A simple method for in vitro preparation of natural killer cells from cord blood.

BACKGROUND: Cord Blood (CB) has been considered a promising source of natural killer (NK) cells for cellular immunotherapy. However, it is difficult to expand the large numbers of highly pure NK cells from CB without cell sorting and feeder cells/multiple cytokines. In this study, we try to develop a simple, safe and economical method for ex vivo expansion and purification of NK cells from CB without cell sorting and feeder cells/multiple cytokines. RESULTS: The large numbers (mean: 1.59 × 1010) of highly pure (≥90%) NK cells from CB could be obtained through interleukin-2, group A streptococcus and zoledronate stimulation of mononuclear cells using the 21-day culture approach. When compared to resting NK cells, expanded NK cells were a higher expression of activating receptors CD16, NKG2D, NKp30, NKp44, NKp46 and activating markers CD62L and CD69, while the inhibitory receptors, CD158a and CD158b remained largely unchanged. In addition, these cells showed a higher concentration of IFN-γ, TNF-α and GM-CSF secretion and cytotoxicity to K562 cells and acute myeloid leukemia targets than resting NK cells. CONCLUSION: We develop a simple, safe and economical method to obtain high yield, purity, and functionality NK cells from CB without cell sorting and feeder cells/multiple cytokines.

Peptidyl-prolyl isomerase Pin1 deficiency attenuates angiotensin II-induced abdominal aortic aneurysm formation in ApoE-/- mice.

Peptidyl-prolyl isomerase Pin1 has been reported to be associated with endothelial dysfunction. However, the role of smooth muscle Pin1 in the vascular system remains unclear. Here, we examined the potential function of Pin1 in smooth muscle cells (SMCs) and its contribution to abdominal aortic aneurysm (AAA) pathogenesis. The level of Pin1 expression was found to be elevated in human AAA tissues and mainly localized to SMCs. We constructed smooth muscle-specific Pin1 knockout mice to explore the role of this protein in AAA formation and to elucidate the underlying mechanisms. AAA formation and elastin degradation were hindered by Pin1 depletion in the angiotensin II-induced mouse model. Pin1 depletion reversed the angiotensin II-induced pro-inflammatory and synthetic SMC phenotype switching via the nuclear factor (NF)-κB p65/Klf4 axis. Moreover, Pin1 depletion inhibited the angiotensin II-induced matrix metalloprotease activities. Mechanically, Pin1 deficiency destabilized NF-κB p65 by promoting its polyubiquitylation. Further, we found STAT1/3 bound to the Pin1 promoter, revealing that activation of STAT1/3 was responsible for the increased expression of Pin1 under angiotensin II stimulation. Thus, these results suggest that Pin1 regulates pro-inflammatory and synthetic SMC phenotype switching and could be a novel therapeutic target to limit AAA pathogenesis.

Irisin inhibits high glucose-induced endothelial-to-mesenchymal transition and exerts a dose-dependent bidirectional effect on diabetic cardiomyopathy.

Emerging evidence indicates that irisin provides beneficial effects in diabetes. However, whether irisin influences the development of diabetic cardiomyopathy (DCM) remains unclear. Therefore, we investigated the potential role and mechanism of action of irisin in diabetes-induced myocardial dysfunction in mice. Type 1 diabetes was induced in mice by injecting streptozotocin, and the diabetic mice were administered recombinant r-irisin (low or high dose: 0.5 or 1.5 µg/g body weight/day, I.P.) or PBS for 16 weeks. Irisin treatment did not alter blood glucose levels in the diabetic mice. However, the results of echocardiographical and histopathological assays indicated that low-dose irisin treatment alleviated cardiac fibrosis and left ventricular function in the diabetic mice, whereas high-dose irisin failed to mitigate the ventricular function impairment and increased collagen deposition. The potential mechanism underlying the effect of low-dose irisin involved irisin-mediated inhibition of high glucose-induced endothelial-to-mesenchymal transition (EndMT); conversely, high-dose irisin treatment enhanced high glucose-induced MMP expression by stimulating MAPK (p38 and ERK) signalling and cardiac fibroblast proliferation and migration. Low-dose irisin alleviated DCM development by inhibiting high glucose-induced EndMT. By contrast, high-dose irisin disrupted normal MMP expression and induced cardiac fibroblast proliferation and migration, which results in excess collagen deposition. Thus, irisin can inhibit high glucose-induced EndMT and exert a dose-dependent bidirectional effect on DCM.

The Role of Psychological Stress on Heart Autophagy in Mice With Heart Failure.

OBJECTIVE: Psychological stress in chronic heart failure (CHF) is associated with systemic neurohormonal and immune system responses and increased mortality. Autophagy refers to the biological process of degradation and recycling of dysfunctional cellular components. We investigated the role of psychological stress on autophagy function in CHF mice. METHODS: C57BL/6 mice underwent transverse aortic constriction, with or without combined acoustic and restraint stress, and cardiac function was assessed by echocardiography analysis. Serum corticosterone and angiotensin II (Ang II) were determined using enzyme-linked immunosorbent assay (ELISA). Autophagy and oxidative stress were measured with immunohistochemistry and quantitative polymerase chain reaction, and chloroquine and rapamycin were used to detect autophagy flux. In vivo, cardiomyocytes were cultured with or without Ang II or N-acetylcysteine, and autophagy and oxidative stress were also detected. RESULTS: A 1-week stress exposure significantly increased serum levels of corticosterone and Ang II (p = .000), increased levels of oxidative stress, induced overt heart failure, and increased mortality (p = .002). Furthermore, stress exposure unregulated messenger RNA expression of Bcl-2-interacting coiled-coil protein 1 (10.891 [3.029] versus 4.754 [1.713], p = .001), cysteine-rich domain containing beclin-1 interacting (6.403 [1.813] versus 3.653 [0.441], p = .006), and autophagy 7 (111.696 [4.049] versus 6.189 [1.931], p = .017), increased expression of autophagosomal, and decreased clearance of autophagosomes. In vitro, Ang II significantly increased autophagy flux in cultured cardiomyocytes, which could be partly inhibited by N-acetylcysteine. CONCLUSIONS: Psychological stress may contribute to the development of CHF by enhancing heart oxidative stress and impairing autophagy flux.

Association of Topoisomerase II (TOP2A) and Dual-Specificity Phosphatase 6 (DUSP6) Single Nucleotide Polymorphisms with Radiation Treatment Response and Prognosis of Lung Cancer in Han Chinese.

BACKGROUND Mutations of DNA topoisomerase II (TOP2A) are associated with chemotherapy resistance, whereas dual-specificity phosphatase 6 (DUSP6) negatively regulates members of the mitogen-activated protein (MAP) kinase superfamily to control cell proliferation. This study assessed TOP2A and DUSP6 single nucleotide polymorphisms (SNPs) in non-small cell lung cancer (NSCLC) patients for association with chemoradiotherapy responses and prognosis. MATERIAL AND METHODS A total of 140 Chinese patients with histologically confirmed NSCLC were enrolled and subjected to genotyping of TOP2A rs471692 and DUSP6 rs2279574 using Taqman PCR. An independent sample t test was used to analyze differences in tumor regression after radiotherapy versus SNP risk factors. Kaplan-Meier curves analyzed overall survival, followed by the log-rank test and Cox proportional hazard models. RESULTS There were no significant associations of TOP2A rs471692 and DUSP6 rs2279574 polymorphisms or clinicopathological variables with response to chemoradiotherapy (p>0.05). Comparing overall survival of 87 patients with stage I-III NSCLC treated with radiotherapy or chemoradiotherapy to clinicopathological variables, the data showed that tumor regression, weight loss, clinical stage, and cigarette smoking were independent prognostic predictors (p=0.009, 0.043, 0.004, and 0.025, respectively). Tumor regression rate >0.34 was associated with patent survival versus tumor regression rate ≤0.34 (p=0.007). CONCLUSIONS TOP2A rs471692 and DUSP6 rs2279574 SNPs were not associated with chemoradiotherapy response, whereas tumor regression, weight loss, clinical stage, and cigarette smoking were independent prognostic predictors for these Chinese patients with NSCLC.

Dual Specificity Phosphatase 6 (DUSP6) Polymorphism Predicts Prognosis of Inoperable Non-Small Cell Lung Cancer after Chemoradiotherapy.

BACKGROUND: Dual-specificity phosphatase 6 (DUSP6) inactivates different target kinases to regulate cell proliferation and differentiation. Altered DUSP6 expressions or gene polymorphisms are associated with human cancer development including non-small cell lung cancer (NSCLC). DNA topoisomerase II alpha (TOP2A) regulates chromosome condensation and chromatid separation, and altered TOP2A expressions are associated with drug resistance development. This study assessed DUSP6 and TOP2A single nucleotide polymorphisms (SNPs) associated with NSCLC patient survival. METHODS: This study included 152 surgically resected NSCLC patients and 277 chemoradiotherapy treated inoperable cases. DNA samples from each patient were genotyped for DUSP6 and TOP2A SNPs. Kaplan-Meier survival analysis, log-rank test, and Cox proportional hazard model were used to evaluate the association between these variants and NSCLC overall survival. RESULTS: DUSP6 rs2279574 A/A genotype was associated with significantly poor inoperable NSCLC patient overall survival (A/A vs. C/C, adjusted HR = 1.549, 95% CI = 1.019-2.355). Stratification analysis against clinical stage, histology, weight loss, and ECOG performance status revealed that the DUSP6 rs2279574 A/A variant homozygous genotype is associated with a decrease in survival of stage IV NSCLC patients compared to those with the C/C genotype (log-rank, p = 0.003). No association was found among histology, weight loss, and ECOG performance status. Moreover, there was no association of TOP2A SNPs between clinicopathological and survival data. CONCLUSIONS: Data obtained from the current study demonstrated that functional DUSP6 rs2279574 polymorphism was able to predict inoperable NSCLC patient survival after chemoradiotherapy.

Rapamycin Combined with Immature Dendritic Cells Attenuates Obliterative Bronchiolitis in Trachea Allograft Rats by Regulating the Balance of Regulatory and Effector T Cells.

BACKGROUND: Obliterative bronchiolitis (OB) ranks as the major obstacle for long-term survival of lung transplantation patients. Rapamycin (Rapa) has recently been confirmed as an immunosuppressant for antirejection due to its suppressive role in T cell activation. Here, we explore the effect of Rapa combined with immature dendritic cells (imDCs) on OB in trachea allograft rats. METHODS: The effect of bone marrow-derived imDCs or Rapa-imDCs on lymphocyte cells and CD4+ T cells were evaluated by methyl thiazolyl tetrazolium and flow cytometry. Tracheal transplantation was performed from Lewis rats to Wistar recipients. Recipient rats received Rapa+imDCs for 10 consecutive days after implantation. Allograft rejection was assessed by micro-CT image, hematoxylin/eosinHE staining and flow cytometry. The underlying mechanism was also investigated. RESULTS: Rapa-imDCs inhibited lymphocyte and CD4+ T cell growth. Furthermore, Rapa-imDC treatment induced T cell hyporesponsiveness by attenuating T cell differentiation into IFN-x03B3;-producing T cells (Th1), but increased CD4+CD25+Foxp3+ T cell (Treg) contents. Importantly, Rapa-imDC administration ameliorated airway obliteration symptoms and CD4+ and CD8+ T cell infiltration. Furthermore, the proinflammatory factor levels of IL-6, TNF-α, IFN-x03B3; and IL-17 were decreased, concomitant with the upregulation of immunosuppressive cytokines IL-10 and TGF-ß1. Further analysis confirmed that Rapa-imDC treatment attenuated the amounts of infiltrated IL-17+CD4+ T cells (Th17 cells) and Th1 cells, but increased Treg contents in the spleens of recipients. CONCLUSIONS: This research may corroborate a protective role of Rapa-imDCs in OB by regulating the balance between effector T cells and Tregs, suggesting a potential applicable strategy to treat OB after lung transplantation.

Period 2 is essential to maintain early endothelial progenitor cell function in vitro and angiogenesis after myocardial infarction in mice.

Cellular therapeutic neovascularization has been successfully performed in clinical trials for patients with ischaemia diseases. Despite the vast knowledge of cardiovascular disease and circadian biology, the role of the circadian clock in regulating angiogenesis in myocardial infarction (MI) remains poorly understood. In this study, we aimed to investigate the role and underlying mechanisms of Period 2 (Per2) in endothelial progenitor cell (EPC) function. Flow cytometry revealed lower circulating EPC proportion in per2(-/-) than in wild-type (WT) mice. PER2 was abundantly expressed in early EPCs in mice. In vitro, EPCs from per2(-/-) mice showed impaired proliferation, migration, tube formation and adhesion. Western blot analysis demonstrated inhibited PI3k/Akt/FoxO signalling and reduced C-X-C chemokine receptor type 4 (CXCR4) protein level in EPCs of per2(-/-) mice. The impaired proliferation was blocked by activated PI3K/Akt/FoxO signalling. Direct interaction of CXCR4 and PER2 was detected in WT EPCs. To further study the effect of per2 on in vivo EPC survival and angiogenesis, we injected saline or DiI-labelled WT or per2(-/-) EPC intramyocardially into mice with induced MI. Per2(-/-) reduced the retention of transplanted EPCs in the myocardium, which was associated with significantly reduced DiI expression in the myocardium of MI mice. Decreased angiogenesis in the myocardium of per2(-/-) EPC-treated mice coincided with decreased LV function and increased infarct size in the myocardium. Per2 may be a key factor in maintaining EPC function in vitro and in therapeutic angiogenesis in vivo.

Resveratrol-enhanced autophagic flux ameliorates myocardial oxidative stress injury in diabetic mice.

Autophagic dysfunction is observed in diabetes mellitus. Resveratrol has a beneficial effect on diabetic cardiomyopathy. Whether the resveratrol-induced improvement in cardiac function in diabetes is via regulating autophagy remains unclear. We investigated the mechanisms underlying resveratrol-mediated protection against heart failure in diabetic mice, with a focus on the role of sirtuin 1 (SIRT1) in regulating autophagic flux. Diabetic cardiomyopathy in mice was induced by streptozotocin (STZ). Long-term resveratrol treatment improved cardiac function, ameliorated oxidative injury and reduced apoptosis in the diabetic mouse heart. Western blot analysis revealed that resveratrol decreased p62 protein expression and promoted SIRT1 activity and Rab7 expression. Inhibiting autophagic flux with bafilomycin A1 increased diabetic mouse mortality and attenuated resveratrol-induced down-regulation of p62, but not SIRT1 activity or Rab7 expression in diabetic mouse hearts. In cultured H9C2 cells, redundant or overactive H2O2 increased p62 and cleaved caspase 3 expression as well as acetylated forkhead box protein O1 (FOXO1) and inhibited SIRT1 expression. Sirtinol, SIRT1 and Rab7 siRNA impaired the resveratrol amelioration of dysfunctional autophagic flux and reduced apoptosis under oxidative conditions. Furthermore, resveratrol enhanced FOXO1 DNA binding at the Rab7 promoter region through a SIRT1-dependent pathway. These results highlight the role of the SIRT1/FOXO1/Rab7 axis in the effect of resveratrol on autophagic flux in vivo and in vitro, which suggests a therapeutic strategy for diabetic cardiomyopathy.

Aberrant promoter methylation of the SFRP1 gene may contribute to colorectal carcinogenesis: a meta-analysis.

This meta-analysis of published cohort studies was conducted to evaluate whether promoter methylation of the secreted frizzled-related protein 1 (SFRP1) gene contributes to colorectal carcinogenesis. The Web of Science (1945 ~ 2013), the Cochrane Library Database (Issue 12, 2013), PubMed (1966 ~ 2013), EMBASE (1980 ~ 2013), CINAHL (1982 ~ 2013), and the Chinese Biomedical Database (CBM) (1982 ~ 2013) were searched without language restrictions. Meta-analysis was conducted using the STATA 12.0 software. We calculated odds ratio (OR) and its 95 % confidence interval (95 % CI) to estimate the correlations between SFRP1 promoter methylation and colorectal carcinogenesis. In the present meta-analysis, 8 cohort studies with a total of 942 patients with colorectal cancer (CRC) were included. The pooled results revealed that the frequency of SFRP1 promoter methylation in cancer tissues were significantly higher than those of normal, adjacent, and benign tissues (cancer tissues vs. normal tissues: OR = 31.49, 95 % CI = 17.57 ~ 56.44, P < 0.001; cancer tissues vs. adjacent tissues: OR = 5.95, 95 % CI 3.12 ~ 10.00, P < 0.001; cancer tissues vs. benign tissues: OR = 3.01, 95 % CI 1.72 ~ 5.27, P < 0.001; respectively). Furthermore, ethnicity-stratified analysis indicated that SFRP1 promoter methylation was strongly correlated with colorectal carcinogenesis among both Asians and Caucasians (all P < 0.05). Our findings provide empirical evidence that SFRP1 promoter methylation may be correlated with the pathogenesis of CRC.

Relationships between genetic polymorphisms in inflammation-related factor gene and the pathogenesis of nasopharyngeal cancer.

Our study aims to discuss the association between inflammation-related factors such as single nucleotide polymorphisms (SNPs) with susceptibility and recurrence in nasopharyngeal carcinoma. We used Taqman real-time polymerase chain reaction (PCR) to characterize the genetic variation of five SNPs in 194 nasopharyngeal carcinoma patients and 231 healthy subjects. All statistical analysis is performed with statistical product and service solutions v13.0; odds ratio (OR) value and 95 % confidence interval (CI) were calculated. There is no relationship between TGFß1 -869 T/C, IL-6 -634C/G, TGFß1 -509C/T, IL1 -511C/T and nasopharyngeal carcinoma susceptibility. Both single factor and multiple factors analysis showed that IL1a -889 T/T genotype is significantly associated with nasopharyngeal carcinoma in decreasing the risk of nasopharyngeal carcinoma. A highly significant association was found between IL1a -889 T/T genotype and protective genotype as defined by various pathological types. This is more obvious in the protective genotype of the non-keratin-type squamous carcinoma undifferentiated type. We also discovered that genotype G/G and C/G + G/G of IL6 -634 gene are associated with reduced recurrence of nasopharyngeal carcinoma. IL1a -889 gene polymorphism and susceptibility is related to nasopharyngeal carcinoma and can potentially decrease the risk of nasopharyngeal carcinoma in the Han Chinese population in north China. IL1-889 TT genotype is protective genotype for nasopharyngeal carcinoma. We have provided evidence that the GG genotype of the IL6 -634 gene is associated with recurrent risk of nasopharyngeal carcinoma. The G allele is the protective gene of nasopharyngeal carcinoma recurrence.

Single-nucleotide polymorphisms of LIG1 associated with risk of lung cancer.

To investigate the association of LIG1 with the risk of lung cancer, all subjects of unrelated ethnic Han Chinese in Liaoning Province were involved in a hospital-based case-control study. The case group consisted of 370 histologically diagnosed lung cancer patients; 314 controls were selected from cancer-free patients during Dec. 2009 to Dec. 2011. LIG1 rs1050298SNP were analyzed by TaqMan real-time PCR method. All statistical analyses were performed with Statistical Product and Service Solution sv13.0 (SPSS). The genotype distribution frequency of LIG1 rs1050298 SNP displayed significant difference between the case and the control group. Individuals carrying the LIG1 rs1050298 T genotype had higher risks of lung cancer, especially those with squamous cell carcinoma.

Angiotensin-(1-7) dose-dependently inhibits atherosclerotic lesion formation and enhances plaque stability by targeting vascular cells.

OBJECTIVE: To test the hypothesis that chronic infusion of angiotensin-(1-7) [Ang-(1-7)] may dose-dependently inhibit atherosclerotic lesion formation by targeting vascular smooth muscle cells and a large dose of Ang-(1-7) may stabilize mature plaque by targeting macrophages. APPROACH AND RESULTS: In vivo, the effects of Ang-(1-7) on atherogenesis and plaque stability were observed in ApoE(-/-) mice fed a high-fat diet and chronic angiotensin II infusion. In vitro, the effects of Ang-(1-7) on vascular smooth muscle cells' proliferation and migration, and macrophage inflammatory cytokines were examined. Ang-(1-7) dose-dependently attenuated early atherosclerotic lesions and inhibited vascular smooth muscle cells' proliferation and migration via suppressing extracellular regulated protein kinase/P38 mitogen-activated protein kinase and janus kinase/signal transducers and activators of transcription activities and enhancing smooth muscle 22α and angiotensin II type 2 receptor expression. Ang-(1-7) treatment resulted in high contents of collagen and vascular smooth muscle cells, and low contents of macrophages and lipids in carotid mature plaques. Ang-(1-7) lowered the expression levels of proinflammatory cytokines and activities of matrix metalloproteinases in mature plaques. CONCLUSIONS: Ang-(1-7) treatment inhibits early atherosclerotic lesions and increases plaque stability in ApoE(-/-) mice, thus providing a novel and promising approach to the treatment of atherosclerosis.

Genetic polymorphisms in the CYP1A1 and CYP1B1 genes and susceptibility to bladder cancer: a meta-analysis.

The current meta-analysis of case-control studies was conducted to evaluated the relationships of genetic polymorphisms in the CYP1A1 and CYP1B1 genes with the susceptibility to bladder cancer, aiming at determine whether these polymorphisms may contribute to the pathogenesis of bladder cancer. Related articles were determined via searching the following electronic databases without any language restrictions: PubMed, CISCOM, CINAHL, Web of Science, Google Scholar, EBSCO, Cochrane Library, and CBM databases for relevant articles published before November 1st, 2013. STATA 12.0 software was also selected to deal with statistical data. The relationships were evaluated using the pooled odds ratios (ORs) and their 95% confidence intervals (CI). Eleven case-control studies with a total of 2,609 bladder cancer patients and 2,634 healthy subjects met the inclusion criteria. The results of our meta-analysis demonstrated that CYP1A1 genetic polymorphisms were associated with increased risks of bladder cancer (allele model: RR = 1.18, 95% CI 1.07-1.30, P = 0.001; dominant model: RR = 1.15, 95% CI 1.05-1.27, P = 0.003; respectively), especially among 11599G>C, 2455A>G, 3810T>C, and 113T>C polymorphisms. A subgroup analysis by ethnicity was conducted to investigate its effect on susceptibility to bladder cancer. The subgroup analysis results revealed positive significant correlations between CYP1A1 genetic polymorphisms and bladder cancer risk among Asians (allele model: RR = 1.26, 95% CI 1.10-1.44, P = 0.001; dominant model: RR = 1.22, 95% CI 1.08-1.38, P = 0.001), but not among Caucasians (all P < 0.05). Nevertheless, we observed no significant correlations between CYP1B1 genetic polymorphisms and bladder cancer risk (all P > 0.05). Our meta-analysis indicates that CYP1A1 genetic polymorphisms may be involved in the pathogenesis of bladder cancer, especially among 11599G>C, 2455A>G, 3810T>C, and 113T>C polymorphisms. However, CYP1B1 genetic polymorphisms may not be important determinants of bladder cancer susceptibility.

Abnormal glutamate metabolism in the retina of aquaporin 4 (AQP4) knockout mice upon light damage.

Glutamate is a major excitatory neurotransmitter in the retina. Glutamate neurotoxicity has been implicated in the pathogenesis of several ocular diseases. Aquaporin 4 (AQP4) is a water-selective membrane transport protein, and its knockout could alter retinal neuron excitability. However, the effect of AQP4 knockout on glutamate metabolism is still unclear in the retina. Here, we reported that the retinas in AQP4 knockout mice showed higher glutamate levels than that in wild-type mice upon light damage. AQP4 knockout could result in accelerated apoptosis of retinal cells, increased reactive gliosis, and attenuated survival of RGCs in response to light damage. Moreover, AQP4 knockout could affect the expression pattern of glutamate metabolism-related proteins such as GLAST and GS. Taken together, this study revealed a novel role of AQP4 in regulating glutamate metabolism. Pharmacological manipulation of AQP4 function may represent as a potent therapeutic target in the treatment of neurological ocular disorders.

Chronic psychological stress induces vascular inflammation in rabbits.

Psychological stress is associated with a systemic inflammatory response. It is unclear, however, whether psychological stress contributes to vascular inflammation. Here, we examined the effects of unpredictable chronic mild stress (UCMS) on vascular inflammation in rabbits. One hundred rabbits were randomly divided into control and stress groups. UCMS was induced by a set of defined adverse conditions applied in a shuffled order for 4, 8, 12, or 16 weeks, and rabbits were killed 24 h after the end of the UCMS protocol. Expression of different inflammatory molecules was analyzed by real-time polymerase chain reaction, immunohistochemistry, or enzyme-linked immunosorbent assay. UCMS resulted in depression-like behaviors, decreased body weight gain, and hypertension with no significant effects on serum lipids. Aortic mRNA and protein expression for tumor necrosis factor-α (TNF-α), C-reactive protein (CRP), monocyte chemoattractant protein-1 (MCP-1), macrophage migration inhibitory factor, and expression of intercellular adhesion molecule-1 (ICAM-1) protein were increased. UCMS increased circulating concentrations of corticosterone, TNF-α, and CRP throughout. Moreover, stress downregulated the expression of endothelial nitric oxide synthase. At 16 weeks of UCMS, macrophage infiltration and lipid accumulation in the subendothelial space were detected in the aorta. In cultured murine vascular smooth muscle cells, treatment with serum from stressed rabbits significantly increased phosphorylation of p38 and c-Jun N-terminal kinase (JNK), and upregulated expression of MCP-1 and ICAM-1 mRNAs, in which the effect was blunted by a TNF-α neutralizing antibody or p38 and JNK inhibitors. Our results indicate that chronic psychological stress induces vascular inflammation via TNF-α and p38/JNK pathways, which may contribute to the development of atherosclerosis.

Hepcidin destabilizes atherosclerotic plaque via overactivating macrophages after erythrophagocytosis.

OBJECTIVE: To explore a direct and causal relationship between vascular hepcidin and atherosclerotic plaque stability. METHODS AND RESULTS: Accelerated atherosclerotic lesions were established by perivascular collar placement in apolipoprotein E-deficient (ApoE(-/-)) mice. Adenoviral overexpression of hepcidin in the carotid artery during plaque formation enhanced intraplaque macrophage infiltration and suppressed the contents of collagen and vascular smooth muscle cells, whereas hepcidin shRNA treatment exerts opposite effects. The overexpression or knockdown of hepcidin did not affect plaque lipid deposition but increased or decreased oxidized low-density lipoprotein (ox-LDL) levels within intraplaque macrophages. In cultured macrophages, ox-LDL not only increased reactive oxygen species formation, inflammatory cytokine production, and apoptosis but also upregulated hepcidin expression. However, hepcidin did not exaggerate the ox-LDL-induced activation of macrophages until an onset of erythrophagocytosis. Whereas hepcidin was critical for the upregulation of L-ferritin and H-ferritin in both ox-LDL-treated erythrophagocytosed macrophages and atherosclerotic plaques, the adding of iron chelators suppressed the intracellular lipid accumulation, reactive oxygen species formation, inflammatory cytokine expression, and apoptosis in erythrophagocytosed macrophages. CONCLUSIONS: Hepcidin promotes plaque destabilization partly by exaggerating inflammatory cytokine release, intracellular lipid accumulation, oxidative stress, and apoptosis in the macrophages with iron retention.

Psychological stress, vascular inflammation, and atherogenesis: potential roles of circulating cytokines.

Cardiovascular disease is a major cause of morbidity and mortality worldwide. Epidemiological studies have clearly demonstrated that chronic psychosocial stress increases the risk of atherosclerotic cardiovascular disease and this may involve multiple mediators and regulating pathways, whereas the precise mechanisms underlying the effects of stress on development of atherosclerosis are not completely understood. In this mini review, we summarize current information from various animal studies suggesting that stress may promote atherogenesis by stimulating vascular inflammation via elevating the level of circulating proinflammatory cytokines (such as tumor necrosis factor α and interleukin 6). Although circulating cytokines can serve as reliable biomarkers of systemic inflammation, in light of the emerging evidence, we propose that these molecules may also have a causal role in mediating stress-triggered vascular inflammatory reaction and atherogenesis. Further studies are warranted to clarify whether targeting circulating cytokines may be an effective approach to reduce the detrimental effects of chronic stress.

Angiotensin-converting enzyme 2 attenuates atherosclerotic lesions by targeting vascular cells.

Angiotensin-converting enzyme 2 (ACE2) is a newly discovered homolog of ACE whose actions oppose those of angiotensin II (AngII). However, the underlying mechanisms by which ACE2 effectively suppresses early atherosclerotic lesions remain poorly understood. Here, we show, both in vitro and in vivo, that ACE2 inhibited the development of early atherosclerotic lesions by suppressing the growth of vascular smooth muscle cells (VSMCs) and improving endothelial function. In a relatively large cohort animal study (66 rabbits), aortic segments transfected by Ad-ACE2 showed significantly attenuated fatty streak formation, neointimal macrophage infiltration, and alleviation of impaired endothelial function. Segments also showed decreased expression of monocyte chemoattractant protein 1, lectin-like oxidized low-density lipoprotein receptor 1, and proliferating cell nuclear antigen, which led to the delayed onset of atherosclerotic lesions. At the cellular level, ACE2 significantly modulated AngII-induced growth and migration in human umbilical vein endothelial cells and VSMCs. The antiatherosclerotic effect of ACE2 involved down-regulation of the ERK-p38, JAK-STAT, and AngII-ROS-NF-kappaB signaling pathways and up-regulation of the PI3K-Akt pathway. These findings revealed the molecular mechanisms of the antiatherosclerotic activity of ACE2 and suggested that modulation of ACE2 could offer a therapeutic option for treating atherosclerosis.