Zufeng Ding

Oxidant stress in mitochondrial DNA damage, autophagy and inflammation in atherosclerosis

Our studies in HUVECs show that ox-LDL induced autophagy and damaged mtDNA leading to TLR9 expression. LOX-1 antibody or the ROS inhibitor apocynin attenuated ox-LDL-mediated autophagy, mtDNA damage and TLR9 expression, suggesting that these events are LOX-1 and ROS-dependent phenomena. Experiments using siRNA to DNase II indicated that DNase II digests mtDNA to protect the tissue from inflammation. Next, we studied and found intense autophagy, TLR9 expression and inflammatory signals (CD45 and CD68) in the aortas of LDLR knockout mice fed high cholesterol diet. Deletion of LOX-1 (LDLR/LOX-1 double knockout mice) attenuated autophagy, TLR9 expression as well as CD45 and CD68. Damaged mtDNA signal was also very high in LDLR knockout mice aortas, and this signal was attenuated by LOX-1 deletion. Thus, it appears that oxidative stress-mediated damaged mtDNA that escapes autophagy induces a potent inflammatory response in atherosclerosis.

Regulation of autophagy and apoptosis in response to ox-LDL in vascular smooth muscle cells, and the modulatory effects of the microRNA hsa-let-7g

OBJECTIVES Regulation of autophagy and apoptosis during treatment of vascular smooth muscle cells (VSMCs) with pro-atherogenic stimuli, such as oxidized low density lipoprotein (ox-LDL), remains unclear. METHODS AND RESULTS We examined the expression of autophagy and apoptosis upon treatment of VSMCs with ox-LDL. Exposure to ox-LDL in modest amounts (10-40 μg/ml) enhanced autophagy (expression of beclin-1, LC3-II/LC3-1 ratio and Atg5) and apoptosis (expression of caspase-3, Bax, Bcl-2 and Bcl-xL); however, exposure to higher concentrations (≥ 60 μg/ml) induced high levels of apoptosis but autophagy declined. Pretreatment of VSMCs with the miRNA hsa-let-7 g inhibited autophagy, as LOX-1 expression and apoptosis declined. Hsa-let-7 g treatment also resulted in a decrease in intracellular ROS generation. Treatment with LOX-1 antibody had similar effects as hsa-let-7 g. Next, we studied autophagy and apoptosis in aortic segments from wild-type and LOX-1 knockout mice fed a high cholesterol diet, and observed increased autophagy as well as apoptosis in lipid-rich sections of aortas from wild-type mice and LOX-1 knockout mice (vs. corresponding controls); however, both autophagy and apoptosis in lipid-rich areas in aortic sections of LOX-1 knockout mice were less than in WT mice. These in vivo data are in keeping with in vitro data showing enhanced autophagy and apoptosis of VSMCs exposed to modest amount of ox-LDL. CONCLUSION This study provides first set of data on the regulation of autophagy and apoptosis in ox-LDL-treated VSMCs. Our observations also suggest that hsa-let-7 g acts as a critical regulator of autophagy and apoptosis by modulating LOX-1.

Current Concepts of the Role of Oxidized LDL Receptors in Atherosclerosis.

Atherosclerosis is characterized by accumulation of lipids and inflammatory cells in the arterial wall. Oxidized low-density lipoprotein (ox-LDL) plays important role in the genesis and progression of atheromatous plaque. Various scavenger receptors have been recognized in the past two decades that mediate uptake of ox-LDL leading to formation of foam cells. Inhibition of scavenger receptor A and CD36 has been shown to affect progression of atherosclerosis by decreasing foam cell formation. Lectin-type oxidized LDL receptor 1 (LOX-1) participates at various steps involved in the pathogenesis of atherosclerosis, and in experimental studies its blockade has been shown to affect the progression of atherosclerosis at multiple levels. In this review, we summarize the role of ox-LDL and scavenger receptors in the formation of atheroma with emphasis on effects of LOX-1 blockade.

LOX-1, mtDNA damage, and NLRP3 inflammasome activation in macrophages: implications in atherogenesis.

AIMS Lectin-like ox-LDL scavenger receptor-1 (LOX-1) and mitochondrial DNA (mtDNA) damage play a key role in a variety of cardiovascular diseases, including atherosclerosis, hypertension, and inflammation. We posited that damaged mtDNA could trigger autophagy and NLRP3 inflammasome activation, and LOX-1 may play a critical role in this process. METHODS AND RESULTS In order to examine this hypothesis, cultured human THP-1 macrophages exposed to lipopolysaccharide (LPS) were applied to study the link between LOX-1, mtDNA damage, autophagy, and NLRP3 inflammasome expression. Our data showed that LPS markedly induced LOX-1 expression, reactive oxygen species (ROS) generation, autophagy, mtDNA damage, and NLRP3 inflammasome. LOX-1 inhibition with a binding antibody or siRNA inhibited ROS generation, autophagy and mtDNA damage, and a decreased expression of NLRP3 inflammasome. To study the LOX-1-NLRP3 inflammasome signalling, we performed studies using ROS inhibitors and an autophagy inducer, and found that both decreased the expression of NLRP3. On the other hand, autophagy inhibitor enhanced the expression of NLRP3 inflammasome. Knockdown of DNase II inhibited autophagy and NLRP3 inflammasome, providing further support for our hypothesis. Finally, we confirmed the relationship between LOX-1, ROS, mtDNA damage, autophagy, and NLRP3 inflammasome activation in primary macrophages. CONCLUSIONS This study based on THP-1 macrophages and primary macrophages indicates that LOX-1-mediated autophagy and mtDNA damage play an essential role in NLRP3 inflammasome activation in inflammatory disease states.

DPP-4 Inhibitors Repress NLRP3 Inflammasome and Interleukin-1beta via GLP-1 Receptor in Macrophages Through Protein Kinase C Pathway

BackgroundAnti-atherosclerotic effects of dipeptidyl peptidase-4 (DPP-4) inhibitors have been shown in many studies. Since inflammation and immune response play a key role in atherogenesis, we examined the effect of DPP-4 inhibitors on the expression of nod-like receptor family, pyrin domain containing 3 (NLRP3) Inflammasome and Interleukin-1beta (IL-1β) in human macrophages.Methods and ResultsTHP-1 macrophages were incubated with oxidized low density lipoprotein (ox-LDL) with or without DPP-4 inhibitors (sitagliptin and NVPDPP728). The effects of DPP-4 inhibitors on the expression of NLRP3, toll-like receptor 4 (TLR4) and pro-inflammatory cytokine IL-1β were studied. Both DPP-4 inhibitors induced a significant reduction in NLRP3, TLR4 and IL-1β expression; concurrently, there was an increase in glucagon like peptide 1 receptor (GLP-1R) expression. Simultaneously, DPP-4 inhibitors reduced phosphorylated-PKC, but not PKA, levels. To determine the role of PKC activation in the effects of DPP-4 inhibitors, cells were treated with PMA- which blocked the effect of DPP-4 inhibitors on NLRP3 and IL-1β as well as TLR4 and GLP-1R. Over-expression of GLP-1R in macrophages with its agonist liraglutide also blocked the effects of PMA.ConclusionDPP-4 inhibitors suppress NLRP3, TLR4 and IL-1β in human macrophages through inhibition of PKC activity. This study provides novel insights into the mechanism of inhibition of inflammatory state and immune response in atherosclerosis by DPP-4 inhibitors.

Cross-talk between LOX-1 and PCSK9 in vascular tissues

AIMS Lectin-like ox-LDL receptor-1 (LOX-1) plays an important role in inflammatory diseases, such as atherosclerosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) modulates LDL receptor degradation and influences serum LDL levels. The present study was designed to investigate the possible interaction between PCSK9 and LOX-1. METHODS AND RESULTS In the first set of experiments, human vascular endothelial cells and smooth muscle cells were studied at baseline and after lipopolysaccharide (LPS) treatment (to create an inflammatory state). Both PCSK9 and LOX-1 were strongly induced by LPS treatment. To define the role of PCSK9 in LOX-1 expression, cells were transfected with siRNA against PCSK9, which resulted in reduced LOX-1 expression and function. On the other hand, cells exposed to recombinant hPCSK9 revealed enhanced LOX-1 expression (P < 0.05). To determine whether LOX-1 also regulates PCSK9, cultured cells in which LOX-1 was knocked down by siRNA expressed less PCSK9, whereas those transfected with hLOX-1 cDNA showed increased PCSK9 expression. The second set of experiments was carried out in wild-type (WT) and gene knockout (KO; LOX-1 and PCSK9) mice; LOX-1 KO mice showed much less PCSK9 (P < 0.05 vs. WT mice). PCSK9-KO mice showed much less LOX-1 (P < 0.05 vs. WT mice). Furthermore, we observed that mitochondrial reactive oxygen species (mtROS) plays an initiating role in the LOX-1/PCSK9 interaction, since mtROS induction enhanced and its inhibition reduced the expression of both PCSK9 and LOX-1. We also found that both LOX-1 and PCSK9 regulate adhesion molecules vascular cell adhesion molecule-1 expression. Finally, oxidized low-density lipoprotein and tumour necrosis factor-α, pro-inflammatory stimuli besides LPS, regulated PCSK9 expression that is mediated by the NF-κB signalling pathway. CONCLUSIONS These observations suggest that LOX-1 and PCSK9 positively influence each others expression, especially during an inflammatory reaction. mtROS appear to be important initiators of PCSK9/LOX-1 expression.

Impact of Hepatitis C Seropositivity on the Risk of Coronary Heart Disease Events

Chronic infections have been shown to enhance atherogenicity. However, the association between chronic hepatitis C (HCV) and coronary heart disease (CHD) remains controversial. We examined the risk for CHD events in patients with HCV with an emphasis on the risk of CHD events with active infection. We conducted a retrospective cohort study using the Enterprise Data Warehouse at the University of Arkansas for Medical Sciences. HCV positive and negative patients were identified based on serology and incident CHD events were studied. Patient characteristics at entry were compared either by analysis of variance/F-test (continuous variables) or by a Chi-squared test (categorical variables). The joint effect of risk factors for incident CHD was evaluated using logistic regression. A total of 8,251 HCV antibody positive, 1,434 HCV RNA positive and 14,799 HCV negative patients were identified. HCV antibody and RNA positive patients had a higher incidence of hypertension, diabetes mellitus, obesity and chronic lung disease, but lower serum cholesterol levels compared to HCV negative patients (p< 0.001). HCV seropositive patients had a higher incidence of CHD events when compared to controls (4.9% vs. 3.2%, p<0.001). In the HCV cohort, patients with detectable HCV RNA had a significantly higher incidence of CHD events when compared to patients who were only HCV antibody positive with no detectable RNA (5.9% vs. 4.7%, p=0.04). In multivariate logistic regression analysis, both HCV antibody positivity (OR 1.32, 95% CI 1.09-1.60, p<0.001) and HCV RNA positivity (OR 1.59, 95% CI 1.13-2.26, p<0.001) were independent risk factors for incident CHD events. In conclusion, there is increased incidence of CHD events in HCV seropositive patients and the incidence is much higher in patients with detectable HCV RNA when compared to patients with remote infection who are only antibody positive. Lipid profile does not appear to be a good cardiovascular risk stratification tool in HVC patients.

Hemodynamic Shear Stress via ROS Modulates PCSK9 Expression in Human Vascular Endothelial and Smooth Muscle Cells and Along the Mouse Aorta

AIMS To investigate a possible link between hemodynamic shear stress, reactive oxygen species (ROS) generation, and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression. RESULTS Using a parallel-plate flow chamber, we observed that PCSK9 expression in vascular smooth muscle cells (SMCs) and endothelial cells (ECs) reached maximal value at low shear stress (3-6 dynes/cm(2)), and then began to decline with an increase in shear stress. PCSK9 expression increased when cells were treated with lipopolysaccharide. PCSK9 expression was always greater in SMCs than in ECs. ROS generation followed the same pattern as PCSK9 expression. Aortic branching and aorta-iliac bifurcation regions of mouse aorta that express low shear stress were also found to have greater PCSK9 expression (vs. other regions). To determine a relationship between ROS and PCSK9 expression, ECs and SMCs were treated with ROS inhibitors diphenylene-iodonium chloride and apocynin, and both markedly reduced PCSK9 expression. Relationship between PCSK9 and ROS was further studied in p47(phox) and gp91(phox) knockout mice; both mice strains revealed low PCSK9 levels in serum and mRNA levels in aorta-iliac bifurcation regions (vs. wild-type mice). Other studies showed that ROS and NF-κB activation plays a bridging role in PCSK9 expression via lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). INNOVATION Low shear stress induces PCSK9 expression, which is mediated by NADPH oxidase-dependent ROS production. CONCLUSIONS This study provides evidence that low shear stress enhances PCSK9 expression in concert with ROS generation in vascular ECs and SMCs. ROS seem to regulate PCSK9 expression. We propose that PCSK9-ROS interaction may be important in the development of atherosclerosis in arterial channels with low shear stress.

Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis

Autophagy is a conserved process involved in lysosomal degradation of protein aggregates and damaged organelles. The role of autophagy in cancer is a topic of intense debate, and the underlying mechanism is still not clear. The hypoxia-inducible factor 2α (HIF2α), an oncogenic transcription factor implicated in renal tumorigenesis, is known to be degraded by the ubiquitin–proteasome system (UPS). Here, we report that HIF2α is in part constitutively degraded by autophagy. HIF2α interacts with autophagy–lysosome system components. Inhibition of autophagy increases HIF2α, whereas induction of autophagy decreases HIF2α. The E3 ligase von Hippel-Lindau and autophagy receptor protein p62 are required for autophagic degradation of HIF2α. There is a compensatory interaction between the UPS and autophagy in HIF2α degradation. Autophagy inactivation redirects HIF2α to proteasomal degradation, whereas proteasome inhibition induces autophagy and increases the HIF2α–p62 interaction. Importantly, clear-cell renal cell carcinoma (ccRCC) is frequently associated with monoallelic loss and/or mutation of autophagy-related gene ATG7, and the low expression level of autophagy genes correlates with ccRCC progression. The protein levels of ATG7 and beclin 1 are also reduced in ccRCC tumors. This study indicates that autophagy has an anticancer role in ccRCC tumorigenesis, and suggests that constitutive autophagic degradation of HIF2α is a novel tumor suppression mechanism.

MicroRNAs Involved in the Regulation of Postischemic Cardiac Fibrosis

As deaths from acute myocardial infarction have declined dramatically over the past several decades, more and more patients survive with chronic myocardial ischemia.1,2 Hearts of most patients who survive acute myocardial infarction undergo remodeling. Hearts of most patients with sustained hypertension, another common cardiovascular disease, also show some degree of remodeling. The heart consists of 3 primary cell types: cardiomyocytes, fibroblasts, and endothelial cells. Myocytes occupy 75% of cardiac mass, generate the electric impulses, and induce cardiac contraction. Fibroblasts are primarily responsible for the maintenance of extracellular matrix (ECM) that is composed of collagens, fibronectins, elastins, fibrillins, and several other proteins. The capillary microcirculation network composed primarily of endothelial cells that serves the contractile assembly.3 In state of health, renin–angiotensin–aldosterone system is thought to be responsible for sustenance of cardiomyocyte, fibroblast, and endothelial cell biology. However, renin–angiotensin–aldosterone system activation leads to profound changes in cell biology during sustained hypoxia. Cardiomyocytes in the regions adjacent to ischemic area become large to maintain cardiac contraction. Fibroblasts around the cardiomyocytes begin to grow and differentiate into myofibroblasts to compensate for low-cardiac output. This process of cardiomyocyte hypertrophy and cardiac fibroblast growth is commonly known as cardiac remodeling.4–6 Release of large amounts of reactive oxygen species in the ischemic myocardium and the associated inflammatory response are thought to be mechanistic contributors to cardiac remodeling. Local activation of renin–angiotensin–aldosterone system contributes to further reactive oxygen species generation and release of transforming growth factor-β (TGF-β); the latter plays a central role in fibroblast transformation into myofibroblasts and subsequent activation of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and synthesis of collagens.7 MMPs and TIMPs mediate the balance between formation of collagens and their degradation, and their abnormal expression promotes secretion of large amounts of collagens by rapidly …