Targeting microRNAs for cardiovascular therapeutics in coronary artery disease.

PURPOSE OF REVIEW: To discuss the possible therapeutic options for miRNA as targets in coronary heart disease. Noncoding RNAs regulate gene expression at a posttranscriptional state. Modulation of miRNAs might be a new therapeutic option in coronary heart disease. RECENT FINDINGS: Noncoding RNAs (long and short noncoding RNAs) might be used as biomarkers in cardiovascular disease, as they are differentially regulated and released in the pathophysiological situation of coronary heart disease. In acute myocardial infarction already a lot of miRNAs are investigated as biomarkers, still not superior to high-sensitive Troponin T. In rare cardiovascular diseases such as Tako-Tsubo cardiomyopathy or the different stages of heart failure, development of new biomarkers is even more important. In addition, miRNA inhibition via antimiRs is capable of attenuating cardiovascular disease in small and large animal models. Over-expression of 'protective' miRNAs (miR mimics) improved the outcome of cardiovascular disease in vitro and in first small animal models. SUMMARY: Noncoding RNAs are promising new biomarkers for cardiovascular disease. Directly targeting miRNA for disease modulation is possible for the specific inhibition, as well as for overexpression of 'protective' miRNAs. However, additional preclinical and clinical testing has to be performed before this therapy will enter clinical routine.

An HPLC-SEC-based rapid quantification method for vesicular stomatitis virus particles to facilitate process development.

Virus particle (VP) quantification plays a pivotal role in the development of production processes of VPs for virus-based therapies. The yield based on total VP count serves as a process performance indicator for evaluating process efficiency and consistency. Here, a label-free particle quantification method for enveloped VPs was developed, with potential applications in oncolytic virotherapy, vaccine development, and gene therapy. The method comprises size-exclusion chromatography (SEC) separation using high-performance liquid chromatography (HPLC) instruments. Ultraviolet (UV) was used for particle quantification and multi-angle light scattering (MALS) for particle characterization. Consistent recoveries of over 97% in the SEC were achieved upon mobile phase screenings and addition of bovine serum albumin (BSA) as sample stabilizer. A calibration curve was generated, and the method's performance and applicability to in-process samples were characterized. The assay's repeatability variation was <1% and its intermediate precision variation was <3%. The linear range of the method spans from 7.08 × 108 to 1.72 × 1011 VP/mL, with a limit of detection (LOD) of 7.72 × 107 VP/mL and a lower limit of quantification (LLOQ) of 4.20 × 108 VP/mL. The method, characterized by its high precision, requires minimal hands-on time and provides same-day results, making it efficient for process development.

Thymosin ß4 attenuates microcirculatory and hemodynamic destabilization in sepsis.

OBJECTIVE: The actin polymerization regulator Thymosin ß4 (Tß4) has been shown to be involved in angiogenesis, wound healing, cell survival and anti-inflammatory responses. We have previously shown that Tß4 is capable of recruiting pericytes, thus stabilizing the endothelial barrier function. Here, we analyzed whether treatment with Tß4 is able to reduce the pericytes loss in lipopolysaccharides (LPS)-induced sepsis and to improve the hemodynamic function and survival in C57BL/6 mice. METHODS: Fourteen days before LPS injection, the mice were injected with an adeno-associated virus carrying the Tß4 (rAAV.Tß4) or LacZ gene (rAAV.LacZ). A sepsis-severity score was assessed, and non-invasive hemodynamic and permeability measurements were performed. Heart and muscle samples were analyzed for PECAM-1(+) capillaries and NG2(+)pericytes. RESULTS: At 36 h, there was a decrease of sepsis severity score in rAAV.Tß4-treated animals as compared to rAAV.LacZ-treated control. rAAV.Tß4-treated animals displayed lower perivascular leakage and higher blood pressure compared to control. Of note, the rAAV.Tß4 group showed a higher pericyte count in heart and peripheral muscle samples. Finally, Tß4-treatment reduced mortality compared to control. CONCLUSION: The data indicate a preventive role of Tß4 in septic hypercirculation and highlight Tß4 as a potential therapeutic target in severe sepsis.

αß T-cell receptors from multiple sclerosis brain lesions show MAIT cell-related features.

OBJECTIVES: To characterize phenotypes of T cells that accumulated in multiple sclerosis (MS) lesions, to compare the lesional T-cell receptor (TCR) repertoire of T-cell subsets to peripheral blood, and to identify paired α and ß chains from single CD8(+) T cells from an index patient who we followed for 18 years. METHODS: We combined immunohistochemistry, laser microdissection, and single-cell multiplex PCR to characterize T-cell subtypes and identify paired TCRα and TCRß chains from individual brain-infiltrating T cells in frozen brain sections. The lesional and peripheral TCR repertoires were analyzed by pyrosequencing. RESULTS: We found that a TCR Vß1(+) T-cell population that was strikingly expanded in active brain lesions at clinical onset comprises several subclones expressing distinct yet closely related Vα7.2(+) α chains, including a canonical Vα7.2-Jα33 chain of mucosal-associated invariant T (MAIT) cells. Three other α chains bear striking similarities in their antigen-recognizing, hypervariable complementarity determining region 3. Longitudinal repertoire studies revealed that the TCR chains that were massively expanded in brain at onset persisted for several years in blood or CSF but subsequently disappeared except for the canonical Vα7.2(+) MAIT cell and a few other TCR sequences that were still detectable in blood after 18 years. CONCLUSIONS: Our observation that a massively expanded TCR Vß1-Jß2.3 chain paired with distinct yet closely related canonical or atypical MAIT cell-related α chains strongly points to an antigen-driven process in early active MS brain lesions.