Anti-inflammatory effect of soyasaponins through suppressing nitric oxide production in LPS-stimulated RAW 264.7 cells by attenuation of NF-κB-mediated nitric oxide synthase expression

The anti-inflammatory properties of soyasaponins (especially soyasaponins with different chemical structures) have scarcely been investigated. We investigated the inhibitory effects of five structural types of soyasaponins (soyasaponin A¹, A², I and soyasapogenol A, B) on the induction of nitric oxide (NO) and inducible NO synthase (iNOS) in murine RAW 264.7 cells activated with lipopolysaccharide (LPS). Soyasaponin A¹, A² and I (25-200 μg/mL) dose-dependently inhibited the production of NO and tumor necrosis factor α (TNF-α) in LPS-activated macrophages, whereas soyasapogenol A and B did not. Furthermore, soyasaponin A¹, A² and I suppressed the iNOS enzyme activity and down-regulated the iNOS mRNA expression both in a dose-dependent manner. The reporter gene assay revealed that soyasaponin A¹, A² and I decreased LPS-induced nuclear factor kappa B (NF-κB) activity. Soyasaponin A¹, A² and I exhibit anti-inflammatory properties by suppressing NO production in LPS-stimulated RAW 264.7 cells through attenuation of NF-κB-mediated iNOS expression. It is proposed that the sugar chains present in the structures of soyasaponins are important for their anti-inflammatory activities. These results have important implication for using selected soyasaponins towards the development of effective chemopreventive and anti-inflammatory agents.     

Osteopontin regulates α-smooth muscle actin and calponin in vascular smooth muscle cells

vSMCs (vascular smooth muscle cells) lose differentiation markers and gain uncontrolled proliferative activity during the early stages of atherosclerosis. Previous studies have shown that OPN (osteopontin) mRNA and protein levels increase significantly on induction of proliferative activity by allylamine (an atherogenic amine) and that this response can be inhibited by OPN antibodies. We have investigated the role of OPN in vSMC differentiation. Primary cultures of aortic mouse vSMCs were transfected with an OPN expression plasmid and several vSMC differentiation markers including α-SM actin (α-smooth muscle actin), SM22-α, tropomyosin and calponin were monitored in this cellular model. α-SM actin and calponin protein levels were significantly decreased by OPN overexpression. Down-regulation of α-SM actin and calponin was also observed on extracellular treatment of mouse vSMCs with recombinant OPN. In addition, calponin mRNA was significantly decreased under serum-restricted conditions when OPN mRNA was dramatically increased, while α-SM actin mRNA remained unchanged. These data indicate that OPN down-regulates α-SM actin and calponin expression through an extracellular signalling pathway. Functional connectivity between OPN and vSMC differentiation markers has been established. Since vSMCs lose differentiation features during early atherosclerosis, a mechanistic basis for OPN functions as a critical regulator of proliferative cardiovascular disease has been presented.     

ROS directly activates transforming growth factor β type 1 receptor signalling in human vascular smooth muscle cells

BackgroundWidely used NAPDH oxidase (Nox) inhibitor, apocynin is a prodrug that needs to be converted to its pharmacologically active form by myeloperoxidase. In myeloperoxidase deficient non phagocytic cells such as vascular smooth muscle cells (VSMCs) apocynin stimulates the production of ROS. ROS is generated by the activation of many signalling pathways, thus we have used apocynin as a pharmacological tool to characterise the role of endogenous ROS in activating the transforming growth factor beta receptor (TGFBR1) without the activation of other pathways.MethodsThe in vitro study utilized human VSMCs. Western blotting and quantitative real time PCR were performed to assess signalling pathways and gene expression, respectively. Intracellular ROS levels was measured using fluorescence detection assay.ResultsTreatment with apocynin of human VSMCs stimulated ROS production and the phosphorylation of TGFBR1 and subsequent activation of TGFBR1 signalling leading to the formation of phosphorylated Smad2 which consequently upregulates the mRNA expression of glycosaminoglycan synthesizing enzyme.ConclusionsThese findings outline a specific involvement of ROS production in TGFBR1 activation. Furthermore, because apocynin stimulates Nox and ROS production, apocynin must be used with considerable care in vitro as its actions clearly extend beyond the stimulation of Nox enzymes and it has consequences for cellular signalling.General significanceApocynin can stimulate Nox leading to the production of ROS and the outcome is completely dependent upon the redox properties of the cell.     

Thalidomide inhibits interferon-γ-mediated nitric oxide production in mouse vascular endothelial cells

Thalidomide is known as an anti-angiogenic, anti-tumor, and anti-proliferative agent, widely used in the treatment of some immunological disorders and cancers. The effect of thalidomide on interferon (IFN)-γ induced nitric oxide (NO) production in mouse vascular endothelial cells was examined in order to elucidate the anti-angiogenic or anti-inflammatory action. Thalidomide inhibited IFN-γ-induced NO production in mouse END-D cells via reduced expression of an inducible type of NO synthase (iNOS) protein and mRNA. Since thalidomide did not alter the cell surface expression of IFN-γ receptor, the NO inhibition was suggested to be due to the impairment of IFN-γ-induced intracellular event by thalidomide. Thalidomide inhibited the phosphorylation of IRF1, which was required for the iNOS expression. Moreover, it inhibited the phosphorylation of STAT1, an upstream molecule of IRF1, in IFN-γ signaling. Thalidomide did not inhibit the JAK activation in response to IFN-γ. A phosphatase inhibitor, sodium orthovanadate, abolished the inhibitory action of thalidomide. Therefore, thalidomide was suggested to inhibit IFN-γ-induced NO production via impaired STAT1 phosphorylation.     

Endothelial regulation of calmodulin expression and eNOS–calmodulin interaction in vascular smooth muscle

Molecular and Cellular Biochemistry - Calmodulin (CaM) is a Ca2+ sensor protein that is required for numerous vascular smooth muscle cell (VSMC) functions. Since CaM is not expressed enough for its...     

Cyclic stretch induces cyclooxygenase-2 gene expression in vascular endothelial cells via activation of nuclear factor kappa-β

Vascular endothelial cells respond to biomechanical forces, such as cyclic stretch and shear stress, by altering gene expression. Since endothelial-derived prostanoids, such as prostacyclin and thromboxane A₂, are key mediators of endothelial function, we investigated the effects of cyclic stretch on the expression of genes in human umbilical vein endothelial cells controlling prostanoid synthesis: cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS) and thromboxane A₂ synthase (TXAS). COX-2 and TXAS mRNAs were upregulated by cyclic stretch for 24 h. In contrast, PGIS mRNA was decreased and stretch had no effect on COX-1 mRNA expression. We further show that stretch-induced upregulation of COX-2 is mediated by activation of the NF-κβ signaling pathway.     

Protein kinase Cδ mediates MCP-1 mRNA stabilization in vascular smooth muscle cells

Monocyte chemoattractant protein-1 (MCP-1) is an inflammatory chemokine that promotes atherosclerosis and is a mediator of the response to arterial injury. We previously demonstrated that platelet-derived growth factor (PDGF) and angiotensin II (Ang) induce the accumulation of MCP-1 mRNA in vascular smooth muscle cells mainly by increasing mRNA stability. In the present study, we have examined the signaling pathways involved in this stabilization of MCP-1 mRNA. The effect of PDGF (BB isoform) and Ang on MCP-1 mRNA stability was mediated by the PDGF β and angiotensin II receptor AT1R, respectively, and did not involve transactivation between the two receptors. The effect of PDGF-BB was blocked by inhibitors of protein kinase C (PKC), but not by inhibitors of phosphoinositol 3-kinase (PI3K), Src, or NADPH oxidase (NADPHox). In contrast, the effect of Ang was blocked by inhibitors of Src, and PKC, but not by inhibitors of PI3 K, or NADPHox. The effect of PDGF BB on MCP-1 mRNA stability was blocked by siRNA directed against PKCδ and protein kinase D (PKD), whereas the effect of Ang was blocked only by siRNA directed against PKCδ. These results suggest that the enhancement of MCP-1 mRNA stability by PDGF-BB and Ang are mediated by distinct “proximal” signaling pathways that converge on activation of PKCδ. This study identifies a novel role for PKCδ in mediating mRNA stability in smooth muscle cells.     

Caldesmon,calponin and α-smooth muscle actin expression in subcultured smooth muscle cells from human airways

Calponin and caldesmon are two proteins considered to play a regulatory role in smooth muscle contraction, which have never previously been found to be expressed in subcultured cells. In the present study, immunocytochemistry and immunoblotting were performed to identify these proteins in smooth muscle cells (SMC) from human bronchi. It was found that human airway SMC, kept in a non-proliferative state, continued to express caldesmon and calponin at least until the 8th passage. The expression of -smooth muscle actin studied under the same conditions was also shown to be preserved in subcultured bronchial SMC.