Increase in Elastin Gene Expression and Protein Synthesis in Arterial Smooth Muscle Cells Derived From Patients with Moyamoya Disease by Mari Yamamoto,

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Increase in Elastin Gene Expression and Protein Synthesis in Arterial Smooth Muscle Cells Derived From Patients with Moyamoya Disease by Mari Yamamoto, Masaru Aoyagi, Shingo Tajima, Hiroshi Wachi, Naomi Fukai, Yoshiharu Matsushima, and Kiyotaka Yamamoto Stroke Volume 28(9):1733-1738 September 1, 1997 Copyright © American Heart Association, Inc. All rights reserved.

Tropoelastin synthesis in cultured SMCs from moyamoya patients (HMSMC) and control subjects (HCSMC). a, Equal amounts of protein obtained from quiescent SMCs were analyzed by immunoblotting with an antibody against tropoelastin. Tropoelastin synthesis in cultured SMCs from moyamoya patients (HMSMC) and control subjects (HCSMC). a, Equal amounts of protein obtained from quiescent SMCs were analyzed by immunoblotting with an antibody against tropoelastin. Lanes 1 and 2, HCSMC strains. Lanes 3 and 4, HMSMC strains. b, Bands corresponding to 70-kD tropoelastin were scanned with a densitometer. Columns show the mean and SD of three separate experiments. *P<.01, compared with cells from control subjects by independent Student’s t test. Mari Yamamoto et al. Stroke. 1997;28:1733-1738 Copyright © American Heart Association, Inc. All rights reserved.

Effects of TGF-β1 on elastin production in the culture medium of HMSMC and HCSMC strains. Effects of TGF-β1 on elastin production in the culture medium of HMSMC and HCSMC strains. Confluent cultures were treated in the absence (a) or presence (b) of TGF-β1 (10 ng/mL) for 48 hours and labeled with 3H-valine for the final 6 hours. Labeled proteins obtained from the culture medium were resolved on 4% to 15% SDS-polyacrylamide gels and subjected to fluorography. The arrow shows the migration of tropoelastin. Lanes 1 through 4, HCSMC strains. Lanes 5 through 10, HMSMC strains. Mari Yamamoto et al. Stroke. 1997;28:1733-1738 Copyright © American Heart Association, Inc. All rights reserved.

Relative levels of elastin production in the culture medium (a) or cell layer (b). Relative levels of elastin production in the culture medium (a) or cell layer (b). Confluent cultures were treated with TGF-β1 (0 to 10 ng/mL) for 48 hours and labeled with 3H-valine for the final 6 hours. Labeled proteins obtained from the culture medium or cell layer were resolved on 4% to 15% SDS-polyacrylamide gels and subjected to fluorography. Densitometric scanning was performed to compare the relative levels of the 70-kD tropoelastin band. Columns show the means and SDs for HCSMC (n=4) and HMSMC (n=6). *P<.004, **P<.0001, compared with cells from control subjects with the use of independent Student’s t test. **P<.0001 on panels a and b. Mari Yamamoto et al. Stroke. 1997;28:1733-1738 Copyright © American Heart Association, Inc. All rights reserved.

Northern blot analysis of elastin mRNA levels in HCSMC and HMSMC strains. a, RNA (15 μg) isolated from cells treated with or without TGF-β1 (10 ng/mL) for 48 hours, resolved by 1% agarose gel electrophoresis, and blotted onto nitrocellulose membranes. Northern blot analysis of elastin mRNA levels in HCSMC and HMSMC strains. a, RNA (15 μg) isolated from cells treated with or without TGF-β1 (10 ng/mL) for 48 hours, resolved by 1% agarose gel electrophoresis, and blotted onto nitrocellulose membranes. The membranes were hybridized with elastin cDNA probes as described in “Materials and Methods” and subjected to autoradiography. The loading of the gels was determined by visualizing the 28S rRNA band using ethidium bromide (28S). Lanes 1 through 3, HCSMC strains. Lanes 4 through 6, HMSMC strains. b, Autoradiograms were scanned with a densitometer. Columns show the means and SDs for HCSMC and HMSMC strains. *P<.03, **P<.002 compared with cells from control subjects by independent Student’s t test. Mari Yamamoto et al. Stroke. 1997;28:1733-1738 Copyright © American Heart Association, Inc. All rights reserved.

Production of TGF-β1 in the medium of cultured HCSMC and HMSMC Production of TGF-β1 in the medium of cultured HCSMC and HMSMC. SMCs grown to confluence were incubated in MEM containing 0.5% FBS at 37°C for 48 hours. Production of TGF-β1 in the medium of cultured HCSMC and HMSMC. SMCs grown to confluence were incubated in MEM containing 0.5% FBS at 37°C for 48 hours. The medium was collected, and the TGF-β1 secreted into the medium was measured with a TGF-β1 ELISA kit. Columns show the means and SDs for HCSMC and HMSMC strains. *P<.001, compared with cells from control subjects by independent Student’s t test. Mari Yamamoto et al. Stroke. 1997;28:1733-1738 Copyright © American Heart Association, Inc. All rights reserved.