David J. Grainger

Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using 1H-NMR-based metabonomics.

Although a wide range of risk factors for coronary heart disease have been identified from population studies, these measures, singly or in combination, are insufficiently powerful to provide a reliable, noninvasive diagnosis of the presence of coronary heart disease. Here we show that pattern-recognition techniques applied to proton nuclear magnetic resonance (1H-NMR) spectra of human serum can correctly diagnose not only the presence, but also the severity, of coronary heart disease. Application of supervised partial least squares-discriminant analysis to orthogonal signal-corrected data sets allows >90% of subjects with stenosis of all three major coronary vessels to be distinguished from subjects with angiographically normal coronary arteries, with a specificity of >90%. Our studies show for the first time a technique capable of providing an accurate, noninvasive and rapid diagnosis of coronary heart disease that can be used clinically, either in population screening or to allow effective targeting of treatments such as statins.

Transforming Growth Factor β and Atherosclerosis: So Far, So Good for the Protective Cytokine Hypothesis

The role of the anti-inflammatory cytokine transforming growth factor beta (TGF-beta) in atherosclerosis has been the subject of considerable debate for a decade. In the early 1990s, we postulated that TGF-beta played an important role in maintaining normal vessel wall structure and that loss of this protective effect contributed to the development of atherosclerosis. We termed this the protective cytokine hypothesis. This proposal was slow to gain broad acceptance, however, because at that time there were little data available on the role of TGF-beta during the development of atherosclerosis but much information about its role during trauma-induced neointima formation. Because TGF-beta apparently aggravates neointima formation, both by inhibiting endothelial regeneration and by promoting fibrosis, it was difficult to accept that its presence might ameliorate the superficially similar atherogenesis process. But several recent studies revealed beyond doubt the fact that TGF-beta protects against lipid lesion formation, at least in mouse models of atherosclerosis. Therefore, two important questions remain. First, is the role of TGF-beta in vascular biology similar in humans and in mice? Secondly, how important, compared with defects in thrombosis or lipoprotein metabolism, is the protective role of TGF-beta during atherogenesis?

Release and activation of platelet latent TGF–β in blood clots during dissolution with plasmin

Transforming growth factor β1 (TGF–β1) is a platelet–derived cytokine involved in both normal wound healing and scarring. We show that human platelets contain two pools of latent TGF–β1, which constitute more than 95% of the total TGF–β assayed in whole platelets. During clotting, one pool, the large latent TGF–β complex consisting of latent TGF–β binding protein (LTBP), the latency–associated peptide (LAP) and the 25–kD mature TGF–β1 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–β1 is gradually activated and released. If similar mechanisms operate in vivo, the clot will act as a slow–release capsule of TGF–β1 activity during wound healing.

Application of chemometrics to 1H NMR spectroscopic data to investigate a relationship between human serum metabolic profiles and hypertension

The application of chemometric methods to 1H NMR spectroscopic data has been documented for pathophysiological processes. In this study we show the application of 1H NMR-based metabonomics to investigate a relationship between serum metabolic profiles and hypertension. Although hypertension can be defined using blood pressure measurements, the underlying aetiology and metabolic effects are not so readily identified. Serum profiles for patients with low/normal systolic blood pressure (SBP < or = 130 mm Hg; n = 28), borderline SBP (131-149 mm Hg; n = 19) and high SBP (> or = 150 mm Hg; n = 17) were acquired using 1H NMR spectroscopy. Orthogonal signal correction followed by principal components analysis were applied to these NMR data in order to facilitate interpretation, and the resulting chemometric models were validated using Soft Independent Modelling of Class Analogy. Using 1H NMR-based metabonomics, it was possible to distinguish low/ normal SBP serum samples from borderline and high SBP samples. Borderline and high SBP samples, however, were indiscriminate from each other. Our preliminary results showed that there was a relationship between serum metabolic profiles and blood pressure which, in part, was due to lipoprotein particle composition differences between the samples. Furthermore, our results indicated that serum pathology associated with blood pressure is apparent at SBP values > 130 mm Hg, which the WHO and ISH currently define as the limit between normal and high-normal.

TGF-β in blood: a complex problem

Abstract The cytokine transforming growth factor-β (TGF-β) was initially purified from human platelets, a rich source of this protein. In addition to platelets, TGF-β1 is also found in other blood fractions, including plasma and the circulating leukocytes. However, more than 15 years after the initial isolation of TGF-β1, there remains no consensus on how much TGF-β1 is present in normal human plasma. Here we review the difficulties associated with measuring TGF-β concentrations in complex biological fluids, and discuss the current state of knowledge on the distribution of TGF-β isoforms in various blood fractions as well as the nature of the TGF-β-containing protein complexes.

Apolipoprotein E Modulates Clearance of Apoptotic Bodies In Vitro and In Vivo, Resulting in a Systemic Proinflammatory State in Apolipoprotein E-Deficient Mice

Apolipoprotein E (apoE) is a 34-kDa glycoprotein involved in lipoprotein transport through interaction with the low-density lipoprotein receptor and related receptors. Recently, it has become clear that apoE binding to its receptors plays a role both in development and in control of the immune system. In this study, we show that apoE modulates the rate of uptake of apoptotic cells by macrophages. In vitro, apoE-deficient macrophages ingest less apoptotic thymocytes (but not latex beads) than wild-type macrophages, and this defect can be corrected by addition of exogenous apoE protein. In vivo, the number of dying macrophages is increased in a range of tissues, including lung and brain. Possibly in response to the larger numbers of persistent apoptotic bodies, the number of live macrophages in these tissues are also increased compared with those of wild-type control mice. In addition to the significant changes in macrophage population dynamics we observed, levels of the proinflammatory cytokine TNF-α and the positive acute phase reactant fibrinogen are also elevated in the livers from apoE-deficient mice. In contrast, neither deletion of the gene encoding the LDL receptor nor cholesterol feeding of wild-type mice affected either the number of apoptotic bodies or the number of live macrophages. We conclude that apoE deficiency results in impaired clearance of apoptotic cell remnants and a functionally relevant systemic proinflammatory condition in mice, independent of its role in lipoprotein metabolism. Any similar reduction of apoE activity in humans may contribute to the pathogenesis of a wide range of chronic diseases including atherosclerosis, dementia, and osteoporosis.

Active and acid-activatable TGF-β 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.

Neuroprotection in ischemia-reperfusion injury: an antiinflammatory approach using a novel broad-spectrum chemokine inhibitor.

Cerebral ischemia–reperfusion injury is associated with a developing inflammatory response with pathologic contributions from vascular leukocytes and endogenous microglia. Signaling chemokines orchestrate the communication between the different inflammatory cell types and the damaged tissue leading to cellular chemotaxis and lesion occupation. Several therapies aimed at preventing this inflammatory response have demonstrated neuroprotective efficacy in experimental models of stroke, but to date, few investigators have used the chemokines as potential therapeutic targets. In the current study, the authors investigate the neuroprotective action of NR58–3.14.3, a novel broad-spectrum inhibitor of chemokine function (both CXC and CC types), in a rat model of cerebral ischemia–reperfusion injury. Rats were subjected to 90 minutes of focal ischemia by the filament method followed by 72 hours of reperfusion. Both the lesion volume, measured by serial magnetic resonance imaging, and the neurologic function were assessed daily. Intravenous NR58–3.14.3 was administered, 2 mg/kg bolus followed by 0.5 mg/kg · hour constant infusion for the entire 72-hour period. At 72 hours, the cerebral leukocytic infiltrate, tumor necrosis factor-α (TNF-α), and interleukin-8 (IL-8)-like cytokines were analyzed by quantitative immunofluorescence. NR58–3.14.3 significantly reduced the lesion volume by up to 50% at 24, 48, and 72 hours post–middle cerebral artery occlusion, which was associated with a marked functional improvement to 48 hours. In NR58–3.14.3-treated rats, the number of infiltrating granulocytes and macrophages within perilesional regions were reduced, but there were no detectable differences in inflammatory cell numbers within core ischemic areas. The authors reported increased expression of the cytokines, TNF-α, and IL-8–like cytokines within the ischemic lesion, but no differences between the NR58–3.14.3-treated rats and controls were reported. Although chemokines can have pro-or antiinflammatory action, these data suggest the overall effect of chemokine up-regulation and expression in ischemia–reperfusion injury is detrimental to outcome.

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.

Metabolomic analysis of urine and serum in Parkinson’s disease

Objective To investigate the metabolic profile of serum and urine samples from 23 female patients with Parkinson’s disease (PD) and 23 age and sex-matched controls. Methods We used gas chromatography coupled to mass spectrometry to detect metabolites (approximately 1,600 in total), then supervised statistical analysis (using projection to latent structures discriminant analysis) to study the differences between control and PD samples. Results Supervised statistical analysis yielded models that possessed statistically significant predictive value for blind samples on the basis of the metabolic profile of urine but not of serum. However, whilst no individual biomarkers were identified, suggesting that any metabolic disturbance associated with PD is comparatively minor, a multivariate metabolic signature associated with PD was identified in urine. Interpretation There is a relatively subtle, yet distinct, metabolic signature of PD present in the urine of patients with early disease. The signature may itself act as a useful biomarker for PD, although larger studies will be required to validate our present findings.