K. Hamdi

Characterization of an endogenous metalloproteinase in human vitreous

The vitreous is a gel-like connective tissue that undergoes liquefaction during aging and pathological processes. We isolated and characterized a degradative enzyme from the vitreous of different species and identified it to be matrix metalloproteinase-2 (MMP-2). This enzyme was found in a latent form and may be associated with endogenous inhibitors. Vitreous isolated from both non-diabetic and diabetic patients contained MMP-2 in the same concentrations. However, the diabetic samples had an additional gelatinase activity at 92 kDa which may be associated with a compromised vasculature. These results suggest that the normal human vitreous contains an endogenous MMP and the appearance of an additional activity is associated with pathologic conditions.

Alu DNA polymorphism in ACE gene is protective for age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. We report an association between an Alu polymorphism in the angiotensin-converting enzyme (ACE) gene with the dry/atrophic form of AMD. Using the polymerase chain reaction (PCR) on genomic DNA isolated from patients with AMD (n=173), and an age-matched control population (n=189), we amplified a region polymorphic for an Alu element insertion in the ACE gene. The Alu(+/+) genotype occurred 4.5 times more frequently in the control population than the dry/atrophic AMD patient population, (p=0.004). The predominance of the Alu(+/+) genotype within the unaffected control group represents a protective insertion with respect to the human ocular disease, dry/atrophic AMD. This is the first demonstration of an Alu element insertion exerting protective effects against a known human disease.

Cleavage of structural components of mammalian vitreous by endogenous matrix metalloproteinase-2

Our goal was to determine if the major endogenous vitreous matrix metalloproteinase (MMP-2) could digest known collagenous components of the vitreous body. Matrix metalloproteinase-2 and its associated inhibitors were isolated from porcine vitreous by affinity column chromatography. The inhibitors were inactivated by chemical modification with dithiothreitol and iodoacetamide. The latent MMP-2 was then activated with the organomercurial, p-aminophenyl mercuric acetate (APMA). Bovine vitreous fibrillar collagens (types II, V/XI and IX) were isolated by pepsin extraction and differential salt precipitation. Intact type IX collagen was purified by selective salt precipitation followed by ion exchange and size exclusion chromatography. These isolated collagens were incubated for 6 to 24 h with different concentrations of activated MMP-2, and the extent of collagen degradation was analyzed. Activated MMP-2 was also introduced into freshly isolated vitreous gels and the degree of liquefaction was determined. Our results showed that the activated MMP-2 has no apparent effect upon type II collagen but can degrade type V/XI collagen and type IX collagen fragments (COL2 and COL2 + COL3). In addition, when the type IX collagen was in the intact helical form, MMP-2 appeared to selectively digest alpha 3 (IX) chains. This suggested that vitreous MMP-2 preferentially cleaved certain vitreous collagen chains into large fragments rather than small peptides. MMP-2 also disrupted the vitreous gel in vitro, releasing proteins but not hexuronic acid or sulfated glycosaminoglycans into the liquefied supernatant. We conclude that MMP-2 activity should be considered as a potential mechanism of vitreous liquefaction that is seen in aging and various pathological states.

Demystifying the ACE polymorphism: from genetics to biology.

The angiotensin converting enzyme (ACE) I/D polymorphism has been one of the most studied genetic systems. It comprises hundreds of reports and a myriad of disease associations, including cardiovascular, metabolic, immune, cancer, aging, neurodegenerative and psychiatric diseases. Despite the wealth of information on the ACE polymorphism and the well-known functions of ACE, several questions arise. Why does the ACE polymorphism associate with so many diseases? What is its function? In this review, we summarize the current information on the ACE polymorphism and explain its function in the context of cell survival. We also provide a model to understand its role in biology and disease at the organism and population levels.

Proteinase activity in normal human tears: Male-female dimorphism

Recent studies using radial caseinolysis suggested that preoperative plasmin and plasminogen-activators were predictive indicators of surgical outcomes. In this study, zymography was used to determine if other caseinolytic proteases were present in normal human tears. Tear samples were collected and proteins extracted with a buffer of 2% sodium dodecyl sulfate (SDS), 100 mM Tris/HCl, pH 6.8. Tears were run on casein zymograms (4 mg/ml) which were incubated in 50 mM Tris/HCl, 5 mM CaCl2, 0.15 M NaCl and NaN3, (pH 7.5) at 37°C for 48 h. After staining and destaining, the caseinolytic activity was seen as cleared areas. Plasminogen activator activity was analyzed with zymograms of copolymerized casein and plasminogen incubated in phosphate buffer saline (pH 8.0) overnight at room temperature. Results showed that besides a 50 kDa plasminogen activator, human tears contained 3 other caseinolytic activities of molecular weights 90 kDa, 70 kDa and 30 kDa. These were serine proteases with optimal activities at pH...