Short-term inhibition of DPP-4 enhances endothelial regeneration after acute arterial injury via enhanced recruitment of circulating progenitor cells

Background

Endothelial injuries regularly occur in atherosclerosis and during interventional therapies of the arterial occlusive disease. Disturbances in the endothelial integrity can lead to insufficient blood supply and bear the risk of thrombus formation and acute vascular occlusion. At present, effective therapeutics to restore endothelial integrity are barely available.We analyzed the effect of pharmacological DPP-4-inhibition by Sitagliptin on endogenous progenitor cell-based endothelial regeneration via the SDF-1α/CXCR4-axis after acute endothelial damage in a mouse model of carotid injury.

Methods and Results

Induction of a defined endothelial injury was performed in the carotid artery of C57Bl/6 mice which led to a local upregulation of SDF-1α expression. Animals were treated with placebo, Sitagliptin or Sitagliptin + AMD3100. Using mass spectrometry we could prove that Sitagliptin prevented cleavage of the chemokine SDF-1α. Accordingly, increased SDF-1α concentrations enhanced recruitment of systemically applied and endogenous circulating CXCR4 + progenitor cells to the site of vascular injury followed by a significantly accelerated reendothelialization as compared to placebo-treated animals. Improved endothelial recovery, as well as recruitment of circulating CXCR4 + progenitor cells (CD133 +, Flk1 +), was reversed by CXCR4-antagonization through AMD3100. In addition, short-term Sitagliptin treatment did not significantly promote neointimal or medial hyperplasia.

Conclusion

Sitagliptin can accelerate endothelial regeneration after acute endothelial injury. DPP-4 inhibitors prevent degradation of the chemokine SDF-1α and thus improve the recruitment of regenerative circulating CXCR4 + progenitor cells which mediate local endothelial cell proliferation without adversely affecting vessel wall architecture.     

Screening of 20 patients with X-linked mental retardation using chromosome X-specific array-MAPH

The rapid advancement of high-resolution DNA copy number assessment methods revealed the significant contribution of submicroscopic genetic imbalances to abnormal phenotypes, including mental retardation. In order to detect submicroscopic genetic imbalances, we have screened 20 families with X-linked mental retardation (XLMR) using a chromosome X-specific array-MAPH platform with median resolution of 238 kb. Among the 20 families, 18 were experimental, as they were not previously screened with any microarray method, and two were blind controls with known aberrations, as they were previously screened by array-CGH. This study presents the first clinical application of chromosome X-specific array-MAPH methodology. The screening of 20 affected males from 20 unrelated XLMR families resulted in the detection of an unknown deletion, spanning a region of 7–23 kb. Family studies and population screening demonstrated that the detected deletion is an unknown rare copy number variant. One of the control samples, carrying approximately 6-Mb duplication was correctly identified, moreover it was found to be interrupted by a previously unknown 19 kb region of normal copy number. The second control 50 kb deletion was not identified, as this particular region was not covered by array-MAPH probes. This study demonstrates that the chromosome X-specific array-MAPH platform is a valuable tool for screening patients with XLMR, or other X-linked disorders, and emerges the need for introducing new high-resolution screening methods for the detection of genetic imbalances.     

Case-based estimation of the risk of enterobiasis

OBJECTIVE: To introduce an original case-based machine learning (ML) and prediction system Constud and its application on tabular data for estimation of the risk of enterobiasis among nursery school children in Estonia. METHODS AND MATERIALS: The system consists of a software application and a knowledge base of observation data, parameters, and results. The data were obtained from anal swabs for the diagnosis of enterobiasis, from questionnaires for children's parents, observations in nursery schools and interviews with supervisors of the groups. The total number of studied children was 1905. Ten parallel ML processes were conducted to find the best set of weights for features and cases. RESULTS: The best goodness-of-fit according to the true skill statistic (TSS) was 0.381. Approximately equal fit can be reached using different sets of features. Cross-validation TSS of logit-regression and classification tree models was <0.24. In addition to the higher prediction fit, Constud is not sensitive to missing values of explanatory variables. The overall prevalence of enterobiasis was 22.8%; the mean of risk estimations was 47.8%. The overestimation of the prevalence in risk calculations can be interpreted as an inefficacy of the single swab analysis, or may be due to the relative constancy of the risk compared to the lability of infection and the applied objective function. CONCLUSIONS: In addition to the higher prediction fit, Constud is not sensitive to missing values of explanatory variables. The main risk factors of enterobiasis among nursery school children were the child's age, communication partners, habits, and cleanness of rooms in the nursery school. Mixed age groups at nursery schools also enhance the risk.     

A parallel SNP array study of genomic aberrations associated with mental retardation in patients and general population in Estonia

The increasing use of whole-genome array screening has revealed the important role of DNA copy-number variations in the pathogenesis of neurodevelopmental disorders and several recurrent genomic disorders have been defined during recent years. However, some variants considered to be pathogenic have also been observed in phenotypically normal individuals. This underlines the importance of further characterization of genomic variants with potentially variable expressivity in both patient and general population cohorts to clarify their phenotypic consequence. In this study whole-genome SNP arrays were used to investigate genomic rearrangements in 77 Estonian families with idiopathic mental retardation. In addition to this family-based approach, phenotype and genotype data from a cohort of 1000 individuals in the general population were used for accurate interpretation of aberrations found in mental retardation patients. Relevant structural aberrations were detected in 18 of the families analyzed (23%). Fifteen of those were in genomic regions where clinical significance has previously been established. In 3 families, 4 novel aberrations associated with intellectual disability were detected in chromosome regions 2p25.1-p24.3, 3p12.1-p11.2, 7p21.2-p21.1 and Xq28. Carriers of imbalances in 15q13.3, 16p11.2 and Xp22.31 were identified among reference individuals, affirming the variable phenotypic consequence of rare variants in some genomic regions considered as pathogenic.     

Relationship between serum uric acid levels and urinary albumin excretion in patients with heart failure

OBJECTIVES: Hyperuricaemia is a constant finding in patients with heart failure (HF). Upregulated xanthine-oxidase activity seems to contribute to progression of the disease through the production of oxidative stress and the development of vascular and endothelial dysfunction. On this basis we speculated that in HF serum uric acid levels correlated with a reliable marker of endothelial dysfunction as urinary albumin excretion. METHODS: Fifty-three patients with HF underwent assessment of serum uric acid, N-terminal probrain natriuretic peptide (NT-proBNP), glomerular filtration rate (GFR), other metabolic parameters and determination of urinary albumin concentration (UAC) in a morning urine sample. RESULTS: In univariate analysis there is a direct correlation between serum uric acid levels and log UAC (r = 0.43, P < 0.01); uric acid correlates also positively with log NT-proBNP (r = 0.31, P < 0.05) and negatively with log-GFR (r = -0.38, P < 0.01). In stepwise regression analysis serum uric acid emerged as the only predictor of increased UAC (standardized coefficient = 0.42, P = 0.001) independent of other clinical determinants and metabolic factors. CONCLUSION: Serum uric acid represents the strongest predictor of elevated UAC in HF. Regression of albuminuria may be a simple target to verify the efficacy of xanthine-oxidase inhibition in these patients.     

Anti‐cytokine autoantibodies suggest pathogenetic links with autoimmune regulator deficiency in humans and mice

Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is a recessive disorder resulting from mutations in the autoimmune regulator (AIRE). The patients' autoantibodies recognize not only multiple organ‐specific targets, but also many type I interferons (IFNs) and most T helper type 17 (Th17) cell‐associated cytokines, whose biological actions they neutralize in vitro. These anti‐cytokine autoantibodies are highly disease‐specific: otherwise, they have been found only in patients with thymomas, tumours of thymic epithelial cells that fail to express AIRE. Moreover, autoantibodies against Th17 cell‐associated cytokines correlate with chronic mucocutaneous candidiasis in both syndromes. Here, we demonstrate that the immunoglobulin (Ig)Gs but not the IgAs in APECED sera are responsible for neutralizing IFN‐ω, IFN‐α2a, interleukin (IL)‐17A and IL‐22. Their dominant subclasses proved to be IgG1 and, surprisingly, IgG4 without IgE, possibly implicating regulatory T cell responses and/or epithelia in their initiation in these AIRE‐deficiency states. The epitopes on IL‐22 and IFN‐α2a appeared mainly conformational. We also found mainly IgG1 neutralizing autoantibodies to IL‐17A in aged AIRE‐deficient BALB/c mice – the first report of any target shared by these human and murine AIRE‐deficiency states. We conclude that autoimmunization against cytokines in AIRE deficiency is not simply a mere side effect of chronic mucosal Candida infection, but appears to be related more closely to disease initiation.     

Macrolide antibiotics allosterically predispose the ribosome for translation arrest

Translation arrest directed by nascent peptides and small cofactors controls expression of important bacterial and eukaryotic genes, including antibiotic resistance genes, activated by binding of macrolide drugs to the ribosome. Previous studies suggested that specific interactions between the nascent peptide and the antibiotic in the ribosomal exit tunnel play a central role in triggering ribosome stalling. However, here we show that macrolides arrest translation of the truncated ErmDL regulatory peptide when the nascent chain is only three amino acids and therefore is too short to be juxtaposed with the antibiotic. Biochemical probing and molecular dynamics simulations of erythromycin-bound ribosomes showed that the antibiotic in the tunnel allosterically alters the properties of the catalytic center, thereby predisposing the ribosome for halting translation of specific sequences. Our findings offer a new view on the role of small cofactors in the mechanism of translation arrest and reveal an allosteric link between the tunnel and the catalytic center of the ribosome.Expression of several bacterial and eukaryotic genes is controlled by nascent peptide-dependent programmed translation arrest. In the general scenario, ribosome stalling at an upstream regulatory ORF (uORF) triggers isomerization of the mRNA structure, leading to activation of expression of downstream cistron(s). Translation arrest ensues when a distinctive amino acid sequence (the “stalling domain”) of the growing chain assembled in the ribosomal peptidyl transferase center (PTC) is placed in the nascent peptide exit tunnel (NPET). Ribosome stalling may require additional signals, thereby making this gene control mechanism sensitive to the physiological state of the cell or to the chemical composition of the environment. Often the external signal is a small molecule whose binding to the ribosome renders translation responsive to specific nascent peptides (reviewed in refs. 1, 2). In most of the examined cases of cofactor- and nascent peptide-dependent translation arrest, the binding site of the cofactor in the ribosome is unknown, which hampers understanding of the interplay among the cofactor, the nascent peptide, and the ribosome. The exception is the inducible antibiotic resistance, in which ribosome stalling and gene activation rely on binding of an antibiotic to a well-defined site in the ribosome.Expression of macrolide resistance genes is triggered by drug-induced ribosome stalling at a defined codon of the uORF (3–5). Macrolides, from the prototype erythromycin (ERY) to the newest macrolide derivatives—ketolides, e.g., solithromycin (SOL)—bind in the NPET at a short distance from the PTC (6–9) (Fig. 1A). When a nascent peptide grows to 4–7 aa, it reaches the site of antibiotic binding and has to negotiate the drug-obstructed NPET aperture. Subsequent events depend on the properties of the nascent chain (3, 10, 11). Although for many proteins the encounter of the peptide with the antibiotic results in peptidyl–tRNA dropoff, the N-termini of certain nascent peptides can bypass the antibiotic. Translation of some of such proteins can be arrested at specific sites within the gene, resulting in formation of a stable stalled complex (11). Such translation arrest defines the role of macrolides as cofactors of programmed ribosome stalling (3, 10–12).Open in a separate windowFig. 1.Antibiotic and nascent peptide in the ribosomal exit tunnel. (A) The relative locations of the macrolide binding site in the NPET and the PTC active site were rendered by aligning crystallographic structures of Thermus thermophilus 70S ribosome complexed with aminoacylated donor and acceptor tRNA substrates [Protein Data Bank (PDB) ID codes 2WDK, 2WDL (25)] and the vacant ribosome complexed with ERY [PDB ID codes 3OHC, 3OHJ (9)]. The PTC active site, defined as the middistance between the attacking amino group of the acceptor substrate and the carbonyl carbon atom of the donor, is marked by an asterisk. (B) The modeled position of the 9-aa–long ErmCL nascent peptide in the ribosomal tunnel obstructed by ERY (19). In the stalled complex, ErmCL is juxtaposed with the antibiotic in the tunnel.The regulatory leader peptides of macrolide resistance genes have been classified by the structure of their known or presumed stalling domains (4, 5). Translation of ErmAL1 and ErmCL peptides is arrested after the ribosome has polymerized the 8-aa (ErmAL1) or 9-aa (ErmCL) long nascent chains that carry the C-terminal stalling domains Ile-Ala-Val-Val (IAVV) and Ile-Phe-Val-Ile (IFVI), respectively (12–14). The drug-bound ribosome stalls because it cannot catalyze transfer of the peptide from the P-site peptidyl–tRNA to the A-site aminoacyl–tRNA (12, 14). Importantly, although the N-terminal sequences of these peptides are not critical, the N-terminal segments are required for translation arrest (12). The conservation of the distance of the stalling domain from the N-terminus among peptides of these classes (4) corroborates the importance of the nascent chain length for the arrest. The 8–9-aa long ErmAL1 or ErmCL stalling peptides reach far into the NPET and must be juxtaposed with the antibiotic molecule in the NPET; such apposition has been suggested to play a key role in the mechanism of arrest (12) (Fig. 1B). This view agrees with the strict structural requirements of the macrolide cofactor in which removal or modification of the C3 cladinose abolishes stalling, possibly by disrupting drug–peptide interactions (15).The resistance leader peptides of the third major class have been studied to a much lesser extent (16–18). These peptides were grouped together based on the presence of the Arg-Leu-Arg (RLR) motif in their sequence (4) (Table S1), although the role of this motif in programmed arrest has not been verified. Intriguingly, in striking contrast to the IAVV and IFVI classes, the placement of the RLR motif within these peptides is highly variable (4).By analyzing translation arrest controlled by the RLR peptides, we discovered that the N-terminus is dispensable and macrolide antibiotic can block peptide bond formation and halt translation when the nascent chain is only 3-aa long and barely reaches the antibiotic in the NPET. Structural probing and molecular dynamics (MD) modeling showed the existence of an allosteric link between the NPET and the PTC, illuminating how binding of an antibiotic in the NPET predisposes the ribosome for stalling when translating specific amino acid sequences.