Binding and Activation of LRP1-Dependent Cell Signaling in Schwann Cells Using a Peptide Derived from the Hemopexin Domain of MMP-9.

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following a peripheral nervous system injury. The low-density lipoprotein receptor-related protein-1 (LRP1) is significantly upregulated in SCs in response to acute injury, activating cJun and promoting SC survival. Matrix-metalloproteinase-9 (MMP-9) is an LRP1 ligand that binds LRP1 through its hemopexin domain (PEX) and activates SC survival signaling and migration. To identify novel peptide mimetics within the hemopexin domain of MMP-9, we examined the crystal structure of PEX, synthesized four peptides, and examined their potential to bind and activate LRP1. We demonstrate that a 22 amino acid peptide, peptide 2, was the only peptide that activated Akt and ERK1/2 signaling in SCs, similar to a glutathione s-transferase (GST)-fused holoprotein, GST-PEX. Intraneural injection of peptide 2, but not vehicle, into crush-injured sciatic nerves activated cJun greater than 2.5-fold in wild-type mice, supporting that peptide 2 can activate the SC repair signaling in vivo. Peptide 2 also bound to Fc-fusion proteins containing the ligand-binding motifs of LRP1, clusters of complement-like repeats (CCRII and CCRIV). Pulldown and computational studies of alanine mutants of peptide 2 showed that positively charged lysine and arginine amino acids within the peptide are critical for stability and binding to CCRII. Collectively, these studies demonstrate that a novel peptide derived from PEX can serve as an LRP1 agonist and possesses qualities previously associated with LRP1 binding and SC signaling in vitro and in vivo.

Hemopexin reverses activation of lung eIF2α and decreases mitochondrial injury in chlorine-exposed mice.

We assessed the mechanisms by which nonencapsulated heme, released in the plasma of mice after exposure to chlorine (Cl2) gas, resulted in the initiation and propagation of acute lung injury. We exposed adult male and female C57BL/6 mice to Cl2 (500 ppm for 30 min), returned them to room air, and injected them intramuscularly with either human hemopexin (hHPX; 5 µg/g BW in 50-µL saline) or vehicle at 1 h post-exposure. Upon return to room air, Cl2-exposed mice, injected with vehicle, developed respiratory acidosis, increased concentrations of plasma proteins in the alveolar space, lung mitochondrial DNA injury, increased levels of free plasma heme, and major alterations of their lung proteome. hHPX injection mice mitigated the onset and development of lung and mitochondrial injury and the increase of plasma heme, reversed the Cl2-induced changes in 83 of 237 proteins in the lung proteome at 24 h post-exposure, and improved survival at 15 days post-exposure. Systems biology analysis of the lung global proteomics data showed that hHPX reversed changes in a number of key pathways including elF2 signaling, verified by Western blotting measurements. Recombinant human hemopexin, generated in tobacco plants, injected at 1 h post-Cl2 exposure, was equally effective in reversing acute lung and mtDNA injury. The results of this study offer new insights as to the mechanisms by which exposure to Cl2 results in acute lung injury and the therapeutic effects of hemopexin.NEW & NOTEWORTHY Herein, we demonstrate that exposure of mice to chlorine gas causes significant changes in the lung proteome 24 h post-exposure. Systems biology analysis of the proteomic data is consistent with damage to mitochondria and activation of eIF2, the master regulator of transcription and protein translation. Post-exposure injection of hemopexin, which scavenges free heme, attenuated mtDNA injury, eIF2α phosphorylation, decreased lung injury, and increased survival.

Modulation of Heme-Induced Inflammation Using MicroRNA-Loaded Liposomes: Implications for Hemolytic Disorders Such as Malaria and Sickle Cell Disease.

Hemolytic disorders, like malaria and sickle cell disease (SCD), are responsible for significant mortality and morbidity rates globally, specifically in the Americas and Africa. In both malaria and SCD, red blood cell hemolysis leads to the release of a cytotoxic heme that triggers the expression of unique inflammatory profiles, which mediate the tissue damage and pathogenesis of both diseases. MicroRNAs (miRNAs), such as miR-451a and let-7i-5p, contribute to a reduction in the pro-inflammatory responses induced by circulating free hemes. MiR-451a targets both IL-6R (pro-inflammatory) and 14-3-3ζ (anti-inflammatory), and when this miRNA is present, IL-6R is reduced and 14-3-3ζ is increased. Let-7i-5p targets and reduces TLR4, which results in anti-inflammatory signaling. These gene targets regulate inflammation via NFκB regulation and increase anti-inflammatory signaling. Additionally, they indirectly regulate the expression of key heme scavengers, such as heme-oxygenase 1 (HO-1) (coded by the HMOX1 gene) and hemopexin, to decrease circulating cytotoxic heme concentration. MiRNAs can be transported within extracellular vesicles (EVs), such as exosomes, offering insights into the mechanisms of mitigating heme-induced inflammation. We tested the hypothesis that miR-451a- or let-7i-5p-loaded artificial EVs (liposomes) will reduce heme-induced inflammation in brain vascular endothelial cells (HBEC-5i, ATCC: CRL-3245) and macrophages (THP-1, ATCC: TIB-202) in vitro. We completed arginase and nitric oxide assays to determine anti- and pro-inflammatory macrophage presence, respectively. We also assessed the gene expression of IL-6R, TLR4, 14-3-3ζ, and NFκB by RT-qPCR for both cell lines. Our findings revealed that the exposure of HBEC-5i and THP-1 to liposomes loaded with miR-451a or let-7i-5p led to a reduced mRNA expression of IL-6R, TLR4, 14-3-3ζ, and NFκB when treated with a heme. It also resulted in the increased expression of HMOX1 and hemopexin. Finally, macrophages exhibited a tendency toward adopting an anti-inflammatory differentiation phenotype. These findings suggest that miRNA-loaded liposomes can modulate heme-induced inflammation and can be used to target specific cellular pathways, mediating inflammation common to hematological conditions, like malaria and SCD.

Double-edged functions of hemopexin in hematological related diseases: from basic mechanisms to clinical application.

It is now understood that hemolysis and the subsequent release of heme into circulation play a critical role in driving the progression of various diseases. Hemopexin (HPX), a heme-binding protein with the highest affinity for heme in plasma, serves as an effective antagonist against heme toxicity resulting from severe acute or chronic hemolysis. In the present study, changes in HPX concentration were characterized at different stages of hemolytic diseases, underscoring its potential as a biomarker for assessing disease progression and prognosis. In many heme overload-driven conditions, such as sickle cell disease, transfusion-induced hemolysis, and sepsis, endogenous HPX levels are often insufficient to provide protection. Consequently, there is growing interest in developing HPX therapeutics to mitigate toxic heme exposure. Strategies include HPX supplementation when endogenous levels are depleted and enhancing HPX's functionality through modifications, offering a potent defense against heme toxicity. It is worth noting that HPX may also exert deleterious effects under certain circumstances. This review aims to provide a comprehensive overview of HPX's roles in the progression and prognosis of hematological diseases. It highlights HPX-based clinical therapies for different hematological disorders, discusses advancements in HPX production and modification technologies, and offers a theoretical basis for the clinical application of HPX.

Free heme and hemopexin in acute kidney injury after cardiopulmonary bypass and transient renal ischemia.

Free heme is released from hemoproteins during hemolysis or ischemia reperfusion injury and can be pro-inflammatory. Most studies on nephrotoxicity of hemolysis-derived proteins focus on free hemoglobin (fHb) with heme as a prosthetic group. Measurement of heme in its free, non-protein bound, form is challenging and not commonly used in clinical routine diagnostics. In contrast to fHb, the role of free heme in acute kidney injury (AKI) after cardiopulmonary bypass (CPB) surgery is unknown. Using an apo-horseradish peroxidase-based assay, we identified free heme during CPB surgery as predictor of AKI in patients undergoing cardiac valve replacement (n = 37). Free heme levels during CPB surgery correlated with depletion of hemopexin (Hx), a heme scavenger-protein. In mice, the impact of high levels of circulating free heme on the development of AKI following transient renal ischemia and the therapeutic potential of Hx were investigated. C57BL/6 mice were subjected to bilateral renal ischemia/reperfusion injury for 15 min which did not cause AKI. However, additional administration of free heme in this model promoted overt AKI with reduced renal function, increased renal inflammation, and reduced renal perfusion on functional magnetic resonance imaging. Hx treatment attenuated AKI. Free heme administration to sham operated control mice did not cause AKI. In conclusion, free heme is a predictor of AKI in CPB surgery patients and promotes AKI in transient renal ischemia. Depletion of Hx in CPB surgery patients and attenuation of AKI by Hx in the in vivo model encourage further research on Hx therapy in patients with increased free heme levels during CPB surgery.

Accelerated atherosclerosis in beta-thalassemia.

Children with beta-thalassemia (BT) present with an increase in carotid intima-medial thickness, an early sign suggestive of premature atherosclerosis. However, it is unknown if there is a direct relationship between BT and atherosclerotic disease. To evaluate this, wild-type (WT, littermates) and BT (Hbbth3/+) mice, both male and female, were placed on a 3-mo high-fat diet with low-density lipoprotein receptor suppression via overexpression of proprotein convertase subtilisin/kexin type 9 (PCSK9) gain-of-function mutation (D377Y). Mechanistically, we hypothesize that heme-mediated oxidative stress creates a proatherogenic environment in BT because BT is a hemolytic anemia that has increased free heme and exhausted hemopexin, heme's endogenous scavenger, in the vasculature. We evaluated the effect of hemopexin (HPX) therapy, mediated via an adeno-associated virus, to the progression of atherosclerosis in BT and a phenylhydrazine-induced model of intravascular hemolysis. In addition, we evaluated the effect of deferiprone (DFP)-mediated iron chelation in the progression of atherosclerosis in BT mice. Aortic en face and aortic root lesion area analysis revealed elevated plaque accumulation in both male and female BT mice compared with WT mice. Hemopexin therapy was able to decrease plaque accumulation in both BT mice and mice on our phenylhydrazine (PHZ)-induced model of hemolysis. DFP decreased atherosclerosis in BT mice but did not provide an additive benefit to HPX therapy. Our data demonstrate for the first time that the underlying pathophysiology of BT leads to accelerated atherosclerosis and shows that heme contributes to atherosclerotic plaque development in BT.NEW & NOTEWORTHY This work definitively shows for the first time that beta-thalassemia leads to accelerated atherosclerosis. We demonstrated that intravascular hemolysis is a prominent feature in beta-thalassemia and the resulting increases in free heme are mechanistically relevant. Adeno-associated virus (AAV)-hemopexin therapy led to decreased free heme and atherosclerotic plaque area in both beta-thalassemia and phenylhydrazine-treated mice. Deferiprone-mediated iron chelation led to deceased plaque accumulation in beta-thalassemia mice but provided no additive benefit to hemopexin therapy.

Blocking Hemopexin With Specific Antibodies: A New Strategy for Treating Diabetic Retinopathy.

Hemopexin (HPX) is overexpressed in the retina of patients with diabetes and induces the breakdown of the blood-retinal barrier in vitro. The aim of this study was to evaluate whether HPX blockade by specific antibodies (aHPX) could avoid vascular leakage in vivo and microvascular angiogenesis in vitro and ex vivo. For this purpose, the effect of intravitreal (IVT) injections of aHPX on vascular leakage was evaluated in db/db mice and rats with streptozotocin-induced diabetes using the Evans Blue method. Retinal neurodegeneration and inflammation were also evaluated. The antiangiogenic effect of aHPX on human retinal endothelial cells (HRECs) was tested by scratch wound healing and tube formation using standardized methods, as well as by choroidal sprouting assays from retinal explants obtained in rats. We found that IVT injection of aHPX significantly reduced vascular leakage, retinal neurodegeneration, and inflammation. In addition, treatment with aHPX significantly reduced HREC migration and tube formation induced by high glucose concentration and suppressed choroidal sprouting even after vascular endothelial growth factor stimulation, with this effect being higher than obtained with bevacizumab. The antipermeability and antiangiogenic effects of IVT injection of aHPX suggest the blockade or inhibition of HPX as a new strategy for the treatment of advanced stages of diabetic retinopathy. ARTICLE HIGHLIGHTS: Hemopexin (HPX) is the best-characterized permeability factor in steroid-sensitive nephrotic syndrome. We have previously reported that HPX is overexpressed in the retina of patients with diabetes and induces the breakdown of the blood-retinal barrier in vitro. Here, we report that intravitreal injection of anti-HPX antibodies significantly reduces vascular leakage, retinal neurodegeneration, and inflammation in diabetic murine models and that the immunoneutralization of HPX exerts a significant antiangiogenic effect in vitro and in retinal explants. The blockade of HPX can be considered as a new therapy for advanced stages of diabetic retinopathy.

Screening Serum Biomarkers for Rats Preconditioned with Hyperbaric Oxygen: Potential of Predicting Prognosis for Stroke.

BACKGROUND: Stroke is a major health concern and a leading cause of mortality and morbidity. We and other groups have documented that hyperbaric oxygen preconditioning could significantly alleviate neuronal damage in ischemia‒reperfusion models through various mechanisms. However, we found that some of the subjects did not benefit from preconditioning with hyperbaric oxygen. The preconditioning phenomenon is similar to vaccination, in which the endogenous survival system is activated to fight against further injuries. However, with vaccine inoculations, we could test for specific antibodies against the pathogens to determine if the vaccination was successful. Likewise, this experiment was carried out to explore a biomarker that can reveal the effectiveness of the preconditioning before neuronal injury occurs. METHODS: Middle cerebral artery occlusion (MCAO) was used to induce focal cerebral ischemia-reperfusion injury. 2D-DIGE-MALDI-TOF-MS/MS proteomic technique was employed to screen the differentially expressed proteins in the serum of rats among the control (Con) group (MCAO model without hyperbaric oxygen (HBO) preconditioning), hyperbaric oxygen protective (HBOP) group (in which the infarct volume decreased after HBO preconditioning vs. Con), and hyperbaric oxygen nonprotective (HBOU) group (in which the infarct volume remained the same or even larger after HBO preconditioning vs. Con). Candidate biomarkers were confirmed by western blot and enzyme linked immunosorbent assay (ELISA), and the relationship between the biomarkers and the prognosis of cerebral injury was further validated. RESULTS: Among the 15 differentially expressed protein spots detected in the HBOP group by Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), 3 spots corresponding to 3 different proteins (haptoglobin, serum albumin, and haemopexin) products were identified by MALDI-TOF-MS/MS. Serum albumin and haemopexin were upregulated, and haptoglobin was downregulated in the HBOP group (p < 0.05 vs. Con and HBOU groups). After the western blot study, only the changes in haemopexin were validated and exhibited similar changes in subjects from the HBOP group in accordance with MALDI-TOF-MS/MS proteomic analysis and enzyme linked immunosorbent assay (ELISA) analysis. The serum level of the hemopexin (HPX) at 2 h after HBO preconditioning was correlated with the infarct volume ratio after MCAO. CONCLUSIONS: Haemopexin may be developed as a predictive biomarker that indicated the effectiveness of a preconditioning strategy against cerebral ischaemic injury.

Safety and feasibility of the gene transfer of hemopexin for conditions with increased free heme.

Heme is a fundamental molecule for several biological processes, but when released in the extracellular space such as in hemolytic diseases, it can be toxic to cells and tissues. Hemopexin (HPX) is a circulating protein responsible for removing free heme from the circulation, whose levels can be severely depleted in conditions such as sickle cell diseases. Accordingly, increasing HPX levels represents an attractive strategy to mitigate the deleterious effects of heme in these conditions. Gene transfer of liver-produced proteins with adeno-associated virus (AAV) has been shown to be an effective and safety strategy in animal and human studies mainly in hemophilia. Here, we report the feasibility of increasing HPX levels using an AAV8 vector expressing human HPX (hHPX). C57Bl mice were injected with escalating doses of our vector, and expression was assessed by enzyme immunoassay (ELISA), Western blot, and quantitative polymerase chain reaction (qPCR). In addition, the biological activity of transgenic hHPX was confirmed using two different models of heme challenge consisting of serial heme injections or phenylhydrazine-induced hemolysis. Sustained expression of hHPX was confirmed for up to 26 weeks in plasma. Expression was dose-dependent and not associated with clinical signs of toxicity. hHPX levels were significantly reduced by heme infusions and phenylhydrazine-induced hemolysis. No clinical toxicity or laboratory signs of liver damage were observed in preliminary short-term heme challenge studies. Our results confirm that long-term expression of hHPX is feasible and safe in mice, even in the presence of heme overload. Additional studies are needed to explore the effect of transgenic HPX protein in animal models of chronic hemolysis.

Free heme exacerbates colonic injury induced by anti-cancer therapy.

Gastrointestinal inflammation and bleeding are commonly induced by cancer radiotherapy and chemotherapy but mechanisms are unclear. We demonstrated an increased number of infiltrating heme oxygenase-1 positive (HO-1+) macrophages (Mø, CD68+) and the levels of hemopexin (Hx) in human colonic biopsies from patients treated with radiation or chemoradiation versus non-irradiated controls or in the ischemic intestine compared to matched normal tissues. The presence of rectal bleeding in these patients was also correlated with higher HO-1+ cell infiltration. To functionally assess the role of free heme released in the gut, we employed myeloid-specific HO-1 knockout (LysM-Cre : Hmox1flfl), hemopexin knockout (Hx-/-) and control mice. Using LysM-Cre : Hmox1flfl conditional knockout (KO) mice, we showed that a deficiency of HO-1 in myeloid cells led to high levels of DNA damage and proliferation in colonic epithelial cells in response to phenylhydrazine (PHZ)-induced hemolysis. We found higher levels of free heme in plasma, epithelial DNA damage, inflammation, and low epithelial cell proliferation in Hx-/- mice after PHZ treatment compared to wild-type mice. Colonic damage was partially attenuated by recombinant Hx administration. Deficiency in Hx or Hmox1 did not alter the response to doxorubicin. Interestingly, the lack of Hx augmented abdominal radiation-mediated hemolysis and DNA damage in the colon. Mechanistically, we found an altered growth of human colonic epithelial cells (HCoEpiC) treated with heme, corresponding to an increase in Hmox1 mRNA levels and heme:G-quadruplex complexes-regulated genes such as c-MYC, CCNF, and HDAC6. Heme-treated HCoEpiC cells exhibited growth advantage in the absence or presence of doxorubicin, in contrast to poor survival of heme-stimulated RAW247.6 Mø. In summary, our data indicate that accumulation of heme in the colon following hemolysis and/or exposure to genotoxic stress amplifies DNA damage, abnormal proliferation of epithelial cells, and inflammation as a potential etiology for gastrointestinal syndrome (GIS).

Degradomic Identification of Membrane Type 1-Matrix Metalloproteinase as an ADAMTS9 and ADAMTS20 Substrate.

The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative terminomics method, terminal amine isotopic labeling of substrates was used to compare the parental and gene-edited RPE-1 cells and their medium to identify ADAMTS9 substrates. Among differentially abundant neo-amino (N) terminal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr314-Gly315 bond in the ectodomain of the transmembrane metalloprotease membrane type 1-matrix metalloproteinase (MT1-MMP), whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge, that is, between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by reexpression of ADAMTS9 and ADAMTS20 and was dependent on hinge O-glycosylation. A C-terminally semitryptic MT1-MMP peptide with greater abundance in WT RPE-1 medium identified a second ADAMTS9 cleavage site in the MT1-MMP hemopexin domain. Consistent with greater retention of MT1-MMP on the surface of gene-edited cells, pro-MMP2 activation, which requires cell surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell surface activity of MT1-MMP, and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.

Assessment of total and unbound cell-free heme in plasma of patients with sickle cell disease.

Intravascular hemolysis results in the release of cell-free hemoglobin and heme in plasma. In sickle cell disease, the fragility of the sickle red blood cell leads to chronic hemolysis, which can contribute to oxidative damage and activation of inflammatory pathways. The scavenger proteins haptoglobin and hemopexin provide pathways to remove hemoglobin and heme, respectively, from the circulation. Heme also intercalates in membranes of blood cells and endothelial cells in the vasculature and associates with other plasma components such as albumin and lipoproteins. Hemopexin has a much higher affinity and can strip heme from the other pools and detoxify plasma from cell-free circulatory heme. However, due to chronic hemolysis, hemopexin is depleted in individuals with sickle cell disease. Thus, cell-free unbound heme is expected to accumulate in plasma. We developed a methodology for the accurate quantification of the fraction of heme, which is pathologically relevant in sickle cell disease, that does not appear to be sequestered to a plasma compartment. Our data show significant variation in the concentration of unbound heme, and rather unexpectedly, the size of the unbound fraction does not correlate to the degree of hemolysis, as measured by the concentration of bound heme. Very high heme concentrations (>150 µM) were obtained in some plasma with unbound concentrations that were several fold lower than in plasma with much lower hemolysis (<50 µM). These findings underscore the long-term effects of chronic hemolysis on the blood components and of the disruption of the essential equilibrium between release of hemoproteins/heme in the circulation and adaptative response of the scavenging/removal mechanisms. Understanding the clinical implications of this loss of response may provide insights into diagnostic and therapeutic targets in patients with sickle cell disease.

Different-sized extracellular vesicles derived from stored red blood cells package diverse cargoes and cause distinct cellular effects.

BACKGROUND: The formation of extracellular vesicles (EVs) occurs during cold storage of RBCs. Transfusion of EVs may contribute to adverse responses in recipients receiving RBCs. However, EVs are poorly characterized with limited data on whether distinct vesicles are formed, their composition, and potential biological effects. STUDY DESIGN AND METHODS: Stored RBC-derived EVs were purified using protocols that separate larger microvesicle-like EVs (LEVs) from smaller exosome-like vesicles (SEVs). Vesicles were analyzed by electron microscopy, content of hemoglobin, heme, and proteins (by mass spectrometry), and the potential to mediate lipid peroxidation and endothelial cell permeability in vitro. RESULTS: SEVs were characterized by having an electron-dense double membrane whereas LEVs had more uniform electron density across the particles. No differences in hemoglobin nor heme levels per particle were observed, however, due to smaller volumes, SEVs had higher concentrations of oxyHb and heme. Both particles contained antioxidant proteins peroxiredoxin-2 and copper/zinc superoxide dismutase, these were present in higher molecular weight fractions in SEVs suggesting either oxidized proteins are preferentially packaged into smaller vesicles and/or that the environment associated with SEVs is more pro-oxidative. Furthermore, total glutathione (GSH + GSSG) levels were lower in SEVs. Both EVs mediated oxidation of liposomes that were prevented by hemopexin, identifying heme as the pro-oxidant effector. Addition of SEVs, but not LEVs, induced endothelial permeability in a process also prevented by hemopexin. CONCLUSION: These data show that distinct EVs are formed during cold storage of RBCs with smaller particles being more likely to mediate pro-oxidant and inflammatory effects associated with heme.

Association of heme-oxygenase 1, hemopexin, and heme levels with markers of disease severity in COVID-19.

Heme-oxygenase 1 (HO-1) is an enzyme with well-known anti-inflammatory and antioxidant properties, whose levels have been previously associated with disease severity in the context of sterile and infectious diseases. Moreover, the heme/HO-1 pathway has been associated with prothrombotic changes in other diseases. Accordingly, the potential of modulating HO-1 levels for the treatment of COVID-19 was extensively speculated during the COVID-19 pandemic, but very few actual data were generated. The aim of our study was to explore the association of HO-1, heme, and hemopexin (HPX) levels with COVID-19 severity and with markers of inflammation and coagulation activation. The study was conducted in 30 consecutive patients with COVID-19 admitted due to hypoxemia, and 30 healthy volunteers matched by sex, age, and geographic region. HO-1 and HPX levels were measured by enzyme immunoassay (ELISA) and heme levels were measured by a colorimetric method. A comprehensive panel of coagulation and fibrinolysis activation was also used. Patients with COVID-19 presented increased levels of HO-1 when compared to controls (5741 ± 2696 vs 1953 ± 612 pg/mL, respectively, P < 0.0001), as well as a trend toward increased levels of HPX (3.724 ± 0.880 vs 3.254 ± 1.022 mg/mL, respectively; P = 0.06). In addition, HO-1 and HPX levels reduced from admission to day + 4. HO-1 levels were associated with duration of intensive care unit stay and with several markers of coagulation activation. In conclusion, modulation of HO-1 could be associated with the prothrombotic state observed in COVID-19, and HO-1 could also represent a relevant biomarker for COVID-19. New independent studies are warranted to explore and expand these findings.

A multiplexed microflow LC-MS/MS-PRM assay for serologic quantification of IgG N- and HPX O- glycoforms in liver fibrosis.

Targeted quantification of glycoproteins has not reached its full potential because of limitations of the existing analytical workflows. In this study, we introduce a targeted microflow LC-MS/MS-PRM method for the quantification of multiple glycopeptides in unfractionated serum samples. The entire preparation of 16 samples in a batch is completed within 3 h, and the LC-MS quantification of all the glycoforms in a sample is completed in 15 min in triplicate, including online capture and desalting. We demonstrate applicability of the workflow on a multiplexed quantification of eight N-glycoforms of immunoglobulin G (IgG) together with two O-glycoforms of hemopexin (HPX). We applied the assay to a serologic study of fibrotic liver disease in patients of HCV etiology. The results document that specific IgG- and HPX-glycoforms detect efficiently fibrotic disease of different degree, and suggest that the LC-MS/MS-PRM assays may provide rapid and reproducible biomarker assay targeting simultaneously the N- and O-glycoforms of the peptides. We propose that such high throughput multiplexed methods may advance the clinical use of the LC-MS/MS assays.

Association of crystalloid fluid infusion with intravascular hemolysis and organ dysfunction in hematopoietic stem cell transplant patients.

Endothelial cell activation and injury is common after hematopoietic stem cell transplant (HSCT) and is associated with many post-transplant complications. An underexplored mechanism of endothelial cell damage in this population is the infusion of normal saline (NS, 0.9 % sodium chloride) and other crystalloids, as NS use is associated with adverse outcomes in other patient populations. We hypothesized that the infusion of unbalanced crystalloids during HSCT may lead to changes in biomarkers commonly associated with red blood cell (RBC) hemolysis in patients before and after infusion, and that markers of endothelial and end-organ damage during admission may be associated with markers of hemolysis and total crystalloid use. Samples were collected from 97 patients. From pre-fluid infusion to post-fluid infusion, mean haptoglobin decreased (11.7 ug/ml vs 8.4 ug/ml; p < 0.0001), hemopexin decreased (549 vs 512 µg/ml; p = 0.005), and red cell distribution width (RDW) decreased (15.7 vs 15.6; p = 0.0009). During admission (mean 19.4 days, SD 9.9), all markers of tissue and organ damage, including mean creatinine, lactate dehydrogenase (LDH), blood urea nitrogen (BUN), total bilirubin, AST, and ALT, increased from admission to peak levels (p < 0.0001). On linear regression, fluid volume (ml/kg) of crystalloid infusion positively predicted post-fluid infusion cell-free hemoglobin (r(96) = 0.34, p < 0.0001), free heme (r(96) = 0.36, p < 0.0001), and peak LDH during admission (r(75) = 0.23, p = 0.041), and negatively predicted post-fluid infusion hemopexin (r(96) = - 0.34, p < 0.0001). Unbalanced crystalloids may contribute to hemolysis and endothelial damage in HSCT patients. Alternatives such as buffered crystalloid solutions (PlasmaLyte, Lactated Ringer's) may be worth investigating in this population.

Tanshinone increases Hemopexin expression in lung cells and macrophages to protect against cigarette smoke-induced COPD and enhance antiviral responses.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease, while respiratory infections can elicit exacerbations in COPD patients to mediate increased mortality. Administration of Tanshinones (TS) derivatives has been demonstrated to protect against cigarette smoking (CS) and lipopolysaccharide (LPS)-induced COPD progression. However, the underlying molecular mechanisms and the roles of TS in mitigating the severity of viral-mediated exacerbations of COPD have not been elucidated. Here, we found that TS treatments significantly attenuated lung function decline, inflammatory responses and oxidative stress in CS and LPS-induced COPD mice. Subsequent RNA-seq analysis revealed significantly upregulated Hemopexin expression and enriched interferons (IFNs) signaling pathways in lung tissues of COPD mice upon TS treatments. Moreover, TS administration demonstrated Hemopexin-dependent beneficial roles in BEAS-2B lung cells and RAW264.7 macrophages, which was associated with the suppression of oxidative stress and ERK, NF-κB, and NLRP3 inflammasome signaling pathways-mediated inflammation. Furthermore, TS promoted IFN signaling and rescued impaired antiviral responses in CS and LPS-exposed lung cells that were infected by influenza virus. Notably, hemopexin over-expression in lung cells and macrophages recapitulated the pharmacological activities of TS. Taken together, these results indicate that TS administration is a promising and potential therapeutic strategy for treating COPD and preventing COPD exacerbations.

Genomic organization and gene evolution of two warm temperature acclimation proteins (Wap65s) of Micropterus salmoides and their responses to temperature and bacterial/viral infections.

Warm temperature acclimation-related 65-kDa proteins (Wap65s) are fish plasma acute-phase glycoproteins homologous to hemopexin with high affinity and clearance for heme. The study characterized Mswap65-1 and Mswap65-2 genes in Micropterus salmoides. Structural analysis showed MsWap65s contained conserved heme-binding sites. MsWap65-1 had a chloride-binding site similar to hemopexin, while MsWap65-2 had an additional calcium-binding site. Phylogenetic and Ka/Ks analysis showed that fish Wap65s were evolutionarily conserved and underwent strong purifying selection. Functional divergence analysis indicated that fish Wap65-2 retained the putative function of ancestral Wap65, while Wap65-1 underwent neofunctional differentiation. QPCR showed Mswap65s were predominantly expressed in liver, but prolonged hyperthermy inhibited Mswap65-2 expression. Mswap65-2 expression was up-regulated in liver and spleen after Nocardia seriolae infection, while Mswap65-1 was down-regulated. MsWap65-2 may be associated with pathogenesis and play potential role in pathogen resistance. LMBV infection resulted in both significant downregulation of Mswap65s were both significantly down-regulated, with differences observed between sexes. We speculated the immune system might suppress expression after viral infection. Exogenous rMsWap65s were prepared, and injection of rMsWap65s alleviated phenylhydrazine-induced hemolysis and inhibited increases in heme, complement C3 and inflammatory symptoms. Our results contribute to an advanced understanding of the functions and mechanisms of MsWap65s in stress resistance.

Circulating hemopexin modulates anthracycline cardiac toxicity in patients and in mice.

Anthracyclines such as doxorubicin (Dox) are effective chemotherapies, but their use is limited by cardiac toxicity. We hypothesized that plasma proteomics in women with breast cancer could identify new mechanisms of anthracycline cardiac toxicity. We measured changes in 1317 proteins in anthracycline-treated patients (n = 30) and replicated key findings in a second cohort (n = 31). An increase in the heme-binding protein hemopexin (Hpx) 3 months after anthracycline initiation was associated with cardiac toxicity by echocardiography. To assess the functional role of Hpx, we administered Hpx to wild-type (WT) mice treated with Dox and observed improved cardiac function. Conversely, Hpx-/- mice demonstrated increased Dox cardiac toxicity compared to WT mice. Initial mechanistic studies indicate that Hpx is likely transported to the heart by circulating monocytes/macrophages and that Hpx may mitigate Dox-induced ferroptosis to confer cardioprotection. Together, these observations suggest that Hpx induction represents a compensatory response during Dox treatment.

The Search for Associations of Serum Proteins with the Presence of Unstable Atherosclerotic Plaque in Coronary Atherosclerosis.

To study the associations of blood proteins with the presence of unstable atherosclerotic plaques in the arteries of patients with coronary atherosclerosis using quantitative proteomics. The studies involved two groups of men with coronary atherosclerosis (group 1 (St) had only stable atherosclerotic plaques; group 2 (Ns) had only unstable atherosclerotic plaques, according to histological analysis of tissue samples); the average age of patients was 57.95 ± 7.22. Protein concentrations in serum samples were determined using the PeptiQuant Plus Proteomics Kit. The identification of protein fractions was carried out by monitoring multiple reactions on a Q-TRAP 6500 mass spectrometer combined with a liquid chromatograph. Mass spectrometric identification revealed in serum samples from patients with unstable atherosclerotic plaques a reduced concentration of proteins in the blood: α-1-acid glycoprotein, α-1-antichymotrypsin, α-1-antitrypsin, ceruloplasmin, hemopexin, haptoglobin, apolipoprotein B-100, apolipoprotein L1, afamin and complement component (C3, C7, C9). Moreover, at the same time a high concentration complements factor H and attractin. The differences were considered significant at p < 0.05. It was found that the instability of atherosclerotic plaques is associated with the concentration of proteins: afamin, attractin, components of the complement system, hemopexin and haptoglobin. The data of our study showed the association of some blood proteins with the instability of atherosclerotic plaques in coronary atherosclerosis. Their potential role in the development of this disease and the possibility of using the studied proteins as biomarkers requires further research.