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Biomedical subjects

D J Grainger

Publications and source records attributed to D J Grainger.

At least 19 recordsLinked to original sources
PubMed1999

Genetic control of the circulating concentration of transforming growth factor type beta1.

The concentration of transforming growth factor beta (TGF-beta) in plasma has been correlated with the development of several diseases, including atherosclerosis and certain forms of cancer. However, the mechanisms that control the concentration of TGF-beta in plasma are poorly understood. In a study of 170 pairs of female twins (average age 57.7 years) we show that the concentration of active plus acid-activatable latent TGF-beta1 [(a+l) TGF-beta therefore is predominantly under genetic control (heritability estimate 0.54). Single strand conformation polymorphism (SSCP) mapping of the TGF-beta1 gene promoter has identified two single base substitution polymorphisms. The two polymorphisms (G-->A at position -800 bp and C-->T at position -509 bp) are in linkage disequilibrium (correlation coefficient Delta = 0.215, P < 0.01). The C-509T polymorphism is significantly associated with the plasma concentration of (a+l) TGF-beta1, explaining 8.2% of the additive genetic variance of (a+l) TGF-beta1 concentration. It is therefore possible that predisposition to atherosclerosis, bone diseases or various forms of cancer may be correlated with the presence of particular alleles at the TGFB1 locus.

Adult↗
PubMed1998

Increased PAI activity and PAI-1 antigen occurring with an oral fat load: associations with PAI-1 genotype and plasma active TGF-beta levels.

Whether the post-prandial lipemic response is linked to potentially pro-atherogenic and/or prothrombotic changes in plasminogen activator inhibitor (PAI) and transforming growth factor-beta (TGF-beta) is uncertain. The aim of our study was to determine whether PAI-1 antigen and PAI activity were elevated during post-prandial lipemia following a standard fat tolerance test. We also investigated changes in TGF-beta1 antigen and TGF-beta activity, to determine whether changes in TGF-beta activity were associated with changes in PAI measurements. Lastly, the influence of genotype at a common insertion/deletion polymorphism in the PAI-1 promoter on changes in PAI activity and PAI-1 antigen was examined. Fat tolerance tests were undertaken in 57 healthy middle-aged men to investigate associations between plasma concentrations of lipoproteins, PAI (antigen and activity) and TGF-beta. PAI-1 concentration increased by 76% after 8 h (P < 0.0001). PAI activity also increased by 64% (P = 0.0054) and TGF-beta activity decreased by 10% (P < 0.0001). Increases in PAI-I antigen and PAI activity varied markedly between individuals. To investigate these heterogeneous responses we examined whether genotype at the common insertion/deletion polymorphism of the PAI-1 promoter accounted for these differences. Individuals with at least one 4G (deletion) allele showed potentially pro-atherogenic changes in both PAI-1 and TGF-beta, compared to individuals who were homozygous for the 5G (insertion) allele. In conclusion, increased PAI and decreased TGF-beta activity occur during a fat tolerance test and this effect may be modulated by a common insertion/deletion polymorphism in the PAI-1 promoter.

Aged↗
PubMed1998

Transforming growth factor-beta1 gene polymorphisms and coronary artery disease.

1. Transforming growth factor-beta1 is a cytokine with a very wide spectrum of biological activities. Previous studies have shown that it is involved in a number of physiological and pathological processes including heart disease. In our study we aimed to scan the transforming growth factor-beta1 locus for polymorphisms and to identify haplotypes significantly associated with a predisposition to coronary atherosclerosis.2. Two patient groups comprising 244 angiographically normal individuals and 655 patients with coronary artery disease were recruited from London and Sheffield. DNA samples from these subjects were screened for mutations in the transforming growth factor-beta1 locus and all subjects were genotyped by a coupled polymerase chain reaction-restriction enzyme digestion method.3. Five polymorphisms have been identified in the transforming growth factor-beta1 gene at positions G-800A, C-509T in the promoter region, Leu10-->Pro, Arg25-->Pro in exon 1 and Thr263-->Ile in exon 5. No significant difference in frequencies for any of the five polymorphisms was found between controls and patients with coronary artery disease. Similarly, there was no correlation between these polymorphisms and hypertension.4. The genotypes of all the individuals participating in the study were assigned to seven main haplotypes of the transforming growth factor-beta1 locus. Based on species comparison data we propose that GCCGC is the ancestral haplotype in humans.5. Our data suggest that these transforming growth factor-beta1 polymorphisms are not associated with coronary artery disease and therefore their presence alone would not be a genetic risk factor for predisposition to coronary artery disease.

Case-Control Studies↗
PubMed1998

Transforming growth factor-beta dynamically regulates vascular smooth muscle differentiation in vivo.

Variations in the levels of smooth muscle-specific isoforms of contractile proteins have been reported to occur in many different vascular diseases. However, although much work has been done in vitro to investigate the regulation of smooth muscle cell differentiation, the molecular mechanisms which regulate the differentiation of vascular smooth muscle tissue in vivo are unknown. Using quantitative immunofluorescence, we show that in rat arteries levels of smooth muscle differentiation markers correlate with the levels of the cytokine TGF-beta. In young mice with one allele of the TGF-beta1 gene deleted, the levels of both TGF-beta1 and smooth muscle differentiation markers are reduced compared to wild-type controls. This regulation of smooth muscle differentiation by TGF-beta during post-natal development also occurs dynamically in the adult animal. Following various pharmacological or surgical interventions, including treatment of mice with tamoxifen and balloon injury of rat carotid arteries, there is a strong correlation between the changes in the levels of TGF-beta and changes in the levels of smooth muscle differentiation markers (r=0. 9, P<0.0001 for n=26 experiments). We conclude that TGF-beta dynamically regulates smooth muscle differentiation in rodent arteries in vivo.

Animals↗
PubMed1997

Lipoprotein(a) vascular accumulation in mice. In vivo analysis of the role of lysine binding sites using recombinant adenovirus.

Although the mechanism by which lipoprotein(a) [Lp(a)] contributes to vascular disease remains unclear, consequences of its binding to the vessel surface are commonly cited in postulated atherogenic pathways. Because of the presence of plasminogen-like lysine binding sites (LBS) in apo(a), fibrin binding has been proposed to play an important role in Lp(a)'s vascular accumulation. Indeed, LBS are known to facilitate Lp(a) fibrin binding in vitro. To examine the importance of apo(a) LBS in Lp(a) vascular accumulation in vivo, we generated three different apo(a) cDNAs: (a) mini apo(a), based on wild-type human apo(a); (b) mini apo(a) containing a naturally occurring LBS defect associated with a point mutation in kringle 4-10; and (c) human- rhesus monkey chimeric mini apo(a), which contains the same LBS defect in the context of several additional changes. Recombinant adenovirus vectors were constructed with the various apo(a) cDNAs and injected into human apoB transgenic mice. At the viral dosage used in these experiments, all three forms of apo(a) were found exclusively within the lipoprotein fractions, and peak Lp(a) plasma levels were nearly identical (approximately 45 mg/dl). In vitro analysis of Lp(a) isolated from the various groups of mice confirmed that putative LBS defective apo(a) yielded Lp(a) unable to bind lysine-Sepharose. Quantitation of in vivo Lp(a) vascular accumulation in mice treated with the various adenovirus vectors revealed significantly less accumulation of both types of LBS defective Lp(a), relative to wild-type Lp(a). These results indicate a correlation between lysine binding properties of Lp(a) and vascular accumulation, supporting the postulated role of apo(a) LBS in this potentially atherogenic characteristic of Lp(a).

Adenoviridae↗
PubMed1997

Tamoxifen decreases cholesterol sevenfold and abolishes lipid lesion development in apolipoprotein E knockout mice.

BACKGROUND: Apolipoprotein E (apo E) knockout mice develop severe vascular lipid lesions resembling human atherosclerotic plaques, irrespective of the fat content of their diet. METHODS AND RESULTS: Oral tamoxifen (TMX) at a dose of 1.9 mg.kg body wt-1.d-1 abolished lipid lesion development, assayed by oil red O staining, whether the mice were fed a normal diet or a diet with high fat content. The TMX-treated mice showed a sevenfold decrease in total cholesterol. However, the proportion of plasma cholesterol present in VLDL remained unchanged, whereas the proportion in LDL decreased by 37%, and that in HDL increased by 64%. Consistent with the shift from LDL to HDL cholesterol, there was a 62% decrease in total triglycerides. The concentrations of active and acid-activatable latent plus active TGF-beta in the aorta were substantially elevated by TMX (87% and 24% increase, respectively). CONCLUSIONS: Although the mechanism of cardiovascular protection by TMX in apo E knockout mice is unknown, the inhibition of lipid lesion formation may be attributable to the changes in lipoprotein profile and the elevated levels of TGF-beta, both of which are thought to be protective against atherosclerosis in humans and animal models.

Animals↗
PubMed1997

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.

Adult↗
PubMed1996

Feedback mechanism of focal vascular lesion formation in transgenic apolipoprotein(a) mice.

Apolipoprotein(a) (apo(a)), the distinguishing protein of atherogenic lipoprotein(a), directs accumulation of the lipoprotein(a) particle to sites in the arterial wall where atherosclerotic lipid lesions develop in man and in transgenic mice expressing human apo(a). It has been proposed that focal apo(a) accumulation in the transgenic mouse vessel wall causes the observed severe local inhibition of transforming growth factor-beta (TGF-beta) activity and the consequent activation of the smooth muscle cells, which subsequently accumulate lipid to form lesions if the mice are fed a high fat diet. We show that blocking formation of these vascular lesions by two independent mechanisms, tamoxifen treatment and increasing high density lipoprotein, also abolishes apo(a) accumulation, inhibition of TGF-beta activity, and activation of smooth muscle cells. The data are consistent with a feedback mechanism in which an initial accumulation of apo(a) inhibits local TGF-beta activity, leading to further accumulation of apo(a). Breaking the feedback loop prevents smooth muscle cell activation and therefore lipid lesion development.

Animals↗
PubMed1996

Optimization of immunofluorescence methods by quantitative image analysis.

There is a growing trend towards the objective quantification of immunohistochemical staining. However, quantification has not been used previously to optimize the original published immunohistochemical methods. We present a quantitative method for analyzing immunofluorescence staining employing the Applied Imaging MAGISCAN image analysis system, which has then been used to optimize major aspects of the standard immunofluorescent staining protocols. The optimization process resulted in a method that increased specific staining up to fivefold over typical published protocols, with no increase in nonspecific staining. The method is extremely reproducible. For slides stained by a single experimenter in one batch on one day, the coefficient of variation between replicate means is 1.2%. The image analysis protocol gave a linear response with increasing antigen concentration, as determined by using purified antigen dried onto slides. The revisions to the standard protocol presented here can also be applied to nonquantitative staining. It will help users of immunofluorescence to maximize their staining and may enable the detection of previously undetected antigens.

Actins↗
PubMed1995

Cloning and analysis of the promoter region of the rat SM22 alpha gene.

We have cloned and sequenced a 1.9 kb fragment of the 5'-upstream sequence of the smooth-muscle-specific gene SM22 alpha. The region cloned consisted of the SM22 alpha promoter, a 65 bp exon containing most of the 5'-untranslated region and 307 bp of the first intron. A 1.5 kb fragment at the 5' end of this sequence was able to drive the expression of a reporter chloramphenicol acetyltransferase (CAT) gene in both vascular smooth-muscle cells and Rat-1 fibroblasts. This promoter region did not contain a consensus TATAA box but contained the sequence TTTAAA 25 bp from the major start site identified by primer extension. Deletion analysis showed that a fragment of the promoter from +65 to -303 was more active in both cell types than the 1.5 kb fragment suggesting that there are silencer sequences in the region 5' to the core promoter. CAT activity was also observed with fragments containing bases +65 to -193 and +65 to -117 in smooth-muscle cells. In contrast with the smooth-muscle cells, no CAT activity was detected in Rat-1 fibroblasts with the smallest two fragments. The residual promoter activity in the smallest fragment of the SM22 alpha promoter tested suggested that, unlike the smooth-muscle alpha-actin promoter, transcription from the SM22 alpha promoter can occur in smooth-muscle cells in the absence of factors binding to CC(A/Trich)6GG (CArG box) or CANNTG (E box) motifs.

Animals↗
PubMed1995

ID--a dominant negative regulator of skeletal muscle differentiation--is not involved in maturation or differentiation of vascular smooth muscle cells.

Rat aortic vascular smooth muscle cells (VSMCs) expressing the MyoD gene formed myotubes at a low frequency in the presence of serum but at a high frequency in the absence of serum. Expression of an antisense Id gene increased myotube formation in the presence of serum indicating that a reduction in Id levels is a major mechanism by which serum withdrawal promotes myotube formation. The role of Id in the development of VSMCs was investigated by expressing an antisense Id gene in neonatal VSMCs. No evidence was found for the conversion of neonatal VSMCs to adult VSMCs in the presence of the antisense Id gene. Similarly reduction in Id by serum withdrawal also failed to cause conversion of the neonatal VSMCs to the adult phenotype. These data suggest that the maturation of neonatal smooth muscle cells is not controlled by a VSMC homologue of the skeletal muscle basic-helix-loop-helix proteins.

Acetamides↗
PubMed1995

Active and acid-activatable TGF-beta in human sera, platelets and plasma.

Assays which measure active and latent forms of transforming growth factor beta (TGF-beta) separately in human serum and plasma are required to investigate the biological role of TGF-beta in a variety of human diseases. We have developed an enzyme-linked immunosorbent assay (ELISA) using two polyclonal antibodies against TGF-beta which rapidly determines the amount of active plus acid-activatable, latent TGF-beta forms ((a+l)TGF-beta) present in human serum and plasma in the range 4 pmol/l to 2000 pmol/l. To measure active TGF-beta alone, we have developed a second ELISA using the extracellular domain of the TGF-beta type II receptor as the capture reagent which detects active TGF-beta in serum and plasma samples in the range 20 pmol/l to 4000 pmol/l. Both assays detect TGF-beta 1 and TGF-beta 3 with similar sensitivity, are > 10-fold less sensitive to TGF-beta 2 and are not affected by a range of other peptide growth factors. The mean (a+l)TGF-beta present in human serum was 330 pmol/l but the range was very large (< 4 pmol/l to 1400 pmol/l). The mean active TGF-beta present was 230 pmol/l (range < 20 pmol/l to 1400 pmol/l) and the proportion of the (a+l)TGF-beta present which was active [a/(a+l)] varied from < 10% to 100%. The concentration of (a+l)TGF-beta and the proportion of TGF-beta which was active were very similar in the serum and platelet-poor plasma prepared from the same whole blood sample. The clot formed during serum preparation retained all of the TGF-beta which was detected by the (a+l)TGF-beta ELISA in the corresponding platelet releasate, although the PDGF in platelets was released into the serum. In contrast, platelet-poor plasma contained no detectable PDGF demonstrating that the (a+l)TGF-beta assayed in the plasma was not due to platelet degranulation after bleeding. Serum active TGF-beta and (a+l)TGF-beta concentrations therefore provide a reliable estimate of these forms of TGF-beta present in plasma.

Adult↗
PubMed1995

The serum concentration of active transforming growth factor-beta is severely depressed in advanced atherosclerosis.

Recent evidence has led us to propose that transforming growth factor-beta (TGF-beta) is a key inhibitor of atherosclerosis. We show here that a population of patients with advanced atherosclerosis all have less active TGF-beta in their sera than patients with normal coronary arteries, with a fivefold difference in average concentration between the two groups. This correlation with atherosclerosis is much stronger than for other known major risk factors and it may therefore have important diagnostic and prognostic significance. Aspirin medication correlates with an increase in active TGF-beta concentration, indicating that therapeutic interventions for TGF-beta are possible.

Aged↗
PubMed1995

Release and activation of platelet latent TGF-beta in blood clots during dissolution with plasmin.

Transforming growth factor beta 1 (TGF-beta 1) is a platelet-derived cytokine involved in both normal wound healing and scarring. We show that human platelets contain two pools of latent TGF-beta 1, which constitute more than 95% of the total TGF-beta assayed in whole platelets. During clotting, one pool, the large latent TGF-beta complex consisting of latent TGF-beta binding protein (LTBP), the latency-associated peptide (LAP) and the 25-kD mature TGF-beta 1 dimer is released into the serum. A second pool, which contains LAP but not LTBP, is retained in the clot, but can be released by RGD peptide. When the clot is dissolved by plasmin this bound TGF-beta 1 is gradually activated and released. If similar mechanisms operate in vivo, the clot will act as a slow-release capsule of TGF-beta 1 activity during wound healing.

Blood Platelets↗
PubMed1995

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.

Actins↗
PubMed1995

Transforming growth factor-beta: the key to understanding lipoprotein(a)?

Most large studies of the blood parameters that act as risk factors for myocardial infarction, stroke and atherosclerosis have identified elevated circulating levels of lipoprotein(a) as an important risk factor. Lipoprotein(a) consists of an LDL particle that is covalently bound to the distinguishing protein component apolipoprotein(a). Ever since apolipoprotein(a) was cloned in 1987 and the marked sequence homology to plasminogen was noted, mechanisms for the atherogenic activity of lipoprotein(a), based on the competitive inhibition of plasminogen activity, have been proposed. However, with the availability of transgenic mice expressing both human apolipoprotein(a) and lipoprotein(a), recent studies have demonstrated that lipoprotein(a) acts to inhibit plasminogen activation in vivo. One consequence of this is reduced activation of the cytokine transforming growth factor-beta, an important regulator of vessel wall structure.

Animals↗