ABSTRACT
Degradation of extracellular matrix, especially elastin, within the aortic wall is a hallmark of abdominal aortic aneurysms (AAAs). Normal turnover of matrix proteins is mediated by a family of enzymes called matrix metalloproteinases (MMPs). MMP activity is regulated by proteins called tissue inhibitors of metalloproteinases (TIMPs). We analyzed the expression of all known MMPs with established elastolytic activity and TIMPs in human AAA and control tissue. mRNA coding for MMP-9, MMP-2, human macrophage metalloelastase, MMP-7, TIMP-1, and TIMP-2 were amplified by reverse transcriptase-PCR in control and AAA tissue. A Northern blot assay was used to measure the levels of mRNA coding for MMP-2, MMP-9, TIMP-1, and TIMP-2. Control aortic tissue was obtained from patients with occlusive disease and from organ donors. The expression of MMP-7 and human macrophage metalloelastase was not detected in any aortic specimens. By Northern blot analysis the mean level of MMP-2 mRNA was not significantly different between control groups and AAAs (normalized values: occlusive, 1.5 +/- 0.8, n = 3; donor, 4.5 +/- 2.2, n = 6; AAA, 4.0 +/- 0.95, n = 15). There was a significant increase in the level of MMP-9 mRNA in AAA specimens (occlusive, 16.8 +/- 3, n = 3; donor, 5.7 +/- 1.2, n = 6; AAA, 56.7 +/- 11, n = 15, p = 0.0069). The levels of mRNA coding for TIMP-1 were not significantly different. There was a small but statistically significant increase in TIMP-2 mRNA in AAA tissue. These data support the hypothesis that increased activity of MMP-9, but not MMP-2, is an important factor in the etiology of AAAs. This enhanced MMP-9 activity could then result in degradation of the ECM, leading to aneurysmal dilatation.
Subject(s)
Aortic Aneurysm, Abdominal/genetics , Collagenases/genetics , Metalloendopeptidases/genetics , Tissue Inhibitor of Metalloproteinases/genetics , Aorta, Abdominal/enzymology , Aortic Aneurysm, Abdominal/enzymology , Blotting, Northern , Extracellular Matrix/enzymology , Gelatinases/genetics , Gene Expression Regulation, Enzymologic/physiology , Humans , Matrix Metalloproteinase 2 , Matrix Metalloproteinase 9 , Polymerase Chain Reaction , RNA, Messenger/geneticsABSTRACT
Glypicans are a member of a family of glycosylphosphatidylinositol anchored heparan sulfate proteoglycans that are expressed in cell and development specific patterns. Rat GPC1 cDNA probes were used to screen rat genomic libraries. Three overlapping genomic clones that contained the entire rat GPC1 gene were isolated. The rat GPC1 gene is approximately 15kb in length and consists of eight exons interrupted by introns of varying lengths. Two of the introns are quite short, with lengths of 41 and 43 base pairs. Each exon-intron splice junction exhibited the consensus splice site sequence. Exon 1 encodes the putative signal peptide and the serine residue of the first putative heparan sulfate attachment site. The last exon encodes the cluster of three potential COOH-terminal heparan sulfate attachment sites, the putative GPI anchor and polypeptide cleavage site, and the 3'-untranslated region including the polyadenylation signal. One of the genomic clones extended approximately 2.8 kb 5' of the exon 1 coding sequence, and is thus likely to contain sequences that regulate GPC1 gene expression. Sequence analysis of the 5'-flanking sequence revealed a lack of consensus TATA and CAAT boxes. A search for potential transcription factor binding sites revealed a number of such motifs, including Sp1 (GC box), NF-kappaB, and MyoD (E-box). This region of the rat GPC1 gene shows significant sequence homology to the 5'-flanking region of the human GPC3 gene. Functional promoter activity of the rat GPC1 sequence was demonstrated by its ability to drive the expression of a luciferase reporter gene in several cell types.
Subject(s)
Heparan Sulfate Proteoglycans/genetics , Promoter Regions, Genetic/genetics , Animals , Base Sequence , Cells, Cultured , Cloning, Molecular , DNA, Complementary/analysis , Exons , Fibroblasts , Genes , Heparan Sulfate Proteoglycans/chemistry , Introns , Molecular Sequence Data , Rats , Regulatory Sequences, Nucleic Acid , Schwann CellsABSTRACT
The expression of cell surface heparan sulfate proteoglycans in rat heart was investigated by Northern blot analysis with specific cDNA probes. In adult heart syndecan-3 and glypican mRNAs were abundantly expressed. Lower levels of syndecan-2 mRNA and very low levels of syndecan-1 mRNA were also detected. Analysis of RNA isolated from hearts of rats of various ages revealed that syndecan-3 and glypican mRNAs levels increased dramatically at birth, and continued to be expressed at high levels in adult animals. To determine which of these proteoglycans was expressed in cardiomyocytes, primary cultures of cardiomyocytes and nonmyocytes isolated from neonatal rat hearts were analyzed for proteoglycan expression. Glypican mRNA was localized almost exclusively to cardiomyocytes. Syndecan-3 mRNA was not detected in myocytes, but was detected in the nonmyocyte cells. Biochemical characterization of cardiomyocyte glypican revealed that it was a phosphatidylinositol-anchored heparan sulfate proteoglycan. Results of immunofluorescent staining of rat hearts with anti-glypican antibodies were consistent with the Northern blot data, and localized glypican to the lateral regions of myocyte plasma membrane that contact the basement membrane, as well as sites of myocyte adhesion junctions. At the latter site glypican colocalized with vinculin. Visualization of basic fibroblast growth factor binding sites by means of a tissue slice overlay assay also revealed colocalization with glypican. These results demonstrate developmental and cell-type-specific expression of membrane heparan sulfate proteoglycans in the heart. They also show that glypican is a major heparan sulfate proteoglycan expressed on the cardiomyocyte plasma membrane.
