Publications

BACKGROUND

The Pl(A2) polymorphism of GPIIIa has been associated with unstable coronary syndromes in some studies, but the association has remained debated. None of the previous studies have focused on families at high risk. Risk factors tend to cluster within kindreds with high prevalence of premature coronary heart disease (CHD). Therefore, a heightened prevalence of the Pl(A2) polymorphism among siblings of patients with CHD would support the hypothesis that Pl(A2) is linked, directly or indirectly, to CHD.

OBJECTIVES

To measure the prevalence of the Pl(A2) polymorphism among siblings of patients with CHD before the age of 60 years and to seek an association between the Pl(A2) polymorphism and established atherosclerotic and thrombogenic risk factors.

METHODS

From January 1994 to April 1996, we genotyped 116 asymptomatic siblings (60 Caucasians, 56 Afro-Caribbeans) of patients with CHD manifestations before the age of 60 years for the Pl(A) polymorphism (also called HPA-1). A control cohort was used for comparison, consisting of individuals that were matched for race and geographic area but were free of CHD (n = 268, 168 Caucasians and 100 Afro-Caribbeans). In addition, we have characterized the sibling cohort for other atherogenic and thrombogenic risk factors.

RESULTS

The prevalence of Pl(A2)-positive individuals (Pl(A2)[+], Pl(A1/A2) heterozygotes plus Pl(A2/A2) homozygotes) in the sibling cohort was high: 41.4%. When analyzed separately, the prevalence of Pl(A2)(+) siblings was 53.3% among Caucasians and 28.6% among Afro-Caribbeans. There was no association between Pl(A2) and other established atherogenic or thrombogenic risk factors. Interestingly, the clustering of other risk factors was lesser among Pl(A2)(+) siblings than their Pl(A1) counterparts.

CONCLUSIONS

This study supports the hypothesis that the prevalence of Pl(A2)(+) individuals is high in kindreds with premature CHD. Hence, like the established risk factors that tend to cluster in families with premature CHD and contribute strongly to the accelerated atherosclerotic process affecting these individuals, the Pl(A2) polymorphism of GPIIIa may represent an inherited risk that promotes the thromboembolic complications of CHD. That these asymptomatic Pl(A2)(+) siblings had overall less established risk factors than their Pl(A1) counterparts might represent an explanation for why they remained asymptomatic despite their Pl(A2) positivity.

Experimental and clinical studies strongly suggest that gelatinase A plays a central role in the evolution of glomerular injury and sclerosis. The sequences of the 5' flanking regions of the human and rat gelatinase A genes do not share similarities with other members of the matrix metalloproteinase gene family and are regulated in a distinctive manner. The human and rat gelatinase A genes include regions of significant homology (r2 human; RE-1 rat), which have been shown to act as potent cis-activators of transcription. The rat RE-1 sequence interacts specifically with the developmentally regulated transcription factors AP2 and YB-1, resulting in a synergistic activation of gelatinase A transcription. Although the human r2 sequence specifically interacts with AP2 (Mol Cell Biol 10: 6524-6532, 1990), there is no clear evidence for the presence of a canonical YB-1 binding site (Y-box) within this sequence. This study demonstrates, despite the absence of a canonical Y-box sequence in the r2 element, that YB-1 and AP2 specifically interact with r2, yielding synergistic transactivation of the human gelatinase A gene. It is concluded that the r2 element is the conserved functional analog of the RE-1 element, and that interactions of AP2 and YB-1 govern human gelatinase A gene expression.

The alphav integrins are likely to be an important group of molecules for regulating astrocyte behaviour within the central nervous system. Together with their ligand vitronectin, they are expressed by astrocytes in vivo and are further upregulated during neurological disease. Here we have characterised the expression of alphav integrins on primary astrocytes from both rat and mouse, and shown that they express just two members, alphavbeta5 and alphavbeta8. By using RGD peptides and function-blocking antibodies against the beta1 integrins and alphavbeta5, we find that both alphavbeta5 and alphavbeta8 can act as functional receptors for vitronectin. However, while alphavbeta5 is largely responsible for astrocyte adhesion to vitronectin this integrin appears to play no role in migration on vitronectin, with alphavbeta8 playing the dominant role in promoting migration on this substrate. beta1 integrins are not involved in mediating interactions between astrocytes and vitronectin. These results were confirmed in experiments with astrocytes derived from mice in which the beta5 gene had been deleted by homologous recombination. beta5 null astrocytes attached to vitronectin at a reduced rate, but showed no defect in migration on vitronectin relative to wild-type astrocytes. These data provide the first evidence that alphavbeta8 regulates migration and show that astrocyte alphavbeta5 and alphavbeta8 integrins have distinct functions.

PURPOSE

To validate the use of polymerase chain reaction (PCR)-amplified full-length cDNA as a substitute for mRNA in nucleic acid array and gene expression analysis.

METHODS

Total RNA was isolated from age-matched normal autopsy corneas and pseudophakic bullous keratopathy (PBK) corneas. Full-length cDNA was generated and PCR amplified using the Smart cDNA synthesis technology. Southern blot analysis of this cDNA was compared with Northern blot analysis of the RNA. Amplified cDNA was used to probe a commercial gene array. By immunohistochemistry, the expression pattern of several adhesion molecules represented on the array was assessed.

RESULTS

The cDNA produced by the Smart cDNA system gave results very similar to those of northern blot analysis when examined for beta2-microglobulin, Rab geranylgeranyl transferase, and tenascin-C. This cDNA obtained from normal or PBK corneas was labeled and used to probe a 588 gene array (Clontech). Among other differences, beta6 integrin was detected only with the PBK probe, beta-catenin was markedly elevated in PBK, and beta4 integrin appeared to be reduced in PBK. Immunohistochemical patterns of these proteins were consistent with the hybridization signals on the gene array.

CONCLUSIONS

Smart cDNA synthesis and nucleic acid arrays were combined and validated for the first time to identify differential gene expression in normal and diseased corneas. These techniques require very little RNA such as that equivalent to a half of a single cornea, which is useful when the amount of tissue is limiting. Altered expression of adhesive proteins beta6 integrin and beta-catenin may be related to the formation of epithelial bullae and microcystic changes in PBK patients.