Perlecan-Induced Suppression of Smooth Muscle Cell Proliferation Is Mediated Through Increased Activity of the Tumor Suppressor PTEN

We were interested in the elucidation of the interaction between the heparan sulfate proteoglycan, perlecan, and PTEN in the regulation of vascular smooth muscle cell (SMC) growth. We verified serum-stimulated DNA synthesis, and Akt and FAK phosphorylation were significantly reduced in SMCs overexpressing wild-type PTEN. Our previous studies showed perlecan is a potent inhibitor of serum-stimulated SMC growth. We report in the present study, compared with SMCs plated on fibronectin, serum-stimulated SMCs plated on perlecan exhibited increased PTEN activity, decreased FAK and Akt activities, and high levels of p27, consistent with SMC growth arrest. Adenoviral-mediated overexpression of constitutively active Akt reversed perlecan-induced SMC growth arrest while morpholino antisense-mediated loss of endogenous PTEN resulted in increased growth and phosphorylation of FAK and Akt of SMCs on perlecan. Immunohistochemical and Western analyses of balloon-injured rat carotid artery tissues showed a transient increase in phosphoPTEN (inactive) after injury, correlating to high rates of neointimal cell replication; phosphoPTEN was largely limited to actively replicating SMCs. Similarly, in the developing rat aorta, we found increased PTEN activity associated with increased perlecan deposition and decreased SMC replication rates. However, significantly decreased PTEN activity was detected in aortas of perlecan-deficient mouse embryos, consistent with SMC hyperplasia observed in these animals, compared with E17.5 heterozygous controls that produce abundant amounts of perlecan at this developmental time point. Our data show PTEN is a potent endogenously produced inhibitor of SMC growth and increased PTEN activity mediates perlecan-induced suppression of SMC proliferation.Costell Rossello, M.Mercedes, [email protected]

Similarity of Recombinant Human Perlecan Domain 1 by Alternative Expression Systems Bioactive Heterogenous Recombinant Human Perlecan D1

<p>Abstract</p> <p>Background</p> <p>Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human perlecan domain 1 (HSPG2 abbreviated as rhPln.D1) synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology.</p> <p>Results</p> <p>By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high M_rrhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary structure analysis suggested helices and sheets in both recombinant species. rhPln.D1 demonstrated binding to rhFGF-2 with an apparent k_Dof 2 ± 0.2 nM with almost complete susceptibility to digestion by heparinase III in ligand blot analysis but not to chondroitinase digestion. Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs. Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor.</p> <p>Conclusions</p> <p>With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.</p

Biodiversity of CS-proteoglycan sulphation motifs: chemical messenger recognition modules with roles in information transfer, control of cellular behaviour and tissue morphogenesis

Chondroitin sulphate glycosaminoglycan chains on cell and ECM proteoglycans can no longer be regarded as merely hydrodynamic space fillers. Overwhelming evidence over recent years indicates that sulphation motif sequences within the chondroitin sulphate chain structure are a source of significant biological information to cells and their surrounding environment. Chondroitin sulphate sulphation motifs have been shown to interact with a wide variety of bioactive molecules e.g. cytokines, growth factors, chemokines, morphogenetic proteins, enzymes and enzyme inhibitors, as well as structural components within the extracellular milieu. They are therefore capable of modulating a panoply of signalling pathways thus controlling diverse cellular behaviours including proliferation, differentiation, migration and matrix synthesis. Consequently, through these motifs, chondroitin sulphate proteoglycans play significant roles in the maintenance of tissue homeostasis, morphogenesis, development, growth and disease. Here we review (i) the biodiversity of chondroitin sulphate proteoglycans and their sulphation motif sequences and (ii) the current understanding of the signalling roles they play in regulating cellular behaviour during tissue development, growth, disease and repai

A massive cluster of Red Supergiants at the base of the Scutum-Crux arm

We report on the unprecedented Red Supergiant (RSG) population of a massive young cluster, located at the base of the Scutum-Crux Galactic arm. We identify candidate cluster RSGs based on {\it 2MASS} photometry and medium resolution spectroscopy. With follow-up high-resolution spectroscopy, we use CO-bandhead equivalent width and high-precision radial velocity measurements to identify a core grouping of 26 physically-associated RSGs -- the largest such cluster known to-date. Using the stars' velocity dispersion, and their inferred luminosities in conjuction with evolutionary models, we argue that the cluster has an initial mass of $\sim$40,000\msun, and is therefore among the most massive in the galaxy. Further, the cluster is only a few hundred parsecs away from the cluster of 14 RSGs recently reported by Figer et al (2006). These two RSG clusters represent 20% of all known RSGs in the Galaxy, and now offer the unique opportunity to study the pre-supernova evolution of massive stars, and the Blue- to Red-Supergiant ratio at uniform metallicity. We use GLIMPSE, MIPSGAL and MAGPIS survey data to identify several objects in the field of the larger cluster which seem to be indicative of recent region-wide starburst activity at the point where the Scutum-Crux arm intercepts the Galactic bulge. Future abundance studies of these clusters will therefore permit the study of the chemical evolution and metallicity gradient of the Galaxy in the region where the disk meets the bulge.Comment: 49 pages, 22 figures. Accepted for publication in ApJ. Version with hi-res figures can be found at http://www.cis.rit.edu/~bxdpci/RSGC2.pd

Illumination in symbiotic binary stars: Non-LTE photoionization models. II. Wind case

We describe a non-LTE photoionization code to calculate the wind structure and emergent spectrum of a red giant wind illuminated by the hot component of a symbiotic binary system. We consider spherically symmetric winds with several different velocity and temperature laws and derive predicted line fluxes as a function of the red giant mass loss rate, \mdot. Our models generally match observations of the symbiotic stars EG And and AG Peg for \mdot about 10^{-8} \msunyr to 10^{-7} \msunyr. The optically thick cross- section of the red giant wind as viewed from the hot component is a crucial parameter in these models. Winds with cross-sections of 2--3 red giant radii reproduce the observed fluxes, because the wind density is then high, about 10^9 cm^{-3}. Our models favor winds with acceleration regions that either lie far from the red giant photosphere or extend for 2--3 red giant radii.Comment: 51 pages, LaTeX including three tables, requires 15 Encapsulated Postscript figures, to appear in Ap

Critical Differences and Clues in Eta Car's 2009 Event

We monitored Eta Carinae with HST WFPC2 and Gemini GMOS throughout the 2009 spectroscopic event, which was expected to differ from its predecessor in 2003 (Davidson et al. 2005). Here we report major observed differences between events, and their implications. Some of these results were quite unexpected. (1) The UV brightness minimum was much deeper in 2009. This suggests that physical conditions in the early stages of an event depend on different parameters than the "normal" inter-event wind. Extra mass ejection from the primary star is one possible cause. (2) The expected He II 4687 brightness maximum was followed several weeks later by another. We explain why this fact, and the timing of the 4687 maxima, strongly support a "shock breakup" hypothesis for X-ray and 4687 behavior as proposed 5-10 years ago. (3) We observed a polar view of the star via light reflected by dust in the Homunculus nebula. Surprisingly, at that location the variations of emission-line brightness and Doppler velocities closely resembled a direct view of the star; which should not have been true for any phenomena related to the orbit. This result casts very serious doubt on all the proposed velocity interpretations that depend on the secondary star's orbital motion. (4) Latitude-dependent variations of H I, He I and Fe II features reveal aspects of wind behavior during the event. In addition, we discuss implications of the observations for several crucial unsolved problems.Comment: 45 pages, 9 figures, submitted to Ap