Heme Levels Research Articles

Protease Activated Receptor-1 (PAR-1) Inhibition By Parmodulin 2, but Not Vorapaxar, Protects Sickle Cell Mice from Heme-Induced Acute Chest Syndrome

Background: Acute chest syndrome (ACS) is a leading cause of ICU admission and mortality in sickle cell patients, characterized by hypoxemia, fever, and significant chest pain. The main cause for ACS in SCD is hemolytic anemia, which elevates circulating heme levels. A model of ACS identified that excess heme induces acute lung injury (ALI) dependent on TLR4 and endothelial p-selectin in sickle cell mice. Despite increased understanding of the etiology of ACS, current treatments are either supportive or not targeted. We have shown that thrombin-dependent protease activated receptor-1 (PAR-1) signaling contributes to heme-induced vascular stasis via upregulation of endothelial p-selectin. Thrombin/PAR-1 signaling is cytotoxic and pro-inflammatory, whereas activation of PAR-1 by activated protein C (APC) is cytoprotective. We hypothesize that PAR-1 signaling contributes to the pathogenesis of ACS. Objective: To investigate the role of PAR-1 in a mouse model of heme-induced ACS using two PAR-1 inhibitors: vorapaxar or parmodulin-2 (PM2). Methods: Townes wild type (AA) and sickle (SS) mice (3-4 months) were used in three experiments. 1) AA females (n=4), SS males (n=6) and SS females (n=11) received 140 µmol/kg heme via retro-orbital injection. 2) SS females received vehicle (n=6) or Vorapaxar (105 µmolar/kg BW, oral gavage) (n=7) daily for 5 days, then treated with heme (140 µmol/kg). 3) SS females received vehicle (n=5) or PM2 (10 mg/kg, intraperitoneal, n=5) 30 minutes prior to heme (140µmol/kg). In all experiments, mice were monitored for signs of ALI (endpoint criteria: severe discomfort with disability) and lungs were collected for histopathology at endpoint or 120 mins after heme infusion. Results: Heme infusion caused 11/11 of SS females to reach endpoint (median survival = 18 min, p=0.0006 vs AA and SS males), whereas only 2/6 of SS males and 0/4 AA mice reached endpoint. Correlation analysis for RBCs and endpoint time showed that SS females with RBC counts less than 7 x 1012/L were vulnerable to heme-induced ACS; yet even males with significant anemia did not reach endpoint. The lung histology showed alveolar thickening, edema and hemorrhage in heme-treated SS females. Hence, only female SS mice were used for the remaining studies. Interestingly, vorapaxar did not protect SS mice from heme-induced ACS (5/7 mice, median survival 17 min) compared to vehicle treatment (4/6 mice, median survival 16 min). In contrast, PM2 treatment significantly protected the SS mice from heme induced ACS with 0/5 reaching endpoint, compared to 4/5 of vehicle -treated SS mice reaching endpoint (p=0.013), and prevented lung damage observed by histology. Conclusions: We found that female mice are more susceptible to heme-induced ACS than males. Vorapaxar, an irreversible antagonist of PAR-1 which blocks all PAR-1 signaling pathways, did not protect SS mice against heme-induced ACS. In contrast, PM2 completely alleviated heme-induced lung injury and death in SS mice. This could be attributed to the fact that PM2 allosterically blocks thrombin/PAR-1 signaling while preserving cytoprotective signaling similar to that mediated by APC. Targeting PAR-1 by preserving (or inducing) beneficial signaling might be effective in preventing the development of ACS.

Blood Transfusion Ameliorates Free Haem-Induced Loss of Thrombomodulin and Endothelial Cell Activation in Paediatric Sickle Cell Anaemia

Background Sickle cell anaemia (SCA) is responsible for major paediatric morbidity including risk of stroke in up to 10% of children. Mainstays of therapy include hydroxyurea (HU) and chronic blood transfusion (BT), with BT considered gold standard for stroke prevention. However, the biological mechanisms through which BT is significantly superior to HU in reducing stroke risk have not been clearly defined. SCA complications are due in part to free haem-mediated damage, leading to endothelial cell (EC) activation, tissue factor (TF) expression and release of von Willebrand Factor (VWF), which binds platelets and sickle RBCs. Recently loss of EC Thrombomodulin (TM) has been shown to promote EC activation and VWF release and soluble TM (sTM) plays a role in disseminated intravascular coagulation and COVID-19. Hypothesis We hypothesize free haem alters EC TM expression, promoting EC activation and VWF release. Intravascular hemolysis and free haem may be reduced in children on chronic BT, maintaining EC quiescence and enhancing protection against stroke compared with HU. Methods Following ethical approval and informed consent 180 SCA children (median age 12 years, 49% male) were recruited. Steady-state plasma samples were collected prior to BT (n=84) or at outpatient clinic for HU (n=96). Paired crisis samples were obtained in a subset of HU (n=30). EC activation markers and Weibel Palade Body (WPB) proteins were evaluated by ELISA, VWF multimers were assessed by 1.8% agarose gels and ADAMTS13 activity assessed by FRET. Free haem levels were quantified and thrombin generation (TG) assays performed. Factor VIII:C was measured in the National Coagulation Centre at St James's Hospital. Human umbilical vein endothelial cells (HUVEC) were exposed to cell-free haem (Hemin), hydroxyurea, cytokines (IL-1β and TNFα) or patient plasma; VWF was measured in the supernatant and HUVEC TM expression assessed by flow cytometry. Results VWF:Ag and VWF propeptide (VWFpp) levels were significantly increased in HU compared to BT children, suggesting acute EC activation (median plasma VWF:Ag levels 1.29 v 1.03 IU/ml; p=0.0005, median VWFpp levels 1.63 v 1.17 IU/ml p=0.006). Furthermore, plasma levels of other WPB stored proteins were also significantly elevated in the HU cohort, including Angiopoietin-2 (Ang-2) (4214 v 2337 pg/ml, p<0.0001 and Osteoprotegerin (OPG) (1825 v 1507 pg/ml p=0.006). Abnormalities of high-, intermediate- and low molecular weight VWF multimers were noted in HU but not BT. While putative inhibitors of ADAMTS13 were increased in HU, including Platelet Factor 4 and Interleukin-6, ADAMTS13 activity did not differ between groups. FVIII:C levels were significantly elevated in 65% of the total cohort and were higher in HU than BT (1.62 IU/ml v 1.30 IU/ml p<0.0001). Free haem levels were increased in HU versus BT (12.5 v 9.5μM p=0.0003), as well as levels of LDH and reticulocyte %, pointing to increased intravascular hemolysis. EC markers and FVIII:C correlated strongly with hemolytic indices. In addition to increased EC activation, increased endogenous thrombin potential (ETP), peak thrombin and velocity index were observed in HU versus BT, pointing to enhanced TG, possibly driven by elevated circulating TF. TG parameters correlated with plasma FVIII:C levels. Exposure of HUVEC to free haem in vitro resulted in a significant decrease in surface TM expression and increased VWF secretion (p<0.05). Conversely exposure to hydroxyurea or cytokines did not alter TM expression. Exposure of HUVEC to plasma from crisis but not paired steady-state plasma induced EC activation and VWF release compared with control plasma. Importantly, free haem levels ≥20 μM were observed in 25% HU children in steady state and 40% in crisis, compared with 0% of BT. Furthermore, significantly elevated sTM levels in SCA children with a history of cerebrovascular disease compared to those without (5.7 v 4.6 ng/ml p=0.02), suggesting that sTM may predict end-organ dysfunction. Conclusions We have shown in a large cohort of SCA children that EC activation, TG and intravascular haemolysis are increased in HU-treated children compared with BT. Free haem induces loss of EC TM and VWF release in vitro, leading to EC activation. Elevated sTM levels were a biomarker of end-organ damage. These data suggest that reduction of free haem in children undergoing regular BT is a key mechanism through which EC quiescence is maintained in paediatric SCA.

The dual role and mutual dependence of heme/HO-1/Bach1 axis in the carcinogenic and anti-carcinogenic intersection.

In physiological concentrations, heme is nontoxic to the cell and is essential for cell survival and proliferation. Increasing intracellular heme concentrations beyond normal levels, however, will lead to carcinogenesis and facilitate the survival of tumor cells. Simultaneously, heme in an abnormally high quantity is also a potent inducer of tumor cell death, contributing to its ability to generate oxidative stress on the cells by boosting oxidative phosphorylation and suppressing tumors through ferroptosis. During tumorigenesis and progression, therefore, heme works as a double-edged sword. Heme oxygenase 1 (HO-1) is the rate-limiting enzyme in heme catabolism, which converts heme into physiologically active catabolites of carbon monoxide (CO), biliverdin, and ferrous iron (Fe2+). HO-1 maintains redox equilibrium in healthy cells and functions as a carcinogenesis inhibitor. It is widely recognized that HO-1 is involved in the adaptive response to cellular stress and the anti-inflammation effect. Notably, its expression level in cancer cells corresponds with tumor growth, aggressiveness, metastasis, and angiogenesis. Besides, heme-binding transcription factor BTB and CNC homology 1 (Bach1) play a critical regulatory role in heme homeostasis, oxidative stress and senescence, cell cycle, angiogenesis, immune cell differentiation, and autoimmune disorders. Moreover, it was found that Bach1 influences cancer cells' metabolism and metastatic capacity. Bach1 controls heme level by adjusting HO-1 expression, establishing a negative feedback loop. Herein, the authors review recent studies on heme, HO-1, and Bach1 in cancer. Specifically, they cover the following areas: (1) the carcinogenic and anticarcinogenic aspects of heme; (2) the carcinogenic and anticarcinogenic aspects of HO-1; (3) the carcinogenic and anticarcinogenic aspects of Bach1; (4) the interactions of the heme/HO-1/Bach1 axis involved in tumor progression. This review summarized the literature about the dual role of the heme/HO-1/Bach1 axis and their mutual dependence in the carcinogenesis and anti-carcinogenesis intersection.

Acteoside alleviates dextran sulphate sodium‑induced ulcerative colitis via regulation of the HO‑1/HMGB1 signaling pathway.

Ulcerative colitis (UC) is a significant burden on human health, and the elucidation of the mechanism by which it develops has potential for the prevention and treatment of UC. It has been reported that acteoside (ACT) exhibits strong anti‑inflammatory activity. In the present study, it was hypothesized that ACT may exert a protective effect against UC. The effects of ACT on inflammation, oxidative stress and apoptosis were evaluated using dextran sulphate sodium (DSS)‑treated mice and DSS‑treated human colorectal adenocarcinoma Caco‑2 cells, which have an epithelial morphology. The results demonstrated that the ACT‑treated mice with DSS‑induced UC exhibited significantly reduced colon inflammation, as demonstrated by a reversal in body weight loss, colon shortening, disease activity index score, inflammation, oxidative stress and colonic barrier dysfunction. Further invivo experiments demonstrated that ACT inhibited DSS‑induced apoptosis in colon tissues, as demonstrated by the results of the TUNEL assay and the altered protein expression levels of Bax, cleaved caspase‑3 and Bcl‑2. Furthermore, DSS significantly stimulated the protein expression levels of high mobility group box 1 protein (HMGB1), which serves a central role in the initiation and progression of UC, an effect which was markedly inhibited by ACT. Finally, DSS significantly decreased the protein expression levels of heme oxygenase‑1 (HO‑1) in colon tissues and the effect of ACT on GSH, apoptotic proteins and HMGB1 was markedly attenuated in the presence of the HO‑1 inhibitor tin protoporphyrin. In conclusion, ACT ameliorated colon inflammation through HMGB1 inhibition in a HO‑1‑dependent manner.

The role of host heme in bacterial infection.

Heme is an indispensable cofactor for almost all aerobic life, including the human host and many bacterial pathogens. During infection, heme and hemoproteins are the largest source of bioavailable iron, and pathogens have evolved various heme acquisition pathways to satisfy their need for iron and heme. Many of these pathways are regulated transcriptionally by intracellular iron levels, however, host heme availability and intracellular heme levels have also been found to regulate heme uptake in some species. Knowledge of these pathways has helped to uncover not only how these bacteria incorporate host heme into their metabolism but also provided insight into the importance of host heme as a nutrient source during infection. Within this review is covered multiple aspects of the role of heme at the host pathogen interface, including the various routes of heme biosynthesis, how heme is sequestered by the host, and how heme is scavenged by bacterial pathogens. Also discussed is how heme and hemoproteins alter the behavior of the host immune system and bacterial pathogens. Finally, some unanswered questions about the regulation of heme uptake and how host heme is integrated into bacterial metabolism are highlighted.

Generation and characterization of human U-2 OS cell lines with the CRISPR/Cas9-edited protoporphyrinogen oxidase IX gene

In humans, disruptions in the heme biosynthetic pathway are associated with various types of porphyrias, including variegate porphyria that results from the decreased activity of protoporphyrinogen oxidase IX (PPO; E.C.1.3.3.4), the enzyme catalyzing the penultimate step of the heme biosynthesis. Here we report the generation and characterization of human cell lines, in which PPO was inactivated using the CRISPR/Cas9 system. The PPO knock-out (PPO-KO) cell lines are viable with the normal proliferation rate and show massive accumulation of protoporphyrinogen IX, the PPO substrate. Observed low heme levels trigger a decrease in the amount of functional heme containing respiratory complexes III and IV and overall reduced oxygen consumption rates. Untargeted proteomics further revealed dysregulation of 22 cellular proteins, including strong upregulation of 5-aminolevulinic acid synthase, the major regulatory protein of the heme biosynthesis, as well as additional ten targets with unknown association to heme metabolism. Importantly, knock-in of PPO into PPO-KO cells rescued their wild-type phenotype, confirming the specificity of our model. Overall, our model system exploiting a non-erythroid human U-2 OS cell line reveals physiological consequences of the PPO ablation at the cellular level and can serve as a tool to study various aspects of dysregulated heme metabolism associated with variegate porphyria.

Regulation of heme utilization and homeostasis in Candida albicans.

Heme (iron-protoporphyrin IX) is an essential but potentially toxic cellular cofactor. While most organisms are heme prototrophs, many microorganisms can utilize environmental heme as iron source. The pathogenic yeast Candida albicans can utilize host heme in the iron-poor host environment, using an extracellular cascade of soluble and anchored hemophores, and plasma membrane ferric reductase-like proteins. To gain additional insight into the C. albicans heme uptake pathway, we performed an unbiased genetic selection for mutants resistant to the toxic heme analog Ga3+-protoporphyrin IX at neutral pH, and a secondary screen for inability to utilize heme as iron source. Among the mutants isolated were the genes of the pH-responsive RIM pathway, and a zinc finger transcription factor related to S. cerevisiae HAP1. In the presence of hemin in the medium, C. albicans HAP1 is induced, the Hap1 protein is stabilized and Hap1-GFP localizes to the nucleus. In the hap1 mutant, cytoplasmic heme levels are elevated, while influx of extracellular heme is lower. Gene expression analysis indicated that in the presence of extracellular hemin, Hap1 activates the heme oxygenase HMX1, which breaks down excess cytoplasmic heme, while at the same time it also activates all the known heme uptake genes. These results indicate that Hap1 is a heme-responsive transcription factor that plays a role both in cytoplasmic heme homeostasis and in utilization of extracellular heme. The induction of heme uptake genes by C. albicans Hap1 under iron satiety indicates that preferential utilization of host heme can be a dietary strategy in a heme prototroph.

A pseudokinase version of the histidine kinase ChrS promotes high heme tolerance of Corynebacterium glutamicum.

Heme is an essential cofactor for almost all living cells by acting as prosthetic group for various proteins or serving as alternative iron source. However, elevated levels are highly toxic for cells. Several corynebacterial species employ two paralogous, heme-responsive two-component systems (TCS), ChrSA and HrrSA, to cope with heme stress and to maintain intracellular heme homeostasis. Significant cross-talk at the level of phosphorylation between these systems was previously demonstrated. In this study, we have performed a laboratory evolution experiment to adapt Corynebacterium glutamicum to increasing heme levels. Isolated strains showed a highly increased tolerance to heme growing at concentrations of up to 100 μM. The strain featuring the highest heme tolerance harbored a frameshift mutation in the catalytical and ATPase-domain (CA-domain) of the chrS gene, converting it into a catalytically-inactive pseudokinase (ChrS_CA-fs). Reintroduction of the respective mutation in the parental C. glutamicum strain confirmed high heme tolerance and showed a drastic upregulation of hrtBA encoding a heme export system, conserved in Firmicutes and Actinobacteria. The strain encoding the ChrS pseudokinase variant showed significantly higher heme tolerance than a strain lacking chrS. Mutational analysis revealed that induction of hrtBA in the evolved strain is solely mediated via the cross-phosphorylation of the response regulator (RR) ChrA by the kinase HrrS and BACTH assays revealed the formation of heterodimers between HrrS and ChrS. Overall, our results emphasize an important role of the ChrS pseudokinase in high heme tolerance of the evolved C. glutamicum and demonstrate the promiscuity in heme-dependent signaling of the paralogous two-component systems facilitating fast adaptation to changing environmental conditions.

The different facets of heme-oxygenase 1 in innate and adaptive immunity.

Heme oxygenase (HO) enzymes are responsible for the main oxidative step in heme degradation, generating equimolar amounts of free iron, biliverdin and carbon monoxide. HO-1 is induced as a crucial stress response protein, playing protective roles in physiologic and pathological conditions, due to its antioxidant, anti-apoptotic and anti-inflammatory effects. The mechanisms behind HO-1-mediated protection are being explored by different studies, affecting cell fate through multiple ways, such as reduction in intracellular levels of heme and ROS, transcriptional regulation, and through its byproducts generation. In this review we focus on the interplay between HO-1 and immune-related signaling pathways, which culminate in the activation of transcription factors important in immune responses and inflammation. We also discuss the dual interaction of HO-1 and inflammatory mediators that govern resolution and tissue damage. We highlight the dichotomy of HO-1 in innate and adaptive immune cells development and activation in different disease contexts. Finally, we address different known anti-inflammatory pharmaceuticals that are now being described to modulate HO-1, and the possible contribution of HO-1 in their anti-inflammatory effects.

Transcriptional regulation of ergosterol biosynthesis genes in response to iron deficiency.

Iron participates as an essential cofactor in the biosynthesis of critical cellular components, including DNA, proteins and lipids. The ergosterol biosynthetic pathway, which is an important target of antifungal treatments, depends on iron in four enzymatic steps. Our results in the model yeast Saccharomyces cerevisiae show that the expression of ergosterol biosynthesis (ERG) genes is tightly modulated by iron availability probably through the iron-dependent variation of sterol and heme levels. Whereas the transcription factors Upc2 and Ecm22 are responsible for the activation of ERG genes upon iron deficiency, the heme-dependent factor Hap1 triggers their Tup1-mediated transcriptional repression. The combined regulation by both activating and repressing regulatory factors allows for the fine-tuning of ERG transcript levels along the progress of iron deficiency, avoiding the accumulation of toxic sterol intermediates and enabling efficient adaptation to rapidly changing conditions. The lack of these regulatory factors leads to changes in the yeast sterol profile upon iron-deficient conditions. Both environmental iron availability and specific regulatory factors should be considered in ergosterol antifungal treatments.

Enhancement of the cytotoxic effect of dihydroartemisinin in high-risk human papillomavirus-infected cells by aminolevulinic acid via the Bax/Bcl-2-caspase pathway

BackgroundTraditional treatments for human papillomavirus-related cutaneous diseases include 5-aminolevulinic acid photodynamic therapy, cryotherapy, microwave ablation, and surgical resection. These treatment methods involvevarious adverse reactions; therefore, it remains necessary to explore new treatment methods. Dihydroartemisinin shows cytotoxic effects against several malignancies by producing reactive oxygen species, and heme environments reportedly enhance its activity. However, the underlying mechanismsare still unclear. Therefore, we investigated the mechanism of dihydroartemisininin inhuman papillomavirus-infected cells. MethodsHeLa cells were treated with dihydroartemisinin, 5-aminolevulinic acid, and succinylacetone. The cell viability, apoptosis, mitochondrial membrane potential, and reactive oxygen species levels were investigated, and via western blotting analysis and polymerase chain reaction, dihydroartemisinin activity-related pathways were also determined. ResultsDihydroartemisinin inhibited HeLa cell proliferation and promoted cell apoptosis via the Bax/Bcl-2-Caspase pathway in a concentration-dependent manner. The specific cytotoxicity toward HeLa cells was enhanced by the addition of 5-aminolevulinic acid, a clinically used heme-synthesis precursor, owing to an increase in heme levels. Conversely, following the addition of succinylacetone, a heme synthesis blocker, heme levels decreased. Furthermore, dihydroartemisinin significantly increased reactive oxygen species levels as intracellular heme synthesis increased. Moreover, photodynamic therapy following dihydroartemisinin and 5-aminolevulinic acid treatment further enhanced the cytotoxic effect of dihydroartemisinin on high-risk human papillomavirus-infected cells. ConclusionsDihydroartemisinin exerts acytotoxic effect on high-risk human papillomavirus-infected cells by modulating heme levels via the Bax/Bcl–2-Caspase pathway, and the dihydroartemisinin, 5-aminolevulinic acid, photodynamic therapy combination treatment significantly enhanced its cytotoxic effect on human papillomavirus-infected cells.

P03 The effects of haem on perfusion parameters and the activation of innate immune system in human kidneys during ex vivo normothermic machine perfusion

Abstract Introduction Normothermic machine perfusion (NMP) of kidneys, using an oxygenated red blood cell (RBC) based solution has been developed to restore cellular metabolism and minimise effects of cold ischaemia. During NMP RBCs become damaged releasing haem activating inflammatory signalling pathways. This study aimed to measure levels of haem during NMP and determine the association with perfusion parameters and transcriptional changes in gene expression. Methods Levels of haem were measured in the perfusate before and after 1h NMP in 43 transplanted and 15 research human kidneys. Levels of haem were correlated with the age of RBCs and parameters during NMP. Human research kidneys were used to examine the changes in transcriptional gene expression using Nanostring nCounter technology. Results In transplanted and research kidneys, levels of haem increased significantly (P &amp;lt;0.05) during NMP. Older units of pRBCs were associated with higher levels of haem, pre-/post-NMP. High levels of haem were associated with higher levels of potassium and lactate but not functional parameters during NMP. NanoString analysis demonstrated significant upregulation of 17 differentially expressed genes including those associated with apoptosis, inflammation and oxidative stress. qPCR analysis of targeted genes demonstrated increased expression of FOS (P&amp;lt;0.0001), IL-6 (P=0.0001), JUN (P=0.0001) and TLR-4 (P=0.035). Conclusion Older units of RBCs contain high levels of haem which increases during NMP. Although, this did not affect perfusion parameters, transcriptional analysis demonstrated significant upregulation of genes involved with apoptotic, inflammatory and oxidative pathways. Activation of these pathways may be associated with high levels of haem. Take-home message Older units of packed red blood cells release increased levels of haem during normothermic machine perfusion of kidneys. Higher levels of haem are linked to increased activation of damage pathways.

Low-dose carbon monoxide suppresses metastatic progression of disseminated cancer cells

Low-dose carbon monoxide (CO) is under investigation in clinical trials to treat non-cancerous diseases and has an excellent safety profile. Due to early detection and cancer awareness, an increasing number of cancer patients are diagnosed at early stages, when potentially curative surgical resection can be done. However, many patients ultimately experience recurrence. Here, we evaluate the therapeutic effect of CO on metastatic cancer progression. We show that 250 ppm CO inhibits the migration of multiple types of cancer cell lines, including breast, pancreatic, colon, prostate, liver, and lung cancer and reduces the ability to adhere to fibronectin. We demonstrate that in mouse models, 250 ppm inhaled CO inhibits lung metastasis of breast cancer and liver metastasis of pancreatic cancer. Moreover, low-dose CO suppresses recurrence and increases survival after surgical removal of primary pancreatic cancer in mice. Mechanistically, low-dose CO blocks transcription of heme importers, leading to diminished intracellular heme levels and a heme-regulated enzyme, cytochrome P4501B1 (CYP1B1). Either supplementing heme or overexpressing CYP1B1 reverses the anti-migration effect of low-dose CO. Taken together, low-dose CO therapy inhibits cell migration, reduces adhesion to fibronectin, prevents disseminated cancer cells from expanding into gross metastases, and improves survival in pre-clinical mouse models of metastasis.

Genome-scale modeling drives 70-fold improvement of intracellular heme production in Saccharomyces cerevisiae

Heme is an oxygen carrier and a cofactor of both industrial enzymes and food additives. The intracellular level of free heme is low, which limits the synthesis of heme proteins. Therefore, increasing heme synthesis allows an increased production of heme proteins. Using the genome-scale metabolic model (GEM) Yeast8 for the yeast Saccharomyces cerevisiae, we identified fluxes potentially important to heme synthesis. With this model, in silico simulations highlighted 84 gene targets for balancing biomass and increasing heme production. Of those identified, 76 genes were individually deleted or overexpressed in experiments. Empirically, 40 genes individually increased heme production (up to threefold). Heme was increased by modifying target genes, which not only included the genes involved in heme biosynthesis, but also those involved in glycolysis, pyruvate, Fe-S clusters, glycine, and succinyl-coenzyme A (CoA) metabolism. Next, we developed an algorithmic method for predicting an optimal combination of these genes by using the enzyme-constrained extension of the Yeast8 model, ecYeast8. The computationally identified combination for enhanced heme production was evaluated using the heme ligand-binding biosensor (Heme-LBB). The positive targets were combined using CRISPR-Cas9 in the yeast strain (IMX581-HEM15-HEM14-HEM3-Δshm1-HEM2-Δhmx1-FET4-Δgcv2-HEM1-Δgcv1-HEM13), which produces 70-fold-higher levels of intracellular heme.

Increase of HO-1 Expression in Critically Ill COVID-19 Patients Is Associated with Poor Prognosis and Outcome.

Heme-oxygenase (HO)-1 is a cytoprotective enzyme with strong antioxidant and anti-apoptotic properties and previous reports have also emphasized the antiviral properties of HO-1, either directly or via induction of interferons. To investigate the potential role of HO-1 in patients with coronavirus disease 2019 (COVID-19), the present study assessed changes in HO-1 expression in whole blood and tissue samples. Upregulation of HO-1 protein was observed in lung, liver, and skin tissue independently of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presence. A significant increase of blood HO-1 mRNA levels was observed in critically ill COVID-19 patients compared to those in severe COVID-19 patients and healthy controls. This increase was accompanied by significantly elevated levels of serum ferritin and bilirubin in critically ill compared to patients with severe disease. Further grouping of patients in survivors and non-survivors revealed a significant increase of blood HO-1 mRNA levels in the later. Receiver operating characteristic (ROC) analysis for prediction of ICU admission and mortality yielded an AUC of 0.705 (p = 0.016) and 0.789 (p = 0.007) respectively indicating that HO-1 increase is associated with poor COVID-19 progression and outcome. The increase in HO-1 expression observed in critically ill COVID-19 patients could serve as a mechanism to counteract increased heme levels driving coagulation and thrombosis or as an induced protective mechanism.

Reduction Kinetic of Water Soluble Metal Salts by Geobacter sulfurreducens: Fe2+/Hemes Stabilize and Regulate Electron Flux Rates.

Geobacter sulfurreducens is a widely applied microorganism for the reduction of toxic metal salts, as an electron source for bioelectrochemical devices, and as a reagent for the synthesis of nanoparticles. In order to understand the influence of metal salts, and of electron transporting, multiheme c-cytochromes on the electron flux during respiration of G. sulfurreducens, the reduction kinetic of Fe3+, Co3+, V5+, Cr6+, and Mn7+ containing complexes were measured. Starting from the resting phase, each G. sulfurreducens cell produced an electron flux of 3.7 × 105 electrons per second during the respiration process. Reduction rates were within ± 30% the same for the 6 different metal salts, and reaction kinetics were of zero order. Decrease of c-cytochrome concentrations by downregulation and mutation demonstrated that c-cytochromes stabilized respiration rates by variation of their redox states. Increasing Fe2+/heme levels increased electron flux rates, and induced respiration flexibility. The kinetic effects parallel electrochemical results of G. sulfurreducens biofilms on electrodes, and might help to optimize bioelectrochemical devices.

Abstract 1108: Evaluation of heme inhibitory therapy in combination with chemotherapy drugs on SCLC progression

Abstract Lung cancer is the leading cause of cancer-related death in the US. It is mainly divided in two types: small cell lung cancer and non-small cell lung cancer. About 85-90% of cases are classified as non-small cell lung cancer (NSCLC). Small cell lung cancer (SCLC) is an aggressive neuroendocrine lung tumor representing 15% of lung cancers. Despite the advent of targeted therapies and chemotherapies, effective therapies for lung cancer are an unlikely outcome for most patients. SCLC is initially highly responsive to cisplatin and etoposide chemotherapy but, in almost every case, becomes rapidly chemo resistant. However, the mechanisms underlying initial sensitivity and subsequent resistance are not well investigated. Thus, alternative approaches and combination therapy is required for treating SCLCs. Recent studies in our lab show that NSCLC cells demonstrate elevated levels of heme via increased heme uptake and synthesis, and elevated oxidative phosphorylation (OXPHOS) and ATP generation which fuels NSCLC proliferation. Also, limiting heme via inhibition of heme uptake using heme sequestering peptides (HeSP2) can delay growth and progression of NSCLC tumors. This HeSP2 can also effectively inhibit angiogenesis, normalize tumor vasculature, and alleviate tumor hypoxia in NSCLC tumors and thereby inhibit lung tumor growth and progression. Tumor heterogeneity is critical for tumor growth, metastasis, and acquired therapeutic resistance. To understand if heme can effectively reduce tumor cell proliferation and growth and improve tumor microenvironment, we treated chemo resistant SCLCs with HeSP2 and in combination with commonly used chemo drugs cisplatin and etoposide. Our data shows that HesP2 can strongly inhibit cell proliferation and decreases oxygen consumption rate along with ATP generation in SCLCs. To gain insights into mechanism by which the heme sequestering peptides limit tumor growth and progression, we implanted subcutaneous or SCLC cell lines in NOD/SCID mice. Mice were treated with saline (control), HeSP2, HeSP2 and cisplatin, HeSP2 and etoposide. Tumor growth was monitored using caliper measurements. Tumors tissues were harvested, processed and paraffin embedded for immunohistochemistry (IHC) and histology experiments. These results indicate that heme sequestration can be a powerful strategy for suppressing lung tumors likely drug-resistant tumors that rely on oxidative phosphorylation for survival. Citation Format: Adnin Ashrafi, Narges Salamat, Li Zhang. Evaluation of heme inhibitory therapy in combination with chemotherapy drugs on SCLC progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1108.

Extracellular Vesicles From Stored Red Blood Cells Convey Heme and Induce Spic Expression on Human Monocytes.

During cold storage, red blood cells (RBC) undergo irreversible morphological changes, hemolysis, and extracellular vesicles (EVs) release. It is known that reinfused stored RBC triggers immune reactions within T-lymphocytes and increases pro-oxidative and immunomodulatory metabolites in the plasma of healthy individuals. Monocytes/macrophages are the primary cells in charge of both uptake of excess heme and recognition of such EVs; however, the effect of reinfusion of stored RBC has not been investigated upon monocytes. Hence, the purpose of this study was to characterize how EVs released by cold-stored RBC impact monocytes. For this, we adopted an ex vivo protocol to mimic autologous blood transfusion. Colorimetric and metabolomic analyses confirmed that cold-stored RBC released increased heme levels. Transcriptomic analysis demonstrated that cold-stored RBC increased the transcript levels of heme-oxygenase 1 and Spic in monocytes, along with an induction of heme-oxygenase-1, CX3CR1, and CD206- known markers of macrophages M2-phenotype. Finally, we have demonstrated that the EVs released by cold-stored RBC were enriched in heme and conveyed to monocytes, triggering Spic expression and an acute production of reactive oxygen species. Finally, our study demonstrates that hemolysis resulting from autologous blood transfusion modifies gene expression patterns in monocytes towards an M2-like phenotype. Furthermore, we highlight the role of EVs released by cold-stored RBC in this scenario, which may act like a damage-associated molecular pattern inducing Spic in monocytes.

Influence of 5-aminolevulinic acid on physiological and biochemical characteristics of winter wheat varieties with different levels of anthocyanins in coleoptiles

Physiological and biochemical characteristics of two varieties of winter wheat seedlings with red (Et W5) and green (Vladi) coleoptiles have been studied. Variety Et W5 with high content of anthocyanins (on average 412 ± 52 μmol/g fr wt), chlorophylls (Chl) and carotenoids and a low level of heme and proline in coleoptiles had high frost resistance (88 % of surviving plants exposed to temperatures of ‒8 °C for 5 h at the age of 7 days) and a high ability to continue growth and development after undergoing cooling. Variety Vladi with a low content of anthocyanins (on average 12.5 ± 1.2 μmol/g fr wt), Chl and carotenoids, but a higher level of heme and proline in coleoptiles compared to varieties Et W5 had low level of frost resistance (80 % of surviving plants) and was characterized by a low ability to continue growth and development after undergoing cooling. Soaking wheat seeds in a solution of exogenous ALA (50 mg/l) for 2 h before sowing increased the germination of seeds of both varieties, enhanced the length of plants of the Et W5 variety by 10 %, 1.4 times the content of anthocyanins in the coleoptiles of this variety plants and also increased their frost resistance up to 97 %. Under action of ALA frost resistance of Vladi seedlings increased up to 93 %.

The Effects of Free Heme on Functional and Molecular Changes During Ex Vivo Normothermic Machine Perfusion of Human Kidneys.

Normothermic machine perfusion (NMP) is a technique of kidney preservation designed to restore cellular metabolism after cold ischemia. Kidneys are perfused with an oxygenated banked red blood cell (RBC) based solution for 1h at 36°C. During NMP, RBCs can become damaged, releasing free heme into the perfusate. This can act as a damage-associated molecular pattern (DAMP) activating inflammatory signalling pathways. The aim of this study was to measure the levels of free heme during NMP, assess the effect on kidney function and determine any association with inflammatory and stress related gene expression. Levels of free heme were measured in perfusate samples from a series of donation after circulatory death (DCD) kidneys undergoing NMP as part of a randomised controlled trial (RCT). The age of RBCs and levels of free heme were correlated with perfusion parameters. Changes in gene expression were analysed in a series of kidneys declined for transplantation using the NanoString nCounter Organ Transplant Panel and qRT-PCR. Older units of RBCs were associated with higher levels of free heme and levels increased significantly during NMP (Pre 8.56 ± 7.19µM vs 26.29 ± 15.18µM, P<0.0001). There was no association with levels of free heme and perfusion parameters during NMP (P > 0.05). Transcriptional and qPCR analysis demonstrated the upregulation of differentially expressed genes associated with apoptosis (FOS and JUN), inflammatory cytokines (IL-6, SOCS3, ATF3), chemokines (CXCL8, CXCL2, CC3/L1) and oxidative stress (KLF4) after NMP. However, these did not correlate with levels of free heme (P >0.05). A significant amount of free heme can be detected in the perfusate before and after NMP particularly when older units of red cells are used. Although transcriptional analysis demonstrated significant upregulation of genes involved with apoptotic, inflammatory and oxidative pathways these were not associated with high levels of free heme.