Pharmacologic stabilization of HIF-1α increases hematopoietic stem cell quiescence in vivo and accelerates blood recovery after severe irradiation.

UNLABELLED: Quiescent hematopoietic stem cells (HSCs) preferentially reside in poorly perfused niches that may be relatively hypoxic. Most of the cellular effects of hypoxia are mediated by O2-labile hypoxia-inducible transcription factors (HIFs). To investigate the effects of hypoxia on HSCs, we blocked O2-dependent HIF-1α degradation in vivo in mice by injecting 2 structurally unrelated prolyl hydroxylase domain (PHD) enzyme inhibitors: dimethyloxalyl glycine and FG-4497. Injection of either of these 2 PHD inhibitors stabilized HIF-1α protein expression in the BM. In vivo stabilization of HIF-1a with these PHD inhibitors increased the proportion of phenotypic HSCs and immature hematopoietic progenitor cells in phase G0 of the cell cycle and decreased their proliferation as measured by 5-bromo-2'-deoxyuridine incorporation. This effect was independent of erythropoietin, the expression of which was increased in response to PHD inhibitors. Finally, pretreatment of mice with a HIF-1α stabilizer before severe, sublethal 9.0-Gy irradiation improved blood recovery and enhanced 89-fold HSC survival in the BM of irradiated mice as measured in long-term competitive repopulation assays. The results of the present study demonstrate that the levels of HIF-1α protein can be manipulated pharmacologically in vivo to increase HSC quiescence and recovery from irradiation. KEY POINTS: HIF-1α protein stabilization increases HSC quiescence in vivo. HIF-1α protein stabilization increases HSC resistance to irradiation and accelerates recovery.

B-lymphopoiesis is stopped by mobilizing doses of G-CSF and is rescued by overexpression of the anti-apoptotic protein Bcl2.

Osteoblasts are necessary to B lymphopoiesis and mobilizing doses of G-CSF or cyclophosphamide inhibit osteoblasts, whereas AMD3100/Plerixafor does not. However, the effect of these mobilizing agents on B lymphopoiesis has not been reported. Mice (wild-type, knocked-out for TNF-α and TRAIL, or over-expressing Bcl-2) were mobilized with G-CSF, cyclophosphamide, or AMD3100. Bone marrow, blood, spleen and lymph node content in B cells was measured. G-CSF stopped medullar B lymphopoiesis with concomitant loss of B-cell colony-forming units, pre-pro-B, pro-B, pre-B and mature B cells and increased B-cell apoptosis by an indirect mechanism. Overexpression of the anti-apoptotic protein Bcl2 in transgenic mice rescued B-cell colony forming units and pre-pro-B cells in the marrow, and prevented loss of all B cells in marrow, blood and spleen. Blockade of endogenous soluble TNF-α with Etanercept, or combined deletion of the TNF-α and TRAIL genes did not prevent B lymphopoiesis arrest in response to G-CSF. Unlike G-CSF, treatments with cyclophosphamide or AMD3100 did not suppress B lymphopoiesis but caused instead robust B-cell mobilization. G-CSF, cyclophosphamide and AMD3100 have distinct effects on B lymphopoiesis and B-cell mobilization with: 1) G-CSF inhibiting medullar B lymphopoiesis without mobilizing B cells in a mechanism distinct from the TNF-α-mediated loss of B lymphopoiesis observed during inflammation or viral infections; 2) CYP mobilizing B cells but blocking their maturation; and 3) AMD3100 mobilizing B cells without affecting B lymphopoiesis. These results suggest that blood mobilized with these three agents may have distinct immune properties.

Experience with a Clinical Audit Requirement for Interns in the Emergency Department.

BACKGROUND: The clinical audit is an important evaluation tool to ensure quality assurance. A clinical audit requirement for interns during their emergency department (ED) term may be a valuable educational activity. METHODS: The Emergency Audit Initiative (EAI) Program was initiated at Redcliffe Hospital in January 2018. Interns, who were paired with a staff specialist audit mentor, chose a topic of interest and carried out a clinical audit during their 10-week ED term. At the end of term, interns formally presented audit findings in a grand round setting. Interns and staff specialists were surveyed at the end of the intern year regarding aspects of the program. Surveys aimed to assess: (1) value of the program as an educational activity, (2) availability of time and resources to conduct the audit, and (3) perceived impact on practice. RESULTS: During the first year of the program, 27 clinical audits were carried out. 16 interns (59%) and 8 staff specialists (57%) responded to the surveys. Interns and staff specialists reported that the audit was a valuable educational experience (88% and 100%). Interns also reported that they had adequate time (94%) and resources (81%) to conduct the audit. Interns and staff specialists however reported only a modest impact on clinical practice because of the audit program. CONCLUSIONS: Our experience with the EAI program suggests that incorporating a clinical audit requirement into the ED term is possible. Interns and staff specialists reported it to be a beneficial educational and professional development activity.

Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cultured at reduced oxygen tensions.

Human embryonic stem (hES) cells are routinely cultured under atmospheric, 20% oxygen tensions but are derived from embryos which reside in a 3-5% oxygen (hypoxic) environment. Maintenance of oxygen homeostasis is critical to ensure sufficient levels for oxygen-dependent processes. This study investigates the importance of specific hypoxia inducible factors (HIFs) in regulating the hypoxic responses of hES cells. We report that culture at 20% oxygen decreased hES cell proliferation and resulted in a significantly reduced expression of SOX2, NANOG and POU5F1 (OCT4) mRNA as well as POU5F1 protein compared with hypoxic conditions. HIF1A protein was not expressed at 20% oxygen and displayed only a transient, nuclear localisation at 5% oxygen. HIF2A (EPAS1) and HIF3A displayed a cytoplasmic localisation during initial hypoxic culture but translocated to the nucleus following long-term culture at 5% oxygen and were significantly upregulated compared with cells cultured at 20% oxygen. Silencing of HIF2A resulted in a significant decrease in both hES cell proliferation and POU5F1, SOX2 and NANOG protein expression while the early differentiation marker, SSEA1, was concomitantly increased. HIF3A upregulated HIF2A and prevented HIF1A expression with the knockdown of HIF3A resulting in the reappearance of HIF1A protein. In summary, these data demonstrate that a low oxygen tension is preferential for the maintenance of a highly proliferative, pluripotent population of hES cells. While HIF3A was found to regulate the expression of both HIF1A and HIF2A, it is HIF2A which regulates hES cell pluripotency as well as proliferation under hypoxic conditions.

Regional nodal metastasis and 5-year survival in patients with thin melanoma in Queensland: a population-based study.

BACKGROUND: Optimal management of regional lymph nodes for thin cutaneous melanoma is uncertain. We evaluated regional lymph node involvement and 5-year melanoma-specific survival (MSS) in patients with thin (≤1 mm) primary melanoma. METHODS: Patients with a melanoma, American Joint Committee on Cancer Staging 8th Edition pT1a (<0.8 mm) or pT1b (ulceration; and/or 0.8-1.0 mm), diagnosed during 2001-2015 were identified from the Queensland Oncology Repository. We extracted demographic, pathology and clinical details, including sentinel lymph node biopsy (SLNB), regional nodal dissection and nodal recurrence. Poisson regression was used to assess recurrence risk in patients who did not undergo SLNB. The 5-year MSS was calculated using the Kaplan-Maier method with Cox regression to compare survival outcomes according to SLNB performance. RESULTS: Of the 27 824 eligible patients, 240 (0.9%) underwent SLNB. One hundred and seventy-eight patients (0.6%) without SLNB had nodal recurrence. Of the 4848 patients with a pT1b lesion, 166 (3.4%) had SLNB with 12 (7.2%) positive; of the remainder, 99 (2.1%) had clinical recurrence. Risk of recurrence was higher in males, nodular subtype and T1b lesions and lower if patients were aged >60 years. The 5-year MSS was similar for observed and SLNB cohorts (99.66% versus 98.92%) but worse for T1b lesions (98.90%) and clinical nodal recurrence (66.89%). CONCLUSION: Overall prognosis for T1 melanoma is excellent with nodal involvement being rare. However, the American Joint Committee on Cancer 8th Edition T1b melanoma correlates with significantly worse 5-year MSS and increased regional nodal recurrence (notably for 0.8-1.0 mm lesions with ulceration). Further characterization of high-risk groups for nodal positivity that impacts patient outcome is needed for the pT1 melanoma cohort.

Targeting the hypoxia-sensing pathway in clinical hematology.

Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcription factors regulated by oxygen-dependent prolyl hydroxylase domain (PHD) enzymes and are key to cell adaptation to low oxygen. The hematopoietic stem cell (HSC) niche in the bone marrow is highly heterogeneous in terms of microvasculature and thus oxygen concentration. The importance of hypoxia and HIFs in the hematopoietic environment is becoming increasingly recognized. Many small compounds that inhibit PHDs have been developed, enabling HIFs to be pharmacologically stabilized in an oxygen-independent manner. The use of PHD inhibitors for therapeutic intervention in hematopoiesis is being increasingly investigated. PHD inhibitors are well established to increase erythropoietin production to correct anemia in hemodialysis patients. Pharmacological stabilization of HIF-1α protein with PHD inhibitors is also emerging as an important regulator of HSC proliferation and self-renewal. Administration of PHD inhibitors increases quiescence and decreases proliferation of HSCs in the bone marrow in vivo, thereby protecting them from high doses of irradiation and accelerating hematological recovery. Recent findings also show that stabilization of HIF-1α increases mobilization of HSCs in response to granulocyte colony-stimulating factor and plerixafor, suggesting that PHD inhibitors could be useful agents to increase mobilization success in patients requiring transplantation. These findings highlight the importance of the hypoxia-sensing pathway and HIFs in clinical hematology.

Mobilization with granulocyte colony-stimulating factor blocks medullar erythropoiesis by depleting F4/80(+)VCAM1(+)CD169(+)ER-HR3(+)Ly6G(+) erythroid island macrophages in the mouse.

Similarly to other tissues, the bone marrow contains subsets of resident tissue macrophages, which are essential to maintain bone formation, functional hematopoietic stem cell (HSC) niches, and erythropoiesis. Pharmacologic doses of granulocyte colony-stimulating factor (G-CSF) mobilize HSC in part by interfering with the HSC niche-supportive function of BM resident macrophages. Because bone marrow macrophages are key to both maintenance of HSC within their niche and erythropoiesis, we investigated the effect of mobilizing doses of G-CSF on erythropoiesis in mice. We now report that G-CSF blocks medullar erythropoiesis by depleting the erythroid island macrophages we identified as co-expressing F4/80, vascular cell adhesion molecule-1, CD169, Ly-6G, and the ER-HR3 erythroid island macrophage antigen. Both broad macrophage depletion, achieved by injecting clodronate-loaded liposomes, and selective depletion of CD169(+) macrophages, also concomitantly depleted F4/80(+)VCAM-1(+)CD169(+)ER-HR3(+)Ly-6G(+) erythroid island macrophages and blocked erythropoiesis. This more precise phenotypic definition of erythroid island macrophages will enable studies on their biology and function in normal settings and on diseases associated with anemia. Finally, this study further illustrates that macrophages are a potent relay of innate immunity and inflammation on bone, hematopoietic, and erythropoietic maintenance. Agents that affect these macrophages, such as G-CSF, are likely to affect these three processes concomitantly.

Environmental oxygen tension regulates the energy metabolism and self-renewal of human embryonic stem cells.

Energy metabolism is intrinsic to cell viability but surprisingly has been little studied in human embryonic stem cells (hESCs). The current study aims to investigate the effect of environmental O2 tension on carbohydrate utilisation of hESCs. Highly pluripotent hESCs cultured at 5% O2 consumed significantly more glucose, less pyruvate and produced more lactate compared to those maintained at 20% O2. Moreover, hESCs cultured at atmospheric O2 levels expressed significantly less OCT4, SOX2 and NANOG than those maintained at 5% O2. To determine whether this difference in metabolism was a reflection of the pluripotent state, hESCs were cultured at 5% O2 in the absence of FGF2 for 16 hours leading to a significant reduction in the expression of SOX2. In addition, these cells consumed less glucose and produced significantly less lactate compared to those cultured in the presence of FGF2. hESCs maintained at 5% O2 were found to consume significantly less O2 than those cultured in the absence of FGF2, or at 20% O2. GLUT1 expression correlated with glucose consumption and using siRNA and chromatin immunoprecipitation was found to be directly regulated by hypoxia inducible factor (HIF)-2α at 5% O2. In conclusion, highly pluripotent cells associated with hypoxic culture consume low levels of O2, high levels of glucose and produce large amounts of lactate, while at atmospheric conditions glucose consumption and lactate production are reduced and there is an increase in oxidative metabolism. These data suggest that environmental O2 regulates energy metabolism and is intrinsic to the self-renewal of hESCs.

Vascular niche E-selectin regulates hematopoietic stem cell dormancy, self renewal and chemoresistance.

The microenvironment, or niche, surrounding a stem cell largely governs its cellular fate. Two anatomical niches for hematopoietic stem cells (HSCs) have been reported in the bone marrow, but a distinct function for each of these niches remains unclear. Here we report a new role for the adhesion molecule E-selectin expressed exclusively by bone marrow endothelial cells in the vascular HSC niche. HSC quiescence was enhanced and self-renewal potential was increased in E-selectin knockout (Sele(-/-)) mice or after administration of an E-selectin antagonist, demonstrating that E-selectin promotes HSC proliferation and is a crucial component of the vascular niche. These effects are not mediated by canonical E-selectin ligands. Deletion or blockade of E-selectin enhances HSC survival threefold to sixfold after treatment of mice with chemotherapeutic agents or irradiation and accelerates blood neutrophil recovery. As bone marrow suppression is a severe side effect of high-dose chemotherapy, transient blockade of E-selectin is potentially a promising treatment for the protection of HSCs during chemotherapy or irradiation.