Focus: The Science of Stress: Oxidative Stress and Inflammation in Cardiovascular Diseases and Cancer: Role of Non-coding RNAs

High oxidative stress, Th1/Th17 immune response, M1 macrophage inflammation, and cell death are associated with cardiovascular diseases. Controlled oxidative stress, Th2/Treg anti-tumor immune response, M2 macrophage inflammation, and survival are associated with cancer. MiR-21 protects against cardiovascular diseases but may induce tumor growth by retaining the anti-inflammatory M2 macrophage and Treg phenotypes and inhibiting apoptosis. Down-regulation of let-7, miR-1, miR-9, miR-16, miR-20a, miR-22a, miR-23a, miR-24a, miR-26a, miR-29, miR-30a, miR-34a, miR-124, miR-128, miR-130a, miR-133, miR-140, miR-143-145, miR-150, miR-153, miR-181a, miR-378, and miR-383 may aid cancer cells to escape from stresses. Upregulation of miR-146 and miR-223 may reduce anti-tumor immune response together with miR-21 that also protects against apoptosis. MiR-155 and silencing of let-7e, miR-125, and miR-126 increase anti-tumor immune response. MiR expression depends on oxidative stress, cytokines, MYC, and TGF-β, and expression of silencing lncRNAs and circ-RNAs. However, one lncRNA or circ-RNA may have opposite effects by targeting several miRs. For example, PVT1 induces apoptosis by targeting miR-16a and miR-30a but inhibits apoptosis by silencing miR-17. In addition, levels of a non-coding RNA in a cell type depend not only on expression in that cell type but also on an exchange of microvesicles between cell types and tumors. Although we got more insight into the function of a growing number of individual non-coding RNAs, overall, we do not know enough how several of them interact in functional networks and how their expression changes at different stages of disease progression.     

Partial monosomy 11q and trisomy 12q: variable expression in two siblings

Clinical and cytogenetical findings are reported and discussed on two siblings with discordant phenotypes despite having both a terminal 11q deletion and a distal 12q duplication resulting from an unbalanced segregation of a balanced translocation t(11:12)(q23:q24.1) mat. The oldest child, a girl, is the index patient. Her clinical features include intrauterine and postnatal growth retardation, fetal distress, mild hypotonia, early feeding difficulties, moderate developmental delay, especially in language acquisition, a velopharyngeal insufficiency with repeated otorhinopharyngeal infections, facial dysmorphism, heart ventricular septal defect, and abnormal hyperactive behaviour with sometimes autistic tendencies. The facial dysmorphic features notably consist of microcephaly, hypertelorism, large palpebral fissures, large eyes with alternant divergent strabismus, long eyelashes, a long and broad nasal bridge, a short "crested" nose with salient tip, a fishmouth with large spaces between teeth and flat palate, retrognathism, large ears and multiple dimples. The second affected child is a boy showing low birthweight, moderate developmental retardation with mainly no active language at 32 months, behaviour abnormalities with an autistic tendency, and no major physical anomalies apart from a slight facial hypotonia with often open mouth, dimples on the shoulders and right cryptorchidism. The authors stress the variable clinical expression of the chromosomal imbalance in this family resulting in low birthweight, developmental delay, abnormal behaviour, but different degrees of physical features and dysmorphism. The possible contribution of each of the two aneusomies to the phenotype is discussed.     

Atypical presentation of the Prader-Willi syndrome. Mosaic trisomy 15?

We report a female with Prader-Willi syndrome and hemihypertrophy. We discuss the possibility of an undetected mosaicism for trisomy 15 explaining this unusual feature.     

Alpha-helix formation is required for high affinity binding of human apolipoprotein A-I to lipids

Apolipoprotein (apo) A-I is thought to undergo a conformational change during lipid association that results in the transition of random coil to alpha-helix. Using a series of deletion mutants lacking different regions along the molecule, we examined the contribution of alpha-helix formation in apoA-I to the binding to egg phosphatidylcholine (PC) small unilamellar vesicles (SUV). Binding isotherms determined by gel filtration showed that apoA-I binds to SUV with high affinity and deletions in the C-terminal region markedly decrease the affinity. Circular dichroism measurements demonstrated that binding to SUV led to an increase in alpha-helix content, but the helix content was somewhat less than in reconstituted discoidal PC.apoA-I complexes for all apoA-I variants, suggesting that the helical structure of apoA-I on SUV is different from that in discs. Isothermal titration calorimetry showed that the binding of apoA-I to SUV is accompanied by a large exothermic heat and deletions in the C-terminal regions greatly decrease the heat. Analysis of the rate of release of heat on binding, as well as the kinetics of quenching of tryptophan fluorescence by brominated PC, indicated that the opening of the N-terminal helix bundle is a rate-limiting step in apoA-I binding to the SUV surface. Significantly, the correlation of thermodynamic parameters of binding with the increase in the number of helical residues revealed that the contribution of alpha-helix formation upon lipid binding to the enthalpy and the free energy of the binding of apoA-I is -1.1 and -0.04 kcal/mol per residue, respectively. These results indicate that alpha-helix formation, especially in the C-terminal regions, provides the energetic source for high affinity binding of apoA-I to lipids.     

CD36 inhibitors reduce postprandial hypertriglyceridemia and protect against diabetic dyslipidemia and atherosclerosis

CD36 is recognized as a lipid and fatty acid receptor and plays an important role in the metabolic syndrome and associated cardiac events. The pleiotropic activity and the multiple molecular associations of this scavenger receptor with membrane associated molecules in different cells and tissues have however questioned its potential as a therapeutic target. The present study shows that it is possible to identify low molecular weight chemicals that can block the CD36 binding and uptake functions. These inhibitors were able to reduce arterial lipid deposition, fatty acid intestinal transit, plasma concentration of triglycerides and glucose, to improve insulin sensitivity, glucose tolerance and to reduce the plasma concentration of HbAc1 in different and independent rodent models. Correlation between the anti-CD36 activity of these inhibitors and the known pathophysiological activity of this scavenger receptor in the development of atherosclerosis and diabetes were observed at pharmacological doses. Thus, CD36 might represent an attractive therapeutic target.     

Hematopoietic Stem/Progenitor Cell Proliferation and Differentiation Is Differentially Regulated by High-Density and Low-Density Lipoproteins in Mice

Rationale

Hematopoietic stem/progenitor cells (HSPC) are responsible for maintaining the blood system as a result of their self-renewal and multilineage differentiation capacity. Recently, studies have suggested that HDL cholesterol may inhibit and impaired cholesterol efflux may increase HSPC proliferation and differentiation.

Objectives

We hypothesized that LDL may enhance HSPC proliferation and differentiation while HDL might have the opposing effect which might influence the size of the pool of inflammatory cells.

Methods and Results

HSPC number and function were studied in hypercholesterolemic LDL receptor knockout (LDLr^−/−) mice on high fat diet. Hypercholesterolemia was associated with increased frequency of HSPC, monocytes and granulocytes in the peripheral blood (PB). In addition, an increased proportion of BM HSPC was in G₂M of the cell cycle, and the percentage of HSPC and granulocyte-macrophage progenitors (GMP) increased in BM of LDLr^−/− mice. When BM Lin-Sca-1+cKit+ (i.e. “LSK”) cells were cultured in the presence of LDL in vitro we also found enhanced differentiation towards monocytes and granulocytes. Furthermore, LDL promoted lineage negative (Lin−) cells motility. The modulation by LDL on HSPC differentiation into granulocytes and motility was inhibited by inhibiting ERK phosphorylation. By contrast, when mice were infused with human apoA-I (the major apolipoprotein of HDL) or reconstituted HDL (rHDL), the frequency and proliferation of HSPC was reduced in BM in vivo. HDL also reversed the LDL-induced monocyte and granulocyte differentiation in vitro.

Conclusion

Our data suggest that LDL and HDL have opposing effects on HSPC proliferation and differentiation. It will be of interest to determine if breakdown of HSPC homeostasis by hypercholesterolemia contributes to inflammation and atherosclerosis progression.     

Effects of enrichment of fibroblasts with unesterified cholesterol on the efflux of cellular lipids to apolipoprotein A-I.

This study elucidates the factors underlying the enhancement in efflux of human fibroblast unesterified cholesterol and phospholipid (PL) by lipid-free apolipoprotein (apo) A-I that is induced by cholesterol enrichment of the cells. Doubling the unesterified cholesterol content of the plasma membrane by incubation for 24 h with low density lipoprotein and lipid/cholesterol dispersions increases the pools of PL and cholesterol available for removal by apoA-I from about 0.8-5%; the initial rates of mass release of cholesterol and PL are both increased about 6-fold. Expression of the ATP binding cassette transporter A1 (ABCA1) is critical for this increased efflux of lipids, and cholesterol loading of the fibroblasts over 24 h increases ABCA1 mRNA about 12-fold. The presence of more ABCA1 and cholesterol in the plasma membrane results in a 2-fold increase in the level of specific binding of apoA-I to the cells with no change in binding affinity. Characterization of the species released from either control or cholesterol-enriched cells indicates that the plasma membrane domains from which lipids are removed are cholesterol-enriched with respect to the average plasma membrane composition. Cholesterol enrichment of fibroblasts also affects PL synthesis, and this leads to enhanced release of phosphatidylcholine (PC) relative to sphingomyelin (SM); the ratios of PC to SM solubilized from control and cholesterol-enriched fibroblasts are approximately 2/1 and 5/1, respectively. Biosynthesis of PC is critical for this preferential release of PC and the enhanced cholesterol efflux because inhibition of PC synthesis by choline depletion reduces cholesterol efflux from cholesterol-enriched cells. Overall, it is clear that enrichment of fibroblasts with unesterified cholesterol enhances efflux of cholesterol and PL to apoA-I because of three effects, 1) increased PC biosynthesis, 2) increased PC transport via ABCA1, and 3) increased cholesterol in the plasma membrane.     

Characterization of a chimeric plasminogen activator consisting of a single-chain Fv fragment derived from a fibrin fragment D-dimer-specific antibody and a truncated single-chain urokinase

An Mr 57,000 single-chain chimeric plasminogen activator, K12G0S32, consisting of a variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 and of a 33-kDa (amino acids Ala132 to Leu411) recombinant single-chain urokinase-type plasminogen activator (rscu-PA-33k) was studied. K12G0S32, secreted by infected Spodoptera frugiperda insect cells at a rate of 1.5 micrograms/10(6) cells/48 h, was purified to homogeneity by ion-exchange chromatography and gel filtration. It was obtained essentially as a single-chain molecule with a Ka = 5.5 x 10(9) M-1 for immobilized fragment D-dimer, similar to that of MA-15C5. The specific activity of both its single-chain and two-chain forms on fibrin plates was 100,000 IU/mg of urokinase-type plasminogen activator (u-PA) equivalent. Activation of plasminogen by two-chain K12G0S32 obeyed Michaelis-Menten kinetics with Km = 2.9 +/- 0.6 microM and a k2 = 3.7 +/- 0.6 s-1 (mean +/- S.D.; n = 3), as compared to Km = 12 microM and k2 = 4.8 s-1 for rtcu-PA-32k (recombinant low Mr two-chain u-PA consisting of amino acids Leu144 to Leu411). Single-chain K12G0S32 induced a dose- and time-dependent lysis of a 125I-fibrin-labeled human plasma clot immersed in citrated human plasma; 50% lysis in 2 h was obtained with 0.70 +/- 0.07 micrograms/ml (mean +/- S.D.; n = 5), as compared with 8.8 +/- 0.1 micrograms/ml for rscu-PA-32k (recombinant low Mr single-chain u-PA consisting of amino acids Leu144 to Leu411) (mean +/- S.D.; n = 3). With two-chain K12G0S32, 50% clot lysis in 2 h required 0.25 +/- 0.03 micrograms/ml (mean +/- S.D.; n = 3), as compared with only 0.62 +/- 0.04 micrograms/ml (mean +/- S.D.; n = 2) for rtcu-PA-32k. These results indicate that low Mr single-chain u-PA can be targeted to a fibrin clot with a single-chain Fv fragment of a fibrin-specific antibody, resulting in a 13-fold increase of the fibrinolytic potency of the single-chain form and a 2.5-fold increase of the potency of the two-chain form.