Ceramide 1-phosphate induces neointimal formation via cell proliferation and cell cycle progression upstream of ERK1/2 in vascular smooth muscle cells

Ceramide 1-phosphate (C1P) is a novel bioactive sphingolipid formed by ceramide kinase (CERK)-catalyzed phosphorylation of ceramide. It has been implicated in the regulation of such vital pathophysiological functions as phagocytosis and inflammation, but there have been no reports ascribing a biological function to CERK in vascular disorders. Here the potential role of CERK/C1P in neointimal formation was investigated using rat aortic vascular smooth muscle cells (VSMCs) in primary culture and a rat carotid injury model. Exogenous C8-C1P stimulated cell proliferation, DNA synthesis, and cell cycle progression of rat aortic VSMCs in primary culture. In addition, wild-type CERK-transfected rat aortic VSMCs induced a marked increase in rat aortic VSMC proliferation and [³H]-thymidine incorporation when compared to empty vector transfectant. C8-C1P markedly activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) within 5 min, and the activation could be prevented by U0126, a MEK inhibitor. Also, K1, a CERK inhibitor, decreased the ERK1/2 phosphorylation and cell proliferation on platelet-derived growth factor (PDGF)-stimulated rat aortic VSMCs. CERK expression and C1P levels were found to be potently increased during neointimal formation using a rat carotid injury model. However, ceramide levels decreased during the neointimal formation process. These findings suggest that C1P can induce neointimal formation via cell proliferation through the regulation of the ERK1/2 protein in rat aortic VSMCs and that CERK/C1P may regulate VSMC proliferation as an important pathogenic marker in the development of cardiovascular disorders.     

Platelet-derived growth factor-BB induces matrix metalloproteinase-2 expression and rat vascular smooth muscle cell migration via ROCK and ERK/p38 MAPK pathways

Matrix metalloproteinases (MMP) play a pivotal role in the pathogenesis of cardiovascular diseases. Their expressions are altered in response to a variety of stimuli, including growth factors, inflammatory markers, and cytokines. In this study, we demonstrated that platelet-derived growth factor-BB (PDGF-BB) induces a dose- and time-dependent increase in MMP-2 expression in rat vascular smooth muscle cells (VSMC). Treatment with either the Rho-associated protein kinase (ROCK) inhibitor Y-27632 or suppression of ROCK-1/2 by small interfering RNA technology significantly reduced the MMP-2 expression, thus suggesting that ROCK regulates such expression. Similar results were observed when VSMC were pretreated with either U0126 or SB203580, which are selective inhibitors of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, respectively, thus suggesting that these kinases are important for the induction of MMP-2 expression by PDGF-BB. In conclusion, these results described a novel mechanism in atherosclerosis through PDGF-BB signaling in VSMC, in which MMP-2 expression is induced via extracellular signal-regulated kinases and p38 mitogen-activated protein kinase phosphorylation, as well as ROCK.     

Angiotensin II induces matrix metalloproteinase-9 expression via a nuclear factor-kappaB-dependent pathway in vascular smooth muscle cells

Angiotensin II (AngII) is widely recognized as a critical regulator of the development of atherosclerosis. Matrix metalloproteinases (MMPs) are thought to participate in plaque destabilization through degradation of the extracellular matrix. In the present study, we investigated the potential mechanism of AngII-induced MMP-9 expression in vascular smooth muscle cells (VSMC). AngII upregulated the expression of MMP-9 significantly in VSMC obtained from rat aorta. RNAi-mediated knockdown of p65 and losartan, an inhibitor of AngII receptors subtype-1 (AT₁), could abolish AngII-induced MMP-9 expression. In addition, AngII induced the NF-κB binding activity via AT₁ and AT₂ receptors in VSMC, and AngII-induced activation of NF-κB is not associated with significant downregulation of IκB. In summary, this study demonstrates that AngII stimulates NF-κB nuclear translocation in VSMC via AT₁ and AT₂. AngII increases the expression of MMP-9 in VSMC, and AT₁ and NF-κB pathways have an important role in this response.     

Mechanism of inhibition of matrix metalloproteinase-9 induction by NO in vascular smooth muscle cells.

Vascular smooth muscle (VSM) cell migration is a critical step in the development of a neointima after angioplasty. Matrix metalloproteinases (MMPs) degrade the basement membrane and extracellular matrix, facilitating VSM cell migration. Recently, we demonstrated that nitric oxide (NO) inhibits interleukin-1 beta (IL-1 beta)-stimulated MMP-9 induction in rat aortic VSM cells. In this study, we examined the hypothesis that NO inhibits MMP-9 induction by attenuating superoxide generation and extracellular signal-regulated kinase (ERK) activation. Stimulation of VSM cells with IL-1 beta significantly (P < 0.05) increased superoxide production, ERK activation, and MMP-9 induction. Pretreatment of VSM cells with the NO donor DETA NONOate significantly (P < 0.05) decreased IL-1 beta-stimulated superoxide generation. In addition, pretreatment of VSM cells with a specific ERK pathway inhibitor, PD-98059, or DETA NONOate inhibited IL-1 beta-stimulated ERK activation and MMP-9 induction. Direct exposure of VSM cells to increased superoxide levels by treatment with xanthine/xanthine oxidase increased ERK activation and MMP-9 induction, whereas pretreatment of cells with PD-98059 significantly (P < 0.05) inhibited xanthine/xanthine oxidase-stimulated ERK activation and MMP-9 induction. We conclude that NO inhibits IL-1 beta-stimulated MMP-9 induction by inhibiting superoxide generation and subsequent ERK activation.     

Nerve growth factor activation of Erk-1 and Erk-2 induces matrix metalloproteinase-9 expression in vascular smooth muscle cells.

In response to vascular injury, smooth muscle cells migrate from the media into the intima, where they contribute to the development of neointimal lesions. Increased matrix metalloproteinase (MMP) expression contributes to the migratory response of smooth muscle cells by releasing them from their surrounding extracellular matrix. MMPs may also participate in the remodeling of extracellular matrix in vascular lesions that could lead to plaque weakening and subsequent rupture. Neurotrophins and their receptors, the Trk family of receptor tyrosine kinases, are expressed in neointimal lesions, where they induce smooth muscle cell migration. We now report that nerve growth factor (NGF)-induced activation of the TrkA receptor tyrosine kinase induces MMP-9 expression in both primary cultured rat aortic smooth muscle cells and in a smooth muscle cell line genetically manipulated to express TrkA. The response to NGF was specific for MMP-9 expression, as the expression of MMP-2, MMP-3, or the tissue inhibitor of metalloproteinase-2 was not changed. Activation of the Shc/mitogen-activated protein kinase pathway mediates the induction of MMP-9 in response to NGF, as this response is abrogated in cells expressing a mutant TrkA receptor that does not bind Shc and by pretreatment of cells with the MEK-1 inhibitor, U0126. Thus, these results indicate that the neurotrophin/Trk receptor system, by virtue of its potent chemotactic activity for smooth muscle cells and its ability to induce MMP-9 expression, is a critical mediator in the remodeling that occurs in the vascular wall in response to injury.     

Monocyte chemoattractant protein-1 mediates angiotensin II-induced vascular smooth muscle cell proliferation via SAPK/JNK and ERK1/2

Abnormal vascular smooth muscle cells proliferation is the pathophysiological basis of cardiovascular diseases, such as hypertension, atherosclerosis, and restenosis after angioplasty. Angiotensin II can induce abnormal proliferation of vascular smooth muscle cells, but the molecular mechanisms of this process remain unclear. Here, we explored the role and molecular mechanism of monocyte chemotactic protein-1, which mediated angiotensin II-induced proliferation of rat aortic smooth muscle cells. 1,000 nM angiotensin II could stimulate rat aortic smooth muscle cells' proliferation by angiotensin II type 1 receptor (AT(1)R). Simultaneously, angiotensin II increased monocyte chemotactic protein-1 expression and secretion in a dose-and time-dependent manner through activation of its receptor AT(1)R. Then, monocyte chemotactic protein-1 contributed to angiotensin II-induced cells proliferation by CCR2. Furthermore, we found that intracellular ERK and JNK signaling molecules were implicated in angiotensin II-stimulated monocyte chemotactic protein-1 expression and proliferation mediated by monocyte chemotactic protein-1. These results contribute to a better understanding effect on angiotensin II-induced proliferation of rat smooth muscle cells.     

Static pressure drives proliferation of vascular smooth muscle cells via caveolin-1/ERK1/2 pathway

Intimal hyperplasia plays an important role in various types of vascular remodeling. Mechanical forces derived from blood flow are associated with the proliferation of vascular smooth muscle cells (VSMC). This contributes to many vascular disorders such as hypertension, atherosclerosis and restenosis after percutaneous transluminal angioplasty (PTA). In this study, we show that static pressure induces the proliferation of VSMC and activates its related signal pathway. VSMC from a rat aorta were treated with different pressures (0, 60, 90, 120, 150 and 180 mm Hg) in a custom-made pressure incubator for 24 h. The most active proliferation of VSMC was detected at a pressure of 120 mm Hg. VSMC was also incubated under a static pressure of 120 mm Hg for different time intervals (0, 2, 4, 8, 12 and 24 h). We found that static pressure significantly stimulates VSMC proliferation. Extracellular signal-regulated kinases 1/2 (ERK1/2) activation showed a peak at the pressure of 120 mm Hg at 4-h time point. Moreover, caveolin-1 expression was significantly inhibited by rising static pressure. Downregulation of VSMC proliferation could be found after PD98059 (ERK1/2 phosphorylation inhibitor) treatment. Our data also showed that a siRNA-mediated caveolin-1 knock down increased ERK1/2 phosphorylation and VSMC proliferation. These results demonstrate that static pressure promotes VSMC proliferation via the Caveolin-1/ERK1/2 pathway.     

Requirement of an intermediate gene expression for biphasic ERK1/2 activation in thrombin-stimulated vascular smooth muscle cells

The expression of contractile proteins in vascular smooth muscle cells is controlled by still poorly defined mechanisms. A thrombin-inducible expression of smooth muscle-specific alpha-actin and myosin heavy chain requires transactivation of the epidermal growth factor (EGF) receptor and a biphasic activation of ERK1/2. Here we demonstrate that the sustained second phase of ERK1/2 phosphorylation requires de novo RNA and protein synthesis. Depolymerization of the actin cytoskeleton by cytochalasin D or disruption of transit between the endoplasmic reticulum and the Golgi apparatus by brefeldin A prevented the second phase of ERK1/2 phosphorylation. We thus conclude that synthesis and trafficking of a plasma membrane-resident protein may be critical intermediates. Analysis of the expression of protease-activated receptor 1, heparin-binding EGF (HB-EGF), and the EGF receptor revealed that pro-HB-EGF is significantly up-regulated upon thrombin stimulation. The kinetic of HB-EGF expression closely matched that of the second phase of ERK1/2 phosphorylation. Because inhibition of matrix metalloproteases or of the EGF receptor strongly attenuated the late phase of ERK1/2 phosphorylation, the second phase of ERK1/2 activation is primarily relayed by shedding of EGF receptor ligands. The small interfering RNA-mediated knockdown of HB-EGF expression confirmed an important role of HB-EGF expression in triggering the second phase of ERK1/2 activation. Confocal imaging of a yellow fluorescent protein-tagged HB-EGF construct demonstrates the rapid plasma membrane integration of the newly synthesized protein. These data imply that the hormonal control of contractile protein expression relies on an intermediate HB-EGF expression to sustain the signaling strength within the Ras/Raf/MEK/ERK cascade.     

Transforming growth factor-beta1 inhibition of vascular smooth muscle cell activation is mediated via Smad3

Activation of vascular smooth muscle cells (VSMCs) by proinflammatory cytokines is a key feature of atherosclerotic lesion formation. Transforming growth factor (TGF)-beta1 is a pleiotropic growth factor that can modulate the inflammatory response in diverse cell types including VSMCs. However, the mechanisms by which TGF-beta1 is able to mediate these effects remains incompletely understood. We demonstrate here that the ability of TGF-beta1 to inhibit markers of VSMC activation, inducible nitric-oxide synthase (iNOS) and interleukin (IL)-6, is mediated through its downstream effector Smad3. In reporter gene transfection studies, we found that among a panel of Smads, Smad3 could inhibit iNOS induction in an analogous manner as exogenous TGF-beta1. Adenoviral overexpression of Smad3 potently repressed inducible expression of endogenous iNOS and IL-6. Conversely, TGF-beta1 inhibition of cytokine-mediated induction of iNOS and IL-6 expression was completely blocked in Smad3-deficient VSMCs. Previous studies demonstrate that CCAAT/enhancer-binding protein (C/EBP) and NF-kappaB sites are critical for cytokine induction of both the iNOS and IL-6 promoters. We demonstrate that the inhibitory effect of Smad3 occurs via a novel antagonistic effect of Smad3 on C/EBP DNA-protein binding and activity. Smad3 mediates this effect in part by inhibiting C/EBP-beta and C/EBP-delta through distinct mechanisms. Furthermore, we find that Smad3 prevents the cooperative induction of the iNOS promoter by C/EBP and NF-kappaB. These data demonstrate that Smad3 plays an essential role in mediating TGF-beta1 anti-inflammatory response in VSMCs.     

Heparin prevents vascular smooth muscle cell progression through the G1 phase of the cell cycle

To gain insight into the mechanism of the antiproliferative effects of heparin on vascular smooth muscle cells (SMC), the influence of this glycosaminoglycan on cell cycle progression and the expression of the c-fos, c-myc, and c-myb proto-oncogenes and two other growth-regulated genes was examined. SMC, synchronized by a serum-deprivation protocol, enter S phase 12-16 h after serum stimulation. Pretreatment with heparin for 48 h blocked the induction of histone H3 RNA, an S phase-expressed product, and prevented cell replication. Thus, heparin prevents entry of cells into S phase. Conversely, heparin had essentially no effect on changes in expression of the c-fos and c-myc proto-oncogenes during the G0 to G1 transition. Normal increases in c-fos and c-myc RNA were observed 30 min and 2 h following serum addition, respectively. However, the increase in expression of the mRNA of the c-myb proto-oncogene and the mitochondrial ATP/ADP carrier protein, 2F1, which begins to occur 8 h following serum addition to SMC, was completely inhibited by heparin. Two-dimensional polyacrylamide gel electrophoresis of the products of a rabbit reticulocyte cell-free translation of RNA isolated at various times confirmed this temporal assessment of the effects of heparin. These results suggest that heparin does not inhibit cell proliferation by blocking the G0 to G1 transition. Rather, heparin may affect a critical event in the mid-G1 phase of the cell cycle which is necessary for subsequent DNA synthesis.     

M3 receptor modulates extracellular matrix synthesis via ERK1/2 signaling pathway in human bladder smooth muscle cells

Extracellular matrix (ECM) accumulation plays a key role in the progression of bladder outlet obstruction (BOO). Muscarinic receptors have been widely reported to serve as pivotal regulators in lung tissue remodeling. However, the influence of them on human bladder smooth muscle cells (HBSMCs) and the underlying molecular mechanisms have not yet been evaluated. The purposes of the present study are to investigate the effect of muscarinic receptors on the synthesis of ECM in HBSMCs and the involvement of intracellular signal transducers. The results indicated that M₁-M₅ muscarinic receptors were all encoded in HBSMCs. The expression rank order was M₂ > M₁ > M₅ > M₃ > M₄. The gene and protein expression of collagen I (COL1), TIMP-1, and TIMP-2 was carbachol (CCH) concentration-dependently enhanced. The synthesis of COL1 in the supernatant of cell culture medium was significantly elevated by exposure to CCH. The CCH-induced protein expression of COL1, TIMP-1, and TIMP-2, however, was obviously reduced by the pretreatment of muscarinic receptor antagonists, atropine, and M₃-preferring antagonist (1,1-dimethyl-4-diphenyl-acetoxypiperidinium iodide [4-DAMP]). Furthermore, ERK1/2 was activated by 100 µM CCH when compared with the control group and the pretreatment of ERK1/2 inhibitor significantly suppressed the synthesis of COL1 induced by 100 µM CCH. Besides, CCH-induced phosphorylation of ERK1/2 was remarkably restrained by the pretreatment of 4-DAMP. All in all, these findings demonstrated that M₃ receptor can modulate extracellular matrix synthesis via the ERK1/2 signaling pathway, which may provide potential novel therapeutic targets for BOO.     

Disruption of GM2/GD2 synthase gene resulted in overt expression of 9-O-acetyl GD3 irrespective of Tis21

GM2/GD2 synthase gene knockout mice lack all complex gangliosides, which are abundantly expressed in the nervous systems of vertebrates. In turn, they have increased precursor structures GM3 and GD3, probably replacing the roles of the depleted complex gangliosides. In this study, we found that 9-O-acetyl GD3 is also highly expressed as one of the major glycosphingolipids accumulating in the nervous tissues of the mutant mice. The identity of the novel component was confirmed by neuraminidase treatment, thin layer chromatography-immunostaining, two-dimensional thin layer chromatography with base treatment, and mass spectrometry. All candidate factors reported to be possible inducer of 9-O- acetylation, such as bitamine D binding protein, acetyl CoA transporter, or O-acetyl ganglioside synthase were not up-regulated. Tis21 which had been reported to be a 9-O-acetylation inducer was partially down-regulated in the null mutants, suggesting that Tis21 is not involved in the induction of 9-O-acetyl-GD3 and that accumulated high amount of GD3 might be the main factor for the dramatic increase of 9-O-acetyl GD3. The ability to acetylate exogenously added GD3 in the normal mouse astrocytes was examined, showing that the wild-type brain might be able to synthesize very low levels of 9-O-acetyl GD3. Increased 9-O-acetyl GD3, in addition to GM3 and GD3, may play an important role in the compensation for deleted complex gangliosides in the mutant mice.     

Heme oxygenase inhibits human airway smooth muscle proliferation via a bilirubin-dependent modulation of ERK1/2 phosphorylation

The aim of this study was to investigate whether the heme oxygenase (HO) pathway could modulate proliferation of airway smooth muscle (ASM) and the mechanism(s) involved in this phenomenon. In cultured human ASM cells, 10% fetal calf serum or 50 ng/ml platelet-derived growth factor AB induced cell proliferation, extracellular and intracellular reactive oxygen species (ROS) production and ERK1/2 phosphorylation. Pharmacological HO-1 induction (by 10 microm hemin or by 20 microm cobalt-protoporphyrin) and HO inhibition (by 25 microm tin-protoporphyrin or by an antisense oligonucleotide), respectively, reduced and enhanced significantly both cell proliferation and ROS production. Neither the carbon monoxide scavenger myoglobin (5-20 microm) nor the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one could reverse ASM proliferation induced by tin-protoporphyrin, making a role of the CO-cGMP pathway in HO-modulated proliferation unlikely. By contrast, bilirubin (1 microm) and the antioxidant N-acetyl-cysteine (1 mm) significantly reduced mitogen-induced cell proliferation, ROS production, and ERK1/2 phosphorylation. Furthermore, both bilirubin and N-acetyl-cysteine and the ERK1/2 inhibitor PD98059 significantly reversed the effects of HO inhibition on ASM proliferation. These results could be relevant to ASM alterations observed in asthma because activation of the HO pathway prevented the increase in bronchial smooth muscle area induced by repeated ovalbumin challenge in immunized guinea pigs, whereas inhibition of HO had the opposite effect. In conclusion, this study provides evidence for an antiproliferative effect of the HO pathway in ASM in vitro and in vivo through a bilirubin-mediated redox modulation of phosphorylation of ERK1/2.     

ETS-1 protein regulates vascular endothelial growth factor-induced matrix metalloproteinase-9 and matrix metalloproteinase-13 expression in human ovarian carcinoma cell line SKOV-3

Matrix metalloproteinase-mediated degradation of extracellular matrix is a crucial event for invasion and metastasis of malignant cells. The expressions of matrix metalloproteinases (MMPs) are regulated by different cytokines and growth factors. VEGF, a potent angiogenic cytokine, induces invasion of ovarian cancer cells through activation of MMPs. Here, we demonstrate that invasion and scattering in SKOV-3 cells were induced by VEGF through the activation of p38 MAPK and PI3K/AKT pathways. VEGF induced the expression of MMP-2, MMP-9, and MMP-13 and hence regulated the metastasis of SKOV-3 ovarian cancer cells, and the activities of these MMPs were reduced after inhibition of PI3K/AKT and p38 MAPK pathways. Interestingly, VEGF induced expression of ETS-1 factor, an important trans-regulator of different MMP genes. ETS-1 bound to both MMP-9 and MMP-13 promoters. Furthermore, VEGF acted through its receptor to perform the said functions. In addition, VEGF-induced MMP-9 and MMP-13 expression and in vitro cell invasion were significantly reduced after knockdown of ETS-1 gene. Again, VEGF-induced MMP-9 and MMP-13 promoter activities were down-regulated in ETS-1 siRNA-transfected cells. VEGF enriched ETS-1 in the nuclear fraction in a dose-dependent manner. VEGF-induced expression of ETS-1 and its nuclear localization were blocked by specific inhibitors of the PI3K and p38 MAPK pathways. Therefore, based on these observations, it is hypothesized that the activation of PI3K/AKT and p38 MAPK by VEGF results in ETS-1 gene expression, which activates MMP-9 and MMP-13, leading to the invasion and scattering of SKOV-3 cells. The study provides a mechanistic insight into the prometastatic functions of VEGF-induced expression of relevant MMPs.