Interaction of angiogenic microvessels with the extracellular matrix

The extracellular matrix (ECM) plays a critical role in angiogenesis by providing biochemical and positional cues, as well as by mechanically influencing microvessel cell behavior. Considerable information is known concerning the biochemical cues relevant to angiogenesis, but less is known about the mechanical dynamics during active angiogenesis. The objective of this study was to characterize changes in the material properties of a simple angiogenic tissue before and during angiogenesis. During sprouting, there was an overall decrease in tissue stiffness followed by an increase during neovessel elongation. The fall in matrix stiffness coincided with peak matrix metalloproteinase mRNA expression and elevated proteolytic activity. An elevated expression of genes for ECM components and cell-ECM interaction molecules and a subsequent drop in proteolytic activity (although enzyme levels remained elevated) coincided with the subsequent stiffening. The results of this study show that the mechanical properties of a scaffold tissue may be actively modified during angiogenesis by the growing microvasculature.     

Investigation of unusual growth and phenotypic characteristics of plasmid-containing and plasmid-free strains of oligotrophic bacterium Ancylobacter vacuolatus

The oligotrophic bacterium Ancylobacter vacuolatus contains two large plasmids pREV1 and pREV2 (about 150 and 250 kb, respectively). Plasmid pREV1 carries the genes responsible for resistance to chloramphenicol, trimethoprim and y-irradiation. Plasmid pREV2 carries the genes responsible for resistance to beta-lactam antibiotics and formation of gas vacuoles. The ability to grow under oligotrophic conditions did not depend directly on either plasmid and was probably chromosome-encoded. Nevertheless, strains lacking the pREV2 plasmid had an improved capacity for growth in enriched media, as is evident from the following findings: 1) the growth rate of the strains lacking pREV2 was about 60% higher with an induction time of about two times less than those for strains carrying the plasmid; 2) the overall cell yield in rich media and colony size on non-oligotrophic agarized media increased with removal of pREV2; 3) the characteristic change in cell morphology occurring in the wild type ofA. vacuolatus when switched from oligotrophic to eutrophic growth conditions was not observed in the strains lacking pREV2; 4) bacterial strains lacking pREV2 exhibited significantly higher rRNA content than the parent strain. As a possible explanation for these phenomena, we suggest that the pREV2 plasmid carries gene(s) for protein(s) acting as repressor(s) of expression of some enzymes involved in eutrophic metabolism. Such protein(s) probably participate in switching between the oligotrophic and eutrophic types of metabolism in response to changing nutrient supply in the environment.     

Modulation of vascular smooth muscle cells proteoglycan synthesis by the extracellular matrix

In this study, we investigated the effect of the extracellular matrix (ECM) secreted by vascular cells on proteoglycan (PG) synthesis by vascular smooth muscle cells in culture. PG synthesis of human aortic smooth muscle cells plated on plastic or the matrices derived from vascular endothelial cells, vascular smooth muscle cells, or THP-1 macrophages was characterized. Smooth muscle cell and macrophage matrices increased both secreted and cellular smooth muscle cells PG production by 2.5-fold to 3.9-fold, respectively, over plastic and endothelial cell matrix. Macrophage matrix was more potent than smooth muscle cell matrix in this regard. Selective enzymatic removal of chondroitin sulfates, collagen, and elastin from smooth muscle cell matrix enhanced the stimulation of PG synthesis, as did the removal of chondroitin sulfates from macrophage matrix. PG turnover rates were similar for smooth muscle cells plated on the three matrices. The newly synthesized PG from cultures plated on smooth muscle cell-, and macrophage-derived matrices had greater charge density, larger molecular size, and longer glycosaminoglycan chains than those from endothelial cell matrix cultures. These data show that the ECM plays a major role in modulating vascular smooth muscle cell PG metabolism in vitro.     

Investigation of unusual growth and phenotypic characteristics of plasmid-containing and plasmid-free strains of oligotrophic bacterium <Emphasis Type="Italic">Ancylobacter vacuolatus</Emphasis>

The oligotrophic bacterium Ancylobacter vacuolatus contains two large plasmids pREV1 and pREV2 (about 150 and 250 kb, respectively). Plasmid pREV1 carries the genes responsible for resistance to chloramphenicol, trimethoprim and γ-irradiation. Plasmid pREV2 carries the genes responsible for resistance to β-lactam antibiotics and formation of gas vacuoles. The ability to grow under oligotrophic conditions did not depend directly on either plasmid and is probably chromosome-encoded. Nevertheless, strains lacking the pREV2 plasmid had an improved capacity for growth in enriched media, as is evident from the following findings: 1) the growth rate of the strains lacking pREV2 was about 60% higher with an induction time of about two times less than those for strains carrying the plasmid; 2) the overall cell yield in rich media and colony size on non-oligotrophic agarized media increased with removal of pREV2; 3) the characteristic change in cell morphology occurring in the wild type of A. vacuolatus when switched from oligotrophic to eutrophic growth conditions was not observed in the strains lacking pREV2; 4) bacterial strains lacking pREV2 exhibited significantly higher rRNA content than the parent strain. As a possible explanation for these phenomena, we suggest that the pREV2 plasmid carries gene(s) for protein(s) acting as repressor(s) of expression of some enzymes involved in eutrophic metabolism. Such protein(s) probably participate in switching between the oligotrophic and eutrophic types of metabolism in response to changing nutrient supply in the environment.     

Differential expression of extracellular matrix proteins in senescent and young human fibroblasts: A comparative proteomics and microarray study

The extracellular matrix (ECM) provides an essential structural framework for cell attachment, proliferation, and differentiation, and undergoes progressive changes during senescence. To investigate changes in protein expression in the extracellular matrix between young and senescent fibroblasts, we compared proteomic data (LTQ-FT) with cDNA microarray results. The peptide counts from the proteomics analysis were used to evaluate the level of ECM protein expression by young cells and senescent cells, and ECM protein expression data were compared with the microarray data. After completing the comparative analysis, we grouped the genes into four categories. Class I included genes with increased expression levels in both analyses, while class IV contained genes with reduced expression in both analyses. Class II and Class III contained genes with an inconsistent expression pattern. Finally, we validated the comparative analysis results by examining the expression level of the specific gene from each category using Western blot analysis and semiquantitative RT-PCR. Our results demonstrate that comparative analysis can be used to identify differentially expressed genes.     

Changes in expression of extracellular matrix genes, fibrogenic factors, and actin cytoskeletal organization in retinol treated and untreated vocal fold stellate cells.

The regulation of extracellular matrix (ECM) constituency is critical in maintaining vocal cord biomechanical viscoelasticity required for phonation. Recently our laboratory successfully isolated and cultured a novel cell called a vocal fold stellate cell (VFSC), thought to play a central role in laryngeal ECM metabolism, aging, scarring and cancer. Our laboratory has shown that these cells undergo transdifferentiation that is partially reversed by exposure to all-trans retinol (ATROH). Here we make the first report on the expression of various ECM components, MMPs, TIMPs, pro-fibrogenic cytokines, and other ECM modulators in transdifferentiated and deactivated VFSCs. We show that VFSCs maintain an ECM expression pattern similar to laryngeal cancer and scars but distinct from tracheal fibroblasts. Exposure to ATROH differentially affects the VFSC expression of ECM components, matrix-regulating enzymes, and fibrogenic factors suggesting that the inhibitory effects of this synthetic cofactor should be studied further in laryngeal fibrosis and scarring. We also show that increased exposure to retinol induces sequential reorganization of the actin cytoskeleton in activated VFSCs. Our findings demonstrate that VFSCs are capable of regulating vocal fold ECM constituency important throughout normal laryngeal development. Furthermore, our results implicate VFSC activation in ECM misregulation which is a hallmark of several laryngeal pathologies.     

Construction, detection and microarray analysis on theShigella flexneri 2asitC mutant

Bacteria of Shigella spp. are commonly called Ba-cillus dysenteriae. The bacillary dysentery caused byShigella is one of the most serious infectious diseasesthroughout the world, especially in the developing countries. The completion of whole genome sequenc-ing of Shigella flexneri 2a strain 301 (brieflySf301)[1] offered a large amount of information andinvaluable clues for its functional genomic research.Microbial functional genomic research aims at deeplyinterpreting the function of genes …     

Decreased cell adhesion promotes angiogenesis in a Pyk2-dependent manner

Angiogenesis is regulated by both soluble growth factors and cellular interactions with the extracellular matrix (ECM). While cell adhesion via integrins has been shown to be required for angiogenesis, the effects of quantitative changes in cell adhesion and spreading against the ECM remain less clear. Here, we show that angiogenic sprouting in natural and engineered three-dimensional matrices exhibited a biphasic response, with peak sprouting when adhesion to the matrix was limited to intermediate levels. Examining changes in global gene expression to determine a genetic basis for this response, we demonstrate a vascular endothelial growth factor (VEGF)-induced upregulation of genes associated with vascular invasion and remodeling when cell adhesion was limited, whereas cells on highly adhesive surfaces upregulated genes associated with proliferation. To explore a mechanistic basis for this effect, we turned to focal adhesion kinase (FAK), a central player in adhesion signaling previously implicated in angiogenesis, and its homologue, proline-rich tyrosine kinase 2 (Pyk2). While FAK signaling had some impact, our results suggested that Pyk2 can regulate both gene expression and endothelial sprouting through its enhanced activation by VEGF in limited adhesion contexts. We also demonstrate decreased sprouting of tissue explants from Pyk2-null mice as compared to wild type mice as further confirmation of the role of Pyk2 in angiogenic sprouting. These results suggest a surprising finding that limited cell adhesion can enhance endothelial responsiveness to VEGF and demonstrate a novel role for Pyk2 in the adhesive regulation of angiogenesis.     

Transcriptional regulation of fermentative and respiratory metabolism in Saccharomyces cerevisiae industrial bakers' strains

Bakers' yeast-producing companies grow cells under respiratory conditions, at a very high growth rate. Some desirable properties of bakers' yeast may be altered if fermentation rather than respiration occurs during biomass production. That is why differences in gene expression patterns that take place when industrial bakers' yeasts are grown under fermentative, rather than respiratory conditions, were examined. Macroarray analysis of V1 strain indicated changes in gene expression similar to those already described in laboratory Saccharomyces cerevisiae strains: repression of most genes related to respiration and oxidative metabolism and derepression of genes related to ribosome biogenesis and stress resistance in fermentation. Under respiratory conditions, genes related to the glyoxylate and Krebs cycles, respiration, gluconeogenesis, and energy production are activated. DOG21 strain, a partly catabolite-derepressed mutant derived from V1, displayed gene expression patterns quite similar to those of V1, although lower levels of gene expression and changes in fewer number of genes as compared to V1 were both detected in all cases. However, under fermentative conditions, DOG21 mutant significantly increased the expression of SNF1 -controlled genes and other genes involved in stress resistance, whereas the expression of the HXK2 gene, involved in catabolite repression, was considerably reduced, according to the pleiotropic stress-resistant phenotype of this mutant. These results also seemed to suggest that stress-resistant genes control desirable bakers' yeast qualities.     

Protein and other compositional differences of the extracellular material from slimy and non-slimy colonies of non-mucoid Pseudomonas aeruginosa

The composition of extracellular material produced by non-mucoid strains of Pseudomonas aeruginosa differed when grown on surfaces that either did or did not induce the formation of slime under conditions where the medium was identical. The nature of the changes in protein composition indicated that protein expression differed in the course of growth on the two surfaces, and hence that there were physiological consequences associated with growth under conditions which do or do not lead to slime formation. The compositional differences also included elevated levels in extracellular material from the slimy colonies of two virulence factors, protease and rhamnolipids.     

The intracellular form of human MAGP1 elicits a complex and specific transcriptional response

Microfibril-associated glycoprotein-1 (MAGP1) is found associated with microfibrils in the extracellular matrix (ECM). In humans, MAGP1 is expressed as two alternatively spliced isoforms: MAGP1A, the extracellular microfibril-associated form; and MAGP1B, an exclusively intracellular isoform derived from the skipping of exon 3. The biological function of MAGP1B is unknown. We performed gene expression profiling to study the cellular response to MAGP1B using whole-genome genechips. We found that MAGP1B specifically induces the expression of genes linked to cell adhesion, motility, metabolism, gene expression, development and signal transduction. Versican, a gene product involved in the structure and functional regulation of the ECM, showed the highest up-regulation in response to MAGP1B. These studies suggest a dual role for MAGP1, with extracellular MAGP1A involved in ECM function, and intracellular MAGP1B modulating the expression of genes that function in cell adhesion, migration and control of ECM deposition.