Role of newly synthesized fibronectin in vascular smooth muscle cell migration on matrix-metalloproteinase-degraded collagen.

The migration of vascular smooth muscle cells (VSMC) is known to be a key process in the development of a number of vascular lesions, although the precise mechanisms involved have still to be elucidated. In the present study, the production of endogenous fibronectins by VSMC migrating across intact and matrix-metalloproteinase-degraded collagen type I has been explored. Cellular fibronectin seems to play a role in the enhanced migration seen when VSMC are exposed to degraded collagen and platelet-derived growth factor-BB. VSMC were found to synthesize both exon IIIA-containing fibronectin (which predominated) and exon IIIB-containing fibronectin. When these cells were exposed to substrates consisting of recombinant exon IIIA- or exon IIIB-containing fibronectin, rates of migration were not elevated above those seen with undegraded collagen. Endogenous fibronectin production may thus be necessary, but not sufficient, for VSMC migration over degraded collagenous substrates.

Collagen degradation and platelet-derived growth factor stimulate the migration of vascular smooth muscle cells.

Cell migration is a key event in many biological processes and depends on signals from both extracellular matrix and soluble motogenic factors. During atherosclerotic plaque development, vascular smooth muscle cells migrate from the tunica media to the intima through a basement membrane and interstitial collagenous matrix and proliferate to form a neointima. Matrix metalloproteinases have previously been implicated in neointimal formation and in this study smooth muscle cell adhesion and migration on degraded collagen have been evaluated. Vascular smooth muscle cells adhered to native intact collagen type I and to its first degradation by-product, 3/4 fragment (generated by collagenase-3 cleavage), unwound at 35 degrees C to mimic physiological conditions. PDGF-BB pre-treatment induced a fourfold stimulation of smooth muscle cell motility on the collagen 3/4 fragment whereas no increase in smooth muscle cell motility on collagen type I was observed. Cell migration on collagen type I was mediated by alpha2 integrin, whereas PDGF-BB-stimulated migration on the 3/4 collagen fragment was dependent on alphavbeta3 integrin. alphavbeta3 integrin was organised in clusters concentrated at the leading and trailing edges of the cells and was only expressed when cells were exposed to the 3/4 collagen fragment. Tyrphostin A9, an inhibitor of PDGF receptor-beta tyrosine kinase activity, resulted in complete abolition of migration of PDGF-BB treated cells on collagen type I and 3/4 fragment. These results strongly support the hypothesis that the cellular migratory response to soluble motogens can be regulated by proteolytic modification of the extracellular matrix.

Real-time video-shot detection for scene surveillance applications.

In this paper, a surveillance system with automatic video-shot detection and indexing capabilities is presented. The proposed system aims at detecting the presence of abandoned objects in a guarded environment and at automatically performing online semantic video segmentation in order to facilitate the human operator's task of retrieving the cause of an alarm. The former task is performed by operating image segmentation based on temporal rank-order filtering, followed by classification in order to reduce false alarms. The latter task is performed by operating temporal video segmentation when an alarm is detected. In the clips of interest, the key frame is the one depicting a person leaving a dangerous object, and is determined on the basis of a feature indicating the movement around the dangerous region. Experimental results are reported in terms of static region detection, classification, clip and key-frame detection errors versus different levels of complexity of the guarded environment, in order to establish the performance that can be expected from the system in different situations.

Restoration of normal bone development by human homologue of collagen type II (COL2A1) gene in Col2a1 null mice.

Development of the vertebrate skeleton is a highly complex process in which collagen type II plays a vital role in the formation of long bones via endochondral ossification. Collagen type II, which is encoded by a single COL2A1/ Col2a1 gene, is the most abundant structural protein in the cartilage matrix, where it undergoes complex interactions with several other proteins. The sequence of mature collagen type II chains, each with about 1,100 amino acids, is conserved between different mammalian species. There are 37 amino acid positions that are different between mouse and human collagen type II. Previously, we have demonstrated that transgenic mice, in which Col2a1 gene is knocked out, exhibit a lethal phenotype due to the absence of endochondral bone formation. To investigate whether the biological role of collagen type II is conserved between the species, human COL2A1 gene was expressed in Col2a1 null mice by crossing with transgenic mice in which human COL2A1 gene was integrated. The collagen type II from human gene rescued the lethal phenotype in null mice, indicating that the biological function of collagen type II is conserved between human and mouse. The animals exhibited normal endochondral bone formation and a normal growth plate in tibio-tarsal joint. Chondrocytes isolated from the cartilage of these mice secreted human protein, suggesting that the animals incorporated heterologous protein to form cartilage which is essentially "humanized." The animals reached puberty and produced normal progeny. A completely normal phenotype in newborns indicates that human COL2A1 gene is expressed properly both temporally and spatially. These animals may be useful to generate models to study the effect of COL2A1 mutations on skeletal development in humans by introducing mutated gene constructs either into embryos or by crossing with transgenic animals with COL2A1 mutations.

The region encoded by the alternatively spliced exon IIIA in mesenchymal fibronectin appears essential for chondrogenesis at the level of cellular condensation.

Fibronectin in the extracellular matrix of tissues acts as a substrate for cell adhesion and migration during development. Heterogeneity in the structure of fibronectin is largely due to the alternative splicing of at least three exons (IIIB, IIIA, and V) during processing of a single primary transcript. Fibronectin mRNA alternative splicing patterns change from B+A+V+ to B+A-V+ during chondrogenesis. In this report, immunohistochemical analysis demonstrates that while fibronectin protein containing the region encoded by exon IIIB is present throughout the limb at all stages of development, fibronectin protein containing the region encoded by exon IIIA disappears from cartilaginous regions just after condensation in vivo and in high-density mesenchymal micromass cultures in vitro. Treatment of mesenchymal micromass cultures prior to condensation with an antibody specific for the region encoded by exon IIIA disrupts the formation of cellular condensations and inhibits subsequent chondrogenesis in a dose- and time-dependent manner. Furthermore, microinjection of the exon IIIA antibody into embryonic chick limb primordia in vivo results in malformations characterized by smaller limbs and loss of limb skeletal elements. These results strongly suggest that the presence of the region encoded by exon IIIA in mesenchymal fibronectin is necessary for the condensation event that occurs during chondrogenesis.

In vitro characterization of chondrogenic cells isolated from chick embryonic muscle using peanut agglutinin affinity chromatography.

Specific binding to the lectin, peanut agglutinin (PNA), has been reported in embryonic precartilage tissues, including the condensing limb bud blastema and the caudal half of the developing somite. The present study aimed to test the hypothesis that PNA-binding may be a surface characteristic of chondroprogenitor cells residing within noncartilage tissues, such as muscle, which have the potential of being induced to form cartilage, e.g., in the presence of bone matrix-derived factors. Day-14 chick embryonic pectoral muscle, which contained histochemically detectable PNA-binding cells, was dissociated into single cells (TM cells) and fractionated by PNA affinity chromatography into PNA-binding (PNA+) and nonbinding (PNA-) cells by PNA-Sepharose 6 MB affinity chromatography. The differentiation potential of the PNA-affinity fractionated cells in vitro was analyzed as a function of culture plating cell density. Immunohistochemistry of a number of cell-type-specific differentiation markers, including sarcomeric actin, collagen type II, and aggrecan core protein, demonstrated that PNA+ cells, when cultured as a micromass at high density (20 x 10(6) cells/ml), exhibited a chondrocyte-like phenotype, whereas the PNA-cells remained myogenic; however, both PNA+ and PNA- monolayer cultures (4 x 10(4) cells/ml) behaved as myoblastic cells. The expression of collagen type II mRNA was also confirmed by coupled reverse transcription/polymerase chain reaction analysis. These observations suggest that PNA binding, i.e., the presence of specific galactose-containing cell surface moieties, is likely to be one of the characteristics of chondrogenic cells residing in mesenchymally derived embryonic tissues.

Chondrogenic cell subpopulation of chick embryonic calvarium: isolation by peanut agglutinin affinity chromatography and in vitro characterization.

The embryonic skull bone, the calvarium, develops via intramembranous ossification, whereby mesenchymal cells differentiate directly into osteoblasts. However, under certain conditions, such as systemic calcium deficiency, regions of cartilage-like tissue are observed in the chick embryonic calvarium, suggesting the presence of pre-cartilage cells. We have recently identified and isolated a chondrogenic cell subpopulation from chick embryonic calvarium by Percoll gradient centrifugation. Using peanut agglutinin (PNA), which has been shown to bind specifically to chondroprogenitor cells in various developing skeletal elements, we have further examined the chondrogenic characteristics of calvarial cells. Histochemical staining of calvaria sections showed the presence of PNA-binding cells in subcambial regions of the calvarium as a function of embryonic development and calcium status. PNA-binding activity was also used as the basis for affinity chromatography fractionation of calvarial cells isolated from normal and calcium-deficient, shell-less chick embryos. A higher percentage of calvarial cells from the normal embryo bound PNA than those from shell-less embryos. Interestingly, more PNA-binding cells were found in the dense, chondrogenic fractions obtained by prior Percoll gradient fractionation of calvarial cells. The chondrogenic potential of the PNA affinity fractionated cells was assessed in culture based on alcian blue staining, and expression of collagen type II and aggrecan core protein. PNA-binding cells isolated from total calvarial cells and from the dense Percoll fractions exhibited a prominent chondrocyte-like phenotype, and were organized in alcian blue-stained nodules, which immunostained positively for collagen type II and aggrecan. Expression of collagen type II was also detected at the mRNA level by means of coupled reverse transcription/polymerase chain reaction. On the other hand, non-PNA-binding cells, isolated from total calvarial cells and from the lighter Percoll fractions, were primarily fibroblastic in appearance and did not express cartilage-associated characteristics. The presence of distinct PNA-binding cells with chondrogenic potential strongly suggests that these cells may be functionally important in morphogenesis of the embryonic calvarium.

Osteoblastic cells from rat long bone. I. Characterization of their differentiation in culture.

We here report the characterization for osteogenic markers alkaline phosphate (AP), osteocalcin, and mineral deposition of osteoblast cultures derived from cells migrating out from seven-day-old rat tibia fragments. The cells outgrown from bone fragments responded to stimulation with PTH with cAMP increase. We show in these cultures a high level of biosynthesis of type III collagen and osteonectin, and report the stimulatory effect that conditioned serum-free media (CM) from these cultures exert on the migration of endothelial cells (EA hy 926). The cultures were kept for the initial seven days in a Coon's modified F12 medium, then were switched to a medium containing ascorbic acid and beta-glycerophosphate (BGP), and cultured for another 41 days. They showed constitutive and timely restricted osteoblast markers and mineralization. Maximal AP activity occurred in concomitance with cell doubling, then fell to low levels by the time cultures were stationary. 45Ca incorporation in the monolayer increased after four weeks of culture, in concomitance with the appearance of unmineralized nodules, and remained high throughout the phase of mineral deposition. Biosynthesis of collagens type I, type III, and type V was detected at all times; secreted newly synthesized collagens decreased overall, relative to total secreted newly synthesized proteins, and on a per cell basis, with progression of the culture, while the ratio of collagen type III/collagen type I increased. Osteonectin was detected by immunohistochemistry and high amounts of osteonectin were synthesized constitutively. Osteocalcin was detected on virtually all cells tested at 21 and 28 days. A preliminary step in neoangiogenesis is the migration of endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of integrins expression during human osteoblasts "in vitro" differentiation.

We here report the modulation of the adhesion of cultured human osteoblasts on Laminin during the acquisition of differentiated phenotype. We also show that interference with the differentiation program caused by treatment with Retinoic acid of the cultures, causes changes of the capability to adhere to Laminin and type I Collagen. The younger or dedifferentiated cells have lower capability to bind to Laminin or Collagen. The maturation associated changes are specific for the adhesion to the above substrata and do not involve the adhesion to FN or plastic. The alpha subunit(s) of the integrin receptor(s) for these proteins is likely to be responsible for the modulation adhesion to Laminin and Collagen.