Peter J. Grant

RAGE and arthritis: The G82S polymorphism amplifies the inflammatory response

The receptor for advanced glycation end products (RAGE) and its proinflammatory S100/calgranulin ligands are enriched in joints of subjects with rheumatoid arthritis (RA) and amplify the immune/inflammatory response. In a model of inflammatory arthritis, blockade of RAGE in mice immunized and challenged with bovine type II collagen suppressed clinical and histologic evidence of arthritis, in parallel with diminished levels of TNF-alpha, IL-6, and matrix metalloproteinases (MMP) 3, 9 and 13 in affected tissues. Allelic variation within key domains of RAGE may influence these proinflammatory mechanisms, thereby predisposing individuals to heightened inflammatory responses. A polymorphism of the RAGE gene within the ligand-binding domain of the receptor has been identified, consisting of a glycine to serine change at position 82. Cells bearing the RAGE 82S allele displayed enhanced binding and cytokine/MMP generation following ligation by a prototypic S100/calgranulin compared with cells expressing the RAGE 82G allele. In human subjects, a case-control study demonstrated an increased prevalence of the 82S allele in patients with RA compared with control subjects. These data suggest that RAGE 82S upregulates the inflammatory response upon engagement of S100/calgranulins, and, thereby, may contribute to enhanced proinflammatory mechanisms in immune/inflammatory diseases.

Evaluation of angiotensin-converting enzyme (ACE), its homologue ACE2 and neprilysin in angiotensin peptide metabolism

In the RAS (renin-angiotensin system), Ang I (angiotensin I) is cleaved by ACE (angiotensin-converting enzyme) to form Ang II (angiotensin II), which has effects on blood pressure, fluid and electrolyte homoeostasis. We have examined the kinetics of angiotensin peptide cleavage by full-length human ACE, the separate N- and C-domains of ACE, the homologue of ACE, ACE2, and NEP (neprilysin). The activity of the enzyme preparations was determined by active-site titrations using competitive tight-binding inhibitors and fluorogenic substrates. Ang I was effectively cleaved by NEP to Ang (1-7) (kcat/K(m) of 6.2x10(5) M(-1) x s(-1)), but was a poor substrate for ACE2 (kcat/K(m) of 3.3x10(4) M(-1) x s(-1)). Ang (1-9) was a better substrate for NEP than ACE (kcat/K(m) of 3.7x10(5) M(-1) x s(-1) compared with kcat/K(m) of 6.8x10(4) M(-1) x s(-1)). Ang II was cleaved efficiently by ACE2 to Ang (1-7) (kcat/K(m) of 2.2x10(6) M(-1) x s(-1)) and was cleaved by NEP (kcat/K(m) of 2.2x10(5) M(-1) x s(-1)) to several degradation products. In contrast with a previous report, Ang (1-7), like Ang I and Ang (1-9), was cleaved with a similar efficiency by both the N- and C-domains of ACE (kcat/K(m) of 3.6x10(5) M(-1) x s(-1) compared with kcat/K(m) of 3.3x10(5) M(-1) x s(-1)). The two active sites of ACE exhibited negative co-operativity when either Ang I or Ang (1-7) was the substrate. In addition, a range of ACE inhibitors failed to inhibit ACE2. These kinetic data highlight that the flux of peptides through the RAS is complex, with the levels of ACE, ACE2 and NEP dictating whether vasoconstriction or vasodilation will predominate.

The genetics of haemostasis: a twin study

BACKGROUND The concentrations of fibrinogen, factor VII and VIII, von Willebrand factor, plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator have been associated with coronary-heart disease. In addition, polymorphisms in the genes coding for fibrinogen, factor VII, PAI-1, and factor XIII have been reported to affect both protein concentrations and cardiovascular disease risk. METHODS We did a classic twin study to assess heritabilities of these haemostatic factors. We enrolled 1002 female twins; 149 pairs of monozygotic and 352 pairs of dizygotic twins. 89 monozygotic and 196 dizygotic twin pairs were analysed for factor VII. FINDINGS Quantitative genetic model fitting showed that genetic factors contributed to about 41-75% of the variation in concentrations of fibrinogen, factor VII, factor VIII, PAI-1, tissue plasminogen activator, factor XIII A-subunit and B-subunit, and von Willebrand factor. Factor XIII activity showed higher (82%) and factor XIIa lower (38%) heritability. INTERPRETATION We have shown that genetic factors have a major effect on plasma concentrations of haemostatic proteins. Our results stress the importance of research into the genetic regulation of proteins involved in haemostasis and atherothrombotic disorders, including myocardial infarction and stroke.

Identification, classification, and expression of RAGE gene splice variants

The receptor for advanced glycation end‐products (RAGE) is a single‐transmembrane, mul tiligand receptor of the immunoglobulin superfamily. RAGE up‐regulation is implicated in numerous patho logical states including vascular disease, diabetes, can cer, and neurodegeneration. The understanding of the regulation of RAGE is important in both disease patho genesis and normal homeostasis. Here, we demonstrate the characterization and identification of human RAGE splice variants by analysis of RAGE cDNA from tissue and cells. We identified a vast range of splice forms that lead to changes in the protein coding region of RAGE, which we have classified according to the Human Gene Nomenclature Committee (HGNC). These resulted in protein changes in the ligand‐binding domain of RAGE or the removal of the transmembrane domain and cytosolic tail. Analysis of splice variants for premature termination codons reveals~50% of identified variants are targeted to the nonsense‐mediated mRNA decay pathway. Expression analysis revealed the RAGE_v1 variant to be the primary secreted soluble isoform of RAGE. Taken together, identification of functional splice variants of RAGE underscores the biological diversity of the RAGE gene and will aid in the under standing of the gene in the normal and pathological state.—Hudson, B. I., Carter, A. M., Harja, E., Kalea, A. Z., Arriero, M., Yang, H., Grant, P. J., Schmidt, A. M. Identification, classification, and expression of RAGE gene splice variants. FASEB J. 22, 1572–1580 (2008)

Association between polymorphisms in the Clock gene, obesity and the metabolic syndrome in man.

Objective:Accumulating evidence raises the hypothesis that dysregulation of intrinsic clock mechanisms are involved in the development of the metabolic syndrome, type 2 diabetes mellitus and cardiovascular disease. The aim of the present study was to investigate the relationship between three known common polymorphisms in the Clock gene and features of the metabolic syndrome in man.Methods:Genotype and haplotype analysis was carried out in a cohort of 537 individuals from 89 families characterized for inflammatory, atherothrombotic and metabolic risk associated with insulin resistance.Results:Heritability of the metabolic syndrome, defined according to International Diabetes Federation criteria, was 0.40. Haplotype analysis indicated three common haplotypes: CAT, TGT and CGC (rs4864548-rs3736544-rs1801260) with frequencies of 31, 33 and 28%, respectively. The CGC haplotype was less prevalent in subjects with the metabolic syndrome (P=0.0015) and was associated with lower waist circumference (P=0.007), lower hip circumference (P=0.023), lower body mass index (P=0.043) and lower leptin levels (P=0.028). The CAT haplotype was significantly associated with the presence of the metabolic syndrome (P=0.020).Conclusions:These findings suggest that the Clock gene CGC haplotype may be protective for the development of obesity and support the hypothesis that genetic variation in the Clock gene may play a role in the development of the metabolic syndrome, type 2 diabetes and cardiovascular disease.

Elevated Serum Inhibin Concentrations in Postmenopausal Women with Ovarian Tumors

Background Inhibin is an ovarian hormone that inhibits the secretion of follicle-stimulating hormone (FSH) by the anterior pituitary gland. Women with granulosa-cell tumors of the ovary have elevated serum inhibin concentrations, but whether the concentrations are increased in women with other ovarian tumors is unknown. Methods We measured serum inhibin and FSH concentrations before surgery in 212 postmenopausal women with suspected ovarian cancer and after surgery in 210 of them. Results Eighteen of the 22 women (82 percent) with mucinous carcinomas (mucinous cystadenocarcinomas and mucinous borderline cystic tumors) of the ovary had elevated serum inhibin concentrations, whereas only 9 of the 53 women (17 percent) with serous carcinomas (serous cystadenocarcinomas and serous borderline cystic tumors) had elevated levels. Serum inhibin concentrations were also elevated in 2 of 12 women (17 percent) with clear-cell carcinomas, 4 of 26 women (15 percent) with undifferentiated carcinomas, 3 of 3 women (100 ...

Genetic regulation of fibrin structure and function: complex gene-environment interactions may modulate vascular risk

BACKGROUND Polymorphisms in the fibrinogen and factor XIII genes are associated with atherothrombotic risk, but clinical studies have produced inconsistent results and laboratory studies have not explained these findings. We aimed to investigate interactions between polymorphisms in the factor XIII and fibrinogen genes, fibrinogen concentrations, and other cardiovascular risk factors in relation to fibrin structure and function. METHODS We used permeation analysis and electron microscopy to investigate interactions between fibrin structure, factor XIII Val34Leu, fibrinogen Aalpha Thr312Ala, fibrinogen Bbeta Arg448Lys, and fibrinogen concentrations in plasma and purified systems. FINDINGS Increased fibrinogen concentrations were associated with decreases in permeability, with tighter clot structures in the presence of factor XIII 34Val alleles compared with those in the presence of 34Leu alleles. Findings were confirmed by scanning electron microscopy of fibrin. Similar changes in permeability were noted for Aalpha fibrinogen 312Ala compared with that for 312Thr. INTERPRETATION Our results show interactions between coding polymorphisms in fibrinogen and factor XIII and fibrinogen concentrations that modify fibrin and explain the apparent paradox between epidemiological studies of factor XIII 34Leu and reported in-vitro effects on fibrin structure and function. We suggest a potential complexity of gene-gene and gene-environment interactions in determining cardiovascular risk.

Association of the Platelet PlA Polymorphism of Glycoprotein IIb/IIIa and the Fibrinogen Bβ 448 Polymorphism With Myocardial Infarction and Extent of Coronary Artery Disease

Background Platelets and fibrinogen play an integral role in the development of thrombosis and are implicated in the process of atherosclerosis. The fibrinogen Bβ 448 polymorphism and the PlA polymorphism of platelet glycoprotein IIIa are reported to be independently associated with coronary artery disease. The aim of this study was to determine the association of the fibrinogen Bβ 448 and the platelet glycoprotein IIIa PlA polymorphisms in relation to extent of coronary atheroma as characterized by angiography and a past history of myocardial infarction (MI) as assessed by World Health Organization criteria. Methods and Results Caucasian patients (n=405) admitted for routine angiography for investigation of chest pain or suspected coronary artery disease were recruited. Caucasian control subjects (n=216) were recruited from local Family Health Services Authority general practice registers. Fibrinogen levels were higher (P=.04) in male patients (3.24 g/L; CI, 3.14 to 3.35) than male control subjects (3.06...

Altered Fibrin Clot Structure in the Healthy Relatives of Patients With Premature Coronary Artery Disease

Background—A family history of premature coronary artery disease (CAD) is an independent cardiovascular risk factor. Fibrin clots composed of dense fiber networks are found in young CAD patients and may occur in the relatives of such individuals. Methods and Results—The ex vivo fibrin structure of 100 healthy male relatives of patients with premature CAD and 100 age-matched control subjects was assessed by measurement of permeability (Ks), fiber mass-length ratio (&mgr;), and turbidity (lag phase and maximum absorbency [max &Dgr;Abs]). Scanning electron microscopy was performed on selected samples. Relatives and controls shared similar levels of conventional cardiovascular risk factors. Ks was lower in relatives than in controls, 12.2 (11.1 to 13.3) versus 15.2 (14.0 to 16.5) ×10−9 cm2 (P <0.001), associated with a smaller decrease in &mgr;, 8.5 (7.7 to 9.2) versus 9.7 (8.9 to 10.5) × 1013 Da/cm (P <0.05), respectively. Lag phase was shorter in relatives than in controls, 39 (37 to 41) versus 47 (44 to 50) seconds (P <0.001), and max &Dgr;Abs was higher in relatives, 0.78 (0.74 to 0.82) versus 0.71 (0.67 to 0.74) in controls (P =0.02), which indicates the presence of thicker fibers in relatives. After adjustment for fibrinogen levels, lag phase and Ks remained significantly different between relatives and control subjects. Scanning electron microscopy images confirmed increased fiber diameter in relatives, possibly of reduced density. Factor XIII Val34Leu and fibrinogen A&agr; Thr312Ala and B&bgr; -455 G/A showed no association with clot structure. Conclusions—The male relatives of patients with premature CAD form fibrin clots that begin polymerization more quickly, have thicker fibers, and are less permeable than those of control subjects.

Plasminogen Activator Inhibitor-1 Promoter 4G/5G Genotype and Plasma Levels in Relation to a History of Myocardial Infarction in Patients Characterized by Coronary Angiography

To investigate the relationship between an insertion/deletion (4G/5G) polymorphism in the promoter region of the plasminogen activator inhibitor-1 (PAI-1) gene and the phenotypes of PAI-1 levels, coronary atheroma, and a past history of coronary thrombosis, we studied 453 patients (320 men and 133 women) characterized by coronary angiography. Patients were classified as having normal vessels (n = 125) or single-vessel (n = 92) or multivessel (n = 232) coronary disease on the basis of > or = 50% stenosis. PAI-1 antigen levels were highest in patients with the 4G/4G genotype (22.5 ng/mL), with a stepwise decrease in levels as the number of 4G alleles decreased (21.5 ng/mL for 4G/5G and 15.8 ng/mL for 5G/5G, P = .02) after adjusting for age, sex, triglyceride levels, and body mass index (BMI). The association between triglyceride level and PAI-1 was genotype specific, with a steeper slope in subjects with the 4G/4G genotype (P = .004). A gene-environment interaction between BMI, PAI-1, and genotype was observed, with a steeper association in patients with the 5G/5G genotype (P = .02). The 4G/4G genotype was significantly associated with a history of myocardial infarction (P < .03; odds ratio, 2.0; 95% CI, 1.1 to 3.7). This relationship was stronger in subjects with diseased vessels (P = .006). There was no relationship between either PAI-1 genotype or levels and the presence of atheroma. Our data suggest that PAI-1 promoter polymorphism influences the development of myocardial infarction through its effect on thrombus formation in patients with preexisting coronary atheroma.