Heparin-II domain of fibronectin is a vascular endothelial growth factor-binding domain: enhancement of VEGF biological activity by a singular growth factor/matrix protein synergism

We describe extracellular interactions between fibronectin (Fn) and vascular endothelial growth factor (VEGF) that influence integrin-growth factor receptor crosstalk and cellular responses. In previous work, we found that VEGF bound specifically to fibronectin (Fn) but not vitronectin or collagens. Herein we report that VEGF binds to the heparin-II domain of Fn and that the cell-binding and VEGF-binding domains of Fn, when physically linked, are necessary and sufficient to promote VEGF-induced endothelial cell proliferation, migration, and Erk activation. Using recombinant Fn domains, the C-terminal heparin-II domain of Fn (type III repeats 13 to 14) was identified as a key VEGF-binding site. Mutation of the heparin-binding residues on FnIII(13-14) abolished VEGF binding, and peptides corresponding to the heparin-binding sequences in FnIII(13-14) inhibited VEGF binding to Fn. Fn fragments containing both the alpha5beta1 integrin-binding domain (III 9 to 10) and the VEGF-binding domain (III 13 to 14) significantly enhanced VEGF-induced EC migration and proliferation and induced strong phosphorylation of the VEGF receptor and Erk. Neither the cell-binding or VEGF-binding fragment of Fn alone had comparable VEGF-promoting effects. These results suggest that the mechanism of VEGF/Fn synergism is mediated extracellularly by the formation of a novel VEGF/Fn complex requiring both the cell-binding and VEGF-binding domains linked in a single molecular unit. These data also highlight a new function for the Fn C-terminal heparin-binding domain that may have important implications for angiogenesis and tumor growth.     

The type of mutation in the low density lipoprotein receptor gene influences the cholesterol-lowering response of the HMG-CoA reductase inhibitor simvastatin in patients with heterozygous familial hypercholesterolaemia

In a genetically heterogeneous group of 109 patients with a clinical diagnosis of heterozygous familial hypercholesterolaemia (FH), the influence of gender, apolipoprotein (apo) E genotype and the type of molecular defect in the LDL-receptor (LDLR) gene on the reduction of plasma LDL-cholesterol levels to treatment with a HMG-CoA reductase inhibitor (simvastatin) were studied. Response was determined as the percentage fall in LDL-cholesterol from untreated levels and as the proportion of patients where levels fell below 4.9 or 4.1 mmol/l. Of the patients, 86 individuals had tendon xanthomata (TX+) and a diagnosis of 'definite' FH and these individuals presented with a significantly higher untreated LDL-cholesterol compared to the 23 individuals who did not have xanthomas (TX-) and a diagnosis of 'probable' FH (8.14+/-0.19 vs. 6.81+/-0.25, P= 0.001). Overall, HMG-CoA reductase inhibitor doses of 10, 20 or 40 mg/day resulted in a significant fall of LDL-cholesterol levels of 29, 39 and 49%, but at all doses those with TX had significantly higher levels than those without, and significantly fewer TX + patients achieved LDL-cholesterol levels below 4.9 or 4.1 mmol/l than the TX - group (P < 0.05 at each dose). In the TX+ group the response to treatment was of similar magnitude in men and women and in patients with different apoE genotype. In the 'probable' FH probands only three mutations were identified (detection rate 13%), one in the LDLR gene and two in the APOB gene, a detection rate significantly lower (P= 0.02) than in the 'definite' FH probands where 28 mutations were detected (detection rate 37%). In the TX + patients where no mutation was detected, treatment resulted in a greater proportion achieving LDL-cholesterol levels below 4.9 and 4.1 mmol/l compared to those with any LDLR mutation, this difference was close to statistical significance at the 4.9 mmol/l threshold at 10 mg/day (41 vs. 13%, P = 0.058). For the 14 patients with an LDLR mutation that was predicted to be 'severe', fewer achieved LDL-cholesterol levels below 4.9 or 4.1 mmol/l at each dosage compared to the 16 individuals with 'mild' mutations, and this difference was statistically significant at the maximal dosage of 40 mg/day (P = 0.018). Thus although characterisation of the molecular defect in FH patients may not be relevant to their immediate clinical management, those with a particular mutation may need more aggressive lipid-lowering treatment to reach LDL-cholesterol levels recommended to reduce the risk of coronary heart disease (CHD).     

Predictive validity of DSM‐IV oppositional defiant and conduct disorders in clinically referred preschoolers

Background: Diagnostic validity of oppositional defiant and conduct disorders (ODD and CD) for preschoolers has been questioned based on concerns regarding the ability to differentiate normative, transient disruptive behavior from clinical symptoms. Data on concurrent validity have accumulated, but predictive validity is limited. Predictive validity is critical to refuting the hypothesis that diagnosing ODD and CD in young children leads to pathologizing normal behavior. ODD and CD have emerged as gateway disorders to many forms of adult psychopathology. Establishing how early we can identify symptoms and disorders that herald poor prognosis is one of the most important goals for research on etiology and prevention. Methods: Subjects were 3–5‐year‐old consecutive referrals to a child psychiatry clinic (n = 123) and demographically matched children from a pediatric clinic (n = 100). A diagnostic interview was used to assess DSM‐IV ODD and CD in a prospective follow‐up design from preschool to school age. Stability of ODD and CD diagnoses and level of impairment were tested as a function of preschool diagnosis. Results: Over 80% of preschoolers diagnosed with ODD and approximately 60% of preschoolers diagnosed with CD met criteria for the same disorder during follow‐up. Impairment over time varied significantly as a function of stability of diagnosis across three years. Conclusions: These results provide the first evidence of the predictive validity of DSM‐IV ODD and CD in clinically referred preschool children. The findings challenge the assumption that symptoms of disruptive behavior disorders that occur during the preschool period tend to be transient.     

IL-6–174G/C genotype is associated with the bone mineral density response to oestrogen replacement therapy in post-menopausal women

A reduction in interleukin-6 (IL-6) activity may contribute to the beneficial effects of hormone replacement therapy (HRT) on the menopausal decline in bone mineral density (BMD). We have examined this hypothesis using a genetic strategy. The –174C (rather than G) IL-6 gene variant is associated with lower IL-6 expression. As such, we might anticipate the C allele to be associated with a greater response to HRT. We have tested this hypothesis. Mean three-site [spine (L1-L4), neck of femur, and Wards triangle] BMD was measured in 65 women in a 1-year randomised controlled trial of HRT with 0.625 mg oestrogen/day and 0.15 mg norgestrel (n=30). Baseline BMD was genotype-independent for both the control and HRT group. In the control group, the percentage change in BMD after 1 year was similar between genotypes (P=0.45). In contrast, in the HRT group, the rise was genotype-dependent. Those homozygous for the G allele showed a 3.62 (2.14)% increase in BMD compared with 10.44 (4.68)% for the C-homozygous group. Heterozygotes had an intermediate BMD increase of 5.6 (2.82)% [P=0.006 (P value for interaction between HRT and genotype was 0.04)] Although the study was limited by its small sample size, these are the first data to demonstrate the importance of IL-6 genotype in determining response to oestrogen therapy, rather than its physiological withdrawal.     

Use of a T cell-specific monoclonal antibody, T10B9, in a novel allogeneic stem cell transplantation protocol for hematologic malignancy high-risk patients.

To reduce the toxicity of traditional conditioning regimens for allogeneic stem cell transplantation (allo-SCT), we used single-agent chemotherapy conditioning with either busulfan (total cumulative dose, 16 mg/kg) or melphalan (200 to 240 mg/m 2 ), followed by the anti-T cell-specific monoclonal antibody T10B9 (MEDI-500) daily for 3 days. T cell-replete SCT was performed from HLA-identical sibling donors. Acute graft-versus-host disease (aGVHD) prophylaxis consisted of 7 additional days of T10B9 and delayed onset of cyclosporine (ie, on day +4 or +5). Twenty-six high-risk hematologic malignancy patients were entered onto this study. All 24 patients who survived longer than 8 days engrafted, although 1 patient experienced late graft failure. Deaths occurred in 21 of 26 patients because of infection (n = 7), progression/recurrence of primary disease (n = 6), aGVHD (n = 4), regimen-related toxicity (n = 1), and other causes (n = 3). Five of these patients are enjoying disease-free survival with a median survival of 1193 days after allo-SCT. The conditioning regimen induced modulation of surface expression of CD3 (but not CD4 or CD8) and was associated with decreasing tumor necrosis factor-alpha (but not interleukin-6) serum levels. In conclusion, single-agent chemotherapy conditioning with T10B9 produced durable engraftment and long-term survival in some patients who would not have qualified for a traditional allo-SCT.     

Tagging SNP haplotype analysis of the secretory PLA2-V gene, PLA2G5, shows strong association with LDL and oxLDL levels, suggesting functional distinction from sPLA2-IIA: results from the UDACS study

Animal and human studies suggest that both secretory PLA2 (sPLA2)-V and sPLA2-IIA (encoded, respectively, by the neighbouring PLA2G5 and PLA2G2A genes) contribute to atherogenesis. Elevated plasma sPLA2-IIA predicts coronary heart disease (CHD) risk, but no mass assay for sPLA2-V is available. We previously reported that tagging single nucleotide polymorphism (tSNP) haplotypes of PLA2G2A are strongly associated with sPLA2-IIA mass, but not lipid levels. Here, we use tSNPs of the sPLA2-V gene to investigate the association of PLA2G5 with CHD risk markers. Seven PLA2G5 tSNPs genotypes, explaining >92% of the locus genetic variability, were determined in 519 patients with Type II diabetes (in whom PLA2G2A tSNP data was available), and defined seven common haplotypes (frequencies >5%). PLA2G5 and PLA2G2A tSNPs showed linkage disequilibrium (LD). Compared to the common PLA2G5 haplotype, H1 (frequency 34.9%), haplotypes H2-7 were associated with overall higher plasma LDL (P < 0.00004) and total cholesterol (P < 0.00003) levels yet lower oxLDL/LDL (P = 0.006) and sPLA2-IIA mass (P = 0.04), probably reflecting LD with PLA2G2A. Intronic tSNP (rs11573248), unlikely itself to be functional, distinguished H1 from LDL-raising haplotypes and may mark a functional site. In conclusion, PLA2G5 tSNP haplotypes demonstrate an association with total and LDL cholesterol and oxLDL/LDL, not seen with PLA2G2A, thus confirming distinct functional roles for these two sPLA2s.     

Association between plasma IL-6, the IL6 -174G>C gene variant and the metabolic syndrome in type 2 diabetes mellitus

Elevated plasma interleukin-6 (IL-6) is associated with coronary heart disease (CHD), impaired glucose tolerance (IGT), and type 2 diabetes (T2DM). We and others have described an association between the human interleukin-6 -174G>C gene variant and body mass index (BMI). Within our previous sample of subjects with T2DM, we measured plasma IL-6 and grouped subjects by the WHO-defined metabolic syndrome, in order to study the association between the -174G>C gene variant, plasma IL-6 and the metabolic syndrome (and component parts). Genotype was obtained in 571 Caucasian subjects with plasma IL-6 measures. There was a significant association between genotype and plasma IL-6 (GG vs GC vs CC: 3.23+/-0.93 pg/ml vs 3.42+/-0.95 pg/ml vs 4.16+/-1.18 pg/ml, p=0.02; for GG/GC vs CC p=0.008). No interactions were observed between genotype and the individual components of the metabolic syndrome in determining plasma IL-6. Increased plasma IL-6 was also associated with the number of components (none vs 1 vs 2 vs > or =3: 2.67+/-0.71 pg/ml vs 2.97+/-0.94 pg/ml vs 4.07+/-1.13 pg/ml, p<0.0001). Within the sample, 76% of CC compared to 56% of GG subjects had the metabolic syndrome (p=0.007). Further analysis of association between the genotype and the components of the metabolic syndrome revealed no further associations than that with BMI previously described. The association of this gene variant with the metabolic syndrome is intimately linked with obesity per se. Further prospective work is required to explore the effect of this gene variant in relation to obesity, the metabolic syndrome and 'prediabetes'.     

A Rare Congenital Finding in an Elderly Lady—Scimitar Syndrome with Dextrocardia in a 79 Year Old

We present a case of a 79-year-old African-American woman with scimitar syndrome and associated dextrocardia. This case represents, to our knowledge, the oldest living patient described in the literature with scimitar syndrome.     

Awareness of lifestyle risk factors for cancer and heart disease among adults in the UK

Objective

To examine and compare awareness of lifestyle risk factors for cancer and heart disease in a single UK representative sample.

Methods

Two open-ended questions about cancer and heart disease risk factors were included in a population-based survey of 1747 adults. Responses were coded for four lifestyles with established links to both diseases: smoking, eating an unhealthy diet, drinking excessive alcohol and physical inactivity.

Results

Awareness of lifestyle risk factors was low for both diseases, although higher for heart disease than cancer. The average number identified by respondents was 2.1 (heart disease) and 1.4 (cancer). The strongest predictor was education (both p < 0.001). Awareness that physical inactivity is a cancer risk factor was particularly low at 7%.

Conclusion

These findings suggest that public awareness of the impact of lifestyle on commonly feared diseases, especially cancer, is low.

Practice implications

Unhealthy lifestyles make a significant contribution to ill health and mortality. Increased public awareness of the links between lifestyles and commonly feared diseases might help people understand the potential health consequences of their actions and encourage them to make much-needed lifestyle changes. Efforts are needed to improve public health messages about how lifestyle risk factors impact on the chances of developing these important diseases.     

Genetic causes of familial hypercholesterolaemia in patients in the UK: relation to plasma lipid levels and coronary heart disease risk

Aims

To determine the relative frequency of mutations in three different genes (low‐density lipoprotein receptor (LDLR), APOB, PCSK9), and to examine their effect in development of coronary heart disease (CHD) in patients with clinically defined definite familial hypercholesterolaemia in UK.

Patients and methods

409 patients with familial hypercholesterolaemia patients (158 with CHD) were studied. The LDLR was partially screened by single‐strand conformational polymorphism (SSCP) (exons 3, 4, 6–10 and 14) and by using a commercial kit for gross deletions or rearrangements. APOB (p.R3500Q) and PCSK9 (p.D374Y) were detected by specific assays. Coding exons of PCSK9 were screened by SSCP.

Results

Mutations were detected in 253 (61.9%) patients: 236 (57.7%) carried LDLR, 10 (2.4%) carried APOB p.Q3500 and 7 (1.7%) PCSK9 p.Y374. No additional mutations were identified in PCSK9. After adjusting for age, sex, smoking and systolic blood pressure, compared to those with no detectable mutation, the odds ratio of having CHD in those with an LDLR mutation was 1.84 (95% CI 1.10 to 3.06), for APOB 3.40 (0.71 to 16.36), and for PCSK9 19.96 (1.88 to 211.5; p = 0.001 overall). The high risk in patients carrying LDLR and PCSK9 p.Y374 was partly explained by their higher pretreatment cholesterol levels (LDLR, PCSK9 and no mutation, 10.29 (1.85), 13.12 and 9.85 (1.90) mmol/l, respectively, p = 0.001). The post‐statin treatment lipid profile in PCSK9 p.Y374 carriers was worse than in patients with no identified mutation (LDL‐C, 6.77 (1.82) mmol/l v 4.19 (1.26) mmol/l, p = 0.001, HDL‐C 1.09 (0.27) mmol/l v 1.36 (0.36) mmol/l, p = 0.03).

Conclusions

The higher CHD risk in patients carrying PCSK9 p.Y347 or a detected LDLR mutation supports the usefulness of DNA testing in the diagnosis and management of patients with familial hypercholesterolaemia. Mutations in PCSK9 appear uncommon in patients with familial hypercholesterolaemia in UK.Familial hypercholesterolaemia is an autosomal dominant disorder associated with increased risk of coronary heart disease (CHD), with an estimated prevalence in the UK of 1 in 500 to 1 in 600.¹ Roughly half of the men with familial hypercholesterolaemia, if untreated, will have developed clinically evident CHD by the age of 55 years. Affected women from the same families typically develop CHD about 9 years later than their affected male relatives, but again, often remarkably prematurely.² The proportion of patients with familial hypercholesterolaemia identified and being treated in lipid clinics to date in the UK is, at best, 15% of the predicted number, with most of these being young people.¹ Because lipid‐lowering drug treatment with statins substantially reduces coronary morbidity and mortality,³ identification of affected people by screening is crucially important. To this end, the Department of Health has recently funded five pilot sites in the UK to determine the efficiency of cascade testing in the current social structure and the framework of the National Health Service. Cascade testing is a cost‐effective method of finding additional patients with familial hypercholesterolaemia,⁴ and has been used extensively in other countries in Europe, most notably in Holland,⁵ for the past 5 years.

Key points

• Patients with familial hypercholesterolaemia with a detectable LDLR mutation have a higher risk of early CHD than those in whom no mutation was detected.
• Patients with the pD374Y mutation in PCSK9 have the highest pretreatment and post‐treatment levels of plasma cholesterol and the highest risk of early CHD.
• Mutations in PCSK9 appear to be uncommon in patients with familial hypercholesterolaemia in UK.

The extent to which DNA testing for familial hypercholesterolaemia complements cholesterol measurement in cascade screening to identify affected patients is unclear, as is its role in determining the risk of CHD and response to treatment. In the current study, we carried out molecular genetic testing in patients recruited from the Simon Broome familial hypercholesterolaemia register⁶ in the UK as a cross‐sectional cohort study to identify risk factors for premature CHD in patients with familial hypercholesterolaemia.⁷ Primary results from this study indicated that the conventional cardiovascular risk factors of age, sex, smoking, pretreatment cholesterol levels and low levels of high‐density lipids (especially in women) were all associated with higher risk of CHD,⁷ confirming associations reported in other studies, for example.⁸ When this UK study was started, mutations at two loci causing familial hypercholesterolaemia had been identified, with mutations in the low‐density lipoprotein receptor gene (LDLR) accounting for most of the identified mutations,⁹ whereas one particular mutation in the gene encoding the ligand for the low density lipoprotein (LDL) receptor—namely apolipoprotein B (FDB)—occurs in about 5% of patients in the UK.¹⁰ This mutation, which alters a single amino acid (p.R3500Q), has been shown to reduce the affinity of the LDL cholesterol particle,¹⁰ where ApoB is the single‐protein component for the receptor. For the LDLR, currently >100 mutations have been reported in UK patients to date^9,11 (see also www.ucl.ac.uk/FH). A commercially available kit for screening for deletions and rearrangements of the LDLR gene has become available, and it is known that up to 5% of patients with familial hypercholesterolaemia in patients in the UK may have such a deletion.¹²Recently, defects in a third gene causing monogenic hypercholesterolaemia have been identified.¹³ The gene protein convertase subtilisin/kexin type 9 (PCSK9) codes for an enzyme that has also been called “neural apoptosis regulated convertase 1”, which has recently been proposed to be involved in degrading the LDLR protein in the lysosome of the cell and preventing it from recycling.¹⁴ Gain‐ of‐ function mutations in the PCSK9 gene could therefore cause increased degradation of LDLRs, reduced numbers of receptors on the surface of the cell, and monogenic hypercholesterolaemia. An alternative mechanism has also been proposed for the hypercholesterolaemic effect, whereby the gain of function causes increased secretion of apoB‐containing lipoproteins from the liver, with this being supported by in vivo turnover studies in patients carrying PCSK9 missense mutations¹⁵ and by in vitro studies in transiently transfected rat liver cells.¹⁶ One mutation in this gene, p.D374Y, has been reported in several independent families^16,17,18 and we therefore also tested for this cause of familial hypercholesterolaemia in this group of patients, as well as using single‐strand conformational polymorphism (SSCP) analysis and direct sequencing to screen all coding exons of the gene.Although patients with no identified mutation may have a monogenic cause of the disorder in an as yet undiscovered gene, it is also possible that some may have polygenic hypercholesterolaemia and have been misclassified using current clinical diagnostic criteria. These patients would be expected to have a milder degree of hyperlipidaemia, possibly not present from birth but only developing in later life, and would therefore be predicted to have a lower risk of CHD. The hypothesis we set out to test was that patients with identified mutations in the LDLR, PCSK9 or APOB genes would be at greater risk of CHD than patients with no identified mutation.