Neurotrophins are growth factors that promote cell survival, differentiation, and cell death. They are synthesized as proforms that can be cleaved intracellularly to release mature, secreted ligands. Although proneurotrophins have been considered inactive precursors, we show here that the proforms of nerve growth factor (NGF) and the proforms of brain derived neurotrophic factor (BDNF) are secreted and cleaved extracellularly by the serine protease plasmin and by selective matrix metalloproteinases (MMPs). ProNGF is a high-affinity ligand for p75(NTR) with high affinity and induced p75NTR-dependent apoptosis in cultured neurons with minimal activation of TrkA-mediated differentiation or survival. The biological action of neurotrophins is thus regulated by proteolytic cleavage, with proforms preferentially activating p75NTR to mediate apoptosis and mature forms activating Trk receptors to promote survival.
Microvascular networks support metabolic activity and define microenvironmental conditions within tissues in health and pathology. Recapitulation of functional microvascular structures in vitro could provide a platform for the study of complex vascular phenomena, including angiogenesis and thrombosis. We have engineered living microvascular networks in three-dimensional tissue scaffolds and demonstrated their biofunctionality in vitro. We describe the lithographic technique used to form endothelialized microfluidic vessels within a native collagen matrix; we characterize the morphology, mass transfer processes, and long-term stability of the endothelium; we elucidate the angiogenic activities of the endothelia and differential interactions with perivascular cells seeded in the collagen bulk; and we demonstrate the nonthrombotic nature of the vascular endothelium and its transition to a prothrombotic state during an inflammatory response. The success of these microvascular networks in recapitulating these phenomena points to the broad potential of this platform for the study of cardiovascular biology and pathophysiology.tissue engineering | regenerative medicine | microfluidics | cancer | blood T he microvasculature is an extensive organ that mediates the interaction between blood and tissues. It defines the biological and physical characteristics of the microenvironment within tissues and plays a role in the initiation and progression of many pathologies, including cancer (1) and cardiovascular diseases (2, 3). Conventional planar cultures fail to recreate the in vivo physiology of the microvasculature with respect to three-dimensional (3D) geometry (lumens and axial branching points), and interactions of endothelium with perivascular cells, extracellular tissue and blood flow (4). Studies of the microvasculature in vivo allow only limited control of physical, chemical, and biological parameters influencing the microvasculature and present challenges with respect to observation (5). In vitro cultures that produce tubular vessels within 3D matrices will aid in elucidation of the roles of the microvasculature in health and disease. Important progress has been made toward this goal: Biologically derived or synthetic materials have been used to generate macrovessel tubes (6) and endothelialized microtubes (7); cellular self-assembly has been used to generate random microvasculature (8); microfabrication has been used to define complex geometries in hydrogels at the micro-scale (9); and distributions of cells and biochemical factors within 3D scaffolds (10). Of particular note, the group of Tien has pioneered the use of collagen to template the growth of vascular endothelium (7, 11) and demonstrated appropriate permeability (7), response to cyclic AMP (12), and differential properties as a function of the luminal shear stress and composition of the medium (13). Nonetheless, prior methodologies have been unable to produce endothelialized networks that can undergo substantial remodeling via angiogenesis; elucidate the ...
Brain-derived neurotrophic factor (BDNF) is best characterized for critical roles in neuronal survival, differentiation, and synaptic modulation mediated by the TrkB receptor tyrosine kinase. Developmentally regulated death signaling by BDNF has also been demonstrated via activation of p75 NTR . Because recent studies suggest that proNGF, the precursor form of NGF, is more active than mature NGF in inducing apoptosis after binding to p75 NTR and a coreceptor, sortilin, we asked whether the precursor of BDNF (proBDNF) is also a proapoptotic ligand in the nervous system. proBDNF is secreted by cultured neurons, and recombinant proBDNF binds to sortilin. In sympathetic neurons coexpressing sortilin and p75 NTR , we found that proBDNF is an apoptotic ligand that induces death at subnanomolar concentrations. In contrast, mature BDNF, but not proBDNF, is effective in inducing TrkB phosphorylation. proBDNF effects are dependent on cellular coexpression of both p75 NTR and sortilin, because neurons deficient in p75 NTR are resistant to proBDNF-induced apoptosis, and competitive antagonists of sortilin block sympathetic neuron death. Moreover, addition of preformed complexes of soluble sortilin and proBDNF failed to induce apoptosis of cells coexpressing both sortilin and p75 NTR , suggesting that interaction of proBDNF with both receptors on the cell surface is required to initiate cell death. Together with our past findings, these data suggest that the neurotrophin family is capable of modulating diverse biological processes via differential processing of the proneurotrophins.
Recent studies have identified a specialized subset of CD31hiEMCNhi vascular endothelium that positively regulates bone formation. However, it remains unclear how CD31hiEMCNhi endothelium levels are coupled to anabolic bone formation. Mice with an osteoblast-specific deletion of Shn3, which have markedly elevated bone formation, demonstrated an increase in CD31hiEMCNhi endothelium. Transcriptomic analysis identified SLIT3 as an osteoblast-derived, SHN3-regulated proangiogenic factor. Genetic deletion of Slit3 reduced skeletal CD31hiEMCNhi endothelium, resulted in low bone mass due to impaired bone formation and partially reversed the high bone mass phenotype of Shn3−/− mice. This coupling between osteoblasts and CD31hiEMCNhi endothelium is essential for bone healing, as shown by defective fracture repair in SLIT3-mutant mice and enhanced fracture repair in SHN3-mutant mice. Finally, administration of recombinant SLIT3 both enhanced bone-fracture healing and counteracted bone loss in a mouse model of postmenopausal osteoporosis. Thus, drugs that target the SLIT3 pathway may represent a new approach for vascular-targeted osteoanabolic therapy to treat bone loss.
Recent studies indicate that, in addition to its neuropoietic actions, brain derived neurotrophic factor (BDNF) promotes endothelial cell survival and induces neoangiogenesis in ischemic tissues. Unlike many vascular growth factors that act on many vascular beds, BDNF activity is relatively restricted to central arteries and cardiac skeletal muscle, skin and vasculature. Studies of newly described biological mediators that act on large vessel and microvascular beds in these organs will help us to better understand organ-specific vascular development as well as to develop novel therapeutic strategies to improve the condition of patients with cardiac and peripheral vascular disease. In this review we summarize dual pro-angiogenic actions of BDNF, which through local activation of TrkB receptor expressed on a sub-population of endothelial cells (ECs) and additionally by recruitment of bone marrow-derived cells, contribute to neoangiogenesis. Importance of endothelial cells in angiogenesis.The development of a mature vascular bed is a complex and dynamic process, characterized by the coordinated proliferation and recruitment of endothelial and smooth muscle cells and subsequent patterning into capillaries, arteries and veins. It is controlled by locally produced growth factors, adhesion molecules and proteinases (Carmeliet 2000) that establish the primary vascular plexus consisting of endothelial cells. Subsequently, accessory cells such as pericytes and smooth muscle cells are recruited to sculpt the vasculature, by pruning and remodeling. During adulthood, the maintenance of blood vessels is further promoted by a combination of growth factors, cell:matrix interactions and shear stress forces. The molecular mechanisms that are utilized during development to sculpt the immature vascular bed may be utilized during the adaptive neovascularization of the heart that occurs as a consequence of cardiac ischemia.Endothelial cells, comprising the inner layer of cells of the vasculature, are the main regulators of vascular homeostasis. They play important roles in the physiology and maintenance of vascular integrity and tone, as well as in pathological situations such as inflammation and tumor angiogenesis. Although endothelial cells exhibit many common morphological characteristics, they display significant heterogeneity in different organs, to promote organ-specific functions (Garlanda and Dejana 1997). While coronary arteries nourish the myocardium, the highly specialized endothelial cells in the bone marrow sinusoids are intimately involved in the homing of hematopoietic progenitors and play an important role in maturation, proliferation and trafficking of those cells to the blood circulation. These characteristics may be endowed by organ-specific growth factors secreted by parenchymal cells in a paracrine manner.The healthy endothelium is both anti-coagulant and anti-thrombotic, by expressing or secreting numerous proteins that regulate blood coagulation and platelet activation (Robson et al.
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