The nitrite/collagen reaction: non-enzymatic nitration as a model system for age-related damage.

The effects of age seen in long-lived connective tissue proteins are thought to be the result of post-translational modifications by reactive molecules. One such molecule is the nitrite ion. Human nitrite exposure results predominately from endogenous production of nitric oxide as well as inhalation of cigarette smoke and ingestion of cured meats. Although nitrite reactions with various proteins have been studied previously with regard to carcinogenesis, the specific reaction with collagen and its role in age-related damage has never been examined. We describe the reaction of nitrite with type I collagen at neutral pH and body temperature. The incubation of collagen with nitrite results in an increase in cross-linking, the accumulation of a yellow chromophore, and a depletion of tyrosine residues. Similar changes also are found in aged human collagen. In addition, 3-nitro-tyrosine, which has recently been used as a marker for peroxynitrite mediated damage, is produced from this reaction. Thus, we propose non-enzymatic nitration as an in vitro model system for human collagen age-related damage.

The Nitrite/alpha crystallin reaction: a possible mechanism in lens matrix damage.

Recent work has explored the potential deleterious role that nitric oxide (NO) and its derivatives may have in human disease. The many by-products of NO include nitrite ion, which accumulates in the anterior chamber during ocular inflammation and can be derived from cigarette smoking. Cigarette smoking has been strongly linked to nuclear cataract formation, although the mechanism remains unknown. We have previously observed that nitrite reactions with the matrix proteins elastin and collagen produce damaging effects that mimic those observed in age- and smoking-related illnesses. In the present study we report on the reaction of nitrite with alpha crystallin, the major lens matrix protein. Incubations at neutral pH and body temperature of nitrite with alpha crystallin resulted in protein modifications indicative of oxidative damage and similar to changes seen in the aging lens as well as cataracts. These include increased fluorescence, yellowing and protein cross-linking. L-kynurenine, a tryptophan derivative, was identified as a reaction product. L-kynurenine was also formed from the reaction of nitrite with free tryptophan. Thus, this non-enzymatic nitration of alpha crystallin provides a novel mechanism by which lens proteins may be damaged in vivo. Since human exposure to nitrite is increased by cigarette smoking, this reaction could provide an explanation for the association between nuclear cataracts and smoking.

The nitrite/elastin reaction: implications for in vivo degenerative effects.

Nitrite ion is a by-product of nitrogen oxides (nitric oxide and nitrogen dioxide) from cigarette smoke and is used as a preservative for curing meats. Therefore, study of the reaction of nitrite with elastin in vitro was undertaken. By colorimetric assay, reactivity of nitrite with insoluble elastin at neutral pH, 37 degrees C, and physiologic concentration was confirmed. In histochemical studies on in situ human aortic elastin, nitrite-treated sections displayed marked structural disruptions. Determinations of fluorescence and absorbance on nitrite-treated soluble bovine elastin revealed marked alterations of fluorescence, and increased UV and visible absorbance. Amino acid analysis confirmed that it reacted with tyrosine. The findings indicate that non-enzymatic nitration by nitrite may have deleterious effects on elastin in vivo and may provide insights into the pathogenesis of chronic elastin degenerative processes, including aortic aneurysms, pulmonary emphysema, and premature skin wrinkling, all of which have been well known to have associations with cigarette smoking.

Complement activation and subclassification of tissue immunoglobulin G in the abdominal aortic aneurysm.

Features of autoimmunity in abdominal aortic aneurysm (AAA) have been described, including increases in IgG content. The present experiments were carried out to determine (1) whether the increases in IgG are subclass specific and (2) whether the IgG complex is associated with an increase in the isoforms of complement C3. Seven AAA, four athero-occlusive (AOD), and two normal (NL) aortic tissue extracts were evaluated for immunoreactive complement (C3) components, both by ELISA and by Western immunoblots (probed with a polyclonal goat anti-human C3). The extracts were also assayed for each of the four subclasses of IgG by ELISA (monoclonal mouse anti-human IgGs). Compared to the amounts of IgG by subclass in normal aorta, AAAs had increases of 193-fold in IgG1, 160-fold in IgG2, 389-fold in IgG3, and 627-fold in IgG4. Increases relative to AOD by subclass were smaller, but each subclass was statistically significantly elevated (P < 0.01) over NL or over AOD. There was a 125-fold increase in immunoreactive C3 by ELISA in AAA vs NL, and Western immunoblotting techniques revealed the presence of multiple C3 degradation products. Increases in IgG1, 2, and 3 may be responsible for activation of complement in AAA by the classical pathway. Since the complement system is one of the major effector pathways of inflammation, the presence of complement-fixing IgG subclasses along with increased C3 in the aneurysm wall may be an important mechanism promoting matrix proteolysis in AAA.

Incidence and pathogenesis of aneurysmal disease of the abdominal aorla.

Abdominal aortic aneurysms (AAAs) are a major cause of illness and death in the United States and abroad. Along with progress in the surgical management of this condition, numerous advances have been made in understanding the pathogenesis of AAAs. Since the time of Scarpa (1804), AAA disease has been associated with, and attributed to, atherosclerotic vessel changes. Excluding patients with Marfan's Syndrome and Ehlers-Danlos type IV diseases, virtually all human AAA specimens contain some degree of atherosclerosis. However, atherosclerotic changes are associated with diverse arteriopathies (ie, aorto-occlusive disease versus AAAs). Over the last 25 years, considerable research has been performed comparing aneurysmal, occlusive, and normal aortae. AAA disease is a unique process with pathogenic mechanisms that may operate independently of atherosclerosis. In this chapter we discuss the incidence of AAA as well as its pathogenesis concerning genetics, molecular biology, biochemistry, and immunology.