Heme Levels Research Articles

ROCK1 promotes B-cell differentiation and proteostasis under stress through the heme-regulated proteins, BACH2 and HRI.

The mechanisms utilized by differentiating B cells to withstand highly damaging conditions generated during severe infections, like the massive hemolysis that accompanies malaria, are poorly understood. Here we demonstrate that ROCK1 regulates B cells differentiation in hostile environments replete with PAMPs (pathogen-associated molecular patterns) and high levels of heme by controlling two key heme-regulated molecules, BACH2 and Heme-regulated eIF2a kinase (HRI). ROCK1 phosphorylates BACH2 and protects it from heme-driven degradation. As B cells differentiate, furthermore, ROCK1 restrains their proinflammatory potential and helps them handle the heightened stress imparted by the presence of PAMPs and heme by controlling HRI, a key regulator of the integrated stress response and cytosolic proteotoxicity. ROCK1 controls the interplay of HRI with HSP90 and limits the recruitment of HRI and HSP90 to unique p62/SQSTM1 complexes that also contain critical kinases like mTORC1 and TBK1, and proteins involved in RNA metabolism, oxidative damage, and proteostasis like TDP-43. Thus, ROCK1 helps B cells cope with intense pathogen-driven destruction by coordinating the activity of key controllers of B cell differentiation and stress responses. These ROCK1-dependent mechanisms may be widely employed by cells to handle severe environmental stresses, and these findings may be relevant for immune-mediated and age-related neurodegenerative disorders.

A Review on MERS-CoV, The Coronavirus Linked to Middle Eastern Respiratory illness

Middle Eastern respiratory syndromes (MERS-CoV) are a novel coronavirus discovered in Saudi Arabia in 2012, with several outbreaks connected to the Arabian Peninsula. The virus has a high fatality rate and no effective antiviral medication or vaccination. It is known to spread mainly among bats and mammals, with close contact being the most likely way to spread the disease. MERS-CoV, SARS-CoV, and SARS-CoV-2 are all initially transmitted via mammals to humans. Medications like Lopinavir and ritonavir are used to treat COVID-19 caused by SARS-CoV-2. Lopinavir inhibits the HIV-1 protease enzyme, promoting the formation of immature virus particles. Acyclovir is used to treat acute herpetic keratitis, varicella zoster, viral labialis, and herpes labialis, while Ribavirin is used to treat hemorrhagic fever caused by viruses and hepatitis C. Chloroquine, a synthetic guanosine nucleoside, was removed from FDA authorization for emergency therapy for COVID-19 in June 2020. It inhibits heme to hemozoin transformation in malarial trophozoites, destroying the parasite when harmful heme levels in Plasmodium species grow. Chloroquine is used to avoid and cure malaria, treats extra intestinal amebiasis, and in some hospitalized individuals for COVID-19 coronavirus treatment. To ensure safety, visitors should wash their hands before and after touching animals and avoid handling dromedary camels, consuming uncooked meat, or consuming camel milk or urine.

Heme alters biofilm formation in Mycobacterium abscessus.

Mycobacterium abscessus (Mabs) is commonly found in the cystic fibrosis (CF) lung. During infection, Mabs can form biofilms in the lung which reduce both the ability of the immune response to clear infection and the effectiveness of antibiotic therapy. In the CF lung, heme and hemoglobin levels are increased and may provide both iron and heme to Mabs cells. In this work, we show that exogenous heme altered Mabs biofilm formation and measured the effects of exogenous heme on protein level and metabolism in Mabs. Our findings suggest that heme impacts iron homeostasis in Mabs and affects other aspects of its metabolism, highlighting the potential role of heme as a critical nutrient for Mabs growth and biofilm formation.IMPORTANCEMycobacterium abscessus (Mabs) is commonly found in the cystic fibrosis (CF) lung, where Mabs can form biofilms that can reduce the efficacy of antibiotics. During infection, the CF lung can have more than 10 times the extracellular heme than that of a healthy lung. We have found that extracellular heme can change the way Mabs cells grow and form biofilms, which may have implications for pathogenesis.

Monitoring the Biochemical Activity of Single Anammox Granules with Microbarometers.

Granule-based anaerobic ammonium oxidation (Anammox) is a promising biotechnology for wastewater treatments with extraordinary performance in nitrogen removal. However, traditional analytical methods often delivered an average activity of a bulk sample consisting of millions and even billions of Anammox granules with distinct sizes and components. Here, we developed a novel technique to monitor the biochemical activity of individual Anammox granules in real-time by recording the production rate of nitrogen gas with a microbarometer in a sealed chamber containing only one granule. It was found that the specific activity of a single Anammox granule not only varied by tens of folds among different individuals with similar sizes (activity heterogeneity) but also revealed significant breath-like dynamics over time (temporal fluctuation). Statistical analysis on tens of individuals further revealed two subpopulations with distinct color and specific activity, which were subsequently attributed to the different expression levels of heme c content and hydrazine dehydrogenase activity. This study not only provides a general methodology for various kinds of gas-producing microbial processes but also establishes a bottom-up strategy for exploring the structural-activity relationship at a single sludge granule level, with implications for developing a better Anammox process.

Biosensor-based growth-coupling as an evolutionary strategy to improve heme export in Corynebacterium glutamicum

The iron-containing porphyrin heme is of high interest for the food industry for the production of artificial meat as well as for medical applications. Recently, the biotechnological platform strain Corynebacterium glutamicum has emerged as a promising host for animal-free heme production. Beyond engineering of complex heme biosynthetic pathways, improving heme export offers significant yet untapped potential for enhancing production strains. In this study, a growth-coupled biosensor was designed to impose a selection pressure on the increased expression of the hrtBA operon encoding an ABC-type heme exporter in C. glutamicum. For this purpose, the promoter region of the growth-regulating genes pfkA (phosphofructokinase) and aceE (pyruvate dehydrogenase) was replaced with that of PhrtB, creating biosensor strains with a selection pressure for hrtBA activation. Resulting sensor strains were used for plate-based selections and for a repetitive batch f(luorescent)ALE using a fully automated laboratory platform. Genome sequencing of isolated clones featuring increased hrtBA expression revealed three distinct mutational hotspots: (i) chrS, (ii) chrA, and (iii) cydD. Mutations in the genes of the ChrSA two-component system, which regulates hrtBA in response to heme levels, were identified as a promising target to enhance export activity. Furthermore, causal mutations within cydD, encoding an ABC-transporter essential for cytochrome bd oxidase assembly, were confirmed by the construction of a deletion mutant. Reversely engineered strains showed strongly increased hrtBA expression as well as increased cellular heme levels. These results further support the proposed role of CydDC as a heme transporter in bacteria. Mutations identified in this study therefore underline the potential of biosensor-based growth coupling and provide promising engineering targets to improve microbial heme production.

RESISTANCE TO PHYTOPHTHORA1 promotes cytochrome b559 formation during early photosystem II biogenesis in Arabidopsis.

As an essential intrinsic component of photosystem II (PSII) in all oxygenic photosynthetic organisms, heme-bridged heterodimer cytochrome b559 (Cyt b559) plays critical roles in the protection and assembly of PSII. However, the underlying mechanisms of Cyt b559 assembly are largely unclear. Here, we characterized the Arabidopsis (Arabidopsis thaliana) rph1 (resistance to Phytophthora1) mutant, which was previously shown to be susceptible to the oomycete pathogen Phytophthora brassicae. Loss of RPH1 leads to a drastic reduction in PSII accumulation, which can be primarily attributed to the defective formation of Cyt b559. Spectroscopic analyses showed that the heme level in PSII supercomplexes isolated from rph1 is significantly reduced, suggesting that RPH1 facilitates proper heme assembly in Cyt b559. Due to the loss of RPH1-mediated processes, a covalently bound PsbE-PsbF heterodimer is formed during the biogenesis of PSII. In addition, rph1 is highly photosensitive and accumulates elevated levels of reactive oxygen species under photoinhibitory-light conditions. RPH1 is a conserved intrinsic thylakoid protein present in green algae and terrestrial plants, but absent in Synechocystis, and it directly interacts with the subunits of Cyt b559. Thus, our data demonstrate that RPH1 represents a chloroplast acquisition specifically promoting the efficient assembly of Cyt b559, probably by mediating proper heme insertion into the apo-Cyt b559 during the initial phase of PSII biogenesis.

Hemoglobin homeostasis in abdominal aortic aneurysm: diagnostic and prognostic potential of hemoglobin/heme and scavenger molecules

BackgroundThere is increasing evidence implicating hemoglobin/heme and their scavengers in oxidative stress-mediated pathologies, but information is limited in abdominal aortic aneurysm (AAA).Methods and resultsIn this case-control study, we assessed heme/heme-related markers in 142 men with AAA and 279 men with a normal aortic diameter consecutively recruited from an ultrasound screening program in Sweden. Enzyme-linked immunosorbent assays (ELISAs) were used to measure heme oxygenase-1 (HO-1) and hemopexin (Hpx) plasma levels, colorimetric assays for cell-free heme and whole blood hemoglobin (Hb) levels, and droplet digital PCR (ddPCR) and real-time PCR to determine haptoglobin (Hp) (pheno)type and genotype, respectively. Hpx and heme plasma levels at baseline were elevated, while HO-1 levels were lower in men with AAA (p < 0.001) and were significantly associated with AAA prevalence independently of potential confounders. A combination of heme and HO-1 showed the best diagnostic potential based on the area under the curve (AUC): 0.76, sensitivity: 80%, specificity: 48%. Additionally, when previously described inflammatory biomarker interleukin-6 (IL-6), was added to our model it significantly improved the diagnostic value (AUC: 0.87, sensitivity: 80%, specificity: 79%) compared to IL-6 alone (AUC: 0.73, sensitivity: 80%, specificity: 49%). Finally, Hb (positively) and Hpx (negatively) levels at baseline were associated with AAA growth rate (mm/year), and their combination showed the best prognostic value for discriminating fast and slow-growing AAA (AUC: 0.76, sensitivity: 80%, specificity: 62%).ConclusionsThis study reports the distinct disruption of heme and related markers in both the development and progression of AAA, underscoring their potential in aiding risk stratification and therapeutic strategies.

Active Heme Metabolism Suppresses Macrophage Phagocytosis via the TLR4/Type I IFN Signaling/CD36 in Uterine Endometrial Cancer.

Uterine endometrial cancer (UEC) is a common gynecological estrogen-dependent carcinoma, usually accompanied by intermenstrual bleeding. Active heme metabolism frequently plays an increasingly important role in many diseases, especially in cancers. Tumor-associated macrophages (TAMs) are the major population in the immune microenvironment of UEC. However, the roles of heme metabolisms in the crosstalk between UEC cells (UECCs) and macrophages are unclear. In our study, by using TCGA database analysis, integration analysis of the protein-protein interaction (PPI) network and sample RNA transcriptome sequencing were done. The expression level of both heme-associated molecules and iron metabolism-related molecules were measured by quantitative real-time polymerase chain reaction. Heme level detection was done through dehydrohorseradish peroxidase assay. In addition to immunohistochemistry, phagocytosis assay of macrophages, immunofluorescence staining, intracellular ferrous iron staining, as well as enzyme-linked immune sorbent assay were performed. In the study, we verified that heme accumulation in UECCs is apparently higher than in endometrial epithelium cells. Low expression of succinate dehydrogenase B under the regulation of estrogen contributes to over-production of succinate and heme accumulation in UECC. More importantly, excessive heme in UECCs impaired macrophage phagocytosis by regulation of CD36. Mechanistically, this process is dependent on toll-like receptor (TLR4)/type I interferons alpha (IFN Iα) regulatory axis in macrophage. Collectively, these findings elucidate that active heme metabolism of UECCs directly decreases phagocytosis by controlling the secretion of TLR4-mediated IFN Iα and the expression of CD36, and further contributing to the immune escape of UEC.

Betaine activates the Nrf2‐Keap1‐ARE pathway by increasing the methylation level of Keap1 DNA promoter

SummaryBetaine is a natural antioxidant lacking the ability to scavenge free radicals. Although it has been shown to exert antioxidant function by enhancing the expression of antioxidant enzymes, its mechanism has not yet been elucidated. This study explored the role and mechanism of the transcription factor NF‐E2‐associated factor 2 (Nrf2)‐Kelch‐like epichlorohydrin‐associated Protein 1 (Keap1) antioxidant reaction element (ARE) pathway in betaine‐mediated enhancement of antioxidant enzyme expression. Results derived from real‐time quantitative PCR, Western blot, quantitative methylation‐specific PCR, and inhibitors assay experiments showed that betaine activated the Nrf2‐Keap1‐ARE pathway, leading to an increase in the mRNA level of superoxide dismutase, glutathione peroxidase, and heme oxygenase‐1. The mechanism may involve betaine's promotion of DNA methyltransferase expression, resulting in increased methylation of Keap1 DNA promoter, which ultimately reduces the level of Keap1 mRNA. Therefore, betaine can directly activate the Nrf2‐Keap1‐ARE pathway by increasing the methylation level of Keap1 DNA promoter, thereby improving the expression of antioxidant enzymes. This study will contribute to unveiling a new antioxidant mechanism for betaine.

Characterizing the 70-gene MammaPrint risk stratification in Chinese luminal type breast cancer and its correlation with clinicopathological characteristics, immune and metabolic functions.

e12559 Background: The 70-gene MammaPrint is endorsed by the NCCN guidelines for predicting 5-year distant metastasis risk in HR+, HER2-, with 0-3 lymph nodes, early invasive breast cancer. Similarly, it is recommended by the CSCO breast cancer guidelines for prognostic assessment post breast cancer surgery. However, no prior studies have investigated its application in Chinese luminal-type breast patients or its correlation with immune and metabolic functions. Methods: This analysis encompasses 76 early-stage invasive Luminal breast cancer patients, registered at the First People's Hospital of Foshan between 2020 and 2023, with recorded clinical characteristics under informed consent. Patient-reported data include clinical features, pathology, and the 70-gene MammaPrint risk of recurrence. Additionally, RNA-seq analysis was conducted in 24 patients. Results: Among 76 enrolled patients,55.3% were categorized as MammaPrint low risk (MP-Low), and 44.7% as MammaPrint high risk (MP-High). Patients over 50 were more likely MP-Low (p=0.028, OR=2.860), while those with &gt;20% Ki67-stained cells were prone to MP-High (p&lt;0.001, OR=0.160). Comparing with MINDACT, there was 52.3% consistency, with 55.6% of MP-Low patients being C-Low (MINDACT risk Low) and 47.1% of MP-High patients being C-High (MINDACT risk High). Notably,21% of C-Low patients showed MP-High risk, and 27% of C-High patients exhibited MP-Low risk, of which 83.3% were ≥50 years old. Based on MINDACT and ISPY2 trial outcomes, patients were further categorized into four groups: MP-Ultralow, MP-Low but not ultralow (LNUL), MP-High but not ultrahigh (HNUH) and MP-Ultrahigh. The study identified an 18% MP-Ultralow population, suggesting that excessive adjuvant therapy might be avoided in these patients. Additionally, 41% of patients were identified as LNUL group, indicating potential benefit from adjuvant treatment or endocrine prolongation therapy. 4% were identified as MP-Ultrahigh group, suggesting potential benefits from neoadjuvant targeted therapy, albeit a poorer long-term benefit in the non-pCR portion. Further transcriptome analysis in 24 patients revealed significantly higher levels of Th2 cells in the MP-High group, with no significant difference observed in CD8 T cells between the two groups. Meanwhile, metabolic-related signatures also differed, with significantly higher levels of Dopamine, Norepinephrine, Heme, and Epinephrine biosynthesis in the MP-Low group. Conclusions: This study elucidates the intricate relationships between MammaPrint prediction results of 70 genes and clinical characteristics, immune and metabolic signatures in Chinese Luminal type breast cancer patients. The refined risk classification provides valuable insights for precision treatment, advancing personalized therapeutic strategies.

Lifespan regulation by targeting heme signaling in yeast

Heme is an essential prosthetic group that serves as a co-factor and a signaling molecule. Heme levels decline with age, and its deficiency is associated with multiple hallmarks of aging, including anemia, mitochondrial dysfunction, and oxidative stress. Dysregulation of heme homeostasis has been also implicated in aging in model organisms suggesting that heme may play an evolutionarily conserved role in controlling lifespan. However, the underlying mechanisms and whether heme homeostasis can be targeted to promote healthy aging remain unclear. Here, we used Saccharomyces cerevisiae as a model to investigate the role of heme in aging. For this, we have engineered a heme auxotrophic yeast strain expressing a plasma membrane-bound heme permease from Caenorhabditis elegans (ceHRG-4). This system can be used to control intracellular heme levels independently of the biosynthetic enzymes by manipulating heme concentration in the media. We observed that heme supplementation leads to a significant extension of yeast replicative lifespan. Our findings revealed that the effect of heme on lifespan is independent of the Hap4 transcription factor. Surprisingly, heme-supplemented cells had impaired growth on YPG medium, which requires mitochondrial respiration to be used, suggesting that these cells are respiratory deficient. Together, our results demonstrate that heme homeostasis is fundamentally important for aging biology, and manipulating heme levels can be used as a promising therapeutic target for promoting longevity.

Export of heme by the feline leukemia virus C receptor regulates mitochondrial biogenesis and redox balance in the hematophagous insect Rhodnius prolixus.

Heme is a prosthetic group of proteins involved in vital physiological processes. It participates, for example, in redox reactions crucial for cell metabolism due to the variable oxidation state of its central iron atom. However, excessive heme can be cytotoxic due to its prooxidant properties. Therefore, the control of intracellular heme levels ensures the survival of organisms, especially those that deal with high concentrations of heme during their lives, such as hematophagous insects. The export of heme initially attributed to the feline leukemia virus C receptor (FLVCR) has recently been called into question, following the discovery of choline uptake by the same receptor in mammals. Here, we found that RpFLVCR is a heme exporter in the midgut of the hematophagous insect Rhodnius prolixus, a vector for Chagas disease. Silencing RpFLVCR decreased hemolymphatic heme levels and increased the levels of intracellular dicysteinyl-biliverdin, indicating heme retention inside midgut cells. FLVCR silencing led to increased expression of heme oxygenase (HO), ferritin, and mitoferrin mRNAs while downregulating the iron importers Malvolio 1 and 2. In contrast, HO gene silencing increased FLVCR and Malvolio expression and downregulated ferritin, revealing crosstalk between heme degradation/export and iron transport/storage pathways. Furthermore, RpFLVCR silencing strongly increased oxidant production and lipid peroxidation, reduced cytochrome c oxidase activity, and activated mitochondrial biogenesis, effects not observed in RpHO-silenced insects. These data support FLVCR function as a heme exporter, playing a pivotal role in heme/iron metabolism and maintenance of redox balance, especially in an organism adapted to face extremely high concentrations of heme.

Quantitative analysis of heme and hemoglobin for the detection of intravascular hemolysis

BackgroundIntravascular hemolysis is associated with massive release of hemoglobin and consequently labile heme into the blood, resulting in prothrombotic and proinflammatory events in patients. Though heme is well-known to participate in these adverse effects, it is not monitored. Instead, haptoglobin and hemoglobin serve as clinical biomarkers. The quantification of labile heme together with hemoglobin, however, should be considered in clinical diagnosis as well, to obtain a complete picture of the hemolytic state in patients. So far, quantification techniques for labile heme were not yet systematically analyzed and compared for their clinical application potential, especially in the presence of hemoglobin. ResultsTwo commercial assays (Heme Assay Kit®, Hemin Assay Kit®) and five common approaches (pyridine hemochromogen assay, apo-horseradish peroxidase-based assay, UV/Vis spectroscopy, HPLC, mass spectrometry) were analyzed concerning their linearity, accuracy, and precision, as well as their ability to distinguish between hemoglobin-bound heme and labile heme. Further, techniques for the quantification of hemoglobin (Harboe method, SLS method, Hemastix®) were included to study their selectivity for hemoglobin and potential interference by the presence of labile heme. Both, indirect and direct approaches were suitable for the determination of a wide concentration of heme (∼0.02–45 μM) and hemoglobin (∼0.002–17 μM). A clear distinction between hemoglobin-bound heme and labile heme with one method was not possible. Thus, a novel combined approach is presented and applied to human and porcine plasma samples for the determination of hemoglobin and labile heme. SignificanceOur results demonstrate the need to develop improved techniques to differentiate labile and protein-bound heme for early detection of intravascular hemolysis. Here, we present a novel strategy by combining two spectroscopic methods, which is most reliable as an easy-to-use tool for the determination of hemoglobin and heme levels in plasma samples for the diagnosis of intravascular hemolysis and in basic biomedical research.

Hemoglobin scavenger receptor CD163 as a potential biomarker of hemolysis-induced hepatobiliary injury in sickle cell disease.

Sickle cell disease (SCD)-associated chronic hemolysis promotes oxidative stress, inflammation, and thrombosis leading to organ damage, including liver damage. Hemoglobin scavenger receptor CD163 plays a protective role in SCD by scavenging both hemoglobin-haptoglobin complexes and cell-free hemoglobin. A limited number of studies in the past have shown a positive correlation of CD163 expression with poor disease outcomes in patients with SCD. However, the role and regulation of CD163 in SCD-related hepatobiliary injury have not been fully elucidated yet. Here we show that chronic liver injury in SCD patients is associated with elevated levels of hepatic membrane-bound CD163. Hemolysis and increase in hepatic heme, hemoglobin, and iron levels elevate CD163 expression in the SCD mouse liver. Mechanistically we show that heme oxygenase-1 (HO-1) positively regulates membrane-bound CD163 expression independent of nuclear factor erythroid 2-related factor 2 (NRF2) signaling in SCD liver. We further demonstrate that the interaction between CD163 and HO-1 is not dependent on CD163-hemoglobin binding. These findings indicate that CD163 is a potential biomarker of SCD-associated hepatobiliary injury. Understanding the role of HO-1 in membrane-bound CD163 regulation may help identify novel therapeutic targets for hemolysis-induced chronic liver injury.

Prognostic value and potential function of a novel heme-related LncRNAs signature in gastric cancer

Heme is a coordination complex formed by the binding of iron ions and porphyrin rings. Its metabolic processes are associated with various cancers, including gastric cancer (GC). In recent years, long non-coding RNAs (LncRNAs) have been identified as key regulatory factors in GC. However, the role of LncRNAs associated with heme metabolism in GC and their relationship with prognosis have not been reported. In this study, we constructed a novel LncRNAs signature related to heme metabolism (HMlncSig) and validated its prognostic value for predicting the survival of GC patients through training, test, and entire cohorts. Kaplan-Meier analysis demonstrated that patients in the high-risk group had shorter survival times. Univariate and multivariate Cox regression analysis showed that HMlncSig was an independent prognostic indicator for GC patients, regardless of other clinical pathological features. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis and gene set variation analysis pathways showed that the activation of these markers may be involved in tumor progression, influencing the survival of GC patients. The nomogram, based on HMlncSig score and clinical features, demonstrated the strong predictive ability of this signature. Additionally, significant differences were observed between the high-risk and low-risk groups in terms of immune cell subtypes, expression of immune checkpoint genes, and response to chemotherapy and immunotherapy. Through clinical validation, we found that the risk score and heme levels of GC patients were both significantly elevated and correlated with the degree of malignancy. Furthermore, we found that AP000692.1, a key gene in this signature, promoted the proliferation, migration, and invasion of GC cells. In conclusion, our HMlncSig model has significant predictive value for the prognosis of GC patients and can provide clinical guidance for personalized immunotherapy.

Abstract 4132: Low-dose carbon monoxide therapies suppress cancer metastasis

Abstract Metastasis is the primary cause of cancer-related deaths. Novel systemic therapies for metastatic cancer are urgently needed. Carbon monoxide (CO) is an endogenously produced signaling molecule in the human body with demonstrated pharmacological activities, including organ protection and anti-inflammation. We determined the therapeutic effect of low-dose CO and CO prodrugs on cancer progression in this study. We found that low-dose CO inhibits the migration of cancer cell lines including breast, pancreatic, colon, prostate, liver, and lung cancer. We showed that low-dose CO inhibits lung metastasis of breast cancer and liver metastasis of pancreatic cancer in preclinical mouse models. Further, to provide safer and more reliable CO delivery, we developed metal-free, organic CO prodrugs and showed that CO prodrugs inhibit tumor growth and metastasis. We demonstrated that low-dose CO blocks the transcription of heme importers, leading to diminished intracellular heme levels. Altogether, our work lays a strong foundation for the development of CO-based systemic cancer therapies. Citation Format: Tiantian Zhang, Cheryl Zhang, Xiang Chen, Xiaoxiao Yang, Qiyue Mao, George Zhang, Zhengming Chen, Adrian Tan, Jenny Zhaoying Xiang, Erika Hissong, Yao-Tseng Chen, Binghe Wang, Nancy Du. Low-dose carbon monoxide therapies suppress cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4132.

Alpha1-antitrypsin improves survival in murine abdominal sepsis model by decreasing inflammation and sequestration of free heme.

Excessive inflammation, hemolysis, and accumulation of labile heme play an essential role in the pathophysiology of multi-organ dysfunction syndrome (MODS) in sepsis. Alpha1-antitrypsin (AAT), an acute phase protein with heme binding capacity, is one of the essential modulators of host responses to inflammation. In this study, we evaluate the putative protective effect of AAT against MODS and mortality in a mouse model of polymicrobial abdominal sepsis. Polymicrobial abdominal sepsis was induced in C57BL/6N mice by cecal ligation and puncture (CLP). Immediately after CLP surgery, mice were treated intraperitoneally with three different forms of human AAT-plasma-derived native (nAAT), oxidized nAAT (oxAAT), or recombinant AAT (recAAT)-or were injected with vehicle. Sham-operated mice served as controls. Mouse survival, bacterial load, kidney and liver function, immune cell profiles, cytokines/chemokines, and free (labile) heme levels were assessed. In parallel, in vitro experiments were carried out with resident peritoneal macrophages (MPMΦ) and mouse peritoneal mesothelial cells (MPMC). All AAT preparations used reduced mortality in septic mice. Treatment with AAT significantly reduced plasma lactate dehydrogenase and s-creatinine levels, vascular leakage, and systemic inflammation. Specifically, AAT reduced intraperitoneal accumulation of free heme, production of cytokines/chemokines, and neutrophil infiltration into the peritoneal cavity compared to septic mice not treated with AAT. In vitro experiments performed using MPMC and primary MPMΦ confirmed that AAT not only significantly decreases lipopolysaccharide (LPS)-induced pro-inflammatory cell activation but also prevents the enhancement of cellular responses to LPS by free heme. In addition, AAT inhibits cell death caused by free heme in vitro. Data from the septic CLP mouse model suggest that intraperitoneal AAT treatment alone is sufficient to improve sepsis-associated organ dysfunctions, preserve endothelial barrier function, and reduce mortality, likely by preventing hyper-inflammatory responses and by neutralizing free heme.

Cobalt protoporphyrin promotes heme oxygenase 1 expression and ameliorates cardiac dysfunction in long-term fasting mice

BackgroundThe association between malnutrition and cardiac dysfunction has been reported. Heme oxygenase (HO)-1 played protective roles in the animals functioning as a myocardial infarction, heart failure, or cardiomyopathy model. We hypothesized that the administration of HO-1 inducer, cobalt protoporphyrin (CoPP) reduces oxidative stress and ameliorates cardiac systolic dysfunction in long-term fasting mice. MethodsC57BL/6 J mice were classified into three groups: fed mice (fed group), 48-h fasting mice with a single intraperitoneal injection of the corresponding vehicle (fasting group), and 48-h fasting mice with a single intraperitoneal injection of 5 mg/kg CoPP (CoPP group). ResultsThe fasting group showed a significant increase in heme and 4-hydroxy-2-nonenal (4HNE) protein in the heart tissue, and reduced left ventricular ejection fraction (LVEF) when compared with the fed group. The CoPP group showed significantly increased protein levels of nuclear factor-erythroid 2-related factor 2 and HO-1, and increased mRNA expression levels of HO-1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, forkhead box protein O1, sirtuin-1, cyclooxygenase 2, and superoxide dismutase 2, and reduced levels of heme and 4HNE protein when compared with the fasting group. LVEF were significantly higher in the CoPP group than in the fasting group. ConclusionsAdministration of CoPP reduced heme accumulation and oxidative stress, and ameliorated cardiac systolic dysfunction in long-term fasting mice. This study suggests that heme accumulation may be associated with impaired cardiac function induced by long-term fasting and that HO-1 may be a key factor or therapeutic target.

Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO

Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45–65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology.

Endoperoxide-enhanced self-assembled ROS producer as intracellular prodrugs for tumor chemotherapy and chemodynamic therapy.

Prodrug-based self-assembled nanoparticles (PSNs) with tailored responses to tumor microenvironments show a significant promise for chemodynamic therapy (CDT) by generating highly toxic reactive oxygen species (ROS). However, the insufficient level of intracellular ROS and the limited drug accumulation remain major challenges for further clinical transformation. In this study, the PSNs for the delivery of artesunate (ARS) are demonstrated by designing the pH-responsive ARS-4-hydroxybenzoyl hydrazide (HBZ)-5-amino levulinic acid (ALA) nanoparticles (AHA NPs) with self-supplied ROS for excellent chemotherapy and CDT. The PSNs greatly improved the loading capacity of artesunate and the ROS generation from endoperoxide bridge using the electron withdrawing group attached directly to C10 site of artesunate. The ALA and ARS-HBZ could be released from AHA NPs under the cleavage of hydrazone bonds triggered by the acidic surroundings. Besides, the ALA increased the intracellular level of heme in mitochondria, further promoting the ROS generation and lipid peroxidation with ARS-HBZ for excellent anti-tumor effects. Our study improved the chemotherapy of ARS through the chemical modification, pointing out the potential applications in the clinical fields.