Carl Franzblau

The Protein and Lipid Composition of Arterial Elastin and Its Relationship to Lipid Accumulation in the Atherosclerotic Plaque

Elastin preparations from intimal layers and the media of normal and atherosclerotic human aortae were analyzed for protein and lipid content. In atherosclerotic aortae, elastin from plaques was compared with elastin from adjacent normal appearing areas of the same aorta. Arterial elastin purified by alkaline extraction appeared to be a protein-lipid complex containing free and ester cholesterol, phospholipids, and triglycerides. The lipid component of normal arterial elastin was small (1-2%). With increasing severity of atherosclerosis, there was a progressive accumulation of lipid in intimal elastin from plaques, reaching a mean lipid content of 37% in severe plaques. The increase in the lipid content of plaque elastic preparations was mainly due to large increases in cholesterol, over 80% of which was cholesteryl ester. This deposition of cholesterol in plaque elastin accounted for 20-34% of the total cholesterol content of the plaque. The increased lipid deposition in plaque elastin was associated with alterations in the amino acid composition of plaque elastin. In elastin from plaque intima, the following polar amino acids were increased significantly: aspartic acid, threonine, serine, glutamic acid, lysine, histidine, and arginine; whereas, cross-linking amino acids: desmosine, isodesmosine, and lysinonorleucine were decreased significantly. The amino acid and lipid composition of elastin from normal appearing aortic areas was comparable to that of normal arterial elastin except for intimal elastin directly adjacent to and medial elastin directly below the most severe plaques.The data indicate that the focal lipid deposition in early atherosclerotic plaques is due to a large extent to lipid accumulations in altered elastin protein of localized intimal areas. Continued lipid deposition in altered elastin appears to contribute substantially to the progressive lipid accumulation in the plaque. The study suggests that elastin of intimal elastic membranes may play an important role in the pathogenesis and progression of atherosclerosis.

Identification of a new crosslinking amino acid in elastin

Abstract Recently, Partridge et al . (1963) described the occurrence in elastin of two hitherto unrecognized amino acids for which evidence has now accrued to show their origin from lysine residues in a “pre-elastin” (Partridge et al ., 1964; Miller et al ., 1964). These substances, called desmosine and isodesmosine, are in fact quaternary pyridinium compounds with four side-chain substituents that function in crosslinking of elastin chains (Thomas et al ., 1964). An examination of peptide fractions of elastin selected for enrichment with respect to the desmosines revealed the presence of an additional amino acid, called X 4′ also previously undescribed (Franzblau et al ., 1965). The present communication presents data to show that X 4 can be represented by the formula, N ϵ -(5-amino 5-carboxypentanyl)-lysine.

Anticardiolipin Antibodies and Risk of Ischemic Stroke and Transient Ischemic Attack The Framingham Cohort and Offspring Study

Background and Purpose— The role of anticardiolipin antibodies (aCLs) as novel risk factors for ischemic stroke and transient ischemic attacks (TIAs) has been a matter of debate. Prior cohort studies included only selected subjects, mostly men. We related serum concentrations of aCLs to incident first ischemic stroke/TIA among men and women in the Framingham Heart Study cohort and offspring. Methods— There were a total of 2712 women (mean age, 59.3 years) and 2262 men (mean age, 58.3 years) free of stroke/TIA at the time of their baseline examinations. An enzyme immunoassay was used to measure aCLs. Optical density of the sample serum compared with the reference serum was defined as the aCL screening ratio (aCL SR). Analyses were based on sex-specific aCL SR quartiles and individual ratios. Results— During the 11-year follow-up, 222 ischemic strokes/TIAs occurred. In multivariate analysis, after adjustment for age, prior cardiovascular disease, systolic blood pressure, diabetes, smoking, C-reactive protein, and total and high-density lipoprotein cholesterol levels, an aCL SR of >0.4 (78% of sample) was significantly associated with an increased risk of ischemic stroke/TIA for women (hazard ratio [HR], 2.6; 95% confidence interval [CI], 1.3 to 5.4; absolute risk, 3.2%, 95% CI, 2.2 to 4.3) but not in men (HR, 1.3; 95% CI, 0.7 to 2.4; absolute risk, 4.5%; 95% CI, 3.0 to 6.0). Similar results were obtained when the higher 3 aCL SR quartiles were compared with the lowest. Conclusions— Elevated serum concentrations of aCLs, independently of other cardiovascular risk factors, significantly predict the risk of future ischemic stroke and TIA in women but not in men.

The synthesis of connective tissue protein in smooth muscle cells

The synthesis of elastin by smooth muscle cells was clearly demonstrated by amino acid analyses and the presence of lysine-derived crosslinks. The values obtained were compatible with those found in amorphous elastin isolated from rabbit aortic tissue. Collagen synthesis by these same cells was monitored by the appearance of [14C] hydroxyproline when the cells were grown in the presence of [14C] proline. When the cells were pulsed with [14C] lysine, one could detect [14C] hydroxylysine and [14C] glucosylgalactosylhydroxylysine. Further evidence for the synthesis of elastin and collagen was the finding of radiolabelled epsilon-hydroxynorleucine and the reduced aldol condensate of two residues of allysine after reduction of [14C] lysine pulsed cells with NaBH4.

Alteration of the extracellular matrix of smooth muscle cells by ascorbate treatment.

The protein composition in the extracellular matrix of cultured neonatal rat aortic smooth muscle cells has been monitored over time in culture. The influence of ascorbate on insoluble elastin and collagen has been described. In the absence of ascorbate, the cells accumulate an insoluble elastin component which can account for as much as 50% of the total protein in the extracellular matrix. In the presence of ascorbate, the amount of insoluble collagen increases, while the insoluble elastin content is significantly less. When ascorbate conditions are varied at different times during the culture, the extracellular matrices are altered with respect to collagen and elastin ratios. The decrease in elastin accumulation in the presence of ascorbate may be explained by an overhydroxylation of tropoelastin. Approximately 1/3 of the prolyl residues in the soluble elastin fractions isolated from cultures grown in the presence of ascorbate are hydroxylated. Since the insoluble elastin accumulated in these cultures contain the unique lysine-derived cross-links in amounts comparable to aortic tissue, this culture system proves ideal for studying the influence of extracellular matrix elastin on cell growth and metabolism.

Elastin and Elastic Tissue

Elastic fibers have been shown to contain two proteins, insoluble elastin and the elastic fiber microfibril, a glycoprotein. The microfibril has been suggested to playa morphogenetic role in determining the presumptive shape and direction of the forming elastic fiber. The principal alteration seen in individuals with the disease Pseudoxanthoma Elasticum is in insoluble elastin which loses its amorphous appearance and affinity for anionic stains, and takes on a finely granular appearance and shows increased affinity for cationic stains. Normal elastic fiber microfibrils are sometimes associated with this material; although, in general, these structures are not present in the elastic fibers that are markedly altered in this disease. Recent work on the nature of the elastic fiber has demonstrated that unlike the fibrous protein collagen, the elastic fiber consists of two morphologic entities that represent two discreet proteins (1-3). In mature elastic fibers the bulk of the elastic fiber consists of the well known protein, elastin, which has been recognized by histologists for over 100 years due to its characteristic staining features, its morphology and its marked insolubility. In the electron microscope this material has, until recently, demonstrated an amorphous appearance in which the elastic fiber takes different shapes, depending upon where the fiber is located, and

Direct binding enzyme-linked immunosorbent assay (ELISA) for serum amyloid A (SAA)

A solid-phase, direct binding ELISA for serum amyloid A (SAA) proteins is described, in which noncovalent interactions of SAA with other plasma constituents are disrupted to permit direct coating of the wells of flexible polyvinyl chloride microtitration plates with an amount of SAA antigen proportional to its concentration in plasma. The wells are coated overnight at 60 degrees C with plasma diluted in 3 M potassium bromide and 0.1 M sodium bicarbonate. pH 9.6. The next day, any remaining sites on the wells are blocked by incubation for 1 h at ambient temperature with a 5% solution of dry milk solids and 0.05% Tween 20 in 0.02 M phosphate buffer, pH 7.4. The wells are rinsed and incubated for 90 min at 37 degrees C with polyclonal rabbit or rat anti-human SAA antiserum. Then, the wells are rinsed and incubated with goat anti-rabbit or rat IgG antiserum to which has been conjugated horseradish peroxidase. o-phenylenediamine and hydrogen peroxide substrates are added to the wells, color is allowed to develop, and sulfuric acid is added to stop the enzyme-catalyzed reaction. The amount of SAA coated to wells is quantified by absorbance at 490 nm. Four or more serial three-fold dilutions of plasma samples are assayed simultaneously on separate plates. Each plate contains a set of wells with identical concentrations of SAA standard protein diluted in decreasing concentrations of plasma proteins corresponding to the dilution of sample. The method can detect SAA concentrations in plasma samples ranging from 1 microgram/ml to greater than 1000 micrograms/ml. The method is suited to serial monitoring of SAA concentration in patients undergoing treatment for inflammatory conditions and to the quantitative analysis of large numbers of samples.

A new assay for collagenolytic activity

Abstract A new assay procedure for the determination of collagenolytic activity is presented. The substrate can be prepared by simple reduction of the purified acidsoluble rat tail tendon collagen with NaB3H4. Collagenase activity is determined by measurement of soluble tritiated collagen peptides released. It has proven to be a method with a high degree of sensitivity and reproducibility.

Aging effects on the elastin composition in the extracellular matrix of cultured rat aortic smooth muscle cells.

SummaryElastin accumulation in the extracellular matrix of cultured rat aortic smooth muscle cells was monitored as a function of age. The effect of the animal donor age and time in culture in single or consecutive passages on the cells’ ability to accumulate total protein as well as elastin was evaluated. Smooth muscle cells were obtained from animals ranging in age from 2 d to 36 mo. Protein accumulation by the cells based on DNA content was similar regardless of which of the above aging parameters was examined. Although there were significant amounts of elastin present in the extracellular matrix of those cells originating from the younger animals (2 d and 6 wk old), little or none was detected in cell cultures derived from the oldest animals. A soluble elastin-like fraction which was isolated from the cultures of the 2-d-old rats seemed to be lacking in the cultures of cells from the 36-mo-old animals. This observation may, in part, explain the absence of insoluble elastin in the matrix of some cultures obtained from older animals. The data strongly suggest that the age of the donor animal from which the cells originate has the greatest influence on in vitro elastin accumulation.

Catalytic activity of aortic lysyl oxidase in an insoluble enzyme-substrate complex

Abstract An insoluble form of lysyl oxidase is identified in the saline insoluble fraction of chick embryo aortae previously pulsed with [6- 3 H]lysinc. This enzyme is catalytically functional in this insoluble complex releasing tritium from the pellet and generating aminoadipic semialdehyde identifiable in the hot-alkali insoluble elastin of the aortic pellet after incubation for the expression of the activity of the bound enzyme. Thus, the enzyme may be insoluble because it is bound to its insoluble substrate. The enzyme as its insoluble complex is inhibited by β-aminopropionitrile, copper chelators, carbonyl reagents, and deoxygenation with nitrogen gas, consistent with the known properties of lysyl oxidase of other sources. This enzyme-substrate complex has a temperature optimum at approx. 52 °C and a pH optimum of approx. 8. The existence of such a tight, functional enzyme-substrate complex may be important in the maturation of elastic fibers in vivo. The present data also indicate that the tritiated aortic pellet, normally used as a substrate for assay of exogenous sources of lysyl oxidase, must first be heat-or acid-treated to inactivate the endogenous background enzyme activity to increase the sensitivity of such assays.