Cardiac hypertrophy that affects hyperthyroidism occurs independently of the NLRP3 inflammasome.

Cardiac hypertrophy (CH) is an adaptive response to maintain cardiac function; however, persistent stress responses lead to contractile dysfunction and heart failure. Although inflammation is involved in these processes, the mechanisms that control cardiac inflammation and hypertrophy still need to be clarified. The NLRP3 inflammasome is a cytosolic multiprotein complex that mediates IL-1ß production. The priming step of NLRP3 is essential for increasing the expression of its components and occurs following NF-κB activation. Hyperthyroidism triggers CH, which can progress to maladaptive CH and even heart failure. We have shown in a previous study that thyroid hormone (TH)-induced CH is linked to the upregulation of S100A8, leading to NF-κB activation. Therefore, we aimed to investigate whether the NLRP3 inflammasome is involved in TH-induced CH and its potential role in CH pathophysiology. Hyperthyroidism was induced in NLRP3 knockout (NLRP3-KO), Caspase-1-KO and Wild Type (WT) male mice of the C57Bl/6J strain, aged 8-12 weeks, by triiodothyronine (7 µg/100 g BW, i.p.) administered daily for 14 days. Morphological and cardiac functional analysis besides molecular assays showed, for the first time, that TH-induced CH is accompanied by reduced NLRP3 expression in the heart and that it occurs independently of the NLRP3 inflammasome and caspase 1-related pathways. However, NLRP3 is important for the maintenance of basal cardiac function since NLRP3-KO mice had impaired diastolic function and reduced heart rate, ejection fraction, and fractional shortening compared with WT mice.

Polyethylenimine as a Versatile Simultaneous Reducing and Stabilizing Agent Enabling One-Pot Synthesis of Transition-Metal Nanoparticles: Fundamental Aspects and Practical Implications.

The large surface area of metallic nanoparticles provides them with particular optical, chemical, and biological properties, accordingly enabling their use in a wide array of applications. In this regard, facile and fast synthetic approaches are desirable for ready-to-use functional materials. Following early investigations focused on the direct synthesis of polymer-coated gold nanoparticles, we herein demonstrate that such a strategy can be used to manufacture different types of d-block transition-metal nanoparticles via a one-pot method in aqueous media and mild temperature conditions. Gold (Au3+), palladium (Pd2+), and silver (Ag+) ions could be reduced using only polyethylenimine (PEI) or PEI derivatives acting simultaneously as a reducing and stabilizing agent and without the aid of any other external agent. The process gave rise, for instance, to Pd urchin-like nanostructures with a large surface area which confers to them outstanding catalytic performance compared to AuNPs and AgNPs produced using the same strategy. The polymer-stabilized AgNPs were demonstrated to be biocide against a variety of microorganisms, although AuNPs and PdNPs do not hold such an attribute at least in the probed concentration range. These findings may provide significant advances toward the practical, facile, and ready-to-use manufacturing of transition-metal nanoparticles for a myriad of applications.

Protein coronas coating polymer-stabilized silver nanocolloids attenuate cytotoxicity with minor effects on antimicrobial performance.

Silver nanoparticles are versatile platforms with a variety of applications in the biomedical field. In this framework, their presence in biological media inevitably leads to the interaction with proteins thus conducting to the formation of biomolecular coronas. This feature alters the identity of the nanomaterial and may affect many biological events. These considerations motivated the investigation of protein adsorption onto the surface of polymer-stabilized AgNPs. The metallic colloids were coated by polyethyleneimine (PEI), polyvinylpyrrolidone (PVP), and poly(2-vinyl pyridine)-b-poly(ethylene oxide) (PEO-b-P2VP), and nanoparticle-protein interaction was probed by using a library of analytical techniques. The experimental data revealed a higher extent of protein adsorption at the surface of AgNPs@PVP whereas PEO-b-P2VP coating conducted to the least amount. The main component of the protein coronas was evidenced to be bovine serum albumin (BSA), which is indeed the protein at the highest abundancy in the model biological media. We have further demonstrated reduced cytotoxicity of the silver colloids coated by biomolecular coronas as compared to the pristine counterparts. Nevertheless, the protein coatings did not notably reduce the antimicrobial performance of the polymer-stabilized AgNPs. Accordingly, although the protein-repelling property is frequently targeted towards longer in vivo circulation of nanoparticles, we herein underline that protein coatings, which are commonly treated as artifacts to be avoided, may indeed enhance the biological performance of nanomaterials. These findings are expected to be highly relevant in the design of polymer-stabilized metallic colloids intended to be used in healthcare.

Cardiorenal syndrome: long road between kidney and heart.

Almost 200 years ago, the first evidence described by Robert Bright (1836) showed the strong interaction between the kidneys and heart and, since then, the scientific community has dedicated itself to better understanding the mechanisms involved in the kidney-heart relationship, known in recent decades as cardiorenal syndrome (CRS). This syndrome includes a wide clinical variety that affects the kidneys and heart, in an acute or chronic manner. Moreover, it is well established in the literature that the immune system, the sympathetic nervous system, the renin-angiotensin-aldosterone, and the oxidative stress actively play a strong role in the cellular and molecular processes present in CRS. More recently, uremic molecules and epigenetic factors have been also shown to be key mediators in the development of syndrome. The present review intends to present the state of the art regarding CRS and to show the paths known, until now, in the long road between the kidneys and heart.

Mitochondrial Dysfunction in Cardiorenal Syndrome 3: Renocardiac Effect of Vitamin C.

Cardiorenal syndrome (CRS) is a pathological link between the kidneys and heart, in which an insult in a kidney or heart leads the other organ to incur damage. CRS is classified into five subtypes, and type 3 (CRS3) is characterized by acute kidney injury as a precursor to subsequent cardiovascular changes. Mitochondrial dysfunction and oxidative and nitrosative stress have been reported in the pathophysiology of CRS3. It is known that vitamin C, an antioxidant, has proven protective capacity for cardiac, renal, and vascular endothelial tissues. Therefore, the present study aimed to assess whether vitamin C provides protection to heart and the kidneys in an in vivo CRS3 model. The unilateral renal ischemia and reperfusion (IR) protocol was performed for 60 min in the left kidney of adult mice, with and without vitamin C treatment, immediately after IR or 15 days after IR. Kidneys and hearts were subsequently collected, and the following analyses were conducted: renal morphometric evaluation, serum urea and creatinine levels, high-resolution respirometry, amperometry technique for NO measurement, gene expression of mitochondrial dynamic markers, and NOS. The analyses showed that the left kidney weight was reduced, urea and creatinine levels were increased, mitochondrial oxygen consumption was reduced, NO levels were elevated, and Mfn2 expression was reduced after 15 days of IR compared to the sham group. Oxygen consumption and NO levels in the heart were also reduced. The treatment with vitamin C preserved the left kidney weight, restored renal function, reduced NO levels, decreased iNOS expression, elevated constitutive NOS isoforms, and improved oxygen consumption. In the heart, oxygen consumption and NO levels were improved after vitamin C treatment, whereas the three NOS isoforms were overexpressed. These data indicate that vitamin C provides protection to the kidneys and some beneficial effects to the heart after IR, indicating it may be a preventive approach against cardiorenal insults.

Evidence of protein coronas around soft nanoparticles regardless of the chemical nature of the outer surface: structural features and biological consequences.

The formation of biomolecular coronas around nanoparticles as soon as they come in contact with biological media is nowadays well accepted. The self-developed biological outer surfaces can affect the targeting capability of the colloidal carriers as well as their cytotoxicity and cellular uptake behavior. In this framework, we explored the structural features and biological consequences of protein coronas around block copolymer assemblies consisting of a common pH-responsive core made by poly[2-(diisopropylamino) ethyl methacrylate] (PDPA) and hydrophilic shells of different chemical natures: zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) or highly hydrophilic poly(ethylene oxide) (PEO) and poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA). We demonstrated the presence of ∼50 nm protein coronas around the nanoparticles regardless of the chemical nature of the polymeric shells. The thickness is understood as the sum of the soft and hard layers and it is the actual interface seen by the cells. Although the soft corona composition is difficult to determine because the proteins are loosely bound to the outer surface of the assemblies, the tightly bound proteins (hard corona) could be identified and quantified. The compositional analysis of the hard corona demonstrated that human serum albumin (HSA), immunoglobulin G (IgG) and fibrinogen are the main components of the protein coronas, and serotransferrin is present particularly in the protein corona of the zwitterionic-stabilized assemblies. The protein coronas substantially reduce the cellular uptake of the colloidal particles due to their increased size and the presence of HSA which is known to reduce nanoparticle-cell adhesion. On the other hand, their existence also reduces the levels of cytotoxicity of the polymeric assemblies, highlighting that protein coronas should not be always understood as artifacts that need to be eliminated due to their positive outputs.

The Protein Corona Conundrum: Exploring the Advantages and Drawbacks of its Presence around Amphiphilic Nanoparticles.

The success of targeted drug delivery systems still requires a detailed understanding about the biological consequences of self-developed biomolecular coronas around them, since this is the surface that interacts with living cells. Herein, we report the behavior of carbohydrate-decorated amphiphilic nanoparticles in a plasma environment with regard to the formation and biological consequences of the protein corona. Naked amphiphilic nanoparticles were produced through the self-assembly of azido-PEO900-docosanoate molecules, and the coupling of N-acetylglucosamine via click chemistry enabled the fabrication of the corresponding bioactive glyco-nanostructures. Light scattering measurements, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, liquid chromatography-mass spectrometry, and the Pierce BCA protein assay all confirmed the presence of protein coronas around the self-assembled nanoparticles, regardless of the presence of the sugar residues, although it reduces the amount of adsorbed proteins. The protein coronas were formed mainly by human serum albumin, complement proteins, apolipoproteins, immunoglobulins, and proteins involved in the coagulation cascade (fibrinogen and prothrombin). While the presence of these protein coronas significantly reduced cellular uptake of the amphiphilic assemblies, they also notably reduced the cytotoxic and hemolytic effects that result from the contact of the nanoparticles with living cells. Accordingly, we highlight that protein coronas should not always be treated as artifacts that have to be avoided because they can also provide beneficial effects.

Characterization of the Oxidative Stress in Renal Ischemia/Reperfusion-Induced Cardiorenal Syndrome Type 3.

In kidney disease (KD), several factors released into the bloodstream can induce a series of changes in the heart, leading to a wide variety of clinical situations called cardiorenal syndrome (CRS). Reactive oxygen species (ROS) play an important role in the signaling and progression of systemic inflammatory conditions, as observed in KD. The aim of the present study was to characterize the redox balance in renal ischemia/reperfusion-induced cardiac remodeling. C57BL/6 male mice were subjected to occlusion of the left renal pedicle, unilateral, for 60 min, followed by reperfusion for 8 and 15 days, respectively. The following redox balance components were evaluated: catalase (CAT), superoxide dismutase (SOD), total antioxidant capacity (FRAP), NADPH oxidase (NOX), nitric oxide synthase (NOS), hydrogen peroxide (H2O2), and the tissue bioavailability of nitric oxide (NO) such as S-nitrosothiol (RSNO) and nitrite (NO2 -). The results indicated a process of renoprotection in both kidneys, indicated by the reduction of cellular damage and some oxidant agents. We also observed an increase in the activity of antioxidant enzymes, such as SOD, and an increase in NO bioavailability. In the heart, we noticed an increase in the activity of NOX and NOS, together with increased cell damage on day 8, followed by a reduction in protein damage on day 15. The present study concludes that the kidneys and heart undergo distinct processes of damage and repair at the analyzed times, since the heart is a secondary target of ischemic kidney injury. These results are important for a better understanding of the cellular mechanisms involved in CRS.

Time Course of Gene Expression Profile in Renal Ischemia and Reperfusion Injury in Mice.

Ischemic renal failure is an inflammatory disease that can affect various organs, including the heart. The organ responds to the stimulus and undergoes tissue remodeling that can result in cardiac hypertrophy. This study aimed to characterize the cardiac global gene expression profile in renal ischemia/reperfusion (IR) model using microarray technology. To do that, left kidney ischemia was induced in male C57BL/6 mice for 60 minutes, followed by reperfusion (IR) for 5, 8, 15, or 20 days post ischemia (dpi). Total cardiac tissue RNA was extracted and hybridized to chips with 35,000 mouse genes. The GeneChip Mouse Genome 430 2.0 Array Expression chip (Affymetrix) was used, and CEL files generated were processed with DNA-Chip-Analyzer (dCHIP) software. Subsequent analysis considered only differences among groups of at least 1.2-fold (up or down) expression changes. Analyses of the samples indicated positive modulation of 17,413 genes and 405 pathways and negative modulation of 18,287 genes and 300 pathways. A narrower analysis of genes related to inflammation, metabolism, apoptosis, oxidative stress, and channels/ion transport was performance, and it was correlated with IR injury, corroborating previous data from literature. Renal IR induced a global shift in cardiac tissue gene expression; in particular, genes related to the inflammatory system and cardiomyocyte function were changed. The in-depth study of the cell signaling in the present study could stimulate the development of new therapeutic option to ameliorate the outcome of renal-IR-induced heart damage.

Caspase-1 as Molecular Key in Cardiac Remodeling during Cardiorenal Syndrome Type 3 in the Murine Model.

BACKGROUND: Renal ischemia/reperfusion induces a systemic inflammatory response that is directly related to the development of cardiac hypertrophy due to cardiorenal syndrome type 3. Classic inflammatory pathways have been extensively investigated in cardiovascular diseases, including the participation of inflammasome in caspase-1-dependent IL-1ß cleavage. OBJECTIVE: In this study, we aimed to understand how lack of caspase-1 would impact the hypertrophic and apoptotic response in the heart after renal ischemia/reperfusion. METHODS: Wildtype and caspase-1 knockout animals were submitted to a renal ischemia/reperfusion protocol. Briefly, left kidney ischemia was induced in male C57BL/6 mice for 60 min, followed by reperfusion for 15 days. Gene expression was analysed by Real-Time PCR. Caspase activity was also evaluated. RESULTS: Lack of caspase-1 led to a more pronounced cardiac hypertrophy in mice subjected to renal ischemia-reperfusion. Such hypertrophic process was accompanied by increased activity of caspase3/7 and 9, indicating apoptosis initiation in an IL-1ß- independent manner. CONCLUSION: Our data corroborate important findings on the role of caspase-1 in the development of cardiac hypertrophy and remodeling.

Cardiac Inflammation after Ischemia-Reperfusion of the Kidney: Role of the Sympathetic Nervous System and the Renin-Angiotensin System.

BACKGROUND/AIMS: To investigate the role of the sympathetic nervous system (SNS) and renin-angiotensin system (RAS) in renal ischemia/reperfusion-induced (I/R) cardiac inflammatoryprofile. METHODS: Left kidney ischemia was induced in male C57BL/6 mice for 60 min, followed by reperfusion for 12 days, and treatment with or without atenolol, losartan, or enalapril. The expression of vimentin in kidney and atrial natriuretic factor (ANF) in the heart has been investigated by RT-PCR. In cardiac tissue, levels of ß1-adrenoreceptors, adenylyl cyclase, cyclic AMP-dependent protein kinase (PKA), noradrenaline, adrenaline (components of SNS), type 1 angiotensin II receptors (AT1R), angiotensinogen/Ang II and renin (components of RAS) have been measured by Western blotting and HPLC analysis. A panel of cytokines - tumour necrosis factor (TNF-α), interleukin IL-6, and interferon gamma (IFN-γ) - was selected as cardiac inflammatory markers. RESULTS: Renal vimentin mRNA levels increased by >10 times in I/R mice, indicative of kidney injury. ANF, a marker of cardiac lesion, increased after renal I/R, the values being restored to the level of Sham group after atenolol or enalapril treatment. The cardiac inflammatory profile was confirmed by the marked increase in the levels of mRNAs of TNF-α, IL-6, and IFN-γ. Atenolol and losartan reversed the upregulation of TNF-α expression, whereas enalapril restored IL-6 levels to Sham levels; both atenolol and enalapril normalized IFN-γ levels. I/R mice showed upregulation of ß1-adrenoreceptors, adenylyl cyclase, PKA and noradrenaline. Renal I/R increased cardiac levels of AT1R, which decreased after losartan or enalapril treatment. Renin expression also increased, with the upregulation returning to Sham levels after treatment with SNS and RAS blockers. Angiotensinogen/Ang II levels in heart were unaffected by renal I/R, but they were significantly decreased after treatment with losartan and enalapril, whereas increase in renin levels decreased. CONCLUSION: Renal I/R-induced cardiac inflammatory events provoked by the simultaneous upregulation of SNS and RAS in the heart, possibly underpin the mechanism involved in the development of cardiorenal syndrome.

Cardiac arrhythmias after renal I/R depend on IL-1ß.

AIMS: Cardiac arrhythmias are one of the most important remote complications after kidney injury. Renal ischemia reperfusion (I/R) is a major cause of acute renal injury predisposing to several remote dysfunctions, including cardiac electrical disturbance. Since IL-1ß production dependent on NLRP3 represents a link between tissue malfunctioning and cardiac arrhythmias, here we tested the hypothesis that longer ventricular repolarization and arrhythmias after renal I/R depend on this innate immunity sensor. METHODS AND RESULTS: Nlrp3-/- and Casp1-/- mice reacted to renal I/R with no increase in plasma IL-1ß, different from WT (wild-type) I/R. A prolonged QJ interval and an increased susceptibility to ventricular arrhythmias were found after I/R compared to Sham controls in wild-type mice at 15 days post-perfusion, but not in Nlrp3-/- or CASP1-/- I/R, indicating that the absence of NLRP3 or CASP1 totally prevented longer QJ interval after renal I/R. In contrast with WT mice, we found no renal atrophy and no renal dysfunction in Nlrp3-/- and Casp1-/- mice after renal I/R. Depletion of macrophages in vivo after I/R and a day before IL-1ß peak (at 7 days post-perfusion) totally prevented prolongation of QJ interval, suggesting that macrophages might participate as sensors of tissue injury. Moreover, treatment of I/R-WT mice with IL-1r antagonist (IL-1ra) from 8 to 15 days post perfusion did not interfere with renal function, but reversed QJ prolongation, prevented the increase in susceptibility to ventricular arrhythmias and rescued a close to normal duration and amplitude of calcium transient. CONCLUSION: Taken together, these results corroborate the hypothesis that IL-1ß is produced after sensing renal injury through NRLP3-CASP1, and IL-1ß on its turn triggers longer ventricular repolarization and increase susceptibility to cardiac arrhythmias. Still, they offer a therapeutic approach to treat cardiac arrhythmias that arise after renal I/R.

Renal ischemia/reperfusion-induced cardiac hypertrophy in mice: Cardiac morphological and morphometric characterization.

BACKGROUND: Tissue remodeling is usually dependent on the deposition of extracellular matrix that may result in tissue stiffness and impaired myocardium contraction. OBJECTIVES: We had previously demonstrated that renal ischemia/reperfusion (I/R) is able to induce development of cardiac hypertrophy in mice. Therefore, we aimed to characterize renal I/R-induced cardiac hypertrophy. DESIGN: C57BL/6 J mice were subjected to 60 minutes' unilateral renal pedicle occlusion, followed by reperfusion (I/R) for 5, 8, 12 or 15 days. Gene expression, protein abundance and morphometric analyses were performed in all time points. RESULTS: Left ventricle wall thickening was increased after eight days of reperfusion (p < 0.05). An increase in the number of heart ventricle capillaries and diameter after 12 days of reperfusion (p < 0.05) was observed; an increase in the density of capillaries starting at 5 days of reperfusion (p < 0.05) was also observed. Analyses of MMP2 protein levels showed an increase at 15 days compared to sham (p < 0.05). Moreover, TGF-ß gene expression was downregulated at 12 days as well TIMP 1 and 2 (p < 0.05). The Fourier-transform infrared spectroscopy analysis showed that collagen content was altered only in the internal section of the heart (p < 0.05); such data were supported by collagen mRNA levels. CONCLUSIONS: Renal I/R leads to impactful changes in heart morphology, accompanied by an increase in microvasculature. Although it is clear that I/R is able to induce cardiac remodeling, such morphological changes is present in only a section of the heart tissue.

Proteomic, cellular, and network analyses reveal new DUSP3 interactions with nucleolar proteins in HeLa cells.

DUSP3 (or Vaccinia virus phosphatase VH1-related; VHR) is a small dual-specificity phosphatase known to dephosphorylate c-Jun N-terminal kinases and extracellular signal-regulated kinases. In human cervical cancer cells, DUSP3 is overexpressed, localizes preferentially to the nucleus, and plays a key role in cellular proliferation and senescence triggering. Other DUSP3 functions are still unknown, as illustrated by recent and unpublished results from our group showing that this enzyme mediates DNA damage response or repair processes. In this study, we sought to identify new interactions between DUSP3 and proteins directly or indirectly involved in or correlated with its biological roles in HeLa cells exposed to gamma or UV radiation. By using GST-DUSP as bait, we pulled down interacting proteins and identified them by LC-MS/MS. Of the 46 proteins obtained, six hits were extensively validated by immune techniques; the proteins Nucleophosmin, HnRNP C1/C2, and Nucleolin were the most promising targets found to directly interact with DUSP3. We then analyzed the DUSP3 interactomes using physical protein-protein interaction networks using our hits as the seed list. The validated hits as well as unvalidated hits fluctuated on the DUSP3 interactomes of HeLa cells, independent of the time post radiation, which confirmed our proteomic and experimental data and clearly showed the proximity of DUSP3 to proteins involved in processes intimately related to DNA repair and senescence, such as Ku70 and Tert, via interactions with nucleolar proteins, which were identified in this study, that regulate DNA/RNA structure and functions.