Fibulin-5 regulates keloid-derived fibroblast-like cells through integrin beta-1.

OBJECTIVE: Keloid scar is pathological tissue that appears after skin injury, and that is more aggressive than hypertrophic scars. Keloid scars are characterized by increased proliferation of fibroblast-like cells (FLCs) and the accumulation of extracellular matrix, mainly collagen. Fibulin-5, a glycoprotein secreted by many cell types, is a component of the extracellular matrix. We investigated the effect of fibulin-5 on the adhesion and proliferation of FLCs derived from keloid scars and the role of integrin beta-1 in these activities. METHODS: Fibroblast-like cells were isolated from six keloid scars and cultured on plates coated with fibulin-5 or with gelatin. Cells were incubated for 72-96 h to examine proliferation rates and incubated for 240 min, with washings at 20, 40, 60, 90, 120, 180 min, to assess adhesion rates. To examine the role of integrin beta-1, the anti-human integrin beta-1 (CD29) antibody was added to the culture medium. RESULTS: Fibroblast-like cells from keloids cultured on a fibulin-5-coated surface showed a significantly reduced proliferation rate and a delayed adhesion rate, compared to cells cultured on gelatin-coated dishes. Adherence of these cells to fibulin-5 pre-coated wells was significantly reduced in the presence of anti-human integrin beta-1 (CD29) antibodies. Our current findings are similar to previously observed reduced proliferation in vascular smooth muscle cells overexpressing fibulin-5. We did not test the effects of fibulin-5 on normal fibroblasts. CONCLUSION: This study demonstrates the pivotal role of the extracellular protein, fibulin-5, on the adhesion and proliferation of human keloid-derived cells, through binding to integrin beta-1.

The formation of an anti-restenotic/anti-thrombotic surface by immobilization of nitric oxide synthase on a metallic carrier.

Coronary stenosis due to atherosclerosis, the primary cause of coronary artery disease, is generally treated by balloon dilatation and stent implantation, which can result in damage to the endothelial lining of blood vessels. This leads to the restenosis of the lumen as a consequence of migration and proliferation of smooth muscle cells (SMCs). Nitric oxide (NO), which is produced and secreted by vascular endothelial cells (ECs), is a central anti-inflammatory and anti-atherogenic player in the vasculature. The goal of the present study was to develop an enzymatically active surface capable of converting the prodrug l-arginine, to the active drug, NO, thus providing a targeted drug delivery interface. NO synthase (NOS) was chemically immobilized on the surface of a stainless steel carrier with preservation of its activity. The ability of this functionalized NO-producing surface to prevent or delay processes involved in restenosis and thrombus formation was tested. This surface was found to significantly promote EC adhesion and proliferation while inhibiting that of SMCs. Furthermore, platelet adherence to this surface was markedly inhibited. Beyond the application considered here, this approach can be implemented for the local conversion of any systemically administered prodrug to the active drug, using catalysts attached to the surface of the implant.

Cloning of European eel (Anguilla anguilla) FSH-beta subunit, and expression of FSH-beta and LH-beta in males and females after sex determination.

The European eel (Anguilla anguilla) is a catadromic teleost species with a complex life cycle, both in sea and freshwater environments. The sex determination phase of gonadal development occurs in a freshwater environment. Polymorphism occurs in increasing rates with respect to gender. While males stop growing at approximately 150 g, females continue to grow to being much larger. In this study, we cloned the cDNA FSH-beta subunit of the European eel (A. anguilla), and measured the mRNA levels of FSH-beta and LH-beta in males and females after sex determination. The FSH-beta subunit cDNA consisted of 1068 bp, encoding a 127 amino acid peptide. A comparison between European and Japanese eels of the FSH-beta amino acid sequence showed 98% similarity.

Growth differences and growth hormone expression in male and female European eels [Anguilla anguilla (L.)].

In this study, we examined the growth differences of males and females following a sex reversion, and the growth hormone (GH) expression variation between sexes of European eels [Anguilla anguilla (L.)]. A high percentage of females (88%) was found in the group fed with estradiol 17beta compared to the control group (comprised of only 6% female eels), which was defined as the male population. Significant differences between growth rate and size were found following 480 days of growth, whereby the males reached 60+/-4.3 g (means+/-SE) in size and the females 73.4+/-5.9 (g+/-SE); after 600 days, the males reached 114.1+/-4.3 and the females 171+/-11.7 (g+/-SE). A cDNA coding for the complete growth hormone of the European eel A. anguilla (eeGH) was cloned by RACE PCR using several sets of degenerate oligonucleotides. The eeGH cDNA coding region is 627 bp long. A sequence comparison of eeGH with Anguilla japonica GH (jeGH) cDNA showed a 98% identical base. Comparison of the deduced amino acid sequence revealed 99% identical residues, meaning that a difference exists in only two of the 209 residues. In both cases, the differing residues in the eeGH amino acid sequence are lysine. We measured the mRNA levels of growth hormone in the pituitaries of male and female eels growing at different rates. A significantly higher expression of eeGH was found in the female eels in comparison to the males. These results show that different levels of GH transcription eeGH can explain the growth rate differences between male and female European eels.

Lysine and threonine metabolism are subject to complex patterns of regulation in Arabidopsis.

To study the regulation of lysine and threonine metabolism in plants, we have transformed Arabidopsis thaliana with chimeric genes encoding the two bacterial enzymes dihydrodipicolinate synthase (DHPS) and aspartate kinase (AK). These bacterial enzymes are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts. Transgenic plants expressing the bacterial DHPS overproduced lysine, but lysine levels were quite variable within and between transgenic genotypes and there was no direct correlation between the levels of free lysine and the activity of DHPS. The most lysine-overproducing plants also exhibited abnormal phenotypes. However, these phenotypes were detected only at early stages of plant growth, while at later stages, new buds emerged that looked completely normal and set seeds. Wild-type plants exhibited relatively high levels of free threonine, suggesting that in Arabidopsis AK regulation may be more relaxed than in other plants. This was also supported by the fact that expression of the bacterial AK did not cause any dramatic elevation in this amino acid. Yet, the relaxed regulation of threonine synthesis in Arabidopsis was not simply due to a reduced sensitivity of the endogenous AK to feedback inhibition by lysine and threonine because growth of wild-type plants, but not of transgenic plants expressing the bacterial AK, was arrested in media containing these two amino acids. The present results, combined with previous studies from our laboratory, suggest that the regulation of lysine and threonine metabolism is highly variable among plant species and is subject to complex biochemical, physiological and environmental controls. The suitability of these transgenic Arabidopsis plants for molecular and genetic dissection of lysine and threonine metabolism is also discussed.