Tanshinone IIA from Salvia miltiorrhiza BUNGE inhibits human aortic smooth muscle cell migration and MMP-9 activity through AKT signaling pathway

Smooth muscle cell (SMC) migration plays an important role in normal angiogenesis and is relevant to disease-related vascular remodeling in conditions such as brain arteriovenous malformations, pulmonary hypertension, arteriosclerosis, and restenosis after angioplasty. In this present study, we showed that tanshinone IIA, the major lipid-soluble pharmacological constituent of Salvia miltiorrhiza BUNGE, inhibits human aortic smooth muscle cell (HASMC) migration and MMP-9 activity. Tanshinone IIA significantly inhibited IkappaBalpha phosphorylation and p65 nuclear translocation through inhibition of AKT phosphorylation. Tanshinone IIA inhibited TNF-alpha-induced ERK and c-jun phosphorylation, but not other MAPKs such as JNK and p38. Tanshinone IIA also inhibited NF-kappaB and AP-1 DNA-binding. Moreover, tanshinone IIA inhibited the migration of TNF-alpha-induced HASMCs. Our results provide evidence that tanshinone IIA has multiple effects in the inhibition of HASMC migration and may offer a therapeutic approach to block HASMC migration.     

Salvianolic acid B attenuates MMP-2 and MMP-9 expression in vivo in apolipoprotein-E-deficient mouse aorta and in vitro in LPS-treated human aortic smooth muscle cells

Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a Chinese medicinal herb, is believed to have multiple therapeutic and preventive against human vascular diseases, including atherosclerosis and restenosis. To elucidate the underlying cellular mechanisms, we produced hypercholesterolemia by feeding apo-E-deficient mice a 0.15% cholesterol diet and inflammation in human aortic smooth muscle cells (HASMCs) with the endotoxin lipopolysaccharide (LPS), focusing on the metallopreteinases MMP-2 and MMP-9, the relevant signal transduction pathways and the effects of Sal B. Immunohistochemical analyses indicated apo-E-deficient mice fed a 0.15% cholesterol diet for 12 weeks exhibited thickened intima and elevated levels of MMP-2 and MMP-9 in aortic sections, both of which were attenuated by 0.3% Sal B dietary supplement. Western blotting and zymography analyses indicated that unstimulated HASMCs exhibited basal levels of protein and activity levels for MMP-2 and barely detectable levels for MMP-9, both of which were markedly upregulated by LPS, which also induced cell migration. Sal B significantly attenuated upregulations of both MMPs as well as the LPS-induced cell migration through the inactivation of MMP-2 and MMP-9 protein synthesis as well as the downregulation of the extracellular-signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). These results demonstrate that Sal B has anti-migration properties on smooth muscle cells and may explain its anti-atherosclerotic properties. This novel mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis.     

Ascochlorin suppresses oxLDL-induced MMP-9 expression by inhibiting the MEK/ERK signaling pathway in human THP-1 macrophages

The critical initiating event in atherogenesis involves the invasion of monocytes through the endothelial walls of arteries and the transformation of monocytes from macrophages into foam cells. Human THP-1 monocytic cells can be induced to differentiate into macrophages by phorbol myristate acetate (PMA) and can then be converted into foam cells by exposure to oxidized low-density lipoprotein (oxLDL). Also, during a chronic inflammatory response, monocytes/macrophages produce the 92-kDa matrix metalloproteinase-9 (MMP-9) that may contribute to the extravasation, migration, and tissue remolding capacities of the phagocytic cells. Here, we investigate the effect of ascochlorin (ASC), a prenylphenol antiviral compound from the fungus Ascochyta viciae, on oxLDL-induced MMP-9 expression and activity in human THP-1 macrophages. ASC reduced oxLDL-induced MMP-9 expression and activity in a time-dependent and dose-dependent manner. Also, an analysis of MMP-9 activity using pharmacologic inhibitors showed that ASC inhibits MMP-9 activity via the extracellular signal-regulated kinase 1 and kinase 2 pathways. Our results suggest that ASC may be useful as a potent clinical antiatherogenic agent, a topic of considerable interest in the biological chemistry of chemotherapeutic agents.     

Cyclic strain-induced endothelial MMP-2: role in vascular smooth muscle cell migration

Matrix metalloproteinases (MMPs) play a vital role in vasculature response to hemodynamic stimuli via the degradation of extracellular matrix substrates. In this study, we investigated the putative role of cyclic strain-induced endothelial MMP-2 (and MMP-9) expression and release in modulating bovine aortic smooth muscle cell (BASMC) migration in vitro. Equibiaxial cyclic strain of bovine aortic endothelial cells (BAECs) leads to elevation in cellular MMP-2 (and MMP-9) expression, activity, and secretion into conditioned media, events which were time- and force-dependent. Subsequent incubation of BASMCs with conditioned media from chronically strained BAECs (5%, 24 h) significantly reduces BASMC migration (38+/-6%), an inhibitory effect which could be completely reversed by targeted siRNA 'knock-down' of MMP-2 (but not MMP-9) expression and activity in BAECs. Moreover, inhibition of strain-mediated MMP-2 expression in BAECs by protein tyrosine kinase (PTK) blockade with genistein (50 microM) was also found to completely reverse this inhibitory effect on BASMC migration. Finally, direct supplementation of recombinant MMP-2 into the BASMC migration assay was found to have no significant effect on migration. However, the effect on BASMC migration of MMP-2 siRNA transfection in BAECs could be reversed by supplementation of recombinant MMP-2 into BAEC media prior to (and for the duration of) strain. These findings reveal a potentially novel role for strain-induced endothelial MMP-2 in regulating vascular SMC migration.     

Hypoxia induces epidermal keratinocyte matrix metalloproteinase-9 secretion via the protein kinase C pathway

Hypoxia promotes keratinocyte migration on wound bed connective tissues and is a profound biological signal that transforms a basal keratinocyte, destined to differentiate, into a motile cell that is essential for re-epithelialization. In this study, we examined the effect of hypoxia on keratinocyte-derived collagenases associated with keratinocyte migration. Cells plated on various connective tissue matrices under normoxic and hypoxic conditions, demonstrated a two-fold increase in the 92 kDa, type IV collagenase (MMP-9) when examined by quantitative zymography and ELISA. Western blotting and ELISA demonstrated a two-fold increase in tissue inhibitor of metalloproteinase (TIMP-1), an enzyme that binds to MMP-9 and inhibits its activity. The hypoxia-induced increase in cell motility could be inhibited by a neutralizing antibody to MMP-9. Northern blotting demonstrated that MMP-9 and TIMP-1 mRNA increased 2.5- to 4-fold, 2-12 h after the cells were made hypoxic. The hypoxia-induced changes in MMP-9 and TIMP-1 were inhibited by staurosporine and bisindolylmaleimide, inhibitors of protein kinase C (PKC), but not by inhibitors of tyrosine phosphorylation and the mitogen-activated protein kinase pathway. Inhibition of PKC also inhibited hypoxia-induced keratinocyte migration on type I collagen. These data provide evidence that hypoxia-induced keratinocyte migration is mediated by increased cellular secretion of MMP-9 via the PKC pathway.     

Phosphatidic acid as a regulator of matrix metalloproteinase-9 expression via the TNF-alpha signaling pathway

Phosphatidic acid (PA) is implicated in pathophysiological processes associated with cellular signaling events and inflammation, which include the expressional regulation of numerous genes. Here, we show that PA stimulation increases matrix metalloproteinase-9 (MMP-9) expression in macrophages through tumor necrosis factor (TNF)-alpha signaling. We performed antibody array analysis on proteins from macrophages stimulated with PA. PA was found to induce the production of TNF-alpha, but not of TNF receptor (TNFR)1 and TNFR2 in a time-dependent manner and stimulated significant, though delayed, MMP-9 expression. PA induced the phosphorylations of both ERK1/2 and p38, but not of c-jun amino-terminal kinase. Moreover, only ERK1/2 inhibition by U0126 suppressed PA-induced TNF-alpha production and MMP-9 expression. Neutralizing TNF-alpha, TNFR1 or TNFR2 antibodies significantly suppressed PA-induced MMP-9 expression, suggesting that the production of TNF-alpha in response to PA preceded the expression of MMP-9. Moreover, lipopolysaccharide-induced PA also led to TNF-alpha release and resulted in MMP-9 expression. Taken together, these observations suggest that PA may play a role in MMP-9 regulation through ERKs/TNF-alpha/TNFRs-dependent signaling pathway.     

Possible involvement of Cyclic-GMP-dependent protein kinase on matrix metalloproteinase-2 expression in rat aortic smooth muscle cells

Degradation and resynthesis of the extracellular matrix (ECM) are essential during tissue remodeling. Expansion of the vascular intima in atherosclerosis and restenosis following injury is dependent upon smooth muscle cell (SMC) proliferation and migration. The migration of SMC from media to intima critically depends on degradation of ECM protein by matrix metalloproteinases (MMPs). MMP inhibitors and eNOS gene transfer have been shown to inhibit SMC migration in vitro and neointima formation in vivo. Nitric oxide (NO) and cyclic-GMP have been implicated in the inhibition of VSMC migration. But, there are few studies addressing the role of NO signaling pathways on the expression of MMPs. Here we reported the involvement of cyclic-GMP-dependent protein kinase (PKG) (an important mediator of NO and cGMP signaling pathway in VSMC) on MMP-2 expression in rat aortic SMC. The goal of the present study was to gain insight into the possible involvement of PKG on MMP-2 in rat aortic SMC. MMP-2 protein and mRNA level and activity were downregulated in PKG-expressing cells as compared to PKG-deficient cells. In addition, the secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2) was increased in PKG-expressing cells as compared to PKG-deficient cells. PKG-specific membrane permeable peptide inhibitor (DT-2) reverses the process. Interestingly, little or no changes of MMP-9 were observed throughout the study. Taken together our data suggest the possible role of PKG in the suppression of MMP-2.     

Activation of toll-like receptor-9 induces matrix metalloproteinase-9 expression through Akt and tumor necrosis factor-alpha signaling

CpG oligodeoxunucleotide (ODN) plays an important role in immune cell function. The present study examined whether temporal control of toll-like receptor (TLR)-9 by CpG ODN can regulate the expression of matrix metalloproteinase-9 (MMP-9). CpG ODN induced the release of tumor necrosis factor (TNF)-alpha and the expression of TNF receptor (TNFR)-II, but not of TNFR-I, in a time-dependent manner and stimulated significant, though delayed, MMP-9 expression. The endosomal acidification inhibitors, chloroquine or bafilomycin A, inhibited CpG ODN-induced TNF-alpha, TNFR-II, and MMP-9 expression. CpG ODN induced the phosphorylation of Akt, and the inhibition of Akt by LY294002 suppressed CpG ODN-induced TNF-alpha, TNFR-II, and MMP-9 expressions. Moreover, neutralizing TNF-alpha antibody significantly suppressed CpG ODN-induced MMP-9 expression, suggesting the involvement of TNF-alpha. These observations suggest that CpG ODN may play important roles in macrophage activation by regulating the expression of MMP-9 via a TLR-9/Akt/TNF-alpha-dependent signaling pathway.     

Statins for the prevention of vein graft stenosis: a role for inhibition of matrix metalloproteinase-9

Saphenous vein (SV) grafts are commonly used to bypass coronary arteries that are diseased due to atherosclerosis. However, the development of intimal hyperplasia in such grafts can lead to patency-threatening stenosis and re-occlusion of the vessel. The proliferation and migration of smooth muscle cells (SMC) play key roles in the development of intimal hyperplasia, and an agent that inhibits both of these processes therefore has therapeutic potential. A prerequisite for SMC proliferation and migration in vivo is degradation of the basement membrane, achieved by secretion of the matrix-degrading gelatinases matrix metalloproteinase-2 (MMP-2) and MMP-9. Statins are cholesterol-lowering drugs that also have direct effects on SMC function. Here we report that neointima formation in organ-cultured human SV segments is inhibited by simvastatin, an effect that is associated with reduced MMP-9 activity. Additionally, our work shows that simvastatin not only inhibits proliferation, but importantly also inhibits invasion (migration through a matrix barrier), of cultured human SV SMC. Thus simvastatin treatment appears to inhibit neointima formation as a result of combined inhibition of SMC proliferation and invasion. The potential intracellular mechanisms by which statins affect SMC proliferation and migration, and thus attenuate intimal hyperplasia, are discussed, with particular emphasis on the role of MMP-9.