RESUMO
Oxygen (O2) binds to hemoglobin (Hb) in the lungs and is then released (dissociated) in the tissues. The Bohr effect is a physiological mechanism that governs the affinity of Hb for O2 based on pH, where a lower pH results in a lower Hb-O2 affinity and higher Hb-O2 dissociation. Hb-O2 affinity and dissociation are crucial for maintaining aerobic metabolism in cells and tissues. Despite its vital role in human physiology, Hb-O2 dissociation measurement is underutilized in basic research and in clinical laboratories, primarily due to the technical complexity and limited throughput of existing methods. We present a rapid Hb-O2 dissociation measurement approach by leveraging the Bohr effect and detecting the optical shift in the Soret band that corresponds to the light absorption by the heme group in Hb. This new method reduces Hb-O2 dissociation measurement time from hours to minutes. We show that Hb deoxygenation can be accelerated chemically at the optimal pH of 6.9. We show that time and pH-controlled deoxygenation of Hb results in rapid and distinct conformational changes in its tertiary structure. These molecular conformational changes are manifested as significant, detectable shifts in Hb's optical absorption spectrum, particularly in the characteristic Soret band (414 nm). We extensively validated the method by testing human blood samples containing normal Hb and Hb variants. We show that rapid Hb-O2 dissociation can be used to screen for and detect Hb-O2 affinity disorders and to evaluate the function and efficacy of Hb-modifying therapies. The ubiquity of optical absorption spectrophotometers positions this approach as an accessible, rapid, and accurate Hb-O2 dissociation measurement method for basic research and clinical use. We anticipate this method's broad adoption will democratize the diagnosis and prognosis of Hb disorders, such as sickle cell disease. Further, this method has the potential to transform the research and development of new targeted and genome-editing-based therapies that aim to modify or improve Hb-O2 affinity.
Assuntos
Hemoglobinas , Óptica e Fotônica , Oxigênio , Humanos , Hemoglobinas/química , Hemoglobinas/metabolismo , Hemoglobinas/análise , Concentração de Íons de Hidrogênio , Oxigênio/metabolismo , Oxigênio/química , Óptica e Fotônica/métodosRESUMO
Individuals with sickle cell disease (SCD) have persistently elevated thrombin generation that results in a state of systemic hypercoagulability. Antithrombin-III (ATIII), an endogenous serine protease inhibitor, inhibits several enzymes in the coagulation cascade, including thrombin. Here, we utilize a biomimetic microfluidic device to model the morphology and adhesive properties of endothelial cells (ECs) activated by thrombin and examine the efficacy of ATIII in mitigating the adhesion of SCD patient-derived red blood cells (RBCs) and EC retraction. Microfluidic devices were fabricated, seeded with ECs, and incubated under physiological shear stress. Cells were then activated with thrombin with or without an ATIII pretreatment. Blood samples from subjects with normal haemoglobin (HbAA) and subjects with homozygous SCD (HbSS) were used to examine RBC adhesion to ECs. Endothelial cell surface adhesion molecule expression and confluency in response to thrombin and ATIII treatments were also evaluated. We found that ATIII pretreatment of ECs reduced HbSS RBC adhesion to thrombin-activated endothelium. Furthermore, ATIII mitigated cellular contraction and reduced surface expression of von Willebrand factor and vascular cell adhesion molecule-1 (VCAM-1) mediated by thrombin. Our findings suggest that, by attenuating thrombin-mediated EC damage and RBC adhesion to endothelium, ATIII may alleviate the thromboinflammatory manifestations of SCD.
Assuntos
Anemia Falciforme , Trombina , Anticoagulantes/farmacologia , Antitrombinas/metabolismo , Antitrombinas/farmacologia , Adesão Celular , Células Endoteliais , Endotélio Vascular/metabolismo , Eritrócitos , Humanos , Trombina/metabolismo , Trombina/farmacologiaRESUMO
Red blood cell (RBC) deformability is a valuable hemorheological biomarker that can be used to assess the clinical status and response to therapy of individuals with sickle cell disease (SCD). RBC deformability has been measured by ektacytometry for decades, which uses shear or osmolar stress. However, ektacytometry is a population based measurement that does not detect small-fractions of abnormal RBCs. A single cell-based, functional RBC deformability assay would complement ektacytometry and provide additional information. Here, we tested the relative merits of the OcclusionChip, which measures RBC deformability by microcapillary occlusion, and ektacytometry. We tested samples containing glutaraldehyde-stiffened RBCs for up to 1% volume fraction; ektacytometry detected no significant change in Elongation Index (EI), while the OcclusionChip showed significant differences in Occlusion Index (OI). OcclusionChip detected a significant increase in OI in RBCs from an individual with sickle cell trait (SCT) and from a subject with SCD who received allogeneic hematopoietic stem cell transplant (HSCT), as the sample was taken from normoxic (pO2:159 mmHg) to physiologic hypoxic (pO2:45 mmHg) conditions. Oxygen gradient ektacytometry detected no difference in EI for SCT or HSCT. These results suggest that the single cell-based OcclusionChip enables detection of sickle hemoglobin (HbS)-related RBC abnormalities in SCT and SCD, particularly when the HbS level is low. We conclude that the OcclusionChip is complementary to the population based ektacytometry assays, and providing additional sensitivity and capacity to detect modest abnormalities in red cell function or small populations of abnormal red cells.
RESUMO
Understanding the cancer stem cell (CSC) landscape in diffuse intrinsic pontine glioma (DIPG) is desperately needed to address treatment resistance and identify novel therapeutic approaches. Patient-derived DIPG cells demonstrated heterogeneous expression of aldehyde dehydrogenase (ALDH) and CD133 by flow cytometry. Transcriptome-level characterization identified elevated mRNA levels of MYC, E2F, DNA damage repair (DDR) genes, glycolytic metabolism, and mTOR signaling in ALDH+ compared with ALDH-, supporting a stem-like phenotype and indicating a druggable target. ALDH+ cells demonstrated increased proliferation, neurosphere formation, and initiated tumors that resulted in decreased survival when orthotopically implanted. Pharmacologic MAPK/PI3K/mTOR targeting downregulated MYC, E2F, and DDR mRNAs and reduced glycolytic metabolism. In vivo PI3K/mTOR targeting inhibited tumor growth in both flank and an ALDH+ orthotopic tumor model likely by reducing cancer stemness. In summary, we describe existence of ALDH+ DIPGs with proliferative properties due to increased metabolism, which may be regulated by the microenvironment and likely contributing to drug resistance and tumor recurrence. IMPLICATIONS: Characterization of ALDH+ DIPGs coupled with targeting MAPK/PI3K/mTOR signaling provides an impetus for molecularly targeted therapy aimed at addressing the CSC phenotype in DIPG.