Previous studies have shown that the type I collagen of tendon and demineralized bone both calcify rapidly in serum. The speed, collagen matrix-type specificity, and extent of the re-calcification of demineralized bone in serum suggest that the serum calcification activity identified in these studies may participate in normal biomineralization. Because of its presence in serum and its long history of association with the normal mineralization of the collagen matrix of bone, tissue-nonspecific alkaline phosphatase (TNAP) is an obvious candidate for a protein that could be a component of serum calcification activity, and experiments were therefore carried out to test this possibility. These experiments show that the inactivation of TNAP in serum prevents collagen calcification, and that the addition of physiological levels of purified TNAP restores the ability of TNAP-deficient serum to calcify collagen. Additional experiments show that the role of TNAP in collagen calcification is to activate a serum nucleator of apatite crystal formation. Based on these and earlier studies, the mechanism of collagen calcification in serum requires at least four elements as follows. Our goal is to understand the biochemical mechanism responsible for the calcification of collagen fibrils in normal bone formation. In the course of our investigations, we have discovered that purified type I collagen and demineralized bone matrix both calcify rapidly when incubated in serum in the absence of cells (1-4). The calcification of collagen is because of the presence of a serum calcification activity, one sufficiently potent that collagen calcifies when incubated in media containing as little as 1.5% serum but not in serum-free media alone (1-4). This serum calcification activity consists of one or more proteins that are 50 -150 kDa in size (3, 4).Although serum-driven collagen calcification is an in vitro, cell-free assay, there are several reasons to believe that it could be relevant to understanding mechanisms by which collagen fibrils are mineralized in nature. 1) The assay conditions are physiologically relevant; collagen added to serum calcifies when incubated at the temperature and pH of mammalian blood, without the need to add anything to serum to promote mineralization, such as -glycerophosphate or phosphate (see Ref. 1 and references therein). 2) Serum is relevant to bone mineralization; osteoblasts form bone in a vascular compartment (5), and proteins in serum have direct access to the site of collagen fibril formation and mineralization, whereas proteins secreted by the osteoblast appear rapidly in serum. 3) Serum-driven calcification is evolutionarily conserved; the serum calcification activity appeared in animals at the time vertebrates acquired the ability to form calcium phosphate mineral structures, with no evidence for a similar activity in the serum of invertebrates (2). 4) Serum-driven calcification is specific; calcification is restricted to those structures that were calcified in bone prior to demineralization, with n...
Objective-To determine whether serum contains an activity that induces artery calcification. Methods and Results-The elastic lamellae of devitalized rat aortas calcify rapidly in rat or bovine serum, or in human serum provided [Pi] Ն2 mmol/L. This calcification is attributable to a potent serum calcification factor (SCF), one that causes devitalized aortas to calcify when incubated in DMEM containing as little as 1.5% serum but not in DMEM alone. The SCF that initiates medial elastin calcification has the same 50-to 150-kDa size and protease sensitivity as the SCF shown previously to initiate calcification of type I collagen. Our working hypothesis is that the same SCF initiates calcification of collagen and elastin, and that this SCF arises from sites of normal bone mineralization and, like alkaline phosphatase, is released into general circulation. The SCF does not initiate medial elastin calcification in living arteries, which suggests that vascular cells may prevent this calcification. This hypothesis is supported by the observations that living arteries secrete the calcification inhibitor matrix Gla protein (MGP); that inactivation of MGP with warfarin causes living arteries to calcify; and that addition of MGP to medium containing warfarin prevents this calcification. Key Words: medial artery calcification Ⅲ elastic lamellae Ⅲ serum calcification factor Ⅲ matrix Gla protein Ⅲ devitalized and living arteries T wo major types of arterial calcification have been observed in human patients. 1,2 One affects the intimal layer of arteries and occurs within atherosclerotic plaques. The other involves the artery media and initially occurs within the elastic lamellae. This second type of vascular calcification is common in patients with chronic kidney disease and in patients with diabetes mellitus. Each type of arterial calcification has different physiological consequences, with clearcut evidence for adverse hemodynamic changes attributable to medial wall calcification but not to atherosclerotic plaque calcification, and the possible contribution of atherosclerotic plaque calcification to plaque rupture and subsequent thrombosis, an issue that does not apply to medial wall calcification. Conclusion-TheOur long-term goal is to understand the mechanisms that initiate calcification of the elastic lamellae of the artery media and the mechanisms that inhibit this calcification. In the course of our investigations, we became intrigued with the evidence for an association between bone metabolism and artery calcification, 3 an association that led us to propose that medial artery calcification is linked to bone resorption. One prediction of this hypothesis is that inhibitors of bone resorption should inhibit artery calcification. 3 In previous studies, we tested this prediction using 3 different types of bone resorption inhibitors, each with an entirely different mode of action on the osteoclast, the amino bisphosphonates alendronate and ibandronate, 3-5 the cytokine osteoprotegerin, 6 and the V-H ϩ -ATPase inhibitor SB...
Proliferation of endogenous neural stem/progenitor cells (NSPCs) has been identified in both normal and injured adult mammalian spinal cord. Yet the signaling mechanisms underlying the regulation of adult spinal cord NSPCs proliferation and commitment toward a neuronal lineage remain undefined. In this study, the role of three growth factor-mediated signaling pathways in proliferation and neuronal differentiation was examined. Adult spinal cord NSPCs were enriched in the presence of fibroblast growth factor 2 (FGF2). We observed an increase in the number of cells expressing the microtubule-associated protein 2 (MAP2) over time, indicating neuronal differentiation in the culture. Inhibition of the mitogen-activated protein kinase or extracellular signal-regulated kinase (ERK) kinase 1 and 2/ERK 1 and 2 (MEK/ERK1/2) or the phosphoinositide 3-kinase (PI3K)/Akt pathways suppressed active proliferation in adult spinal cord NSPC cultures; whereas neuronal differentiation was negatively affected only when the ERK1/2 pathway was inhibited. Inhibition of the phospholipase Cγ (PLCγ) pathway did not affect proliferation or neuronal differentiation. Finally, we demonstrated that the blockade of either the ERK1/2 or PLCγ signaling pathways reduced neurite branching of MAP2+ cells derived from the NSPC cultures. Many of the MAP2+ cells expressed synaptophysin and had a glutamatergic phenotype, indicating that over time adult spinal cord NSPCs had differentiated into mostly glutamatergic neurons. Our work provides new information regarding the contribution of these pathways to the proliferation and neuronal differentiation of NSPCs derived from adult spinal cord cultures, and emphasizes that the contribution of these pathways is dependent on the origin of the NSPCs.
Spinal cord motor neuron cultures are an important tool for the study of mechanisms involved in motor neuron survival, degeneration and regeneration, volatile anesthetic-induced immobility, motor neuron disorders such as amyotrophic lateral sclerosis or spinal muscular atrophy as well as in spinal cord injury. Embryonic spinal cord motor neurons derived from rats have been successfully cultured; unfortunately, the culture of adult motor neurons has been problematic due to their short-term survival. Recently, by using a cocktail of target-derived factors, neurotrophins (brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor) and a permeable cyclic adenosine monophosphate analog, we have established a reproducible protocol for long-term cultures of healthy and functional adult motor neurons (Exp Neurol 220:303-315, 2009). Here, we now describe in detail the steps that we used for the optimization of the process of isolation and maintenance of adult rat ventral horn motor neurons in vitro.
In contrast to the adult brain, the adult spinal cord is a non-neurogenic environment. Understanding how to manipulate the spinal cord environment to promote the formation of new neurons is an attractive therapeutic strategy for spinal cord injury and disease. The cannabinoid 1 receptor (CB1R) has been implicated as a modulator of neural progenitor cell proliferation and fate specification in the brain; however, no evidence exists for modulation of adult spinal cord progenitor cells. Using adult rat spinal cord primary cultures, we demonstrated that CB1R antagonism with AM251 significantly decreased the number of Nestin(+) cells, and increased the number of βIII tubulin(+) and DCX(+) cells, indicative of neuronal differentiation. AM251’s effect was blocked by co-application of the CB1R agonists, WIN 55, 212-2, or ACEA. Consistent with our hypothesis, cultures, and spinal cord slices derived from CB1R knock-out (CB1−/−) mice had significantly higher levels of DCX(+) cells compared to those derived from wild type (CB1+/+) mice, indicative of enhanced neuronal differentiation in CB1−/− spinal cords. Moreover, AM251 promoted neuronal differentiation in CB1+/+, but not in CB1−/− cultures. Since CB1R modulates synaptic transmission, and synaptic transmission has been shown to influence progenitor cell fate, we evaluated whether AM251-induced neuronal differentiation was affected by chronic inactivity. Either the presence of the voltage-dependent sodium channel blocker tetrodotoxin (TTX), or the removal of mature neurons, inhibited the AM251-induced increase in DCX(+) cells. In summary, antagonism or absence of CB1R promotes neuronal differentiation in adult spinal cords, and this action appears to require TTX-sensitive neuronal activity. Our data suggest that the previously detected elevated levels of endocannabinoids in the injured adult spinal cord could contribute to the non-neurogenic environment and CB1R antagonists could potentially be used to enhance replacement of damaged neurons.
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