Indoxyl sulphate-initiated activation of cardiac fibroblasts is modulated by aryl hydrocarbon receptor and nuclear factor-erythroid-2-related factor 2.

In the last decade, extensive attention has been paid to the uremic toxin indoxyl sulphate (IS) as an inducer of cardiac fibroblast (cFib) activation and cardiac fibrosis in chronic kidney disease. At cellular level, IS engages aryl hydrocarbon receptor (AhR) and regulates many biological functions. We analysed how AhR inhibition by CH-223191 (CH) and overexpression of non-functional (dominant negative, DN) nuclear factor-erythroid-2-related factor 2 (NRF2), a transcription factor recruited by AhR, modulate the response of neonatal mouse (nm) cFib to IS. We also evaluated nm-cardiomyocytes after incubation with the conditioned medium (CM) of IS±CH-treated nm-cFib. IS induced activation, collagen synthesis, TLR4 and-downstream-MCP-1, and the genes encoding angiotensinogen, angiotensin-converting enzyme, angiotensin type 1 receptor (AT1r) and neprilysin (Nepr) in nm-cFib. CH antagonized IS-initiated nm-cFib activation, but did not affect or even magnified the other features. IS promoted NRF2 nuclear translocation and expression the NRF2 target Nqo1. Both pre-incubation with CH and transfection of DN-NRF2 resulted in loss of NRF2 nuclear localization. Moreover, DN-NRF2 overexpression led to greater TLR4 and MCP-1 levels following exposure to IS. The CM of IS-primed nm-cFib and to a larger extent the CM of IS+CH-treated nm-cFib upregulated AT1r, Nepr and TNFα and myostatin genes in nm-cardiomyocytes. Hence, IS triggers pro-inflammatory activation of nm-cFib partly via AhR, and AhR-NRF2 counteract it. Strategies other than AhR inhibition are needed to target IS detrimental actions on cardiac cells.

Renal Ischemia/Reperfusion Early Induces Myostatin and PCSK9 Expression in Rat Kidneys and HK-2 Cells.

During visceral interventions, the transient clampage of supraceliac aorta causes ischemia/reperfusion (I/R) in kidneys, sometime resulting in acute renal failure; preclinical studies identified redox imbalance as the main driver of I/R injury. However, in humans, the metabolic/inflammatory responses seem to prevail on oxidative stress. We investigated myostatin (Mstn) and proprotein convertase subtilisin/kexin type 9 (PCSK9), proatherogenic mediators, during renal I/R. Compared to sham-operated animals, the kidneys of rats who had experienced ischemia (30 min) had higher Mstn and PCSK9 expression after 4 h of reperfusion. After 24 h, they displayed tubular necrosis, increased nitrotyrosine positivity, and nuclear peroxisome proliferator-activated receptor gamma coactivator-1alpha relocation, markers of oxidative stress and mitochondria imbalance. Mstn immunopositivity was increased in tubuli, while PCSK9 immunosignal was depleted; systemically, PCSK9 was higher in plasma from I/R rats. In HK-2 cells, both ischemia and reperfusion enhanced reactive oxygen species production and mitochondrial dysfunction. H2O2 upregulated Mstn and PCSK9 mRNA after 1 and 3.5 h, respectively. Accordingly, ischemia early induced Mstn and PCSK9 mRNA; during reperfusion Mstn was augmented and PCSK9 decreased. Mstn treatment early increased PCSK9 expression (within 8 h), to diminish over time; finally, Mstn silencing restrained ischemia-induced PCSK9. Our study demonstrates that renal I/R enhances Mstn and PCSK9 expression and that Mstn induces PCSK9, suggesting them as therapeutic targets for vascular protection during visceral surgery.

Glibenclamide Mimics Metabolic Effects of Metformin in H9c2 Cells.

BACKGROUND: Sulfonylureas, such as glibenclamide, are antidiabetic drugs that stimulate beta-cell insulin secretion by binding to the sulfonylureas receptors (SURs) of adenosine triphosphate-sensitive potassium channels (KATP). Glibenclamide may be also cardiotoxic, this effect being ascribed to interference with the protective function of cardiac KATP channels for which glibenclamide has high affinity. Prompted by recent evidence that glibenclamide impairs energy metabolism of renal cells, we investigated whether this drug also affects the metabolism of cardiac cells. METHODS: The cardiomyoblast cell line H9c2 was treated for 24 h with glibenclamide or metformin, a known inhibitor of the mitochondrial respiratory chain. Cell viability was evaluated by sulforodhamine B assay. ATP and AMP were measured according to the enzyme coupling method and oxygen consumption by using an amperometric electrode, while Fo-F1 ATP synthase activity assay was evaluated by chemiluminescent method. Protein expression was measured by western blot. RESULTS: Glibenclamide deregulated energy balance of H9c2 cardiomyoblasts in a way similar to that of metformin. It inhibited mitochondrial complexes I, II and III with ensuing impairment of oxygen consumption and ATP synthase activity, ATP depletion and increased AMPK phosphorylation. Furthermore, glibenclamide disrupted mitochondrial subcellular organization. The perturbation of mitochondrial energy balance was associated with enhanced anaerobic glycolysis, with increased activity of phosphofructo kinase, pyruvate kinase and lactic dehydrogenase. Interestingly, some additive effects of glibenclamide and metformin were observed. CONCLUSIONS: Glibenclamide deeply alters cell metabolism in cardiac cells by impairing mitochondrial organization and function. This may further explain the risk of cardiovascular events associated with the use of this drug, alone or in combination with metformin.

Inside the Mechanism of Action of Three Pyrazole Derivatives in Human Platelets and Endothelial Cells.

In the effort to obtain multitarget compound interfering with inflammation, oxidative stress, and tumorigenesis, we synthesized a small library of pyrazole compounds, selecting 4a, 4f, and 4g as the most noteworthy being IC50 against platelet ROS production induced by thrombin of about 10 µM. The in vitro antioxidant potential of the three molecules was evaluated, and since they show a remarkable antioxidative activity, their effect on several parameter indicative of oxidative status and on the efficiency of the aerobic metabolism was tested. The three molecules strongly inhibit superoxide anion production, lipid peroxidation, NADPH oxidase activity and almost restore the oxidative phosphorylation efficiency in thrombin-stimulated platelet, demonstrating a protective effect against oxidative stress. This effect was confirmed in endothelial cell in which 4a, 4f, and 4g show an interesting inhibition activity on H2O2-stimulated EA.hy926 cells. At last, antiproliferative activity of 4a, 4f, and 4g was submitted to a large screening at the NCI. The molecules show interesting anticancer activity, among them the most remarkable is 4g able to strongly inhibit the proliferation of both solid tumor and leukemia cells lines. In conclusion, all the three newly synthetized pyrazoles show remarkable antioxidant and antiproliferative effect worthy of further study.

Scouting Different Phosphodiesterase 4 Inhibitor Chemotypes in Silico To Guide the Design of Anti-inflammatory/Antioxidant Agents.

During the last years, we developed a large library of new selective phosphodiesterase 4D inhibitors, maintaining the catechol portion of the well-known PDE4 inhibitor Rolipram, featuring different substitutions in place of the lactam group of this reference compound. Based on the X-ray analysis of PDE4 inhibitors (PDE4Is) previously synthesized by us and of naphthyridine- and naphthyridinone-containing derivatives exhibiting PDE4 inhibitory ability described in the literature, we designed and synthesized new compounds 1-3. All of them were screened in silico as putative PDE4Is, via molecular docking studies to exploit structural variation at the catechol group to gain further contacts especially with the flat aromatic residues (Phe506 and Phe538) of enzyme. Subsequent in silico prediction of ADMET properties and in vitro biological assays on platelets and endothelial cells are in good agreement with our previous data concerning the antioxidant/anti-inflammatory activity exhibited by our previous PDE4Is and similarly to other well-known PDE4Is.

Soluble form of the endothelial adhesion molecule CD146 binds preferentially CD16+ monocytes.

The cell adhesion molecule CD146 is normally located at the endothelial cell-to-cell junction and colocalizes with actin cytoskeleton. The soluble form of CD146 (sCD146) has been identified in the endothelial cell supernatant and in normal human plasma, and is increased in pathologic conditions with altered endothelial function. Soluble CD146 binding to monocytes promotes their transendothelial migration, which represents a central step in the development of atherosclerotic plaque. Since peripheral blood monocytes are characterized by a phenotypic and functional heterogeneity, with different transendothelial migration capacity, we hypothesized that monocyte subsets differently bind sCD146. Based on surface CD14 and CD16 expression monocytes were distinguished by flow cytometry (FACS) into three subsets: CD14++/CD16-, CD14++/CD16+ and CD14+/CD16+. CD16+ monocytes have been found to possess higher transendothelial migration ability. FACS analysis on blood monocytes from 30 healthy subjects revealed that higher percentages of CD14++/CD16+ (median, first and third quartile: 2.26, 1.62-3.87) and of CD14+/CD16+ (2.59, 1.28-4.80) were positive for CD146 (both p < 0.01), in comparison to CD14++/CD16- (0.66, 0.47-1.01). Moreover, in vitro treatment of ficoll separated monocytes with recombinant CD146 showed that both CD16+ subsets increased their percentage of CD146-positive events compared to CD16- monocytes (p < 0.01). Soluble CD146 levels were evaluated by ELISA in plasma samples of subjects from our study group and showed a correlation with percentage of CD146-positive CD14+/CD16+ monocyte subset. In this work we have demonstrated that monocyte subsets behave differently with regard to their sCD146 binding activity; because binding of CD146 influences transendothelial migration of monocytes, modulation of monocyte-CD146 interaction may represent a potential target to limit atherosclerotic plaque development.

Inhibition of the Cell Uptake of Delta and Omicron SARS-CoV-2 Pseudoviruses by N-Acetylcysteine Irrespective of the Oxidoreductive Environment.

The binding of SARS-CoV-2 spikes to the cell receptor angiotensin-converting enzyme 2 (ACE2) is a crucial target both in the prevention and in the therapy of COVID-19. We explored the involvement of oxidoreductive mechanisms by investigating the effects of oxidants and antioxidants on virus uptake by ACE2-expressing cells of human origin (ACE2-HEK293). The cell uptake of pseudoviruses carrying the envelope of either Delta or Omicron variants of SARS-CoV-2 was evaluated by means of a cytofluorimetric approach. The thiol N-acetyl-L-cysteine (NAC) inhibited the uptake of both variants in a reproducible and dose-dependent fashion. Ascorbic acid showed modest effects. In contrast, neither hydrogen peroxide (H2O2) nor a system-generating reactive oxygen species (ROS), which play an important role in the intracellular alterations produced by SARS-CoV-2, were able to affect the ability of either Delta or Omicron SARS-CoV-2 pseudoviruses to be internalized into ACE2-expressing cells. In addition, neither H2O2 nor the ROS generating system interfered with the ability of NAC to inhibit that mechanism. Moreover, based on previous studies, a preventive pharmacological approach with NAC would have the advantage of decreasing the risk of developing COVID-19, irrespective of its variants, and at the same time other respiratory viral infections and associated comorbidities.

Resveratrol counteracts systemic and local inflammation involved in early abdominal aortic aneurysm development.

BACKGROUND: Monocyte activation, macrophage infiltration, vascular oxidative stress and matrix proteolysis are inflammatory key steps contributing to abdominal aortic aneurysm (AAA) development. A phenotypical and functional heterogeneity is recognizable in monocytes by the differential expression of surface molecules: CD62L- subset corresponds to activated monocytes, while CD143/ACE surface expression increases during their differentiation into macrophages. In this work, Resveratrol, which is an antioxidant polyphenol with vasoprotective properties, has been evaluated for its potential to limit aneurysm development and monocyte-dependent inflammatory response in a model of elastase-induced AAA. METHODS: Male Sprague-Dawley rats received Resveratrol (10 mg/kg/die) (Rsv group, n=15) or vehicle (ethanol) alone (Et-OH group, n=15) continuously from 7 d before until 14 d after the AAA induction with elastase; five littermates were used as untreated control group (Ctr group, n=5). At the end of treatment, CD143 and CD62L monocyte expression was analyzed by flow cytometry, serum antioxidant capacity was evaluated using the TRAP method and circulating TNFα, and MMP-9 were measured with ELISA and gel zymography, respectively. Aortas were subjected to histology and immunohistochemistry for morphological analysis, macrophage infiltration, and MMP-9, TNFα, and VEGF expression. RESULTS: Resveratrol counteracted the CD62L-monocyte subset expansion, CD143 monocyte expression, and circulating levels of MMP-9 activity and TNFα associated to AAA induction. Similarly, treatment with Resveratrol significantly attenuated AAA expansion, vessel wall macrophage infiltration and MMP-9, VEGF, and TNFα expression, compared with AAA from Et-OH group. CONCLUSIONS: Resveratrol limited the monocyte-dependent inflammatory response, macrophage differentiation and aortic lumen enlargement in elastase-induced AAA. These data suggest that Resveratrol might be tested in selected patients with small AAA to modulate the early systemic and local inflammatory response associated to AAA progression.

Enhanced myostatin expression and signalling promote tubulointerstitial inflammation in diabetic nephropathy.

Myostatin (MSTN), a family member of the transforming growth factor (TGF)-ß super family, has been detected in the tubuli of pig kidney, but its role in the human kidney is not known. In this study we observed upregulation of MSTN mRNA (~8 to 10-fold increase) both in the glomeruli and tubulointerstitium in diabetic nephropathy (DN). In DN, immunoreactive MSTN was mainly localized in the tubuli and interstitium (∼4-8 fold increase), where it colocalized in CD45+ cells. MSTN was also upregulated in the glomeruli and the arterial vessels. Tubulointerstitial MSTN expression was directly related to interstitial fibrosis (r = 0.54, p < 0.01). In HK-2 tubular epithelial cells, both high (30 mmol) glucose and glycated albumin upregulated MSTN mRNA and its protein (p < 0.05-0.01). MSTN-treated HK-2 cells underwent decreased proliferation, together with NF-kB activation and CCL-2 and SMAD 2,3 overexpression. In addition, MSTN induced intracellular ROS release and upregulated NADPH oxidase, effects which were mediated by ERK activation. In conclusion, our data show that MSTN is expressed in the human kidney and overexpressed in DN, mainly in the tubulointerstitial compartment. Our results also show that MSTN is a strong inducer of proximal tubule activation and suggest that MSTN overexpression contributes to kidney interstitial fibrosis in DN.

Doxorubicin induces senescence or apoptosis in rat neonatal cardiomyocytes by regulating the expression levels of the telomere binding factors 1 and 2.

Low or high doses of doxorubicin induce either senescence or apoptosis, respectively, in cardiomyocytes. The mechanism by which different doses of doxorubicin may induce different stress-response cellular programs is not well understood. A recent study showed that the level of telomere dysfunction may induce senescence or apoptosis. We investigated the pathways to both apoptosis and senescence in neonatal rat cardiomyocytes and in H9c2 cells exposed to a single pulsed incubation with low or high doses of doxorubicin. High-dose doxorubicin strongly reduces TRF2 expression while enhancing TRF1 expression, and it determines early apoptosis. Low-dose doxorubicin induces downregulation of both TRF2 and TRF1, and it also increases the senescence-associated-beta-galactosidase activity, downregulates the checkpoint kinase Chk2, induces chromosomal abnormalities, and alters the cell cycle. The involvement of TRF1 and TRF2 with apoptosis and senescence was assessed by short interfering RNA interference. The cells maintain telomere dysfunction and a senescent phenotype over time and undergo late death. The increase in the phase>4N and the presence of micronuclei and anaphase bridges indicate that cells die by mitotic catastrophe. p38 modulates TRF2 expression, whereas JNK and cytoplasmic p53 regulate TRF1. Pretreatment with specific inhibitors of MAPKs and p53 may either attenuate the damage induced by doxorubicin or shift the cellular response to stress from senescence to apoptosis. In conclusion, various doses of doxorubicin induce differential regulation of TRF1 and TRF2 through p53 and MAPK, which is responsible for inducing either early apoptosis or senescence and late death due to mitotic catastrophe.

Cell-cell bond modulates vascular smooth muscle cell responsiveness to Angiotensin II.

Cell attachment is provided by cell-matrix and cell-cell bonds, and acts as a regulator of vascular smooth muscle cell (VSMC) survival, activity and homeostasis, as well as of VSMCs response to pathogenic stimuli. In this work we elicited an exclusive cell-cell contact by culturing A7r5 VSMCs on agarose-coated wells to form floating cell clusters, and we demonstrated that a steady state with a reduced response to the vasoactive peptide Angiotensin II (ATII) was induced. We found that clustered VSMCs showed subcortical stabilization of beta-catenin and Caveolin 1 (Cav1), unlike adherent confluent counterparts. We demonstrated that beta-catenin and Cav1 stabilization at the membrane level hampers the molecular cross-talk induced by ATII-activated AT1 receptor (AT1R), thereby impeding the phosphorylation of Cav1 and IGF1R, the NADPH oxidase activity, and counteracting ATII-dependent hypertrophy. Thus, elective cell-cell bond might modulate the proatherogenic activity of ATII, reducing the adverse vascular remodelling associated with AT1R activation.

Increased neutrophil lifespan in patients with congestive heart failure.

AIMS: Congestive heart failure (CHF) can be thought of as a state of chronic immune activation. Polymorphonuclear neutrophil (PMN) apoptosis is one of the mechanisms responsible for the resolution of inflammation. A reduced PMN apoptotic rate in CHF patients may generate a persistent inflammatory response and hence mediate tissue damage in this group of patients. We aimed to measure levels of spontaneous apoptosis of circulating PMNs in CHF patients and in controls, and to examine whether NYHA class, left ventricular ejection fraction (LV-EF), and laboratory parameters of inflammation, endothelial damage, and of liver and renal function, could predict the rate of PMN apoptosis in CHF patients. METHODS AND RESULTS: A total of 29 CHF patients and 26 controls were studied. Propidium iodide and flow cytometry were used to assess PMN apoptosis. Delay in PMN apoptosis was expressed as percentage (expressed as median, first and third quartiles) of surviving PMNs in the study subjects. We found an increased percentage of surviving PMNs [38(27.1-47.1)] in CHF patients compared with controls [19.4 (15.8-25.2)] (P < 0.05). The PMN survival rate in the CHF group was correlated to NYHA class, and plasma levels of C-reactive protein and alkaline phosphatase, while it was inversely correlated to LV-EF and protein levels. A positive relationship between PMN survival and increased ex vivo endothelial apoptosis was found. CONCLUSION: Increased PMN lifespan in patients with worsening CHF could be used as a novel measurement of tissue and endothelial damage in this group of patients.

Indoxyl Sulfate Induces Renal Fibroblast Activation through a Targetable Heat Shock Protein 90-Dependent Pathway.

Indoxyl sulfate (IS) accumulation occurs early during chronic kidney disease (CKD) progression and contributes to renal dysfunction by inducing fibrosis, inflammation, oxidative stress, and tissue remodeling. Renal toxicity of high IS concentrations (250 µM) has been widely explored, particularly in resident tubular and glomerular cells, while the effect of a moderate IS increase on kidneys is still mostly unknown. To define the effects of IS accumulation on renal fibroblasts, we first analyzed kidneys of C57BL/6 mice receiving IS (0.1%) in drinking water for 12 weeks. As a next step, we treated renal fibroblasts (NRK-49F) with IS (20 µM) with or without the HSP90 inhibitor 17-AAG (1 µM). In mouse kidneys, IS increased the collagen deposition and HSP90 and α-SMA expression (immunohistochemistry) in interstitial fibroblasts and caused tubular necrosis (histological H&E and picrosirius red staining). In NRK-49F cells, IS induced MCP1, TGF-ß, collagen I, α-SMA, and HSP90 gene/protein expression and Smad2/3 pathway activation. IS had no effects on fibroblast proliferation and ROS production. 17-AAG counteracted IS-induced MCP1, TGF-ß, collagen I, and α-SMA expression and Smad2/3 phosphorylation. Our study demonstrates that the IS increase promotes renal fibroblast activation by a HSP90-dependent pathway and indicates HSP90 inhibition as a potential strategy to restrain IS-induced kidney inflammation and fibrosis in CKD.

Caveolin-1 down-regulation inhibits insulin-like growth factor-I receptor signal transduction in H9C2 rat cardiomyoblasts.

Caveolin (Cav)-1, the major caveolar protein, directly interacts with IGF-I receptor (IGF-IR) and its intracellular substrates. To determine the role of Cav-1 in IGF-IR signaling, we transfected H9C2 cells with small interfering RNA specific for Cav-1-siRNA. The selective down-regulation of Cav-1 (90%) was associated with a smaller reduction of Cav-2, whereas Cav-3 expression was unaffected. A significant reduction of IGF-IR tyrosine phosphorylation in Cav-1-siRNA H9C2 cells was found compared with H9C2 control cells (Ctr-siRNA). The reduced IGF-IR autophosphorylation resulted in a decrease of insulin receptor substrate-1, Shc, and Akt activation. In addition, in Cav-1-siRNA H9C2 cells, IGF-I did not prevent apoptosis, suggesting that Cav-1 is required to mediate the antiapoptotic effect of IGF-I in cardiomyoblasts. The down-regulation of Cav-1 decreased IGF-IR activation and affected the ability of IGF-I to prevent apoptosis after serum withdrawal also in human umbilical vein endothelial cells. These results demonstrate that: 1) Cav-1 down-regulation negatively affects IGF-IR tyrosine phosphorylation; 2) this effect causes a reduced activation of insulin receptor substrate-1, Shc, and Akt; and 3) Cav-1 is involved in IGF-IR antiapoptotic signaling after serum deprivation.

Brain microglia activation induced by intracranial administration of oligonucleotides and its pharmacological modulation.

Oligonucleotide overloading results in type I interferonopathies such as the Aicardi-Goutiéres Syndrome, a progressive encephalopathy determined by an immune response against endogenous DNA/RNA molecules. No therapy targeting pathogenic mechanisms is available for affected patients. Accordingly, we set up an in vitro/in vivo experimental model aimed at reproducing the pathogenic mechanisms of type I interferonopathies, in order to develop an effective pharmacological modulation and toxicological alterations caused by intracranial delivery of encapsulated CpG. The in vitro model used Aicardi-Goutiéres Syndrome immortalized lymphocytes activated by interferon I and co-cultured with human astrocytes; lymphocyte neurotoxicity was attenuated by the calcineurin-inhibitor Tacrolimus and by the anti-interferon monoclonal antibody Sifalimumab. The in vivo model was set up in mice by subcutaneous injection of encapsulated CpG oligonucleotides; the immune-stimulating activity was demonstrated by cytometric analysis in the spleen. To mime pathogenesis of type I interferonopathies in the central nervous system, CpG oligonucleotides were administered intracranially in mice. In the brain, CpG overload induced a rapid activation of macrophage-like microglial cells and focal accumulation mononuclear cells. The subcutaneous administration of Tacrolimus and, more potently, Sifalimumab attenuated CpG-induced brain alterations. These findings shed light on molecular mechanisms triggered by oligonucleotides to induce brain damage. Monoclonal antibodies inhibiting interferon seem a promising therapeutic strategy to protect brain in type I interferonopathies.

microRNA-494 Favors HO-1 Expression in Neuroblastoma Cells Exposed to Oxidative Stress in a Bach1-Independent Way.

Heme oxygenase 1 (HO-1) is crucially involved in cell adaptation to oxidative stress and has been demonstrated to play an important role in cancer progression and resistance to therapies. We recently highlighted that undifferentiated neuroblastoma (NB) cells are prone to counteract oxidative stress through the induction of HO-1. Conversely, differentiated NB cells were more sensitive to oxidative stress since HO-1 was scarcely upregulated. In this work, we investigated the role played by miR-494, which has been proved to be involved in cancer biology and in the modulation of oxidative stress, in the upregulation of HO-1. We showed that NB differentiation downregulates miR-494 level. In addition, endogenous miR-494 inhibition in undifferentiated cells impairs HO-1 induction in response to exposure to 500 µM H2O2, reducing the number of viable cells. The analysis of Bach1 expression did not reveal any significant modifications in any experimental conditions tested, proving that the impairment of HO-1 induction observed in cells treated with miR-494 inhibitor and exposed to H2O2 is independent from Bach1. Our results underline the role played by miR-494 in favoring HO-1 induction and cell adaptation to oxidative stress and contribute to the discovery of new potential pharmacological targets to improve anticancer therapies.

Matrix metalloproteinase-2 and -9 are induced differently by doxorubicin in H9c2 cells: The role of MAP kinases and NAD(P)H oxidase.

OBJECTIVE: Dysregulation of myocardial metalloproteinases (MMPs) is now regarded as an early contributory mechanism for the initiation and progression of heart failure. Doxorubicin is a strongly cardiotoxic anticancer drug. This study investigates the effects of doxorubicin on myocardial MMP-2 and MMP-9 activation. METHODS: After pre-treatment with or without carvedilol or dexrazoxane, we exposed H9c2 cardiomyocytes to doxorubicin to evaluate reactive oxygen species (ROS) formation and MMP-2 and MMP-9 expression and activation. To investigate the signaling pathways leading to doxorubicin-induced MMP activation, we also examined the phosphorylation of three members of the MAPK family (ERK1/2, p38, and JNK), the effects of selective inhibitors of ERK1/2, p38, and JNK on MMP transcription and activity, the transcription of the NAD(P)H oxidase subunit Nox1, and the effects of the NAD(P)H oxidase inhibitor DPI on MMP activation. RESULTS: Doxorubicin induces a significant increase in ROS formation and a rapid increase of MMP expression and activation. Pre-treatment with carvedilol or dexrazoxane prevented these effects. We also found that p38 is the MAPK that is mainly responsible for MMP-9 activation through an NAD(P)H-independent mechanism. ERK and JNK modulate the transcription of the NAD(P)H oxidase subunit Nox1, while the JNK/ERK NAD(P)H oxidase cascade is an important pathway that mediates doxorubicin signaling to MMP-2. Inhibition of NAD(P)H oxidase attenuates the increase in MMP-2, but augments the doxorubicin-induced increase in MMP-9. CONCLUSIONS: Enhancement of MMP-2 and MMP-9 in cardiac myocytes in response to doxorubicin is mediated by the cooperation of ERK, JNK, and p38 kinase pathways, most of which are redox dependent.

Advanced Oxidation Protein Products-Modified Albumin Induces Differentiation of RAW264.7 Macrophages into Dendritic-Like Cells Which Is Modulated by Cell Surface Thiols.

Local accumulation of Advanced Oxidation Protein Products (AOPP) induces pro-inflammatory and pro-fibrotic processes in kidneys and is an independent predictor of renal fibrosis and of rapid decline of eGFR in patients with chronic kidney disease (CKD). In addition to kidney damage, circulating AOPP may be regarded as mediators of systemic oxidative stress and, in this capacity, they might play a role in the progression of atherosclerotic damage of arterial walls. Atherosclerosis is a chronic inflammatory disease that involves activation of innate and adaptive immunity. Dendritic cells (DCs) are key cells in this process, due to their role in antigen presentation, inflammation resolution and T cell activation. AOPP consist in oxidative modifications of proteins (such as albumin and fibrinogen) that mainly occur through myeloperoxidase (MPO)-derived hypochlorite (HOCl). HOCl modified proteins have been found in atherosclerotic lesions. The oxidizing environment and the shifts in cellular redox equilibrium trigger inflammation, activate immune cells and induce immune responses. Thus, surface thiol groups contribute to the regulation of immune functions. The aims of this work are: (1) to evaluate whether AOPP-proteins induce activation and differentiation of mature macrophages into dendritic cells in vitro; and (2) to define the role of cell surface thiol groups and of free radicals in this process. AOPP-proteins were prepared by in vitro incubation of human serum albumin (HSA) with HOCl. Mouse macrophage-like RAW264.7 were treated with various concentrations of AOPP-HSA with or without the antioxidant N-acetyl cysteine (NAC). Following 48 h of HSA-AOPP treatment, RAW264.7 morphological changes were evaluated by microscopic observation, while markers of dendritic lineage and activation (CD40, CD86, and MHC class II) and allogeneic T cell proliferation were evaluated by flow cytometry. Cell surface thiols were measured by AlexaFluor-maleimide binding, and ROS production was assessed as DCF fluorescence by flow cytometry. HSA-AOPP induced the differentiation of RAW264.7 cells into a dendritic-like phenotype, as shown by morphological changes, by increased CD40, CD86 and MHC class II surface expression and by induction of T cell proliferation. The cell surface thiols dose dependently decreased following HSA-AOPP treatment, while ROS production increased. NAC pre-treatment enhanced the amount of cell surface thiols and prevented their reduction due to treatment with AOPP. Both ROS production and RAW264.7 differentiation into DC-like cells induced by HSA-AOPP were reduced by NAC. Our results highlight that oxidized plasma proteins modulate specific immune responses of macrophages through a process involving changes in the thiol redox equilibrium. We suggest that this mechanism may play a role in determining the rapid progression of the atherosclerotic process observed in CKD patients.

Matrine in association with FD­2 stimulates F508del­cystic fibrosis transmembrane conductance regulator activity in the presence of corrector VX809.

Cystic fibrosis is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the predominant mutation is termed Phe508del (F508del). Therapy for F508del­CFTR patients is based on the use of Orkambi®, a combination of VX809 and VX770. However, though Orkambi leads to an improvement in the lung function of patients, a progressive reduction in its efficacy has been observed. In order to overcome this effect, the aim of the present study was to investigate the role of matrine and the in­house compound FD­2 in increasing the action of VX809 and VX770. Fischer rat thyroid cells overexpressing F508del­CFTR were treated with matrine, VX809 (corrector) and/or with a number of potentiators (VX770, FD­1 and FD­2). The results demonstrated that matrine was able to stimulate CFTR activity and, in association with FD­2, increased the functionality of the channel in the presence of VX809. Based on these results, it may be hypothesized that FD­2 may be a novel and more effective potentiator compared with VX770.

Sphingomyelin as a myelin biomarker in CSF of acquired demyelinating neuropathies.

Fast, accurate and reliable methods to quantify the amount of myelin still lack, both in humans and experimental models. The overall objective of the present study was to demonstrate that sphingomyelin (SM) in the cerebrospinal fluid (CSF) of patients affected by demyelinating neuropathies is a myelin biomarker. We found that SM levels mirror both peripheral myelination during development and small myelin rearrangements in experimental models. As in acquired demyelinating peripheral neuropathies myelin breakdown occurs, SM amount in the CSF of these patients might detect the myelin loss. Indeed, quantification of SM in 262 neurological patients showed a significant increase in patients with peripheral demyelination (p = 3.81 * 10 - 8) compared to subjects affected by non-demyelinating disorders. Interestingly, SM alone was able to distinguish demyelinating from axonal neuropathies and differs from the principal CSF indexes, confirming the novelty of this potential CSF index. In conclusion, SM is a specific and sensitive biomarker to monitor myelin pathology in the CSF of peripheral neuropathies. Most importantly, SM assay is simple, fast, inexpensive, and promising to be used in clinical practice and drug development.