Cryptotanshinone against vascular dementia through inhibition of Aβ aggregation and inflammatory responses in cerebrovascular endothelial cells

Abnormal aggregation of amyloid-β (Aβ) peptides and associated inflammation and apoptosis in cerebrovascular endothelial cells are prelude to inhibition of onset of vascular dementia (VaD). Although small molecules have been widely used to mitigate the cell damage induced by aggregated species of Aβ, its molecular mechanism based on anti-amyloid properties and corresponding mitigation of cytotoxicity against cerebrovascular endothelial cells have not been elucidated. Herein, we used cryptotanshinone as the major bioactive compound from the root of Salvia miltiorrhiza Bunge to effectively inhibit Aβ fibrillation and associated cytotoxicity. Thoflavin T (ThT) and 1-Anilino-8-naphthalene sulfonate (ANS) fluorescence, Congo red, and circular dichroism (CD) analyses indicted that cryptotanshinone potentially inhibit Aβ_1-42 aggregation through elongation of nucleation phase, apparent decrease in the slope of the growth phase, and the final fluorescence intensity in a concentration-dependent manner. Also, cell viability, inflammation and capsae-3 assays showed that co-incubation of Aβ_1-42 peptide with cryptotanshinone in the aggregation buffer not only mitigated their cytotoxicity, but also reduced the levels of TNF-α, IL-1β, IL-6 and caspase-3 activity in cerebrovascular endothelial cells induced by Aβ_1-42. This study suggested that cryptotanshinone may show a great promise in the development of small molecule-based platforms for the treatment of VaD.     

Investigation of calcium antagonist–L-type calcium channel interactions by a vascular smooth muscle cell membrane chromatography method

The dissociation equilibrium constant (K _D) is an important affinity parameter for studying drug–receptor interactions. A vascular smooth muscle (VSM) cell membrane chromatography (CMC) method was developed for determination of the K _D values for calcium antagonist–L-type calcium channel (L-CC) interactions. VSM cells, by means of primary culture with rat thoracic aortas, were used for preparation of the cell membrane stationary phase in the VSM/CMC model. All measurements were performed with spectrophotometric detection (237 nm) at 37 °C. The K _D values obtained using frontal analysis were 3.36 × 10⁻⁶ M for nifedipine, 1.34 × 10⁻⁶ M for nimodipine, 6.83 × 10⁻⁷ M for nitrendipine, 1.23 × 10⁻⁷ M for nicardipine, 1.09 × 10⁻⁷ M for amlodipine, and 8.51 × 10⁻⁸ M for verapamil. This affinity rank order obtained from the VSM/CMC method had a strong positive correlation with that obtained from radioligand binding assay. The location of the binding region was examined by displacement experiments using nitrendipine as a mobile-phase additive. It was found that verapamil occupied a class of binding sites on L-CCs different from those occupied by nitrendipine. In addition, nicardipine, amlodipine, and nitrendipine had direct competition at a single common binding site. The studies showed that CMC can be applied to the investigation of drug–receptor interactions.     

Extending foldamer design beyond α-helix mimicry: α/β-peptide inhibitors of vascular endothelial growth factor signaling

Diverse strategies have been explored to mimic the surface displayed by an α-helical segment of a protein, with the goal of creating inhibitors of helix-mediated protein-protein interactions. Many recognition surfaces on proteins, however, are topologically more complex and less regular than a single α-helix. We describe efforts to develop peptidic foldamers that bind to the irregular receptor-recognition surface of vascular endothelial growth factor (VEGF). Our approach begins with a 19-residue α-peptide previously reported by Fairbrother et al. (Biochemistry 1998, 37, 17754) to bind to this surface on VEGF. Systematic evaluation of α→β replacements throughout this 19-mer sequence enabled us to identify homologues that contain up to ~30% β residues, retain significant affinity for VEGF, and display substantial resistance to proteolysis. These α/β-peptides can block VEGF-stimulated proliferation of human umbilical vein endothelial cells.     

Inhibition of NF-κB prevents high glucose-induced proliferation and plasminogen activator inhibitor-1 expression in vascular smooth muscle cells

Recent epidemiologic studies clearly showed that early intensive glucose control has a legacy effect for preventing diabetic macrovascular complications. However, the cellular and molecular processes by which high glucose leads to macrovascular complications are poorly understood. Vascular smooth muscle cell (VSMC) dysfunction due to high glucose is a characteristic of diabetic vascular complications. Activation of nuclear factor-κB (NF-κB) may play a key role in the regulation of inflammation and proliferation of VSMCs. We examined whether VSMC proliferation and plasminogen activator inhibitor-1 (PAI-1) expression induced by high glucose were mediated by NF-κB activation. Also, we determined whether selective inhibition of NF-κB would inhibit proliferation and PAI-1 expression in VSMCs. VSMCs of the aorta of male SD rats were treated with various concentrations of glucose (5.6, 11.1, 16.7, and 22.2 mM) with or without an inhibitor of NF-κB or expression of a recombinant adenovirus vector encoding an IκB-α mutant (Ad-IκBαM). VSMC proliferation was examined using an MTT assay. PAI-1 expression was assayed by real-time PCR and PAI-1 protein in the media was measured by ELISA. NF-κB activation was determined by immunohistochemical staining, NF-κB reporter assay, and immunoblotting. We found that glucose stimulated VSMC proliferation and PAI-1 expression in a dose-dependent manner up to 22.2 mM. High glucose (22.2 mM) alone induced an increase in NF-κB activity. Treatment with inhibitors of NF-κB such as MG132, PDTC or expression of Ad-IκB-αM in VSMCs prevented VSMC proliferation and PAI-1 expression induced by high glucose. In conclusion, inhibition of NF-κB activity prevented high glucose-induced VSMC proliferation and PAI-1 expression.     

A vascular smooth muscle/cell membrane chromatography–offline-gas chromatography/mass spectrometry method for recognition,separation and identification of active components from traditional Chinese medicines

We describe an analytical method of vascular smooth muscle cell membrane chromatography (VSM/CMC) combined with gas chromatography/mass spectrometry (GC/MS) for recognition, separation and identification of active components from traditional Chinese medicines (TCMs). VSM cells by means of primary culture with rat thoracic aortas were used for preparation of the stationary phase in the CMC model. Retention components by the VSM–CMC model were collected and then analyzed by GC/MS under the optimized conditions in offline conditions. After investigating the suitability and reliability of the VSM/CMC–offline-GC/MS method using nifedipine and nitrendipine as standard compounds, this method was applied in screening active components from the extracts of TCMs such as Radix Angelicae Dahuricae (RAD), Rhizomza Seu Radix Notopterygii (RSRN), Radix Glehniae (RG) and Fructus Cnidii (FC). Retention components from the extracts in the VSM–CMC model were imperatorin and osthole identified by the GC/MS method. In vitro pharmacological trials indicated that imperatorin and osthole could concentration dependently relax the rat thoracic artery pre-contracted by KCl (P < 0.05). The maximum relaxation effects (R_max) were 63 ± 5% and 40 ± 6% for imperatorin and osthole, respectively. The VSM/CMC–offline-GC/MS method is an effective screening system that can rapidly detect and enrich target components from a complex sample and then accurately identify them.