Identification of tropoelastin as a ligand for the 65-kD FK506-binding protein, FKBP65, in the secretory pathway.

The folding and trafficking of tropoelastin is thought to be mediated by intracellular chaperones, although the identity and role of any tropoelastin chaperone remain to be determined. To identify proteins that are associated with tropoelastin intracellularly, bifunctional chemical cross-linkers were used to covalently stabilize interactions between tropoelastin and associated proteins in the secretory pathway in intact fetal bovine auricular chondrocytes. Immunoprecipitation of tropoelastin from cell lysates after cross-linking and analysis by SDS-PAGE showed the presence of two proteins of approximately 74 kD (p74) and 78 kD (p78) that coimmunoprecipitated with tropoelastin. Microsequencing of peptide fragments from a cyanogen bromide digest of p78 identified this protein as BiP and sequence analysis identified p74 as the peptidyl-prolyl cis-trans isomerase, FKPB65. The appearance of BiP and FKBP65 in the immunoprecipitations could be enhanced by the addition of brefeldin A (BFA) and N-acetyl-leu-leu-norleucinal (ALLN) to the culture medium for the final 4 h of labeling. Tropoelastin accumulates in the fused ER/Golgi compartment in the presence of BFA if its degradation is inhibited by ALLN (Davis, E.C., and R.P. Mecham. 1996. J. Biol. Chem. 271:3787-3794). The use of BFA and other secretion-disrupting agents suggests that the association of tropoelastin with FKBP65 occurs in the ER. Results from this study provide the first identification of a ligand for an FKBP in the secretory pathway and suggest that the prolyl cis-trans isomerase activity of FKBP65 may be important for the proper folding of the proline-rich tropoelastin molecule before secretion.

Structure and expression of fibrillin-2, a novel microfibrillar component preferentially located in elastic matrices.

During the previous cloning of the fibrillin gene (FBN1), we isolated a partial cDNA coding for a fibrillin-like peptide and mapped the corresponding gene (FBN2) to human chromosome 5. (Lee, B., M. Godfrey, E. Vitale, H. Hori, M. G. Mattei, M. Sarfarazi, P. Tsipouras, F. Ramirez, and D. W. Hollister. 1991. Nature [Lond.]. 352:330-334). The study left, however, unresolved whether or not the FBN2 gene product is an extracellular component structurally related to fibrillin. Work presented in this report clarifies this important point. Determination of the entire primary structure of the FBN2 gene product demonstrated that this polypeptide is highly homologous to fibrillin. Immunoelectron microscopy localized both fibrillin proteins to elastin-associated extracellular microfibrils. Finally, immunohistochemistry revealed that the fibrillins co-distribute in elastic and non-elastic connective tissues of the developing embryo, with preferential accumulation of the FBN2 gene product in elastic fiber-rich matrices. These results support the original hypothesis that the fibrillins may have distinct but related functions in the formation and maintenance of extracellular microfibrils. Accordingly, we propose to classify the FBN1 and FBN2 gene products as a new family of extracellular proteins and to name its members fibrillin-1 and fibrillin-2, respectively.

Defective angiogenesis in mice lacking endoglin.

Endoglin is a transforming growth factor-beta (TGF-beta) binding protein expressed on the surface of endothelial cells. Loss-of-function mutations in the human endoglin gene ENG cause hereditary hemorrhagic telangiectasia (HHT1), a disease characterized by vascular malformations. Here it is shown that by gestational day 11.5, mice lacking endoglin die from defective vascular development. However, in contrast to mice lacking TGF-beta, vasculogenesis was unaffected. Loss of endoglin caused poor vascular smooth muscle development and arrested endothelial remodeling. These results demonstrate that endoglin is essential for angiogenesis and suggest a pathogenic mechanism for HHT1.

Differential expression of two tropoelastin genes in zebrafish.

Elastin is the extracellular matrix protein responsible for properties of extensibility and elastic recoil in large blood vessels, lung and skin of most vertebrates. Elastin is synthesized as a monomer, tropoelastin, but is rapidly transformed into its final polymeric form in the extracellular matrix. Until recently information on sequence and developmental expression of tropoelastins was limited to mammalian and avian species. We have recently identified and characterized two expressed tropoelastin genes in zebrafish. This was the first example of a species with multiple tropoelastin genes, raising the possibility of differential expression and function of these tropoelastins in elastic tissues of the zebrafish. Here we have investigated the temporal expression and tissue distribution of the two tropoelastin genes in developing and adult zebrafish. Expression was detected early in skeletal cartilage structures of the head, in the developing outflow tract of the heart, including the bulbus arteriosus and the ventral aorta, and in the wall of the swim bladder. While the temporal pattern of expression was similar for both genes, the upregulation of eln2 was much stronger than that of eln1. In general, both genes were expressed and their gene products deposited in most of the elastic tissues examined, with the notable exception of the bulbus arteriosus in which eln2 expression and its gene product was predominant. This finding may represent a sub-specialization of eln2 to provide the unique architecture of elastin and the specific mechanical properties required by this organ.

The role of vascular injury and hemodynamics in rat pulmonary artery remodeling.

Vascular remodeling in adult human elastic pulmonary arteries is characterized by diffuse neointimal lesions containing smooth muscle cells expressing extracellular matrix genes. Recent studies suggest vascular injury is needed to initiate remodeling and that growth factor mediators participate in the repair response. However, because neointimal formation is only observed in patients with pulmonary artery blood pressures approaching systemic levels, it has been hypothesized that systemic-like hemodynamic conditions are also necessary. To test that hypothesis, subclavian-pulmonary artery anastomoses were created in Sprague-Dawley rats under three different experimental conditions: no accompanying injury, or after monocrotaline or balloon endarterectomy injury. Pulmonary vascular remodeling was not induced by the subclavian-pulmonary artery anastomosis alone. A non-neointimal pattern of remodeling after mild monocrotaline-induced injury was converted into a neointimal pattern in the presence of the anastomosis. Neointima was also observed after severe, balloon endarterectomy-induced injury even in the absence of anastomosis. Tropoelastin, type I procollagen and TGF-beta gene expression, and angiotensin converting enzyme immunoreactivity, was confined to the neointima resembling the pattern of gene expression and immunoreactivity in human hypertensive elastic pulmonary artery neointimal lesions. These observations introduce the concepts that the type of injury and the associated hemodynamic conditions can modify the elastic pulmonary artery response to injury.

Novel arterial pathology in mice and humans hemizygous for elastin.

Obstructive vascular disease is an important health problem in the industrialized world. Through a series of molecular genetic studies, we demonstrated that loss-of-function mutations in one elastin allele cause an inherited obstructive arterial disease, supravalvular aortic stenosis (SVAS). To define the mechanism of elastin's effect, we generated mice hemizygous for the elastin gene (ELN +/-). Although ELN mRNA and protein were reduced by 50% in ELN +/- mice, arterial compliance at physiologic pressures was nearly normal. This discrepancy was explained by a paradoxical increase of 35% in the number of elastic lamellae and smooth muscle in ELN +/- arteries. Examination of humans with ELN hemizygosity revealed a 2. 5-fold increase in elastic lamellae and smooth muscle. Thus, ELN hemizygosity in mice and humans induces a compensatory increase in the number of rings of elastic lamellae and smooth muscle during arterial development. Humans are exquisitely sensitive to reduced ELN expression, developing profound arterial thickening and markedly increased risk of obstructive vascular disease.

Bovine latent transforming growth factor beta 1-binding protein 2: molecular cloning, identification of tissue isoforms, and immunolocalization to elastin-associated microfibrils.

Monoclonal antibodies to fibrillin 1 (MP340), a component of elastin-associated microfibrils, were used to screen cDNA libraries made from bovine nuchal ligament mRNA. One of the selected clones (cL9; 1.2 kb) hybridized on Northern (RNA) blotting with nuchal ligament mRNA to two abundant mRNAs of 9.0 and 7.5 kb, which were clearly distinct from fibrillin mRNA (10 kb). Further library screening and later reverse transcription PCR by the rapid amplification of cDNA ends (RACE) technique resulted in the isolation of additional overlapping cDNAs corresponding to about 6.7 kb of the mRNA. The encoded protein exhibited sequence similarity of around 80% with a recently identified human protein named latent transforming growth factor beta 1 (TGF-beta 1)-binding protein 2 (LTBP-2), indicating that the new protein was bovine LTBP-2. This was confirmed by the specific localization of bovine LTBP-2 cDNA probes to human chromosome 14q24.3, which is the locus of the human LTBP-2 gene. The domain structure of bovine LTBP-2 is very similar to that of the human LTBP-2, containing 20 examples of 6-cysteine epidermal growth factor-like repeats, 16 of which have the consensus sequence for calcium binding, together with 4 examples of 8-cysteine motifs characteristic of fibrillins and LTBP-1. A 4-cysteine sequence which is unique to bovine LTBP-2 and which has similarity to the 8-cysteine motifs was also present. Antibodies raised to two unique bovine LTBP-2 peptides specifically localized in tissue sections to the elastin-associated microfibrils, indicating that LTBP-2 is closely associated with these structures. Immunoblotting experiments identified putative LTBP-2 isoforms as a 260-kDa species released into the medium by cultured elastic tissue cells and as larger 290- and 310-kDa species in tissue extracts. A major proportion of tissue-derived LTBP-2 required treatment with 6 M guanidine for solubilization, indicating that the protein was strongly bound to the microfibrils. Most of the guanidine-solubilized LTBP-2 appeared to be monomeric, indicating that it was not involved in disulfide-bonded aggregation either with itself or with latent TGF-beta. Additional LTBP-2 was resistant to solubilization with 6 M guanidine but was readily extracted with a reductive saline solution. This treatment is relatively specific for solubilization of microfibrillar constituents including fibrillin 1 and microfibril-associated glycoprotein. Therefore, it can be inferred that some LTBP-2 is bound covalently to the microfibrils by reducible disulfide linkages. The evidence suggests that LTBP-2 has a direct role in elastic fiber structure and assembly which may be independent of its growth factor-binding properties. Thus, LTBP-2 appears to share functional characteristics with both LTBP-1 and fibrillins.

Developmental regulation of FKBP65. An ER-localized extracellular matrix binding-protein.

FKBP65 (65-kDa FK506-binding protein) is a member of the highly conserved family of intracellular receptors called immunophilins. All have the property of peptidyl-prolyl cis-trans isomerization, and most have been implicated in folding and trafficking events. In an earlier study, we identified that FKBP65 associates with the extracellular matrix protein tropoelastin during its transport through the cell. In the present study, we have carried out a detailed investigation of the subcellular localization of FKBP65 and its relationship to tropoelastin. Using subcellular fractionation, Triton X-114 phase separation, protease protection assays, and immunofluorescence microscopy (IF), we have identified that FKBP65 is contained within the lumen of the endoplasmic reticulum (ER). Subsequent IF studies colocalized FKBP65 with tropoelastin and showed that the two proteins dissociate before reaching the Golgi apparatus. Immunohistochemical localization of FKBP65 in developing lung showed strong staining of vascular and airway smooth muscle cells. Similar areas stained positive for the presence of elastic fibers in the extracellular matrix. The expression of FKBP65 was investigated during development as tropoelastin is not expressed in adult tissues. Tissue-specific expression of FKBP65 was observed in 12-d old mouse tissues; however, the pattern of expression of FKBP65 was not restricted to those tissues expressing tropoelastin. This suggests that additional ligands for FKBP65 likely exist within the ER. Remarkably, in the adult tissues examined, FKBP65 expression was absent or barely detectable. Taken together, these results support an ER-localized FKBP65-tropoelastin interaction that occurs specifically during growth and development of tissues.

Characterization of an in vitro model of elastic fiber assembly.

Elastic fibers consist of two morphologically distinct components: elastin and 10-nm fibrillin-containing microfibrils. During development, the microfibrils form bundles that appear to act as a scaffold for the deposition, orientation, and assembly of tropoelastin monomers into an insoluble elastic fiber. Although microfibrils can assemble independent of elastin, tropoelastin monomers do not assemble without the presence of microfibrils. In the present study, immortalized ciliary body pigmented epithelial (PE) cells were investigated for their potential to serve as a cell culture model for elastic fiber assembly. Northern analysis showed that the PE cells express microfibril proteins but do not express tropoelastin. Immunofluorescence staining and electron microscopy confirmed that the microfibril proteins produced by the PE cells assemble into intact microfibrils. When the PE cells were transfected with a mammalian expression vector containing a bovine tropoelastin cDNA, the cells were found to express and secrete tropoelastin. Immunofluorescence and electron microscopic examination of the transfected PE cells showed the presence of elastic fibers in the matrix. Biochemical analysis of this matrix showed the presence of cross-links that are unique to mature insoluble elastin. Together, these results indicate that the PE cells provide a unique, stable in vitro system in which to study elastic fiber assembly.

Intracellular trafficking of tropoelastin.

Elastin is secreted as soluble tropoelastin monomers which are then cross-linked in the presence of extracellular microfibrils to form insoluble elastic fibers. Although the secretion of tropoelastin is thought to be mediated and targeted by an intracellular chaperone complex, the intracellular route taken by this protein and the role of such a chaperone complex remain undefined. In the present study, the specific pathway of tropoelastin secretion was investigated in fetal bovine chondrocytes and ligamentum nuchae fibroblasts by immunofluorescence staining and immunoprecipitation of tropoelastin following treatment with secretion-disrupting agents. In untreated cells, tropoelastin is secreted in approximately 30 min. In both cell types, brefeldin A and monensin inhibited secretion of tropoelastin and caused an intracellular accumulation of the protein in the fused ER/Golgi compartment or in the Golgi stacks, respectively. Incubations of longer than 1 h in the presence of brefeldin A result in eventual degradation of tropoelastin in the ER/Golgi compartment (Davis and Mecham, 1996). In contrast, the tropoelastin trapped in the Golgi as a result of monensin treatment steadily accumulated. Agents that elevate intracellular pH, such as ammonium chloride and chloroquine, also caused an intracellular accumulation of tropoelastin which appeared by immunofluorescence staining to be localized in secretory vesicles and/or endosomes. Since weak bases and ionophores alter the morphology of vacuolar compartments, the effect of bafilomycin A1 on tropoelastin secretion was also investigated. This vacuolar H+-ATPase inhibitor prevents acidification of the trans-Golgi network and endosomal compartments without disrupting intracellular organelle formation. When the elastogenic cells were treated with bafilomycin A1, tropoelastin secretion was diminished and an intracellular accumulation of tropoelastin was detected in the trans-Golgi network and small secretory vesicles. These results suggest that tropoelastin may be diverted from the constitutive pathway after exiting the Golgi and instead targeted to an acidic compartment prior to transport to the cell surface. The identity and role of such a compartment in the sorting and/or trafficking of tropoelastin has yet to be determined.

Elastic lamina growth in the developing mouse aorta.

The growth and development of elastic laminae in the mouse aortic media were investigated by light and electron microscopic autoradiography after a single SC injection of L-[3,4-3H]-valine. Because of the remarkable stability of elastin, radiolabel incorporated into the elastic laminae during early stages of aortic development can be identified in the mature vessel. Light microscopic autoradiographs of aortae from mice injected with radiolabeled valine at 3, 14, or 21 days of postnatal age and sacrificed at 4 months of age showed silver grains evenly distributed around the circumference of the vessel, suggesting uniform elastic lamina growth. Electron microscopic autoradiographs of aortae from mice injected at 3 and 14 days' postnatal age and killed at 4 months of age showed the elastin initially deposited at 3 days to be in the center of the lamina, whereas the elastin deposited at 14 days remained peripherally located. These observations suggest that elastin deposited early in development does not undergo any significant redistribution during growth of the vessel. Because the aorta continues to increase in diameter after the elastic laminae are essentially complete, the fenestrations in the laminae were investigated as possible sites of further expansion of the laminae. In aortae from mice injected at 3 days and sacrificed at 4 days of postnatal age, the edges of the elastic lamina that border on fenestrations showed a large number of silver grains. Regions of the elastic lamina at some distance from the fenestration, however, appeared to be associated with fewer grains. Results from this study not only present unique observations of elastin deposition in developing elastic laminae but also provide evidence that the fenestrations in the elastic laminae may play a role in their continued expansion during later stages of aortic development.

The role of apoptosis in sexual differentiation of the rat sexually dimorphic nucleus of the preoptic area.

The sexually dimorphic nucleus of the preoptic area (SDN-POA) in the rat hypothalamus is larger in volume in males than in females due to a larger number of cells in the nucleus. Although the SDN-POA, and its development, have been extensively studied, the actual mechanism of its sexual differentiation has not been established. The results of previous studies have not supported a role for gonadal steroids in the regulation of neurogenesis or the determination of the migratory pathway perinatally. In this study, the role of cell death in the development of the sexual dimorphism in the SDN-POA was investigated using in situ end-labeling to visualize fragmented DNA in apoptotic cells. In the experiments described here, the incidence of apoptosis was determined in part of the SDN-POA, the central division of the medial preoptic nucleus (MPNc), over the first 13 days postnatally in male and female rats. There was a sex difference in the incidence of apoptosis in the MPNc between postnatal days 7 and 10; the incidence was higher in females. The role of testosterone (T) in regulating the incidence of apoptosis in the developing MPNc was examined in neonatally castrated males following T or vehicle injection. Testosterone had a profound inhibitory effect on the incidence of apoptosis between days 6 and 10. In a control region within the lateral preoptic area, there was no sex difference in the incidence of apoptosis, nor was there an effect of T. Thus, the data indicate that the regulation of apoptosis by T is one mechanism involved in the sexual differentiation of the SDN-POA.

Extant diversity of bryophytes emerged from successive post-Mesozoic diversification bursts.

Unraveling the macroevolutionary history of bryophytes, which arose soon after the origin of land plants but exhibit substantially lower species richness than the more recently derived angiosperms, has been challenged by the scarce fossil record. Here we demonstrate that overall estimates of net species diversification are approximately half those reported in ferns and ∼30% those described for angiosperms. Nevertheless, statistical rate analyses on time-calibrated large-scale phylogenies reveal that mosses and liverworts underwent bursts of diversification since the mid-Mesozoic. The diversification rates further increase in specific lineages towards the Cenozoic to reach, in the most recently derived lineages, values that are comparable to those reported in angiosperms. This suggests that low diversification rates do not fully account for current patterns of bryophyte species richness, and we hypothesize that, as in gymnosperms, the low extant bryophyte species richness also results from massive extinctions.

Mutations in fibrillin-1 cause congenital scleroderma: stiff skin syndrome.

The predisposition for scleroderma, defined as fibrosis and hardening of the skin, is poorly understood. We report that stiff skin syndrome (SSS), an autosomal dominant congenital form of scleroderma, is caused by mutations in the sole Arg-Gly-Asp sequence-encoding domain of fibrillin-1 that mediates integrin binding. Ordered polymers of fibrillin-1 (termed microfibrils) initiate elastic fiber assembly and bind to and regulate the activation of the profibrotic cytokine transforming growth factor-beta (TGFbeta). Altered cell-matrix interactions in SSS accompany excessive microfibrillar deposition, impaired elastogenesis, and increased TGFbeta concentration and signaling in the dermis. The observation of similar findings in systemic sclerosis, a more common acquired form of scleroderma, suggests broad pathogenic relevance.

The role of caveolin-1 in pulmonary matrix remodeling and mechanical properties.

Caveolin-1 (cav1) is a 22-kDa membrane protein essential to the formation of small invaginations in the plasma membrane, called caveolae. The cav1 gene is expressed primarily in adherent cells such as endothelial and smooth muscle cells and fibroblasts. Caveolae contain a variety of signaling receptors, and cav1 notably downregulates transforming growth factor (TGF)-beta signal transduction. In pulmonary pathologies such as interstitial fibrosis or emphysema, altered mechanical properties of the lungs are often associated with abnormal ECM deposition. In this study, we examined the physiological functions and the deposition of ECM in cav1(-/-) mice at various ages (1-12 mo). Cav1(-/-) mice lack caveolae and by 3 mo of age have significant reduced lung compliance and increased elastance and airway resistance. Pulmonary extravasation of fluid, as part of the cav1(-/-) mouse phenotype, probably contributed to the alteration of compliance, which was compounded by a progressive increase in deposition of collagen fibrils in airways and parenchyma. We also found that the increased elastance was caused by abundant elastic fiber deposition primarily around airways in cav1(-/-) mice at least 3 mo old. These observed changes in the ECM composition probably also contribute to the increased airway resistance. The higher deposition of collagen and elastic fibers was associated with increased tropoelastin and col1alpha2 and col3alpha1 gene expression in lung tissues, which correlated tightly with increased TGF-beta/Smad signal transduction. Our study illustrates that perturbation of cav1 function may contribute to several pulmonary pathologies as the result of the important role played by cav1, as part of the TGF-beta signaling pathway, in the regulation of the pulmonary ECM.

Stability of elastin in the developing mouse aorta: a quantitative radioautographic study.

Elastic lamina growth during development and the ultimate stability of elastin in the mouse aortic media was investigated by light and electron microscopic radioautography. Following a single subcutaneous injection of L-[3,4-3H]valine at 3 days of age, animals were killed at 9 subsequent time intervals up to 4 months of age. One day after injection, radioautographic silver grains were primarily observed over the elastic laminae; however, silver grains were also seen over the smooth muscle cells and extracellular matrix. By 21 to 28 days of age, the silver grains were almost exclusively located over the elastic laminae. From 28 days to 4 months of age, the distribution of silver grains appeared relatively unchanged. Quantitation of silver grain number/micron2 of elastin showed a steady decrease in the concentration of silver grains associated with the elastic laminae from 4 to 21 days of age. After this time, no significant difference in silver grain concentration was observed. Since the initial decrease in grains/micron2 of elastin corresponds to a period of rapid post-natal growth, the decrease is likely to be a result of dilution of the radiolabel due to new elastin synthesis. With the assumption that little or no significant turnover occurs during this time, a constant growth rate of 4.3% per day was predicted by linear regression analysis. Since no significant difference in the concentration of silver grains was observed from 28 to 118 days of age, no new growth or turnover of elastin can be said to occur during this time period. This is supported by the observation that animals injected with radiolabeled valine at 28 days and 8 months of age showed no significant incorporation of radiolabel into the elastic laminae. The results from this study present the first long-term radioautographic evidence of the stability of aortic elastin and emphasize that initial deposition of elastin and proper assembly of elastic laminae is a critical event in vessel development.

Immunolocalization of microfibril and microfibril-associated proteins in the subendothelial matrix of the developing mouse aorta.

In the developing aorta, endothelial cell connecting filaments extend from the abluminal surface of the endothelial cell to the subjacent elastic lamina. The connecting filaments are in alignment with intracellular stress fibers and are oriented parallel to the direction of blood flow. In the present study, the composition of the endothelial cell connecting filaments was investigated by indirect immunogold labeling with antibodies to the microfibril proteins, MP340 (fibrillin) and MAGP, and to fibronectin and heparan sulfate proteoglycan (HSPG). In the subendothelial matrix of both 15-day gestational and 5-day post-natal mouse aortae, the connecting filaments showed moderate immunoreactivity with anti-MP340; however, no significant immunoreaction was seen with anti-MAGP. Anti-fibronectin strongly labeled the connecting filaments and a weak immunoreaction was seen with anti-HSPG. In contrast, the adjacent 'elastin-associated microfibrils' showed a very strong immunoreaction with anti-MP340 and a moderate reaction with anti-MAGP. Little or no reaction was seen with anti-fibronectin or anti-HSPG. The filaments that connect endothelial cells to the subjacent elastic lamina during aortic development are thus microfibrillar in nature and related to elastin-associated microfibrils as evidenced by their positive immunoreaction with anti-MP340. The absence of labeling with anti-MAGP, however, suggests that either these fibrillin-containing filaments do not contain MAGP or that the immunoreactive epitopes are blocked by the proteins that coat the connecting filaments such as fibronectin. These results suggest that microfibrils not in association with elastin may play a role in cell anchorage and, more specifically, in the aorta may be involved in maintaining the structural integrity of the endothelial cell layer during early development of the vessel wall. Furthermore, the absence of immunoreactivity with anti-MAGP on the fibrillin-containing endothelial cell connecting filaments raises the possibility that microfibrils may consist of a family of related filaments rather than a single structural entity.

Smooth muscle cell to elastic lamina connections in developing mouse aorta. Role in aortic medial organization.

BACKGROUND: The structural and functional intigration of smooth muscle cells and elastic laminae in the aortic media is not well established. Detailed information concerning normal ultrastructural features of the aortic media will provide a better understanding of the medial changes that occur in vascular diseases such as hypertension and aortic aneurysms. EXPERIMENTAL DESIGN: The ultrastructural development and organization of connections between smooth muscle cells and elastic laminae in the mouse aortic media were studied by light and electron microscopy. RESULTS: Early in development, the smooth muscle cells become linked to the elastic laminae by bundles of microfibrils. These microfibrils become progressively infiltrated with elastin so as to form extensions of elastin from the elastic laminae in the adult media. Each elastin extension spans obliquely from the elastic lamina to the surface of the smooth muscle cell where it attaches in a region of membrane occupied by an intracellular membrane-associated dense plaque. On the cytoplasmic face of the plaque, a contractile filament bundle penetrates and anchors in an orientation similar to that of the extracellular elastin extension. The contractile filament bundle traverses the cell obliquely and anchors in a dense plaque on the opposite side of the cell that is in turn linked to the next elastic lamina by another elastin extension. The extracellular elastin extensions and the intracellular contractile filament bundles thus form a "contractile-elastic unit," a continuous line of structures that links adjacent elastic laminae. The oblique orientation of the contractile-elastic units reverses direction in successive smooth muscle cell layers in a herringbone-like pattern. Thus, tension transmitted to one elastic lamina by the smooth muscle cells on either side results in a uniform force exerted on the elastic lamina in one circumferential direction, that on the adjacent elastic laminae being in the opposite direction. CONCLUSIONS: Results from this study demonstrate the presence of smooth muscle cell to elastic lamina connections that form early in development as contractile-elastic units; basic units of aortic medial ultrastructure. The overall organization of the contractile-elastic units within the aortic media is proposed to provide a means for coordinating contractile and elastic tensions in response to mechanical stresses imposed on the vessel wall.

Endothelial cell connecting filaments anchor endothelial cells to the subjacent elastic lamina in the developing aortic intima of the mouse.

The ultrastructural association of endothelial cells with the subjacent elastic lamina was investigated in the developing mouse aorta by electron microscopy. In the 5-day postnatal aorta, extensive filament bundles extend along the subendothelial matrix connecting the endothelial cells to the underlying elastic lamina. The connecting filaments form lateral associations with the abluminal surface of the endothelial cells in regions of membrane occupied by membrane-associated dense plaques. On the intracellular face of each plaque, the termini of stress fibers penetrate and anchor to the cell membrane in alignment with the extracellular connecting filaments. Both the stress fibers and the connecting filaments are oriented parallel to the longitudinal axis of the vessel. High magnification electron micrographs of individual endothelial cell connecting filaments reveal features similar to those of elastin-associated microfibrils. Each connecting filament consists of a 9-10 nm linear core with an electron-lucent center and peripheral spike-like projections. From the filaments, small thread-like extensions span laterally, linking the filaments into a loose bundle and anchoring them to the endothelial cell membrane and the surface of the elastic lamina. The filaments also appear heavily coated with electron-dense material; often with some degree of periodicity along the filament length. During development, the number of endothelial cell connecting filaments decreases as the elastic lamina expands and the subendothelial matrix is reduced. In the aortic intima of mature mice, the elastic lamina is closely apposed to the abluminal surface of the endothelial cell and no connecting filaments are seen.(ABSTRACT TRUNCATED AT 250 WORDS)