondrial dynamics.     

Crosstalk between Akt and NF‐κB pathway mediates inhibitory effect of gas6 on monocytes‐endothelial cells interactions stimulated by P. gingivalis‐LPS

Correlation between periodontitis and atherosclerosis is well established, and the inherent mechanisms responsible for this relationship remain unclear. The biological function of growth arrest‐specific 6 (gas6) has been discovered in both atherosclerosis and inflammation. Inhibitory effects of gas6 on the expression of inflammatory factors in human umbilical vein endothelial cells (HUVECs) stimulated by Porphyromonas gingivalis lipopolysaccharide (P. gingivalis‐LPS) were reported in our previous research. Herein, the effects of gas6 on monocytes‐endothelial cells interactions in vitro and their probable mechanisms were further investigated. Gas6 protein in HUVECs was knocked down with siRNA or overexpressed with plasmids. Transwell inserts and co‐culturing system were introduced to observe chemotaxis and adhering affinity between monocytes and endothelial cells in vitro. Expression of gas6 was decreased in inflammatory periodontal tissues and HUVECs challenged with P. gingivalis‐LPS. The inhibitory effect of gas6 on chemotaxis and adhesion affinity between monocytes and endothelial cells was observed, and gas6 promoted Akt phosphorylation and inhibited NF‐κB phosphorylation. To our best knowledge, we are first to report that gas6 inhibit monocytes‐endothelial cells interactions in vitro induced by P. gingivalis‐LPS via Akt/NF‐κB pathway. Additionally, inflammation‐mediated inhibition of gas6 expression is through LncRNA GAS6‐AS2, rather than GAS6‐AS1, which is also newly reported.     

AKAP1 mediates high glucose-induced mitochondrial fission through the phosphorylation of Drp1 in podocytes

Increasing evidence suggests that mitochondrial dysfunction plays a critical role in the development of diabetic kidney disease (DKD), however, its specific pathomechanism remains unclear. A-kinase anchoring protein (AKAP) 1 is a scaffold protein in the AKAP family that is involved in mitochondrial fission and fusion. Here, we show that rats with streptozotocin (STZ)-induced diabetes developed podocyte damage accompanied by AKAP1 overexpression and that AKAP1 closely interacted with the mitochondrial fission enzyme dynamin-related protein 1 (Drp1). At the molecular level, high glucose (HG) promoted podocyte injury and Drp1 phosphorylation at Ser637 as proven by decreased mitochondrial membrane potential, elevated reactive oxygen species generation, reduced adenosine triphosphate synthesis, and increased podocyte apoptosis. Furthermore, the AKAP1 knockdown protected HG-induced podocyte injury and suppressed HG-induced Drp1 phosphorylation at Ser637. AKAP1 overexpression aggravated HG-induced mitochondrial fragmentation and podocyte apoptosis. The coimmunoprecipitation assay showed that HG-induced Drp1 interacted with AKAP1, revealing that AKAP1 could recruit Drp1 from the cytoplasm under HG stimulation. Subsequently, we detected the effect of drp1 phosphorylation on Ser637 by transferring several different Drp1 mutants. We demonstrated that activated AKAP1 promoted Drp1 phosphorylation at Ser637, which promoted the transposition of Drp1 to the surface of the mitochondria and accounts for mitochondrial dysfunction events. These findings indicate that AKAP1 is the main pathogenic factor in the development and progression of HG-induced podocyte injury through the destruction of mitochondrial dynamic homeostasis by regulating Drp1 phosphorylation in human podocytes.     

Cadmium induced Drp1-dependent mitochondrial fragmentation by disturbing calcium homeostasis in its hepatotoxicity

Mitochondria are critical targets in the hepatotoxicity of cadmium (Cd). Abnormal mitochondrial dynamics have been increasingly implicated in mitochondrial dysfunction in pathophysiological conditions. Therefore, our study aimed to investigate the effects and underlying mechanism of Cd on mitochondrial dynamics during hepatotoxicity. In the L02 liver cell lines, 12 μM cadmium chloride (CdCl₂) exposure induced excessive mitochondrial fragmentation as early as 3 h post-treatment with Cd, which preceded the mitochondrial dysfunction such as reactive oxygen species (ROS) overproduction, mitochondrial membrane potential (ΔΨm) loss and ATP reduction. Concurrent to mitochondrial fragmentation, CdCl₂ treatment increased the protein levels of dynamin-related protein (Drp1) and promoted the recruitment of Drp1 into mitochondria. Strikingly, mitochondrial fragmentation also occurred in the liver tissue of rats exposed to CdCl₂, accompanied by enhanced recruitment of Drp1 into mitochondria. Moreover, in L02 cells, Drp1 silencing could effectively reverse Cd-induced mitochondrial fragmentation and mitochondrial dysfunction. Furthermore, the increased expression and mitochondrial recruitment of Drp1 were tightly related to the disturbance of calcium homeostasis, which could be prevented by both chelating [Ca²⁺]_i and inhibiting [Ca²⁺]_m uptake. Overall, our study indicated that Cd induced Drp1-dependent mitochondrial fragmentation by disturbing calcium homeostasis to promote hepatotoxicity. Manipulation of Drp1 may be the potential avenue for developing novel strategies to protect against cadmium-induced hepatotoxicity.