Lucia Prezioso

Diabetes Impairs Hematopoietic Stem Cell Mobilization by Altering Niche Function

Impaired mobilization of hematopoietic stem cells in diabetic mice is due to sympathetic nervous system dysregulation of CXCL12 distribution. Boosting Stem Cell Mobilization Transplantation of hematopoietic stem cells (HSCs) from the bone marrow is a successful approach for treating blood diseases and certain cancers. Usually, the patient’s own (autologous) HSCs are used for transplant, but in some patients, their HSCs cannot be mobilized in sufficient numbers using the growth factor G-CSF (granulocyte colony-stimulating factor) to enable a successful transplant. In a new study, Ferraro and colleagues set out to discover the causes of this poor HSC mobilization. The investigators discovered by analyzing data from a number of bone marrow transplant patients that patients with diabetes showed poorer mobilization of HSCs in response to G-CSF than did those patients who did not have diabetes. The authors then confirmed in mouse models of type 1 and type 2 diabetes that HSCs were poorly mobilized from the bone marrow in response to G-CSF in these mice but not healthy control animals. The authors discovered that there was a defect in the bone marrow microenvironment of the diabetic mice rather than a problem with the HSCs themselves. Specifically, in diabetic (but not control) mice, the researchers observed mislocalization of HSCs in the bone marrow and an increase in the number of perivascular sympathetic nerve fibers in the niche with a concomitant inability of bone marrow mesenchymal stem cells to down-modulate production of the chemokine CXCL12 (a molecule known to mediate HSC localization). Finally, the authors were able to overcome the defect in HSC mobilization using a clinically approved drug called AMD3100 that interrupts the interaction of CXCL12 with its receptor CXCR4. The authors suggest that AMD3100 could be used to boost HSC mobilization in diabetic patients who require a bone marrow transplant. Success with transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) in patients depends on adequate collection of these cells after mobilization from the bone marrow niche by the cytokine granulocyte colony-stimulating factor (G-CSF). However, some patients fail to achieve sufficient HSPC mobilization. Retrospective analysis of bone marrow transplant patient records revealed that diabetes correlated with poor mobilization of CD34+ HSPCs. In mouse models of type 1 and type 2 diabetes (streptozotocin-induced and db/db mice, respectively), we found impaired egress of murine HSPCs from the bone marrow after G-CSF treatment. Furthermore, HSPCs were aberrantly localized in the marrow niche of the diabetic mice, and abnormalities in the number and function of sympathetic nerve termini were associated with this mislocalization. Aberrant responses to β-adrenergic stimulation of the bone marrow included an inability of marrow mesenchymal stem cells expressing the marker nestin to down-modulate the chemokine CXCL12 in response to G-CSF treatment (mesenchymal stem cells are reported to be critical for HSPC mobilization). The HSPC mobilization defect was rescued by direct pharmacological inhibition of the interaction of CXCL12 with its receptor CXCR4 using the drug AMD3100. These data suggest that there are diabetes-induced changes in bone marrow physiology and microanatomy and point to a potential intervention to overcome poor HSPC mobilization in diabetic patients.

Diabetes Mellitus Induces Bone Marrow Microangiopathy

Objective—The impact of diabetes on the bone marrow (BM) microenvironment was not adequately explored. We investigated whether diabetes induces microvascular remodeling with negative consequence for BM homeostasis. Methods and Results—We found profound structural alterations in BM from mice with type 1 diabetes with depletion of the hematopoietic component and fatty degeneration. Blood flow (fluorescent microspheres) and microvascular density (immunohistochemistry) were remarkably reduced. Flow cytometry verified the depletion of MECA-32+ endothelial cells. Cultured endothelial cells from BM of diabetic mice showed higher levels of oxidative stress, increased activity of the senescence marker &bgr;-galactosidase, reduced migratory and network-formation capacities, and increased permeability and adhesiveness to BM mononuclear cells. Flow cytometry analysis of lineage− c-Kit+ Sca-1+ cell distribution along an in vivo Hoechst-33342 dye perfusion gradient documented that diabetes depletes lineage− c-Kit+ Sca-1+ cells predominantly in the low-perfused part of the marrow. Cell depletion was associated to increased oxidative stress, DNA damage, and activation of apoptosis. Boosting the antioxidative pentose phosphate pathway by benfotiamine supplementation prevented microangiopathy, hypoperfusion, and lineage− c-Kit+ Sca-1+ cell depletion. Conclusion—We provide novel evidence for the presence of microangiopathy impinging on the integrity of diabetic BM. These discoveries offer the framework for mechanistic solutions of BM dysfunction in diabetes.

Doxorubicin induces senescence and impairs function of human cardiac progenitor cells

The increasing population of cancer survivors faces considerable morbidity and mortality due to late effects of the antineoplastic therapy. Cardiotoxicity is a major limiting factor of therapy with doxorubicin (DOXO), the most effective anthracycline, and is characterized by a dilated cardiomyopathy that can develop even years after treatment. Studies in animals have proposed the cardiac progenitor cells (CPCs) as the cellular target responsible for DOXO-induced cardiomyopathy but the relevance of these observations to clinical settings is unknown. In this study, the analysis of the DOXO-induced cardiomyopathic human hearts showed that the majority of human CPCs (hCPCs) was senescent. In isolated hCPCs, DOXO triggered DNA damage response leading to apoptosis early after exposure, and telomere shortening and senescence at later time interval. Functional properties of hCPCs, such as migration and differentiation, were also negatively affected. Importantly, the differentiated progeny of DOXO-treated hCPCs prematurely expressed the senescence marker p16INK4a. In conclusion, DOXO exposure severely affects the population of hCPCs and permanently impairs their function. Premature senescence of hCPCs and their progeny can be responsible for the decline in the regenerative capacity of the heart and may represent the cellular basis of DOXO-induced cardiomyopathy in humans.

Resident Cardiac Stem Cells

The introduction of stem cells in cardiology provides new tools in understanding the regenerative processes of the normal and pathologic heart and opens new options for the treatment of cardiovascular diseases. The feasibility of adult bone marrow autologous and allogenic cell therapy of ischemic cardiomyopathies has been demonstrated in humans. However, many unresolved questions remain to link experimental with clinical observations. The demonstration that the heart is a self-renewing organ and that its cell turnover is regulated by myocardial progenitor cells offers novel pathogenetic mechanisms underlying cardiac diseases and raises the possibility to regenerate the damaged heart. Indeed, cardiac stem progenitor cells (CSPCs) have recently been isolated from the human heart by several laboratories although differences in methodology and phenotypic profile have been described. The present review points to the potential role of CSPCs in the onset and development of congestive heart failure and its reversal by regenerative approaches aimed at the preservation and expansion of the resident pool of progenitors.

Noncanonical Fungal Autophagy Inhibits Inflammation in Response to IFN-γ via DAPK1

Summary Defects in a form of noncanonical autophagy, known as LC3-associated phagocytosis (LAP), lead to increased inflammatory pathology during fungal infection. Although LAP contributes to fungal degradation, the molecular mechanisms underlying LAP-mediated modulation of inflammation are unknown. We describe a mechanism by which inflammation is regulated during LAP through the death-associated protein kinase 1 (DAPK1). The ATF6/C/EBP-β/DAPK1 axis activated by IFN-γ not only mediates LAP to Aspergillus fumigatus but also concomitantly inhibits Nod-like receptor protein 3 (NLRP3) activation and restrains pathogenic inflammation. In mouse models and patient samples of chronic granulomatous disease, which exhibit defective autophagy and increased inflammasome activity, IFN-γ restores reduced DAPK1 activity and dampens fungal growth. Additionally, in a cohort of hematopoietic stem cell-transplanted patients, a genetic DAPK1 deficiency is associated with increased inflammation and heightened aspergillosis susceptibility. Thus, DAPK1 is a potential drugable player in regulating the inflammatory response during fungal clearance initiated by IFN-γ.

Cancer treatment-induced cardiotoxicity: a cardiac stem cell disease?

Cardiovascular diseases and cancer represent respectively the first and second cause of death in industrialized countries. These two conditions may become synergistic when cardiovascular complications of anti-cancer therapy are considered. More than 70% of childhood and 50% of adult cancer patients can be cured, however this important success obtained by the biological and medical research is obfuscated by emerging findings of early and late morbidity due to cardiovascular events. Although anthracyclines are effective drugs against cancer a dose-dependent cardiotoxic effects whose mechanism has not been elucidated resulting in failure of therapeutic interventions limit their use. Unexpectedly, tyrosine/kinase inhibitors (TKIs) aimed at molecularly interfering with oncogenic pathways, have been implicated in cardiac side effects. Possible explanations of this phenomenon have been ambiguous, further strengthening the need to deepen our understanding on the mechanism of cardiotoxicity. In addition to a detailed description of anthracyclines and TKIs-related cardiovascular effects, the present review highlights recent observations supporting the hypothesis that the cellular target of anthracyclines and TKIs may include myocardial compartments other than parenchymal cells. The demonstration that the adult mammalian heart possesses a cell turnover regulated by primitive cells suggests that this cell population may be implicated in the onset and development of cardiovascular effects of anti-cancer strategies. The possibility of preventing cardiotoxicity by preservation and/or expansion of the resident stem cell pool responsible for cardiac repair may open new therapeutic options to unravel an unsolved clinical issue.

An immunomodulatory activity of micafungin in preclinical aspergillosis

OBJECTIVES Micafungin inhibits 1,3-β-D-glucan synthase and interferes with fungal cell wall synthesis. Clinically, micafungin has been shown to be efficacious for the treatment of invasive fungal infections. However, little is known about the immunomodulatory activity of micafungin in these infections. METHODS We evaluated the immunomodulatory activity of escalating doses of micafungin in murine and human polymorphonuclear neutrophils (PMNs) in vitro and in vivo in different preclinical models of invasive aspergillosis, including mice deficient for selected innate immune receptors. RESULTS Micafungin was able to regulate PMN cytokine response to Aspergillus fumigatus conidia by decreasing the expression of tumour necrosis factor-α and increasing that of interleukin-10 (IL-10). In vivo, the therapeutic efficacy of micafungin was strictly dose-dependent, with the maximum activity observed at the highest dose, concomitant with reduced inflammatory pathology. The anti-inflammatory activity of micafungin required IL-10 and occurred through signalling via the TLR2/dectin-1 and TLR3/TRIF pathways. CONCLUSION Together, these findings suggest that the therapeutic efficacy of micafungin in aspergillosis is orchestrated by the activation of innate immune receptors affecting the inflammatory/anti-inflammatory balance during infection.

Oxidative Stress and Cellular Response to Doxorubicin: A Common Factor in the Complex Milieu of Anthracycline Cardiotoxicity

The production of reactive species is a core of the redox cycling profile of anthracyclines. However, these molecular characteristics can be viewed as a double-edged sword acting not only on neoplastic cells but also on multiple cellular targets throughout the body. This phenomenon translates into anthracycline cardiotoxicity that is a serious problem in the growing population of paediatric and adult cancer survivors. Therefore, better understanding of cellular processes that operate within but also go beyond cardiomyocytes is a necessary step to develop more effective tools for the prevention and treatment of progressive and often severe cardiomyopathy experienced by otherwise successfully treated oncologic patients. In this review, we focus on oxidative stress-triggered cellular events such as DNA damage, senescence, and cell death implicated in anthracycline cardiovascular toxicity. The involvement of progenitor cells of cardiac and extracardiac origin as well as different cardiac cell types is discussed, pointing to molecular signals that impact on cell longevity and functional competence.

Imatinib mesylate-induced cardiomyopathy involves resident cardiac progenitors

Graphical abstract Figure. No Caption available. ABSTRACT Cardiovascular complications are included among the systemic effects of tyrosine kinase inhibitor (TKI)‐based therapeutic strategies. To test the hypothesis that inhibition of Kit tyrosine kinase that promotes cardiac progenitor cell (CPC) survival and function may be one of the triggering mechanisms of imatinib mesylate (IM)‐related cardiovascular effects, the anatomical, structural and ultrastructural changes in the heart of IM‐treated rats were evaluated. Cardiac anatomy in IM‐exposed rats showed a dose‐dependent, restrictive type of remodeling and depressed hemodynamic performance in the absence of remarkable myocardial fibrosis. The effects of IM on rat and human CPCs were also assessed. IM induced rat CPC depletion, reduced growth and increased cell death. Similar effects were observed in CPCs isolated from human hearts. These results extend the notion that cardiovascular side effects are driven by multiple actions of IM. The identification of cellular mechanisms responsible for cardiovascular complications due to TKIs will enable future strategies aimed at preserving concomitantly cardiac integrity and anti‐tumor activity of advanced cancer treatment.

Implication of MAPK1/MAPK3 signalling pathway in t(8;9)(p22;24)/PCM1-JAK2 myelodysplastic/myeloproliferative neoplasms.

Translocations involving the JAK2 ‘Janus-activated kinase 2’ gene have been described in both lymphoid and myeloid haematological malignancies. Among them, t(8;9) leading to PCM1 ‘pericentriolar material 1’-JAK2 fusion events are extremely rare with <30 reported clinical cases (Bousquet et al, 2005; Heiss et al, 2005; Murati et al, 2005 Reiter et al, 2005; Patnaik et al, 2010; Dargent et al, 2011; Lierman et al, 2012). Although the clinical onset of these disorders is widely heterogeneous, most of them present with a myelodysplastic/ myeloproliferative disease with striking dysplastic features of the erythroid compartment (Heiss et al, 2005; Dargent et al, 2011). Given that allogenic stem cell transplant is the only curative option for eligible patients, several therapeutic approaches (interferon-a, hydroxycarbamide, conventional chemotherapy) have been employed to act as a bridge to transplantation or for use in unfit patients (Murati et al, 2005). Very recently, Lierman et al (2012) showed that ruxolitinb, a JAK1/2-inhibitor approved for high-risk myelofibrosis, could inhibit in-vitro growth and JAK2/STAT5 phosphorylation of the PCM1-JAK2-transformed Ba/F3 murine cell line. However, the status of JAK/STAT signalling has not been assessed in primary cells from PCM1-JAK2 patients. Here, for the first time, we characterize: (i) the erythroid differentiation capacity of ex-vivo expanded CD34 cells, and (ii) the signalling pathways activated in circulating neoplastic cells (CNCs) from a patient diagnosed with atypical chronic myeloid leukaemia carrying t(8;9)(p22;p24)/PCM1-JAK2. A 29-year-old Caucasian male presented with the typical stigmata of a chronic myeloproliferative disorder: leucocytosis (67 1 9 10/l) with circulating myeloid progenitors, mild anaemia (124 g/l), thrombocytopenia (130 9 10/l) and splenomegaly. Bone marrow (BM) histology was hypercellular. The most prominent feature was represented by a severe erythroid dyplasia, showing abundant large peritrabecular clusters of proerythroblasts with marked reduction of the mature erythroid compartment (Fig 1A,B). Cytogenetics showed 46,XY,t(8;9)(p22;p24) in 87% of metaphases and follow-up break-apart FISH assays for JAK2/ 9p24, RP11-125K10 (5′JAK2) and RP11-39K24 (3′JAK2) and for PCM1/8p22, RP11-156K13 (5′PCM1) and RP11-484L21 (3′PCM1) confirmed the PCM1-JAK2 rearrangement (Fig 1C). Chimeric PCM1-JAK2 fusion transcript was also detected in CNCs by nested reverse transcription polymerase chain reaction (RT-PCR) (Fig 1D, lane 1) performed as previously described (Reiter et al, 2005). Specificity of the assay was confirmed by the absence of PCM1-JAK2 transcript signal in a healthy subject (HS) (Fig 1D, lane 8). The patient underwent two courses of acute myeloid leukaemia-like chemotherapy followed by allogenic bone marrow transplant from a human leucocyte antigen (HLA)identical sibling donor. While no molecular response was achieved with the chemotherapy only (Fig 1D, lanes 2–5), no pathological transcript was detectable by nested RT-PCR in both bone marrow mononuclear cells and peripheral blood mononuclear cells (PBMCs) at day 100 post-transplantation (Fig 1D, lanes 6–7). Haematopoietic progenitors taken from polycythaemia vera (PV) patients that bear the constitutively-active JAK2 V617F mutation display cytokine hypersensitivity (Ugo et al, 2004). To test whether this was the case in our patient, we isolated primary CD34 cells from our PCM1-JAK2 fusion patient, a JAK2 V617F PV and a granulocyte colonystimulating factor-mobilized individual (M) using the CD34 cell isolation kit (Miltenyi Biotech, Gladbach, Germany). CD34 cells were plated at a cell density of 1 9 10 cells/ml and then cultured for 14 d in serum-free X-vivo medium supplemented with 3 ng/ml recombinant human interleukin-3 (rIL3), 50 ng/ml stem cell factor (SCF, also known as KITLG) and 5 U/ml erythropoietin (EPO). At days 9, 11 and 14 of culture, cell number and viability was determined by trypan-blue staining. On the 14th day of culture, we stained cells with a Rphycoerythrin (RPE)-conjugated anti-Glycophorin A (Gly-A) antibody (Dako, Glostrup, Denmark) and assessed erythroid differentiation by flow cytometry. Surprisingly, CD34 from the PCM1-JAK2 fusion patient displayed impaired growth capacity [fold increase (FI) = 0 6] in erythroid differentiation medium, as compared to both PV and M CD34 cells (FI = 8 and 6 5 respectively) (Fig 2B). Additionally, CD34 from the PCM1-JAK2 fusion patient cells showed impaired erythroid differentiation capacity, as demonstrated by the low percentage of Gly-A cells (3 1%) compared to both PV and M CD34 cells (84 5% and 52 6% Gly-A cells, respectively) (Fig 2A). Finally, as described above, histology revealed a prominent dyserythropoietic phenotype in the bone marrow (Fig 1A,B). Collectively, these observations suggested that the ex-vivo erythroid differentiation capacity of haematopoietic progenitors from this t(8;9)(p22;p24)/PCM1-JAK2 fusion case was Correspondence