Translating Evidence from Clonal Hematopoiesis to Cardiovascular Disease: A Systematic Review

Some random mutations can confer a selective advantage to a hematopoietic stem cell. As a result, mutated hematopoietic stem cells can give rise to a significant proportion of mutated clones of blood cells. This event is known as “clonal hematopoiesis.” Clonal hematopoiesis is closely associated with age, and carriers show an increased risk of developing blood cancers. Clonal hematopoiesis of indeterminate potential is defined by the presence of clones carrying a mutation associated with a blood neoplasm without obvious hematological malignancies. Unexpectedly, in recent years, it has emerged that clonal hematopoiesis of indeterminate potential carriers also have an increased risk of developing cardiovascular disease. Mechanisms linking clonal hematopoiesis of indeterminate potential to cardiovascular disease are only partially known. Findings in animal models indicate that clonal hematopoiesis of indeterminate potential-related mutations amplify inflammatory responses. Consistently, clinical studies have revealed that clonal hematopoiesis of indeterminate potential carriers display increased levels of inflammatory markers. In this review, we describe progress in our understanding of clonal hematopoiesis in the context of cancer, and we discuss the most recent findings linking clonal hematopoiesis of indeterminate potential and cardiovascular diseases

Fermentation of Vaccinium floribundum Berries with Lactiplantibacillus plantarum Reduces Oxidative Stress in Endothelial Cells and Modulates Macrophages Function

Accumulating evidence suggests that high consumption of natural antioxidants promotes health by reducing oxidative stress and, thus, the risk of developing cardiovascular diseases. Similarly, fermentation of natural compounds with lactic acid bacteria (LAB), such as Lactiplantibacillus plantarum, enhances their beneficial properties as regulators of the immune, digestive, and cardiovascular system. We investigated the effects of fermentation with Lactiplantibacillus plantarum on the antioxidant and immunomodulatory effects of Pushgay berries (Vaccinium floribundum, Ericaceae family) in human umbilical vein endothelial cells (HUVECs) and macrophage cell line RAW264.7. Polyphenol content was assayed by Folin-Ciocalteu and HPLC-MS/MS analysis. The effects of berries solutions on cell viability or proliferation were assessed by WST8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt and Lactate dehydrogenase (LDH) release, Trypan blue exclusion test, and Alamar blue assay. Antioxidant activity was evaluated by a cell-based chemiluminescent probe for the detection of intracellular H2O2 production in HUVECs. Heme oxygenase-1 (HO-1) expression levels were investigated by RT-qPCR. Glutathione reductase (GR), glutathione peroxidase (Gpx), superoxide dismutase (SOD), and catalase (CAT) activities, as markers of intracellular antioxidant defense, were evaluated by spectrophotometric analysis. The immunomodulatory activity was examined in RAW 264.7 by quantification of inducible nitric oxide synthase (iNOS) and Tumor Necrosis Factor-alpha (TNF alpha) by RT-qPCR. Data showed that fermentation of Pushgay berries (i) enhances the content of quercetin aglycone, and (ii) increases their intracellular antioxidant activity, as indicated by the reduction in H2O2-induced cell death and the decrease in H2O2-induced HO-1 gene expression in HUVECs treated for 24 h with fermented berries solution (10 mu g/mL). Moreover, treatment with Pushgay berries for 72 h (10 mu g/mL) promotes cells growth in RAW 264.7, and only fermented Pushgay berries increase the expression of iNOS in the same cell line. Taken together, our results show that LAB fermentation of Pushgay berries enhances their antioxidant and immunomodulatory properties

Substitution of histidine 30 by asparagine in manganese superoxide dismutase alters biophysical properties and supports proliferation in a K562 leukemia cell line.

We have generated a mutant of C. elegans manganese superoxide dismutase at histidine 30 by site-directed mutagenesis. The structure was solved at a resolution of 1.52 Å by X-ray crystallography (pdb: 6S0D). His30 was targeted, as it forms as a gateway residue at the top of the solvent access funnel to the active site, together with Tyr34. In the wild-type protein, these gateway residues are involved in the hydrogen-bonding network providing the protons necessary for the catalytic reaction at the metal center. However, biophysical characterization and cell viability experiments reveal that a mutation from histidine to asparagine in the H30N mutant modifies metal selectivity in the protein, favoring the uptake of iron over manganese in minimal media conditions, alters active-site coordination from the characteristic trigonal bipyramidal to octahedral geometry, and encourages cellular proliferation in K562 cells, when added exogenously to the cells

COVID-19 in the heart and the lungs: could we “Notch” the inflammatory storm?

From January 2020, coronavirus disease (COVID-19) originated in China has spread around the world. The disease is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The presence of myocarditis, cardiac arrest, and acute heart failure in COVID-19 patients suggests the existence of a relationship between SARS-CoV-2 infection and cardiac disease. The Notch signalling is a major regulator of cardiovascular function and it is also implicated in several biological processes mediating viral infections. In this report we discuss the possibility to target Notch signalling to prevent SARS-CoV-2 infection and interfere with the progression of COVID-19- associated heart and lungs disease

Estrogen-mediated protection against coronary heart disease: The role of the Notch pathway

Estrogen regulates a plethora of biological processes, under physiological and pathological conditions, by affecting key pathways involved in the regulation of cell proliferation, fate, survival and metabolism. The Notch receptors are mediators of communication between adjacent cells and are key determinants of cell fate during development and in postnatal life. Crosstalk between estrogen and the Notch pathway intervenes in many processes underlying the development and maintenance of the cardiovascular system. The identification of molecular mechanisms underlying the interaction between these types of endocrine and juxtacrine signaling are leading to a deeper understanding of physiological conditions regulated by these steroid hormones and, potentially, to novel therapeutic approaches to prevent pathologies linked to reduced levels of estrogen, such as coronary heart disease, and cardiotoxicity caused by hormone therapy for estrogen-receptor-positive breast cancer

AQP8 facilitates Nox-produced hydrogen peroxide transport in leukaemia cells

It is known that an increase in the intracellular level of reactive oxygen species (ROS), in particolar hydrogen peroxide (H2O2) is able to affect signalling pathways regulating proliferation and cancer development. H2O2 has been long thought to freely permeate across biological membranes, but recently, new evidence demonstrated that membrane H2O2-permeability is limited and that specific acquaporin (AQP) isoforms are able to funnel H2O2 across membranes. Data here reported show that AQP inhibition cause a decrease in intracellular H2O2 accumulation in leukaemia cells. Furthermore, AQP8 overexpression or silencing by means of siRNA resulted in the modulation of VRGF ability of triggering H2O2 intracellular level increase or decrease, respectively. Finally, we show that AQP8-facilitated H2O2 transport is able to increase cell proliferation through a mechanism dependent on PI3K and p38MAPK. In summary, our findings indicate that AQP8 is able to modulate H2O2 transport through the plasma membrane affecting redox signalling linked to leukaemia cell proliferation. Therefore, the development of new drugs targeting specific AQP isoforms might be and interesting novel anti-cancer strategy

AQP8 modulates hydrogen peroxide signalling in leukaemia cells

Aquaporins (AQP) are channels that transport water and other small solutes across plasma membranes. In human, 13 different AQP isoforms have been discovered and three of them (AQP1, AQP3 and AQP8) have recently been reported to channel hydrogen peroxide (H2O2). Interestingly, the three AQP isoforms that possess the capacity of channel H2O2 are also widely expressed in blood cells. It is known that H2O2 can act as a messenger in cell signalling pathways and that an increase in intracellular ROS level supports pro-survival pathways contributing to cancer development. We previously demonstrated that H2O2 produced by NAD(P)H oxidases (Nox) promotes cellular proliferation in leukaemia cells. The aim of this work was to assess whether specific AQP isoforms can channel Nox-produced H2O2 across the plasma membrane of leukaemia cells affecting downstream pathways linked to cell proliferation. In the present study we report that specific AQP isoforms (AQP8 and AQP3, in a lesser extent) constitute a possible way through which Nox-derived H2O2 can enter leukaemia cells. Moreover, we demonstrate that AQP8 overexpression is able to enhance intracellular H2O2 accumulation induced by VEGF. Furthermore, we show that AQP8 is capable of amplifying phosphorylation triggered by H2O2 of both PI3K and p38 MAPK. In summary, results here reported indicate that AQP8 is able to modulate H2O2 transport through plasma membrane contributing to the aberrant proliferation of leukaemia cells

Nox4 inhibitory activity of new synthesized oxindole derivatives

Recently, the Nox4 inhibitory activity shown by some oxindole derivatives has been described. in This study, a panel of new synthesized derivatives against different leukemia cell lines in the NCI assays were tested for the ability to inhibit Nox4 in B1647 cells, a human acute myeloid leukemia cell line. Nox4 is known to be costitutively active and to play the major role in the generation of ROS requird for sustaining cellular growth and proliferation of B1647 cells. MTT assay confirmes the anti-proliferative activity of alla the tested molecules in a dose dependent manner at a concentration of 5 and 10 microM. ROS production was measured in B1647 cells after an acute treatment (30 min) with the selected compounds. Results indicate that two compounds (C5 and C7) are able to inhibit ROS generation in a dose-dependent manner. As B1647 cells express Nox2 and Nox4, in order to rule out the role of Nox2-derived ROS, the acute inhibition of ROS production was measured following RNA silencing of Nox2. Data show that the two selected compounds were able to inhibit ROS production at the same extent either in Nox2 silenced cells or in the control. Finally, Wester blotting analysis was performed to investigate Nox4 expression inB1647 cells after 24 h-incubation with the compounds. Data show that treatments with C5 and C7 were able to downregulate Nox4. In conclusion, results here reported indicate that Nox4 could be a target for two of the new synthesized oxindole derivatives and the Nox4 inhibition seems to contribute to the anti-proliferative effect of the compounds in acute leukemia B1647 cells and to be a promising strategy for a novel antileukemic therapy

Cholesterol depletion modifies plasma membrane lipid raft integrity and affects glucose uptake in a human leukemic cell line

Cholesterol depletion modifies plasma membrane lipid raft integrity and affects glucose uptake in a human leukemic cell line C. Caliceti, L. Zambonin, C. Prata, F. Vieceli Dalla Sega, G. Hakim and D. Fiorentini Dipartimento di Biochimica “G. Moruzzi”, Università di Bologna - Italy In hemopoietic M07e cells, stem cell factor (SCF) activates glucose transport through GLUT1 translocation from intracellular stores. Recently, it has been reported that in many cell types GLUT1 is localized in part in detergent-resistant membrane (DRM) domains and a role for lipid rafts in GLUT1 stimulation has been suggested. We investigated whether a plasma membrane cholesterol depletion might play a role in GLUT1 activity. Cell exposure to 10 mM methyl--cyclodextrin (MBCD) for 20 min resulted in a loss of cholesterol from plasma membrane, without affecting viability. MBCD-treated cells exhibited a rise in glucose uptake, higher than that observed upon SCF treatment. The combined cell treatment with SCF and MBCD determined a further increase in glucose uptake, showing an additive effect. Membrane fractionation by flotation on sucrose density gradient showed that in untreated cells most of GLUT1 is distributed in the high-density region, but a little amount is colocalized with lipid raft marker proteins in low-density regions, corresponding to DRM. Upon MBCD treatment, GLUT1 resulted totally confined to the high-density region. DRM exhibited also the highest cholesterol content. The cholesterol distribution profile of the MBCD-treated samples shows that the cholesterol preferentially decreased in DRM, confirming its selective depletion. Cell membrane isolation by biotinylation and immunofluorescence staining indicated that MBCD treatment, like SCF stimulation, greatly enhanced the amount of GLUT1 at cell surface, promoting a translocation from intracellular stores. MBCD increases Akt-pS473, while SCF positively regulates PLCγ signaling cascade, both involved in GLUT translocation and in glucose uptake control. Therefore, these two stimuli could proceed through distinct signalling pathways. In summary, a mild lipid environment alteration in lipid rafts enhances GLUT1 activity in M07e cells via a cholesterol-dependent mechanism, possibly involving Akt