Dynamin-related protein 1 mediates the therapeutic effect of isoliquiritigenin in diabetic intimal hyperplasia via regulation of mitochondrial fission.

Phenotypic shift of vascular smooth muscle cells (VSMCs) plays a key role in intimal hyperplasia, especially in patients with diabetes mellitus (DM). This study aimed to investigate the role of dynamin-related protein 1 (DRP1) in mitochondrial fission-mediated VSMC phenotypic shift and to clarify whether DRP1 is the therapeutic target of isoliquiritigenin (ISL). Wire injury of carotid artery or platelet-derived growth factor treatment was performed in DM mice or high-glucose cultured human aortic smooth muscle cells (HASMCs), respectively. The effects of DRP1 silencing on DM-induced intimal hyperplasia were investigated both in vivo and in vitro. Phenotypic shift of HASMCs was evaluated by detection of reactive oxygen species (ROS) generation, cell viability, and related protein expressions. The effects of ISL on DM-induced intimal hyperplasia were evaluated both in vivo and in vitro. DRP1 silencing and ISL treatment attenuated DM-induced intimal hyperplasia with reduced ROS generation, cell viability, and VSMC dedifferentiation. The GTPase domain of DRP1 protein played a critical role in mitochondrial fission in DM-induced VSMC phenotypic shift. Cellular experiments showed that ISL inhibited mitochondrial fission and reduced the GTPase activity of DRP1, which was achieved by the directly binding to K216 of the DRP1 GTPase domain. ISL attenuated mouse intimal hyperplasia by reducing GTPase activity of DRP1 and inhibiting mitochondrial fission in vivo. In conclusion, increased GTPase activity of DRP1 aggregated DM-induced intimal hyperplasia by increasing mitochondrial fission-mediated VSMC phenotypic shift. ISL attenuated mouse intimal hyperplasia by reducing DRP1 GTPase activity and inhibiting mitochondrial fission of VSMCs.

Glucagon-like peptide-1 receptor agonist exendin 4 ameliorates diabetes-associated vascular calcification by regulating mitophagy through the AMPK signaling pathway.

BACKGROUND: Vascular calcification (VC) is a complication in diabetes mellitus (DM) patients. Osteogenic phenotype switching of vascular smooth muscle cells (VSMCs) plays a critical role in diabetes-related VC. Mitophagy can inhibit phenotype switching in VSMCs. This study aimed to investigate the role of the glucagon-like peptide-1 receptor (GLP-1R) agonist exendin 4 (EX4) in mitophagy-induced phenotype switching. MATERIALS AND METHODS: The status of VC in T2DM mice was monitored using Von Kossa and Alizarin Red S (ARS) staining in mouse aortic tissue. Human aortic smooth muscle cells were cultured in high glucose (HG) and ß-glycerophosphate (ß-GP) conditioned medium. Accumulation of LC3B and p62 was detected in the mitochondrial fraction. The effect of EX4 in vitro and in vivo was investigated by knocking down AMPKα1. RESULTS: In diabetic VC mice, EX4 decreased the percentage of von Kossa/ARS positive area. EX4 inhibited osteogenic differentiation of HG/ß-GP-induced VSMCs. In HG/ß-GP-induced VSMCs, the number of mitophagosomes was increased, whereas the addition of EX4 restored mitochondrial function, increased the number of mitophagosome-lysosome fusions, and reduced p62 in mitochondrial frictions. EX4 increased the phosphorylation of AMPKα (Thr172) and ULK1 (Ser555) in HG/ß-GP-induced VSMCs. After knockdown of AMPKα1, ULK1 could not be activated by EX4. The accumulation of LC3B and p62 could not be reduced after AMPKα1 knockdown. Knockdown of AMPKα1 negated the therapeutic effects of EX4 on VC of diabetic mice. CONCLUSION: EX4 could promote mitophagy by activating the AMPK signaling pathway, attenuate insufficient mitophagy, and thus inhibit the osteogenic phenotype switching of VSMCs.

CDKN2B-AS1 mediates proliferation and migration of vascular smooth muscle cells induced by insulin.

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the intimal hyperplasia in type 2 diabetes mellitus (T2DM) patients after percutaneous coronary intervention. We aimed to investigate the role of lncRNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in VSMC proliferation and migration, as well as the underlying mechanism. T2DM model mice with carotid balloon injury were used in vivo and mouse aortic vascular smooth muscle cells (MOVAS) stimulated by insulin were used in vitro to assess the role of CDKN2B-AS1 in VSMC proliferation and migration following vascular injury in T2DM state. To investigate cell viability and migration, MTT assay and Transwell assay were conducted. To elucidate the underlying molecular mechanisms, the methylation-specific polymerase chain reaction, RNA immunoprecipitation, RNA-pull down, co-immunoprecipitation, and chromatin immunoprecipitation were performed. In vivo, CDKN2B-AS1 was up-regulated in common carotid artery tissues. In vitro, insulin treatment increased CDKN2B-AS1 level, enhanced MOVAS cell proliferation and migration, while the promoting effect was reversed by CDKN2B-AS1 knockdown. CDKN2B-AS1 forms a complex with enhancer of zeste homolog 2 (EZH2) and DNA methyltransferase (cytosine-5) 1 (DNMT1) to regulate smooth muscle 22 alpha (SM22α) methylation levels. In insulin-stimulated cells, SM22α knockdown abrogated the inhibitory effect of CDKN2B-AS1 knockdown on cell viability and migration. Injection of lentivirus-sh-CDKN2B-AS1 relieved intimal hyperplasia in T2DM mice with carotid balloon injury. Up-regulation of CDKN2B-AS1 induced by insulin promotes cell proliferation and migration by targeting SM22α through forming a complex with EZH2 and DNMT1, thereby aggravating the intimal hyperplasia after vascular injury in T2DM.

Mucin 1 Inhibits Ferroptosis and Sensitizes Vitamin E to Alleviate Sepsis-Induced Acute Lung Injury through GSK3ß/Keap1-Nrf2-GPX4 Pathway.

Background: Ferroptosis is a nonapoptotic form of programmed cell death, which may be related to the occurrence and development of sepsis-induced acute respiratory distress syndrome (ARDS)/acute lung injury (ALI). Mucin 1 (MUC1) is a kind of macromolecule transmembrane glycoprotein. Previous studies have shown that MUC1 could relieve ALI in sepsis and predict whether sepsis patients would develop into ARDS. However, the role of MUC1 in the ferroptosis of sepsis-induced ALI/ARDS remains unclear. Materials and Methods: Sera samples from 50 patients with sepsis/septic shock were used to detect iron metabolism-related markers. Western blot and qRT-PCR were conducted to detect the expression levels of ferroptosis-related genes. Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate inflammatory factors. Transmission electron microscopy (TEM) was used to assess morphological changes of cells. Results: The results showed that the iron metabolism-related indicators in sepsis-induced ARDS patients changed significantly, suggesting the iron metabolism disorder. The expression levels of ferroptosis-related genes in lung tissues of sepsis had marked changes, and the lipid peroxidation levels increased, while Ferrostatin-1 (Fer-1) could reverse the above results, which confirmed the occurrence of ferroptosis. In terms of mechanism studies, inhibition of MUC1 dimerization could increase the expression level of Keap1, reduce the phosphorylation level of GSK3ß, inhibit the entry of Nrf2 into the nucleus, further inhibit the expression level of GPX4, enhance the lipid peroxidation level of lung tissues, trigger ferroptosis, and aggravate lung injury. Besides, inhibiting MUC1 reversed the alleviating effect of vitamin E on ALI caused by sepsis, increased the aggregation of inflammatory cells in lung tissues, and aggravated alveolar injury and edema. Conclusions: Our study was the first to explore the changes of iron metabolism indicators in ALI/ARDS of sepsis, clarify the important role of ferroptosis in ALI/ARDS induced by sepsis, and reveal the effects and specific mechanisms of MUC1 in regulating ferroptosis, as well as the sensitization on vitamin E.

Establishment of a novel risk score for in-hospital mortality in adult sepsis patients.

Background: Existing scoring systems have limitations in predicting the in-hospital mortality of adult sepsis patients. We aimed to develop and validate a novel risk score for predicting the in-hospital mortality of adult sepsis patients. Methods: The clinical data of 1,335 adult sepsis inpatients were retrospectively analyzed. Enrolled patients were randomly divided into a modeling group and a validation group at a 3:2 ratio. The modeling group (n=801) was used to develop the risk score by univariate and multivariate logistic regression analyses. The score's performance was validated in the validation group (n=534). We classified patients into four risk levels according to the novel risk score. Results: Age, central vein catheterization, mechanical ventilation, vasopressin, Charlson comorbidity index (CCI), respiratory rate (RR), heart rate (HR), Glasgow coma scale (GCS) score, platelet (PLT), hematocrit (HCT), aspartate aminotransferase (AST), and activated partial thrombin time (APTT) were independent risk factors for in-hospital death in adult sepsis patients. Continuous variables were converted into classified variables to develop the risk score, with a total score of 39 points. Adult sepsis patients with low, lower medium, higher medium, and high risk levels had in-hospital mortality rates of 9.8%, 24.7%, 55.8%, and 83.5%, respectively. Conclusions: Compared with the Acute Physiology and Chronic Health Evaluation II scoring system (APACHE II) and the Modified Early Warning Score (MEWS), the novel risk score showed good predictive performance for in-hospital mortality in adult sepsis patients.

M6A methylation-mediated elevation of SM22α inhibits the proliferation and migration of vascular smooth muscle cells and ameliorates intimal hyperplasia in type 2 diabetes mellitus.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) induced by insulin resistance facilitates intimal hyperplasia of type 2 diabetes mellitus (T2DM) and N6-methyladenosine (m6A) methylation modification mediates the VSMC proliferation. This study aimed to reveal the m6A methylation modification regulatory mechanism. In this study, m6A demethylase FTO was elevated in insulin-treated VSMCs and T2DM mice with intimal injury. Functionally, FTO knockdown elevated m6A methylation level and further restrained VSMC proliferation and migration induced by insulin. Mechanistically, FTO knockdown elevated Smooth muscle 22 alpha (SM22α) expression and m6A-binding protein IGF2BP2 enhanced SM22α mRNA stability by recognizing and binding to m6A methylation modified mRNA. In vivo studies confirmed that the elevated m6A modification level of SM22α mRNA mitigated intimal hyperplasia in T2DM mice. Conclusively, m6A methylation-mediated elevation of SM22α restrained VSMC proliferation and migration and ameliorated intimal hyperplasia in T2DM.

lncRNA KCNQ1OT1 Suppresses the Inflammation and Proliferation of Vascular Smooth Muscle Cells through IκBa in Intimal Hyperplasia.

Inflammation and proliferation of vascular smooth muscle cells (VSMCs) are the key events in intimal hyperplasia. This study aimed to explore the mechanism by which long non-coding RNA (lncRNA) KCNQ1OT1 affects VSMC inflammation and proliferation in this context. A vein graft (VG) model was established in mice to introduce intimal hyperplasia. Isolated normal VSMCs were induced with platelet-derived growth factor type BB (PDGF-BB), and the cell proliferation, migration, and secretion of inflammatory factors were determined. The results showed that KCNQ1OT1 was downregulated in the VSMCs from mice with intimal hyperplasia and in the PDGF-BB-treated VSMCs, and such downregulation of KCNQ1OT1 resulted from the increased methylation level in the KCNQ1OT1 promoter. Overexpressing KCNQ1OT1 suppressed PDFG-BB-induced VSMC proliferation, migration, and secretion of inflammatory factors. In VSMCs, KCNQ1OT1 bound to the nuclear transcription factor kappa Ba (IκBa) protein and increased the cellular IκBa level by reducing phosphorylation and promoting ubiquitination of the IκBa protein. Meanwhile, KCNQ1OT1 promoted the expression of IκBa by sponging miR-221. The effects of KCNQ1OT1 knockdown on promoting VSMC proliferation, migration, and secretion of inflammatory factors were abolished by IκBa overexpression. The roles of KCNQ1OT1 in reducing the intimal area and inhibiting IκBa expression were proved in the VG mouse model after KCNQ1OT1 overexpression. In conclusion, KCNQ1OT1 attenuated intimal hyperplasia by suppressing the inflammation and proliferation of VSMCs, in which the mechanism upregulated IκBa expression by binding to the IκBa protein and sponging miR-221.

Determination of Risk Factors and Establishment of a Prediction Model for Immediate Technical Failure during Endovascular Treatment of Femoropopliteal Occlusive Disease.

BACKGROUND: For long femoropopliteal occlusive lesions, the immediate technical failure (ITF) of endovascular treatment (EVT) is relatively high. Therefore, this study aims to reveal risk factors and establish a prediction model of ITF of EVT in femoropopliteal occlusive disease (FPOD) patients based on preoperative clinical date that may be helpful to the clinical procedures. METHODS: A retrospective analysis of 1,563 FPOD patients who underwent above-the-knee EVT was undertaken. Univariate analysis with chi-squared test was used to screen risk factors from preoperative clinical data. Multivariable analysis with logistic regression was used to generate a model for predicting the ITF rate of EVT, which was evaluated through the receiver operating characteristic curve and another independent cohort of 242 FPOD patients. RESULTS: Risk factors for ITF during EVT in FPOD included age (>80 years, X1), the absence of diabetes mellitus (X2), low-density lipoprotein (>160 mg/dL, X3), lesion calcification (X4), lesion length (>20 cm, X5), ostial occlusion of superficial femoral artery (SFA) (X6), and SFA lesion involving the popliteal artery (X7). A logistic regression model was established based on the equation: -6.504 + 1.236X1 + 0.945X2 + 1.406X3 + 1.136X4 + 1.059X5 + 2.307X6 + 2.194X7. Scores were given to risk factors as follows: X1 (yes = 12, no = 0), X2 (yes = 9, no = 0), X3 (yes = 14, no = 0), X4 (yes = 11, no = 0), X5 (yes = 11, no = 0), X6 (yes = 23, no = 0), and X7 (yes = 22, no = 0). We determined that the optimal comprehensive score for predicting EVT failure was 39, with a sensitivity of 0.847 and a specificity of 0.8. Among these 242 peripheral arterial disease patients, 12 of 14 patients who had failed EVT had a comprehensive score of >39. CONCLUSIONS: We identified a number of risk factors of ITF during the above-the-knee EVT and established a prediction model that may be used for guidance in clinical practice.

Zinc Finger E-Box Binding Protein 2 (ZEB2) Suppress Apoptosis of Vascular Endothelial Cells Induced by High Glucose Through Mitogen-Activated Protein Kinases (MAPK) Pathway Activation.

BACKGROUND Hyperglycemia has been confirmed to damage endothelial function of vascular and microvascular. The regulation of zinc finger E-box binding protein 2 (ZEB2) on vascular endothelial cells (VECs) is reported rarely. Our study investigates the role of ZEB2 on the apoptosis of VECs induced by high glucose through MAPK pathway. MATERIAL AND METHODS Downregulated and upregulated expression of ZEB2 in human umbilical vein endothelial cells (HUVECs) were performed by plasmids transfection. HUVECs are respectively treated with different concentrations of glucose (5.5 mM, 33 mM). The expression of mRNA and protein were detected by real-time quantified PCR and western blotting. Apoptotic cells were measured by flow cytometry. Proliferation and migration of HUVECs were detected by MTT assay and wound healing assay. RESULTS The apoptosis of HUVECs detected by flow cytometry and western blot revealed that ZEB2 overexpression distinctly suppressed the apoptosis of HUVECs induced by high glucose. ZEB2 overexpression promoted the proliferative and migration activity of HUVECs. Besides, ZEB2 overexpression specifically accelerated the phosphorylation level of JNK, and suppressed the apoptosis and promoted the proliferative of VECs via JNK pathway. CONCLUSIONS ZEB2 suppress apoptosis of VECs induced by high glucose through MAPK pathway activation, which provides a novel insight and therapeutic target for endothelial injury.

Induction of autophagy by salidroside through the AMPK-mTOR pathway protects vascular endothelial cells from oxidative stress-induced apoptosis.

Vascular endothelial cells are highly sensitive to oxidative stress, and this is one of the mechanisms by which widespread endothelial dysfunction is induced in most cardiovascular diseases and disorders. However, how these cells can survive in oxidative stress environments remains unclear. Salidroside, a traditional Chinese medicine, has been shown to confer vascular protective effects. We aimed to understand the role of autophagy and its regulatory mechanisms by treating human umbilical vein endothelial cells (HUVECs) with salidroside under oxidative stress. HUVECs were treated with salidroside and exposed to hydrogen peroxide (H2O2). The results indicated that salidroside exerted cytoprotective effects in an H2O2-induced HUVEC injury model and suppressed H2O2-induced apoptosis of HUVECs. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased oxidative stress-induced HUVEC apoptosis, while the autophagy activator rapamycin induced anti-apoptosis effects in HUVECs. Salidroside increased autophagy and decreased apoptosis of HUVECs in a dose-dependent manner under oxidative stress. Moreover, 3-MA attenuated salidroside-induced HUVEC autophagy and promoted apoptosis, whereas rapamycin had no additional effects compared with salidroside alone. Salidroside upregulated AMPK phosphorylation but downregulated mTOR phosphorylation under oxidative stress; however, administration of compound C, an AMPK inhibitor, abrogated AMPK phosphorylation and increased mTOR phosphorylation and apoptosis compared with salidroside alone. These results suggest that autophagy is a protective mechanism in HUVECs under oxidative stress and that salidroside might promote autophagy through activation of the AMPK pathway and downregulation of mTOR pathway.

The Use of the Angiosome Concept for Treating Infrapopliteal Critical Limb Ischemia through Interventional Therapy and Determining the Clinical Significance of Collateral Vessels.

BACKGROUND: The purpose of this study was to evaluate the clinical significance of the involvement of collateral vessels in interventional therapy based on the angiosome concept for infrapopliteal critical limb ischemia (CLI). METHODS: We enrolled 486 patients with unilateral infrapopliteal CLI (Rutherford Stage 5 or 6) treated at 2 hospitals from January 2005 to December 2014. Using the angiosome concept, the patients were categorized into 3 groups: the direct revascularization (DR) group, the indirect revascularization through collaterals (IR-tc) group, and the indirect revascularization without collaterals (IR-wc) group. The data from 1 year of follow-up after the initial surgery were analyzed for all 3 groups. RESULTS: During the 1-year follow-up, the unhealed ulcer rate of the IR-wc group was significantly higher than that of the DR group (83.4% vs. 31.7%, P < 0.001) and the IR-tc group (83.4% vs. 34.8%, P < 0.001). Furthermore, the limb salvage rate of the IR-wc group was significantly lower than that of the DR group (70.4% vs. 89.2%, P < 0.001) and the IR-tc group (70.4% vs. 85.4%, P = 0.0013). However, there were no differences in the unhealed ulcer rate or the limb salvage rate between the DR group and the IR-tc group (31.7% vs. 34.8%, P = 0.096 and 89.2% vs. 85.4%, P = 0.2834, respectively). In addition, within the IR-wc group, the unhealed ulcer rate of diabetic patients was higher than that of patients without diabetes (90.0% vs. 74.6%, P = 0.0116), but there was no difference in the limb salvage rate between diabetic and nondiabetic patients. No differences in the unhealed ulcer rate or the limb salvage rate were found between diabetic and nondiabetic patients in the other 2 groups. CONCLUSIONS: Following the angiosome model of perfusion for endovascular therapy, directly revascularizing the feeding artery and indirectly achieving revascularization through collaterals can effectively prompt the healing of ulcers and decrease the amputation rate in patients with infrapopliteal CLI. Collateral vessels play a crucial role in the treatment of ischemic foot.

Sodium butyrate down-regulates tristetraprolin-mediated cyclin B1 expression independent of the formation of processing bodies.

Butyrate regulates multiple host cellular events including the cell cycle; however, little is known about the molecular mechanism by which butyrate induces a global down-regulation of the expression of genes associated with the cell cycle. Here, we demonstrate that treating HEK293T cells and the non-small-cell lung cancer cell line A549 with a high concentration of sodium butyrate reduces cyclin B1 expression. The underlying mechanism is related to the destabilization of its mRNA by tristetraprolin, which is up-regulated in response to sodium butyrate. Specifically, the sodium butyrate stimulation reduces the mRNA and protein expression of cyclin B1 and, conversely, upregulates tristetraprolin expression. Importantly, the overexpression of tristetraprolin in HEK293T decreases the mRNA and protein expression of cyclin B1; in contrast, knockdown of tristetraprolin mediated by small interfering RNA increases its expression in response to sodium butyrate treatment for both HEK293T and A549 cells. Furthermore, results from luciferase reporter assays and RNA immunoprecipitation indicate that sodium butyrate accelerates 3' UTR-dependent cyclin B1 decay by enhancing the binding of tristetraprolin to the 3' untranslated region of cyclin B1. Surprisingly, the overexpression of tristetraprolin prevents the formation of processing bodies, and the siRNA-mediated silencing of EDC4 does not restore the sodium butyrate-induced reduction of cyclin B1 expression. Thus, we confirm that NaBu regulates ZFP36-mediated cyclin B1 expression in a manner that is independent of the formation of P-bodies. The above findings disclose a novel mechanism of sodium butyrate-mediated gene expression regulation and might benefit its application in tumor treatment.

Incidence and risk factors of early deep venous thrombosis after varicose vein surgery with routine use of a tourniquet.

INTRODUCTION: The incidence of early deep venous thrombosis (DVT) following varicose vein surgery (traditional open stripping) with routine use of a tourniquet remains unknown. MATERIALS AND METHODS: A retrospective analysis of all patients who underwent varicose vein surgery with a tourniquet in the authors' unit between 1 January 2012 and 30 November 2013 was undertaken. Cases of postoperative DVT were identified from the unit database, and re-assessments conducted 1, 3 and 6 months after the initial diagnosis were recorded from the outpatient department. RESULTS: Out of 1461 patients, 113 (7.7%) developed postoperative DVT. Nineteen (1.3%) patients had proximal DVT, and 94 (6.4%) patients had isolated distal DVT. The risk factors for postoperative DVT included old age (≥65 years), female sex and gastrocnemius vein dilation (GVD). GVD was found to be a significant independent risk factor for the occurrence of DVT, with an odds ratio of 2.437 (95% confidence interval 1.644-3.611). Five patients with distal DVT (5.7%) and eight patients with proximal DVT (44.4%) still exhibited a thrombus at 6-month follow-up, but with decreased size and at various stages of resolution. CONCLUSIONS: This study found a higher incidence of postoperative DVT (7.7%) with routine use of a tourniquet during varicose vein surgery than has been reported previously. Among the factors examined, GVD had the highest predictive power for postoperative DVT. Both distal and proximal DVT were associated with acceptable outcomes.

Femorofemoral bypass allowed limb preservation after late diagnosis of left common iliac artery thrombosis due to blunt trauma: A case report.

OBJECTIVE: Acute common iliac artery occlusion which results from blunt abdominal trauma is rare and potentially leads to a late diagnosis. METHODS: We report a case of a 58-year-old patient who suffered a late diagnosed acute left common iliac artery occlusion secondary to abdominal trauma. An emergency exploratory laparotomy was performed to stop intra-abdominal bleeding, while his left limb ischemia was not noticed until 32 h later and femorofemoral bypass was then successfully performed for revascularization. Compartment syndrome was observed postoperatively, and fasciotomy was performed promptly. The wound was temporarily covered with Vaccum Sealing Drainage due to high skin tension. Patient underwent skin-grafting after leg swelling subsided. RESULTS: The follow-up turned out that these managements were valid in the preservation of the limb viability. CONCLUSIONS: This case highlights the prudent recognition of the acute lower extremity ischemia in the abdominal trauma and immediate remedy for acute iliac artery occlusion after a late diagnosis.