Anthony J. Valente

Interleukin-18-induced human coronary artery smooth muscle cell migration is dependent on NF-κB- and AP-1-mediated matrix metalloproteinase-9 expression and is inhibited by atorvastatin

The proliferation and migration of arterial smooth muscle cells (SMCs) are key events in the vascular restenosis that frequently follows angioplasty. Furthermore, SMC migration and neointimal hyperplasia are promoted by degradation of the extracellular matrix by matrix metalloproteinases (MMPs). Because we demonstrated previously that the proinflammatory and proatherogenic cytokine interleukin-18 (IL-18) stimulates SMC proliferation (Chandrasekar, B., Mummidi, S., Valente, A. J., Patel, D. N., Bailey, S. R., Freeman, G. L., Hatano, M., Tokuhisa, T., and Jensen, L. E. (2005) J. Biol. Chem. 280, 26263–26277), we investigated whether IL-18 induces SMC migration in an MMP-dependent manner and whether the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor atorvastatin can inhibit this response. IL-18 treatment increased both mRNA and protein expression of MMP9 in human coronary artery SMCs. Gel shift, enzyme-linked immunosorbent, and chromatin immunoprecipitation assays revealed a strong induction of IL-18-mediated AP-1 (c-Fos, c-Jun, and Fra-1) and NF-κB (p50 and p65) activation and stimulation of MMP9 promoter-dependent reporter gene activity in an AP-1- and NF-κB-dependent manner. Ectopic expression of p65, c-Fos, c-Jun, and Fra-1 induced MMP9 promoter activity. Specific antisense or small interfering RNA reagents for these transcription factors reduced IL-18-mediated MMP9 transcription. Furthermore, IL-18 stimulated SMC migration in an MMP9-dependent manner. Atorvastatin effectively suppressed IL-18-mediated AP-1 and NF-κB activation, MMP9 expression, and SMC migration. Together, our results indicate for the first time that the proatherogenic cytokine IL-18 induces human coronary artery SMC migration in an MMP9-dependent manner. Atorvastatin inhibits IL-18-mediated aortic SMC migration and has therapeutic potential for attenuating the progression of atherosclerosis and restenosis.

A modern view of atherogenesis

Two key events in the atherogenic cascade are the focal influx and accumulation of low-density lipoprotein (LDL) cholesterol at arterial sites having a predilection for atherosclerotic lesion development and the recruitment of blood monocytes to these lesion-prone sites. Both processes are enhanced in the setting of hyperlipidemia and dyslipoproteinemia. The monocytes recruited to the endothelial surface subsequently migrate to the subendothelial space under the directed guidance of chemoattractants, such as monocyte chemotactic protein-1 and oxidatively modified LDL. These cells then undergo activation-differentiation to become macrophages. At the same time, LDL, and probably other lipoproteins such as the small dense LDL particles and lipoprotein (a), traverse the endothelium and undergo oxidative modification by reactive oxygen species. These oxidatively modified lipoproteins are recognizable by the non-down-regulating macrophage scavenger receptor. Their uptake by these receptors results in the formation of the foam cell characteristic of early-stage atherosclerosis. As monocyte recruitment and lipoprotein influx continue, the lesion grows and develops into the fatty streak. Subsequent foam cell necrosis due to the influence of cytotoxic oxidatively modified LDL and increased collagen synthesis by intimal smooth muscle cells lead to the established atherosclerotic lesion referred to as the fibrous plaque. As our understanding of the mechanisms involved in the pathogenesis of atherosclerosis has evolved over the past few years, novel strategies for intervention in the atherogenic process have emerged.

Interleukin-17 Stimulates C-reactive Protein Expression in Hepatocytes and Smooth Muscle Cells via p38 MAPK and ERK1/2-dependent NF-κB and C/EBPβ Activation

Elevated systemic levels of the acute phase C-reactive protein (CRP) are predictors of future cardiovascular events. There is evidence that CRP may also play a direct role in atherogenesis. Here we determined whether the proinflammatory interleukin (IL)-17 stimulates CRP expression in hepatocytes (Hep3B cell line and primary hepatocytes) and coronary artery smooth muscle cells (CASMC). Our results demonstrate that IL-17 potently induces CRP expression in Hep3B cells independent of IL-1β and IL-6. IL-17 induced CRP promoter-driven reporter gene activity that could be attenuated by dominant negative IκBα or C/EBPβ knockdown and stimulated both NF-κB and C/EBP DNA binding and reporter gene activities. Targeting NF-κB and C/EBPβ activation by pharmacological inhibitors, small interfering RNA interference and adenoviral transduction of dominant negative expression vectors blocked IL-17-mediated CRP induction. Overexpression of wild type p50, p65, and C/EBPβ stimulated CRP transcription. IL-17 stimulated p38 MAPK and ERK1/2 activation, and SB203580 and PD98059 blunted IL-17-mediated NF-κB and C/EBP activation and CRP transcription. These results, confirmed in primary human hepatocytes and CASMC, demonstrate for the first time that IL-17 is a potent inducer of CRP expression via p38 MAPK and ERK1/2-dependent NF-κB and C/EBPβ activation and suggest that IL-17 may mediate chronic inflammation, atherosclerosis, and thrombosis.

Pathogenesis of the Atherosclerotic Lesion: Implications for diabetes mellitus

In this review, we have highlighted pivotal cellular and molecular events in the initiation and progression of atherosclerosis. Key components of lesion initiation are an enhanced focal intimal influx and accumulation of lipoproteins, including LDL in hemodynamically determined lesion-prone areas, focal monocyte-macrophage recruitment, intimal generation of ROS, and oxidative modification of lipoproteins (including LDL [Ox-LDL]). Modified lipoproteins are taken up by the non-downregulating macrophage scavenger receptor, with foam cell formation and the development of the so-called fatty streak. One transitional event in lesion progression is foam cell necrosis, likely attributable to the cytotoxicity of both intimal free radicals and Ox-LDL, with development of an extracellular metabolically inert lipid core. Another is the migration to and proliferation within the intima of medial SMCs, leading to the synthesis of plaque collagens, elastin, and proteoglycans. Mural thrombosis plays a significant role in the late-stage progression of lesions. Regression of lesions is considered a function of the dynamic balance among components of initiation, progression, plaque stabilization, and removal of plaque constituents—the so-called regression quartet. Here, we critically examine how components of diabetes mellitus might impact not only lesion development, but also lesion regression. It is concluded that some components of diabetes mellitus augment key mechanisms in lesion initiation and progression and will likely retard the processes of plaque regression. Specifically, we focus on the various influences of diabetes mellitus on lipoprotein influx and accumulation, free radical generation and Ox-LDL, monocyte-macrophage recruitment, thrombosis and impaired fibrinolysis, and the reverse cholesterol transport system. The importance of nonenzymatic protein glycosylation in modifying a number of these processes is emphasized.

Novel Regulation of Parkin Function Through c-Abl-Mediated Tyrosine Phosphorylation: Implications for Parkinson's Disease

Mutations in parkin, an E3 ubiquitin ligase, are the most common cause of autosomal-recessive Parkinsons disease (PD). Here, we show that the stress-signaling non-receptor tyrosine kinase c-Abl links parkin to sporadic forms of PD via tyrosine phosphorylation. Under oxidative and dopaminergic stress, c-Abl was activated in cultured neuronal cells and in striatum of adult C57BL/6 mice. Activated c-Abl was found in the striatum of PD patients. Concomitantly, parkin was tyrosine-phosphorylated, causing loss of its ubiquitin ligase and cytoprotective activities, and the accumulation of parkin substrates, AIMP2 (aminoacyl tRNA synthetase complex-interacting multifunctional protein 2) (p38/JTV-1) and FBP-1.STI-571, a selective c-Abl inhibitor, prevented tyrosine phosphorylation of parkin and restored its E3 ligase activity and cytoprotective function both in vitro and in vivo. Our results suggest that tyrosine phosphorylation of parkin by c-Abl is a major post-translational modification that leads to loss of parkin function and disease progression in sporadic PD. Moreover, inhibition of c-Abl offers new therapeutic opportunities for blocking PD progression.

Thrombin stimulates proliferation of liver fat-storing cells and expression of monocyte chemotactic protein-1: Potential role in liver injury☆

Liver fat‐storing cells (FSC) proliferate and secrete extracellular matrix in experimental models of liver injury. In this study, we determined if thrombin, a serine protease produced during acute and chronic tissue injury, modulates the functions of FSC. Thrombin stimulated DNA synthesis and proliferation of FSC, as assessed by [3H]‐thymidine incorporation assay and measurement of cell number, respectively. Thrombin also increased the secretion of monocyte chemotactic protein‐1 (MCP‐1) in a time‐and dose‐dependent fashion. The effect of thrombin on both DNA synthesis and MCP‐1 secretion was neutralized by pretreatment of thrombin with hirudin. The increased MCP‐1 secretion was associated with increased steady‐state levels of MCP‐1 messenger RNA. Pretreatment of FSC with 5 μmol/L retinol for 48 hours inhibited the mitogenic effects of thrombin but not the induction of MCP‐1 secretion. FSC express specific transcripts encoding for the human thrombin receptor, as shown by Northern blot analysis of poly (A)+ RNA. Proteolytic activation of the thrombin receptor results in the formation of a new N‐terminus that functions as a tethered ligand. We studied the effects of a thrombin receptor activating peptide (TRAP) corresponding to the newly formed N‐terminus, on FSC. TRAP mimicked the effects of thrombin on [3H]‐thymidine incorporation, MCP‐1 secretion, and MCP‐1 gene expression. This study suggests that thrombin may be involved in modulating FSC proliferation and monocyte chemotaxis during human liver disease, through proteolytic activation of its receptor. (Hepatology 1995; 22:780–787.)

Adiponectin blocks interleukin-18-mediated endothelial cell death via APPL1-dependent AMP-activated protein kinase (AMPK) activation and IKK/NF-κB/PTEN suppression

The adipocyte-derived cytokine adiponectin is known to exert anti-inflammatory and anti-apoptotic effects. In patients with atherosclerotic cardiovascular disease, circulating levels of adiponectin correlate inversely with those of the proinflammatory, proapoptotic cytokine interleukin (IL)-18. The opposing actions of IL-18 and adiponectin on both cell survival and inflammation led us to investigate whether adiponectin signaling antagonizes IL-18-mediated endothelial cell death and to identify the underlying molecular mechanisms. Treatment with IL-18 suppressed Akt phosphorylation and its associated kinase activity, induced IκB kinase (IKK)-NF-κB-dependent PTEN activation, and promoted endothelial cell death. Pretreatment with adiponectin stimulated APPL1-dependent AMPK activation, reversed Akt inhibition in a phosphatidylinositol 3-kinase-dependent manner, blocked IKK-NF-κB-PTEN signaling, reduced caspase-3 activity, blocked Bax translocation, and inhibited endothelial cell death. The cytoprotective effect of adiponectin signaling was recapitulated by treatment with the pharmacological AMPK activator 5-aminoimidazole-4-carboxamide-1-β-riboside. Collectively, these results demonstrated that adiponectin reverses IL-18-mediated endothelial cell death through an AMPK-associated mechanism, which may thus have therapeutic potential for diminishing IL-18-dependent vascular injury and inflammation.

The potential role of platelet-derived growth factor as an autocrine or paracrine factor for human bone cells

Platelet-derived growth factor, PDGF, is a potent mitogen for cells of mesenchymal origin such as fibroblasts, smooth muscle cells and glial cells. PDGF is thought to have the potential to act as both a paracrine and an autocrine factor. Studies described here extend these observations to human bone-derived cells. Exogenous PDGF induces biologic activity in two human osteogenic sarcoma cell lines and in one of these, the two PDGF genes, PDGF-1 and PDGF-2/c-sis are expressed. In addition, PDGF stimulates proliferation of normal osteoblastic cells derived from adult human cancellous bone. The expression of the PDGF-1 gene but not the PDGF-2/c-sis gene is demonstrated in normal human adult bone-derived cells by Northern blot analysis and synthesis of PDGF is shown by immunoprecipitation with PDGF antisera. These studies indicate that PDGF has the potential to act as a paracrine or autocrine regulator of bone cells.

Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion

We recently reported that prolonged exposure of human aortic endothelial cells (HAEC) to low shear stress flow patterns is associated with a sustained increase in the activated form of the transcriptional regulator nuclear factor-kappaB (NF-kappaB). Here we investigate the hypothesis that low shear-induced activation of NF-kappaB is responsible for enhanced expression of vascular cell adhesion molecule (VCAM-1) resulting in augmented endothelial cell-monocyte (EC-Mn) adhesion and that this activation is dependent on intracellular oxidant activity. Before exposure to low shear (2 dyn/cm2) for 6 h, HAEC were preincubated with or without the antioxidants pyrrolidine dithiocarbamate (PDTC) or N-acetyl-L-cysteine (NAC). PDTC strongly inhibited low shear-induced activation of NF-kappaB, expression of VCAM-1, and EC-Mn adhesion. Paradoxically, NAC exerted a positive effect on low shear-induced VCAM-1 expression and EC-Mn adhesion and only slightly downregulated NF-kappaB activation. However, cytokine-induced NF-kappaB activation and VCAM-1 expression are blocked by both PDTC and NAC. These data suggest that NF-kappaB plays a key role in low shear-induced VCAM-1 expression and that pathways mediating low shear- and cytokine-induced EC-Mn adhesion may be differentially regulated.

Resveratrol inhibits high glucose-induced PI3K/Akt/ERK-dependent interleukin-17 expression in primary mouse cardiac fibroblasts

We investigated the expression of the proinflammatory cytokine interleukin (IL)-17 in cardiac fibroblasts and its induction by high glucose (HG). Our results show that primary mouse cardiac fibroblasts (mCFs) secrete low basal levels of IL-17 and that HG (25 mM D-glucose) as opposed to low glucose (5 mM D-glucose + 20 mM mannitol) significantly enhances its secretion. HG induces IL-17 mRNA expression by both transcriptional and posttranscriptional mechanisms. HG induces phosphoinositide 3- kinase [PI3K; inhibited by adenoviral (Ad).dominant negative (dn)PI3Kp85], Akt (inhibited by Ad.dnAkt1), and ERK (inhibited by PD-98059) activation and induces IL-17 expression via PI3K-->Akt-->ERK-dependent signaling. Moreover, mCFs express both IL-17 receptors A and C, and although IL-17RA is upregulated, HG fails to modulate IL-17RC expression. Furthermore, IL-17 stimulates net collagen production by mCFs. Pretreatment with the phytoalexin resveratrol blocks HG-induced PI3K-, Akt-, and ERK-dependent IL-17 expression. These results demonstrate that 1) cardiac fibroblasts express IL-17 and its receptors; 2) HG upregulates IL-17 and IL-17RA, suggesting a positive amplification loop in IL-17 signaling in hyperglycemia; 3) IL-17 enhances net collagen production; and 4) resveratrol can inhibit these HG-induced changes. Thus, in hyperglycemic conditions, IL-17 may potentiate myocardial inflammation, injury, and remodeling through autocrine and paracrine mechanisms, and resveratrol has therapeutic potential in ameliorating this effect.