Cardiovascular efficacy and safety of dipeptidyl peptidase-4 inhibitors: A meta-analysis of cardiovascular outcome trials

BACKGROUND Dipeptidyl peptidase-4(DPP-4) inhibitors are a generally safe and well tolerated antidiabetic drug class with proven efficacy in type 2 diabetes mellitus(T2 DM). Recently, a series of large, randomized controlled trials(RCTs) addressing cardiovascular outcomes with DPP-4 inhibitors have been published.AIM To pool data from the aforementioned trials concerning the impact of DPP-4 inhibitors on surrogate cardiovascular efficacy outcomes and on major cardiac arrhythmias.METHODS We searched PubMed and grey literature sources for all published RCTs assessing cardiovascular outcomes with DPP-4 inhibitors compared to placebo until October 2020. We extracted data concerning the following "hard" efficacy outcomes: fatal and non-fatal myocardial infarction, fatal and non-fatal stroke, hospitalization for heart failure, hospitalization for unstable angina, hospitalization for coronary revascularization and cardiovascular death. We also extracted data regarding the risk for major cardiac arrhythmias, such as atrial fibrillation, atrial flutter, ventricular fibrillation and ventricular tachycardia.RESULTS We pooled data from 6 trials in a total of 52520 patients with T2 DM assigned either to DPP-4 inhibitor or placebo. DPP-4 inhibitors compared to placebo led to a non-significant increase in the risk for fatal and non-fatal myocardial infarction [risk ratio(RR) = 1.02, 95%CI: 0.94-1.11, I2 = 0%], hospitalization for heart failure(RR = 1.09, 95%CI: 0.92-1.29, I2 = 65%) and cardiovascular death(RR = 1.02, 95%CI: 0.93-1.11, I2 = 0%). DPP-4 inhibitors resulted in a non-significant decrease in the risk for fatal and non-fatal stroke(RR = 0.96, 95%CI: 0.85-1.08, I2 = 0%) and coronary revascularization(RR = 0.99, 95%CI: 0.90-1.09, I2 = 0%), Finally, DPP-4 inhibitors demonstrated a neutral effect on the risk for hospitalization due to unstable angina(RR = 1.00, 95%CI: 0.85-1.18, I2 = 0%). As far as cardiac arrhythmias are concerned, DPP-4 inhibitors did not significantly affect the risk for atrial fibrillation(RR = 0.95, 95%CI: 0.78-1.17, I2 = 0%), while they were associated with a significant increase in the risk for atrial flutter, equal to 52%(RR = 1.52, 95%CI: 1.03-2.24, I2 = 0%). DPP-4 inhibitors did not have a significant impact on the risk for any of the rest assessed cardiac arrhythmias.CONCLUSION DPP-4 inhibitors do not seem to confer any significant cardiovascular benefit for patients with T2 DM, while they do not seem to be associated with a significant risk for any major cardiac arrhythmias, except for atrial flutter. Therefore, this drug class should not be the treatment of choice for patients with established cardiovascular disease or multiple risk factors, except for those cases when newer antidiabetics(glucagon-like peptide-1 receptor agonists and sodium-glucose cotransporter-2 inhibitors) are not tolerated, contraindicated or not affordable for the patient.     

Acute myocardial infarction with normal coronary arteries in a patient with Hodgkin's disease: a late complication of irradiation and chemotherapy

We describe the case of a 31-year-old man who experienced an acute myocardial infarction 16 years after undergoing radiation and vinca alkaloid therapy for Hodgkin's disease. Even though coronary artery disease is a well-established complication after mediastinal radiation therapy, this adult patient had normal coronary angiographic results, with no traditional risk factors for coronary artery disease, and no hematologic or other abnormality associated with hypercoagulability.     

Noninvasive detection of tumor-infiltrating T cells by PET reporter imaging

Adoptive transfer of tumor-reactive T cells can successfully reduce tumor burden; however, in rare cases, lethal on-target/off-tumor effects have been reported. A noninvasive method to track engineered cells with high sensitivity and resolution would allow observation of correct cell homing and/or identification of dangerous off-target locations in preclinical and clinical applications. Human deoxycytidine kinase triple mutant (hdCK3mut) is a nonimmunogenic PET reporter that was previously shown to be an effective tool to monitor whole-body hematopoiesis. Here, we engineered a construct in which hdCK3mut is coexpressed with the anti-melanoma T cell receptor F5, introduced this construct into human CD34 cells or PBMCs, and evaluated this approach in multiple immunotherapy models. Expression of hdCK3mut allowed engrafted cells to be visualized within recipient bone marrow, while accumulation of [¹⁸F]-L-FMAU in hdCK3mut-expressing T cells permitted detection of intratumoral homing. Animals that received T cells coexpressing hdCK3mut and the anti-melanoma T cell receptor had demonstrably higher signals in HLA-matched tumors compared with those in animals that received cells solely expressing hdCK3mut. Engineered T cells caused cytotoxicity in HLA/antigen-matched tumors and induced IFN-γ production and activation. Moreover, hdCK3mut permitted simultaneous monitoring of engraftment and tumor infiltration, without affecting T cell function. Our findings suggest that hdCK3mut reporter imaging can be applied in clinical immunotherapies for whole-body detection of engineered cell locations.     

Phosphorylation of the CENP-A amino-terminus in mitotic centromeric chromatin is required for kinetochore function

The role of the mitotic phosphorylation of the amino (NH₂) terminus of Centromere Protein A (CENP-A), the histone variant epigenetic centromeric marker, remains elusive. Here, we show that the NH₂ terminus of human CENP-A is essential for mitotic progression and that localization of CENP-C, another key centromeric protein, requires only phosphorylation of the CENP-A NH₂ terminus, and is independent of the CENP-A NH₂ terminus length and amino acid sequence. Mitotic CENP-A nucleosomal complexes contain CENP-C and phosphobinding 14-3-3 proteins. In contrast, mitotic nucleosomal complexes carrying nonphosphorylatable CENP-A–S7A contained only low levels of CENP-C and no detectable 14-3-3 proteins. Direct interactions between the phosphorylated form of CENP-A and 14-3-3 proteins as well as between 14-3-3 proteins and CENP-C were demonstrated. Taken together, our results reveal that 14-3-3 proteins could act as specific mitotic “bridges,” linking phosphorylated CENP-A and CENP-C, which are necessary for the platform function of CENP-A centromeric chromatin in the assembly and maintenance of active kinetochores.Histone variants are nonallelic isoforms of conventional histones. It is widely accepted that the incorporation of histone variants generally confers novel structural and functional properties to the nucleosome (1). Centromere Protein A (CENP-A) is a histone variant, which replaces the canonical histone H3 at the centromere (2) and marks epigenetically the centromeres and the kinetochores (for reviews see refs. 3 and 4). The presence of CENP-A is required for the assembly of active kinetochores and its depletion results in numerous mitotic problems, such as chromosome misalignments and segregation defects, generation of chromosome bridges, aneuploidy, etc. (5). The resulting mitotic defects, following CENP-A depletion, were associated also with notable alterations in the composition and organization of the kinetochore, including the delocalization of the inner kinetochore proteins CENP-C, CENP-I, and CENP-H as well as the outer kinetochore components Highly Expressed in Cancer protein 1 (HEC1), Mitotic Arrest Deficient 2-like protein (Mad2), and CENP-E (5).During recent years, the studies of CENP-A were focused mainly on its histone-fold domain. The NH₂ terminus of CENP-A, which is not required for centromeric targeting (6, 7) appeared, however, to play an important role in both mitosis and meiosis. In yeast, the NH₂ tail of Chromosome Segregation Protein 4 (Cse4p) (the homolog of mammalian CENP-A) has an essential function distinct from that of the histone-fold domain in chromosome assembly and segregation (8). The reported data in Arabidopsis thaliana suggested the existence of a meiosis-specific loading pathway for CENP-A, requiring its NH₂ terminus (9). In addition, human CENP-A is phosphorylated in its NH₂ terminus at serine 7 in mitosis but the role of this phorphorylation is far from being clear (10, 11).Sequence alignments of the NH₂ termini of CENP-A from different species show very low sequence conservation in terms of amino acid composition, sequence, and length (Fig. 1A). However, expression of CENP-A with its NH₂ terminus deleted is lethal in yeast (12) and plants (13). These data create a paradox because on one hand, the NH₂ terminus of CENP-A, in certain organisms, is required for their survival and on the other hand, it appears to be nonessential because there is no evolutionary pressure to conserve at least some specific sequence elements.Open in a separate windowFig. 1.The NH₂ tail of CENP-A is required for mitosis and the H3 swapped NH₂ tail CENP-A chimera rescues the CENP-A null cell phenotype. (A) Sequence alignment of CENP-A NH₂ termini from different species. Names of species are indicated at Left. Serine residues are indicated in blue. Conservation between different species is shown (Lower). (B) Cell cycle visualization, after CENP-A suppression by siRNA treatment of naïve HeLa cells (second row) or HeLa cells stably expressing siRNA-resistant full-length GFP–CENP-A (third row) or tailless GFP–ΔN–CENP-A (fourth row) or GFP–H3–CENP-A swapped tail mutant (fifth row). First row shows naïve cells not treated with siRNA. A CREST antibody was used to visualize the centromeres in naïve cells; GFP fluorescence was used to visualize CENP-A in GFP fusion-expressing cells. An antibody against inner centromere protein (INCENP) and antilamina antibody were used to detect the midbody during cytokinesis and the nuclear envelope in interphase cells (shown in red). Blue, DNA; white arrowheads point to misaligned or lagging chromosomes and to chromatin bridges. (Scale bar, 5 µm.) (C) Detection of the GFP–CENP-A fusions and endogenous CENP-A in control (−) and siRNA treated (+) cells at 72 h posttransfection by Western blot. Cells used are indicated (Upper). Arrows indicate the positions of the GFP–CENP-A fusions or endogenous CENP-A. (D) Histograms showing the percentage of mitotic defects at 72 h posttransfection with siRNA against CENP-A in the indicated cell lines. HeLa, naïve cell line. (E) Same as D, but showing the percentage of multinucleate cells. For each experiment, at least 400 cells were counted. Data are means and SEM of five independent experiments.Here, we have analyzed the mitotic function of the NH₂ terminus of human CENP-A and its phosphorylation. We show the mere phosphorylation of CENP-A, but not its length and amino acid sequence, is required for the localization of CENP-C, a key mediator between centromeric chromatin and the outer kinetochore components. Our data reveal that in mitosis, the phosphorylated CENP-A nucleosomes are “bridged” to CENP-C via the phosphobinding 14-3-3 proteins. These 14-3-3 mitotic bridges are essential for the assembly of active kinetochores.     

Erythrocyte membrane cholesterol and lipid core growth in a rabbit model of atherosclerosis: Modulatory effects of rosuvastatin

Background

Lipid core expansion is partly responsible for the conversion of a stable atherosclerotic lesion to a rupture-prone plaque. Intraplaque hemorrhage contributes to the accumulation of cholesterol within unstable plaques. In the present study, we investigated, using a rabbit model of atherosclerosis, the extent to which diet-induced increases in cholesterol content of erythrocyte membranes (CEM) contribute to lipid core expansion and the modulatory effect of rosuvastatin use.

Methods and results

Rabbits fed with atherogenic diet (0.75% cholesterol) for 5 months exhibited advanced atherosclerotic lesions (mean plaque area, 0.39 ± 0.03 mm²), and lipid core size was associated with the concentration–time integral (CTI) of CEM levels (r = 0.567, P = 0.004) independent of other established predictors of lipid core size. Further experiments were performed by feeding rabbits atherogenic diet (1% cholesterol) for 3 months, followed by either normal diet or normal diet plus rosuvastatin for the next 3 months. Although no differences were observed in total plaque area between both groups, administration of rosuvastatin was associated with significantly smaller lipid cores, fewer macrophages within the lipid core, less microvessels as well as with lower CTI of CEM levels compared to normal diet alone. Moreover, intraplaque erythrocyte membranes covered a smaller lipid core area in rabbits under rosuvastatin plus normal diet as opposed to rabbits under diet alone.

Conclusions

Increased CEM levels, induced by high-cholesterol diet, are associated with lipid core growth. Ingestion of a potent HMG-CoA reductase inhibitor (rosuvastatin) may decrease CEM levels, and this effect may contribute to regression of the lipid core.     

Diverse associations of microalbuminuria with C-reactive protein, interleukin-18 and soluble CD 40 ligand in male essential hypertensive subjects

BACKGROUND: Microalbuminuria (MA) and low-grade inflammation constitute emerging markers of subclinical atherosclerosis. We investigated whether urinary albumin excretion, expressed as the albumin-to-creatinine ratio (ACR), is associated with high sensitivity C-reactive protein (hs-CRP), interleukin (IL)-18, and soluble CD40 ligand (sCD40L), in hypertensive subjects. METHODS: The study population consisted of 108 nondiabetic male patients with newly diagnosed untreated stage I to II essential hypertension (aged 44.6 years, office blood pressure [BP] 148/95 mm Hg). According to ACR values determined as the average of two nonconsecutive overnight spot urine samples, subjects were divided into microalbuminurics (n = 28) (mean ACR = 30 to 300 mg/g) and normoalbuminurics (n = 80) (mean ACR <30 mg/g). RESULTS: Although microalbuminurics as compared to normoalbuminuric hypertensives had greater hs-CRP levels (2.55 +/- 1.18 v 1.45 +/- 0.52 mg/L, P < .0001), independently of confounding factors, these two groups did not differ regarding IL-18 and sCD40L values (P = not significant [NS] for both cases). In the entire population, ACR exhibited a positive correlation with hs-CRP (r = 0.623, P < .0001), whereas there was no association with both IL-18 and sCD40L (P = NS for both cases). When multiple linear regression analysis was performed, it was revealed that age, body mass index, office systolic BP, total cholesterol, and hs-CRP levels were significant independent predictors of the ACR (P < .05). CONCLUSIONS: In essential hypertensive subjects, MA is accompanied by elevated hs-CRP levels, but not by augmented IL-18 and sCD40L concentrations, suggesting activation of different inflammatory pathways in the progression of renal and cardiovascular atherosclerotic disease. The pathophysiologic mechanisms of these associations remain to be further elucidated in future studies.     

The response of NG2-expressing oligodendrocyte progenitors to demyelination in MOG-EAE and MS

Remyelination of primary demyelinated lesions is a common feature of experimental models of multiple sclerosis (MS) and is also suggested to be the normal response to demyelination during the early stages of MS itself. Many lines of evidence have shown that remyelination is preceded by the division of endogenous oligodendrocyte precursor cells (OPCs) in the lesion and its borders. It is suggested that this rapid response of OPCs to repopulate the lesion site and their subsequent differentiation into new oligodendrocytes is the key to the rapid remyelination. Antibodies to the NG2 chondroitin sulphate proteoglycan have proved exceedingly useful in following and quantitating the response of endogenous OPCs to demyelination. Here we review the literature on the response of NG2-expressing OPCs to demyelination and provide some new evidence on their response to the chronic inflammatory demyelinating environment seen in recombinant myelin oligodendrocyte glycoprotein (MOG) induced experimental allergic encephalomyelitis (EAE) in the DA rat. NG2-expressing OPCs responded to the inflammatory demyelination in this model by becoming reactive and increasing in number in a very focal manner. Evidence of NG2⁺OPCs in lesioned areas beginning to express the oligodendrocyte marker CNP was also seen. The response of OPCs appeared to occur following successive relapses but did not always lead to remyelination, with areas of chronic demyelination observed in the spinal cord. The presence of OPCs in the adult human CNS is clearly of vital importance for repair in multiple sclerosis (MS). As in rat tissue, the antibody labels an evenly distributed cell population present in both white and grey matter, distinct from HLA-DR⁺microglia. NG2⁺cells are sparsely distributed in the centre of chronic MS lesions. These cells apparently survive demyelination and exhibit a multi-processed or bipolar morphology in the very hypocellular environment of the lesion.     

T2 mapping with magnetization‐prepared 3D TSE based on a modified BIR‐4 T2 preparation

The purpose of this work was to investigate the performance of the modified BIR‐4 T₂ preparation for T₂ mapping and propose a method to remove T₂ quantification errors in the presence of large B₁ and B₀ offsets. The theoretical investigation of the magnetization evolution during the T₂ preparation in the presence of B₁ and B₀ offsets showed deviations from a mono‐exponential T₂ decay (two parameter fit). A three parameter fit was used to improve T₂ accuracy. Furthermore, a two parameter fit with an additional saturation preparation scan was proposed to improve T₂ accuracy and precision. These three fitting methods were compared based on simulations, phantom measurements and an in vivo healthy volunteer study of the neck musculature using a 3D TSE readout. The results based upon the pure two parameter fit overestimated T₂ in regions with high B₀ offsets (up to 40% in phantoms). The three parameter fit T₂ values were robust to B₀ offsets but with higher standard deviation (up to 40% in simulations). The two parameter fit with the saturation preparation yielded high robustness towards B₀ offsets with a noise performance comparable to that of the two parameter fit. In the volunteer study the T₂ values obtained by the pure two parameter fit showed a dependence on the field inhomogeneities, whereas the T₂ values from the proposed fitting approach were shown to be insensitive to B₀ offsets. The proposed method enabled accurate and precise T₂ mapping in the presence of large B₁ and B₀ offsets.     

Clostridium difficile toxins A and B directly stimulate human mast cells

Clostridium difficile toxins A and B (TcdA and TcdB) are the causative agents of antibiotic-associated pseudomembranous colitis. Mucosal mast cells play a crucial role in the inflammatory processes underlying this disease. We studied the direct effects of TcdA and TcdB on the human mast cell line HMC-1 with respect to degranulation, cytokine release, and the activation of proinflammatory signal pathways. TcdA and TcdB inactivate Rho GTPases, the master regulators of the actin cytoskeleton. The inactivation of Rho GTPases induced a reorganization of the actin cytoskeleton accompanied by morphological changes of cells. The TcdB-induced reorganization of the actin cytoskeleton in HMC-1 cells reduced the number of electron-dense mast cell-specific granules. Accordingly, TcdB induced the release of hexosaminidase, a marker for degranulation, in HMC-1 cells. The actin rearrangement was found to be responsible for degranulation since latrunculin B induced a comparable hexosaminidase release. In addition, TcdB as well as latrunculin B induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 and also resulted in a p38 MAPK-dependent increased formation of prostaglandins D(2) and E(2). The autocrine stimulation of HMC-1 cells by prostaglandins partially contributed to the degranulation. Interestingly, TcdB-treated HMC-1 cells, but not latrunculin B-treated HMC-1 cells, showed a strong p38 MAPK-dependent increase in interleukin-8 release. Differences in the mast cell responses to TcdB and latrunculin B are probably due to the presence of functionally inactive Rho GTPases in toxin-treated cells. Thus, the HMC-1 cell line is a promising model for studying the direct effects of C. difficile toxins on mast cells independently of the tissue context.     

Immunohistochemical and molecular analysis of PI3K/AKT/mTOR pathway in esophageal carcinoma

Among the numerous signaling pathways involved in tumorigenesis, PI3K‐AKT‐mTOR is a key one that regulates diverse cellular functions. However, its prognostic value in esophageal carcinoma remains unclear. In our study, we examined the immunohistochemical expression of phosphorylated (p‐) AKT, mTOR, p70S6K and 4E‐BP1 along with the mutational status of PIK3CA and AKT1 genes by High Resolution Melting Analysis and Pyrosequencing in 44 esophageal carcinomas. The results were correlated with the clinicopathological characteristics of the patients in an effort to define their possible prognostic significance. Total p‐mTOR cytoplasmic expression, assessed in 10 random areas, was positively correlated with tumor stage (Kruskal–Wallis ANOVA, I/II vs III/IV, p = 0.0500). Μoreover, maximum p‐mTOR cytoplasmic immunoexpression, estimated in hot spot areas, was positively associated with tumor grade (Mann–Whitney U test, I/II vs III, p = 0.0565). Interestingly, p‐4E‐BP1 immunoreactivity was negatively correlated with tumor histological grade (Mann–Whitney U test, I/II vs III, p = 0.0427). No mutation was observed in exons 9 and 20 of PIK3CA gene and in exon 4 of AKT1 gene. In conclusion, our findings depict the presence of activated PI3K/AKT/mTOR pathway in esophageal cancer bringing forward p‐mTOR and p‐4E‐BP1 for their potential role in esophageal carcinogenesis. Additional studies are warranted to validate our findings.