Loss of Atg7 in Endothelial Cells Enhanced Cutaneous Wound Healing in a Mouse Model.

BACKGROUND: Emerging evidence has linked autophagy to skin wound healing; however, the underlying cellular and molecular mechanisms remain poorly understood. The present study was designed to determine the role of autophagy in endothelial cell (EC)-mediated skin wound healing in mice. METHODS: Autophagy-related gene (Atg7) in mouse ECs was inactivated by the Cre-loxP system under the control of an EC-specific VE-Cadherin (Cdh5) promoter (Atg7EC-/- mice). Full-thickness skin wounds were created on the dorsum of wild-type (WT), Cdh5-Cre+, floxed Atg7 (Atg7F/F), and Atg7EC-/- mice. Autophagic activity was determined by autophagic flux assay in the primary culture of ECs isolated from these mice. The wound re-epithelialization and angiogenesis was examined by histological analyses. The angiogenic activity of ECs was evaluated by tube formation assay in vitro. EC proliferation was examined by a cell count CCK-8 kit. EC-originated intercellular communication with dermal fibroblasts and keratinocytes was assessed by measuring the effect of EC conditional medium on the growth of keratinocytes and fibroblasts. The levels of VEGF, EGF, bFGF in EC conditional medium were measured by ELISA. RESULTS: Autophagy deficiency in ECs markedly enhanced the re-epithelialization and the wound closure during skin wound healing. However, it has minimal impact on angiogenesis in the wounded skin. Notably, autophagy deficiency in ECs did not affect their proliferation and migration or angiogenic activity per se but enhanced the EC conditional medium-induced proliferation and migration of keratinocytes and fibroblasts. CONCLUSIONS: These results demonstrate for the first time an inhibitory role of autophagy in the EC-originated paracrine regulation of skin wound healing.

Comparison of 2013 and 2009 versions of Caprini risk assessment models for predicting VTE in Chinese cancer patients: a retrospective study.

This study aimed to compare the predictive value of 2009 and 2013 version of Caprini risk assessment models (RAM) for venous thromboembolism (VTE) in cancer patients by receiver operating characteristic (ROC) analysis. This retrospective study reviewed a total of 1439 VTE and 1439 non-VTE Chinese cancer inpatients. The baseline demographic data of these patients were recorded. 2009 and 2013 versions Caprini RAMs were applied, and cumulative risk scores were obtained by adding the scores of each risk factor. The specificity, sensitivity, positive predictive value and negative predictive value of these two models were analyzed. ROC curve was drawn to calculate the area under the curve (AUC) and the Youden index. Significant differences were observed in the risk factors between VTE and non-VTE Group. The specificity and negative predictive value of 2013 version were higher than those of 2009 version (P < 0.05). No significant differences were found in the sensitivity or positive predictive value between 2009 and 2013 versions of the Caprini RAM (P > 0.05). The AUC and Youden index of 2013 Caprini RAM were significantly higher than those of 2009 Caprini RAM (P < 0.001), whereas the Youden index of the 2009 Caprini RAM at critical point 4 was higher than that at critical point 3 (0.362 vs 0.067, P < 0.05). Compared with 2009 version, 2013 version of the Caprini RAM provides a more accurate and efficacious method for the risk assessment of VTE in Chinese cancer patients.

Palmitic acid dysregulates the Hippo-YAP pathway and inhibits angiogenesis by inducing mitochondrial damage and activating the cytosolic DNA sensor cGAS-STING-IRF3 signaling mechanism.

Impaired angiogenesis and wound healing carry significant morbidity and mortality in diabetic patients. Metabolic stress from hyperglycemia and elevated free fatty acids have been shown to inhibit endothelial angiogenesis. However, the underlying mechanisms remain poorly understood. In this study, we show that dysregulation of the Hippo-Yes-associated protein (YAP) pathway, an important signaling mechanism in regulating tissue repair and regeneration, underlies palmitic acid (PA)-induced inhibition of endothelial angiogenesis. PA inhibited endothelial cell proliferation, migration, and tube formation, which were associated with increased expression of mammalian Ste20-like kinases 1 (MST1), YAP phosphorylation/inactivation, and nuclear exclusion. Overexpression of YAP or knockdown of MST1 prevented PA-induced inhibition of angiogenesis. When searching upstream signaling mechanisms, we found that PA dysregulated the Hippo-YAP pathway by inducing mitochondrial damage. PA treatment induced mitochondrial DNA (mtDNA) release to cytosol, and activated cytosolic DNA sensor cGAS-STING-IRF3 signaling. Activated IRF3 bound to the MST1 gene promoter and induced MST1 expression, leading to MST1 up-regulation, YAP inactivation, and angiogenesis inhibition. Thus, mitochondrial damage and cytosolic DNA sensor cGAS-STING-IRF3 signaling are critically involved in PA-induced Hippo-YAP dysregulation and angiogenesis suppression. This mechanism may have implication in impairment of angiogenesis and wound healing in diabetes.

STING-IRF3 Triggers Endothelial Inflammation in Response to Free Fatty Acid-Induced Mitochondrial Damage in Diet-Induced Obesity.

OBJECTIVE: Metabolic stress in obesity induces endothelial inflammation and activation, which initiates adipose tissue inflammation, insulin resistance, and cardiovascular diseases. However, the mechanisms underlying endothelial inflammation induction are not completely understood. Stimulator of interferon genes (STING) is an important molecule in immunity and inflammation. In the present study, we sought to determine the role of STING in palmitic acid-induced endothelial activation/inflammation. APPROACH AND RESULTS: In cultured endothelial cells, palmitic acid treatment activated STING, as indicated by its perinuclear translocation and binding to interferon regulatory factor 3 (IRF3), leading to IRF3 phosphorylation and nuclear translocation. The activated IRF3 bound to the promoter of ICAM-1 (intercellular adhesion molecule 1) and induced ICAM-1 expression and monocyte-endothelial cell adhesion. When analyzing the upstream signaling, we found that palmitic acid activated STING by inducing mitochondrial damage. Palmitic acid treatment caused mitochondrial damage and leakage of mitochondrial DNA into the cytosol. Through the cytosolic DNA sensor cGAS (cyclic GMP-AMP synthase), the mitochondrial damage and leaked cytosolic mitochondrial DNA activated the STING-IRF3 pathway and increased ICAM-1 expression. In mice with diet-induced obesity, the STING-IRF3 pathway was activated in adipose tissue. However, STING deficiency (Stinggt/gt ) partially prevented diet-induced adipose tissue inflammation, obesity, insulin resistance, and glucose intolerance. CONCLUSIONS: The mitochondrial damage-cGAS-STING-IRF3 pathway is critically involved in metabolic stress-induced endothelial inflammation. STING may be a potential therapeutic target for preventing cardiovascular diseases and insulin resistance in obese individuals.

[Progress in circular RNAs of plants].

Circular RNAs (circRNAs) are covalently closed, conserved single-stranded transcripts that are produced from precursor mRNA (pre-mRNA) back-splicing. They could function as microRNA sponges, interfere with splicing and bind to protein to regulate the expression of parental genes and linear mRNAs. Next-generation RNA sequencing (RNA-seq) has recently shown that the expression of circRNAs is widespread in plants. circRNAs participate in multiple biological processes such as floral development, fruit ripening, and biotic and abiotic stress responses by cell type-specific and tissue-specific expression patterns, indicating that they may play an important role in plant development. In this review, we summarize the current knowledge of plant circRNAs in recent years, including the biogenesis, detection, databases, expression pattern, and potential functions in comparison with animal results to provide new insights for functional research interests of circRNAs in the future.

Evaluating the Efficacy, Safety, and Tolerability of Serelaxin When Added to Standard Therapy in Asian Patients With Acute Heart Failure: Design and Rationale of RELAX-AHF-ASIA Trial.

BACKGROUND: Acute heart failure (AHF), a common and growing health concern worldwide, is associated with high risk of post-discharge rehospitalization and mortality. Existing evidence indicates potential therapeutic benefits of serelaxin in Caucasian AHF patients, but corresponding data in Asians remain scarce. RELAX-AHF-ASIA, a multinational, randomized, double-blind, placebo-controlled, phase III trial, will evaluate the effects of serelaxin on symptom relief and clinical outcomes in Asian AHF patients, with the use of novel assessments. METHODS AND RESULTS: Patients with AHF, systolic blood pressure ≥125 mm Hg, and mild to moderate renal dysfunction will be randomized within 16 hours of presentation to receive 48-hour intravenous infusion of 30 µg ⋅ kg-1 ⋅ d-1 serelaxin or placebo in addition to standard therapy. The composite primary end point includes: (1) treatment success (moderate/marked improvement in patient-reported dyspnea and physician-assessed signs of congestion on day 2); (2) treatment failure (in-hospital worsening of signs and/or symptoms of heart failure [HF] requiring intensification of intravenous HF therapy or mechanical ventilation, renal/circulatory support, rehospitalization due to HF/renal-failure, or death through day 5); and (3) unchanged status. Secondary end points include time to in-hospital worsening HF through day 5 and all-cause and cardiovascular deaths through day 180. CONCLUSIONS: RELAX-AHF-ASIA, the largest randomized clinical trial in Asian AHF patients to date, has a novel composite primary end point and the potential to become a hallmark of AHF trials.

[Research of reversal effect of PESV to multi-drug resistance of leukemia stem cell].

Using the BALB/c mouse multidrug resistance model of leukemia, the effect of peptide extract from scorpion venom (PESV) to the upstream signal factors of P-gp of MDR leukemia stem cells on the mouse tumor block was observed, and the mechanism of PESV to reverse the MDR of LSC was studied. At the same time, the expression of P-gp, MDR1 mRNA and PI3-K, NF-κB were respectively detected through flow cytometry, RT-PCR, Western blot and Elisa, and the mouse liver, spleen were examined via histopathological methods. The results of the experiment were as follows: mice of the control group didn't show any obvious changes, while mice of the other six groups all showed arched back, emaciation, liver swell, and inflammation was found in all liver tissue. The expression level of P-gp and PI3K on the LSC membrane of mouse tumor block was down-regulated; the expression of MDR1 mRNA in the cytoplasm was obviously down in the PESV low dose group, and which was inordinately up in the middle dose group and the high dose group. The expression level of NF-κB in the leukemia stem cell nucleus remarkably decreased. PESV had a outstanding role of down-regulating PI3K, NF-κb, MDR1 which were all upstream factors of P-gp, and to a certain degree enhanced the sensitivity of LSC to ADM. Therefore, this experiment explained one of the mighty mechanism of PESV to reverse MDR of LSC, and provided a foundation to further study of combinational anti-cancer effects of PESV.

A critical role of cardiac fibroblast-derived exosomes in activating renin angiotensin system in cardiomyocytes.

Chronic activation of the myocardial renin angiotensin system (RAS) elevates the local level of angiotensin II (Ang II) thereby inducing pathological cardiac hypertrophy, which contributes to heart failure. However, the precise underlying mechanisms have not been fully delineated. Herein we report a novel paracrine mechanism between cardiac fibroblasts (CF)s and cardiomyocytes whereby Ang II induces pathological cardiac hypertrophy. In cultured CFs, Ang II treatment enhanced exosome release via the activation of Ang II receptor types 1 (AT1R) and 2 (AT2R), whereas lipopolysaccharide, insulin, endothelin (ET)-1, transforming growth factor beta (TGFß)1 or hydrogen peroxide did not. The CF-derived exosomes upregulated the expression of renin, angiotensinogen, AT1R, and AT2R, downregulated angiotensin-converting enzyme 2, and enhanced Ang II production in cultured cardiomyocytes. In addition, the CF exosome-induced cardiomyocyte hypertrophy was blocked by both AT1R and AT2R antagonists. Exosome inhibitors, GW4869 and dimethyl amiloride (DMA), inhibited CF-induced cardiomyocyte hypertrophy with little effect on Ang II-induced cardiomyocyte hypertrophy. Mechanistically, CF exosomes upregulated RAS in cardiomyocytes via the activation of mitogen-activated protein kinases (MAPKs) and Akt. Finally, Ang II-induced exosome release from cardiac fibroblasts and pathological cardiac hypertrophy were dramatically inhibited by GW4869 and DMA in mice. These findings demonstrate that Ang II stimulates CFs to release exosomes, which in turn increase Ang II production and its receptor expression in cardiomyocytes, thereby intensifying Ang II-induced pathological cardiac hypertrophy. Accordingly, specific targeting of Ang II-induced exosome release from CFs may serve as a novel therapeutic approach to treat cardiac pathological hypertrophy and heart failure.

CAT-1 as a novel CAM stabilizes endothelial integrity and mediates the protective actions of L-Arg via a NO-independent mechanism.

Interendothelial junctions play an important role in the maintenance of endothelial integrity and the regulation of vascular functions. We report here that cationic amino acid transporter-1 (CAT-1) is a novel interendothelial cell adhesion molecule (CAM). We identified that CAT-1 protein localized at cell-cell adhesive junctions, similar to the classic CAM of VE-cadherin, and knockdown of CAT-1 with siRNA led to an increase in endothelial permeability. In addition, CAT-1 formed a cis-homo-dimer and showed Ca(2+)-dependent trans-homo-interaction to cause homophilic cell-cell adhesion. Co-immunoprecipitation assays showed that CAT-1 can associate with ß-catenin. Furthermore, we found that the sub-cellular localization and function of CAT-1 are associated with cell confluency, in sub-confluent ECs CAT-1 proteins distribute on the entire surface and function as L-Arg transporters, but most of the CAT-1 in the confluent ECs are localized at interendothelial junctions and serve as CAMs. Further functional characterization has disclosed that extracellular L-Arg exposure stabilizes endothelial integrity via abating the cell junction disassembly of CAT-1 and blocking the cellular membrane CAT-1 internalization, which provides the new mechanisms for L-Arg paradox and trans-stimulation of cationic amino acid transport system (CAAT). These results suggest that CAT-1 is a novel CAM that directly regulates endothelial integrity and mediates the protective actions of L-Arg to endothelium via a NO-independent mechanism.

Deubiquitinating enzyme CYLD mediates pressure overload-induced cardiac maladaptive remodeling and dysfunction via downregulating Nrf2.

Ubiquitin proteasome system (UPS) consists of ubiquitin, ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), ubiquitin ligases (E3s), proteasomes, and deubiquitinating enzymes (DUBs). Ubiquitin, E1s, several E2s, E3s, and proteasomes play an important role in the regulation of cardiac homeostasis and dysfunction; however, less is known about the role of DUBs in the heart. Here, we uncovered a crucial role of cyclindromatosis (CYLD), a DUB, in mediating cardiac maladaptive remodeling and dysfunction. CYLD expression was dramatically upregulated in the cardiomyocytes of hypertrophic and failing human and murine hearts. Knockout of CYLD improved survival rate and alleviated cardiac hypertrophy, fibrosis, apoptosis, oxidative stress, and dysfunction in mice that were subjected to sustained pressure overload induced by transverse aortic constriction. Deep sequencing and gene array analyses revealed that the most dramatically changed genes are those involving in the free radical scavenging pathway and cardiovascular disease, including fos, jun, myc, and nuclear factor erythroid-2 related factor 2 (Nrf2) in the heart. Moreover, knockdown of CYLD enhanced mitogen-activated protein kinase (MAPK) ERK- and p38-mediated expression of c-jun, c-fos, and c-myc, which govern Nrf2 expression in cardiomyocytes. The CYLD deficiency-induced suppression of reactive oxygen species (ROS) formation, death and hypertrophy in cardiomyocytes was blocked by additional knockdown of Nrf2. Taken together, our findings demonstrate for the first time that CYLD mediates cardiac maladaptive remodeling and dysfunction, most likely via enhancing myocardial oxidative stress in response to pressure overload. At the molecular level, CYLD interrupts the ERK- and p38-/AP-1 and c-Myc pathways to suppress Nrf2-operated antioxidative capacity, thereby enhancing oxidative stress in the heart.

Inhibitory role of reactive oxygen species in the differentiation of multipotent vascular stem cells into vascular smooth muscle cells in rats: a novel aspect of traditional culture of rat aortic smooth muscle cells.

Proliferative or synthetic vascular smooth muscle cells (VSMCs) are widely accepted to be mainly derived from the dedifferentiation or phenotypic modulation of mature contractile VSMCs, i.e., a phenotype switch from a normally quiescent and contractile type into a proliferative or synthetic form. However, this theory has been challenged by recent evidence that synthetic VSMCs predominantly originate instead from media-derived multipotent vascular stem cells (MVSCs). To test these hypotheses further, we re-examine whether the conventional rat aortic SMC (RASMC) culture involves the VSMC differentiation of MVSCs or the dedifferentiation of mature VSMCs and the potential mechanism for controlling the synthetic phenotype of RASMCs. We enzymatically isolated RASMCs and cultured the cells in both a regular growth medium (RGM) and a stem cell growth medium (SCGM). Regardless of culture conditions, only a small portion of freshly isolated RASMCs attaches, survives and grows slowly during the first 7 days of primary culture, while expressing both SMC- and MVSC-specific markers. RGM-cultured cells undergo a process of synthetic SMC differentiation, whereas SCGM-cultured cells can be differentiated into not only synthetic SMCs but also other somatic cells. Notably, compared with the RGM-cultured differentiated RASMCs, the SCGM-cultured undifferentiated cells exhibit the phenotype of MVSCs and generate greater amounts of reactive oxygen species (ROS) that act as a negative regulator of differentiation into synthetic VSMCs. Knockdown of phospholipase A2, group 7 (Pla2g7) suppresses ROS formation in the MVSCs while enhancing SMC differentiation of MVSCs. These results suggest that cultured synthetic VSMCs can be derived from the SMC differentiation of MVSCs with ROS as a negative regulator.

AKT2 confers protection against aortic aneurysms and dissections.

RATIONALE: Aortic aneurysm and dissection (AAD) are major diseases of the adult aorta caused by progressive medial degeneration of the aortic wall. Although the overproduction of destructive factors promotes tissue damage and disease progression, the role of protective pathways is unknown. OBJECTIVE: In this study, we examined the role of AKT2 in protecting the aorta from developing AAD. METHODS AND RESULTS: AKT2 and phospho-AKT levels were significantly downregulated in human thoracic AAD tissues, especially within the degenerative medial layer. Akt2-deficient mice showed abnormal elastic fibers and reduced medial thickness in the aortic wall. When challenged with angiotensin II, these mice developed aortic aneurysm, dissection, and rupture with features similar to those in humans, in both thoracic and abdominal segments. Aortas from Akt2-deficient mice displayed profound tissue destruction, apoptotic cell death, and inflammatory cell infiltration that were not observed in aortas from wild-type mice. In addition, angiotensin II-infused Akt2-deficient mice showed significantly elevated expression of matrix metalloproteinase-9 (MMP-9) and reduced expression of tissue inhibitor of metalloproteinase-1 (TIMP-1). In cultured human aortic vascular smooth muscle cells, AKT2 inhibited the expression of MMP-9 and stimulated the expression of TIMP-1 by preventing the binding of transcription factor forkhead box protein O1 to the MMP-9 and TIMP-1 promoters. CONCLUSIONS: Impaired AKT2 signaling may contribute to increased susceptibility to the development of AAD. Our findings provide evidence of a mechanism that underlies the protective effects of AKT2 on the aortic wall and that may serve as a therapeutic target in the prevention of AAD.

Nrf2 enhances myocardial clearance of toxic ubiquitinated proteins.

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a master transcription factor that controls the basal and inducible expression of a battery of antioxidant genes and other cytoprotective phase II detoxifying enzymes. While knockout of Nrf2 exaggerates cardiac pathological remodeling and dysfunction in diverse pathological settings, pharmacological activation of Nrf2 protects against cardiomyocyte injury and cardiac dysfunction. In contrast, there is also a concern that the chronic activation of Nrf2 secondary to oxidative stress is a contributing mechanism for the reductive stress-mediated heart failure. However, a direct link between cardiac specific activation of Nrf2 and cardiac protection or dysfunction in vivo remains to be established. Therefore, we investigated the effect of cardiomyocyte-specific transgenic activation of Nrf2 (Nrf2(ctg)) on cardiac pathological remodeling and dysfunction. We found that the cardiomyocyte-specific activation of Nrf2 suppressed myocardial oxidative stress as well as cardiac apoptosis, fibrosis, hypertrophy, and dysfunction in a setting of sustained pressure overload induced by transverse aortic arch constriction (TAC) in mice. Notably, the constitutive activation of Nrf2 increased the steady level of autophagosomes while decreasing the ubiquitinated protein aggregates in the heart after TAC. Nrf2 gene gain- and loss-of-function approaches revealed that Nrf2 enhances autophagosome formation and autophagic flux in cardiomyocytes. Unexpectedly, while Nrf2 minimally regulated apoptosis, it suppressed significantly the proteotoxic necrosis in cardiomyocytes. In addition, Nrf2 attenuated the proteocytotoxicity presumably via enhancing autophagy-mediated clearance of ubiquitinated protein aggregates in cardiomyocytes. Taken together, we demonstrated for the first time that cardiac specific activation of Nrf2 suppresses cardiac maladaptive remodeling and dysfunction most likely by enhancing autophagic clearance of toxic protein aggregates in the heart.

Dihydro-CDDO-trifluoroethyl amide suppresses inflammatory responses in macrophages via activation of Nrf2.

Nuclear factor erythroid 2-related factor (Nrf2) is the major regulator of cellular defenses against various pathological stresses in a variety of organ systems, thus Nrf2 has evolved to be an attractive drug target for the treatment and/or prevention of human disease. Several synthetic oleanolic triterpenoids including dihydro-CDDO-trifluoroethyl amide (dh404) appear to be potent activators of Nrf2 and exhibit chemopreventive promises in multiple disease models. While the pharmacological efficacy of Nrf2 activators may be dependent on the nature of Nrf2 activation in specific cell types of target organs, the precise role of Nrf2 in mediating biological effects of Nrf2 activating compounds in various cell types remains to be further explored. Herein we report a unique and Nrf2-dependent anti-inflammatory profile of dh404 in inflamed macrophages. In lipopolysaccharide (LPS)-inflamed RAW264.7 macrophages, dh404 dramatically suppressed the expression of pro-inflammatory cytokines including inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1 beta (MIP-1ß), while minimally regulating the expression of interleulin-6 (IL-6), IL-1ß, and tumor necrosis factor alpha (TNFα). Dh404 potently activated Nrf2 signaling; however, it did not affect LPS-induced NF-κB activity. Dh404 did not interrupt the interaction of Nrf2 with its endogenous inhibitor Kelch-like ECH associating protein 1 (Keap1) in macrophages. Moreover, knockout of Nrf2 blocked the dh404-induced anti-inflammatory responses in LPS-inflamed macrophages. These results demonstrated that dh404 suppresses pro-inflammatory responses in macrophages via an activation of Nrf2 independently of Keap1 and NF-κB, suggesting a unique therapeutic potential of dh404 for specific targeting a Nrf2-mediated resolution of inflammation.

YAP/TAZ Signaling Enhances Angiogenesis of Retinal Microvascular Endothelial Cells in a High-Glucose Environment.

PURPOSE: Diabetic retinopathy (DR) is a major cause of irreversible blindness in the working-age population. Neovascularization is an important hallmark of advanced DR. There is evidence that Yes-associated protein (YAP)/transcriptional co-activator with a PDZ binding domain (TAZ) plays an important role in angiogenesis and that its activity is regulated by vascular endothelial growth factor (VEGF). Therefore, the aim of this study was to investigate the effect of YAP/TAZ-VEGF crosstalk on the angiogenic capacity of human retinal microvascular endothelial cells (hRECs) in a high-glucose environment. METHODS: The expression of YAP and TAZ of hRECs under normal conditions, hypertonic conditions and high glucose were observed. YAP overexpression (OE-YAP), YAP silencing (sh-YAP), VEGF overexpression (OE-VEGF) and VEGF silencing (sh-VEGF) plasmids were constructed. Cell counting kit-8 assay was performed to detect cells proliferation ability, transwell assay to detect cells migration ability, and tube formation assay to detect tube formation ability. The protein expression of YAP, TAZ, VEGF, matrix metalloproteinase (MMP)-8, MMP-13, vessel endothelium (VE)-cadherin and alpha smooth muscle actin (α-SMA) was measured by western blot. RESULTS: The proliferation of hRECs was significantly higher in the high glucose group compared with the normal group, as well as the protein expression of YAP and TAZ (p < 0.01). YAP and VEGF promoted the proliferation, migration and tube formation of hRECs in the high glucose environment (p < 0.01), and increased the expression of TAZ, VEGF, MMP-8, MMP-13 and α-SMA while reducing the expression of VE-cadherin (p < 0.01). Knockdown of YAP effectively reversed the above promoting effects of OE-VEGF (p < 0.01) and overexpression of YAP significantly reversed the inhibition effects of sh-VEGF on above cell function (p < 0.01). CONCLUSION: In a high-glucose environment, YAP/TAZ can significantly promote the proliferation, migration and tube formation ability of hRECs, and the mechanism may be related to the regulation of VEGF expression.

Repertoire of endothelial progenitor cells mobilized by femoral artery ligation: a nonhuman primate study.

To determine in the baboon model the identities and functional characteristics of endothelial progenitor cells (EPCs) mobilized in response to artery ligation, we collected peripheral blood mononuclear cells (PBMNCs) before and 3 days after a segment of femoral artery was removed. Our goal was to find EPC subpopulations with highly regenerative capacity. We identified 12 subpopulations of putative EPCs that were altered >1.75-fold; two subpopulations (CD146+/CD54-/CD45- at 6.63-fold, and CD146+/UEA-1-/CD45- at 12.21-fold) were dramatically elevated. To investigate the regenerative capacity of putative EPCs, we devised a new assay that maximally resembled their in vivo scenario, we purified CD34+ and CD146+ cells and co-cultured them with basal and mobilized PBMNCs; both cell types took up Dil-LDL, but purified CD146+ cells exhibited accelerated differentiation by increasing expression of CD31 and CD144, and by exhibiting more active cord-like structure formation by comparison to the CD34+ subpopulation in a co-culture with mobilized PBMNCs. We demonstrate that ischaemia due to vascular ligation mobilizes multiple types of cells with distinct roles. Baboon CD146+ cells exhibit higher reparative capacity than CD34+ cells, and thus are a potential source for therapeutic application.

A pro-inflammatory role of deubiquitinating enzyme cylindromatosis (CYLD) in vascular smooth muscle cells.

CYLD, a deubiquitinating enzyme (DUB), is a critical regulator of diverse cellular processes, ranging from proliferation and differentiation to inflammatory responses, via regulating multiple key signaling cascades such as nuclear factor kappa B (NF-κB) pathway. CYLD has been shown to inhibit vascular lesion formation presumably through suppressing NF-κB activity in vascular cells. However, herein we report a novel role of CYLD in mediating pro-inflammatory responses in vascular smooth muscle cells (VSMCs) via a mechanism independent of NF-κB activity. Adenoviral knockdown of Cyld inhibited basal and the tumor necrosis factor alpha (TNFα)-induced mRNA expression of pro-inflammatory cytokines including monocyte chemotactic protein-1 (Mcp-1), intercellular adhesion molecule (Icam-1) and interleukin-6 (Il-6) in rat adult aortic SMCs (RASMCs). The CYLD deficiency led to increases in the basal NF-κB transcriptional activity in RASMCs; however, did not affect the TNFα-induced NF-κB activity. Intriguingly, the TNFα-induced IκB phosphorylation was enhanced in the CYLD deficient RASMCs. While knocking down of Cyld decreased slightly the basal expression levels of IκBα and IκBß proteins, it did not alter the kinetics of TNFα-induced IκB protein degradation in RASMCs. These results indicate that CYLD suppresses the basal NF-κB activity and TNFα-induced IκB kinase activation without affecting TNFα-induced NF-κB activity in VSMCs. In addition, knocking down of Cyld suppressed TNFα-induced activation of mitogen activated protein kinases (MAPKs) including extracellular signal-activated kinases (ERK), c-Jun N-terminal kinase (JNK), and p38 in RASMCs. TNFα-induced RASMC migration and monocyte adhesion to RASMCs were inhibited by the Cyld knockdown. Finally, immunochemical staining revealed a dramatic augment of CYLD expression in the injured coronary artery with neointimal hyperplasia. Taken together, our results uncover an unexpected role of CYLD in promoting inflammatory responses in VSMCs via a mechanism involving MAPK activation but independent of NF-κB activity, contributing to the pathogenesis of vascular disease.

Interleukin 6 destabilizes atherosclerotic plaques by downregulating prolyl-4-hydroxylase α1 via a mitogen-activated protein kinase and c-Jun pathway.

Interleukin 6 (IL-6) is a pivotal cytokine that regulates extracellular matrix metabolism by ameliorating the modification of collagen content, important in fibrous caps of atherosclerotic plaque. Prolyl-4-hydroxylase α1 (P4Hα1) is a key intracellular enzyme required for synthesis of collagen in animals. We investigated the relationship of IL-6 and P4Hα1 in atherosclerosis-prone mice and human aortic smooth muscle cells (HASMCs). Apolipoprotein E (ApoE)-/- mice were fed a high-fat diet and a perivascular constrictive silica collar was placed on the right common carotid artery to induce atherosclerotic lesions, then mice were divided into two groups for transfection with empty lentivirus or IL-6 lentivirus. HASMCs were transfected with small interfering RNA or treated with recombinant human IL-6. IL-6 significantly downregulated collagen, P4Hα1 and smooth muscle cell contents in atherosclerotic mouse arteries. Macrophage and lipid contents in the atherosclerotic area were significantly increased with IL-6 treatment. IL-6 significantly downregulated P4Hα1 expression in HASMCs through an RAF-MEK1/2-ERK1/2 mitogen-activated protein kinase (MAPK) pathway, and c-Jun was involved in the process. Our findings highlight IL-6 destabilize atherosclerotic plaques in mice by downregulating P4Hα1 via an RAF-MEK1/2-ERK1/2 MAPK and c-Jun pathway.

Genetic regulation of endothelial inflammatory responses in baboons.

OBJECTIVE: To investigate the genetic contributions to the expression of cell surface adhesion molecules on endothelial cells (ECs) and to the release by ECs of chemokines, which are responsible for local inflammation. METHODS AND RESULTS: Monocyte adhesion to ECs and transmigration across the endothelial barrier are the key steps in the formation of atherosclerotic plaques and the rupture of the existing plaques. Biopsy specimens were obtained from the femoral arteries of 131 pedigreed baboons (65 males and 66 females) aged 10.4+/-1.5 years (mean+/-SD); arterial ECs were harvested and cultured up to the second passage and then subjected to in vitro challenge with tumor necrosis factor (TNF) alpha, 10 ng/mL, or vehicle for 4 hours. Endothelial surface adhesion molecules were measured using flow cytometry, and chemokines released by the ECs were measured by immunoassay. In response to TNF-alpha treatment, interleukin 8 and monocyte chemoattractant protein-1 released by ECs were increased 3.4- and 26-fold, respectively (P<0.001). The expressions of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 were increased 12.2-, 41.4-, and 3.5-fold, respectively (P<0.001). The quantitative levels of several traits were heritable after TNF-alpha stimulation: h(2)=0.24 (P=0.02) for interleukin 8 and h(2)=0.28 (P=0.003) for E-selectin in culture medium; h(2)=0.21 (P=0.03) for intercellular adhesion molecule-1; and h(2)=0.37 (P<0.001) for vascular cell adhesion molecule-1 expression on EC surfaces. Furthermore, significant heritability was observed for lysate protein level, which is a measure of cell growth rate, with (h(2)=0.64, P<0.001) or without (h(2)=0.51, P<0.001) TNF-alpha stimulation. CONCLUSIONS: This study reports on the heritability of adhesion molecules in ECs when activated by TNF-alpha. This finding suggests genetic regulation of key arterial wall inflammatory processes that are responsible for the initiation of atherosclerotic lesions and the plaque rupture of existing atheromas.

Meta-analysis of aldehyde dehydrogenase 2 gene polymorphism and Alzheimer's disease in East Asians.

BACKGROUND: The association of genetic polymorphism of mitochondrial aldehyde dehydrogenase 2 (ALDH2) and Alzheimer's disease (AD) has been controversial and has been investigated only in several small-sample studies. In the present study, we performed a meta-analysis to evaluate the cross-sectional association of ALDH2 variants and AD risk in East Asian populations. METHODS: Trials were retrieved through MEDLINE, EMBASE, J-STAGE and the China National Knowledge Internet databases (from January 1, 1994 to November 1, 2010) without any restriction on language. Data were abstracted by a standardized protocol. RESULTS: We found four studies of 821AD patients and 1380 healthy controls that qualified for the analysis. The variant ALDH2 genotype GA/AA was not associated with increased AD risk (odds ratio (OR) = 1.35; 95% confidence interval (CI) = 0.75-2.42; p = 0.32), even after stratification for the status of apolipoprotein E epsilon 4 allele. However, in the subgroup analyses, the association was significant for men (OR = 1.72; 95% CI = 1.10-2.67; p = 0.02). CONCLUSIONS: This study adds to the evidence that ALDH2 GA/AA genotype increases the risk of AD among East Asian men, although the effect size is moderate.