Objective-TGF- plays a significant role in vascular injury-induced stenosis. This study evaluates the efficacy of a novel, small molecule inhibitor of ALK5/ALK4 kinase, in the rat carotid injury model of vascular fibrosis. Methods and Results-The small molecule, SM16, was shown to bind with high affinity to ALK5 kinase ATP binding site using a competitive binding assay and biacore analysis. SM16 blocked TGF- and activin-induced Smad2/3 phosphorylation and TGF--induced plasminogen activator inhibitor (PAI)-luciferase activity in cells. Good overall selectivity was demonstrated in a large panel of kinase assays, but SM16 also showed nanomolar inhibition of ALK4 and weak (micromolar) inhibition of Raf and p38. In the rat carotid injury model, SM16 dosed once daily orally at 15 or 30 mg/kg SM16 for 14 days caused significant inhibition of neointimal thickening and lumenal narrowing. SM16 also prevented induction of adventitial smooth muscle ␣-actin-positive myofibroblasts and the production of intimal collagen, but did not decrease the percentage of proliferative cells. Key Words: TGF beta Ⅲ restenosis Ⅲ fibrosis Ⅲ neointimal formation Ⅲ activin T GF- promotes key cellular events in vascular fibrosis including the induction of adventitial myofibroblasts and activated smooth muscle cells as well as their proliferation, survival, contraction, and increased collagen deposition. 1 TGF- is overexpressed in animal models of vascular fibrosis as well as human diseases such as restenosis, cardiac hypertrophy, primary pulmonary hypertension, and organ transplant vasculopathy. [1][2][3][4][5][6][7] Activin, another TGF- superfamily member, is also upregulated during vascular remodeling after balloon injury of the rat carotid artery. 8,9 The association of overexpression of these 2 cytokines with inflammation and fibrotic responses to injury suggest that inhibition of either or both of these TGF- superfamily pathways may prevent vascular fibrosis. [1][2][3][4][5][6][7][8][9][10] TGF- and activin signal through binding to their respective type II receptors, TGF-RII and ActRII. The subsequent interaction of this complex with their respective type I receptors, ALK5 and ALK4, allows the interaction with and phosphorylation of type I receptor-associated signaling proteins, Smad2 and Smad3. 11,12 Blockade of TGF- signaling with either the soluble TGF- type II receptor extracellular domain-Fc fusion protein (sTGF-RII:Fc), 5,7 antibodies to TGF-1, 13 decorin, 14 TGF- antisense oligonucleotides, 15-17 or adenovirus Smad7 18 was shown previously to inhibit vascular fibrosis in animal models. Here, we identified SM16, a potent, selective and orally active ALK5/ALK4 kinase inhibitor which potently inhibits TGF--induced phosphorylation of Smad2 and Smad3 in cells and neointimal thickening and vascular remodeling in the rat carotid balloon injury model. This orally active, small molecule TGF-/activin signaling inhibitor prevents neointimal thickening and lumenal loss primarily through inhibition of myofibroblast induction a...
Nerve growth factor (NGF) acutely modulates synaptic transmission between sympathetic neurons and their cardiac myocyte targets. NGF also has developmental effects in establishing the level of synaptic transmission between sympathetic neurons and myocytes in culture, although little is known about the mechanisms by which NGF influences this synaptic connectivity. Here we report that NGF acts in conjunction with factors produced by cardiac myocytes to promote neuronal contact with the target and the extension of synaptic vesicle-containing growth cones. In conjunction with previously published results showing that NGF has long-term effects on synaptic transmission between sympathetic neurons and myocytes, this work suggests that NGF acts to promote sympathetic neurotransmission by increasing the number of sympathetic fibers establishing target contact. Further, we found that developmental changes in cardiac myocytes led to an increase in the density of synaptic vesicle-containing variocosities along sympathetic fibers, a process regulated by NGF. Thus, as myocytes mature they produce factors that promote the formation of sympathetic presynaptic structures. These results argue that multiple target interactions regulate the extent of synapse formation between sympathetic neurons and cardiac cells and suggest that NGF promotes presynaptic development by increasing neuronal contact with myocyte-derived cell surface or matrix-associated factors.
Nerve growth factor (NGF) acutely modulates synaptic transmission between sympathetic neurons and their cardiac myocyte targets. NGF also has developmental effects in establishing the level of synaptic transmission between sympathetic neurons and myocytes in culture, although little is known about the mechanisms by which NGF influences this synaptic connectivity. Here we report that NGF acts in conjunction with factors produced by cardiac myocytes to promote neuronal contact with the target and the extension of synaptic vesicle‐containing growth cones. In conjunction with previously published results showing that NGF has long‐term effects on synaptic transmission between sympathetic neurons and myocytes, this work suggests that NGF acts to promote sympathetic neurotransmission by increasing the number of sympathetic fibers establishing target contact. Further, we found that developmental changes in cardiac myocytes led to an increase in the density of synaptic vesicle–containing variocosities along sympathetic fibers, a process regulated by NGF. Thus, as myocytes mature they produce factors that promote the formation of sympathetic presynaptic structures. These results argue that multiple target interactions regulate the extent of synapse formation between sympathetic neurons and cardiac cells and suggest that NGF promotes presynaptic development by increasing neuronal contact with myocyte‐derived cell surface or matrix‐associated factors. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 460–476, 2000
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