Gholamreza Fazeli

Angiotensin II induces DNA damage via AT1 receptor and NADPH oxidase isoform Nox4

Epidemiological studies revealed increased renal cancer incidences and higher cancer mortalities in hypertensive individuals. Activation of the renin-angiotensin-aldosterone system leads to the formation of reactive oxygen species (ROS). In vitro, in renal cells, and ex vivo, in the isolated perfused mouse kidney, we could show DNA-damaging potential of angiotensin II (Ang II). Here, the pathway involved in the genotoxicity of Ang II was investigated. In kidney cell lines with properties of proximal tubulus cells, an activation of NADPH oxidase and the production of ROS, resulting in the formation of DNA strand breaks and micronuclei induction, was observed. This DNA damage was mediated by the Ang II type 1 receptor (AT1R), together with the G protein G ( α-q/11 ) . Subsequently, phospholipase C (PLC) was activated and intracellular calcium increased. Both calcium stores of the endoplasmic reticulum and extracellular calcium were involved in the genotoxicity of Ang II. Downstream, a role for protein kinase C (PKC) could be detected, because its inhibition hindered Ang II from damaging the cells. Although PKC was activated, no involvement of its known target, the NADPH oxidase isoform containing the Nox2 subunit, could be found, as tested by small-interfering RNA down-regulation. Responsible for the DNA-damaging activity of Ang II was the NADPH oxidase isoform containing the Nox4 subunit. In summary, in kidney cells the DNA-damaging activity of Ang II depends on an AT1R-mediated activation of NADPH oxidase via PLC, PKC and calcium signalling, with the NADPH subunit Nox4 playing a crucial role.

Superoxide anion and hydrogen peroxide-induced signaling and damage in angiotensin II and aldosterone action

Abstract The formation of reactive oxygen species (ROS) can be induced by xenobiotic substances, such as redox cycling molecules, but also by endogenous substances such as hormones and cytokines. Recent research shows the importance of ROS in cellular signaling. Here, the signaling pathways of the two blood pressure-regulating hormones angiotensin II and aldosterone are presented, focusing on both their physiological effects and the change of signaling owing to the action of increased concentrations or prolonged exposure. When present in high concentrations, both angiotensin II and aldosterone, as various other endogenous substances, activate NADPH oxidase, which produces superoxide. In this review the generation of superoxide anions and hydrogen peroxide in cells stimulated with angiotensin II or aldosterone, as well as the subsequently induced signaling processes and DNA damage is discussed.

The Role of the Dopamine Transporter in Dopamine-Induced DNA Damage

The neurotransmitter dopamine causes DNA damage, oxidative stress and is involved in the pathology of neurological diseases. To elucidate this potential link we investigated the mechanism of dopamine‐induced DNA damage. We studied the role of the dopamine transporter (DAT) in MDCK and MDCK‐DAT cells, containing the human DAT gene. After treatment with dopamine, only MDCK‐DAT cells showed elevated chromosomal damage and dopamine uptake. Although stimulation of dopamine type 2 receptor (D2R) with quinpirole in the absence of dopamine did not induce genotoxicity in rat neuronal PC12 cells, interference with D2R signaling by inhibition of G‐proteins, phosphoinositide 3 kinase and extracellular signal‐regulated kinases reduced dopamine‐induced genotoxicity and affected the ability of DAT to take up dopamine. Furthermore, the D2R antagonist sulpiride inhibited the dopamine‐induced migration of DAT from cytosol to cell membrane. To determine whether oxidation of dopamine by monoamine oxidase (MAO) is relevant in its genotoxicity, we inhibited MAO, which reduced the formation of micronuclei and of the oxidative DNA adduct 8‐oxodG. Overall, dopamine exerted its genotoxicity in vitro upon transport into the cells and oxidation by MAO. D2R signaling was involved in the genotoxicity of dopamine by affecting activation and cell surface expression of DAT and hence modulating dopamine uptake.

Neuronal Activation in the Central Nervous System of Rats in the Initial Stage of Chronic Kidney Disease-Modulatory Effects of Losartan and Moxonidine

The effect of mild chronic renal failure (CRF) induced by 4/6-nephrectomy (4/6NX) on central neuronal activations was investigated by c-Fos immunohistochemistry staining and compared to sham-operated rats. In the 4/6 NX rats also the effect of the angiotensin receptor blocker, losartan, and the central sympatholyticum moxonidine was studied for two months. In serial brain sections Fos-immunoreactive neurons were localized and classified semiquantitatively. In 37 brain areas/nuclei several neurons with different functional properties were strongly affected in 4/6NX. It elicited a moderate to high Fos-activity in areas responsible for the monoaminergic innervation of the cerebral cortex, the limbic system, the thalamus and hypothalamus (e.g. noradrenergic neurons of the locus coeruleus, serotonergic neurons in dorsal raphe, histaminergic neurons in the tuberomamillary nucleus). Other monoaminergic cell groups (A5 noradrenaline, C1 adrenaline, medullary raphe serotonin neurons) and neurons in the hypothalamic paraventricular nucleus (innervating the sympathetic preganglionic neurons and affecting the peripheral sympathetic outflow) did not show Fos-activity. Stress- and pain-sensitive cortical/subcortical areas, neurons in the limbic system, the hypothalamus and the circumventricular organs were also affected by 4/6NX. Administration of losartan and more strongly moxonidine modulated most effects and particularly inhibited Fos-activity in locus coeruleus neurons. In conclusion, 4/6NX elicits high activity in central sympathetic, stress- and pain-related brain areas as well as in the limbic system, which can be ameliorated by losartan and particularly by moxonidine. These changes indicate a high sensitivity of CNS in initial stages of CKD which could be causative in clinical disturbances.

Genotoxicity of the neurotransmitter dopamine in vitro.

Alterations in dopamine levels play a role in several human pathological conditions and their pharmacological treatment. Here we describe an induction of genomic damage detected as micronucleus formation by concentrations in the low micromolar range (6.25-25 microM) in three cell lines in vitro. Rat neuronal PC12 cells exhibited a more pronounced induction (about 10-fold over control at 100 microM) than human lymphoblastoid TK6 cells and rat kidney NRK cells (about 2-fold over control at 100 microM). The role of transporters and receptors in the formation of genomic damage was investigated in PC12 cells, in which the effect of dopamine was reduced by addition of the antioxidants TEMPOL and dimethylthiourea, by inhibitors of the dopamine transporter (GBR 12909 and nomifensine) and by a D2 antagonist (sulpiride). Antioxidative effects of nomifensine and sulpiride, but not of GBR 12909, were excluded, since they did not protect oxidative stress sensitive HL-60 cells from hydrogen peroxide-induced damage in the comet assay. Thus, the transport of dopamine into the cell and the signalling upon binding to D2 receptors was required for the genotoxic effect of dopamine in PC12 cells, which was mediated by intracellular dopamine oxidation products and/or reactive oxygen species.

C. elegans midbodies are released, phagocytosed and undergo LC3-dependent degradation independent of macroautophagy

ABSTRACT In animals, the midbody coordinates the end of cytokinesis when daughter cells separate through abscission. The midbody was thought to be sequestered by macroautophagy, but recent evidence suggests that midbodies are primarily released and phagocytosed. It was unknown, however, whether autophagy proteins play a role in midbody phagosome degradation. Using a protein degradation assay, we show that midbodies are released in Caenorhabditis elegans. Released midbodies are known to be internalized by actin-driven phagocytosis, which we show requires the RAB-5 GTPase to localize the class III phosphoinositide 3-kinase (PI3K) complex at the cortex. Autophagy-associated proteins, including the Beclin 1 homolog BEC-1 and the Atg8/LC3-family members LGG-1 and LGG-2, localize around the midbody phagosome and are required for midbody degradation. In contrast, proteins required specifically for macroautophagy, such as UNC-51 and EPG-8 (homologous to ULK1/Atg1 and Atg14, respectively) are not required for midbody degradation. These data suggest that the C. elegans midbody is degraded by LC3-associated phagocytosis (LAP), not macroautophagy. Our findings reconcile the two prevailing models on the role of phagocytic and autophagy proteins, establishing a new non-canonical role for autophagy proteins in midbody degradation. Summary: Autophagy proteins are required for the degradation of midbodies. In C. elegans, Atg8/LC3-family proteins act during phagosome maturation rather than during macroautophagy.

25-hydroxyvitamin d and advanced glycation endproducts in healthy and hypertensive subjects: are there interactions?

Advanced glycation endproducts (AGEs) accumulate during aging. Skin is the single organ of vitamin D synthesis, induced by ultraviolet B light. Accumulation of AGEs in the skin could interfere with synthesis of the vitamin, whereas the microinflammation and oxidative stress (associated with hypovitaminosis D) could amplify both the toxic effects of AGEs and their production. Clinical data on potential interactions between vitamin D3 deficiency and AGE accumulation are sparse. Here we investigated potential associations between levels of circulating vitamin D3 and those of AGEs in blood and skin with regard to markers of inflammation and oxidative stress in nondiabetic subjects. In a cross-sectional study, 146 subjects (119 healthy persons and 27 hypertensive patients; 73 male and 73 female; mean age, 57.0 ± 15.5 years) were included. Skin autofluorescence (SAF) and plasma levels of vitamin D3, AGE-associated fluorescence, high-sensitivity C-reactive protein level, and advanced oxidation protein products as well as renal function (estimated glomerular filtration rate) were determined. In a subgroup of 61 patients, N(ε)-carboxymethyllysine, soluble receptor of AGEs, and soluble vascular adhesion protein-1 were additionally analyzed. Vitamin D3 level averaged 22.5 ± 8.9 ng/mL. Prevalence of vitamin D insufficiency (20-29 ng/mL) was 43%, and that of deficiency (<20 ng/mL) 37%. The age-dependent rise in SAF was steeper in smokers and in subjects presenting arterial hypertension. No association between SAF and hypovitaminosis D was revealed. Among smokers, an inverse relationship manifested between vitamin D3 and plasma AGE-associated fluorescence as well as soluble vascular adhesion protein-1. Our data suggest that in nondiabetic adults, hypovitaminosis D does not enhance toxicity and accumulation of AGEs. Only in smokers interactions are conceivable.

Safely removing cell debris with LC3-associated phagocytosis: LAP in cell debris removal

Phagocytosis and autophagy are two distinct pathways that degrade external and internal unwanted particles. Both pathways lead to lysosomal degradation inside the cell, and over the last decade, the line between them has blurred; autophagy proteins were discovered on phagosomes engulfing foreign bacteria, leading to the proposal of LC3‐associated phagocytosis (LAP). Many proteins involved in macroautophagy are used for phagosome degradation, although Atg8/LC3 family proteins only decorate the outer membrane of LC3‐associated phagosomes, in contrast to both autophagosome membranes. A few proteins distinguish LAP from autophagy, such as components of the autophagy pre‐initiation complex. However, most LAP cargo is wrapped in multiple layers of membranes, making them similar in structure to autophagosomes. Recent evidence suggests that LC3 is important for the degradation of internal membranes, explaining why LC3 would be a vital part of both macroautophagy and LAP. In addition to removing invading pathogens, multicellular organisms also use LAP to degrade cell debris, including cell corpses and photoreceptor outer segments. The post‐mitotic midbody remnant is another cell fragment, which results from each cell division, that was recently added to the growing list of LAP cargoes. Thus, LAP plays an important role during the normal physiology and homoeostasis of animals.

Extracellular vesicle budding is inhibited by redundant regulators of TAT-5 flippase localization and phospholipid asymmetry

Significance Cells must interact with their environment to survive. The lipids and proteins of the plasma membrane send and receive signals at the cell surface to respond to stimuli. When the lipid bilayer of the plasma membrane is damaged, cells release membrane-bound extracellular vesicles to repair the membrane. Cells also release signals on extracellular vesicles to communicate at a distance. Here, we identify proteins that regulate the formation of extracellular vesicles from the plasma membrane, providing additional tools to control their release that can be used to test potential functions of extracellular vesicles. Furthermore, we reveal that proteins regulating the asymmetric localization of the lipid phosphatidylethanolamine are critical for extracellular vesicle release, implicating this abundant but understudied lipid. Cells release extracellular vesicles (EVs) that mediate intercellular communication and repair damaged membranes. Despite the pleiotropic functions of EVs in vitro, their in vivo function is debated, largely because it is unclear how to induce or inhibit their formation. In particular, the mechanisms of EV release by plasma membrane budding or ectocytosis are poorly understood. We previously showed that TAT-5 phospholipid flippase activity maintains the asymmetric localization of the lipid phosphatidylethanolamine (PE) in the plasma membrane and inhibits EV budding by ectocytosis in Caenorhabditis elegans. However, no proteins that inhibit ectocytosis upstream of TAT-5 were known. Here, we identify TAT-5 regulators associated with retrograde endosomal recycling: PI3Kinase VPS-34, Beclin1 homolog BEC-1, DnaJ protein RME-8, and the uncharacterized Dopey homolog PAD-1. PI3Kinase, RME-8, and semiredundant sorting nexins are required for the plasma membrane localization of TAT-5, which is important to maintain PE asymmetry and inhibit EV release. PAD-1 does not directly regulate TAT-5 localization, but is required for the lipid flipping activity of TAT-5. PAD-1 also has roles in endosomal trafficking with the GEF-like protein MON-2, which regulates PE asymmetry and EV release redundantly with sorting nexins independent of the core retromer. Thus, in addition to uncovering redundant intracellular trafficking pathways, our study identifies additional proteins that regulate EV release. This work pinpoints TAT-5 and PE as key regulators of plasma membrane budding, further supporting the model that PE externalization drives ectocytosis.

Rab GTPases mature the LC3-associated midbody phagosome

ABSTRACT Post-mitotic midbody remnants have recently been added to the list of structures degraded via LC3-associated phagocytosis (LAP). LAP involves proteins of the autophagy pathway to degrade phagosomal contents, mingling 2 pathways that were thought to be distinct. To better characterize how similar LAP is to classical phagocytosis, we asked whether the midbody LAPosome (LC3-associated phagosome) matures using Rab GTPases in C. elegans embryos. We found that RAB-5 and RAB-7 appear transiently on midbody LAPosomes and that RAB-7 is required for midbody degradation, suggesting that RAB-5 and RAB-7 direct LAPosome maturation similar to classical phagosomes. Further, we observed that the Rab2 homolog UNC-108 and the major proton pump, the V-type ATPase, are required for acidification of the midbody LAPosome, demonstrating that phagosomes and LAPosomes acidify via a common pathway. Together, these data reveal that Rab GTPases play similar roles during LAPosome maturation and phagosome maturation.