Under conventional culture conditions, smooth muscle cells display their phenotypic modulation from a differentiated to a dedifferentiated state. Here, we established a primary culture system of smooth muscle cells maintaining a differentiated phenotype, as characterized by expression of smooth muscle-specific marker genes such as h-caldesmon and calponin, cell morphology, and ligand-induced contractility. Laminin retarded the progression of dedifferentiation of smooth muscle cells. Insulin-like growth factors (IGF-I and IGF-II) and insulin markedly prolonged the differentiated phenotype, with IGF-I being the more potent. In contrast, serum, epidermal growth factor, transforming growth factors, and platelet-derived growth factors potently induced dedifferentiation compared with angiotensin II, arginine-vasopressin, and basic fibroblast growth factor. Using the present culture system, we investigated signaling pathways regulating a phenotype of smooth muscle cells. In cultured cells, IGF-I specifically activated phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, protein kinase B, but not mitogen-activated protein kinases. Specific inhibitors of PI3-kinase (wortmannin and LY294002) induced dedifferentiation of smooth muscle cells even when they were cultured on laminin under IGF-I-stimulated conditions. The sole effect of laminin to retard the dedifferentiation was completely blocked by anti-IGF-I antibody, and laminin promoted the endogenous expression of IGF-I in cultured cells. The reduced promoter activity of the caldesmon gene induced by platelet-derived growth factor BB was overcome by the forced expression of the constitutive active form of PI3-kinase p110␣ catalytic subunit. These findings suggest that an IGF-I signaling pathway through PI3-kinase plays a critical role in maintaining a differentiated phenotype of smooth muscle cells.The smooth muscle cells (SMCs) 1 play roles in the control of blood pressure, enteric peristalsis, and bronchial, uterus, and bladder contraction. Although the precursor cells of SMC seem to undergo phenotypic modulation into matured cells, the origin of precursor cells has not been well characterized. During embryogenesis, three different lineages (cardiac neural crest, nodosa placode, and lateral mesoderm) have been, in part, proven as precursors of SMC. In the case of vasculogenesis (1, 2), angioblasts originated from mesoderm differentiate into endothelial cells, which then coalesce to form a single layer of endothelial tubes. It has been proposed that these primitive vessels are involved in the recruitment of neighboring mesenchymal cells and the subsequent differentiation of SMCs. However, the precise mechanism regarding recruitment of SMC precursors has remained unknown. Phenotypic modulation of SMCs is also associated with pathological conditions, such as atherosclerosis, hypertension, and leiomyogenic tumorigenicity. In the progression of these diseases, SMCs change their phenotype from a differentiated to a dedifferentiated state (3). Recent studi...

Isoform diversity of tropomyosin is generated from the limited genes by a combination of differential transcription and alternative splicing. In the case of the ␣-tropomyosin (␣-TM) gene, exon 2a rather than exon 2b is specifically spliced in ␣-TM-SM mRNA, which is one of the major tropomyosin isoforms in smooth muscle cells. Here we demonstrate that expressions of ␣-tropomyosin and caldesmon isoforms are coordinately regulated in association with phenotypic modulation of smooth muscle cells. Molecular cloning and Western and Northern blottings have revealed that in addition to the downregulation of ␤-TM-SM, ␣-TM-SM converted to ␣-TM-F1 and ␣-TM-F2 by a selectional change from exon 2a to exon 2b during dedifferentiation of smooth muscle cells in culture. Simultaneously, a change of caldesmon isoforms from high M r type to low M r type was also observed by alternative selection between exons 3b and 4 in the caldesmon gene during this process. In contrast, cultured smooth muscle cells maintaining a differentiated phenotype continued to express ␣-TM-SM, ␤-TM-SM, and high M r caldesmon. In situ hybridization revealed specific coexpression of ␣-TM-SM and high M r caldesmon in smooth muscle in developing embryos. These results suggest a common splicing mechanism for phenotype-dependent expression of tropomyosin and caldesmon isoforms in both visceral and vascular smooth muscle cells.It is important to elucidate the molecular mechanism of phenotypic modulation of smooth muscle cells (SMCs) 1 such as vasculogenesis, enterogenesis, atherosclerosis, hypertension, and leiomyogenic tumorigenesis. The SMCs are derived from mesodermal precursors, but the intracellular and extracellular factors determining the SMC lineage and its phenotype remain unclear. The search for molecular parameters indicating SMC phenotype is a first step in analyzing phenotypic modulation of SMCs. Several cytoskeletal and contractile proteins are such candidates. Among them, changes of actin (1, 2), caldesmon (CaD) (1, 3, 4), myosin heavy chain (5, 6), and vinculin/metavinculin (1, 7) isoforms are closely associated with phenotypic modulation of SMCs. Recent studies have focused on the gene regulation of such parameters (8 -14). In addition to these isoform changes, expression of ␣-smooth muscle actin (␣-SM actin) (15, 16), CaD (1, 3, 4), myosin heavy and light chains (5, 6, 17), meta-vinculin (1, 7), SM22, and calponin (18 -20) have been reported to be up-regulated during differentiation of SMCs, but down-regulated during dedifferentiation, suggesting the involvement of SMC phenotype-dependent transcriptional regulation in the SMC-specific parameter genes. In fact, the transcriptional machineries of ␣-SM actin (8, 9), CaD (12), myosin heavy chain (13), SM22 (21), and calponin (22) have been partially characterized.Tropomyosin (TM) is a predominant helical protein that binds to actin groves. Recent evidence suggests that Ca 2ϩ -dependent actin-myosin interaction in smooth and nonmuscle cells is controlled by myosin-and actin-linked dual regulation....

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We have developed a new method of preparing acellular vascular grafts. Cellular components, including cell membranes and proteins in cytosol, were efficiently extracted from the vessels in a concentrated aqueous solution of poly(ethylene glycol), an amphiphilic biocompatible polymer. The residual DNA was digested by deoxyribonuclease I treatment after extraction with poly(ethylene glycol). The two-step extraction process proved quite effective at removing the cellular components while causing little damage to the extracellular matrices. We did not use any detergent that would damage the extracellular matrices. Therefore, vascular endothelial cells grew well on the acellular vessels after recellularization, promising longi-patent cardiovascular grafts.

The cathodic reaction of Ti has been studied in a CaF 2 -MgF 2 bath with CaO and TiO 2 at 1373K. Cyclic voltammograms were measured in the baths of various compositions, and the dependence of the cathodic reaction of Ti on the bath composition was discussed. With reference to the results of the cyclic voltammetry, Ti metal deposition by potentio-static electrolysis was attempted. It was shown that the reduction sequence of Ti consisted of three steps, and Ti metal was deposited under the suitable condition. The cathodic behavior of Ti was influenced by the molar ratio of TiO 2 to CaO in the melt. Considering the previous results of Ti electrolysis by using DC-ESR unit, it is strongly suggested that a binuclear complex ion of Ti with O, such as Ti 2 O 7