scholarly journals Linagliptin improves high glucose-induced glomerular endothelial hyper- permeability through AMPK activation and RhoA/ROCK suppression

2021 ◽  
Author(s):  
Jianhui Chen ◽  
Xianfan Li ◽  
Zengpu Yu ◽  
Xiaolin Chen
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Endothelial Dysfunction ◽  
High Glucose ◽  
Fluorescence Probe ◽  
Endothelial Barrier ◽  
Protective Effects ◽  
Ampk Activation ◽  
Barrier Dysfunction ◽  
Rock Inhibitor

Abstract OBJECTIVEHyperglycemia is a major cause of albuminuria relevant to glomerular endothelial dysfunction of the kidney in diabetes mellitus. Linagliptin, dipeptidyl peptidase-4 (DPP-4) inhibitor, was shown to have pleiotropic protective effects to improve albuminuria and endothelial dysfunction in diabetic conditions. The purpose of this research was to investigate the molecular mechanism by which linagliptin improves human renal glomerular endothelial cells (HRGECs) barrier dysfunction caused by hyperglycemia.MATERIALS AND METHODSWe examined the DPP-4 activity, the transendothelial electrical resistance of the monolayer in HRGECs, and the horseradish peroxidase–albumin leakage after the treatment with high glucose (HG) in the presence or absence of linagliptin preincubation. The distribution of F-actin remodeling was detected by a fluorescence probe. The phosphorylation of myosin light chain (MLC), AMPK, and RhoA activity were evaluated in the endothelial cells by Western blotting. The effects of inhibition AMPK or RhoA/ROCK pathway on linagliptin-mediated protective effect on the endothelial barrier in HG status were observed.RESULTSHG caused MLC-dependent reorganization of F-actin, leading to endothelial barrier disruption. However, these changes in HRGECs were attenuated by the Rho-dependent kinase (ROCK) inhibitor Y-27632 or linagliptin. The phospho-MLC (pMLC) activated in HRGECs was regulated by RhoA/ROCK signaling. AMPK activation participated in the repair process of HG-induced RhoA-dependent endothelial cell hyperpermeability by linagliptin . Similarly , AMPK activation by linagliptin attenuated HG-induced RhoA dependent F-actin rearrangement and endothelial cell barrier dysfunction, whereas compoud C diminished the effect of linagliptin. CONCLUSIONSOur data showed that HG led to increases in the activity of RhoA and DPP-4. These changes resulted in the rearrangement of endothelial cell cytoskeleton and the dysfunction of the glomerular endothelial barrier, which were mediated by MLC. The present study revealed a novel mechanism of linagliptin-mediated AMPK activation in preventing RhoA-dependent F-actin cytoskeleton rearrangement and reduced glomerular endothelial barrier permeability in diabetic conditions.

scholarly journals Bone Morphogenetic Protein-2 Induces Non-Canonical Inflammatory and Oxidative Pathways in Human Retinal Endothelial Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Mohamed Al-Shabrawey ◽  
Khaled Hussein ◽  
Fang Wang ◽  
Ming Wan ◽  
Khaled Elmasry ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Bone Morphogenetic Protein ◽  
P38 Mapk ◽  
High Glucose ◽  
Bone Morphogenetic Protein 2 ◽  
Barrier Dysfunction ◽  
Retinal Endothelial Cells ◽  
Bmp Receptors ◽  
Morphogenetic Protein

The mechanisms of diabetic retinopathy (DR), are not yet fully understood. We previously demonstrated an upregulation of retinal bone morphogenetic protein-2 (BMP2) in experimental diabetes and in retinas of diabetic human subjects. The purpose of current study was to investigate the role of non-canonical inflammatory pathway in BMP2-induced retinal endothelial cell (REC) barrier dysfunction. For this purpose, we used RT-PCR and western blotting to evaluate the levels of BMP2 signaling components (BMP2, BMP4, BMP receptors), VEGF, phosphorylated p38 MAPK and NFκB, and oxidative stress markers in cultured human retinal endothelial cells (HRECs) subjected to BMP2 (50ng/ml) for up to 24 h. Also, effect of high glucose (HG, 30mM D-glucose) on the expression of BMP2 and its downstream genes was examined in HRECs. H2-DCF is a fluorogenic dye that measures the levels of cellular reactive oxygen species (ROS) was used to measure the pro-oxidative effect of BMP2. Moreover, we evaluated the effect of inhibiting p38 and VEGF signaling on BMP2-induced HRECs barrier dysfunction by measuring the trans-endothelial cell electrical resistance (TER) using electric cell-substrate impedance sensing (ECIS). We also tested the effect of HG on the integrity of HRECs barrier in the presence or absence of inhibitors of BMP2 signaling. Our data reveals that BMP2 and high glucose upregulates BMP components of the BMP signaling pathway (SMAD effectors, BMP receptors, and TGFβ ligand itself) and induces phosphorylation of p38 MAPK and NFκB with nuclear translocation of NFκB. Inhibition of p38 or NFκB attenuated BMP2-induced VEGF expression and barrier dysfunction in HRECs. Also, inhibition of VEGFR2 attenuated BMP2-induced barrier dysfunction. Moreover, BMP2 induces generation of ROS and endothelial nitric oxide synthase (eNOS) expression and activity in HRECs. Finally, HG upregulated BMP2 and its downstream genes (SMAD, BMP4, ALKs, and TGF-β) in HRECs and BMP2 inhibitors attenuated HG-induced HRECs barrier dysfunction. Our results suggest that in addition to the regular canonical SMAD signaling BMP2 induces non-canonical inflammatory pathway in HRECs via activation of p38/NFκB pathway that causes the upregulation of VEGF and the disruption of HRECs. Inhibition of BMP2 signaling is a potential therapeutic intervention to preserve endothelial cell barrier function in DR.

Rhodiola crenulata Suppresses High Glucose-Induced Matrix Metalloproteinase Expression and Inflammatory Responses by Inhibiting ROS-Related HMGB1-TLR4 Signaling in Endothelial Cells

2020 ◽  
Vol 48 (01) ◽  
pp. 91-105
Author(s):  
Li-Yen Huang ◽  
I-Chuan Yen ◽  
Wei-Cheng Tsai ◽  
Shih-Yu Lee
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Adhesion Molecule ◽  
High Glucose ◽  
Inflammatory Responses ◽  
Umbilical Vein ◽  
Mechanisms Of Action ◽  
Primary Response ◽  
Protective Effects ◽  
Rhodiola Crenulata

Rhodiola crenulata, a popular folk medicine for anti-altitude sickness in Tibet, has been shown to have protective effects against high glucose (HG)-induced endothelial cell dysfunction in human umbilical vein endothelial cells (HUVECs). However, its mechanisms of action are unclear. Here, we aimed to examine the effects and the mechanisms of action of Rhodiola crenulata extract (RCE) on matrix metalloproteinases (MMPs) and inflammatory responses under HG conditions. HUVECs were pretreated with RCE or untreated and then exposed to 33[Formula: see text]mM glucose medium for 24[Formula: see text]h. The levels of oxidative stress markers, MMPs, endogenous tissue inhibitors of MMPs (TIMPs), and adhesion molecules were determined. Zymography assays were also carried out. We found that RCE significantly decreased HG-induced increases in reactive oxygen species (ROS) and activation of MAPK and NF-[Formula: see text]B pathways. In addition, RCE not only significantly reduced the expression and activities of MMPs but also upregulated TIMP protein levels. Consistently, HG-induced activation of the toll-like receptor 4 (TLR4)/myeloid differentiation primary response protein (MyD88) signaling pathway, intracellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and high mobility group box 1 (HMGB1) as well as endothelial cell apoptosis was inhibited by RCE treatment. RCE exerts protective effects on endothelial cells against HG insult, partially by suppressing the HMGB1/TLR4 axis. These findings indicate that Rhodiola crenulata may be a potential therapeutic agent for diabetes-associated vascular diseases.

Non-Canonical PAR3 Activation Induces Tie2-Dependent Endothelial Barrier Protective Effects

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2802-2802
Author(s):  
Fabian Stavenuiter ◽  
Laurent O. Mosnier
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tight Junction ◽  
Barrier Function ◽  
Protein C ◽  
Endothelial Barrier ◽  
Protective Effects ◽  
Maximal Activation ◽  
Tethered Ligand ◽  
Blocking Antibodies

Abstract Introduction: Endothelial barrier protective effects of activated protein C (APC) require the endothelial protein C receptor (EPCR), protease activated receptor 1 (PAR1), and PAR3. In contrast, PAR1 and PAR3 activation by thrombin results in barrier disruption. Non-canonical PAR1 and PAR3 activation by APC versus canonical activation by thrombin provide an explanation for the functional selectivity of these proteases. APC induces non-canonical PAR3 activation at Arg41 and synthetic peptides representing the tethered-ligand sequence of PAR3 after non-canonical cleavage (P3R) induce barrier protective effects in vitro and vascular integrity in vivo. However, signaling mechanisms employed by PAR3 remain undefined. To obtain better insights into the relation between coagulation proteases with endothelial barrier protective effects and canonical/non-canonical PAR1 and PAR3 activation, the PAR proteolysis analysis was extended to factor Xa (FXa). Similar to APC, FXa-mediates endothelial barrier protective effects that involve both PAR1 and EPCR. To date, however, no role for PAR3 in FXa-induced barrier integrity has been implicated. Results: In the presence of EPCR, FXa cleaved PAR1 at Arg41 similar to thrombin and not at Arg46 alike APC, whereas FXa cleaved PAR3 at the non-canonical Arg41 similar to APC but not at the canonical Lys38 corresponding to cleavage by thrombin. Surprisingly, changes in electric cell-substrate impedance sensing (ECIS) using the iCelligence system showed FXa induced an immediate drop in endothelial cell index (~60%) comparable to that induced by thrombin, indicating that FXa induced a loss of cell barrier function. Notwithstanding, after incubation of endothelial cells with FXa for 3 hours, FXa protected (~40%) against TRAP-induced loss of barrier function, similar to that induced by APC, confirming barrier protective effects of FXa. PAR1 blocking antibodies prevented the early FXa-mediated loss of barrier function, indicating that PAR1 cleavage at Arg41 was responsible for this.In contrast,a combinationofPAR1 and PAR2 blocking antibodies was needed to inhibit late (3h) FXa-mediated barrier protection. Blocking antibodies against PAR3 confirmed that canonical PAR3 activation enhanced PAR1-mediated barrier disruptive effects of thrombin (~15%). PAR3 blocking antibodies also significantly reduced the barrier protective effect of FXa (~15%), indicating a functional role for non-canonical PAR3 activation by FXa. Neither canonical (P3K) nor non-canonical (P3R) PAR3 tethered-ligand peptides directly induced significant phosphorylation of ERK1/2 or Akt in endothelial cells. The P3K however, but not the P3R peptide, enhanced TRAP induced ERK1/2 phosphorylation. No Akt phosphorylation was observed in endothelial cells treated with TRAP in the presence of either P3K or P3R. Interestingly, both APC and FXa but not thrombin induced prolonged activation of the endothelial cell specific Tie2 receptor, determined by phosphorylation of Y992 and S1119. Tie2 activation by FXa required PAR3 and EPCR with a partial contribution of PAR1 and PAR2. P3R induced potent activation of Tie2 achieving maximal activation at ~0.8 µM P3R, whereas P3K failed to do so. Additionally, neither (non-)canonical PAR1 nor PAR2 tethered-ligand peptides induced activation of Tie2. Activation of Tie2 by P3R was relatively fast and reached half-maximal activation in about 5 minutes. Blocking antibodies against Tie2 reduced FXa-mediated barrier protective effects by approximately 34%, whereas inhibition of Tie2 did not affect thrombin mediated barrier disruption. Immunohistochemistry indicated that Tie2 activation by FXa and P3R resulted in clustering of activated Tie2 at the cell borders. Accordingly, Tie2 activation by FXa and P3R resulted in changes in the cellular distribution of the tight-junction-associated protein zona occludens (ZO-1) in time. Conclusion: Here we identified a novel pathway for Tie2 activation by non-canonical PAR3 activation that promoted tight-junction formation and endothelial barrier protective effects. In contrast, canonical activation of PAR3 enhanced PAR1-mediated barrier disruptive effects by thrombin. These results exemplify the novel dimensions that non-canonical activation of PARs provides for the possible molecular mechanisms that are responsible for the functional selectivity of protease signaling. Disclosures No relevant conflicts of interest to declare.

scholarly journals Selective HDAC6 inhibition prevents TNF-α-induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema

2016 ◽  
Vol 311 (1) ◽  
pp. L39-L47 ◽  
Author(s):  
Jinyan Yu ◽  
Zhongsen Ma ◽  
Sreerama Shetty ◽  
Mengshi Ma ◽  
Jian Fu
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Pulmonary Edema ◽  
Fiber Formation ◽  
Cell Junctions ◽  
Endothelial Barrier ◽  
Barrier Dysfunction ◽  
Barrier Disruption ◽  
Endothelial Barrier Dysfunction ◽  
Tnf Α

Lung endothelial damage contributes to the pathogenesis of acute lung injury. New strategies against lung endothelial barrier dysfunction may provide therapeutic benefits against lung vascular injury. Cell-cell junctions and microtubule cytoskeleton are basic components in maintaining endothelial barrier integrity. HDAC6, a deacetylase primarily localized in the cytoplasm, has been reported to modulate nonnuclear protein function through deacetylation. Both α-tubulin and β-catenin are substrates for HDAC6. Here, we examined the effects of tubastatin A, a highly selective HDAC6 inhibitor, on TNF-α induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema. Selective HDAC6 inhibition by tubastatin A blocked TNF-α-induced lung endothelial cell hyperpermeability, which was associated with increased α-tubulin acetylation and microtubule stability. Tubastatin A pretreatment inhibited TNF-α-induced endothelial cell contraction and actin stress fiber formation with reduced myosin light chain phosphorylation. Selective HDAC6 inhibition by tubastatin A also induced β-catenin acetylation in human lung endothelial cells, which was associated with increased membrane localization of β-catenin and stabilization of adherens junctions. HDAC6 knockdown by small interfering RNA also prevented TNF-α-induced barrier dysfunction and increased α-tubulin and β-catenin acetylation in endothelial cells. Furthermore, in a mouse model of endotoxemia, tubastatin A was able to prevent endotoxin-induced deacetylation of α-tubulin and β-catenin in lung tissues, which was associated with reduced pulmonary edema. Collectively, our data indicate that selective HDAC6 inhibition by tubastatin A is a potent approach against lung endothelial barrier dysfunction.

PKA inhibits RhoA activation: a protection mechanism against endothelial barrier dysfunction

2003 ◽  
Vol 284 (6) ◽  
pp. L972-L980 ◽  
Author(s):  
Jing Qiao ◽  
Fei Huang ◽  
Hazel Lum
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Rho Gtpases ◽  
Endothelial Barrier ◽  
Intracellular Camp ◽  
Protective Effects ◽  
Barrier Dysfunction ◽  
Similar Time ◽  
Endothelial Barrier Dysfunction ◽  
Rhoa Activation

Much evidence indicates that cAMP-dependent protein kinase (PKA) prevents increased endothelial permeability induced by inflammatory mediators. We investigated the hypothesis that PKA inhibits Rho GTPases, which are regulator proteins believed to mediate endothelial barrier dysfunction. Stimulation of human microvascular endothelial cells (HMEC) with thrombin (10 nM) increased activated RhoA (RhoA-GTP) within 1 min, which remained elevated approximately fourfold over control for 15 min. The activation was accompanied by RhoA translocation to the cell membrane. However, thrombin did not activate Cdc42 or Rac1 within similar time points, indicating selectivity of activation responses by Rho GTPases. Pretreatment of HMEC with 10 μM forskolin plus 1 μM IBMX (FI) to elevate intracellular cAMP levels inhibited both thrombin-induced RhoA activation and translocation responses. FI additionally inhibited thrombin-mediated dissociation of RhoA from guanine nucleotide dissociation inhibitor (GDI) and enhanced in vivo incorporation of32P by GDI. HMEC pretreated in parallel with FI showed >50% reduction in time for the thrombin-mediated resistance drop to return to near baseline and inhibition of ∼23% of the extent of resistance drop. Infection of HMEC with replication-deficient adenovirus containing the protein kinase A inhibitor gene (PKA inhibitor) blocked both the FI-mediated protective effects on RhoA activation and resistance changes. In conclusion, the results provide evidence that PKA inhibited RhoA activation in endothelial cells, supporting a signaling mechanism of protection against vascular endothelial barrier dysfunction.

scholarly journals High stretch induces endothelial dysfunction accompanied by oxidative stress and actin remodeling in human saphenous vein endothelial cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
T. Girão-Silva ◽  
M. H. Fonseca-Alaniz ◽  
J. C. Ribeiro-Silva ◽  
J. Lee ◽  
N. P. Patil ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Endothelial Dysfunction ◽  
Saphenous Vein ◽  
Saphenous Vein Graft ◽  
Artery Bypass ◽  
Bypass Graft ◽  
Human Saphenous Vein ◽  
Actin Remodeling

AbstractThe rate of the remodeling of the arterialized saphenous vein conduit limits the outcomes of coronary artery bypass graft surgery (CABG), which may be influenced by endothelial dysfunction. We tested the hypothesis that high stretch (HS) induces human saphenous vein endothelial cell (hSVEC) dysfunction and examined candidate underlying mechanisms. Our results showed that in vitro HS reduces NO bioavailability, increases inflammatory adhesion molecule expression (E-selectin and VCAM1) and THP-1 cell adhesion. HS decreases F-actin in hSVECs, but not in human arterial endothelial cells, and is accompanied by G-actin and cofilin’s nuclear shuttling and increased reactive oxidative species (ROS). Pre-treatment with the broad-acting antioxidant N-acetylcysteine (NAC) supported this observation and diminished stretch-induced actin remodeling and inflammatory adhesive molecule expression. Altogether, we provide evidence that increased oxidative stress and actin cytoskeleton remodeling play a role in HS-induced saphenous vein endothelial cell dysfunction, which may contribute to predisposing saphenous vein graft to failure.

scholarly journals Protective Effects ofPanax notoginsengSaponins against High Glucose-Induced Oxidative Injury in Rat Retinal Capillary Endothelial Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Yue Fan ◽  
Yuan Qiao ◽  
Jianmei Huang ◽  
Minke Tang
Keyword(s):  
Endothelial Cells ◽  
Diabetic Retinopathy ◽  
High Glucose ◽  
Microvascular Dysfunction ◽  
Panax Notoginseng ◽  
Protective Effects ◽  
Pronounced Increase ◽  
Nadph Oxidase 4 ◽  
Retinal Capillary ◽  
Capillary Endothelial Cells

Diabetic retinopathy, a leading cause of visual loss and blindness, is characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for diabetic retinopathy and is associated with increased oxidative stress in the retina. In this study, we investigated the potential protective effects ofPanax notoginsengSaponins (PNS) in retinal capillary endothelial cells (RCECs) exposed to high glucose conditions. We found a pronounced increase in cell viability in rat RCECs incubated with both PNS and high glucose (30 mM) for 48 h or 72 h. The increased viability was accompanied by reduced intracellular hydrogen peroxide (H2O2) and superoxide (O2-), decreased mitochondrial reactive oxygen species (ROS), and lowered malondialdehyde (MDA) levels. PNS also increased the activities of total superoxide dismutase (SOD), MnSOD, catalase (CAT), and glutathione peroxidase (GSH-PX). The glutathione (GSH) content also increased after PNS treatment. Furthermore, PNS reduced NADPH oxidase 4 (Nox4) expression. These results indicate that PNS exerts a protective effect against high glucose-induced injury in RCECs, which may be partially attributed to its antioxidative function.

Antibodies to kidney endothelial cells contribute to a “leaky” glomerular barrier in patients with chronic kidney diseases

2012 ◽  
Vol 302 (7) ◽  
pp. F884-F894 ◽  
Author(s):  
Nidia Maritza Hernandez ◽  
Anna Casselbrant ◽  
Meghnad Joshi ◽  
Bengt R. Johansson ◽  
Suchitra Sumitran-Holgersson
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Endothelial Dysfunction ◽  
Kidney Diseases ◽  
Clinical Importance ◽  
Human Kidney ◽  
Cell Permeability ◽  
Chronic Kidney Diseases ◽  
Esrd Patients

Anti-endothelial cell antibodies (AECA) have been reported to cause endothelial dysfunction, but their clinical importance for tissue-specific endothelial cells is not clear. We hypothesized that AECA reactive with human kidney endothelial cells (HKEC) may cause renal endothelial dysfunction in patients with chronic kidney diseases. We report that a higher fraction (56%) of end-stage renal disease (ESRD) patients than healthy controls (5%) have AECA reactive against kidney endothelial cells ( P <0.001). The presence of antibodies was associated with female gender ( P < 0.001), systolic hypertension ( P < 0.01), and elevated TNF-α ( P < 0.05). These antibodies markedly decrease expression of both adherens and tight junction proteins VE-cadherin, claudin-1, and zonula occludens-1 and provoked a rapid increase in cytosolic free Ca2+and rearrangement of actin filaments in HKEC compared with controls. This was followed by an enhancement in protein flux and phosphorylation of VE-cadherin, events associated with augmented endothelial cell permeability. Additionally, kidney biopsies from ESRD patients with AECA but not controls demonstrated a marked decrease in adherens and tight junctions in glomerular endothelium, confirming our in vitro data. In summary, our data demonstrate a causal link between AECA and their capacity to induce alterations in glomerular vascular permeability.

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