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.

Background— The neurotrophin nerve growth factor (NGF) regulates neuron survival and differentiation. Implication in neovascularization is supported by statement of NGF and its high-affinity receptor at vascular level and by NGF property of stimulating vascular endothelial cell proliferation. The present study investigated the involvement of endogenous NGF in spontaneous reparative response to ischemia. Mechanisms and therapeutic potential of NGF-induced neovascularization were examined. Methods and Results— Unilateral limb ischemia was produced in CD1 mice by femoral artery resection. By ELISA and immunohistochemistry, we documented that statement of NGF and its high-affinity receptor is upregulated in ischemic muscles. The functional relevance of this phenomenon was assessed by means of NGF-neutralizing antibody. Chronic NGF blockade abrogated the spontaneous capillarization response to ischemia and augmented myocyte apoptosis. Then we tested whether NGF administration may exert curative effects. Repeated NGF injection into ischemic adductors increased capillary and arteriole density, reduced endothelial cell and myofiber apoptosis, and accelerated perfusion recovery, without altering systemic hemodynamics. In normoperfused muscles, NFG-induced capillarization was blocked by vascular endothelial growth factor–neutralizing antibodies, dominant-negative Akt, or NO synthase inhibition. Conclusions— These results indicate that NGF plays a functional role in reparative neovascularization. Furthermore, supplementation of the growth factor promotes angiogenesis through a vascular endothelial growth factor-Akt-NO–mediated mechanism. In the setting of ischemia, potentiation of NGF pathway stimulates angiogenesis and arteriogenesis, thereby accelerating hemodynamic recovery. NGF might be envisaged as a utilitarian target for the treatment of ischemic vascular disease.

We evaluated the healing potential of human fetal aorta-derived CD133 ⁺ progenitor cells and their conditioned medium (CD133 ⁺ CCM) in a new model of ischemic diabetic ulcer. Streptozotocin-induced diabetic mice underwent bilateral limb ischemia and wounding. One wound was covered with collagen containing 2×10 ⁴ CD133 ⁺ or CD133 ⁻ cells or vehicle. The contralateral wound, covered with only collagen, served as control. Fetal CD133 ⁺ cells expressed high levels of wingless ( Wnt ) genes, which were downregulated following differentiation into CD133 ⁻ cells along with upregulation of Wnt antagonists secreted frizzled-related protein (sFRP)-1, -3, and -4. CD133 ⁺ cells accelerated wound closure as compared with CD133 ⁻ or vehicle and promoted angiogenesis through stimulation of endothelial cell proliferation, migration, and survival by paracrine effects. CD133 ⁺ cells secreted high levels of vascular endothelial growth factor (VEGF)-A and interleukin (IL)-8. Consistently, CD133 ⁺ CCM accelerated wound closure and reparative angiogenesis, with this action abrogated by coadministering the Wnt antagonist sFRP-1 or neutralizing antibodies against VEGF-A or IL-8. In vitro, these effects were recapitulated following exposure of high-glucose-primed human umbilical vein endothelial cells to CD133 ⁺ CCM, resulting in stimulation of migration, angiogenesis-like network formation and induction of Wnt expression. The promigratory and proangiogenic effect of CD133 ⁺ CCM was blunted by sFRP-1, as well as antibodies against VEGF-A or IL-8. CD133 ⁺ cells stimulate wound healing by paracrine mechanisms that activate Wnt signaling pathway in recipients. These preclinical findings open new perspectives for the cure of diabetic ulcers.

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 β-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.

Rationale : Nerve growth factor (NGF) promotes angiogenesis and cardiomyocyte survival, which are both desirable for postinfarction myocardial healing. Nonetheless, the NGF potential for cardiac repair has never been investigated. Objective : To define expression and localization of NGF and its high-affinity receptor TrkA (tropomyosin-related receptor A) in the human infarcted heart and to investigate the cardiac roles of both endogenous and engineered NGF using a mouse model of myocardial infarction (MI). Methods and Results : Immunostaining for NGF and TrkA was performed on heart samples from humans deceased of MI or unrelated pathologies. To study the post-MI functions of endogenous NGF, a NGF-neutralizing antibody (Ab-NGF) or nonimmune IgG (control) was given to MI mice. To investigate the NGF therapeutic potential, human NGF gene or control (empty vector) was delivered to the murine periinfarct myocardium. Results indicate that NGF is present in the infarcted human heart. Both cardiomyocytes and endothelial cells (ECs) possess TrkA, which suggests NGF cardiovascular actions in humans. In MI mice, Ab-NGF abrogated native reparative angiogenesis, increased EC and cardiomyocyte apoptosis and worsened cardiac function. Conversely, NGF gene transfer ameliorated EC and cardiomyocyte survival, promoted neovascularization and improved myocardial blood flow and cardiac function. The prosurvival/proangiogenic Akt/Foxo pathway mediated the therapeutic benefits of NGF transfer. Moreover, NGF overexpression increased stem cell factor (the c-kit receptor ligand) expression, which translated in higher myocardial abundance of c-kit ^pos progenitor cells in NGF -engineered hearts. Conclusions : NGF elicits pleiotropic beneficial actions in the post-MI heart. NGF should be considered as a candidate for therapeutic cardiac regeneration.