Sylvie Shen

Glycogen Synthase Kinase-3β Inhibition Preserves Hematopoietic Stem Cell Activity and Inhibits Leukemic Cell Growth

Ex vivo expansion of cord blood cells generally results in reduced stem cell activity in vivo. Glycogen synthase kinase‐3β (GSK‐3β) regulates the degradation of β‐catenin, a critical regulator of hematopoietic stem cells (HSCs). Here we show that GSK‐3β inhibition activates β‐catenin in cord blood CD34+ cells and upregulates β‐catenin transcriptional targets c‐myc and HoxB4, both known to regulate HSC self‐renewal. GSK‐3β inhibition resulted in delayed ex vivo expansion of CD34+ cells, yet enhanced the preservation of stem cell activity as tested in long‐term culture with bone marrow stroma. Delayed cell cycling, reduced apoptosis, and increased adherence of hematopoietic progenitor cells to bone marrow stroma were observed in these long‐term cultures treated with GSK‐3β inhibitor. This improved adherence to stroma was mediated via upregulation of CXCR4. In addition, GSK‐3β inhibition preserved severe combined immunodeficiency (SCID) repopulating cells as tested in the nonobese diabetic/SCID mouse model. Our data suggest the involvement of GSK‐3β inhibition in the preservation of HSC and their interaction with the bone marrow environment. Methods for the inhibition of GSK‐3β may be developed for clinical ex vivo expansion of HSC for transplantation. In addition, GSK‐3β inhibition suppressed leukemic cell growth via the induction of apoptosis mediated by the downregulation of survivin. Modulators of GSK‐3β may increase the range of novel drugs that specifically kill leukemic cells while sparing normal stem cells.

Blockade of KCa3.1 Ameliorates Renal Fibrosis Through the TGF-β1/Smad Pathway in Diabetic Mice

The Ca2+-activated K+ channel KCa3.1 mediates cellular signaling processes associated with dysfunction of vasculature. However, the role of KCa3.1 in diabetic nephropathy is unknown. We sought to assess whether KCa3.1 mediates the development of renal fibrosis in two animal models of diabetic nephropathy. Wild-type and KCa3.1−/− mice, and secondly eNOS−/− mice, had diabetes induced with streptozotocin and then were treated with/without a selective inhibitor of KCa3.1 (TRAM34). Our results show that the albumin-to-creatinine ratio significantly decreased in diabetic KCa3.1−/− mice compared with diabetic wild-type mice and in diabetic eNOS−/− mice treated with TRAM34 compared with diabetic mice. The expression of monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule 1 (ICAM1), F4/80, plasminogen activator inhibitor type 1 (PAI-1), and type III and IV collagen significantly decreased (P < 0.01) in kidneys of diabetic KCa3.1−/− mice compared with diabetic wild-type mice. Similarly, TRAM34 reduced the expression of the inflammatory and fibrotic markers described above in diabetic eNOS−/− mice. Furthermore, blocking the KCa3.1 channel in both animal models led to a reduction of transforming growth factor-β1 (TGF-β1) and TGF-β1 type II receptor (TβRII) and phosphorylation of Smad2/3. Our results provide evidence that KCa3.1 mediates renal fibrosis in diabetic nephropathy through the TGF-β1/Smad signaling pathway. Blockade of KCa3.1 may be a novel target for therapeutic intervention in patients with diabetic nephropathy.

Towards a Clinically Relevant Lentiviral Transduction Protocol for Primary Human CD34+ Hematopoietic Stem/Progenitor Cells

Background Hematopoietic stem cells (HSC), in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multi-potency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34+ HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin. Methodology/Principal Findings Using commercially available G-CSF mobilized peripheral blood (PB) CD34+ cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, pre-stimulation time, multiplicity of infection (MOI), transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin. Conclusions/Significance This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34+ cells.

Inhibition of kidney proximal tubular glucose reabsorption does not prevent against diabetic nephropathy in type 1 diabetic eNOS knockout mice.

Background and Objective Sodium glucose cotransporter 2 (SGLT2) is the main luminal glucose transporter in the kidney. SGLT2 inhibition results in glycosuria and improved glycaemic control. Drugs inhibiting this transporter have recently been approved for clinical use and have been suggested to have potential renoprotective benefits by limiting glycotoxicity in the proximal tubule. We aimed to determine the renoprotective benefits of empagliflozin, an SGLT2 inhibitor, independent of its glucose lowering effect. Research Design and Methods We induced diabetes using a low dose streptozotocin protocol in 7–8 week old endothelial nitric oxide (eNOS) synthase knockout mice. We measured fasting blood glucose on a monthly basis, terminal urinary albumin/creatinine ratio. Renal histology was assessed for inflammatory and fibrotic changes. Renal cortical mRNA transcription of inflammatory and profibrotic cytokines, glucose transporters and protein expression of SGLT2 and GLUT1 were determined. Outcomes were compared to diabetic animals receiving the angiotensin receptor blocker telmisartan (current best practice). Results Diabetic mice had high matched blood glucose levels. Empagliflozin did not attenuate diabetes-induced albuminuria, unlike telmisartan. Empagliflozin did not improve glomerulosclerosis, tubular atrophy, tubulointerstitial inflammation or fibrosis, while telmisartan attenuated these. Empagliflozin did not modify tubular toll-like receptor-2 expression in diabetic mice. Empagliflozin did not reduce the upregulation of macrophage chemoattractant protein-1 (MCP-1), transforming growth factor β1 and fibronectin mRNA observed in the diabetic animals, while telmisartan decreased transcription of MCP-1 and fibronectin. Empagliflozin increased GLUT1 mRNA expression and telmisartan increased SGLT2 mRNA expression in comparison to untreated diabetic mice. However no significant difference was found in protein expression of GLUT1 or SGLT2 among the different groups. Conclusion Hence SGLT2 inhibition does not have renoprotective benefits independent of glucose lowering.

KCa3.1 mediates activation of fibroblasts in diabetic renal interstitial fibrosis

Background Fibroblast activation plays a critical role in diabetic nephropathy (DN). The Ca2+-activated K+ channel KCa3.1 mediates cellular proliferation of many cell types including fibroblasts. KCa3.1 has been reported to be a potential molecular target for pharmacological intervention in a diverse array of clinical conditions. However, the role of KCa3.1 in the activation of myofibroblasts in DN is unknown. These studies assessed the effect of KCa3.1 blockade on renal injury in experimental diabetes. Methods As TGF-β1 plays a central role in the activation of fibroblasts to myofibroblasts in renal interstitial fibrosis, human primary renal interstitial fibroblasts were incubated with TGF-β1 +/− the selective inhibitor of KCa3.1, TRAM34, for 48 h. Two streptozotocin-induced diabetic mouse models were used in this study: wild-type KCa3.1+/+ and KCa3.1−/− mice, and secondly eNOS−/− mice treated with or without a selective inhibitor of KCa3.1 (TRAM34). Then, markers of fibroblast activation and fibrosis were determined. Results Blockade of KCa3.1 inhibited the upregulation of type I collagen, fibronectin, α-smooth muscle actin, vimentin and fibroblast-specific protein-1 in renal fibroblasts exposed to TGF-β1 and in kidneys from diabetic mice. TRAM34 reduced TGF-β1-induced phosphorylation of Smad2/3 and ERK1/2 but not P38 and JNK MAPK in interstitial fibroblasts. Conclusions These results suggest that blockade of KCa3.1 attenuates diabetic renal interstitial fibrogenesis through inhibiting activation of fibroblasts and phosphorylation of Smad2/3 and ERK1/2. Therefore, therapeutic interventions to prevent or ameliorate DN through targeted inhibition of KCa3.1 deserve further consideration.

Cassette deletion in multiple shRNA lentiviral vectors for HIV-1 and its impact on treatment success

BackgroundMultiple short hairpin RNA (shRNA) gene therapy strategies are currently being investigated for treating viral diseases such as HIV-1. It is important to use several different shRNAs to prevent the emergence of treatment-resistant strains. However, there is evidence that repeated expression cassettes delivered via lentiviral vectors may be subject to recombination-mediated repeat deletion of 1 or more cassettes.ResultsThe aim of this study was to determine the frequency of deletion for 2 to 6 repeated shRNA cassettes and mathematically model the outcomes of different frequencies of deletion in gene therapy scenarios. We created 500+ clonal cell lines and found deletion frequencies ranging from 2 to 36% for most combinations. While the central positions were the most frequently deleted, there was no obvious correlation between the frequency or extent of deletion and the number of cassettes per combination. We modeled the progression of infection using combinations of 6 shRNAs with varying degrees of deletion. Our in silico modeling indicated that if at least half of the transduced cells retained 4 or more shRNAs, the percentage of cells harboring multiple-shRNA resistant viral strains could be suppressed to < 0.1% after 13 years. This scenario afforded a similar protection to all transduced cells containing the full complement of 6 shRNAs.ConclusionDeletion of repeated expression cassettes within lentiviral vectors of up to 6 shRNAs can be significant. However, our modeling showed that the deletion frequencies observed here for 6× shRNA combinations was low enough that the in vivo suppression of replication and escape mutants will likely still be effective.

Plumbagin Ameliorates Diabetic Nephropathy via Interruption of Pathways that Include NOX4 Signalling

NADPH oxidase 4 (Nox4) is reported to be the major source of reactive oxygen species (ROS) in the kidneys during the early stages of diabetic nephropathy. It has been shown to mediate TGFβ1-induced differentiation of cardiac fibroblasts into myofibroblasts. Despite TGFβ1 being recognised as a mediator of renal fibrosis and functional decline role in diabetic nephropathy, the renal interaction between Nox 4 and TGFβ1 is not well characterised. The aim of this study was to investigate the role of Nox4 inhibition on TGFβ1-induced fibrotic responses in proximal tubular cells and in a mouse model of diabetic nephropathy. Immortalised human proximal tubular cells (HK2) were incubated with TGFβ1 ± plumbagin (an inhibitor of Nox4) or specific Nox4 siRNA. Collagen IV and fibronectin mRNA and protein expression were measured. Streptozotocin (STZ) induced diabetic C57BL/6J mice were administered plumbagin (2 mg/kg/day) or vehicle (DMSO; 50 µl/mouse) for 24 weeks. Metabolic, physiological and histological markers of nephropathy were determined. TGFβ1 increased Nox4 mRNA expression and plumbagin and Nox4 siRNA significantly inhibited TGF-β1 induced fibronectin and collagen IV expression in human HK2 cells. STZ-induced diabetic C57BL/6J mice developed physiological features of diabetic nephropathy at 24 weeks, which were reversed with concomitant plumbagin treatment. Histologically, plumbagin ameliorated diabetes induced upregulation of extracellular matrix protein expression compared to control. This study demonstrates that plumbagin ameliorates the development of diabetic nephropathy through pathways that include Nox4 signalling.

A retroviral library genetic screen identifies IRF-2 as an inhibitor of N-ras-induced growth suppression in leukemic cells

Activating mutations of the N-ras gene occur at relatively high frequency in acute myeloid leukemia and myelodysplastic syndrome. Somewhat paradoxically, ectopic expression of activated N-ras in primary hematopoietic cells and myeloid cell lines (in some cases) can lead to inhibition of proliferation. Expression of mutant N-ras in murine hematopoietic stem/progenitor cells is sufficient to induce myeloid malignancies, but these pathologies occur with long latency. This suggests that mutations that disable the growth suppressive properties of N-ras in hematopoietic cells are required for the development of frank malignancy. In the present work, the growth suppression induced by a mutant N-ras gene in U937 myeloid cells was used as the basis to screen a retroviral cDNA library for genes that prevent mutant N-ras-induced growth suppression (i.e., putative cooperating oncogenes). This screen identified the gene for the transcription factor interferon regulatory factor-2 (IRF-2), and as confirmation of the screen, overexpression of this gene in U937 cells was shown to inhibit mutant N-ras-induced growth suppression. Also recovered from the screen were two truncated clones of an uncharacterized gene (interim official symbol: PP2135). Overexpression of this truncated PP2135 gene in U937 cells did not appear to abrogate mutant N-ras-induced growth suppression, but rather appeared to confer an increased sensitivity of U937 cells to retroviral infection, accounting for the recovery of this gene from the genetic screen.

GSK-3β inhibition promotes early engraftment of ex vivo-expanded haematopoietic stem cells.

Umbilical cord blood (UCB) is a source of stem cells used for allogeneic transplantation, in addition to bone marrow and peripheral blood. Limited numbers of stem cells in a single UCB unit is associated with slow haematopoietic recovery and high risk of graft failure, particularly in adult patients. UCB stem cells can be expanded ex vivo; however, rapid differentiation reduces their regenerative potential. We have recently shown that Wnt/β‐catenin signalling is down‐regulated in ex vivo‐expanded stem cells; therefore, we propose that re‐activation of Wnt signalling using GSK‐3β inhibition may act to improve regenerative potential of these ex vivo‐expanded stem cells.

The Role of IRF1 and IRF2 Transcription Factors in Leukaemogenesis

Acute myeloid leukaemia (AML) is the most common form of leukaemia in adults. Although of the order of 75-85% of patients will achieve complete remission after induction chemotherapy, long-term survival is still relatively low. Despite the progress in the rational design of drugs in disorders such as chronic myeloid leukaemia, AML lacks a single specific pathogenomic event to act as a drug target. Interferon regulatory factor 1 (IRF1) is a member of a family of related proteins that act as transcriptional activators or repressors. IRF1 and its functional antagonist IRF2 originally discovered as transcription factors regulating the interferon-beta (IFN-beta) gene, are involved in the regulation of normal haematopoiesis and leukaemogenesis. IRF1 appears to act as a tumour suppressor gene and IRF2 as an oncogene. IRF1 acts to repress IRF2 function through the repression of cyclin-dependent kinase (CDK) inhibitor p21WAF1 critical for cell growth control. It appears that the tumour suppression function of IRF1 is abolished by IRF2. This review focuses on the interaction between IRF1 and IRF2 in myeloid development and leukaemogenesis, particularly in relation to the Ras signalling pathway. IRF2 may be a viable and specific therapeutic target in human leukaemia.