Free Hemin Research Articles

Hemin promotes platelet activation and plasma membrane disintegration regulated by the subtilisin-like proprotein convertase furin.

Platelet activation plays a critical role in thrombosis and hemostasis. Several pathophysiological situations lead to hemolysis, resulting in the liberation of free ferric iron-containing hemin. Hemin has been shown to activate platelets and induce thrombo-inflammation. Classical antiplatelet therapy failed to prevent hemin-induced platelet activation. Thus, the aim of the present study was to characterize the mechanism of hemin-induced platelet death (ferroptosis). We evaluated the invitro effect of hemin on platelet activation, signaling, oxylipins, and plasma membrane destruction using light transmission aggregometry, exvivo thrombus formation, multiparametric flow cytometry, micro-UHPLC mass spectrometry for oxylipin profiling, and scanning ion conductance microscopy (SICM). We found that hemin induces platelet cell death indicated by increased ROS levels, phosphatidyl serine (PS) exposure, and loss of mitochondrial membrane potential (ΔΨm). Further, hemin causes lipid peroxidation and generation of distinct oxylipins, which strongly affects plasma membrane integrity leading to generation of platelet-derived microvesicles. Interestingly, hemin-dependent platelet death (ferroptosis) is specifically regulated by the subtilisin-like proprotein convertase furin. In summary, platelet undergo a non-apoptotic cell death mediated by furin. Inhibition of furin may offer a therapeutic strategy to control hemin-induced thrombosis and thrombo-inflammation at a site of hemolysis.

Hemin-induced platelet activation is regulated by the ACKR3 chemokine surface receptor and has implications for passivation of vulnerable atherosclerotic plaques.

In vulnerable atherosclerotic plaques, intraplaque hemorrhages (IPH) result in hemolysis of red blood cells and release of hemoglobin and free hemin. Hemin activates platelets and leads to thrombosis. Agonism of the inhibitory platelet receptor ACKR3 inhibits hemin-dependent platelet activation and thrombus formation. To characterize the effect of hemin and ACKR3 agonism on isolated human platelets, multi-color flow cytometry and classical experimental setup such as light transmission aggregometry and a flow chamber assay were used. Hemin induces platelet aggregation and ex vivo platelet-dependent thrombus formation on immobilized collagen under a low shear rate of 500 s-1, indicating that free hemin is a strong activator of platelet-dependent thrombosis. Recently, we described that ACKR3 is a prominent inhibitory receptor of platelet activation. Specific ACKR3 agonists but not conventional antiplatelet compounds such as COX-1 inhibitor (indometacin), ADP-receptor blocker (cangrelor), or PAR1 inhibitor (ML161) inhibit both hemin-dependent aggregation and thrombus formation. To further characterize the effect of hemin on platelet subpopulations, we established a multi-color flow cytometry assay. We found that hemin induces procoagulant (CD42bpos/PAC-1neg/AnnexinVpos), aggregatory (CD42bpos/PAC-1pos/AnnexinVneg), and inflammatory (CD42bpos/CXCR4pos/ACKR3pos/AnnexinVpos) platelet subpopulations. Treatment with ACKR3 agonists significantly decreased the formation of procoagulant and ACKR3pos platelets in response to hemin. We conclude that hemin is a strong activator for the formation of procoagulant platelets and thrombus formation which is dependent on the function of ACKR3. Activation of ACKR3 using specific agonists may offer a therapeutic strategy to regulate the vulnerability of atherosclerotic plaques in areas of IPH.

АНТИБАКТЕРИАЛЬНЫЕ СВОЙСТВА ПОЛИМЕРНОЙ ФОРМЫ ГЕМИНА

Porphyrins are promising and modern antimicrobial agents. One of the best known representatives of this class of compounds is hemin. There is literature data on its high efficiency both in free form and as part of polymeric matrices. This paper presents an assessment of the purity of the hemin substance used in research, the study of the binding of hemin to a polymeric carrier, as well as analyze of the effectiveness of hemin-poly-N-vinylpyrrolidone (hemin-PVP) complex as an antimicrobial agent. It was found that the hemin-PVP and free form hemin had activity against S. aureus and E. coli. However, hemin-PVP compared to hemin substance had the advantage of prolonged action and increased antimicrobial activity. The results obtained indicate the prospect of using the hemin polymeric form as the alternative antimicrobial agent.

Trypanosoma cruzi heme responsive gene (TcHRG) plays a central role in orchestrating heme uptake in epimastigotes.

Trypanosoma cruzi, a heme auxotrophic parasite, can control intracellular heme content by modulating heme responsive gene (TcHRG) expression when a free heme source is added to an axenic culture. Herein, we explored the role of TcHRG protein in regulating the uptake of heme derived from hemoglobin in epimastigotes. We demonstrate that the endogenous TcHRG (protein and mRNA) responded similarly to bound (hemoglobin) and free (hemin) heme. Endogenous TcHRG was found in the flagellar pocket boundaries and partially overlapping with the mitochondrion. On the other hand, endocytic null parasites were able to develop and exhibited a similar heme content compared to wild-type when fed with hemoglobin, indicating that endocytosis is not the main entrance pathway for hemoglobin-derived heme in this parasite. Moreover, the overexpression of TcHRG led to an increase in heme content when hemoglobin was used as the heme source. Taken together, these results suggest that the uptake of hemoglobin-derived heme likely occurs through extracellular proteolysis of hemoglobin via the flagellar pocket, and this process is governed by TcHRG. In sum, T. cruzi epimastigotes control heme homeostasis by modulating TcHRG expression independently of the available source of heme.

The interaction of perfluorooctane sulfonate with hemoproteins and its relevance to molecular toxicology

Perfluorooctane sulfonate (PFOS), a representative of perfluorinated compounds in industrial and commercial products, has posed a great threat to animals and humans via environmental exposure and dietary consumption. Herein, we investigated the effects of PFOS binding on the redox state and stability of two hemoproteins (hemoglobin (Hb) and myoglobin (Mb)). Fluorescence spectroscopy, circular dichroism and UV–vis absorption spectroscopy demonstrated that PFOS could induce the conformational changes of proteins along with the exposure of heme cavity and generation of hemichrome, which resulted in the increased release of free hemin. After that, free hemin liberated from hemoproteins led to reactive oxygen species formation, lipid peroxidation, cell membrane damage and loss of cell viability in vascular endothelial cells, while neither Hb nor Mb did show cytotoxicity. Chemical inhibitors of ferroptosis effectively mitigated hemin-caused toxicity, identifying the hemin-dependent ferroptotic cell death mechanisms. These data demonstrated that PFOS posed a potential threat of toxicity through a mechanism which involved its binding to hemoproteins, decreased oxygen transporting capacity, and increased hemin release. Altogether, our findings elucidate the binding mechanisms of PFOS with two hemoproteins, as well as possible risks on vascular endothelial cells, which would have important implications for the human and environmental toxicity of PFOS.

Optimization and Enhancement of the Peroxidase-like Activity of Hemin in Aqueous Solutions of Sodium Dodecylsulfate.

Iron porphyrins play several important roles in present-day living systems and probably already existed in very early life forms. Hemin (= ferric protoporphyrin IX = ferric heme b), for example, is the prosthetic group at the active site of heme peroxidases, catalyzing the oxidation of a number of different types of reducing substrates after hemin is first oxidized by hydrogen peroxide as the oxidizing substrate of the enzyme. The active site of heme peroxidases consists of a hydrophobic pocket in which hemin is embedded noncovalently and kept in place through coordination of the iron atom to a proximal histidine side chain of the protein. It is this partially hydrophobic local environment of the enzyme which determines the efficiency with which the sequential reactions of the oxidizing and reducing substrates proceed at the active site. Free hemin, which has been separated from the protein moiety of heme peroxidases, is known to aggregate in an aqueous solution and exhibits low catalytic activity. Based on previous reports on the use of surfactant micelles to solubilize free hemin in a nonaggregated state, the peroxidase-like activity of hemin in the presence of sodium dodecyl sulfate (SDS) at concentrations below and above the critical concentration for SDS micelle formation (critical micellization concentration (cmc)) was systematically investigated. In most experiments, 3,3',5,5'-tetramethylbenzidine (TMB) was applied as a reducing substrate at pH = 7.2. The presence of SDS clearly had a positive effect on the reaction in terms of initial reaction rate and reaction yield, even at concentrations below the cmc. The highest activity correlated with the cmc value, as demonstrated for reactions at three different HEPES concentrations. The 4-(2-hydroxyethyl)-1-piperazineethanesulfonate salt (HEPES) served as a pH buffer substance and also had an accelerating effect on the reaction. At the cmc, the addition of l-histidine (l-His) resulted in a further concentration-dependent increase in the peroxidase-like activity of hemin until a maximal effect was reached at an optimal l-His concentration, probably corresponding to an ideal mono-l-His ligation to hemin. Some of the results obtained can be understood on the basis of molecular dynamics simulations, which indicated the existence of intermolecular interactions between hemin and HEPES and between hemin and SDS. Preliminary experiments with SDS/dodecanol vesicles at pH = 7.2 showed that in the presence of the vesicles, hemin exhibited similar peroxidase-like activity as in the case of SDS micelles. This supports the hypothesis that micelle- or vesicle-associated ferric or ferrous iron porphyrins may have played a role as primitive catalysts in membranous prebiotic compartment systems before cellular life emerged.

General Gel-sol Method to Synthesize Various Highly Fluorescent Nanoclusters and Assay of Nuclease with the Near Infrared-emitting Gold Nanoclusters.

A general egg white gel-sol strategy for fabrication of highly fluorescent Au, Ag, Cu, and Pt nanoclusters (NCs) and the first example of using Au NCs for assay of nuclease activity and inhibition were described. The Au NCs enabled bright red fluorescence, and the other Ag, Cu, and Pt NCs have highly blue emission. The red-emitting Au NCs were further applied in assay of S1 nuclease activity and inhibition. Free hemin efficiently quenches the emission of Au NCs by photoinduced electron transfer due to the formation of Au NCs-hemin conjugates. However, G-quadruplex/hemin exerts negligible effect on its fluorescence due to no Au NCs-hemin conjugate formed. There are stronger electrostatic repulsion effects between both negatively charged G-quadruplex and Au NCs. Therefore, a novel G-quadruplex/hemin-based Au NCs fluorescent sensor for S1 nuclease was designed. A known G-rich oligonucleotide (ODN) serves as not only substrate for S1 nuclease but also for the construction of G-quadruplex/hemin. The G-rich ODN is hydrolyzed into fragments by S1 nuclease resulting in no G-quadruplex/hemin formation. Therefore, the free hemin quenches Au NCs fluorescence remarkably and the assay of S1 nuclease activity and inhibition has accomplished. Both the fluorescent NCs syntheses and the detection of S1 nuclease are facile and efficient.

Self-assembled hemin-conjugated heparin with dual-enzymatic cascade reaction activities for acute kidney injury

Nanozymes have prominent catalytic activities with high stability as a substitute for unstable and expensive natural enzymes. However, most nanozymes are metal/inorganic nanomaterials, facing difficulty in clinical translation due to their unproven biosafety and limited biodegradability issues. Hemin, an organometallic porphyrin, was newly found to possess superoxide dismutase (SOD) mimetic activity along with previously known catalase (CAT) mimetic activity. However, hemin has poor bioavailability due to its low water solubility. Therefore, a highly biocompatible and biodegradable organic-based nanozyme system with SOD/CAT mimetic cascade reaction activity was developed by conjugating hemin to heparin (HepH) or chitosan (CS-H). Between them, Hep-H formed a smaller (<50 nm) and more stable self-assembled nanostructure and even possessed much higher and more stable SOD and CAT activities as well as the cascade reaction activity compared to CS-H and free hemin. Hep-H also showed a better cell protection effect against reactive oxygen species (ROS) compared to CS-H and hemin in vitro. Furthermore, Hep-H was selectively delivered to the injured kidney upon intravenous administration at the analysis time point (24 h) and exhibited excellent therapeutic effects on an acute kidney injury model by efficiently removing ROS, reducing inflammation, and minimizing structural and functional damage to the kidney.

The Subtilisin-Like Protease Furin Regulates Hemin-Dependent Ectodomain Shedding of Glycoprotein VI.

Hemolysis results in release of free hemoglobin and hemin liberation from erythrocytes. Hemin has been described to induce platelet activation and to trigger thrombosis. We evaluated the effect of hemin on platelet function and surface expression of the platelet collagen receptor glycoprotein VI (GPVI). Isolated platelets were stimulated with increasing concentrations of hemin. We found that hemin strongly enhanced platelet activation, aggregation, and aggregate formation on immobilized collagen under flow. In contrast, we found that surface expression of GPVI was significantly reduced upon hemin stimulation with high hemin concentrations indicating that hemin-induced loss of surface GPVI does not hinder platelet aggregation. Loss of hemin-induced surface expression of GPVI was caused by shedding of the ectodomain of GPVI as verified by immunoblotting and is independent of the GPVI or CLEC-2 mediated ITAM (immunoreceptor-tyrosine-based-activation-motif) signaling pathway as inhibitor studies revealed. Hemin-induced GPVI shedding was independent of metalloproteinases such as ADAM10 or ADAM17, which were previously described to regulate GPVI degradation. Similarly, concentration-dependent shedding of CD62P was also induced by hemin. Unexpectedly, we found that the subtilisin-like proprotein convertase furin controls hemin-dependent GPVI shedding as shown by inhibitor studies using the specific furin inhibitors SSM3 and Hexa-D-arginine. In the presence of SSM3 and Hexa-D-arginine, hemin-associated GPVI degradation was substantially reduced. Further, SSM3 inhibited hemin-induced but not CRP-XL-induced platelet aggregation and thrombus formation, indicating that furin controls specifically hemin-associated platelet functions. In summary, we describe a novel mechanism of hemin-dependent GPVI shedding and platelet function mediated by furin.

Release of hepatic xanthine oxidase (XO) to the circulation is protective in intravascular hemolytic crisis

Xanthine oxidase (XO) catalyzes the catabolism of hypoxanthine to xanthine and xanthine to uric acid, generating oxidants as a byproduct. Importantly, XO activity is elevated in numerous hemolytic conditions including sickle cell disease (SCD); however, the role of XO in this context has not been elucidated. Whereas long-standing dogma suggests elevated levels of XO in the vascular compartment contribute to vascular pathology via increased oxidant production, herein, we demonstrate, for the first time, that XO has an unexpected protective role during hemolysis. Using an established hemolysis model, we found that intravascular hemin challenge (40 μmol/kg) resulted in a significant increase in hemolysis and an immense (20-fold) elevation in plasma XO activity in Townes sickle cell phenotype (SS) sickle mice compared to controls. Repeating the hemin challenge model in hepatocyte-specific XO knockout mice transplanted with SS bone marrow confirmed the liver as the source of enhanced circulating XO as these mice demonstrated 100% lethality compared to 40% survival in controls. In addition, studies in murine hepatocytes (AML12) revealed hemin mediates upregulation and release of XO to the medium in a toll like receptor 4 (TLR4)-dependent manner. Furthermore, we demonstrate that XO degrades oxyhemoglobin and releases free hemin and iron in a hydrogen peroxide-dependent manner. Additional biochemical studies revealed purified XO binds free hemin to diminish the potential for deleterious hemin-related redox reactions as well as prevents platelet aggregation. In the aggregate, data herein reveals that intravascular hemin challenge induces XO release by hepatocytes through hemin-TLR4 signaling, resulting in an immense elevation of circulating XO. This increased XO activity in the vascular compartment mediates protection from intravascular hemin crisis by binding and potentially degrading hemin at the apical surface of the endothelium where XO is known to be bound and sequestered by endothelial glycosaminoglycans (GAGs).

Ferric heme b in aqueous micellar and vesicular systems: state-of-the-art and challenges.

Ferric heme b (= ferric protoporphyrin IX = hemin) is an important prosthetic group of different types of enzymes, including the intensively investigated and widely applied horseradish peroxidase (HRP). In HRP, hemin is present in monomeric form in a hydrophobic pocket containing among other amino acid side chains the two imidazoyl groups of His170 and His42. Both amino acids are important for the peroxidase activity of HRP as an axial ligand of hemin (proximal His170) and as an acid/base catalyst (distal His42). A key feature of the peroxidase mechanism of HRP is the initial formation of compound I under heterolytic cleavage of added hydrogen peroxide as a terminal oxidant. Investigations of free hemin dispersed in aqueous solution showed that different types of hemin dimers can form, depending on the experimental conditions, possibly resulting in hemin crystallization. Although it has been recognized already in the 1970s that hemin aggregation can be prevented in aqueous solution by using micelle-forming amphiphiles, it remains a challenge to prepare hemin-containing micellar and vesicular systems with peroxidase-like activities. Such systems are of interest as cheap HRP-mimicking catalysts for analytical and synthetic applications. Some of the key concepts on which research in this fascinating and interdisciplinary field is based are summarized, along with major accomplishments and possible directions for further improvement. A systematic analysis of the physico-chemical properties of hemin in aqueous micellar solutions and vesicular dispersions must be combined with a reliable evaluation of its catalytic activity. Future studies should show how well the molecular complexity around hemin in HRP can be mimicked by using micelles or vesicles. Because of the importance of heme b in virtually all biological systems and the fact that porphyrins and hemes can be obtained under potentially prebiotic conditions, ideas exist about the possible role of heme-containing micellar and vesicular systems in prebiotic times.

Fluorescent silicon nanoparticle-based quantitative hemin assay.

Hemin with functions such as oxygen carrying, oxygen storing, promoting redox, and performing electron transfer is important for the health of organisms. In this paper, green synthetic silicon nanoparticles (Si-NPs) were synthesized and used for free hemin detection in serum (a low limit of detection (LOD), 29.5 nM). The quenching mechanism was investigated by UV-vis absorption spectra, time-resolved luminescence decay curve, and circular dichroism (CD) spectra. It was confirmed that multiple redox centers of hemin led to intensified effective collision and increased the electron transfer rate, therefore enhancing the dynamic quenching, and it was undeniable that the inner filter effect (IFE) also played a role in the quenching.

Intracellular hemin is a potent inhibitor of the voltage-gated potassium channel Kv10.1

Heme, an iron-protoporphyrin IX complex, is a cofactor bound to various hemoproteins and supports a broad range of functions, such as electron transfer, oxygen transport, signal transduction, and drug metabolism. In recent years, there has been a growing recognition of heme as a non-genomic modulator of ion channel functions. Here, we show that intracellular free heme and hemin modulate human ether à go-go (hEAG1, Kv10.1) voltage-gated potassium channels. Application of hemin to the intracellular side potently inhibits Kv10.1 channels with an IC50 of about 4 nM under ambient and 63 nM under reducing conditions in a weakly voltage-dependent manner, favoring inhibition at resting potential. Functional studies on channel mutants and biochemical analysis of synthetic and recombinant channel fragments identified a heme-binding motif CxHx8H in the C-linker region of the Kv10.1 C terminus, with cysteine 541 and histidines 543 and 552 being important for hemin binding. Binding of hemin to the C linker may induce a conformational constraint that interferes with channel gating. Our results demonstrate that heme and hemin are endogenous modulators of Kv10.1 channels and could be exploited to modulate Kv10.1-mediated cellular functions.

Soy protein isolate (SPI)-hemin complex nanoparticles as a novel water-soluble iron-fortifier: Fabrication, formation mechanism and in vitro bioavailability

Hemin is recognized as a superior biological iron-fortifier in the prevention and treatment of iron deficiency anemia. Nevertheless, free hemin could have low bioavailability due to its insolubility in intestinal fluid after passing through the digestive tract, so hemin as a direct fortifier is not as effective as it should be. In this study, a novel hemin-delivery system with good water-solubility was developed by fabricating soy protein isolate (SPI)-hemin complex nanoparticles through pH-driven method, and the formation mechanism and bioavailability were also investigated. Hemin could bind to SPI and form nanoparticles with a diameter ranging from 100 to 300 nm in the process of pH dropping from 12.0 to 7.0, and thereby significantly improve the water-solubility of hemin. It was proved that the nanoparticles were produced mainly through hydrophobic interaction between SPI and hemin, and driven by entropy. The results from in vitro simulated gastrointestinal digestion experiments indicated that bioavailability of hemin was also significantly improved (P < 0.05), and the value in SPI-hemin nanoparticles reached nearly 80% at the concentrations of 1.0% (w/v) SPI and 0.25% (w/v) hemin. SPI-hemin nanoparticles could be an innovation for improving the solubility and bioavailability of hemin and an easily acceptable candidate for the design of novel iron-fortified foods.

Pro-oxidative activity of trout and bovine hemoglobin during digestion using a static in vitro gastrointestinal model

The degradation of trout and bovine hemoglobin (Hb) and their pro-oxidant activities in washed cod muscle mince (WCM) were studied using simple pH-shifts to simulate gastrointestinal (GI) conditions (pH 7 → 6 → 3 → 7), as well as full static in vitro GI digestion. Following gastric acidification to pH 6, metHb formation increased, especially for trout Hb. Subsequent acidification to pH 3 promoted Hb unfolding and partial or complete heme group-loss. During full GI digestion, polypeptide/peptide analyses revealed more extensive Hb-degradation in the gastric than duodenal phase, without any species-differences. When digesting WCM +/-Hb, both Hbs strongly promoted malondialdehyde (MDA), 4-hydroxy-2-hexenal (HHE), and 4-hydroxy-2-nonenal (HNE) formation, peaking at the end of the gastric phase. Trout-Hb stimulated MDA and HHE more than bovine Hb in the first gastric phase. Altogether, partially degraded Hb, and/or free hemin -both mammal and fish-derived- stimulated oxidation of PUFA-rich lipids under GI-conditions, especially gastric ones.

Protein nitration induced by Hemin/NO: A complementary mechanism through the catalytic functions of hemin and NO-scavenging

Hemin and heme-peroxidases have been considered essential catalysts for the nitrite/hydrogen peroxide (H2O2)-mediated protein nitration in vitro, understood as one of the main pathways for protein modification in biological systems. However, the role of nitric oxide (●NO) in the heme/hemin-induced protein nitration has not been studied in-depth. This is despite its reductive nitrosylating effects following binding to hemin and the possible involvement of the reactive nitrogen species in the nitration of various functional proteins. Here, the ●NO-binding affinity of hemin has been studied along with the influence of ●NO on the internalization of hemin into MDA-MB-231 cells and the accompanying changes in the profile of intracellular nitrated proteins. Moreover, to further understand the mechanism involved, bovine serum albumin (BSA) nitration was studied after treatment with hemin and ●NO, with an investigation of the effects of pH of the reaction medium, generation of H2O2, and the oxidation of the tyrosine residues as the primary sites for the nitration. We demonstrated that hemin nitrosylation enhanced its cellular uptake and induced the one-electron oxidation and nitration of different intracellular proteins along with its ●NO-scavenging efficiency. Moreover, the hemin/NO-mediated BSA nitration was proved to be dependent on the concentration of ●NO and the pH of the reaction medium, with a vital role being played by the scavenging effects of protein for the free hemin molecules. Collectively, our results reaffirm the involvement of hemin and ●NO in the nitration mechanism, where the nitrosylation products can induce protein nitration while promoting the effects of the components of the nitrite/H2O2-mediated pathway.

Insertion of Hemin into Metal-Organic Frameworks: Mimicking Natural Peroxidase Microenvironment for the Rapid Ultrasensitive Detection of Uranium.

Constructing enzyme-like active sites in mimic enzyme systems is critical for achieving catalytic performances comparable to natural enzymes and can shed light on the natural development of enzymes. In this study, we described a specific hemin-based mimetic enzyme, which was facilely synthesized by the assembly of zeolitic imidazolate framework-l (ZIF-l) and hemin. The obtained hemin-based mimetic enzyme (denoted as ZIF-l-hemin) displayed enhanced peroxidase activity compared to free hemin in solution. Such excellent activity originated from the ZIF-l framework mimicking the active site cavity microenvironment of horseradish peroxidase in terms of axially coordinated histidine and distal histidine. Additionally, the constructed peroxidase mimetic was extremely resistant to a variety of severe circumstances that would normally denature natural enzymes. These characteristics made ZIF-l-hemin a potential platform for the colorimetric sensor of uranium (UO22+) with wide linear ranges (0.25-40 μM) and low limits of detection (0.079 μM). Moreover, the detection mechanism demonstrated that the coordination of uranyl ion with imidazole of ZIF-l-hemin reduced the catalytic efficiency of ZIF-l-hemin. The current work not only proposed a novel approach for fabricating artificial peroxidase but also offered facile colorimetric methods for selective radionuclide detection.

Biomaterial and tissue-engineering strategies for the treatment of brain neurodegeneration.

The incidence of neurodegenerative diseases is increasing due to changing age demographics and the incidence of sports-related traumatic brain injury is tending to increase over time. Currently approved medicines for neurodegenerative diseases only temporarily reduce the symptoms but cannot cure or delay disease progression. Cell transplantation strategies offer an alternative approach to facilitating central nervous system repair, but efficacy is limited by low in vivo survival rates of cells that are injected in suspension. Transplanting cells that are attached to or encapsulated within a suitable biomaterial construct has the advantage of enhancing cell survival in vivo. A variety of biomaterials have been used to make constructs in different types that included nanoparticles, nanotubes, microspheres, microscale fibrous scaffolds, as well as scaffolds made of gels and in the form of micro-columns. Among these, Tween 80-methoxy poly(ethylene glycol)-poly(lactic-co-glycolic acid) nanoparticles loaded with rhynchophylline had higher transport across a blood-brain barrier model and decreased cell death in an in vitro model of Alzheimer’s disease than rhynchophylline or untreated nanoparticles with rhynchophylline. In an in vitro model of Parkinson’s disease, trans-activating transcriptor bioconjugated with zwitterionic polymer poly(2-methacryoyloxyethyl phosphorylcholine) and protein-based nanoparticles loaded with non-Fe hemin had a similar protective ability as free non-Fe hemin. A positive effect on neuron survival in several in vivo models of Parkinson’s disease was associated with the use of biomaterial constructs such as trans-activating transcriptor bioconjugated with zwitterionic polymer poly(2-methacryoyloxyethyl phosphorylcholine) and protein-based nanoparticles loaded with non-Fe hemin, carbon nanotubes with olfactory bulb stem cells, poly(lactic-co-glycolic acid) microspheres with attached DI-MIAMI cells, ventral midbrain neurons mixed with short fibers of poly-(L-lactic acid) scaffolds and reacted with xyloglucan with/without glial-derived neurotrophic factor, ventral midbrain neurons mixed with Fmoc-DIKVAV hydrogel with/without glial-derived neurotrophic factor. Further studies with in vivo models of Alzheimer’s disease and Parkinson’s disease are warranted especially using transplantation of cells in agarose micro-columns with an inner lumen filled with an appropriate extracellular matrix material.

Free radical-mediated acetaldehyde formation by model reactions of dietary components: effects of meat, wine, cooking oil and coffee

BackgroundAlcohol consumption and the ingestion of red meat and oxidized cooking oil are risk factors of gastric and colorectal cancers. We reported that acetaldehyde (AcAld) is generated from Heme/Mb/Meat-Linoleate-EtOH model reaction mixtures, and thus could be a new plausible mechanism for the carcinogenesis (Kasai and Kawai, ACS Omega, 2021).ResultsIn this study, we investigated the effects of wine and coffee, in addition to meat components, on this reaction. Depending on the conditions, such as pH, reaction time and choice of free hemin, myoglobin (Mb), as well as meat extracts (raw meat, baked meat, salami), wine and coffee enhanced AcAld formation. Polyphenols in red wine and coffee may stimulate AcAld formation by acting as pro-oxidants in the presence of Heme/Mb/Meat. In a model reaction of Mb + EtOH + H2O2, we observed time-dependent AcAld formation. In support of these in vitro data, after the consumption of a red meat-rich diet with red wine, the fecal AcAld level significantly increased as compared to the levels associated with a diet of fish + wine, or red meat without alcohol.ConclusionsThese results suggested that AcAld generation from dietary components may be an important mechanism of gastrointestinal tract carcinogenesis.

Neutrophil Extracellular Traps Promote Lung Arteriole Occlusion in Sickle Cell Disease

Introduction:Sickle cell disease (SCD) is an autosomal-recessive-genetic disorder, which leads to red blood cell sickling and hemolysis. Acute systemic painful vaso-occlusive crisis (VOC) is the predominant pathophysiology requiring emergency medical care by SCD patients. 10-20% of SCD patients hospitalized with VOC tend to develop acute chest syndrome (ACS), a type of lung injury within next few days, suggesting a role for pulmonary vaso-occlusion in ACS. The cellular, molecular and biophysical mechanism of pulmonary vaso-occlusion has just started to unfold and a profound understanding is needed for the development of rescue therapies to halt the progression of VOC to ACS. Our recent findings using quantitative fluorescence intravital lung microscopy (qFILM) revealed that lung vaso-occlusion is enabled by the entrapment of embolic neutrophil-platelet aggregates in the pulmonary arterioles of transgenic humanized SCD mice. Neutrophil extracellular traps (NETs) are web-like structures of decondensed nuclear DNA decorated with citrunilated-histones and neutrophil granule proteins. NETs are released by activated neutrophils under inflammatory conditions and are known to possess pro-inflammatory and pro-thrombotic properties. Recently, hemolysis driven release of cell free hemoglobin or hemin has been shown to promote NETs formation in the pulmonary microcirculation of SCD mice. Nevertheless, how these NETs can serve to facilitate pulmonary vaso-occlusion and promote ACS is still unclear.Materials and Methods:Townes knock-in humanized SS (hα/hα:βS/βS) and AS (hα/hα:βA/βS) mice were used as SCD and control mice, respectively. SS and AS mice were intravascularly (IV) challenged with Oxy-hemoglobin (Oxy-Hb) and hemin to model VOC. Fluorescently-conjugated anti-mouse CD49b and Ly6G mAbs, Sytox Orange, and FITC dextran were administered IV to visualize platelets, neutrophils, extracellular DNA and lung vasculature in the mouse, respectively. Lung microcirculation was visualized in real time in vivo using qFILM and analyzed as described elsewhere. Also, SCD or control human blood with or without treatment with Oxy-Hb or hemin was perfused through in vitro microfluidic microchannels presenting a combination of P-selectin, ICAM-1 and IL-8, and neutrophil-platelet interactions as well as NET formation was visualized using Quantitative Microfluidic Fluorescence Microscopy (qMFM). Fluorescent anti-human mAbs against CD49b, CD16, citrullinated histones (H3-Cit) and neutrophil elastase were added to human blood to visualize platelets, neutrophils, histones, and neutrophil elastase, respectively.Results and Discussion:Our initial findings using qFILMreveal that non-lethal dose of 20 µmole/kg IV hemin or 10 µmole/kg IV Oxy-Hb led to entrapment of neutrophil-platelet embolic aggregates in the lung arteriolar bottlenecks of SCD but not control mice. These aggregates resulted loss of blood perfusion in the lung, followed by shedding of NETs from neutrophils into the blood circulation.Conclusion: Erythroid DAMPs, hemin and Oxy-Hb promote occlusion of lung arterioles by neutrophil-platelet aggregates in SCD mice. These aggregates promote loss of blood flow and release of NETs from neutrophils in the lung arterioles. DisclosuresKato:Bayer: Research Funding; Global Blood Therapeutics: Consultancy; Novartis: Consultancy; MAST Therapeutics: Research Funding.