Transforming growth factor-beta 1 inhibits human arterial smooth-muscle cell proliferation in a growth-rate-dependent manner.

BACKGROUND: Proliferation of arterial smooth-muscle cells is central in the development of both atherosclerosis and intimal hyperplasia. The cytokine transforming growth factor-beta 1 (TGF-beta 1) is known to have variable effects on smooth-muscle cell proliferation. Using human arterial smooth-muscle cells, we sought (1) to define the serum concentrations required to maintain cellular proliferation; and (2) to define the effects of TGF-beta 1 on smooth-muscle cell proliferation. METHODS: Smooth-muscle cell cultures were established from the normal aorta of transplant donors. Cells were grown to subconfluent and confluent densities, then incubated in either serum-free media, or 1% or 10% fetal bovine serum (FBS) enhanced media. Cellular proliferation was assayed by cell counting at 24, 48, and 96 hours to establish growth rate. Identical experiments with the addition of recombinant human TGF-beta 1 (5 ng/mliters) were also performed. Studies were done in triplicate for each group, and results expressed as the mean +/- SE. Groups were compared by analysis of variance. RESULTS: In subconfluent cultures, only smooth-muscle cells in 10% FBS proliferated, whereas growth arrest occurred in serum-free media and 1% FBS. In confluent cultures, cells in all media conditions proliferated. TGF-beta 1 had an inhibitory effect in actively proliferating cultures. There was a positive correlation between the inhibitory effects of TGF-beta 1 and smooth-muscle cell growth rate (r = .65; P = 0.005). CONCLUSIONS: When confluent, human arterial smooth-muscle cells continue to proliferate after serum deprivation, suggesting that these cells are capable of conditioning their own medium. TGF-beta 1 inhibits smooth-muscle cell proliferation in a growth-rate-dependent manner. These data suggest that TGF-beta 1 may have a growth-regulatory role in vascular disease by counteracting states of arterial smooth-muscle cell proliferation.

Improved high-performance liquid chromatographic analysis of intracellular deoxyribonucleoside triphosphate levels.

The ability to measure intracellular deoxyribonucleoside triphosphate (dNTP) pool sizes is important for understanding the intracellular metabolism of DNA synthesis and repair. We have developed an improved method for measuring intracellular dNTP pool size by high-performance liquid chromatography (HPLC). Previous methods have enabled accurate measurement of dNTPs only in concentrations greater than approximately 10 pmol per 10(6) cells due to the inability to partially purify cell extracts, to the inability to apply extracts from extremely large numbers of cells, to the lack of efficient columns, to the presence of incompatible solvents, and to the inability to inject large volumes. We have modified a low-pressure strong anion-exchange column pre-step developed by others to concentrate and partially purify oxidized cell extracts while at the same time eluting them in a more compatible solvent for HPLC injection. The HPLC column is a YMC ODS-AQ column operating in a combined hydrophobic-interaction chromatography-reversed-phase chromatography mode. The injection and elution solvents are both phosphate-based. Using this method it is possible to measure intracellular dNTP levels well below 0.5 pmol per 10(6) cells or at the sensitivity of the DNA polymerase assay.

Soluble factors modulate changes in collagen gene expression in abdominal aortic aneurysms.

BACKGROUND: Although increased procollagen gene expression and synthesis have been implicated in the progression of abdominal aortic aneurysms (AAA), factors modulating this change have not been identified. Furthermore, it is not known whether the increase in AAA procollagen expression is specific to this disease or also occurs in tissue affected by atherosclerotic occlusive disease (AOD). If paracrine rather than autocrine factors are responsible for increased gene expression in AAA, this effect should be transferable to target smooth muscle cells through conditioned media. Our objectives were to determine 1 alpha (I) procollagen messenger RNA levels in AOD tissue compared with normal and AAA and to determine whether differences noted in tissue procollagen gene expression could be transferred through conditioned media from normal, AOD, and AAA tissues to target smooth muscle cells in primary culture. METHODS: Normal, AOD, and AAA tissue was used for tissue RNA extraction or was minced and washed with serum-free media (4 degrees C) x 30 minutes and the media applied to human aortic smooth muscle cells (SMC) in primary culture for 36 hours. Total RNA from tissue and SMC exposed to conditioned media was analyzed by Northern and dot blot analysis for 1 alpha (I) procollagen. RESULTS: Relative tissue 1 alpha (I) procollagen levels were not increased in AOD (0.23 +/- 0.05) as compared with normal (0.17 +/- 0.03); both were decreased compared with AAA (0.53 +/- 0.07; p < 0.01). The 1 alpha (I) procollagen levels in SMC exposed to conditioned media from AAA (1.73 +/- 0.15) were increased (p < 0.05) compared with AOD (1.10 +/- 0.12) and normal (1.16 +/- 0.16). CONCLUSIONS: There is no increase in tissue AOD procollagen gene expression. The ability to transfer the same relative patterns of gene expression from tissue to target SMC with conditioned media suggests that paracrine, rather than autocrine, factors modulate procollagen expression in AAA tissues.