Use of bifemelane hydrochloride in improving and maintaining the visual field of patients with glaucoma

Recently we demonstrated a large induction of activin expression in fibroblasts and keratinocytes after cutaneous injury in mice. To identify possible mediators of activin induction during skin repair, we have now analyzed the regulation of this factor in cultured keratinocytes and fibroblasts. Here we show that activin A mRNA and protein levels are low in quiescent keratinocytes and fibroblasts but expression is strongly induced upon serum treatment. The stimulatory effect of serum on activin expression is likely to be a combinatorial effect of different growth factors, since platelet-poor plasma serum and several purified serum growth factors also stimulated activin expression, although to a lesser extent than complete serum. Furthermore, we found increased expression of activin in keratinocytes and fibroblasts after addition of the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha. Taken together, our data suggest that serum growth factors which are released upon hemorrhage as well as proinflammatory cytokines derived from neutrophils and macrophages might be responsible for induction of activin expression after injury.     

Hepatocyte growth factor releases epithelial and endothelial cells from growth arrest induced by transforming growth factor-beta1

Human lung fibroblasts and Mv1Lu mink lung epithelial cells were used as a model to study the role of extracellular matrix in epithelial-mesenchymal interactions. Extracellular matrices of fibroblasts were found to contain growth promoting activity that reduced the sensitivity of Mv1Lu cells to the growth inhibitory effects of transforming growth factor-beta (TGF-beta). The majority of the activity was identified as hepatocyte growth factor/scatter factor (HGF) by inhibition with specific antibodies and by reconstitution of the effect by recombinant HGF. HGF induced cell proliferation when contact-inhibited Mv1Lu cells were trypsinized and plated in the presence of TGF-beta1. The effect was valid also in assays where Madin-Darby canine kidney epithelial cells or bovine capillary endothelial cells were used. The multiplication of chronically TGF-beta1 inhibited Mv1Lu cells was also induced by HGF. In addition, HGF induced anchorage independent growth of Mv1Lu cells that was refractory to TGF-beta1 growth inhibition. Immunoprecipitation analysis indicated that HGF prevented the suppression of Cdk4 and Cdk2, but not the induction of p21, by TGF-beta1. Since both TGF-beta1 and HGF require proteolysis for activation, the results imply that proteolytic activity of epithelial and endothelial cells directs their responses to signals from mesenchymal-type extracellular matrices, and that during development, matrix-bound growth and invasion promoting and suppressing factors are activated in a coordinated manner.     

Autoxidation of pyridoxalated hemoglobin polyoxyethylene conjugate

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which stimulates angiogenesis. VEGF is regulated by multiple factors such as hypoxia, phorbol esters, and growth factors. However, data concerning the expression of VEGF in the different vascular cell types and its regulation by cAMP are not available. In the present study, we have investigated the effect of adenylate cyclase activation on VEGF mRNA expression in rat vascular cells in primary culture. Basal VEGF expression is greater in smooth muscle cells than in endothelial cells and fibroblasts. A 4-h treatment with forskolin (10(-5) M) induced a 2-fold stimulation of VEGF mRNA expression in smooth muscle cells and fibroblasts, but, in contrast, did not affect VEGF expression in endothelial cells. In smooth muscle cells, a pharmacologically induced increase in intracellular cAMP levels using iloprost or isoprenaline led to a rise in VEGF mRNA expression comparable to that induced by forskolin. Adenosine, which increases cAMP levels in smooth muscle cells, also increases VEGF expression. Moreover, the 2.2-fold stimulation of VEGF expression by adenosine was enhanced following a cotreatment with cobalt chloride (a hypoxia miming agent). The observed additive effect (4.3-fold increase) suggests that these two factors, hypoxia and adenosine, regulate VEGF mRNA expression in smooth muscle cells by independent mechanisms.     

Evidence for an impaired ability to determine semantic relations in Alzheimer''s disease patients

Gingival and periodontal ligament (PDL) fibroblasts are the major cellular components of periodontal soft connective tissues, but the precise differences between these cells are not yet known. In the present study, we have therefore examined the phenotypic and functional features of the cells obtained from gingival and PDL biopsy samples. Spindle-shaped cells characteristic of fibroblasts were the main cell type observed in vitro, although epithelial cells were also present in primary gingival cell cultures. Flow cytometry was used to measure the size and granularity of the cultured cells, and showed that the gingival fibroblasts were smaller and less granular compared with the PDL cells. The expression of certain key extracellular matrix (ECM) proteins, fibronectin, collagen type I, and tenascin was measured by flow cytometry. Analysis of the fluorescence profiles of these cultures showed that the majority of cells expressed fibronectin and that the average fluorescence intensity of this antigen in the PDL cells was higher than that in the gingival fibroblasts. Moreover, the fibronectin-positive PDL cells apparently comprised two subpopulations which expressed fibronectin at different levels, suggesting that the cells in the PDL cultures were functionally heterogeneous. The level of collagen type I was also found to be up-regulated in the PDL compared with the gingival cells and, as with fibronectin, was expressed at two different levels by subsets of the PDL cells. In contrast, tenascin was expressed at very similar levels by both the gingival fibroblasts and PDL cells. In addition, measurement of alkaline phosphatase, a marker enzyme for mineralized tissue-forming cells, showed that the PDL cells had higher activity than the gingival fibroblasts and that the alkaline phosphatase activity in the PDL cells was far more markedly up-regulated by dexamethasone. Our findings demonstrate that, despite their similar spindle-shaped appearance, fibroblasts derived from gingival and PDL tissues appear to display distinct functional activities which are likely to play a vital part in the maintenance of tissue integrity and regenerative processes.     

Glucose modulates growth of gingival fibroblasts and periodontal ligament cells: correlation with expression of basic fibroblast growth factor

Diabetes mellitus is a systemic disease with profound effects on oral health and periodontal wound healing. Uncontrolled diabetes adversely affects surgical wound healing and is often associated with abnormal proliferation of fibroblasts, excessive angiogenesis and poor bone regeneration. Human gingival fibroblasts and periodontal ligament cells from both diabetics and non-diabetics were evaluated for growth responses following culture in 20 mM glucose, a concentration compatible with blood glucose levels in uncontrolled diabetics. Gingival fibroblasts derived from 9 non-diabetic patients and 3 insulin-dependent diabetics either proliferated or showed little change of growth in elevated glucose. Enhanced proliferation was observed following 1 wk of culture in glucose. Growth of periodontal ligament cells from 5 non-diabetic patients was inhibited by 20 mM glucose. Fibroblasts that were markedly growth stimulated were probed for expression of basic fibroblast growth factor (bFGF) using a reverse-transcribed polymerase chain reaction (RT-PCR). Results indicate that fibroblasts exhibiting the greatest increase in growth in response to high glucose also exhibited increased expression of bFGF. No changes were observed in mRNA expression for platelet-derived growth factor-AA, platelet-derived growth factor-BB, insulin-like growth factor and transforming growth factor-beta 1. Mitogenic effects induced by the cytosol of fibroblasts exhibiting increases of growth in 20 mM glucose were abrogated by neutralizing antibodies to bFGF. In addition, some periodontal ligament cells that were growth inhibited by high glucose had reduced expression of bFGF. These data suggest that bFGF may play a role in the abnormal wound healing associated with periodontal surgery of uncontrolled diabetics.     

Signaling through the ARK tyrosine kinase receptor protects from apoptosis in the absence of growth stimulation

ARK (AXL) is the prototype of a distinctive family of receptor tyrosine kinases which contain in their extracellular domains features reminiscent of cell adhesion molecules. ARK is capable of homophilic binding, which results in a degree of receptor activation, but can also be activated by a heterophilic ligand, Gas6, a member of the family of vitamin K dependent proteins that is preferentially expressed in quiescent cells. Since a number of tissues and cell lines express both ARK and Gas6, we studied the effect of endogenous and exogenous Gas6 on the phenotype of ARK expressing cells. Here we show that constitutive expression of Gas6 in an NIH3T3 cell line that does not spontaneously express this protein does not result in cell transformation or uncontrolled growth, but protects from apoptosis induced by serum deprivation. Recombinant exogenous Gas6 was also capable of protecting cells from apoptosis at concentrations that did not result in significant induction of DNA synthesis. Activation of ARK phosphorylation and a weak but significant induction of MAP kinase activity accompanied the increased survival of cells treated with Gas6. The antiapoptotic effect of ARK signaling was confirmed by studies using fibroblasts from ARK knock-out mice, that showed that the absence of ARK resulted in higher levels of serum deprivation-induced apoptosis, that could not be rescued by the addition of Gas6. Interestingly ARK signaling protects from apoptosis induced by serum deprivation, myc overexpression, or by TNF alpha but not from u.v. irradiation or Staurosporine. These results suggest that a major function of Gas6-ARK signaling is that of increasing cell survival under conditions which do not allow cell proliferation.     

Differential development of CD4 and CD8 cytotoxic T cells (CTL) in PBMC across the leprosy spectrum; IL-6 with IFN-gamma or IL-2 generate CTL in multibacillary patients

Cation-exchange chromatography effectively concentrates the cell growth activity present in whey and we have used this process as a basis to characterise further the growth factors present in bovine milk. Under neutral conditions, total bioactivity in the growth factor-enriched cation-exchange fraction chromatographed with an apparent molecular mass of 80-100 kDa. In contrast, acid gel-filtration chromatography resolved two peaks of cell growth activity. A peak at 15-25 kDa contained the bulk of growth activity for Balb/c 3T3 fibroblasts while bio-activity for L6 myoblasts and skin fibroblasts eluted with a molecular mass of 6 kDa. A peak of inhibitory activity for Mv1Lu and MDCK cells also eluted at 15-25 kDa. Both IGF-I and IGF-II were purified from fractions that eluted at 6 kDa, although the IGF peptides alone did not account for the total bioactivity recovered. Platelet-derived growth factor (PDGF), identified by radioreceptor assay, eluted at a slightly higher molecular mass than the peak of growth activity for Balb/c 3T3 cells, and an anti-PDGF antibody was without effect on the growth of Balb/c 3T3 cells in response to the whey-derived factors. Further purification of the inhibitory activity for epithelial cells yielded a sequence for transforming growth factor beta (TGF-beta), and all inhibitory activity for Mv1Lu cells was immunoneutralised by an antibody against TGF-beta. In contrast, this antibody decreased the growth of Balb/c 3T3 fibroblasts in the whey-derived extract by only 10%. Finally, a cocktail of recombinant growth factors containing IGF-I, IGF-II, PDGF, TGF-beta and fibroblast growth factor 2 stimulated growth of Balb/c 3T3 cells to a level equivalent to only 51% of that observed in the milk-derived growth factor preparation. We conclude that: (i) cell growth activity recovered from bovine whey is present in acid-labile high molecular weight complexes; (ii) all cell growth inhibitory activity for epithelial cells can be accounted for by TGF-beta; (iii) IGF-I and IGF-II co-elute with the major peak of activity for L6 myoblasts and skin fibroblasts, although the IGF peptides alone do not explain the growth of these cells in the whey-derived extract; and (iv) neither PDGF nor TGF-beta account for the 15-25 kDa peak of Balb/c 3T3 growth activity. These data suggest the presence of additional mitogenic factors in bovine milk.     

Paracrine inducers of uterine endometrial spermidine/spermine N1-acetyltransferase gene expression during early pregnancy in the pig

The endogenous factors that underlie the transient induction of the gene encoding spermidine/spermine N1-acetyltransferase (SSAT), the rate-limiting enzyme in cellular polyamine catabolism, in pig uterine endometrium during periimplantation are not known. The present study examined a number of peptide growth factors and regulatory molecules that are present within the uterine environment at early pregnancy, coincident with maximal SSAT gene expression, for their ability to manifest endogenous SSAT gene-inducing activity. Basal SSAT expression in luminal epithelial cells was higher (p < 0. 01) than that for glandular epithelial (GE) or stromal (ST) cells. Recombinant human insulin-like growth factor-I (IGF-I; 50 ng/ml) had no effect on steady-state SSAT mRNA levels, but it increased mitogenesis in all three cell types. In contrast, IGF-I caused a marked induction (p < 0.01) of SSAT mRNA levels in the human endometrial carcinoma cell line Hec-1-A. Uterine explants incubated with interleukin-6, transforming growth factor alpha, epidermal growth factor (each at 1, 10, and 100 ng/ml), retinoic acid and retinol (each at 0.01, 0.1, and 1 microM), and estradiol-17beta (10 nM) had SSAT mRNA levels similar to controls. By contrast, leukemia inhibitory factor (LIF; at 10 and 100 ng/ml) caused a modest, but significant (p < 0.05), increase in SSAT mRNA levels over those of untreated explants. This effect of LIF, however, did not approach the level of induction observed in GE or ST cells after addition of medium conditioned by Day 12 or 17 porcine conceptuses and in endometrial explants supplemented with medium conditioned by Day 21 porcine conceptuses or a continuous cell line (Jag-1) derived from Day 14 porcine trophoblast. We suggest that transient induction of endometrial SSAT gene expression at implantation is mediated by the functional interactions of specific conceptus-derived regulatory factors, distinct from estrogen, with endometrial-derived factor(s) such as LIF. These complex interactions are probably requisite for the transient, yet dramatic, induction of SSAT gene expression and may be critical for successful implantation.     

Supramalleolar osteotomy for ankle valgus in myelomeningocele

Oral fibroblasts stimulated invasion of oral-carcinoma cells into the collagen matrix. The mechanisms of the fibroblast-induced stimulation of invasiveness was further investigated by examining cell motility and proteolytic activity of tumor cells, using mainly an adenoid-cystic-carcinoma cell line (ACCS) and normal fibroblasts from gingival tissues. Conditioned medium from the fibroblasts grown in serum-free medium was fractionated on a Superdex 200 pg column, and Peak 1 eluted at 200 to 300 kDa and Peak 2 eluted at 50 to 100 kDa were found to contain different specific activity. Treatment of ACCS cells with Peak 1 resulted in an increase in the production of proteolytic enzymes. Peak 2 stimulated both chemotaxis and chemokinesis of ACCS cells. A chemotactic factor was purified from the heparin-unbound fraction of Peak 2 by anion exchange and hydrophobic chromatography, and was named "fibroblast-derived motility factor (FDMF)". At 1 microg/ml, FDMF stimulated chemotaxis of ACCS cells by 4-fold compared with unstimulated controls. Characterization of the physicochemical properties of FDMF suggested that it might be different from any known motility factors. Exposure of ACCS cells to FDMF resulted in reduced amounts of actin stress fiber in the cytoplasm and induction of tyrosine phosphorylation of several cellular proteins detectable 30 to 60 min after treatment. These FDMF-induced changes were blocked by pre-treatment either with genistein or with pertussis toxin. These findings suggest that FDMF may be a novel protein which stimulates cell motility via a signaling pathway mediated by a pertussis-toxin-sensitive G protein and tyrosine phosphorylation.     

FGF-10 is a chemotactic factor for distal epithelial buds during lung development

BACKGROUND: We investigated the ability of a variety of growth factors to regulate the differentiation of prostatic fibroblasts into smooth muscle cells. METHODS: Smooth muscle actin levels were monitored by immunoblot analysis and immunocytochemistry. Proliferation was measured in clonal growth assays and by cell counts. RESULTS: We determined that TGFbeta inhibited proliferation and induced smooth muscle differentiation of stromal cells derived from prostatic adenocarcinomas, as we previously reported for cells derived from the normal peripheral zone. Basic FGF, EGF, TGFalpha, and PDGF, but not IGF, retinoic acid, 1,25-dihydroxyvitamin D3, or androgen, attenuated induction of differentiation by TGFbeta, by a mechanism apparently unrelated to proliferation. CONCLUSIONS: Regulation of growth and differentiation occurs equivalently in prostatic stromal cells derived from adenocarcinomas and normal peripheral zone. TGFbeta is a potent inducer of the smooth muscle phenotype. Basic FGF, EGF and/or TGFalpha, and PDGF attenuate TGFbeta's activity, and promote a fibroblastic phenotype. Our studies provide an in vitro model system in which fibroblastic or smooth muscle cells can be promoted, maintained, and investigated in a defined manner. The results suggest that the ratio of fibroblasts to smooth muscle cells in the stroma reflects the relative levels of growth factors, which may be altered in diseased states.     

Studies of human diploid fibroblast growth. I. Responses of normal and hypopituitary cells to fibroblast growth factor, insulin, and serum

We have assayed the growth stimulating activity of bovine insulin, fibroblast growth factor (FGF), and fetal bovine serum (FBS) in diploid human fibroblasts from normal and idiopathic hypopituitary donors. All three factors stimulated DNA synthesis in cells arrested by serum starvation. FGF was active at concentrations as low as 5 ng/ml with maximum effect at 100 ng/ml. FGF stimualted DNA synthesis at lower concentrations than did insulin and also produced a greater maximum response. Only serum was capable of supporting cell division and growth, but FGF accellerated this growth rate when it was added to serum-containing medium. Hydrocortisone, actinomycin D, and cycloheximide inhibit FGF stimulation. There was no significant difference between fibroblasts from normal and hypopituitary donors.     

Expression of tissue factor pathway inhibitor in vascular smooth muscle cells and its regulation by growth factors

Tissue factor pathway inhibitor (TFPI) in vivo is thought to be synthesized mainly by endothelial cells. To date, no significant regulator of TFPI synthesis has been described. Vascular smooth muscle cells (VSMC) express tissue factor in vitro and in vivo, which may contribute to vascular thrombosis. We hypothesized that VSMC might also express TFPI. To determine this, we examined growth-arrested coronary VSMC in culture and found that VSMC secreted an amount of TFPI similar to that seen in endothelial cells. Immunohistochemistry of normal human coronary arteries showed TFPI staining throughout the media and intima of the vessel with localization to VSMC and endothelial cells. To determine regulation of TFPI expression in VSMC, we examined the effects of serum stimulation on TFPI secretion and found that FBS induced a 5-fold increase in TFPI antigen and activity levels in conditioned medium at 48 hours (P<0.001) when compared with serum-free conditions. A similar stimulatory effect was seen with 10% pooled human serum. Moreover, epidermal growth factor and platelet-derived growth factor-B increased TFPI secretion by 4- to 5-fold and 2- to 3-fold, respectively (P<0.05), and these growth factors accounted for approximately 50% of the TFPI secretion effects of human serum. The serum effect was associated with a 3-fold increase in TFPI mRNA 24 hours after release from growth arrest and a 50% decrease in TFPI secretion after treatment with actinomycin D. Taken together, this study suggests that there is significant TFPI expression in VSMC in culture and in VSMC within the intima and media of the normal coronary artery wall. We present the first evidence for TFPI regulation by serum in VSMC and more specifically by its constituent growth factors, epidermal growth factor and platelet-derived growth factor-B.