Tamoxifen elevates transforming growth factor-beta and suppresses diet-induced formation of lipid lesions in mouse aorta.

When C57B16 male mice are fed a high-fat diet, they develop significant fatty streak lesions in the aorta. Addition of tamoxifen (TMX) to a high-fat diet, equivalent to a dose of approximately 1 mg TMX per kg body weight per day, suppressed the diet-induced increase in the area of lipid staining in the aortic sinus of the mice by 88% and in the average number of lesions by 86%. The TMX-treated mice had 11% +/- 5% less total plasma cholesterol, with most of the reduction in the high density lipoprotein fraction, whereas plasma triglycerides were significantly elevated, and circulating concentrations of 17 beta-estradiol and testosterone were unaffected. Both circulating and aortic concentrations of active and latent transforming growth factor-beta (TGF-beta) were substantially elevated by TMX. The inhibition of lesion formation may be due, at least in part, to cardiovascular protection by TGF-beta.

Heparin decreases the rate of proliferation of rat vascular smooth muscle cells by releasing transforming growth factor beta-like activity from serum.

OBJECTIVES: Various heparins have been reported to inhibit the proliferation of vascular smooth muscle cells (VSMCs). The effects of eight chemically distinct heparins on the cell cycle and differentiation of primary and passaged cultures of rat aortic VSMCs have been characterised and the mechanism of heparin action investigated. METHODS: VSMCs from adult rat aorta were prepared by enzyme dispersion and stimulated to enter the cell cycle with 10% serum in the presence or absence of heparin. Progressions through S phase and M phase were measured by [3H]-thymidine incorporation and cell counting respectively. Flow cytometry was used to confirm the effects of heparin on VSMC cell cycle progression. The effect of heparin on VSMC differentiation was investigated by analysing smooth muscle specific myosin heavy chain content of the cells after heparin treatment. RESULTS: Eight heparins at concentrations between 5 micrograms.ml-1 and 100 micrograms.ml-1 partially inhibited VSMC proliferation (27% to 76% 96 hours after addition of heparin), but did not affect the entry of the cells into S phase. Flow cytometry confirmed that VSMC populations in the presence of heparin contained significantly (p < 0.005) more cells in the G2/M phase of the cell cycle than control populations. Heparin also blocked the dedifferentiation of primary cultures of VSMCs stimulated by serum. These effects of heparin were completely reversed by the presence of a neutralising antiserum to transforming growth factor beta (TGF beta) and heparin attached to agarose beads was as effective as free heparin as a growth inhibitor of VSMCs. CONCLUSIONS: Heparins of varying molecular weight and anticoagulant properties all partially inhibited VSMC proliferation predominantly by extending the G2/M phase of the cell cycle. Heparin also inhibited dedifferentiation of primary cultures of VSMCs. Heparin (< 100 micrograms.ml-1) acted extracellularly to release TGF beta from serum, which accounted for the effects of heparin on proliferation and differentiation.

Mitogens for adult rat aortic vascular smooth muscle cells in serum-free primary culture.

OBJECTIVE: When vascular smooth muscle cells are dispersed into culture in the presence of serum they modulate their phenotype. The aim of this study was to determine the range of growth factors likely to stimulate replication of medial vascular smooth muscle cells in vivo by examining their responses when cultured in the absence of serum. METHODS: Freshly dispersed vascular smooth muscle cells from the healthy aortic media of adult rats were prepared and plated out in cell culture in the absence of fetal calf serum. DNA synthesis by these cells when plated onto fibronectin in response to various growth factors and vasoconstrictors was analysed by [3H]-thymidine incorporation. RESULTS: Less than 5% of cells plated on culture plastic spread, but 15-25% of cells plated onto fibronectin spread and survived for at least 8 d in culture. These cells responded to platelet derived growth factor BB homodimer (PDGF-BB), basic fibroblast growth factor, and epidermal growth factor by stimulating DNA synthesis at least 10-fold compared with cells in the absence of growth factors. Maximum rate of DNA synthesis occurred 36-42 h after addition of 10% fetal calf serum, whereas maximum rate of DNA synthesis occurred 80-88 h after stimulation with PDGF-BB or basic fibroblast growth factor. By contrast, PDGF-AA homodimer, transforming growth factor type beta, insulin-like growth factor I, angiotensin II, and endothelin I did not stimulate DNA synthesis by more than threefold. CONCLUSIONS: Freshly dispersed vascular smooth muscle cells plated onto fibronectin in the absence of serum proliferate in response to PDGF-BB, basic fibroblast growth factor, and epidermal growth factor by stimulating DNA synthesis. The range of mitogens and the time course of entry into DNA synthesis under these culture conditions suggest that serum-free culture provides a good model for the responses of medial vascular smooth muscle cells in vivo.

The endothelin peptides ET-1, ET-2, ET-3 and sarafotoxin S6b are co-mitogenic with platelet-derived growth factor for vascular smooth muscle cells.

We have investigated whether any of the three isoforms of endothelin (ET) ET-1, ET-2 and ET-3 or the structurally similar peptide sarafotoxin S6b is mitogenic on its own for rat vascular smooth muscle cells in culture. DNA synthesis was determined by a peroxidase-linked double antibody technique to detect bromodeoxyuridine incorporation into the nucleus and stained nuclei were counted by image analysis. None of the ET peptides or sarafotoxin S6b (up to 100 nM) was capable of initiating DNA synthesis in the absence of platelet derived growth factor (PDGF) or fetal calf serum. All the peptides potentiated the mitogenic effect of low concentrations of PDGF. ET-1 and ET-2 (10 nM) caused a 2-fold increase in the number of stained nuclei induced by 5 nM and 10 nM PDGF, whereas ET-3 and sarafotoxin S6b were less potent. These findings demonstrate that ET is a co-mitogen for rat vascular smooth muscle cells. The release of ET at sites of endothelial injury may therefore enhance the mitogenic action of locally acting PDGF on vascular smooth muscle cells and potentiate the proliferative response.

Chronic headaches in adults with spina bifida and associated hydrocephalus.

INTRODUCTION: Adults with spina bifida and associated hydrocephalus are exposed to multiple risk factors for the development of chronic headache. The management of these patients can be complex and misdiagnosis can precipitate unnecessary shunt revision. This study aims to evaluate the usefulness of intracranial pressure (ICP) monitoring as a diagnostic tool in these cases and to look at the causes of chronic headaches and treatment outcomes for this patient population. METHODS: All patients over the age of 18 years with a diagnosis of spina bifida and shunted hydrocephalus who had undergone inpatient or outpatient neurosurgical review within the last 10 years were identified in our hospital database. Case notes were then retrospectively reviewed to identify all patients who had undergone either inpatient or outpatient evaluation of chronic headaches (defined as a headache of at least one month's duration) occurring in the absence of any other symptoms or signs suggestive of raised intracranial pressure (ICP). The incidence, causes, management and outcome of chronic headache in these patients was determined. RESULTS: 42 patients were identified, mean age 30 years (range 18 - 59). All had undergone lifelong follow-up. All had previously undergone shunt insertion for hydrocephalus. 16 had undergone endoscopic third ventriculostomy (ETV). 11 had undergone choroid plexus coagulation. 55 % (23/42) of patients underwent investigation for 1 or more episodes of chronic headache. Recurrent hydrocephalus due to shunt malfunction or ETV failure was excluded by ICP monitoring using either an intraparenchymal transducer or monitoring via a ventricular access device. All patients underwent repeat imaging, using CT and/or MR imaging. Identified causes of headache included: shunt blockage; shunt overdrainage; ETV failure and symptomatic Arnold-Chiari malformation. A history of choroid plexus coagulation (CPC) as an infant was associated with a decreased risk of chronic headache in later life (p = 0.02). In 8 patients no definite cause for headaches was identified, in 4 of these patients symptoms resolved spontaneously, the remainder required specialist pain management. CONCLUSIONS: The aetiology of chronic headaches in this patient group is multifactorial. In the absence of other clinical symptoms or signs of raised ICP, ICP monitoring is an invaluable adjunct to management. 10 % of hydrocephalic adult spina bifida patients required specialist pain management for control of chronic idiopathic headache.

Transforming growth factor beta is sequestered into an inactive pool by lipoproteins.

Elevated plasma concentrations of low density lipoprotein (LDL) and very-low density lipoprotein (VLDL) have been correlated with the development of atherosclerosis. These lipoproteins may promote atherogenesis by direct deposition of lipid in the vessel wall. In addition, previous data suggested that there was an inverse correlation between serum LDL-cholesterol concentration and the proportion of transforming growth factor beta (TGF-beta) in an active form (Grainger et al. 1995. Nature Med. 1:74). Here we have investigated whether lipoproteins can affect the activity of TGF-beta1 in plasma and show that TGF-beta can associate with the lipoprotein fraction. In the plasma of healthy males, 16 +/- 5% (mean +/- standard deviation; n = 57) of the total plasma TGF-beta1 was associated with the lipoprotein fraction, with the major proportion (64 +/- 15%) in the HDL-3 subfraction. However, in ten diabetic subjects with moderately poor glucose control (Hb alc > 8.0), the proportion of total plasma TGF-beta in the lipoprotein fraction was 68 +/- 21%. This large increase in TGF-beta1 associated with the lipoprotein fraction was mainly due to association with VLDL, chylomicrons, and LDL. The lipoprotein fraction inhibits TGF-beta1 binding to the type II TGF-beta receptor extracellular domain in an ELISA and inhibits TGF-beta1 activity in the mink lung cell bioassay. We propose that sequestration of TGF-beta into lipoproteins represents a novel mechanism by which TGF-beta activity in circulation may be regulated. Lipoprotein sequestration of TGF-beta may therefore contribute to the severe depression of TGF-beta activity in advanced atherosclerosis.

Transforming growth factor beta decreases the rate of proliferation of rat vascular smooth muscle cells by extending the G2 phase of the cell cycle and delays the rise in cyclic AMP before entry into M phase.

Transforming growth factor beta 1 (TGF-beta 1) decreased the rate of proliferation of rat aortic vascular smooth muscle cells (VSMCs) stimulated with serum showing a maximal effect at > 5 ng/ml (200 pM). However, it did not reduce the proportion of cells which passed through S phase (> 90%) and entry into S phase was delayed by less than 3 h. The proportion of cells passing through M phase (> 90%) was also unaffected, but entry into mitosis was delayed by approx. 24 h. This increase in cell cycle time was therefore due mainly to an increase in the G2 to mitotic metaphase period. Addition of TGF-beta 1 late in G1 or late in S phase failed to delay the onset of mitosis, but the presence of TGF-beta 1 between 0 and 12 h after the addition of serum to quiescent cells was sufficient to cause the maximal delay in mitosis of approx. 24 h. The role of cyclic AMP in the mechanism of the TGF-beta 1 effects on the cell cycle was examined. Entry into mitosis was preceded by a transient 2-fold increase in cyclic AMP concentration and TGF-beta 1 delayed both this increase in cyclic AMP and entry into mitosis to the same extent. Addition of forskolin or 8-(4-chlorophenylthio)-cyclic AMP to cells 30 h after stimulation with serum completely reversed the increase in duration of G2 in the presence of TGF-beta 1, suggesting that the rise in cyclic AMP levels which precedes mitosis might trigger entry of the VSMCs into M phase. Addition of forskolin late in S phase (26 h after stimulation with serum) advanced the entry of the cells into M phase and they divided prematurely. This effect was unaffected by the addition of cycloheximide with the forskolin; however, the effect of forskolin on cell division was completely inhibited when cycloheximide was added late in G1. TGF-beta 1 prevented the loss of smooth-muscle-specific myosin heavy chain (SM-MHC), which occurs in primary VSMC cultures in the presence or absence of serum, and the cells proliferated while maintaining a differentiated phenotype. However, TGF-beta 1 did not cause re-differentiation of subcultured VSMCs which contained very low amounts of SM-MHC and the effect of TGF-beta 1 in extending the G2 phase of the cell cycle is exerted independently of its effect on differentiation.