Fibulin-2 and fibulin-5 cooperatively function to form the internal elastic lamina and protect from vascular injury.

OBJECTIVE: Recent findings on the role of fibulin-5 (Fbln5) have provided substantial progress in understanding the molecular mechanism of elastic fiber assembly in vitro. However, little is known about differential roles of fibulins in the elastogenesis of blood vessels. Here, we generated double knockout mice for Fbln5 and Fbln2 (termed DKO) and examined the role of fibulins-2 and -5 in development and injury response of the blood vessel wall. METHODS AND RESULTS: Fibulin-2 is distinctly located in the subendothelial matrix, whereas fibulin-5 is observed throughout the vessel wall. All of the elastic laminae, including the internal elastic lamina (IEL), were severely disorganized in DKO mice, which was not observed in single knockout mice for Fbln2 or Fbln5. Furthermore, DKO vessels displayed upregulation of vascular adhesion molecules, tissue factor expression, and thrombus formation with marked dilation and thinning of the vessel wall after carotid artery ligation-injury. CONCLUSIONS: Fibulin-2 and fibulin-5 cooperatively function to form the IEL during postnatal development by directing the assembly of elastic fibers, and are responsible for maintenance of the adult vessel wall after injury. The DKO mouse will serve as a unique animal model to test the effect of vessel integrity during various pathological insults.

Molecular adaptation to an extreme environment: origin of the thermal stability of the pompeii worm collagen.

The annelid Alvinella pompejana is probably the most heat-tolerant metazoan organism known. Previous results have shown that the level of thermal stability of its interstitial collagen is significantly greater than that of coastal annelids and of vent organisms, such as the vestimentiferan Riftia pachyptila, living in colder parts of the deep-sea hydrothermal environment. In order to investigate the molecular basis of this thermal behavior, we cloned and sequenced a large cDNA molecule coding the fibrillar collagen of Alvinella, including one half of the helical domain and the entire C-propeptide domain. For comparison, we also cloned the 3' part of the homologous cDNA from Riftia. Comparison of the corresponding helical domains of these two species, together with that of the previously sequenced domain of the coastal lugworm Arenicola marina, showed that the increase in proline content and in the number of stabilizing triplets correlate with the outstanding thermostability of the interstitial collagen of A. pompejana. Phylogenetic analysis showed that triple helical and the C-propeptide parts of the same collagen molecule evolve at different rates, in favor of an adaptive mechanism at the molecular level.

Mouse fibulin-2 gene. Complete exon-intron organization and promoter characterization.

Fibulin-2, an extracellular matrix protein containing tandem arrays of calcium-binding epidermal growth factor-like motifs, is present in the basement membrane and stroma of many tissues. Its expression pattern suggested an essential role in organogenesis, particularly in embryonic heart development. In this study, we cloned the extreme 5' end of the mouse fibulin-2 cDNA, isolated phage and cosmid clones encoding the entire gene, and functionally characterized the promoter. The gene was found to consist of 18 exons spanning 55 kb of DNA. The exon-intron organization reflected the modular structure of the protein. Exon 9 was subjected to alternative splicing. All splice junctions conformed to the GT/AG rule, except that GC instead of GT was found in the splice donor site of exon 4. The gene lacked TATA and CAAT boxes but contained an initiator element (Inr) and several consensus Sp1 binding sites surrounding the transcription start sites. By transient transfection of promoter deletion constructs, a 0.46-kb region containing the clustered Sp1 sites was found to confer a high promoter activity.

Cloning of an annelid fibrillar-collagen gene and phylogenetic analysis of vertebrate and invertebrate collagens.

Arenicola marina possesses cuticular and interstitial collagens, which are mostly synthesised by its epidermis. A cDNA library was constructed from the body wall. This annelid cDNA library was screened with a sea-urchin-collagen cDNA probe, and several overlapping clones were isolated. Nucleotide sequencing of these clones revealed an open reading frame of 2052 nucleotides. The translation product exhibits a triple helical domain of 138 Gly-Xaa-Yaa repeats followed by a 269-residue-long C-terminal non-collagenous domain (C-propeptide). The triple helical domain exhibits an imperfection that has been previously described in a peptide produced by cyanogen bromide digestion (CNBr peptide) of A. marina interstitial collagen. This imperfection occurs at the same place in the interstitial collagen of the vestimentiferan Riftia pachyptila. This identifies the clone as coding for the C-terminal part of a fibrillar collagen chain. It was called FAm1alpha, for fibrillar collagen 1alpha chain of A. marina. The non-collagenous domain possesses a structure similar to carboxy-terminal propeptides of fibrillar pro-alpha chains. Only six conserved cysteine residues are observed in A. marina compared with seven or eight in all other known C-propeptides. This provides information on the importance of disulfide bonds in C-propeptide interactions and in the collagen-assembly process. Phylogenetic studies indicate that the fibrillar collagen 1alpha chain of A. marina is homologous to the R. pachyptila interstitial collagen and that the FAm1alpha gene evolved independently from the other alpha-chain genes. Complementary analyses indicate that the vertebrate fibrillar collagen family is composed of two monophyletic subgroups with a specific position of the collagen type-V chains.

Immunological properties and tissue localization of two different collagen types in annelid and vestimentifera species.

Rabbit antisera against cuticle and interstitial collagens from shallow sea water and hydrothermal vent annelids (Arenicola marina, and the pompeii worm Alvinella pompejana) and the vestimentiferan tube worm Riftia pachyptila showed a clear distinction between the two types of collagens, a broad cross-reactivity among the worm collagens and no reactions with various mammalian collagens. The antibodies reacted with various epitopes found on both triple helical and unfolded collagens. The cuticle collagens were localized by immunofluorescence to the outer surface of the epidermis and in annelids additionally to the anterior part of the digestive tract. The interstitial collagen was detected underneath the epidermis and between distinct muscle layers. Both collagens were also detected in the anterior obturaculum, a tissue unique to vestimentifera. They were located either in the periphery of the tissue (cuticle collagen) or in the central part (interstitial collagen), which appeared to be a large extracellular matrix. Both collagens, however, showed a different supramolecular organization in the obturaculum when compared to the posterior body wall collagens. The identity of the interstitial collagens from the two locations was verified by biochemical analysis. These data demonstrate a very special and rigid matrix structure in the obturaculum, which may adapt it to specific physiological functions.