herbimycin and Hypertrophy

Increased intraluminal pressure of the rat portal vein in vivo causes hypertrophy and altered contractility in 1 to 7 days. We have used organ cultures to investigate mechanisms involved in this adaptation to mechanical load. Strips of rat portal vein were cultured for 3 days, either undistended or loaded by a weight. Length-force relations were shifted toward longer length in stretched cultured veins compared with freshly dissected veins, whereas the length-force relations of unstretched cultured veins were shifted in the opposite direction. This occurred after culture either with or without 10% FCS to promote growth. The wet weight of loaded veins increased by 56% in the presence of FCS, whereas that of undistended control veins increased by 24%. No weight increase was seen in serum-free culture. The dry/wet weight ratio decreased during culture with FCS but was not affected by stretch. Electron microscopy revealed increased cell cross-sectional area in stretched relative to unstretched veins, and protein contents were greater, as were [(3)H]thymidine and [(3)H]leucine incorporation rates. Growth responses were associated with the activation of stretch-sensitive extracellular signal-regulated kinases 1 and 2 and were inhibited by herbimycin A and PD 98059, inhibitors of extracellular signal-regulated kinases 1 and 2. The results demonstrate that by culture of whole vascular tissue, smooth muscle cells are maintained in the contractile phenotype and respond to stretch with a physiological adaptation involving hypertrophy/hyperplasia and remodeling of the contractile system, similar to that in vivo. Mechanical stimulation and growth factors are both required for functionally significant growth.

Topics: Animals; Benzoquinones; Cattle; Culture Media; Culture Media, Serum-Free; DNA Replication; Enzyme Activation; Enzyme Inhibitors; Female; Fetal Blood; Flavonoids; Hyperplasia; Hypertrophy; Lactams, Macrocyclic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Muscle Contraction; Muscle Development; Muscle Proteins; Muscle, Smooth, Vascular; Neovascularization, Physiologic; Organ Culture Techniques; Organ Size; Phenotype; Portal Vein; Quinones; Rats; Rats, Sprague-Dawley; Rifabutin; Signal Transduction; Stress, Mechanical

We have shown previously that thromboxane A2 stimulates hypertrophy of cultured rat aortic smooth muscle cells defined as protooncogene expression and protein synthesis without DNA synthesis or cellular proliferation (Dorn, G.W., II, Becker, M.W., Davis, M.G. (1992) J. Biol. Chem. 267, 24897-24905). Since endogenous growth modulators could possibly regulate vascular smooth muscle growth to this vasoconstrictor, we tested the hypothesis that thromboxane-stimulated vascular smooth muscle hypertrophy was due to increased expression of endogenously produced basic fibroblast growth factor (bFGF). The thromboxane mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619) (1 microM) increased cultured rat aorta derived smooth muscle cell immunoreactive bFGF content by 331 +/- 40% over untreated controls after 24 h. Co-incubation of vascular smooth muscle cells with a specific antisense oligodeoxynucleotide (AS) against codon 60 of bFGF coding sequence reduced thromboxane-stimulated bFGF expression by 72 +/- 5% and prevented thromboxane-stimulated hypertrophy (nonsense oligonucleotide had no effects). Addition of exogenous bFGF (20 ng/ml) restored growth to AS-treated/thromboxane-stimulated vascular smooth muscle cells. Furthermore, addition to the culture medium of neutralizing antibody against bFGF inhibited U46619-stimulated vascular smooth muscle hypertrophy by 69 +/- 17%, whereas nonimmune IgG had no effect. Since protein tyrosine phosphorylation is a cell signal associated with growth, thromboxane-stimulated tyrosine phosphorylation was also examined. Exposure to 1 microM U46619 for 10 min increased vascular smooth muscle immunoreactive phosphotyrosine content of 130-144-, 86-, 80-, 75-, and 58-kDa proteins. The tyrosine kinase inhibitor herbimycin A (5 microM) prevented thromboxane-stimulated tyrosine phosphorylation, but not thromboxane-stimulated hypertrophy, suggesting that tyrosine phosphorylation was not required for thromboxane-stimulated vascular smooth muscle growth. These results indicate that increased expression and release of endogenous bFGF, but not direct tyrosine phosphorylation, mediates the hypertrophic vascular smooth muscle response to thromboxane.

Topics: Animals; Base Sequence; Benzoquinones; DNA; Fibroblast Growth Factor 2; Humans; Hypertrophy; Lactams, Macrocyclic; Molecular Sequence Data; Muscle, Smooth, Vascular; Phosphorylation; Protein Kinase Inhibitors; Quinones; Rats; Rifabutin; Thromboxane A2; Tyrosine; Up-Regulation