Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.

Knockout of transforming growth factor (TGF)-beta1 or components of its signaling pathway leads to embryonic death in mice due to impaired yolk sac vascular development before significant smooth muscle cell (SMC) maturation occurs. Thus the role of TGF-beta1 in SMC development remains unclear. Embryonic stem cell (ESC)-derived embryoid bodies (EBs) recapitulate many of the events of early embryonic development and represent a more physiological context in which to study SMC development than most other in vitro systems. The present studies showed induction of the SMC-selective genes smooth muscle alpha-actin (SMalphaA), SM22alpha, myocardin, smoothelin-B, and smooth muscle myosin heavy chain (SMMHC) within a mouse ESC-EB model system. Significantly, SM2, the SMMHC isoform associated with fully differentiated SMCs, was expressed. Importantly, the results showed that aggregates of SMMHC-expressing cells exhibited visible contractile activity, suggesting that all regulatory pathways essential for development of contractile SMCs were functional in this in vitro model system. Inhibition of endogenous TGF-beta with an adenovirus expressing a soluble truncated TGF-beta type II receptor attenuated the increase in SMC-selective gene expression in the ESC-EBs, as did an antibody specific for TGF-beta1. Of interest, the results of small interfering (si)RNA experiments provided evidence for differential TGF-beta-Smad signaling for an early vs. late SMC marker gene in that SMalphaA promoter activity was dependent on both Smad2 and Smad3 whereas SMMHC activity was Smad2 dependent. These results are the first to provide direct evidence that TGF-beta1 signaling through Smad2 and Smad3 plays an important role in the development of SMCs from totipotential ESCs.

Cyclic nucleotide-gated (CNG) channels are Ca2+-permeable, nonspecific cation channels that can be activated through direct interaction with cAMP and/or cGMP. Recent electrophysiological evidence for these channels in cultured hippocampal neurons prompted us to investigate the expression of CNG channel genes in hippocampus. PCR amplification detected the expression of transcripts for subunit 1 of both the rod photoreceptor (RCNGC1) and the olfactory receptor cell (OCNGC1) subtype of CNG channel in adult rat hippocampus. In situ hybridization detected expression of both channel subtypes in most principal neurons, including pyramidal cells of the CAl through CA3 regions and granule cells of the dentate gyrus. From the hybridization patterns, we conclude that the two genes are colocalized in individual neurons. Comparison of the patterns of expression of type 1 cGMP-dependent protein kinase and the CNG channels suggests that hippocampal neurons can respond to changes in cGMP levels with both rapid changes in CNG channel activity and slower changes induced by phosphorylation. Future models of hippocampal function should include CNG channels and their effects on both electrical responses and intracellular Ca2+ levels.cAMP and cGMP regulate the activity of neurons throughout the central nervous system (CNS), controlling metabolic processes, electrical signaling, and synaptic physiology (1-3). In the hippocampus, both neurotransmitters and diffusible messengers have been shown to modulate cAMP and cGMP levels (2, 4-6). Both nucleotides may have roles in certain forms of use-dependent alteration of synaptic strength which are thought to underlie information storage, such as long-term potentiation (for reviews, see refs. 7-9). It has been generally assumed that cAMP and cGMP act at hippocampal synapses by activating specific protein kinases. Although abundant evidence supports a role for these kinases (10), recent findings have suggested an additional pathway by which cAMP and cGMP can influence neuronal activity, namely through the direct gating of a class of Ca2+-permeable, nonselective cation channels.Cyclic nucleotide-gated (CNG) channels were originally detected in rod and cone photoreceptors (11, 12) and olfactory receptor cells (13), where they mediate the transduction of sensory stimuli into neuronal activity. The principal subunit (subunit 1 or a) of each subtype of CNG channel is encoded by a separate gene (14-18). Native CNG channels are likely to be heterooligomers and additional subunits (subunit 2 or f3) have been discovered that are incapable of forming functional CNG channels by themselves but do alter the kinetics of channel opening and the selectivity of cyclic nucleotide responsiveness when coexpressed with subunit 1 (19-23). The three subtypes of CNG channels clearly form a gene family, and the predicted amino acid sequences of subunit 1 show extensive homology over the C-terminal 75% of the molecules (for review, see ref. 24). This region contains the proposed six transmembrane spans, the por...

Background-Extracellular matrix (ECM) remodeling is central to the development of restenosis after coronary angioplasty (PTCA). As a regulator of ECM deposition by vascular cells, substantial evidence implicates transforming growth factor-␤1 (TGF-␤1) in the pathogenesis of restenosis. We investigated the effects of intracoronary expression of a transgenic antagonist of TGF-␤1 on luminal loss after PTCA. Methods and Results-Porcine coronary arteries were randomized to receive a recombinant adenovirus expressing a secreted form of TGF-␤ type II receptor (Ad5-RIIs), an adenovirus expressing ␤-galactosidase (Ad5-lacZ), or vehicle only by intramural injection at the site of PTCA. Computerized morphometry 28 days after angioplasty revealed a greater minimum luminal area in Ad5-RIIs-injected arteries (1.71Ϯ0.12 mm 2 ) than in the Ad5-lacZ (1.33Ϯ0.13 mm 2 ) or vehicle-only (1.08Ϯ0.17 mm 2 ; Pϭ0.010 by ANOVA) groups. This was accompanied by greater areas within the internal (Pϭ0.013) and external (Pϭ0.031) elastic laminae in Ad5-RIIs-treated vessels. Adventitial collagen content at the site of injury was increased in the Ad5-RIIs group, in contrast to decreases in the Ad5-lacZ and vehicle-only groups (Pϭ0.004). Conclusions-Adenovirus-mediated