Differential Regulation of Matrix Metalloproteinase-2 and -9 Expression and Activity in Adult Rat Cardiac Fibroblasts in Response to Interleukin-1β

Matrix metalloproteinases (MMPs), a family of endoproteinases, are implicated in cardiac remodeling. Interleukin-1β (IL-1β), which is increased in the heart following myocardial infarction, increases expression and activity of MMP-2 (gelatinase A) and -9 (gelatinase B) in cardiac fibroblasts. Previously, we have shown that IL-1β activates ERK1/2, JNKs, and protein kinase C (PKC). However, signaling pathways involved in the regulation of MMP-2 and -9 expression and activity are not yet well understood. Using adult rat cardiac fibroblasts, we show that inhibition of ERK1/2 and JNKs inhibits IL-1β-stimulated increases in MMP-9, not MMP-2, expression and activity. Chelerythrine, an inhibitor of PKC, inhibited activation of ERK1/2 and JNKs and expression and activity of both MMPs. Selective inhibition of PKC-α/β1 using Gö6976 inhibited JNKs activation and the expression and activity of MMP-9, not MMP-2. Inhibition of PKC-θ and PKC-ζ using pseudosubstrates inhibited IL-1β-stimulated activation of ERK1/2 and JNKs and the expression and activity of MMP-2 and -9. Inhibition of PKC-ε had no effect. IL-1β activated NF-κB pathway as measured by increased phosphorylation of IKKα/β and Inhibition of ERK1/2, JNKs, and PKC-α/β1 had no effect on NF-κB activation, whereas inhibition of PKC-θ and PKC-ζ inhibited IL-1β-stimulated activation of NF-κB. SN50, NF-κB inhibitor peptide, inhibited IL-1β-stimulated increases in MMP-2 and -9 expression and activity. These observations suggest that 1) activation of ERK1/2 and JNKs plays a critical role in the regulation of MMP-9, not MMP-2, expression and activity; 2) PKC-α/β1 act upstream of JNKs, not ERK1/2; 3) PKC-ζ and -θ, not PKC-ε, act upstream of JNKs, ERK1/2, and NF-κB; and 4) activation of NF-κB stimulates expression and activity of MMP-2 and -9

Osteopontin Inhibits Interleukin-1β-Stimulated Increases in Matrix Metalloproteinase Activity in Adult Rat Cardiac Fibroblasts: Role of Protein Kinase C-ζ

We have shown that osteopontin (OPN), an extracellular matrix protein, plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Interleukin-1β (IL-1β), increased in the heart following MI, increases matrix metalloproteinase (MMP) activity in cardiac fibroblasts in vitro. Here, we show that OPN alone has no effect on MMP activity or expression. However, it reduces IL-1β-stimulated increases in MMP activity and expression in adult rat cardiac fibroblasts. Pretreatment with bovine serum albumin had no effect on MMP activity or protein content, whereas GRGDS (glycine-arginine-glycine-aspartic acid-serine)- pentapeptide (which interrupts binding of RGD-containing proteins to cell surface integrins) and monoclonal antibody m7E3 (a rat β3 integrins antagonist) inhibited the effects of OPN. Inhibition of PKC using chelerythrine inhibited the activities of both MMP-2 and MMP-9. Stimulation of cells using IL-1β increased phosphorylation and translocation of PKC to membrane fractions, which was inhibited by OPN. OPN inhibited IL-1β-stimulated increases in translocation of PKC-ζ from cytosolic to membrane fractions. Furthermore, the levels of phospho-PKC-ζ were lower in the cytosolic fractions of OPN knock-out mice hearts as compared with wild type 6 days post-MI. Inhibition of PKC-ζ using PKC-ζ pseudosubstrate inhibited IL-1β-stimulated increases in MMP-2 and MMP-9 activities. These observations suggest that OPN, acting via 3 integrins, inhibits IL-1β-stimulated increases in MMP-2 and MMP-9 activity, at least in part, via the involvement of PKC-ζ. Thus, OPN may play a key role in collagen deposition during myocardial remodeling following MI by modulating cytokine-stimulated MMP activity

Upregulation of Mitochondrial Uncoupling Protein-2 by the AMP-Activated Protein Kinase in Endothelial Cells Attenuates Oxidative Stress in Diabetes

OBJECTIVE—Recent evidence suggests that the AMP-activated protein kinase (AMPK) is an important therapeutic target for diabetes. The present study was conducted to determine how AMPK activation suppressed tyrosine nitration of prostacyclin synthase in diabetes

7-Ketocholesterol Induces Autophagy in Vascular Smooth Muscle Cells through Nox4 and Atg4B

Oxidized lipoproteins stimulate autophagy in advanced atherosclerotic plaques. However, the mechanisms underlying autophagy induction and the role of autophagy in atherogenesis remain to be determined. This study was designed to investigate the mechanisms by which 7-ketocholesterol (7-KC), a major component of oxidized lipoproteins, induces autophagy. This study was also designed to determine the effect of autophagy induction on apoptosis, a central event in the development of atherosclerosis. Exposure of human aortic smooth muscle cells to 7-KC increased autophagic flux. Autophagy induction was suppressed by treating the cells with either a reactive oxygen species scavenger or an antioxidant. Administration of 7-KC concomitantly up-regulated Nox4 expression, increased intracellular hydrogen peroxide levels, and inhibited autophagy-related gene 4B activity. Catalase overexpression to remove hydrogen peroxide or Nox4 knockdown with siRNA reduced intracellular hydrogen peroxide levels, restored autophagy-related gene 4B activity, and consequently attenuated 7-KC–induced autophagy. Moreover, inhibition of autophagy aggravated both endoplasmic reticulum (ER) stress and cell death in response to 7-KC. In contrast, up-regulation of autophagic activity by rapamycin had opposite effects. Finally, activation of autophagy by chronic rapamycin treatment attenuated ER stress, apoptosis, and atherosclerosis in apolipoprotein E knockout (ApoE−/−) mouse aortas. In conclusion, we demonstrate that up-regulation of autophagy is a cellular protective response that attenuates 7-KC–induced cell death in human aortic smooth muscle cells

PRKAA1/AMPKα1 is required for autophagy-dependent mitochondrial clearance during erythrocyte maturation

AMP-activated protein kinase α1 knockout (prkaa1−/−) mice manifest splenomegaly and anemia. The underlying molecular mechanisms, however, remain to be established. In this study, we tested the hypothesis that defective autophagy-dependent mitochondrial clearance in prkaa1−/− mice exacerbates oxidative stress, thereby enhancing erythrocyte destruction. The levels of ULK1 phosphorylation, autophagical flux, mitochondrial contents, and reactive oxygen species (ROS) were examined in human erythroleukemia cell line, K562 cells, as well as prkaa1−/− mouse embryonic fibroblasts and erythrocytes. Deletion of Prkaa1 resulted in the inhibition of ULK1 phosphorylation at Ser555, prevented the formation of ULK1 and BECN1- PtdIns3K complexes, and reduced autophagy capacity. The suppression of autophagy was associated with enhanced damaged mitochondrial accumulation and ROS production. Compared with wild-type (WT) mice, prkaa1−/− mice exhibited a shortened erythrocyte life span, hemolytic destruction of erythrocytes, splenomegaly, and anemia, all of which were alleviated by the administration of either rapamycin to activate autophagy or Mito-tempol, a mitochondria-targeted antioxidant, to scavenge mitochondrial ROS. Furthermore, transplantation of WT bone marrow into prkaa1−/− mice restored mitochondrial removal, reduced intracellular ROS levels, and normalized hematologic parameters and spleen size. Conversely, transplantation of prkaa1 −/− bone marrow into WT mice recapitulated the prkaa1−/− mouse phenotypes. We conclude that PRKAA1-dependent autophagy-mediated clearance of damaged mitochondria is required for erythrocyte maturation and homeostasis

X-Ray Induced Photodynamic Therapy: A Combination of Radiotherapy and Photodynamic Therapy

Conventional photodynamic therapy (PDT)'s clinical application is limited by depth of penetration by light. To address the issue, we have recently developed X-ray induced photodynamic therapy (X-PDT) which utilizes X-ray as an energy source to activate a PDT process. In addition to breaking the shallow tissue penetration dogma, our studies found more efficient tumor cell killing with X-PDT than with radiotherapy (RT) alone. The mechanisms behind the cytotoxicity, however, have not been elucidated. In the present study, we investigate the mechanisms of action of X-PDT on cancer cells. Our results demonstrate that X-PDT is more than just a PDT derivative but is essentially a PDT and RT combination. The two modalities target different cellular components (cell membrane and DNA, respectively), leading to enhanced therapy effects. As a result, X-PDT not only reduces short-term viability of cancer cells but also their clonogenecity in the long-run. From this perspective, X-PDT can also be viewed as a unique radiosensitizing method, and as such it affords clear advantages over RT in tumor therapy, especially for radioresistant cells. This is demonstrated not only in vitro but also in vivo with H1299 tumors that were either subcutaneously inoculated or implanted into the lung of mice. These findings and advances are of great importance to the developments of X-PDT as a novel treatment modality against cancer

Integrative metabolomic and transcriptomic analysis reveals difference in glucose and lipid metabolism in the longissimus muscle of Luchuan and Duroc pigs

Luchuan pig, an obese indigenous Chinese porcine breed, has a desirable meat quality and reproductive capacity. Duroc, a traditional western breed, shows a faster growth rate, high feed efficiency and high lean meat rate. Given the unique features these two porcine breeds have, it is of interest to investigate the underlying molecular mechanisms behind their distinctive nature. In this study, the metabolic and transcriptomic profiles of longissimus dorsi muscle from Duroc and Luchuan pigs were compared. A total of 609 metabolites were identified, 77 of which were significantly decreased in Luchuan compared to Duroc, and 71 of which were significantly elevated. Most differentially accumulated metabolites (DAMs) upregulated in Luchuan were glycerophospholipids, fatty acids, oxidized lipids, alcohols, and amines, while metabolites downregulated in Luchuan were mostly amino acids, organic acids and nucleic acids, bile acids and hormones. From our RNA-sequencing (RNA-seq) data we identified a total of 3638 differentially expressed genes (DEGs), 1802 upregulated and 1836 downregulated in Luchuan skeletal muscle compared to Duroc. Combined multivariate and pathway enrichment analyses of metabolome and transcriptome results revealed that many of the DEGs and DAMs are associated with critical energy metabolic pathways, especially those related to glucose and lipid metabolism. We examined the expression of important DEGs in two pathways, AMP-activated protein kinase (AMPK) signaling pathway and fructose and mannose metabolism, using Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). Genes related to glucose uptake, glycolysis, glycogen synthesis, fatty acid synthesis (PFKFB1, PFKFB4, MPI, TPI1, GYS1, SLC2A4, FASN, IRS1, ULK1) are more activated in Luchuan, while genes related to fatty acid oxidation, cholesterol synthesis (CPT1A, HMGCR, FOXO3) are more suppressed. Energy utilization can be a decisive factor to the distinctive metabolic, physiological and nutritional characteristics in skeletal muscle of the two breeds we studied. Our research may facilitate future porcine breeding projects and can be used to reveal the potential molecular basis of differences in complex traits between various breeds

Genotypic and Environmental Effects on the Volatile Chemotype of Valeriana jatamansi Jones

Valeriana jatamansi Jones is an aromatic medicinal herb and important alternative to V. officinalis, which is utilized for medicinal purposes in China and India and also as spices in India. Bioactive ingredients of V. jatamansi vary in different regions. However, no information is currently available on influence of genotype and environmental factors in the volatile compounds, especially when germplasms and planting locations need to be selected. Based on the results of SNP and volatile constituents from GC-MS analysis, this study found various genotypes and chemotypes of V. jatamansi for wild plants from seven regions in China and common-garden samples; correlations between genotype and chemotype were revealed for the plants. Two distinct populations (PX, FY) were distinguishable from five others (GJ, YL, SY, DD, DY) according to their genotypes and volatile profiles, the consistency of which was observed showing that genotype could significantly influence chemotype. Wild populations and common-garden samples were also separated in their volatile profiles, demonstrating that environmental factors strongly affected their chemotypes. Compounds contributing to the discrimination were identified as discriminatory compounds. This investigation has explored and provided essential information concerning the correlation between genotype and chemotype as well as environmental factors and chemotype of V. jatamansi in some regions of China. Feasible plantation and conservation strategies of V. jatamansi could be further explored based on these results