Synergistic interaction between thromboxane A2 and mildly oxidized low density lipoproteins on vascular smooth muscle cell proliferation

Low density lipoprotein (LDL) and mildly oxidized low density lipoprotein (mox-LDL) are known mitogens for vascular smooth muscle cell (VSMC). Since aggregating platelets at sites of atherosclerotic injury release thromboxane A2(TXA2), a known mitogen for VSMC, we examined whether TXA2 can act synergistically with mox-LDL or its oxidative components in inducing VSMC proliferation. Growth arrested primary aortic rabbit VSMCs in 1st or 2nd passage were incubated with different concentrations of LDL or mox-LDL or lysophosphatidylcholine (LPC) or H2O2 or 4-hydroxy-2-nonenel (HNE) for 24 h followed by incubation with TXA2 mimetic U46619 for another 24 h. The amount of 3[H]-thymidine incorporated into the DNA was measured. Both LDL and mox-LDL at a concentration of 120 microg/ml induced proliferation of VSMC (168% or 184% respectively) when compared to the control. U46619 induced VSMC proliferation was observed at a concentration of 5 microm/L. As compared to native LDL, the mitogenic effect of mox-LDL on VSMC proliferation was markedly potentiated by U46619 to 301% or 316% at 0.5 or 5 microm/L U46619 respectively. LPC, H2O2 and HNE induced DNA synthesis was also marked by enhanced by U46619. These results suggest that even low concentration of TXA2 released from aggregating platelets may potentiate the mitogenic effect of mox-LDL at sites of vascular damage. The mitogenic effect of mox-LDL may be mediated via its oxidation products LPC, H2O2 (reactive oxygen species donor), and HNE.     

Human neutrophil-derived elastase induces airway smooth muscle cell proliferation

Neutrophils and their derived elastase are abundant in chronic inflammatory responses of asthma. This study aimed to investigate the mitogenic effect of elastase on airway smooth muscle (ASM) cells and the implicated signal transduction pathway. Near confluent cultured human ASM cells were treated with human neutrophil elastase (HNE, 0.01 to 0.5 microg/ml) or vehicle for 24 hours with or without extracellular signal-regulated kinase (ERK) inhibitor (PD98059, 30 microM), p38 kinase inhibitor (SB203580, 10 microM) or elastase inhibitor II (100 microg/ml). The ASM cell numbers were counted by a hemocytometer and DNA synthesis was assessed by flowcytometry. Western blots analysis for the expression of ERK, p38 and cyclin D1 was determined. HNE dose-dependently increased ASM cell numbers and the percentage of cells entering S-phase of cell cycle. This response was abolished by neutrophil elastase inhibitors and attenuated by PD98059, but not SB203580. HNE increased ERK phosphorylation and cyclin D1 expression. Pretreatment with PD98059 significantly inhibited elastase-induced cyclin D1 activity. The increased ASM cellular gap and cell shape change by proteolytic activity of HNE may be contributory to ERK activation and therefore cell proliferation. Our results demonstrate that HNE is mitogenic for ASM cells by increasing cyclin D1 activity through ERK signaling pathway.     

Soluble DPP4 induces inflammation and proliferation of human smooth muscle cells via protease-activated receptor 2

DPP4 is an ubiquitously expressed cell-surface protease that is shedded to the circulation as soluble DPP4 (sDPP4). We recently identified sDPP4 as a novel adipokine potentially linking obesity to the metabolic syndrome. The aim of this study was to investigate direct effects of sDPP4 on human vascular smooth muscle cells (hVSMCs) and to identify responsible signaling pathways. Using physiological concentrations of sDPP4, we could observe a concentration-dependent activation of ERK1/2 (3-fold) after 6 h, which remained stable for up to 24 h. Additionally, sDPP4 treatment induced a 1.5-fold phosphorylation of the NF-κB subunit p65. In accordance with sDPP4-induced stress and inflammatory signaling, sDPP4 also stimulates hVSMC proliferation. Furthermore we could observe an increased expression and secretion of pro-inflammatory cytokines like interleukin (IL)-6, IL-8 and MCP-1 (2.5-, 2.4- and 1.5-fold, respectively) by the sDPP4 treatment. All direct effects of sDPP4 on signaling, proliferation and inflammation could completely be prevented by DPP4 inhibition. Bioinformatic analysis and signaling signature induced by sDPP4 suggest that sDPP4 might be an agonist for PAR2. After the silencing of PAR2, the sDPP4-induced ERK activation as well as the proliferation was totally abolished. Additionally, the sDPP4-induced upregulation of IL-6 and IL-8 could completely be prevented by the PAR2 silencing. In conclusion, we show for the first time that sDPP4 directly activates the MAPK and NF-κB signaling cascade involving PAR2 and resulting in the induction of inflammation and proliferation of hVSMC. Thus, our in vitro data might extend the current view of sDPP4 action and shed light on cardiovascular effects of DPP4-inhibitors.     

Platelet and vascular smooth muscle thromboxane A2/prostaglandin H2 receptors

Thromboxane A2 (TxA2) and prostaglandin H2 (PGH2) aggregate platelets and contract vascular smooth muscle. Inasmuch as both compounds produce the same effects and presumably through the same receptor, their receptors have been referred to as TxA2/PGH2 receptors. Pharmacological studies of stable agonists and antagonists of the TxA2/PGH2 receptors have shown different rank order potencies for these compounds in platelets compared with blood vessels. These studies have provided evidence to support the hypothesis that the platelet TxA2/PGH2 receptor is different from the one found in vascular tissue. The vascular receptor has been named [TxA2/PGH2]tau and the platelet receptor has been named [TxA2/PGH2]alpha. In the past few years several radiolabeled antagonists and agonists have been developed and used in radioligand-binding studies, primarily in platelets. One of these ligands, 125I-labeled PTA-OH, a TxA2/PGH2 receptor antagonist, has been extensively used to characterize the human platelet TxA2/PGH2-binding site. It has been found to have a Kd of approximately 20 nM and a Bmax of 2500 binding sites/platelet. Through the combination of pharmacological and biochemical approaches, it should be possible to characterize platelet and vascular TxA2/PGH2 receptors.     

Human eosinophil-airway smooth muscle cell interactions

Eosinophils are present throughout the airway wall of asthmatics. The nature of the interaction between human airway smooth muscle cells (ASMC) and eosinophils was investigated in this study. We demonstrated, using light microscopy, that freshly isolated eosinophils from healthy donors rapidly attach to ASMC in vitro. Numbers of attached eosinophils were highest at 2 h, falling to 50% of maximum by 20 h. Eosinophil attachment at 2 h was reduced to 72% of control by anti-VCAM-1, and to 74% at 20 h by anti-ICAM-1. Pre-treatment of ASMC for 24h with TNF-alpha, 10 nM, significantly increased eosinophil adhesion to 149 and 157% of control after 2 and 20 h. These results provide evidence that eosinophil interactions with ASMC involve VCAM-1 and ICAM-1 and are modulated by TNF-alpha.     

Regulation of vascular smooth muscle cell proliferation by nuclear orphan receptor Nur77

Endothelium-derived nitric oxide (NO) is a cytoprotective molecule to prevent endothelial cells (ECs) from apoptosis. CREB-binding protein (CBP) is involved in the apoptotic pathway in several tumor cells, however, little is known whether CBP is associated with apoptosis in ECs and the apoptotic effect of CBP on ECs is regulated by NO. Therefore, the purpose of the present study was to investigate whether silencing CBP expression could affect the sensitivity of ECs toward apoptotic stimuli and determined the role of NO. In this study, we found that when CBP expression was silenced by RNA interference, ECs were more prone to apoptosis under serum deprivation, whereas the apoptosis was not significantly induced in the serum-containing condition. The increased apoptosis is paralleled by a reduction of NO, and the apoptosis was reversed by NO donors, suggesting an important role of NO. Furthermore, CBP silencing decreased NO production by downregulating the endothelial NO synthase (eNOS) expression in a dose-dependent manner. These results indicated that CBP silencing is associated with decreased eNOS expression and NO production, and therefore concomitantly increased the sensitivity of ECs toward apoptosis.     

麻黄碱对支气管平滑肌细胞增殖的影响

在前人工作的基础上,采用一种更简省的方法分离培养支气管平滑肌细胞;并且利用显微镜观察和MTT的方法,研究了麻黄碱对支气管平滑肌细胞形态和增殖的影响.结果表明:麻黄碱对支气管平滑肌细胞的形态没有明显影响,但是对平滑肌细胞的增殖有一定的抑制作用,并进一步阐述了麻黄碱治疗哮喘等呼吸系统疾病的作用机理.     

Angiotensin II AT(2) receptor inhibits smooth muscle cell migration via fibronectin cell production and binding

To explore the vascular function of the angiotensin II (ANG II) AT(2) receptor subtype (AT(2)R), we generated a vascular smooth muscle cell (SMC) line expressing the AT(2)R (SMC-vAT(2)). The involvement of AT(2)R in the motility response of SMCs was examined in SMC-vAT(2) cells and their controls (SMC-v) cultured on either laminin or fibronectin matrix proteins with the agarose drop technique. All experiments were conducted in the presence of a saturating concentration of losartan to inactivate the AT(1)R subtype. Under basal conditions, both cell lines migrated outside drops, but on laminin only. Treatment with ANG II significantly inhibited the migration of SMC-vAT(2) but not SMC-v cells, and this effect was prevented by the AT(2)R antagonist CGP-42112A. The decreased migration of SMC-vAT(2) was not associated with changes in cell growth, cytoskeleton stiffness, or smooth muscle actin, desmin, and tenascin expression. However, it was correlated with increased synthesis and binding of fibronectin. Both responses were prevented by incubation with selective AT(2)R antagonists. Addition of GRGDTP peptide, which prevents cell attachment of fibronectin, reversed the AT(2)R inhibitory effect on SMC-vAT(2) migration. These results suggest that activated ANG II AT(2)R inhibits SMC migration via cellular fibronectin synthesis and associated cell binding.     

Human heat shock protein 60 stimulates vascular smooth muscle cell proliferation through Toll-like receptors 2 and 4

Heat shock proteins (HSPs) of endogenous and exogenous origin are suspected contributors to the initiation and aggravation of vascular pathologies like atherosclerosis and restenosis. Toll-like receptors (TLRs) 2 and 4 are well-known receptors for exogenous pathogen-associated molecular patterns and have recently been thought to play a role in HSP60-induced cellular activation. We hypothesized that human HSP60 directly stimulates venous smooth muscle cell (VSMC) proliferation through a TLR-dependent mechanism. Localization of HSP60, TLR2 and TLR4 was studied in failed venous grafts and normal venous tissue by double immunostaining. In vitro VSMCs were incubated for 48 h with recombinant human HSP60. In other experiments, VSMCs were pre-incubated for 30 min with specific anti-TLR2 and anti-TLR4 antibodies. VSMC proliferation was determined by Ki67 immunoreactivity, and mean values were compared between experimental and control groups. In addition, human embryonic kidney (HEK) cells transfected with human TLR2 or TLR4/MD-2 were exposed to HSP60 for 48 h, and proliferation was determined by using a hemocytometer. Co-localization of HSP60 and TLRs was detected in all neointimal lesions but was virtually absent in normal veins. Human HSP60 stimulated VSMC proliferation in a concentration-dependent fashion. In addition, TLR2 and TLR4 antibodies attenuated VSMC proliferation. The role of TLR-mediated stimulation of cell proliferation by HSP60 was supported by the significant increase in proliferation of transfected HEK cells. These findings provide supporting evidence for the role of HSP60 and TLR2 and TLR4 in vascular disease. Moreover, our data surpass the infection- and autoimmunity-based hypotheses of cardiovascular disease and suggest an additional HSP60-related autocrine process.     

Perlecan up-regulation of FRNK suppresses smooth muscle cell proliferation via inhibition of FAK signaling

We previously reported that fully assembled basement membranes are nonpermissive to smooth muscle cell (SMC) replication and that perlecan (PN), a basement membrane heparan sulfate proteoglycan, is a dominant effector of this response. We report here that SMC adhesion to basement membranes, and perlecan in particular, up-regulate the expression of focal adhesion kinase-related nonkinase (FRNK), a SMC-specific endogenous inhibitor of FAK, which subsequently suppresses FAK-mediated, ERK1/2-dependent growth signals. Up-regulation of FRNK by perlecan is actively and continuously regulated. Relative to the matrix proteins studied, the effects are unique to perlecan, because plating of SMCs on several other basement membrane proteins is associated with low levels of FRNK and corresponding high levels of FAK and ERK1/2 phosphorylation and SMC growth. Perlecan supports SMC adhesion, although there is reduced cell spreading compared with fibronectin (FN), laminin (LN), or collagen type IV (IV). Despite the reduction in cell spreading, we report that perlecan-induced up-regulation of FRNK is independent of cell shape changes. Growth inhibition by perlecan was rescued by overexpressing a constitutively active FAK construct, but overexpressing kinase-inactivated mutant FAK or FRNK attenuated fibronectin-stimulated growth. These data indicate that perlecan functions as an endogenously produced inhibitor of SMC growth at least in part through the active regulation of FRNK expression. FRNK, in turn, may control SMC growth by downregulating FAK-dependent signaling events.     

Human tribbles-1 controls proliferation and chemotaxis of smooth muscle cells via MAPK signaling pathways

Migration and proliferation of smooth muscle cells are key to a number of physiological and pathological processes, including wound healing and the narrowing of the vessel wall. Previous work has shown links between inflammatory stimuli and vascular smooth muscle cell proliferation and migration through mitogen-activated protein kinase (MAPK) activation, although the molecular mechanisms of this process are poorly understood. Here we report that tribbles-1, a recently described modulator of MAPK activation, controls vascular smooth muscle cell proliferation and chemotaxis via the Jun kinase pathway. Our findings demonstrate that this regulation takes place via direct interactions between tribbles-1 and MKK4/SEK1, a Jun activator kinase. The activity of this kinase is dependent on tribbles-1 levels, whereas the activation and the expression of MKK4/SEK1 are not. In addition, tribbles-1 expression is elevated in human atherosclerotic arteries when compared with non-atherosclerotic controls, suggesting that this protein may play a role in disease in vivo. In summary, the data presented here suggest an important regulatory role for trb-1 in vascular smooth muscle cell biology.     

Ouabain-induced signaling and vascular smooth muscle cell proliferation

The hypothesis of this study is that the sodium pump complex acts as an intracellular signal-transducing molecule in canine vascular smooth muscle cells through its interaction with other membrane and cytoskeletal proteins. We have demonstrated that 1 nm ouabain induced transactivation of the epidermal growth factor receptor (EGFR), resulting in increased proliferation and bromodeoxyuridine (BrdUrd) uptake. Immunoprecipitation and Western blotting showed that the EGFR and Src were phosphorylated within 5 min of 10(-9) m ouabain stimulation. Both ouabain-induced DNA synthesis (BrdUrd uptake) and MAPK42/44 phosphorylation were inhibited by the Src inhibitor PP2, the EGFR kinase inhibitor AG1478, the tyrosine kinase inhibitor genistein, and the MEK1 inhibitor PD98059. Ouabain concentrations higher than 1 nm had little or no stimulating effect on proliferation or BrdUrd uptake but did minimally activate ERK1/2. Thus, low concentrations of ouabain, which do not inhibit the sodium pump sufficiently to perturb the resting cellular ionic milieu, initiate a transactivational signaling cascade leading to vascular smooth muscle cell proliferation.     

Lactosylceramide stimulates aortic smooth muscle cell proliferation.

We have investigated the effects of various sphingolipids on aortic smooth muscle cell proliferation employing viable cell counting, [3H] thymidine incorporation into DNA and the release of lactate dehydrogenase. Assays for UDP Gal: GlcCer Bl-4 galactosyltransferase (GalT-2) in control and treated cells were pursued simultaneously. Lactosylceramide stimulated cell proliferation in the order of 5 fold. Antibody against LacCer but not GbOse3Cer blocked the proliferative effects of LacCer in these cells. This phenomena was specific for aortic smooth muscle cells as LacCer decreased cell viability of aortic endothelial cells and had no effect on pulmonary endothelial cells. D-PDMP inhibited the activity of GalT-2 in smooth muscle cells and markedly prevented cell proliferation. In contrast, L-PDMP stimulated the activity of GalT-2 in smooth muscle cells and stimulated cell proliferation. Antibody against GalT-2 inhibited cell proliferation. Our findings suggest that the activation of GalT-2 leads to increased LacCer levels, which in turn, may be involved in aortic smooth muscle cell proliferation.     

PDGF-induced vascular smooth muscle cell proliferation is associated with dysregulation of insulin receptor substrates

In vascular smooth muscle cells (VSMCs), platelet-derived growth factor (PDGF) plays a major role in inducing phenotypic switching from contractile to proliferative state. Importantly, VSMC phenotypic switching is also determined by the phosphorylation state/expression levels of insulin receptor substrate (IRS), an intermediary signaling component that is shared by insulin and IGF-I. To date, the roles of PDGF-induced key proliferative signaling components including Akt, p70S6kinase, and ERK1/2 on the serine phosphorylation/expression of IRS-1 and IRS-2 isoforms remain unclear in VSMCs. We hypothesize that PDGF-induced VSMC proliferation is associated with dysregulation of insulin receptor substrates. Using human aortic VSMCs, we demonstrate that prolonged PDGF treatment led to sustained increases in the phosphorylation of protein kinases such as Akt, p70S6kinase, and ERK1/2, which mediate VSMC proliferation. In addition, PDGF enhanced IRS-1/IRS-2 serine phosphorylation and downregulated IRS-2 expression in a time- and concentration-dependent manner. Notably, phosphoinositide 3-kinase (PI 3-kinase) inhibitor (PI-103) and mammalian target of rapamycin inhibitor (rapamycin), which abolished PDGF-induced Akt and p70S6kinase phosphorylation, respectively, blocked PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. In contrast, MEK1/ERK inhibitor (U0126) failed to block PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. PDGF-induced IRS-2 downregulation was prevented by lactacystin, an inhibitor of proteasomal degradation. Functionally, PDGF-mediated IRS-1/IRS-2 dysregulation resulted in the attenuation of insulin-induced IRS-1/IRS-2-associated PI 3-kinase activity. Pharmacological inhibition of PDGF receptor tyrosine kinase with imatinib prevented IRS-1/IRS-2 dysregulation and restored insulin receptor signaling. In conclusion, strategies to inhibit PDGF receptors would not only inhibit neointimal growth but may provide new therapeutic options to prevent dysregulated insulin receptor signaling in VSMCs in nondiabetic and diabetic states.     

The prostacyclin receptor induces human vascular smooth muscle cell differentiation via the protein kinase A pathway

Recent studies of cyclooxygenase-2 (COX-2) inhibitors suggest that the balance between thromboxane and prostacyclin is a critical factor in cardiovascular homeostasis. Disruption of prostacyclin signaling by genetic deletion of the receptor or by pharmacological inhibition of COX-2 is associated with increased atherosclerosis and restenosis after injury in animal models and adverse cardiovascular events in clinical trials (Vioxx). Human vascular smooth muscle cells (VSMC) in culture exhibit a dedifferentiated, migratory, proliferative phenotype, similar to what occurs after arterial injury. We report that the prostacyclin analog iloprost induces differentiation of VSMC from this synthetic, proliferative phenotype to a quiescent, contractile phenotype. Iloprost induced expression of smooth muscle (SM)-specific differentiation markers, including SM-myosin heavy chain, calponin, h-caldesmon, and SM alpha-actin, as determined by Western blotting and RT-PCR analysis. Iloprost activated cAMP/protein kinase A (PKA) signaling in human VSMC, and the cell-permeable cAMP analog 8-bromo-cAMP mimicked the iloprost-induced differentiation. Both myristoylated PKA inhibitor amide-(14-22) (PKI, specific PKA inhibitor), as well as ablation of the catalytic subunits of PKA by small interfering RNA, opposed the upregulation of contractile markers induced by iloprost. These data suggest that iloprost modulates VSMC phenotype via G(s) activation of the cAMP/PKA pathway. These studies reveal regulation of VSMC differentiation as a potential mechanism for the cardiovascular protective effects of prostacyclin. This provides important mechanistic insights into the induction of cardiovascular events with the use of selective COX-2 inhibitors.     

Inositol trisphosphate receptor calcium release is required for cerebral artery smooth muscle cell proliferation

Vascular damage signals smooth muscle cells to proliferate, often exacerbating existing pathologies. Although the role of changes in "global" Ca2+ in vascular smooth muscle (VSM) cell dedifferentiation has been studied, the role of specific Ca2+ signals in determining VSM phenotype remains relatively unexplored. Earlier work with cultured VSM cells suggests that inositol 1,4,5-trisphosphate receptor (IP3R) expression and sarcoplasmic reticulum (SR) Ca2+ release may be linked to VSM cell proliferation in native tissue. Thus we hypothesized that SR Ca2+ release through IP3Rs in the form of discrete transient signals is necessary for VSM cell proliferation. To investigate this hypothesis, we used mouse cerebral arteries to design an organ culture system that permitted examination of Ca2+ dynamics in native tissue. Explanted arteries were cultured in normal medium with 10% FBS, and appearance of individual VSM cells migrating from explanted arteries (outgrowth cells) was tracked daily. Initial exposure to 10% FBS increased Ca2+ waves in myocytes in the arteries that were blocked by the IP3R antagonist 2-aminoethoxydiphenylborate (2-APB). Inhibition of IP3R opening (via 100 microM 2-APB, 10 microM xestospongin C, or 25 microM U-73122) dramatically reduced outgrowth cell number compared with untreated or ryanodine-treated (10 microM) arteries. Consistent with this finding, 2-APB inhibited cell proliferation, as measured by reduced proliferating cell nuclear antigen immunostaining within 48 h of culture but did not inhibit cell migration. These results indicate that activation of IP3R Ca2+ release is required for VSM cell proliferation in these arteries.