A core function of CHARM’s grant focuses on expanding and enhancing the research advances made by the Howard University Research Scientist Program in clinical and molecular aspects of sickle cell disease, iron metabolism, and oxygen sensing.
The following projects are being conducted using CHaRM support:
Molecular mechanisms of increased iron stores caused by the ferroportin Q248H mutation, which is unique to Africans and African Americans
Ferroportin is a critical transporter in iron metabolism, and provides the only mechanism for excreting iron out of cells. Iron overload is a common and potentially fatal clinical complication of hemoglobinopathy patients who require packed red blood cell transfusions, both intermittently and regularly. Its onset is often insidious in sickle cell patients. We previously showed that ferroportin Q248H mutation is moderately resistant to hepcidin, which may have a protective effect in sickle cell disease. We used mass spectrometry to analyze the effect of the ferroportin Q248H mutation on its ubiquitination, which occurs at nearby amino acids, and may be impaired by this mutation. We are also developing vectors for stable ferroportin Q248H expression in macrophages. We obtained IRB approval to isolate primary epithelial cells from patients’ urine to generate iPSC, which we plan to convert to macrophages to analyze the functional effects of the ferroportin Q248H mutation. These tools will allow us to determine the effects of the mutation on iron overload in sickle cell disease, and how it affects sickle cell patients, as a potential biomarker for risks.
Analysis of the hypoxic response in sickle cell disease
We conducted a gene array analysis of genes differentially expressed in SCD patients using sample collection with an existing IRB approval for the study of pulmonary hypertension (PH), a serious complication of sickle cell disease with a high mortality rate. We also conducted real-time PCR validation and pathway analysis using Ingenuity software. We detected previously unrecognized genes involved in heme and iron regulatory pathways which are candidates for contributing to development of PH, which we plan to investigate further as potential biomarkers of patients at differing levels of risk for PH. We are focusing on heme-regulatory pathway initially, as increased hemolysis has been shown to increase the risks of PH.
Analysis of the hypoxic response and HIV-1
Findings in this work have relevance to hypoxia and cells containing sickle hemoglobin. We used global proteomic approach to analyze the phosphoproteome of murine liver with conditional phosphatase knock out, which we observed to be downregulated under ischemic conditions. We utilized a new software, SIEVE 2.1 which allows label-free quantification of raw spectral data and peptides identification with Proteome Discover (Thermo).
Potential protective effect of sickle cell disease against HIV-1 infection
Obtained IRB and analyzed expression of HIV-1 restricting genes including heme oxygenase-1 (HO-1), ferroportin, HIF-1, CDK2, CDK9, and PP1. We showed for the first time that ferroportin is expressed in SCD PBMCs and that hepcidin can alleviate the HIV-1 inhibition in SCD-derived PBMCs. Developed several knock-out cell lines including HIF-1, ferroportin, EGR-1 to analyze their effect HIV-1. Dr. Nekhai applied for RO1 to expand the experiments initially proposed under CHaRM.
Analysis of novel iron chelators
We developed in house novel iron chelators that we found to induce p21 and an NF-kB inhibitor, IKBα, and inhibit HIV-1.
This is potentially quite exciting as there is no “perfect” or universally tolerated iron chelator, and many patients still cannot maintain the 24/7 coverage that is needed to protect the heart. Additional chelators are needed.
Urinary markers of renal disease in SCD patients
Obtained IRB and collected urine samples from several SCD patients. Also obtained urine samples from Dr. Gordeuk’s study at UIC in which hemoglobin was shown to be a predictor of chronic renal disease in SCD patients. We conducted global proteomic analysis of several urine samples and identified several protein candidate factors that may serve as potential biomarkers. In collaboration with Dr. Jerebtsova at Children’s National Medical Center, we analyzed the effect of previously identified factor, PEDF (pigmental epithelium-derived factor), in mouse injected with LPS to determine its effect on proteinurea.
Acute lung dysfunction in HIV-1 infection and SCD
We recently showed that pigmental epithelium derived factor (PEDF) promotes growth of primary endothelial cells. We analyzed the effect of PEDF on endothelial cells in vitro and mice and showed that it prevents apoptosis of endothelial cells. We then analyzed the effect of PEDF on the lungs in WT and HIV-1 transgenic mice injected with LPS. We observed increased HIV-1 expression in the lungs in accord with our earlier observations that high oxygen levels induce HIV-1 replication. This work was submitted as a UO1 application where feedback was obtained. We plan to resubmit to NHLBI as an RO1 in response to a specific RFA.
Effect of protein phosphatase-1 (PP1) inhibition on hydration and sickling of SCD RBCs
Dehydration is a critical factor in the pathophysiology of sickle polymerization and most of its complications. PPI inhibition is a potential approach to preventing this damaging process. We developed PP1-targeted inhibitors which were analyzed for the binding to PP1 in collaboration with Dr. Aykut Uren (Georgetown University). We tested some of these compounds on HIV-1 and Ebola viruses (in collaboration with UTMB). The Ebola project was funded as part of U19 grant (Chris Basler, Mt. Sinai University – PI). These unrelated viruses utilize PP1 during transcription. Developing PP1-targeting molecules may be beneficial in future studies of sickle cell disease as anti-sickling agents and also to alleviate the negative effect of PP1 during hypoxia.
Increasing awareness of recruitment and retention strategies in sickle cell disease research
Enrollment and retention of patients is a critical problem of all clinical trials, but is especially limiting to all research in rare diseases. Trial participation is especially onerous for patients with limited socio-economic means. Determining factors that negatively influence enrollment and retention can assist the entire field. After reviewing the current literature, we have developed two initial questionnaires for patients and investigators to determine factors that affect investigators presenting studies to patients, patients accepting studies, and staying on studies. Howard University IRB approved the study. We recruited 15 sickle cell patients from Howard and 2 investigators from Howard and Case Western Reserve University to complete the pilot survey. Data from pilot surveys were used to reformat the questionnaires. The questionnaires were reviewed by two experts in the field and final questionnaires are ready for IRB approval and participants’ recruitment.
Fetal Hemoglobin Expression in Neonates with Sickle Cell Inheritance
Steel factor, the c-kit ligand, has a dramatic enhancement of HbF expression, more than any other growth factor, and likely is important in differential declines in HbF, the major modifier of sickle cell disease severity. We have analyzed the temporal expression of c-Kit in different hemoglobinopathy genotypes. We are currently analyzing transcriptional regulators of hemoglobin switching as mediated by c-Kit. We collected 106 neonatal blood samples, identified hemoglobin profiles in erythrocytes using HPLC, and developed a database of newborn clinical parameters. We are in the process of profiling other recognized transcriptional regulators of fetal globin expression (i.e. KLF1, BCL11A, Sox6, FOG, etc.) using microarrays to correlate; (1) the basal expression of cKit as an inhibitor of HbF switching, (2) key clinical parameters with expression profile, and (3) level of HbF with the temporal expression profile.
Clinical trials of HbF inducers
Two therapeutic candidates for clinical trials are being discussed with Dr. Thomas Mellman, for conduct in the Clinical Research Unit (CRU) from the GHUCCTS. One is available now, an agent which has 2 distinct molecular mechanisms and for which an IND is active and a drug supply is available to Dr. Perrine. A second agent, discovered through high throughput screening of an approved medicinal library by Dr. Perrine’s group, was accepted for development by the TRND program. This drug has been used for 40 years for another medical condition in many countries worldwide, has a benign safety profile, and has shown high inducing activity in two in vivo models. An IND, US FDA required toxicology studies, and a drug supply will be prepared with the TRND team.
We are developing quantitative approaches for global proteome analysis and also for phosphoproteome analysis of potential biomarkers and mediators of severity in sickle cell disease. We also provided, as part of this Core, access to various equipment including fluorescence, absorbance and luminescence plate readers, Bio-Rad imaging station, Roche Real time PCR machine and Agilent FACS analyzer for investigators of CHaRM research.