Hemopexin alleviates sterile inflammation in ischemia-reperfusion-induced lung injury.

Introduction: Pulmonary ischemia-reperfusion (IR) injury (IRI) plays a significant role in various lung disorders and is a key factor in the development of primary graft dysfunction following lung transplantation. Hemopexin (Hx) is the major serum scavenger protein for heme, which is a prooxidant and pro-inflammatory compound. In the current study, we hypothesized that Hx could confer beneficial effects in sterile inflammation induced by IR-mediated lung injury. Methods: To examine this hypothesis, we administered Hx in an experimental mouse model of unilateral lung IRI. Results: Our results demonstrate that treatment with Hx alleviated histopathological signs of inflammation in ischemic lungs, as evidenced by a reduction in the number of infiltrating neutrophils and decreased levels of perivascular edema. In addition, thrombotic vaso-occlusion in pulmonary blood vessels of IRI lungs was reduced by Hx. Immunohistochemical analysis revealed that Hx inhibited the up-regulation of heme oxygenase-1, an enzyme highly induced by heme, in ischemic lungs. Finally, Hx administration caused a decrease in the levels of circulating B- and CD8+ T-lymphocytes in the peripheral blood of mice with pulmonary IRI. Conclusion: These findings suggest that the serum heme scavenger protein Hx holds therapeutic promise in alleviating lung IRI-mediated sterile inflammation. Thus, Hx may represent a preemptive therapeutic approach in IR-related lung disorders such as primary graft dysfunction in lung transplantation.

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.

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.

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.

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.

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.

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.

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.

Strongyloidiasis stercoralis coinfection is associated with altered iron status biomarkers in tuberculous lymphadenitis.

Soil-transmitted helminth [mainly Strongyloidiasis stercoralis (Ss)] and tuberculous lymphadenitis (TBL) coinfection in humans is a significant public health problem. We have previously shown that TBL+Ss+ coinfection significantly alters diverse cytokine, matrix metalloproteinase, and tissue inhibitors of metalloproteinase profiles. However, no data is available to understand the influence of Ss coinfection in TBL disease with respect to iron status biomarkers. Hence, we have studied the effect of Ss coinfection on the circulating levels of iron status (ferritin, transferrin [TF], apotransferrin [ApoT], hepcidin, hemopexin) biomarkers in TBL disease. Our results show that TBL+Ss+ and/or TBL+Ss- individuals are associated with significantly altered biochemical and hematological (red blood cell (RBC) counts, hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) were decreased, and platelets were increased) parameters compared to TBL-Ss+ individuals. Our results also show that TBL+Ss+ coinfection is associated with diminished circulating levels of ferritin, ApoT, hepcidin, and hemopexin compared to TBL+Ss- individuals. TBL+Ss+ and TBL+Ss- groups are associated with altered iron status biomarkers (decreased ferritin [TBL+Ss+ alone] and increased TF, ApoT, hepcidin and hemopexin [TBL+Ss- alone]) compared to TBL-Ss+ group. The heat map expression profile and principal component analysis (PCA) analysis of iron status biomarkers were significantly altered in TBL+Ss+ compared to TBL+Ss- and/or TBL-Ss+ individuals. A significant correlation (positive/negative) was obtained among the biochemical and hematological parameters (white blood cells (WBC)/ferritin, TF, and hepcidin, mean corpuscular hemoglobin concentration (MCHC)/ferritin and hemopexin) with iron status biomarkers. Finally, receiver operating characteristic (ROC) analysis revealed that hemopexin was significantly associated with greater specificity and sensitivity in discriminating TBL+Ss+ and TBL+Ss- coinfected individuals. Thus, our data conclude that Ss coinfection is associated with altered iron status biomarkers indicating that coinfection might alter the host-Mtb interface and could influence the disease pathogenesis.

Detecting the PEX Like Domain of Matrix Metalloproteinase-14 (MMP-14) with Therapeutic Conjugated CNTs.

Matrix metalloproteinases (MMPs) are essential proteins acting directly in the breakdown of the extra cellular matrix and so in cancer invasion and metastasis. Given its impact on tumor angiogenesis, monitoring MMP-14 provides strategic insights on cancer severity and treatment. In this work, we report a new approach to improve the electrochemical interaction of the MMP-14 with the electrode surface while preserving high specificity. This is based on the detection of the hemopexin (PEX) domain of MMP-14, which has a greater availability with a stable and low-cost commercial molecule, as a recognition element. This molecule, called NSC-405020, is specific of the PEX domain of MMP-14 within the binding pocket. Through the covalent grafting of the NSC-405020 molecule on carbon nanotubes (CNTs), we were able to detect and quantify MMP-14 using electrochemical impedance spectroscopy with a linear range of detection of 10 ng⋅mL-1 to 100 ng⋅mL-1, and LOD of 7.5 ng⋅mL-1. The specificity of the inhibitory small molecule was validated against the PEX domain of MMP-1. The inhibitor loaded CNTs system showed as a desirable candidate to become an alternative to the conventional recognition bioelements for the detection of MMP-14.

Potential Urine Proteomic Biomarkers for Focal Segmental Glomerulosclerosis and Minimal Change Disease.

Primary focal segmental glomerulosclerosis (FSGS), along with minimal change disease (MCD), are diseases with primary podocyte damage that are clinically manifested by the nephrotic syndrome. The pathogenesis of these podocytopathies is still unknown, and therefore, the search for biomarkers of these diseases is ongoing. Our aim was to determine of the proteomic profile of urine from patients with FSGS and MCD. Patients with a confirmed diagnosis of FSGS (n = 30) and MCD (n = 9) were recruited for the study. For a comprehensive assessment of the severity of FSGS a special index was introduced, which was calculated as follows: the first score was assigned depending on the level of eGFR, the second score-depending on the proteinuria level, the third score-resistance to steroid therapy. Patients with the sum of these scores of less than 3 were included in group 1, with 3 or more-in group 2. The urinary proteome was analyzed using liquid chromatography/mass spectrometry. The proteome profiles of patients with severe progressive FSGS from group 2, mild FSGS from group 1 and MCD were compared. Results of the label free analysis were validated using targeted LC-MS based on multiple reaction monitoring (MRM) with stable isotope labelled peptide standards (SIS) available for 47 of the 76 proteins identified as differentiating between at least one pair of groups. Quantitative MRM SIS validation measurements for these 47 proteins revealed 22 proteins with significant differences between at least one of the two group pairs and 14 proteins were validated for both comparisons. In addition, all of the 22 proteins validated by MRM SIS analysis showed the same direction of change as at the discovery stage with label-free LC-MS analysis, i.e., up or down regulation in MCD and FSGS1 against FSGS2. Patients from the FSGS group 2 showed a significantly different profile from both FSGS group 1 and MCD. Among the 47 significantly differentiating proteins, the most significant were apolipoprotein A-IV, hemopexin, vitronectin, gelsolin, components of the complement system (C4b, factors B and I), retinol- and vitamin D-binding proteins. Patients with mild form of FSGS and MCD showed lower levels of Cystatin C, gelsolin and complement factor I.

Hemopexin accumulates in kidneys and worsens acute kidney injury by causing hemoglobin deposition and exacerbation of iron toxicity in proximal tubules.

Hemopexin, a heme scavenging protein, accumulates in the kidneys during acute kidney injury (AKI). However, the function of this accumulated hemopexin in the kidney is unclear. In both the cisplatin-induced and the unilateral kidney ischemia-reperfusion injury models of AKI, we found accumulation of hemoglobin and hemopexin in the kidneys localized to the proximal tubules. Next, hemopexin wild-type and knockout mice were compared in both AKI models and hemopexin wild type mice had significantly worse kidney injury. Furthermore, there was increased kidney expression of kidney injury molecule-1 (a biomarker of AKI) and heme oxygenase-1 (an indicator of oxidative stress) in hemopexin wild type compared with knockout mice in both models of AKI. Next, the interaction of hemopexin and hemoglobin in vitro was investigated using cultured proximal tubular cells. Co-incubation of hemopexin with hemoglobin resulted in hemoglobin deposition and exaggerated hemoglobin-induced injury. Deferoxamine, an iron chelator, and ferrostatin-1, a ferroptosis inhibitor, inhibited this deleterious effect of hemoglobin and hemopexin in proximal tubular cells, implicating iron toxicity in the mechanism of hemopexin mediated injury. Furthermore, the protective effect of deferoxamine in cisplatin-induced AKI was apparent in hemopexin wild type, but not in hemopexin knockout mice, further implicating hemopexin as a mediator of iron toxicity in AKI. Thus, our findings demonstrate that hemopexin accumulates in the kidneys and worsens kidney injury in AKI by increasing hemoglobin deposition on proximal tubular cells to exaggerate hemoglobin-induced cell injury.

Identification and characterization of warm temperature acclimation proteins (Wap65s) in rainbow trout (Oncorhynchus mykiss).

Hemopexin is a vital glycoprotein for processing excessive iron in blood and functions as an iron scavenger in mammals. Teleosts however, unlike mammals, have two known hemopexin paralogs called warm temperature acclimation-related 65 kDa protein (Wap65-1 and Wap65-2, collectively termed Wap65s). Although Wap65s in rainbow trout have been considered notable biomarkers with significantly higher and/or lower expression under conditions of stress or disease, the individual roles, similarities and differences between the two paralogs are not well known. The aim of this study was to gain an understanding of the characteristics and functions of trout Wap65s from the perspective of iron-metabolism, physiological roles, and relevant immunological responses. The expression of Wap65-1 and -2 in this study was determined in the face of challenges by Aeromonas salmonicida, infectious hematopoietic necrosis virus (IHNV), and iron-dextran. Immuno-histochemistry (IHC) was employed to localize the major cell types for Wap65-2 expression, and trout leukocytes were isolated and incubated with LPS and OxLDL for comprehending the immunological characteristics of Wap65-2. We demonstrate that Wap65-1 is expressed only in the liver but Wap65-2 is systemically expressed in most organs and tissues. Interestingly, Wap65-1 expression was not significantly changed under A. salmonicida and iron-dextran administration, but was significantly decreased under IHNV. In contrast, Wap65-2 was up-regulated in all challenged groups, however with different expression patterns in the blood and liver. These results suggested that the two paralogs may participate in different biological roles. IHC showed that Wap65-2 antibody had high affinity for leukocyte-like cells, and macrophages but not lymphocytes significantly increased expression under LPS and OxLDL stimulation. These results support the conclusion that trout Wap65-2, not Wap65-1 may have conventional hemopexin functions such as reported in mammals including effects on iron metabolism, inflammation, and acute-phase protein.

Hemopexin and Cancer.

Hemopexin is the plasma protein with the highest affinity for heme. Seminal studies have highlighted its role in different kinds of heme-associated disorders, but its implication in cancer has been neglected for a long time. Considering the emerging importance of heme in tumors, the present review proposes an update of the works investigating hemopexin involvement in cancer, with the attempt to stimulate further future studies on this topic.

Skeletal muscle atrophy-induced hemopexin accelerates onset of cognitive impairment in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is an unmet medical need worldwide, and physical inactivity is a risk factor for AD. Performing physical exercise is difficult at old age, and thus, decline in physical movement may be a cause of age-associated lowering of the brain function. This study aimed to elucidate the molecular mechanism and onset of the skeletal muscle atrophy-induced acceleration of AD. METHODS: Pre-symptomatic young 5XFAD or non-transgenic wildtype mice were used. The bilateral hindlimbs were immobilized by placing them in casts for 14 days. Cognitive function was evaluated using the object recognition and spatial memory tests. Further, the hindlimb muscles were isolated for organ culture. Conditioned media (CM) of each muscle was separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein expressions in the CM were analysed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis. The expression levels of candidate proteins were quantified using ELISA. After continuous intracerebroventricular (i.c.v.) infusion of recombinant hemopexin, cognitive function was evaluated. Gene microarray analysis of the hippocampus was performed to investigate the molecules involved in the accelerated memory deficit. Real-time reverse transcription polymerase chain reaction and histological analysis confirmed the expression. RESULTS: Casting for 2 weeks reduced skeletal muscle weight. Object recognition memory in the cast-attached 5XFAD mice (n = 7, training vs. test, P = 0.3390) was impaired than that in age-matched wildtype (n = 7, training vs. test, P = 0.0523) and non-cast 5XFAD mice (n = 7, training vs. test, P = 0.0473). On 2D-PAGE, 88 spots were differentially expressed in muscle CM. The most increased spot in the cast-attached 5XFAD CM was hemopexin. Hemopexin levels in the skeletal muscle (n = 3, P = 0.0064), plasma (n = 3, P = 0.0386), and hippocampus (n = 3, P = 0.0164) were increased in cast-attached 5XFAD mice than those in non-cast 5XFAD mice. Continuous i.c.v. infusion of hemopexin for 2 weeks induced memory deficits in young 5XFAD mice (n = 4, training vs. test, P = 0.6764). Lipocalin-2 (Lcn2) messenger RNA (mRNA), neuroinflammation-associated factor, was increased in the hippocampus in hemopexin-infused 5XFAD mice than in control mice. LCN2 protein in the hippocampus was localized in the neurons, but not glial cells. Lcn2 mRNA levels in the hippocampus were also increased by cast-immobilization of the hindlimbs (n = 6, P = 0.0043). CONCLUSIONS: These findings provide new evidence indicating that skeletal muscle atrophy has an unbeneficial impact on the occurrence of memory impairment in young 5XFAD mice, which is mediated by the muscle secreted hemopexin.

Cuprizone and EAE mouse frontal cortex proteomics revealed proteins altered in multiple sclerosis.

Two pathophysiological different experimental models for multiple sclerosis were analyzed in parallel using quantitative proteomics in attempts to discover protein alterations applicable as diagnostic-, prognostic-, or treatment targets in human disease. The cuprizone model reflects de- and remyelination in multiple sclerosis, and the experimental autoimmune encephalomyelitis (EAE, MOG1-125) immune-mediated events. The frontal cortex, peripheral to severely inflicted areas in the CNS, was dissected and analyzed. The frontal cortex had previously not been characterized by proteomics at different disease stages, and novel protein alterations involved in protecting healthy tissue and assisting repair of inflicted areas might be discovered. Using TMT-labelling and mass spectrometry, 1871 of the proteins quantified overlapped between the two experimental models, and the fold change compared to controls was verified using label-free proteomics. Few similarities in frontal cortex between the two disease models were observed when regulated proteins and signaling pathways were compared. Legumain and C1Q complement proteins were among the most upregulated proteins in cuprizone and hemopexin in the EAE model. Immunohistochemistry showed that legumain expression in post-mortem multiple sclerosis brain tissue (n = 19) was significantly higher in the center and at the edge of white matter active and chronic active lesions. Legumain was associated with increased lesion activity and might be valuable as a drug target using specific inhibitors as already suggested for Parkinson's and Alzheimer's disease. Cerebrospinal fluid levels of legumain, C1q and hemopexin were not significantly different between multiple sclerosis patients, other neurological diseases, or healthy controls.

Simultaneous targeting of CD44 and MMP9 catalytic and hemopexin domains as a therapeutic strategy.

Crosstalk of the oncogenic matrix metalloproteinase-9 (MMP9) and one of its ligands, CD44, involves cleavage of CD44 by the MMP9 catalytic domain, with the CD44-MMP9 interaction on the cell surface taking place through the MMP9 hemopexin domain (PEX). This interaction promotes cancer cell migration and invasiveness. In concert, MMP9-processed CD44 induces the expression of MMP9, which degrades ECM components and facilitates growth factor release and activation, cancer cell invasiveness, and metastasis. Since both MMP9 and CD44 contribute to cancer progression, we have developed a new strategy to fully block this neoplastic process by engineering a multi-specific inhibitor that simultaneously targets CD44 and both the catalytic and PEX domains of MMP9. Using a yeast surface display technology, we first obtained a high-affinity inhibitor for the MMP9 catalytic domain, which we termed C9, by modifying a natural non-specific MMP inhibitor, N-TIMP2. We then conjugated C9 via a flexible linker to PEX, thereby creating a multi-specific inhibitor (C9-PEX) that simultaneously targets the MMP9 catalytic and PEX domains and CD44. It is likely that, via its co-localization with CD44, C9-PEX may compete with MMP9 localization on the cell surface, thereby inhibiting MMP9 catalytic activity, reducing MMP9 cellular levels, interfering with MMP9 homodimerization, and reducing the activation of downstream MAPK/ERK pathway signaling. The developed platform could be extended to other oncogenic MMPs as well as to other important target proteins, thereby offering great promise for creating novel multi-specific therapeutics for cancer and other diseases.

Scavenging of Labile Heme by Hemopexin Is a Key Checkpoint in Cancer Growth and Metastases.

Hemopexin (Hx) is a scavenger of labile heme. Herein, we present data defining the role of tumor stroma-expressed Hx in suppressing cancer progression. Labile heme and Hx levels are inversely correlated in the plasma of patients with prostate cancer (PCa). Further, low expression of Hx in PCa biopsies characterizes poorly differentiated tumors and correlates with earlier time to relapse. Significantly, heme promotes tumor growth and metastases in an orthotopic murine model of PCa, with the most aggressive phenotype detected in mice lacking Hx. Mechanistically, labile heme accumulates in the nucleus and modulates specific gene expression via interacting with guanine quadruplex (G4) DNA structures to promote PCa growth. We identify c-MYC as a heme:G4-regulated gene and a major player in heme-driven cancer progression. Collectively, these results reveal that sequestration of labile heme by Hx may block heme-driven tumor growth and metastases, suggesting a potential strategy to prevent and/or arrest cancer dissemination.

Stromal expression of hemopexin is associated with lymph-node metastasis in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma is one of the most aggressive types of cancer. Certain proteins in the tumor microenvironment have attracted considerable attention owing to their association with tumor invasion and metastasis. Here, we used proteomics to identify proteins associated with lymph-node metastasis, which is one of the prognostic factors. We selected lymph node metastasis-positive and -negative patients (n = 5 each) who underwent pancreatectomy between 2005 and 2015 and subjected to comprehensive proteomic profiling of tumor stroma. A total of 490 proteins were detected by mass spectrometry. Software analysis revealed that nine of these proteins were differentially expressed between the two patient groups. We focused on hemopexin and ferritin light chain based on immunohistochemistry results. We assessed the clinicopathological data of 163 patients and found that hemopexin expression was associated with UICC N2 (p = 0.0399), lymph node ratio (p = 0.0252), venous invasion (p = 0.0096), and lymphatic invasion (p = 0.0232). Notably, in vitro assays showed that hemopexin promotes invasion of the pancreatic cancer cells. Our findings suggest that hemopexin is a lymph node metastasis-associated protein that could potentially serve as a useful therapeutic target or biomarker of pancreatic ductal adenocarcinoma.

The Role of Circulating Cell-Free Hemoglobin in Sepsis-Associated Acute Kidney Injury.

Sepsis is a heterogeneous clinical syndrome that is complicated commonly by acute kidney injury (sepsis-AKI). Currently, no approved pharmacologic therapies exist to either prevent sepsis-AKI or to treat sepsis-AKI once it occurs. A growing body of evidence supports a connection between red blood cell biology and sepsis-AKI. Increased levels of circulating cell-free hemoglobin (CFH) released from red blood cells during hemolysis are common during sepsis and can contribute to sepsis-AKI through several mechanisms including tubular obstruction, nitric oxide depletion, oxidative injury, and proinflammatory signaling. A number of potential pharmacologic therapies targeting CFH in sepsis have been identified including haptoglobin, hemopexin, and acetaminophen, and early phase clinical trials have suggested that acetaminophen may have beneficial effects on lipid peroxidation and kidney function in patients with sepsis. Bedside measurement of CFH levels may facilitate predictive enrichment for future clinical trials of CFH-targeted therapeutics. However, rapid and reliable bedside tests for plasma CFH will be required for such trials to move forward.