MiR-3571 modulates the proliferation and migration of vascular smooth muscle cells by targeting claudin 1.

Background and aims: The miRNA-based post-transcription modification has been extensively studied in hypertension. It however remains elusive how miRNA expression is regulated in this pathological process. We hypothesize that hydroxymethylation in the promoter regions tightly controls the levels of key miRNAs, which in turn affects the development of hypertension. Methods: The levels of hydroxymethylation in the promoter regions from thoracic aortic tissues were compared between spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto rats (WKYs), using hydroxymethylcytosine DNA immunoprecipitation (hMeDIP) sequencing. The altered hydroxymethylation level of miR-3571 was confirmed by glucosylation-coupled hydroxymethylation-sensitive qPCR. We further identified claudin 1(CLDN1) as a key target of miR-3571 via bioinformatic prediction (targetscan) and dual-luciferase activity assays. Finally, we analyzed the contribution of miR-3571/CLDN1 axis in the proliferation and migration of vascular smooth muscle cells (VSMCs). Results: The hydroxymethylation level of miR-3571 promoter region in thoracic aortic tissue from spontaneously hypertensive rats was lower than that from normotensive Wistar-Kyoto rats. Accordingly, the expression of miR-3571 was lower during hypertension, with up-regulated CLDN1 protein levels. More importantly, we found that miR3571 overexpression led to phenotypic changes of VSMCs, and inhibited the proliferation and migration of muscle cells via suppressing CLDN1 as well. Our findings further suggested that CLDN1 up-regulation increase the activity of ERK1/2 in VSMCs. Conclusions: Our study suggested that hydroxymethylation in the promoter regions controlled the level of miR-3571 and revealed the important roles of miR-3571 and CLDN1 in VSMCs during the development of hypertension. In addition, our results also indicated that miR-3571/CLDN1 axis regulated the functions of VSMCs via the ERK1/2 pathway. Taken together, our findings support miR-3571 as a novel biomarker for the diagnosis and prevention of hypertension.

P-selectin (CD62P) and soluble TREM-like transcript-1 (sTLT-1) are associated with coronary artery disease: a case control study.

BACKGROUND: Platelet activation plays a crucial role in the pathogenesis of coronary artery disease (CAD). Platelet P-selectin (CD62P) is a classic platelet activation indicator on the platelet surface, and soluble TREM-like transcript-1 (sTLT-1) is a new indicator. However, the relationship between these two markers and CAD, especially in acute coronary syndrome (ACS), has not been elucidated. This study aimed to investigate CD62P expression on the platelet surface and sTLT-1 expression in serum, as well as to assess their relationship with CAD. METHODS: We measured the levels of CD62P and sTLT-1 in 83 patients with CAD compared to 49 controls. The association of these indicators with age, blood pressure, lipid profile, body mass index, and liver injury marker level were also examined. RESULTS: CD62P concentration was higher in CAD patients than in the control group (P < 0.01), especially in acute myocardial infarction (AMI) patients (P < 0.01). Serum sTLT-1 concentration was higher in the AMI and unstable angina pectoris (UAP) groups than in the normal control (NC) group (P < 0.01). CONCLUSIONS: The consistency of sTLT-1 and CD62P expression levels in CAD patients indicates that sTLT-1 level, the same as CD62P, may be a new marker of platelet activation that is positively related to CAD.

Effects and mechanisms of the secondary structure on the antimicrobial activity and specificity of antimicrobial peptides.

A 15-mer cationic α-helical antimicrobial peptide HPRP-A1 was used as the parent peptide to study the effects of peptide secondary structure on the biophysical properties and biological activities. Without changing the amino acid composition of HPRP-A1, we designed two α-helical peptides with either higher or lower helicity compared with the parent peptide, a ß-sheet peptide and a random coiled peptide using de novo design approach. The secondary structures were confirmed by circular dichroism spectroscopy. The three α-helical peptides exhibited comparable antibacterial activities, but their hemolytic activity varied from extreme hemolysis to no hemolysis, which is correlated with their helicity. The ß-sheet peptide shows poor antibacterial and strong hemolytic activities. More interestingly, the random coil peptide shows no antibacterial activity against Gram-negative bacteria, weak antibacterial activity against Gram-positive bacteria, and extremely weak hemolytic activity. Bacterial membrane permeabilization was also testified on peptides with different secondary structures. Tryptophan fluorescence experiment revealed that the peptide binding preference to the lipid vesicles for mimicking the prokaryotic or eukaryotic membranes was consistent with their biological activities. With the de novo design approach, we proved that it is important to maintain certain contents of amphipathic secondary structure for a desirable biological activity. We believe that the de novo design approach of relocation of the amino acids within a template sequence could be an effective approach in optimizing the specificity of an antimicrobial peptide.

Effects of single amino acid substitution on the biophysical properties and biological activities of an amphipathic α-helical antibacterial peptide against Gram-negative bacteria.

An antimicrobial peptide, known as V13K, was utilized as the framework to study the effects of charge, hydrophobicity and helicity on the biophysical properties and biological activities of α-helical peptides. Six amino acids (Lys, Glu, Gly, Ser, Ala, and Leu) were individually used to substitute the original hydrophobic valine at the selected sixteenth location on the non-polar face of V13K. The results showed that the single amino acid substitutions changed the hydrophobicity of peptide analogs as monitored by RP-HPLC, but did not cause significant changes on peptide secondary structures both in a benign buffer and in a hydrophobic environment. The biological activities of the analogs exhibited a hydrophobicity-dependent behavior. The mechanism of peptide interaction with the outer membrane and cytoplasmic membrane of Gram-negative bacteria was investigated. We demonstrated that this single amino acid substitution method has valuable potential for the rational design of antimicrobial peptides with enhanced activities.

An Aligned Patterned Biomimetic Elastic Membrane Has a Potential as Vascular Tissue Engineering Material.

Cardiovascular disease is the leading cause of death worldwide, with an annual mortality incidence predicted to rise to 23.3 million worldwide by 2030. Synthetic vascular grafts as an alternative to autologous vessels have shown satisfactory long-term results for replacement of large- and medium-diameter arteries, but have poor patency rates when applied to small-diameter vessels. Nanoparticles with low toxicity, contrasting agent properties, tailorable characteristics, targeted/stimuli- response delivery potential, and precise control over behavior (via external stimuli such as magnetic fields) have made possible their use for improving engineered tissues. Poly (styrene-block-butadiene-block-styrene) (SBS) is a kind of widely used thermoplastic elastomer with good mechanical properties and biocompatibility. Here, we synthesized anthracene-grafted SBS (SBS-An) by the method for the fabrication of a biomimetic elastic membrane with a switchable Janus structure, and formed the patterns on the surface of SBS-An under ultraviolet (UV) light irradiation. By irradiating the SBS-An film at different times (0, 10, 20, 30, 60, and 120 s), we obtained six well-ordered surface-patterned biomimetic elastic film with SBS-An at different heights in the thickness direction and the same distances of intervals (named sample-0, 10, 20, 30, 60, and 120 s). The structural effects of the SBS-An films on the adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) were studied, and the possible mechanism was explored. When the HUVECs were cultured on the SBS-An films at different heights in the thickness direction, the sample-30 s with approximately 4 µm height significantly promoted adhesion of the HUVECs at the early stage and proliferation during the culture period compared with the samples of the lower (0, 10, and 20 s) and higher (60 and 120 s) heights. Consistent with this, the sample 30 s showed a higher stimulatory effect on the proliferation- and angiogenesis-related genes. These results suggest that SBS-An with appropriate height could efficiently control bioactivities of the biomimetic elastic membrane and might have great potential in vascular tissue engineering application.

Long noncoding RNA-dependent regulation of vascular smooth muscle cell proliferation and migration in hypertension.

Hypertension is one of the main risks causing cardiovascular diseases. Long noncoding RNAs (lncRNAs) play a critical role in many physiological and pathological conditions. However, their role in hypertension is still unclear. In this study, 460 lncRNAs and 522 mRNA were identified to have different expressions in the thoracic aortas of spontaneously hypertensive rats compared with normotensive Wistar-Kyoto rats through next-generation sequencing. Several randomly selected lncRNAs including NONRATT011842 were validated by qRT-PCR and their potential functions were predicted by co-expression analysis with the help of bioinformatics. Moreover, this study focused on the function of lncRNA NONRATT011842 in the vascular smooth muscle cells (VSMCs) during hypertension and confirmed that NONRATT011842 could interact with PABPC1 to regulate the functions of VSMCs. Therefore, the intervention or utilization of certain lncRNAs could be a new biomarker for the diagnosis and prevention of hypertension.

MicroRNA-26a targets MAPK6 to inhibit smooth muscle cell proliferation and vein graft neointimal hyperplasia.

Neointima formation is the major reason for vein graft failure. However, the underlying mechanism is unclear. The aim of this study was to determine the role of miR-26a in the development of neointimal hyperplasia of autogenous vein grafts. Using autologous jugular vein grafts in the rat carotid artery as a model, we found that miR-26a was significantly downregulated in grafted veins as well as proliferating vascular smooth muscle cells (VSMCs) stimulated with platelet-derived growth factor-BB (PDGF-BB). Overexpression of miR-26a reduced the proliferation and migration of VSMCs. Further analysis revealed that the effects of miR-26a in VSMCs were mediated by targeting MAPK6 at the mRNA and protein levels. Luciferase assays showed that miR-26a repressed wild type (WT) MAPK6-3'-UTR-luciferase activity but not mutant MAPK6-3'-UTR-luciferease reporter. MAPK6 deficiency reduced proliferation and migration; in contrast, overexpression of MAPK6 enhanced the proliferation and migration of VSMCs. This study confirmed that neointimal hyperplasia in vein grafts was reduced in vivo by up-regulated miR-26a expression. In conclusion, our results showed that miR-26a is an important regulator of VSMC functions and neointimal hyperplasia, suggesting that miR-26a may be a potential therapeutic target for autologous vein graft diseases.

The study of single anticancer peptides interacting with HeLa cell membranes by single molecule force spectroscopy.

To determine the effects of biophysical parameters (e.g. charge, hydrophobicity, helicity) of peptides on the mechanism of anticancer activity, we applied a single molecule technique-force spectroscopy based on atomic force microscope (AFM)-to study the interaction force at the single molecule level. The activity of the peptide and analogs against HeLa cells exhibited a strong correlation with the hydrophobicity of peptides. Our results indicated that the action mode between α-helical peptides and cancer cells was largely hydrophobicity-dependent.

Inhibitory effects and mechanisms of physiological conditions on the activity of enantiomeric forms of an α-helical antibacterial peptide against bacteria.

Enantiomeric amphipathic α-helical antibacterial peptides were synthesized and their biophysical and biological properties under different physiological conditions were studied. In the absence of physiological factors, the L- and D-peptides exhibited similar antimicrobial activities against a broad spectrum of bacteria, even against clinical isolates with resistance to traditional antibiotics. However, in the presence of NaCl, CaCl2 or human serum albumin (HSA) at physiological concentrations, the enantiomers revealed bacterium-species dependent attenuations in antibacterial activity. In the presence of salts the electrostatic interaction between the peptides and the biomembrane was inhibited. Salts, especially CaCl2 weakened the ability of the peptides to permeabilize the outer membrane of Gram-negative bacteria, as determined by a 1-N-phenylnaphthylamine uptake assay. HSA exhibited variable inhibitory effects on the activity of the peptides when incubated with different bacterial strains. The peptides showed different binding association abilities to HSA at different molar ratios, regardless of their chirality, resulting in reduced peptide biological activity. The D-peptide performed better than its L-enantiomer in all conditions tested because of its resistance to proteolysis, and may therefore represent a promising candidate for development as a therapeutic agent.