Potential Actions of Baicalein for Preventing Vascular Calcification of Smooth Muscle Cells In Vitro and In Vivo.

Vascular calcification (VC) is associated with cardiovascular disease. Baicalein, a natural flavonoid extract of Scutellaria baicalensis rhizome has several biological properties which may inhibit VC. We investigated whether baicalein suppresses Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP-2) and upregulates smooth muscle 22-alpha (SM22-α) and alpha-smooth muscle actin (α-SMA). In an in vitro experiment, primary rat aortic vascular smooth muscle cells (VSMCs) were pretreated with 0.1, 1, and 5 µM baicalein, followed by ß-glycerophosphate (ß-GP) to induce calcification. In an in vivo experiment, VC was generated by vitamin D3 plus nicotine (VDN) administration to male Sprague Dawley (SD) rats randomly assigned into a control group, a VC group, a VC group pretreated with baicalein, and a baicalein alone group. Each group comprised 10 rats. Left ventricular (LV) morphology, function and performance were assessed by echocardiography. Calcium content was measured by Alizarin red S staining and alkaline phosphatase (ALP) activity assays. Apoptotic VSMCs were detected by flow cytometry. Protein levels and superoxide changes were evaluated using Western blotting and immunofluorescence assays respectively. Plasma malondialdehyde (MDA) was assayed. Baicalein pretreatment significantly reduced calcium content in calcified VSMCs (p < 0.001) as well as in VC rat aortic smooth muscle (p < 0.001). Additionally, ALP activity was decreased in calcified VSMCs and VC rat aortic smooth muscle (p < 0.001). Apoptosis was significantly attenuated by 1 µM baicalein pretreatment in calcified VSMCs. Runx2 and BMP-2 expressions were downregulated by the baicalein in calcified VSMCs. Baicalein pretreatment increased typical VSMCs markers SM22-α and α-SMA in calcified VSMCs. Baicalein pretreatment was associated with adverse changes in LV morphometry. Markers of oxidative stress declined, and endogenous antioxidants increased in VC rats pretreated with baicalein. Baicalein mitigates VC through the inhibition of Runx2/BMP-2 signaling pathways, enhancement of vascular contractile phenotype and oxidative stress reduction. However, our study is of basic experimental design; more advanced investigations to identify other molecular regulators of VC and their mechanisms of action is required.

Inhibition of Neointima Hyperplasia, Inflammation, and Reactive Oxygen Species in Balloon-Injured Arteries by HVJ Envelope Vector-Mediated Delivery of Superoxide Dismutase Gene.

Extracellular superoxide dismutase (EC-SOD) has been implicated in regulation of vascular function but its underlying molecular mechanism is largely unknown. These two-step experiments investigate whether hemagglutinating virus of Japan envelope (HVJ-E) vector-mediated EC-SOD gene delivery might protect against neointima formation, vascular inflammation, and reactive oxygen species (ROS) generation, and also explore cell growth signaling pathways. The first in-vitro experiment was performed to assess the transfection efficacy and safety of HVJ-E compared to lipofectamine®. Results revealed that HVJ-E has higher transfection efficiency and lower cytotoxicity than those of lipofectamine®. Another in-vivo study initially used balloon denudation to rat carotid artery, then delivered EC-SOD cDNA through the vector of HVJ-E. Arterial section with H&E staining from the animals 14 days after balloon injury showed a significant reduction of intima-to-media area ratio in EC-SOD transfected arteries when compared with control (empty vector-transfected arteries) (p < 0.05). Arterial tissue with EC-SOD gene delivery also exhibited lower levels of ROS, as assessed by fluorescent microphotography with dihydroethidium staining. Quantitative RT-PCR revealed that EC-SOD gene delivery significantly diminished mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß (p < 0.05 in all comparisons). An immunoblotting assay from vascular smooth muscle cell (VSMC) cultures showed that the EC-SOD transfected group attenuated the activation of MEK1/2, ERK1/2, and Akt signaling significantly. In conclusion, EC-SOD overexpression by HVJ-E vector inhibits neointima hyperplasia, inflammation, and ROS level triggered by balloon injury. The modulation of cell growth-signaling pathways by EC-SOD in VSMCs might play an important role in these inhibitory effects.

Exogenous Heat Shock Cognate Protein 70 Suppresses LPS-Induced Inflammation by Down-Regulating NF-κB through MAPK and MMP-2/-9 Pathways in Macrophages.

Heat shock cognate protein 70 (HSC70), a molecular chaperone, is constitutively expressed by mammalian cells to regulate various cellular functions. It is associated with many diseases and is a potential therapeutic target. Although HSC70 also possesses an anti-inflammatory action, the mechanism of this action remains unclear. This current study aimed to assess the anti-inflammatory effects of HSC70 in murine macrophages RAW 264.7 exposed to lipopolysaccharides (LPS) and to explain its pathways. Mouse macrophages (RAW 264.7) in 0.1 µg/mL LPS incubation were pretreated with recombinant HSC70 (rHSC70) and different assays (Griess assay, enzyme-linked immune assay/ELISA, electrophoretic mobility shift assay/EMSA, gelatin zymography, and Western blotting) were performed to determine whether rHSC70 blocks pro-inflammatory mediators. The findings showed that rHSC70 attenuated the nitric oxide (NO) generation, tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) expressions in LPS-stimulated RAW264.7 cells. In addition, rHSC70 preconditioning suppressed the activities and expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9. Finally, rHSC70 diminished the nuclear translocation of nuclear factor-κB (NF-κB) and reduced the phosphorylation of extracellular-signal regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPK), and phosphatidylinositol-3-kinase (PI3K/Akt). We demonstrate that rHSC70 preconditioning exerts its anti-inflammatory effects through NO production constriction; TNF-α, and IL-6 suppression following down-regulation of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and MMP-2/MMP-9. Accordingly, it ameliorated the signal transduction of MAPKs, Akt/IκBα, and NF-κB pathways. Therefore, extracellular HSC70 plays a critical role in the innate immunity modulation and mechanisms of endogenous protective stimulation.