Mitochondrial function and mitochondrial DNA maintenance with advancing age

We review the impact of mitochondrial DNA (mtDNA) maintenance and mitochondrial function on the aging process. Mitochondrial function and mtDNA integrity are closely related. In order to create a protective barrier against reactive oxygen and nitrogen species (RONS) attacks and ensure mtDNA integrity, multiple cellular mtDNA copies are packaged together with various proteins in nucleoids. Regulation of antioxidant and RONS balance, DNA base excision repair, and selective degradation of damaged mtDNA copies preserves normal mtDNA quantities. Oxidative damage to mtDNA molecules does not substantially contribute to increased mtDNA mutation frequency; rather, mtDNA replication errors of DNA PolG are the main source of mtDNA mutations. Mitochondrial turnover is the major contributor to maintenance of mtDNA and functionally active mitochondria. Mitochondrial turnover involves mitochondrial biogenesis, mitochondrial dynamics, and selective autophagic removal of dysfunctional mitochondria (i.e., mitophagy). All of these processes exhibit decreased activity during aging and fall under greater nuclear genome control, possibly coincident with the emergence of nuclear genome instability. We suggest that the age-dependent accumulation of mutated mtDNA copies and dysfunctional mitochondria is associated primarily with decreased cellular autophagic and mitophagic activity.     

Comparative analysis of different enzyme immunoassays for assessment of phosphatidylserine-dependent antiprothrombin antibodies

Phosphatidylserine-dependent antiprothrombin antibodies (aPS/PT) were strongly correlated with the presence of lupus anticoagulant showing a high specificity for the diagnosis of antiphospholipid syndrome. However, the main criticism for the clinical applicability of aPS/PT testing is the lack of reproducibility of the results among laboratories. In this study, we measured IgG and IgM aPS/PT using our original in-house enzyme-linked immunosorbent assays (ELISA) and commercial ELISA kits to assess the assay performance and to evaluate the accuracy of aPS/PT results. The study included 111 plasma samples collected from patients and stored at our laboratory for aPS/PT assessment. Sixty-one samples were tested for IgG aPS/PT using two assays: (1) aPS/PT in-house ELISA and (2) QUANTA Lite? aPS/PT IgG ELISA kit (INOVA Diagnostics, Inc., USA). Fifty samples were evaluated for IgM aPS/PT using two assays: (1) aPS/PT in-house ELISA and (2) QUANTA Lite? aPS/PT IgM ELISA kit (INOVA Diagnostics). Ninety-eight percent of samples yielded concordant results for IgG aPS/PT and 82 % for IgM aPS/PT. There was an excellent agreement between the IgG aPS/PT assays (Cohen κ = 0.962) and moderate agreement between the IgM aPS/PT assays (κ = 0.597). Statistically significant correlations in the aPS/PT results were obtained from both IgG and IgM aPS/PT assays (r = 0.749, r = 0.622, p < 0.001, respectively). In conclusion, IgG and IgM detection by ELISA is accurate. The performance of aPS/PT is reliable, and concordant results can be obtained using different ELISA methods.     

Expression of matrix metalloproteinase-2 and 9 in cervical intraepithelial neoplasia and cervical carcinoma among different age groups of premenopausal and postmenopausal women

Purpose

The objective was to study the gelatinolytic activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in preinvasive and invasive carcinoma of the uterine cervix. The expressions were analysed against different age groups, as to demonstrate whether the expression of MMP-2 and MMP-9 is an early or a late event during the progression of cervical cancer. Additionally, the diagnostic accuracy of MMP-2 and MMP-9 was evaluated with ROC curve.

Methods

A total number of 180 samples of cervical tissue were studied for MMP-2 and MMP-9 gelatinolytic activity. The cases were selected as to include 63 normal cases, 94 CIN cases and 23 cervical carcinoma cases. Among 94 CIN cases, 40 were CIN1, 26 were CIN2 and 28 were CIN3, as reported by histopathology. The gelatinolytic activities of MMP-2 and MMP-9 were evaluated by gelatin zymography in premenopausal and postmenopausal groups.

Results

MMP-2 expressions (latent and active) were very low in control samples, followed by increase in CIN1, decrease in CIN2 and further increase in advance stages. MMP-9 had also shown the same expression pattern that of MMP-2. While comparing the expression of MMP-2 and MMP-9 in different age groups, we found initial CIN stages were prevalent in early age that expressed considerable amount of MMP-2 and MMP-9, and advance stages of carcinoma cervix were prevalent at an elderly age.

Conclusion

Both MMP-2 and MMP-9 have role in cancer progression and remodelling of the ectocervix. Although expression level varies intricately, a distinctive ROC curve demonstrated MMP-2 active form and MMP-9 form could be used in diagnostic purpose in detection of cervical lesion and cancer.     

Antidepressant-like effects of the phosphodiesterase-4 inhibitor etazolate and phosphodiesterase-5 inhibitor sildenafil via cyclic AMP or cyclic GMP signaling in mice

Inhibition of phosphodiesterase-4 or 5 (PDE4 or PDE5) increases cyclic adenosine monophosphate (cAMP)- or cyclic guanosine monophosphate (cGMP), respectively, which activates cAMP response element-binding protein (CREB)/brain-derived neurotrophic factor (BDNF)/neuropeptide VGF (non-acryonimic) signaling and produces antidepressant-like effects on behavior. However, causal links among these actions have not been established. In the present study, mice were evaluated for the effects of etazolate and sildenafil, the inhibitor of PDE4 or PDE5, respectively, on depressive-like behavior induced by chronic unpredictable mild stress (CUMS) in the forced-swimming test (FST) and tail suspension test (TST), in the presence or absence of the inhibitor of protein kinase A (PKA) or protein kinase G (PKG) via intracerebroventricular (i.c.v.) infusions. The levels of cAMP, cGMP and expression of pCREB, CREB, BDNF and VGF in both the hippocampus and prefrontal cortex were determined. The results showed that etazolate at 5.0 mg/kg or sildenafil at 30 mg/kg significantly reversed CUMS-induced depressive-like behavior; the effects were paralleled with the increased levels of cAMP/pCREB/BDNF/VGF or cGMP/pCREB/BDNF/VGF signaling, respectively. These effects were completely abolished following inhibition of PKA or PKG, respectively. The results suggest that inhibition of PDE4 by etazolate or PDE5 by sildenafil produced antidepressant-like effects in CUMS-treated animals via cAMP or cGMP signaling, which shares the common downstream signal pathway of CREB/BDNF/VGF.     

MiR-132 Inhibits Expression of SIRT1 and Induces Pro-inflammatory Processes of Vascular Endothelial Inflammation through Blockade of the SREBP-1c Metabolic Pathway

Purpose

Inflammation participates centrally in all stages of atherosclerosis (AS), which begins with pro-inflammatory processes and inflammatory changes in the endothelium, related to lipid metabolism. MicroRNA (miRNA) inhibition of inflammation related to SIRT1 has been shown to be a promising therapeutic approach for AS. However, the mechanism of action is unknown.

Methods

We investigated whether miRNAs regulate the SIRT1 and its downstream SREBP-lipogenesis-cholesterogenesis metabolic pathway in human umbilical vein endothelial cells (HUVECs). HUVECs were transfected with miR-132 mimics and inhibitors, and then treated with or without tumor necrosis factor α (TNFα). The effects of miR-132 on pro-inflammatory processes, proliferation and apoptosis were assessed.

Results

We identified that the relative 3’ UTR luciferase activities of SIRT1 were significantly decreased in miR-132 transfected HUVECs (0.338?±?0.036) compared to control (P?=?0.000). miR-132 inhibited SIRT1 expression of mRNA level in HUVECs (0.53?±?0.06) (P?SIRT1. mRNA expression and protein levels of SREBP (0.45?±?0.07), fatty acid synthase (FASN) (0.55?±?0.09) and 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) (0.62?±?0.08) (P?TNF-α, and inhibited its proliferation, viability and migration.

Conclusions

SIRT1 mRNAs are direct targets of miR-132. miR-132 controls lipogenesis and cholesterogenesis in HUVECs by inhibiting SIRT1 and SREBP-1c expression and their downstream regulated genes, including FASN and HMGCR. Inhibition of SIRT1 by miR-132 was associated with lipid metabolism-dependent pro-inflammatory processes in HUVECs. The newly identified miRNA, miR-132 represents a novel targeting mechanism for AS therapy.     

AZA-Deoxycytidine stimulates proopiomelanocortin gene expression and ACTH secretion in human pituitary ACTH-secreting tumors

Purpose

It is well known that methylation plays an important role in regulating tissue expression of proopiomelanocortin (POMC) and recent studies have shown that demethylation can occur also in vitro in neuroendocrine tumors. Aim of the present study was to evaluate whether inhibition of methylation modulates POMC expression and ACTH secretion by human corticotrope tumors.

Methods

Twenty two ACTH-secreting pituitary tumors were incubated with 5-AZA-2′-deoxycytidine (AZA), an inhibitor of DNA-methyltransferases, with or without 10 nM corticotropin-releasing hormone (CRH). Both dose response (100 nM–10 μM AZA) and time course (4–96 h) experiments were carried out for measurement of ACTH secretion and POMC gene expression.

Results

Incubation with AZA increased constitutive POMC expression and ACTH secretion by human corticotrope adenomas. The effect appeared most notable at 24 and 48 h with 1 μM AZA. Incubation with AZA did not exert an additional stimulatory effect on CRH-stimulated POMC and ACTH.

Conclusions

The present study shows that AZA increases POMC gene expression and ACTH secretion in human pituitary ACTH-secreting tumors. This can be taken to indicate that mechanisms set into motion by AZA play a role in the regulation of ACTH secretion/POMC expression in tumoral corticotropes and paves the way to further studies in Cushing’s disease.     

Anti-PR3 and anti-MPO antibodies are not present in sera of patients with pulmonary tuberculosis

Anti-neutrophil cytoplasmic antibodies (ANCA) are autoantibodies directed to intracellular components of neutrophils and are present in several vasculitic syndromes. Recently, these autoantibodies have been described in other autoimmune disorders as well as in infectious diseases such as tuberculosis (TB). As there are some clinical similarities between TB and granulomatosis with polyangiitis, we searched for ANCA in a group of patients with proven TB. Patients with TB confirmed by chest X-ray and sputum bacilloscopy either before or within 30 days after beginning treatment were included in this study. Anti-MPO and anti-PR3 antibodies were studied using well-standardized ELISA kits (INOVA Diagnostics, Inc.). ANCA were also investigated by indirect immunofluorescence (IIF). Fifty TB patients (26 females, mean age 47.34 ± 17 years) were enrolled in the present study. No patient tested positive for ANCA by IIF, or anti-MPO or anti-PR3 antibodies by ELISA. Although previous studies have shown the presence of ANCA in some infectious diseases, the findings of the present study demonstrated the absence of such antibodies in TB. The discrepancy in the prevalence of ANCA in TB among different studies may be attributed to methodological factors and/or the genetic background of the studied populations.     

PTEN Upregulation May Explain the Development of Insulin Resistance and Type 2 Diabetes with High Dose Statins

Purpose

Statins increase the incidence of new onset diabetes. Prolonged statin therapy upregulates PTEN expression. PTEN levels are also elevated in diabetic animals. Activation of protein kinase A by cAMP decreases PTEN expression. We assessed whether prolonged treatment with rosuvastatin (ROS) induces glucose intolerance by upregulating Phosphatase and Tensin Homologue on Chromosome 10 (PTEN) in mice receiving normal (ND) or Western Diet (WD) and whether concomitant treatment with cilostazol (CIL, a phosphodiesterase-3 inhibitor) attenuates the effects.

Methods

PTEN^loxp/cre or PTEN^+/? mice received ND or WD without or with ROS (10 mg/kg/day). Wild-type mice received ND or WD without or with ROS, CIL (10 mg/kg/day), or ROS+CIL for 30 days. Fasting insulin and glucose tolerance test were measured as well as PTEN and P-AKT levels in skeletal muscle.

Results

Serum glucose after intraperitoneal injection of glucose was higher in PTEN^loxp/cre mice receiving WD or ROS and especially WD+ROS. Levels were lower in PTEN^+/? mice compared to PTEN^loxp/cre in each treatment group. CIL decreased glucose levels in mice receiving WD, ROS and their combination. Insulin levels were higher in the WD+ROS group. CIL decreased insulin in mice receiving WD+ROS. WD, ROS and especially their combination increased PTEN and decreased P-AKT levels. CIL attenuated the effect of WD, ROS and their combination.

Conclusions

Long-term ROS can induce diabetes by upregulating PTEN. CIL attenuates these changes. Partial knockdown of PTEN also ameliorates ROS-induced insulin resistance. Further studies are needed to assess the effects of increasing cAMP levels to prevent the induction of diabetes by statins.     

TLR3 and TLR4 as potential clinically biomarkers of cardiovascular risk in coronary artery disease (CAD) patients

Coronary artery disease (CAD), as a lipid-driven and inflammation-driven disease, has threatened thousands of patients’ lives. Toll-like receptors, the most characterized innate immune receptors, have recently been demonstrated to play a key role in coronary artery disease, particularly Toll-like receptor (TLR) 3 and TLR4. We examined TLR3, TLR4, and associated inflammatory factors expression in monocytes and their signaling pathway proteins in patients with varying degrees of coronary artery atherosclerosis [group S (single diseased vessel), n = 36; group D (double diseased vessels), n = 36; group T (three diseased vessels), n = 33 compared with controls (n = 35)]. In mononuclear cells, TLR3 mRNA and protein, and IRF-3 were significantly down-regulated as the coronary arteries stenosis number increased. However, TLR4 mRNA and protein, and MyD88 were significantly increased in patients with coronary artery stenosis compared with controls, and were associated with the number of stenoses. In serum, there was significant up-regulation in TNF-α, IL-8, and MCP-1 and obvious down-regulation in INF-β and IP-10 with severity of CAD. This study demonstrates differential expression of TLR3 and TLR4 at both the mRNA and protein level in both mononuclear cells and downstream serum readouts of patients with CAD compared with the control. The expression of TLR4 and TLR3 closely correlated with the severity of coronary artery disease as reflected by the number of coronary artery stenoses. TLR3 and TLR4 have the potential to be a clinically useful biomarker of cardiovascular risk.     

Somatostatin Inhibits the Production of Interferon-γ by Intestinal Epithelial Cells During Intestinal Ischemia–Reperfusion in Macaques

Background

Our previous study found that somatostatin (SST) inhibited the intestinal inflammatory injury in a macaque model of intestinal ischemia–reperfusion (IIR); however, the underlying mechanism was unclear.

Aims

The present study was aimed to investigate the effects of SST on IFN-γ and the systemic inflammatory response after IIR.

Methods

Fifteen macaques were randomly divided into controls, IIR and SST+ IIR groups. ELISA was performed to measure IFN-γ in ileum tissues, ileac epithelial cells (IECs) and ileal lymphocytes, as well as the systemic levels of IL-6, IL-1β, TNF-α and IFN-γ in the peripheral circulation and the portal vein. HE staining was performed to evaluate morphological changes in vital organs. Immunohistochemistry was performed to identify the distribution of IFN-γ, CD4, CD8 and CD57 in the ileum.

Results

After IIR, IFN-γ level was significantly increased in the IECs. IL-6, IL-1β and TNF-α were significantly increased in both the portal vein and the peripheral circulation; in contrast, IFN-γ level was increased in the portal vein alone. Prophylactic SST reversed the change in IFN-γ in the IECs and portal vein. SST led to an alleviation of the pathological changes in systemic vital organs. The distribution of CD4⁺, CD57⁺ and CD8⁺ cells was not positively correlated with the secretion of IFN-γ.

Conclusion

IECs are the main source of IFN-γ production after IIR. SST may indirectly lead to mast cell deactivation through the inhibition of IFN-γ production by IECs. Pretreatment with SST may be beneficial for preventing a massive systemic inflammatory response in vital organs after IIR.     

Gut Bacterial Translocation May Aggravate Microinflammation in Hemodialysis Patients

Background/Aims

Bacterial translocation (BT) promotes microinflammation in predialysis patients with end-stage renal disease (ESRD). However, the change in BT has not been reported in ESRD patients undergoing regular hemodialysis treatment. The present study investigated whether hemodialysis promotes gut BT and microinflammation.

Methods

The blood, gut, and dialysate of hemodialysis patients were analyzed using bacterial 16S rDNA amplification and DNA pyrosequencing to determine the presence of bacteria and alteration in gut microbiomes. High-sensitive C-reactive protein (hs-CRP), interleukin-6 (IL-6), and endotoxin were also determined. Plasma d-lactate was tested for gut permeability.

Results

Bacteria were present in the plasma of 12 out of 52 ESRD patients. The majority of the bacteria detected in the blood were also distributed in the gut of ESRD patients on the basis of the phylogenetics of the blood and gut microbial specimens in the patients. In patient, groups treated with and without hemodialysis, the plasma hs-CRP, IL-6, and endotoxin levels differed between the positive and negative plasma bacterial DNA. In patients who were positive in blood bacteria, the bacterial DNA concentration was positively correlated with plasma levels of CRP and IL-6. The ESRD patients who underwent hemodialysis had a different flora and showed slightly higher levels of hs-CRP, IL-6, and plasma endotoxin, compared with those in ESRD patients who did not undergo hemodialysis.

Conclusion

ESRD, rather than hemodialysis, primarily contributes to BT and microinflammation in ESRD patients. Hemodialysis may exaggerate microinflammation in ESRD patients to some extent.     

LOX-1 Deletion Limits Cardiac Angiogenesis in Mice Given Angiotensin II

Lectin-like oxidized low-density lipoprotein (ox-LDL) receptor-1 (LOX-1) is a major receptor for ox-LDL in endothelial cells. Its activation regulates endothelial proliferation, differentiation, migration and apoptosis. Recent in vitro studies show that LOX-1 activation by ox-LDL and angiotensin II (Ang II) induces angiogenesis via activation of NADPH oxidase and subsequent increase in ROS production. In this study, we investigated the effect of LOX-1 gene deletion (LOX-1 knockout or KO mice) on angiogenesis in response to prolonged Ang II infusion in vivo. Our studies showed that Ang II (vs. saline) infusion enhanced capillary formation in subcutaneously injected Matrigel® plugs. Ang II infusion also resulted in marked angiogenesis in the hearts as determined by CD31 immunopositivity. There was an increased expression (RT-PCR and Western blotting) of CD31 and VEGF in the hearts of mice infused with Ang II, indicating pro-angiogenic miliue. More importantly, LOX-1 KO mice reveled markedly limited angiogenesis in the Matrigel® plugs as well as in the hearts despite similar infusion with Ang II (all P?MCP-1 and IL-1β following Ang II Infusion. Lastly, the rise in blood pressure in response to Ang II was less in the LOX-1 KO mice (P?相似文献   

Evaluating the Optimal Timing of Revascularisation in Patients with Transient ST-Segment Elevation Myocardial Infarction: Rationale and Design of the TRANSIENT Trial

Patients with chest pain and a prehospital ST-segment elevation myocardial infarction (STEMI) are preferably treated with immediate percutaneous coronary intervention (PCI). However, patients with normalization of symptoms and ST-segment elevation upon hospital arrival (transient STEMI) received inconsistent therapy due to logistic reasons and the absence of evidence or explicit guidelines. In this trial, the optimal timing of coronary angiography and subsequent revascularisation is investigated in patients presenting with transient STEMI. In this prospective, multicentre, randomized controlled clinical trial, 142 consecutive patients with initially acute chest pain and STEMI, whose symptoms and ST-segment elevation resolve upon admission, are randomized to immediate intervention or a delayed intervention. Primary outcome is infarct size measured at 4 days determined by cardiovascular magnetic resonance. Secondary outcomes are left ventricular function and volumes, myocardial salvage and microvascular injury at baseline; the change in left ventricular function, volumes and infarct size at 4 months; and major adverse cardiac events at 4 and 12 months. The TRANSIENT Trial evaluates whether a delayed invasive strategy (according to NSTEMI-guidelines) is superior to an immediate invasive strategy (according to STEMI-guidelines) in patients with a transient STEMI.     

PPARβ/δ prevents endoplasmic reticulum stress-associated inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism

Aim/hypothesis

Endoplasmic reticulum (ER) stress, which is involved in the link between inflammation and insulin resistance, contributes to the development of type 2 diabetes mellitus. In this study, we assessed whether peroxisome proliferator-activated receptor (PPAR)β/δ prevented ER stress-associated inflammation and insulin resistance in skeletal muscle cells.

Methods

Studies were conducted in mouse C2C12 myotubes, in the human myogenic cell line LHCN-M2 and in skeletal muscle from wild-type and PPARβ/δ-deficient mice and mice exposed to a high-fat diet.

Results

The PPARβ/δ agonist GW501516 prevented lipid-induced ER stress in mouse and human myotubes and in skeletal muscle of mice fed a high-fat diet. PPARβ/δ activation also prevented thapsigargin- and tunicamycin-induced ER stress in human and murine skeletal muscle cells. In agreement with this, PPARβ/δ activation prevented ER stress-associated inflammation and insulin resistance, and glucose-intolerant PPARβ/δ-deficient mice showed increased phosphorylated levels of inositol-requiring 1 transmembrane kinase/endonuclease-1α in skeletal muscle. Our findings demonstrate that PPARβ/δ activation prevents ER stress through the activation of AMP-activated protein kinase (AMPK), and the subsequent inhibition of extracellular-signal-regulated kinase (ERK)1/2 due to the inhibitory crosstalk between AMPK and ERK1/2, since overexpression of a dominant negative AMPK construct (K45R) reversed the effects attained by PPARβ/δ activation.

Conclusions/interpretation

Overall, these findings indicate that PPARβ/δ prevents ER stress, inflammation and insulin resistance in skeletal muscle cells by activating AMPK.