Endothelial ß-catenin upregulation and Y142 phosphorylation drive diabetic angiogenesis via upregulating KDR/HDAC9.

BACKGROUND: Diabetic angiogenesis is closely associated with disabilities and death caused by diabetic microvascular complications. Advanced glycation end products (AGEs) are abnormally accumulated in diabetic patients and are a key pathogenic factor for diabetic angiogenesis. The present study focuses on understanding the mechanisms underlying diabetic angiogenesis and identifying therapeutic targets based on these mechanisms. METHODS: In this study, AGE-induced angiogenesis serves as a model to investigate the mechanisms underlying diabetic angiogensis. Mouse aortic rings, matrigel plugs, and HUVECs or 293T cells were employed as research objects to explore this pathological process by using transcriptomics, gene promoter reporter assays, virtual screening and so on. RESULTS: Here, we found that AGEs activated Wnt/ß-catenin signaling pathway and enhanced the ß-catenin protein level by affecting the expression of ß-catenin degradation-related genes, such as FZDs (Frizzled receptors), LRPs (LDL Receptor Related Proteins), and AXIN1. AGEs could also mediate ß-catenin Y142 phosphorylation through VEGFR1 isoform5. These dual effects of AGEs elevated the nuclear translocation of ß-catenin and sequentially induced the expression of KDR (Kinase Insert Domain Receptor) and HDAC9 (Histone Deacetylase 9) by POU5F1 and NANOG, respectively, thus mediating angiogenesis. Finally, through virtual screening, Bioymifi, an inhibitor that blocks VEGFR1 isoform5-ß-catenin complex interaction and alleviates AGE-induced angiogenesis, was identified. CONCLUSION: Collectively, this study offers insight into the pathophysiological functions of ß-catenin in diabetic angiogenesis.

SENP6-Mediated deSUMOylation of VEGFR2 Enhances Its Cell Membrane Transport in Angiogenesis.

Angiogenesis is a significant pathogenic characteristic of diabetic microangiopathy. Advanced glycation end products (AGEs) are considerably elevated in diabetic tissues and can affect vascular endothelial cell shape and function. Regulation of the vascular endothelial growth factor (VEGF)-VEGF receptor 2 (VEGFR2) signaling pathway is a critical mechanism in the regulation of angiogenesis, and VEGFR2 activity can be modified by post-translational changes. However, little research has been conducted on the control of small ubiquitin-related modifier (SUMO)-mediated VEGFR2 alterations. The current study investigated this using human umbilical vein endothelial cells (HUVECs) in conjunction with immunoblotting and immunofluorescence. AGEs increased Nrf2 translocation to the nucleus and promoted VEGFR2 expression. They also increased the expression of sentrin/SUMO-specific protease 6 (SENP6), which de-SUMOylated VEGFR2, and immunofluorescence indicated a reduction in VEGFR2 accumulation in the Golgi and increased VEGFR2 transport from the Golgi to the cell membrane surface via the coatomer protein complex subunit beta 2. VEGFR2 on the cell membrane was linked to VEGF generated by pericytes, triggering the VEGF signaling cascade. In conclusion, this study demonstrates that SENP6 regulates VEGFR2 trafficking from the Golgi to the endothelial cell surface. The SENP6-VEGFR2 pathway plays a critical role in pathological angiogenesis.

The role of S100B/RAGE-enhanced ADAM17 activation in endothelial glycocalyx shedding after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) remains one of the main causes for disability and death worldwide. While the primary mechanical injury cannot be avoided, the prevention of secondary injury is the focus of TBI research. Present study aimed to elucidate the effects and mechanisms of S100B and its receptor RAGE on mediating secondary injury after TBI. METHODS: This study established TBI animal model by fluid percussion injury in rats, cell model by stretch-injured in astrocytes, and endothelial injury model with conditioned medium stimulation. Pharmacological intervention was applied to interfere the activities of S100B/RAGE/ADAM17 signaling pathway, respectively. The expressions or contents of S100B, RAGE, syndecan-1 and ADAM17 in brain and serum, as well as in cultured cells and medium, were detected by western blot. The distribution of relative molecules was observed with immunofluorescence. RESULTS: We found that TBI could activate the release of S100B, mostly from astrocytes, and S100B and RAGE could mutually regulate their expression and activation. Most importantly, present study revealed an obvious increase of syndecan-1 in rat serum or in endothelial cultured medium after injury, and a significant decrease in tissue and in cultured endothelial cells, indicating TBI-induced shedding of endothelial glycocalyx. The data further proved that the activation of S100B/RAGE signaling could promote the shedding of endothelial glycocalyx by enhancing the expression, translocation and activity of ADAM17, an important sheddase, in endothelial cells. The damage of endothelial glycocalyx consequently aggravated blood brain barrier (BBB) dysfunction and systemic vascular hyper-permeability, overall resulting in secondary brain and lung injury. CONCLUSIONS: TBI triggers the activation of S100B/RAGE signal pathway. The regulation S100B/RAGE on ADAM17 expression, translocation and activation further promotes the shedding of endothelial glycocalyx, aggravates the dysfunction of BBB, and increases the vascular permeability, leading to secondary brain and lung injury. Present study may open a new corridor for the more in-depth understanding of the molecular processes responsible for cerebral and systemic vascular barrier impairment and secondary injury after TBI.

The Apelin-Apelin Receptor Axis Triggers Cholangiocyte Proliferation and Liver Fibrosis During Mouse Models of Cholestasis.

BACKGROUND AND AIMS: Apelin (APLN) is the endogenous ligand of its G protein-coupled receptor, apelin receptor (APJ). APLN serum levels are increased in human liver diseases. We evaluated whether the APLN-APJ axis regulates ductular reaction and liver fibrosis during cholestasis. APPROACH AND RESULTS: We measured the expression of APLN and APJ and serum APLN levels in human primary sclerosing cholangitis (PSC) samples. Following bile duct ligation (BDL) or sham surgery, male wild-type (WT) mice were treated with ML221 (APJ antagonist) or saline for 1 week. WT and APLN-/- mice underwent BDL or sham surgery for 1 week. Multidrug resistance gene 2 knockout (Mdr2-/- ) mice were treated with ML221 for 1 week. APLN levels were measured in serum and cholangiocyte supernatants, and cholangiocyte proliferation/senescence and liver inflammation, fibrosis, and angiogenesis were measured in liver tissues. The regulatory mechanisms of APLN-APJ in (1) biliary damage and liver fibrosis were examined in human intrahepatic biliary epithelial cells (HIBEpiCs) treated with APLN and (2) hepatic stellate cell (HSC) activation in APLN-treated human HSC lines (HHSteCs). APLN serum levels and biliary expression of APLN and APJ increased in PSC samples. APLN levels were higher in serum and cholangiocyte supernatants from BDL and Mdr2-/- mice. ML221 treatment or APLN-/- reduced BDL-induced and Mdr2-/- -induced cholangiocyte proliferation/senescence, liver inflammation, fibrosis, and angiogenesis. In vitro, APLN induced HIBEpiC proliferation, increased nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) expression, reactive oxygen species (ROS) generation, and extracellular signal-regulated kinase (ERK) phosphorylation. Pretreatment of HIBEpiCs with ML221, diphenyleneiodonium chloride (Nox4 inhibitor), N-acetyl-cysteine (NAC, ROS inhibitor), or PD98059 (ERK inhibitor) reduced APLN-induced cholangiocyte proliferation. Activation of HHSteCs was induced by APLN but reduced by NAC. CONCLUSIONS: The APLN-APJ axis induces cholangiocyte proliferation through Nox4/ROS/ERK-dependent signaling and HSC activation through intracellular ROS. Modulation of the APLN-APJ axis may be important for managing cholangiopathies.

Circular Mathieu and Weber autofocusing beams.

In this Letter, the new classes of non-paraxial autofocusing beams are introduced for the first time, to the best of our knowledge. We investigate both numerically and experimentally non-paraxial circular Mathieu and Weber autofocusing beams based on the solutions of the Helmholtz equation in elliptical and parabolic coordinates, respectively. The results show that such beams can significantly shorten the focus distance, and eliminate the intense oscillation effectively after the focusing point. The focal length and the peak intensity can be controlled by tunable parameters. In addition, we further experimentally realize their application of such beams in optical trapping.

Impact of UCP2 depletion on heat stroke-induced mitochondrial function in human umbilical vein endothelial cells.

OBJECTIVE: The incidence rate of heat stroke (HS) has increased, with high morbidity and mortality rates, in recent years. Previous studies have suggested that vascular endothelial cell injury is one of the main pathological features of HS. Uncoupling protein 2 (UCP2) exhibits antioxidant activity under various stress conditions. This study aims to investigate the role of UCP2 in HS-induced vascular endothelial injury. METHOD: To explore the mechanisms mediating vascular endothelial cell injury induced by HS, we established an HS model of HUVECs in vitro. The percentage of cell death and viability induced by HS were assessed using annexin V-FITC/PI staining and CCK8 assays. HS-induced mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining. HS-induced mitochondrial superoxide was measured by MitoSOX staining, and analyzed by flow cytometry. UCP2, Drp1, phosphorylated Drp1, OPA1, and Mfn2 expression levels were measured by western blotting. RESULTS: HS triggered mitochondrial fragmentation and UCP2 upregulation in a time-dependent manner in HUVECs. As a specific Drp1 inhibitor, Mdivi-1 pretreatment significantly promoted mitochondrial fission and apoptosis in HS-induced HUVECs. In addition, siRNA-mediated UCP2 knockdown further aggravated mitochondrial fragmentation and ΔΨm depolarization and increased mitochondrial ROS production and cell apoptosis in HS-induced HUVECs, which were abolished by Drp1 inhibition. CONCLUSION: Our results indicate that UCP2 protects against HS-induced vascular endothelial damage and that it enhances mitochondrial function. These findings reveal that UCP2 can be a potential contributor to mechanism-based therapeutic strategies for HS.

Advanced glycation end products induce endothelial hyperpermeability via ß-catenin phosphorylation and subsequent up-regulation of ADAM10.

Endothelial hyperpermeability is the initial event in the development of diabetic microvascular complications, and advanced glycation end products (AGEs) are suggested to cause much of the endothelial hyperpermeability associated with diabetes mellitus, but the molecular mechanism remains to be characterized. ß-catenin reportedly plays dual functions in maintaining normal endothelial permeability by serving both as an adhesive component and a signal transduction component. Here, we found that AGEs induced the phosphorylation of ß-catenin at residues Y654 and Y142 and the endothelial hyperpermeability was reversed when the two residues were blocked. In mechanism, phosphorylation of Y654 was blocked by Src inactivation, whereas phosphorylation of Y142 was reduced by a focal adhesion kinase inhibitor. ß-catenin Y654 phosphorylation induced by AGEs facilitated the dissociation of vascular endothelial (VE)-cadherin/ß-catenin and the impairment of adherens junctions (AJs), whereas ß-catenin Y142 phosphorylation favoured the dissociation of ß-catenin and α-catenin. Further investigation revealed that ß-catenin Y142 phosphorylation was required for AGEs-mediated ß-catenin nuclear translocation, and this nuclear-located ß-catenin subsequently activated the TCF/LEF pathway. This pathway promotes the transcription of the Wnt target, ADAM10 (a disintegrin and metalloprotease 10), which mediates VE-cadherin shedding and leads to further impairment of AJs. In summary, our study showed the role of ß-catenin Y654 and Y142 phosphorylation in AGEs-mediated endothelial hyperpermeability through VE-cadherin/ß-catenin/α-catenin dissociation and up-regulation of ADAM10, thereby advancing our understanding of the underlying mechanisms of AGEs-induced microvascular hyperpermeability.

Endothelial glycocalyx in traumatic brain injury associated coagulopathy: potential mechanisms and impact.

Traumatic brain injury (TBI) remains one of the leading causes of death and disability worldwide; more than 10 million people are hospitalized for TBI every year around the globe. While the primary injury remains unavoidable and not accessible to treatment, the secondary injury which includes oxidative stress, inflammation, excitotoxicity, but also complicating coagulation abnormalities, is potentially avoidable and profoundly affects the therapeutic process and prognosis of TBI patients. The endothelial glycocalyx, the first line of defense against endothelial injury, plays a vital role in maintaining the delicate balance between blood coagulation and anticoagulation. However, this component is highly vulnerable to damage and also difficult to examine. Recent advances in analytical techniques have enabled biochemical, visual, and computational investigation of this vascular component. In this review, we summarize the current knowledge on (i) structure and function of the endothelial glycocalyx, (ii) its potential role in the development of TBI associated coagulopathy, and (iii) the options available at present for detecting and protecting the endothelial glycocalyx.

Hypertension in Patients Hospitalized with COVID-19 in Wuhan, China.

It is unclear whether patients with hypertension are more likely to be infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) than the general population and whether there is a difference in the severity of coronavirus disease (COVID-19) pneumonia in patients who have taken ACEI/ARB drugs compared with those who have not.This observational study included data from all patients with clinically confirmed COVID-19 admitted to Hankou Hospital, Wuhan, China, between January 5 and March 8, 2020. Data were extracted from clinical and laboratory records. Follow-up was cut off on March 8, 2020.A total of 274 patients, 75 with hypertension and 199 without hypertension, were included in the analysis. Compared with patients without hypertension, patients with hypertension were older and were more likely to have preexisting comorbidities, including chronic renal insufficiency, cardiovascular disease, diabetes mellitus, and cerebrovascular disease. Moreover, patients with hypertension tended to have higher positive rate for SARS-CoV-2 PCR detection. Multivariate logistic regression analysis showed that age (P = 0.005) and gender (P = 0.019) were independent risk factors associated with the severity of pneumonia in patients on admission, whereas ACEI/ARB treatment (P = 0.184) was not.Patients with COVID-19 with hypertension were significantly older and were more likely to have underlying comorbidities, including chronic renal insufficiency, cardiovascular disease, diabetes mellitus, and cerebrovascular disease. ACEI/ARB drugs did not influence the severity of pneumonia in patients with SARS-CoV-2. In future studies, a larger sample size and multi-center clinical data would be needed to support these conclusions.

Polydatin protects against lipopolysaccharide-induced endothelial barrier disruption via SIRT3 activation.

In a previous study, we demonstrated the role of polydatin (PD) in protecting against multiple organ dysfunction in sepsis. The aim of this study is to investigate whether PD protects against lipopolysaccharide (LPS)-induced endothelial barrier disruption through SIRT3 activation and to disclose the underlying mechanisms. Wild-type mice were injected with LPS and Evans Blue assay was performed to evaluate vascular permeability. Primary human umbilical vein endothelial cells (HUVECs) were stimulated with LPS. Endothelial permeability was evaluated by transendothelial electrical resistance (TER) and FITC-dextran leakage. SIRT3 activity was determined by a Deacetylase Fluorometric kit, and protein expression level of SIRT3 was detected by western blotting. Mitochondrial function was evaluated by determination of ROS level, mitochondrial membrane potential and mPTP opening. In endotoxemic mice, PD pretreatment attenuated vascular leakage in multiple organs while SIRT3 inhibition with 3-TYP reversed the effects of PD. PD treatment in late sepsis also exhibited barrier protective effects. In HUVECs, PD alleviated LPS-induced F-actin rearrangement, cadherin-catenin complex dissociation and endothelial hyperpermeability, whereas 3-TYP or SIRT3 siRNA attenuated the protective effects of PD. PD enhanced SIRT3 deacetylase activity, and attenuated LPS-induced decrease in SIRT3 expression as well. Furthermore, gain-of-function and loss-of-function strategies also confirmed the role of SIRT3 in enhancing endothelial barrier integrity. It was further ascertained that PD enhanced SIRT3-mediated deacetylation of SOD2 and cyclophilin D (CypD), thus suppressing mitochondrial dysfunction and subsequent endothelial barrier dysfunction. In addition, it was revealed that RAGE was involved in LPS-regulated SIRT3 signaling. Our results suggest that polydatin protects against LPS-induced endothelial barrier disruption dependent on SIRT3 and can be applied as a potential therapy for sepsis.

Correction to: Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Advanced glycation end products induce immature angiogenesis in in vivo and ex vivo mouse models.

Proliferative diabetic retinopathy (PDR) is a progressive disease predominantly involving pathological angiogenesis and is characterized by the development of immature, fragile, and easily hemorrhagic new vessels. Advanced glycation end products (AGEs) and the receptor for AGEs (RAGE) play important roles in the progression of diabetic retinopathy. Our previous studies demonstrated that AGEs promoted HUVEC angiogenesis by inducing moesin phosphorylation via RhoA/Rho-associated protein kinase (ROCK) pathway. The aim of this study was to further confirm AGE-induced angiogenesis in vivo and the involvement of RAGE, ROCK, and moesin phosphorylation in this process. We performed the study in an AGE-treated mouse model with various angiogenesis assays in multiple in vivo and ex vivo models. The results demonstrated that AGEs promoted significant neovascularization in whole mount retina and mouse aortic ring of adult and postnatal mice and in Matrigel plug as well, which were consistently accompanied by increased moesin phosphorylation. The increase of AGE-evoked neovascularization and moesin phosphorylation were both attenuated by RAGE knockout or ROCK inhibitor Y27632 administration in mice. We also revealed the pathological characteristics of AGE-promoted angiogenesis by demonstrating the decrease of pericyte coverage and the disarranged endothelial alignment in microvessels. In conclusion, this study provides in vivo evidences that AGEs induce immature angiogenesis by binding to RAGE, activating the RhoA/ROCK signal pathway and inducing moesin phosphorylation.NEW & NOTEWORTHY Advanced glycation end product (AGE)-induced formation of neovessels and phosphorylation of moesin in retina and aortic ring required AGE receptors. AGEs increased neovessels and the phosphorylation of moesin in retina and aortic ring via RhoA/ROCK pathway. AGE-induced immature angiogenesis in AGE-treated mouse retina and aortic ring. The AGE-RAGE axis and moesin could be candidate targets for overcoming relative diseases.

Heat stress induces RIP1/RIP3-dependent necroptosis through the MAPK, NF-κB, and c-Jun signaling pathways in pulmonary vascular endothelial cells.

Necroptosis represents a newly defined form of regulated necrosis and participates in various human inflammatory diseases. It remains unclear whether necroptosis is presented in heatstroke-induced lung injury. We show that heat stress(HS) triggered an significant upregulation of receptor-interacting protein 1 (RIP1) and mixed lineage kinase domain-like protein (MLKL) expression in a time-dependent manner, without a significant change of receptor-interacting protein 3 (RIP3). Furthermore, co-immunoprecipitation assays showed that RIP1 binds to RIP3 to form the necrosome in heat stress-induced PMVECs. In vitro, necrostatin-1 (Nec-1) pre-treatment reduced heat stress-induced PMVECs necroptosis, which also inhibited HMGB1 translocation from the nucleus into the cytoplasm. Similarly, inhibition for ERK (PD98059), NF-κB (BAY11-7082) and c-Jun (c-Jun peptide), respectively, also suppressed the HMGB1 cytoplasm translocation. Furthermore, siRNA-mediated RIP1/RIP3 knockdown negatively regulated the release of HMGB1 in HS-induced necroptosis through the ERK, NF-κB, and c-Jun signaling pathways. Our study reveals that HS induces RIP1/RIP3-dependent necroptosis through the MAPK, NF-κB, and c-Jun signaling pathways in PMVECs.

Intestinal Microbiota Mediates the Susceptibility to Polymicrobial Sepsis-Induced Liver Injury by Granisetron Generation in Mice.

Sepsis-induced liver injury is recognized as a key problem in intensive care units. The gut microbiota has been touted as an important mediator of liver disease development; however, the precise roles of gut microbiota in regulating sepsis-induced liver injury are unknown. Here, we aimed to investigate the role of the gut microbiota in sepsis-induced liver injury and the underlying mechanism. Cecal ligation and puncture (CLP) was used to induce polymicrobial sepsis and related liver injury. Fecal microbiota transplantation (FMT) was used to validate the roles of gut microbiota in these pathologies. Metabolomics analysis was performed to characterize the metabolic profile differences between sepsis-resistant (Res; survived to 7 days after CLP) and sepsis-sensitive (Sen; moribund before or approximately 24 hours after CLP) mice. Mice gavaged with feces from Sen mice displayed more-severe liver damage than did mice gavaged with feces from Res mice. The gut microbial metabolic profile between Sen and Res mice was different. In particular, the microbiota from Res mice generated more granisetron, a 5-hydroxytryptamine 3 (5-HT3 ) receptor antagonist, than the microbiota from Sen mice. Granisetron protected mice against CLP-induced death and liver injury. Moreover, proinflammatory cytokine expression by macrophages after lipopolysaccharide (LPS) challenge was markedly reduced in the presence of granisetron. Both treatment with granisetron and genetic knockdown of the 5-HT3A receptor in cells suppressed nuclear factor kappa B (NF-кB) transactivation and phosphorylated p38 (p-p38) accumulation in macrophages. Gut microbial granisetron levels showed a significantly negative correlation with plasma alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels in septic patients. Conclusion: Our study indicated that gut microbiota plays a key role in the sensitization of sepsis-induced liver injury and associates granisetron as a hepatoprotective compound during sepsis development.

ß-Catenin phosphorylation at Y654 and Y142 is crucial for high mobility group box-1 protein-induced pulmonary vascular hyperpermeability.

OBJECTIVE: Endothelial hyperpermeability is a hallmark of acute lung injury in response to sepsis. The imbalance between adherence junction (AJ) mediated cell-cell adherence forces and stress fiber driven contractile forces contributes to increased endothelial permeability. Here, we spotlight the effects of ß-catenin Y654 andY142 phosphorylation on HMGB1-mediated endothelial barrier leakage. APPROACH AND RESULTS: Our results showed that phospho-deficiencies at both ß-catenin Y654and Y142ameliorated pulmonary vascular dysfunction in male C57 mice receiving a cecal ligation and puncture operation. In vitro analysis indicated that high mobility group box-1 protein (HMGB1) triggered ß-catenin Y654 and Y142 phosphorylation, causing ß-catenin translocation and adherence junction (AJ) disruptions as well as cytoskeleton rearrangement. In addition,ß-catenin Y654 dephosphorylation attenuated HMGB1-mediated dissociation of VE-cadherin/ß-catenin and, hence, partially prevented endothelial hyperpermeability. ß-catenin Y142 dephosphorylation abolished HMGB1-induced uncoupling of ß-catenin and α-catenin, suppressed cytoskeletal reassembly and, hence, alleviated endothelial hyperpermeability. Further investigation demonstrated that RAGE and Src were required forß-catenin Y654 phosphorylation in response to HMGB1, while FAK was responsible for HMGB1-triggered ß-catenin Y142 phosphorylation. CONCLUSIONS: In sum, this study revealed the role of ß-catenin Y654 and Y142 phosphorylation in HMGB1-mediated endothelial hyperpermeability through dysregulation between adherence and contractile forces. This result advances understanding of the mechanisms underlying pulmonary vascular hyperpermeability in sepsis.

Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes.

α-Calcitonin gene-related peptide (α-CGRP) is a 37-amino acid neuropeptide involved in several pathophysiological processes. α-CGRP is involved in the regulation of cholangiocyte proliferation during cholestasis. In this study, we aimed to evaluate if α-CGRP regulates bile duct ligation (BDL)-induced liver fibrosis by using a α-CGRP knockout (α-CGRP-/-) mouse model. α-CGRP-/- and wild-type (WT) mice were subjected to sham surgery or BDL for 7 days. Then, liver fibrosis and cellular senescence as well as the expression of kinase such as p38 and C-Jun N-terminal protein kinase (JNK) in mitogen-activated protein kinases (MAPK) signaling pathway were evaluated in total liver, together with measurement of cellular senescence in cholangiocytes or hepatic stellate cells (HSCs). There was enhanced hepatic expression of Calca (coding α-CGRP) and the CGRP receptor components (CRLR, RAMP-1 and RCP) in BDL and in both WT α-CGRP-/- and BDL α-CGRP-/- mice, respectively. Moreover, there was increased CGRP serum levels and hepatic mRNA expression of CALCA and CGRP receptor components in late-stage PSC samples compared to healthy control samples. Depletion of α-CGRP reduced liver injury and fibrosis in BDL mice that was associated with enhanced cellular senescence of hepatic stellate cells and reduced senescence of cholangiocytes as well as decreased activation of p38 and JNK MAPK signaling pathway. Cholangiocyte supernatant from BDL α-CGRP-/- mice inhibited the activation and increased cellular senescence of cultured human HSCs (HHSCs) compared to HHSCs stimulated with BDL cholangiocyte supernatant. Taken together, endogenous α-CGRP promoted BDL-induced cholestatic liver fibrosis through differential changes in senescence of HSCs and cholangiocytes and activation of p38 and JNK signaling. Modulation of α-CGRP/CGRP receptor signaling may be key for the management of biliary senescence and liver fibrosis in cholangiopathies.

Necrostatin-1 accelerates time to death in a rat model of cecal ligation and puncture and massively increases hepatocyte caspase-3 cleavage.

Necroptosis, a form of regulated necrosis, has been reported to be involved in numerous pathologies, including sepsis. However, a protective effect of the selective inhibitor of necroptosis, necrostatin-1 (Nec-1), against sepsis remains to be confirmed. Animals (rats and mice) were subjected to cecal ligation and puncture (CLP) to mimic clinical sepsis. Nec-1 or its vehicle (control) was administered 20 min before CLP. Survival time was observed up to 72 h after CLP. Specimens of liver tissue and serum were obtained at 6 h, 12 h, and 18 h. Expression of necroptosis-related proteins [receptor-interacting protein kinase (RIP)1, RIP3, and mixed lineage kinase domain-like (MLKL)] was determined by Western blot analysis. The RIP1/RIP3 interaction and the recruitment of MLKL to RIP3 were also analyzed. Liver function, histopathological changes, serum inflammation cytokines, TUNEL staining, and the expression of apoptosis-related protein, including caspase-3, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X (Bax), was determined. As expected, Nec-1 administration reduced the expression of necroptosis-related proteins and the RIP1/RIP3 interaction, indicating inhibited necroptosis. Surprisingly, Nec-1 treatment exacerbated the liver injury and shortened survival time of septic rats with increased TUNEL-positive cells, cleaved caspase-3 protein content, and Bax/Bcl-2 ratio. Collectively, these findings show that Nec-1 administration inhibited the hepatocyte necroptosis pathway but accelerated apoptosis via the apoptotic pathway in CLP-induced sepsis rat. NEW & NOTEWORTHY The present study demonstrated that a chemical inhibitor necrostatin-1 (Nec-1) or receptor-interacting protein kinase(RIP1) knock down targeted at necroptosis inhibition accelerated liver injury of following sepsis. For fundamental research, these results warrant further investigation of the potential link between Nec-1 administration and the cellular apoptosis following sepsis induced liver injury. For applied research, these results suggest the potential harmful effect of Nec-1 on future sepsis treatment.

The let-7/Lin28 axis regulates activation of hepatic stellate cells in alcoholic liver injury.

The let-7/Lin28 axis is associated with the regulation of key cellular regulatory genes known as microRNAs in various human disorders and cancer development. This study evaluated the role of the let-7/Lin28 axis in regulating a mesenchymal phenotype of hepatic stellate cells in alcoholic liver injury. We identified that ethanol feeding significantly down-regulated several members of the let-7 family in mouse liver, including let-7a and let-7b. Similarly, the treatment of human hepatic stellate cells (HSCs) with lipopolysaccharide (LPS) and transforming growth factor-ß (TGF-ß) significantly decreased the expressions of let-7a and let-7b. Conversely, overexpression of let-7a and let-7b suppressed the myofibroblastic activation of cultured human HSCs induced by LPS and TGF-ß, as evidenced by repressed ACTA2 (α-actin 2), COL1A1 (collagen 1A1), TIMP1 (TIMP metallopeptidase inhibitor 1), and FN1 (fibronectin 1); this supports the notion that HSC activation is controlled by let-7. A combination of bioinformatics, dual-luciferase reporter assay, and Western blot analysis revealed that Lin28B and high-mobility group AT-hook (HMGA2) were the direct targets of let-7a and let-7b. Furthermore, Lin28B deficiency increased the expression of let-7a/let-7b as well as reduced HSC activation and liver fibrosis in mice with alcoholic liver injury. This feedback regulation of let-7 by Lin28B is verified in hepatic stellate cells isolated by laser capture microdissection from the model. The identification of the let-7/Lin28 axis as an important regulator of HSC activation as well as its upstream modulators and down-stream targets will provide insights into the involvement of altered microRNA expression in contributing to the pathogenesis of alcoholic liver fibrosis and novel therapeutic approaches for human alcoholic liver diseases.

Mdia1 is Crucial for Advanced Glycation End Product-Induced Endothelial Hyperpermeability.

BACKGROUND/AIMS: Disruption of endothelial barrier integrity in response to advanced glycation end products (AEGs) stimulation contributes to vasculopathy associated with diabetes mellitus. Mammalian diaphanous-related formin (mDia1) has been reported to bind to the cytoplasmic domain of the receptor for advanced glycation end products (RAGE), which induces a series of cellular processes. This study directly evaluated the participation of mDia1 in AGE-induced hyperpermeability and revealed the precise intracellular signal transductions of this pathological process. METHODS: Human umbilical vein endothelial cells (HUVECs) were used in the in vitro studies. Trans-endothelial electric resistance and permeability coefficient for dextran (Pd) were measured to analyze cell permeability. Western blotting, immunofluorescence staining and flow cytometry assay were performed to investigate the underlying mechanism. Dextran flux across the mesentery in mice was monitored to investigate in vivo microvascular permeability. RESULTS: we found that AGEs evoked Nox4 membrane translocation, reactive oxygen species production, phosphorylation of Src and VE-cadherin, dissociation of adherens junctions and eventual endothelial hyperpermeability through RAGE-mDia1 binding. Cells overexpressing mDia1 by recombinant adenovirus infection showed stronger cellular responses induced by AGEs. Down-regulation of mDia1 by infection with an adenovirus encoding siRNA or blockade of RAGE-mDia1 binding by transfection with RAGE mutant plasmids into HUVECs abolished these AGE-induced effects. Furthermore, knockdown of mDia1 using an adenovirus or genetical knockout of RAGE in C57 mice rescued AGE-evoked microvascular hyperpermeability. CONCLUSION: Our study revealed that mDia1 plays a critical role in AGE-induced microvascular hyperpermeability through binding to RAGE.

Regulation of Cellular Senescence by miR-34a in Alcoholic Liver Injury.

Alcoholic liver disease remains a major cause of liver-related morbidity and mortality, which ranges from alcoholic steatohepatitis to fibrosis/cirrhosis and hepatocellular carcinoma, and the related mechanisms are understood poorly. In this study, we aimed to investigate the role of miR-34a in alcohol-induced cellular senescence and liver fibrosis. We found that hepatic miR-34a expression was upregulated in ethanol-fed mice and heavy drinkers with steatohepatitis compared with respective controls. Mice treated with miR-34a Vivo-Morpholino developed less severe liver fibrosis than wild-type mice after 5 weeks of ethanol feeding. Further mechanism exploration showed that inhibition of miR-34a increased cellular senescence of hepatic stellate cells (HSCs) in ethanol-fed mice, although it decreased senescence in total liver and hepatocytes, which was verified by the changes of senescence-associated ß-galactosidase and gene expression. Furthermore, enhanced cellular senescence was observed in liver tissues from steatohepatitis patients compared with healthy controls. In addition, the expression of transforming growth factor-ß1, drosophila mothers against decapentaplegic protein 2 (Smad2), and Smad3 was decreased after inhibition of miR-34a in ethanol-fed mice. Our in vitro experiments showed that silencing of miR-34a partially blocked activation of HSCs by lipopolysaccharide and enhanced senescence of HSCs. Furthermore, inhibition of miR-34a decreased lipopolysaccharide-induced fibrotic gene expression in cultured hepatocytes. In conclusion, our data suggest that miR-34a functions as a profibrotic factor that promotes alcohol-induced liver fibrosis by reducing HSC senescence and increasing the senescence of hepatocytes.