Susan B. Deak

Collagen and elastin metabolism in hypertensive pulmonary arteries of rats

We evaluated the processes controlling the accumulation of collagen and elastin in main pulmonary arteries of rats during an episode of hypoxic pulmonary hypertension. Explant cultures of main pulmonary arteries were incubated with [3H]proline to measure collagen and protein synthesis and percent collagen synthesis. Elastin synthesis was measured by [14C]valine incorporation into insoluble elastin. Relative collagen synthesis increased twofold (from 1.1 +/- 0.2 x 10(3) to 2.0 +/- 1.0 x 10(3) disintegrations per minute [14C]hydroxyproline/vessel/hr/mg protein), relative collagen synthesis doubled (from 2% to 4-5% of total protein synthesis), and elastin synthesis increased ninefold (from 0.4 +/- 0.2 x 10(4) to 3.6 +/- 0.6 x 10(4) dpm [14C]valine/vessel/hr/mg protein) in early hypertension. The level of pro alpha l(I) collagen RNA paralleled the relative collagen synthetic rate during the study period. Within 7 days of recovery from hypoxia, collagen and elastin contents were normal. We conclude that collagen and elastin in main pulmonary arteries are synthesized rapidly during an episode of hypoxic pulmonary hypertension and that collagen and elastin are rapidly removed from the hypertensive vessel during normoxic recovery.

The complete derived amino acid sequence of human lysyl oxidase and assignment of the gene to chromosome 5 (extensive sequence homology with the murine ras recision gene).

Lysyl oxidase catalyzes the oxidation of lysine residues to alpha-aminoadipic-delta-semialdehyde. This is the first step in the covalent cross-linking of collagen and tropoelastin and results in the formation of insoluble collagen and elastic fibers in the extracellular matrix. We have characterized the complete nucleotide sequence of human lysyl oxidase (EC 1.4.3.13) and compared the derived amino acid sequence (417-amino acids) to rat lysyl oxidase and the mouse ras recision gene (rrg). 88% of amino acids and 83% of nucleotides were conserved between human and rat lysyl oxidase. The mouse ras recision gene demonstrated 89% conservation of amino acids with human lysyl oxidase. The sequence conservation was not evenly distributed along the molecule. The carboxy terminus of the protein, which contains the putative copper binding sites and is likely to be the catalytically active domain, was more highly conserved than the amino terminus. The 89% amino acid sequence similarity between the murine ras recision gene and human lysyl oxidase suggests that they are the same gene product. Therefore, in addition to cross linking of extracellular matrix proteins, lysyl oxidase may have a direct role in tumor suppression. Northern blot analysis of poly A+RNA from cultured skin fibroblasts revealed at least three-distinct transcripts, sized 4.8 kb, 3.8 kb and 2.0 kb. In addition, using a panel of human mouse cell hybrids, the lysyl oxidase gene was assigned to human chromosome 5.

MAMMALIAN TROPOELASTIN : MULTIPLE DOMAINS OF THE PROTEIN DEFINE AN EVOLUTIONARILY DIVERGENT AMINO ACID SEQUENCE

We have recently derived the complete amino acid sequence of rat tropoelastin from a series of overlapping cDNA clones. Comparison of this protein sequence to bovine and human tropoelastin has revealed significant differences in the rates of evolutionary divergence of the various domains of tropoelastin. The overall rate of divergence of the hydrophobic domains of tropoelastin was twice as fast as the cross-link domains of the protein. Certain hydrophobic domains, however, are as conserved as cross-link regions, particularly the hydrophobic sequence coded for by exon 33, the only exon subject to alternate usage in all three mammalian species and the most conserved domain in rat, bovine and human tropoelastin. This conservation of sequence strongly suggests a more complex function of the hydrophobic region encoded by exon 33, beyond the elastic recoil characteristic of all hydrophobic domains of tropoelastin. A comparison of average rates of divergence of hydrophobic and cross-link domains of tropoelastin to functionally-defined domains of other structural proteins, such as collagen, has also revealed that overall, tropoelastin is a highly divergent amino acid sequence, comparable to proteins such as globin and the fibrino-peptides.

Altered Helical Structure of a Homotrimer of α1(I)Chains Synthesized by Fibroblasts from a Variant of Osteogenesis Imperfecta

Cultured skin fibroblasts from a variant of osteogenesis imperfecta were previously shown to synthesize a type I procollagen which was a homotrimer of pro alpha 1(I) chains. Trimers of alpha 1(I) collagen were isolated by pepsin digestion of culture medium from these fibroblasts. The amino acid composition of the isolated protein indicated that it contained an increased amount of hydroxylysine, apparently because of post-translational over-modification. The thermal stability of the alpha 1(I) trimers was examined by circular dichroism. We found no consistent difference in the melting curve of the alpha 1(I) trimers compared to control type I collagen. We next examined the thermal stability of the alpha 1(I) trimers using digestion with a combination of trypsin and alpha-chymotrypsin as an alternative probe of helical stability. When enzymatic digestions were carried out at 36 degrees to 40 degrees C, the alpha 1(I) chains in the trimers were cleaved to polypeptides which were shortened by approximately 100 amino acids. Vertebrate collagenase digestion of the shortened molecules indicated that the 100 amino acid segment removed from each alpha 1(I) chain was located at the carboxyl-terminus. The decreased thermal stability of the alpha 1(I) trimers was probably explained by the absence of alpha 2(I) chains in the molecules. The results, however, did not exclude the possibility that the post-translational over-modification of the alpha 1(I) chains contributed to the altered helical structure.

Abnormalities in the Biosynthesis of Type III Procollagen in Cultured Skin Fibroblasts from Two Patients with Multiple Aneurysms

We examined the synthesis of collagenous proteins by cultured skin fibroblasts taken from 14 patients with an abdominal aortic aneurysm and either an aneurysm at a second site (8 patients) or a first order relative with an abdominal aortic aneurysm (6 patients). Fibroblasts were labeled with [3H] proline and, following pepsin digestion of media proteins, the ratio of type I/III collagen was examined by denaturing polyacrylamide gel electrophoresis (SDS-PAGE). With the exception of two patients, the ratio of type I/III collagen in the media of fibroblasts from aneurysm patients was similar to control values (6 controls). In two of the patients, the type I/III collagen ratio was greater than 3 standard deviations from the mean of both control ratios and those of other aneurysm patients. mRNA levels coding for type III procollagen, however, were normal in both patients. Patient #1 (ME) showed reduced type III procollagen on SDS-PAGE analysis of intracellular proteins. Intracellular and media type III procollagen levels were normal in patient #2 (HR), but media type III collagen was reduced by over 50% after digestion with a combination of trypsin and alpha-chymotrypsin for 5 minutes at 36 degrees C. Control type III collagen was only reduced after digestion at 39 degrees C. These data suggest an altered thermal stability of the type III collagen trimer synthesized by this patient, probably due to a mutation in the amino acid sequence. The data presented in this paper suggest that some forms of common abdominal aortic aneurysms may be caused by mutations in the gene coding for type III procollagen.

Alternative Splicing of Rat Tropoelastin mRNA is Tissue-Specific and Developmentally Regulated

Sequence analysis of cDNA clones coding for rat tropoelastin previously has identified two variants that potentially corresponded to alternatively spliced tropoelastin mRNAs (Pierce et al., 1990). We have now used S1 nuclease protection analysis of total RNA from aorta, skin and lungs of 10-day and 6-week old rats to localize all sites of alternative splicing in the tropoelastin mRNA and to examine tissue-specific and developmental regulation of the use of these sites. This analysis revealed multiple sites of alternative splicing involving rat tropoelastin coding sequences corresponding to exons 12 through 15 of the bovine tropoelastin gene and a single site of alternative splicing at sequences corresponding to exon 33. Messenger RNAs from all three tissues at both developmental stages were alternatively spliced at the same sites; there was no evidence for the use of an alternative splice site unique to a particular tissue or developmental stage. However, both tissue-specific and developmentally regulated differences were apparent in the proportion of rat tropoelastin mRNA alternatively spliced at exon 33. Tropoelastin mRNA from the aorta and lungs of neonatal rats was alternatively spliced at exon 33 ten time more frequently than tropoelastin mRNA from skin. Between 10 days and 6 weeks of development, the use of this site of alternative splicing decreased by twenty-fold in RNA from skin, ten-fold in RNA from lungs and two-fold in RNA from aorta. In contrast, alternative splicing at exons 12 through 15 occurred in a small percentage of the mRNA and use of these sites exhibited minimal tissue-specific differences or developmental regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple defects in type III collagen synthesis are associated with the pathogenesis of abdominal aortic aneurysms.

Confluent skin fibroblast cultures were prepared from 40 patients diagnosed with and surgically treated for an abdominal aortic aneurysm. An analysis of secreted type I and type III collagen in the media of these fibroblast preparations revealed reduced secretion of type III collagen from six patients. DNA sequence analysis of the entire coding domain of the pro alpha 1 (III) collagen mRNA in skin fibroblast RNA from these six patients revealed a C to T substitution at nucleotide 607 in one of the probands that would result in the replacement of a leucine residue with phenylalanine in the second position of the first tripeptide repeat in the triple-helical domain of type III collagen. Allele-specific hybridization analysis of genomic DNA from this proband and family members indicated that this non-glycine substitution probably contributed to the aneurysmal phenotype in this patient. No coding sequence mutations were found in the other five patients. It is clear from this study, therefore, that aberrant synthesis of type III collagen, as a consequence of both a coding sequence mutation and other factors contributing to reduced secretion of type III procollagen, will result in the development of an aortic aneurysm in a significant percentage of patients with this disease.

Increased Elastin mRNA Levels Associated with Surgically Induced Intimal Injury

Quantitative levels of mRNAs coding for elastin, types I and III procollagen and gamma-actin were measured in porcine vascular material following balloon catheterization. A balloon catheter was introduced into the thoracic aorta and jugular vein of 3-6 week old pigs; following distention and six days of postoperative recovery, tissue samples were obtained for histopathology, electron microscopy, RNA extraction and mRNA quantitation. Using a series of mammalian cDNA clones and the procedure of slot blot hybridization, we have shown that elastin and types I and III procollagen mRNA levels rose significantly during the postoperative period following vascular distention. The increase correlated with an increase in the cell mass present in both the venous and arterial intimal layers. Changes in gamma-actin mRNA levels were also associated with this rapid proliferative response but in arterial tissue only.

The single copy gene coding for human α1 (IV) procollagen is located at the terminal end of the long arm of chromosome 13

SummaryUsing dual-laser sorted chromosomes and spot-blot analysis, we have previously assigned genomic DNA sequences coding for human α1(IV) procollagen to chromosome 13 (Pihlajaniemi et al. 1985). By in situ hybridization to normal chromosomes and chromosomes with 13q deletions, we now report the localization of this gene to the terminal end of the long arm of chromosome 13. In addition, Southern and slot blot hybridization analysis clearly show that these genomic sequences are present only once per haploid genome.

Desmoplasia in benign and malignant breast disease is characterized by alterations in level of mRNAs coding for types I and III procollagen

Desmoplasia, the formation of excessive connective tissue surrounding some neoplasms, is a well documented, but incompletely understood phenomenon. To characterize the fibrotic response in benign breast conditions and malignancy, we examined the steady state levels of mRNA coding for types I and III procollagen in mild fibrocystic changes, in fibroadenoma, and in infiltrating ductal carcinoma. The results indicate that in mild fibrocystic change there is a relative increase in type III procollagen mRNA. In contrast, fibroadenoma and carcinoma are characterized by increased levels of type I procollagen mRNA.