The heterogeneous contents of the CNS interstitial clefts and the configuration of their astrocytic walls may be regionally variable. Astrocytic processes of the glia limitans, in normal midbrain and in astroglial scars, form thin, parallel, concentric sheets comprising the walls of narrow interstitial clefts. There is a critical thickness of about <em>20</em> to 30 nm, below which astrocytic cell process or those of the <em>fibroblast</em>-like cells in the meninges, do not invaginate to form transcytotic vesicles. Large hydrophilic solutes cannot, therefore, pass across the thin portion of a cell process. Consequently, (a) the diffusion and convection paths of interstitial fluid and solutes are lengthened, (b) a solute will remain within the interstitial cleft between thin lamellae for a relatively long time and (c) if a ligand does bind to its receptor on the thin process's cell membrane, there can be no receptor-mediated transcytosis at that site. Interstitial clefts, themselves, vary in size, shape and content, including extracellular matrix and basal lamina. A common constituent of basal lamina and extracellular matrix, presumably including that at ependymal, astroglial and endothelial interfaces of the CNS, is heparan sulfate proteoglycans. As in other organs, these proteoglycans may store <em>growth</em> <em>factors</em>, <em>growth</em> inhibitors, cytokines and other modulators which can then be released enzymatically during, e.g., regeneration. Exogenous heparan sulfate proteoglycan might serve as a natural, intermittent-release matrix for delivery of trophic <em>factors</em>.

The gut microbiota were shown to play critical roles in the development of atherosclerosis, but the detailed mechanism is limited. The purpose of this study is to clarify the influence of gut microbiota on atherogenesis via lipid metabolism and systemic inflammation. Germ-free or conventionally raised (Conv) ApoE-deficient (ApoE-/-) mice were fed chow diet and euthanized at <em>20</em> weeks of age. We found that the lack of gut microbiota in ApoE-/- mice caused a significant increase in the plasma and hepatic cholesterol levels compared with Conv ApoE-/- mice. The absence of gut microbiota changed the bile acid composition in the ileum, which was associated with activation of the enterohepatic <em>fibroblast</em> <em>growth</em> <em>factor</em> 15, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 axis, and reduction of cholesterol 7α-hydroxylase and hepatic bile acid synthesis, resulting in the accumulation of liver cholesterol content. However, we found that the lack of microbiota caused a significant reduction in atherosclerotic lesion formation compared with Conv ApoE-/- mice, which might be associated with the attenuation of lipopolysaccharide-mediated inflammatory responses. Our findings indicated that the gut microbiota affected both hypercholesterolemia and atherogenesis in mice.

Connective tissue <em>growth</em> <em>factor</em> (CTGF; now often referred to as CCN2) is a secreted protein predominantly expressed during development, in various pathological conditions that involve enhanced fibrogenesis and tissue fibrosis, and in several cancers and is currently an emerging target in several early-phase clinical trials. Tissues containing high CCN2 activities often display smaller degradation products of full-length CCN2 (FL-CCN2). Interpretation of these observations is complicated by the fact that a uniform protein structure that defines biologically active CCN2 has not yet been resolved. Here, using DG44 CHO cells engineered to produce and secrete FL-CCN2 and cell signaling and cell physiological activity assays, we demonstrate that FL-CCN2 is itself an inactive precursor and that a proteolytic fragment comprising domains III (thrombospondin type 1 repeat) and IV (cystine knot) appears to convey all biologically relevant activities of CCN2. In congruence with these findings, purified FL-CCN2 could be cleaved and activated following incubation with matrix metalloproteinase activities. Furthermore, the C-terminal fragment of CCN2 (domains III and IV) also formed homodimers that were ∼<em>20</em>-fold more potent than the monomeric form in activating intracellular phosphokinase cascades. The homodimer elicited activation of <em>fibroblast</em> migration, stimulated assembly of focal adhesion complexes, enhanced RANKL-induced osteoclast differentiation of RAW264.7 cells, and promoted mammosphere formation of MCF-7 mammary cancer cells. In conclusion, CCN2 is synthesized and secreted as a preproprotein that is autoinhibited by its two N-terminal domains and requires proteolytic processing and homodimerization to become fully biologically active.

Synovial membrane has been shown to contain mesenchymal stem cells. We hypothesized that an enriched population of synovial <em>fibroblasts</em> would undergo chondrogenic differentiation and secrete cartilage extracellular matrix to a greater extent than would a mixed synovial cell population (MSCP). The optimum doses of transforming <em>growth</em> <em>factor</em> beta 1 (TGF-beta1) and insulin-like <em>growth</em> <em>factor</em> 1 (IGF-1) for chondrogenesis were investigated. CD14-negative isolation was used to obtain a porcine cell population enriched in type-B synovial <em>fibroblasts</em> (SFB) from an MSCP. The positive cell surface markers in SFB were CD90, CD44, and cadherin-11. SFB and MSCP were cultured in the presence of <em>20</em> ng/mL TGF-beta1 for 7 days, and SFB were demonstrated to have higher chondrogenic potential. Further dose-response studies were carried out using the SFB cells and several doses of TGF-beta1 (2, 10, <em>20</em>, and 40 ng/mL) and/or IGF-1 (1, 10, 100, and 500 ng/mL) for 14 days. TGF-beta1 supplementation was essential for chondrogenesis and prevention of cell death, whereas IGF-1 did not have a significant effect on the SFB cell number or glycosaminoglycan production. This study demonstrates that the CD14-negative isolation yields an enhanced cell population SFB that is more potent than MSCP as a cell source for cartilage tissue engineering.

The aim of the present study is to clarify in situ expression of platelet-derived <em>growth</em> <em>factor</em> (PDGF) and its receptors in different phases of inflammatory bowel disease (IBD). Tissues samples were obtained from <em>20</em> patients with ulcerative colitis (UC) and 29 with Crohn's disease (CD) at surgery. In situ hybridization and immunohistochemistry on frozen sections were performed for PDGF-A and -B and its alpha and beta receptors (alphaR and betaR). The area of active inflammation was infiltrated by abundant polymorphonuclear and mononuclear leukocytes, of which the latter expressed mRNA and proteins of PDGF-A, -B, and -alphaR and mRNA for PDGF-betaR. The area of active fibrosis, characterized by activated <em>fibroblasts</em>/ myo<em>fibroblasts</em>, was juxtaposed to ulceration, which is induced as a repair process to tissue destruction. In these areas, activated <em>fibroblasts</em>/myo<em>fibroblasts</em> were positive for mRNA and protein of PDGF-A, -B, -alphaR, and -betaR. The expression of PDGF-A, -B, and -alphaR declined significantly in the scar area. Our results suggest that PDGF is not only important as an inducer of fibrosis in the repair phase but also it is involved in the active inflammatory phase possibly as a chemoattractant for mononuclear inflammatory cells.

Articular cartilage regeneration is insufficient to restore sports injuries or defects that can occur from trauma. Treatment options for cartilage repair include autologous chondrocyte implantation (ACI) by isolation, expansion, and reimplantation of healthy donor chondrocytes. Chondrocyte expansion onto 2D substrates leads to dedifferentiation and loss of the cellular phenotype. We aimed to overcome the state of dedifferentiation by biochemical stimuli with platelet derivatives such as platelet-rich plasma (PRP) and hyperacute serum (HAS) to achieve sufficient cell numbers in combination with variable oxygen tension. Human articular chondrocytes from osteoarthritic (OA) cartilage chondrocytes were switched from 10% FCS supplementation to either 10% PRP or 10% HAS after initial passaging for further experiments under normoxic (<em>20</em>% O2) or hypoxic (1% O2) conditions. An XTT assay measured the effect of PRP or HAS on the cell proliferation at 3, 6, and 9 days. The chondrogenic redifferentiation potential of dedifferentiated chondrocytes was determined with reverse transcriptase quantitative real-time PCR for markers of expression for type II collagen (COL2A1), type I collagen (COL1A1), and matrix metalloproteinases MMP3, matrix metalloproteinase 13 (MMP13) at 24 and 72 h. Measured protein levels of 100% PRP or HAS by multiplex quantification revealed basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, G-CSF, and PDGF were significantly higher in PRP than in HAS (p < 0.05) but LEPTIN levels did not differ. The quantified protein levels did not differ when isolated from same donors at a different time. Chondrocyte proliferation indicated that supplementation of 10% HAS enhanced the proliferation rate compared to 10% PRP or 10% FCS at 6 and 9 days significantly (p < 0.05). mRNA levels for expression of COL1A1 were significantly downregulated (p < 0.05) when cultured with 10% PRP than 10% HAS or 10% FCS under normoxic/hypoxic conditions. COL2A1 was significantly upregulated (p < 0.05) in PRP than 10% HAS or 10% FCS. MMP3 expression was downregulated after 72 h under all conditions. MMP13 was upregulated with 10% PRP at both 24 and 72 h but significantly downregulated under hypoxia (1% O2) for all circumstances. While HAS has its effect on chondrocyte proliferation, PRP enhances both proliferation and redifferentiation of dedifferentiated chondrocytes. PRP can replace standard usage of FCS for chondrogenic priming and expansion as implications for clinical use such as ACI procedures.

In vertebrates, pancreas and liver arise from bipotential progenitors located in the embryonic gut endoderm. Bone morphogenic protein (BMP) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) signaling pathways have been shown to induce hepatic specification while repressing pancreatic fate. Here we show that BMP and FGF <em>factors</em> also play crucial function, at slightly later stages, in the specification of the ventral pancreas. By analyzing the pancreatic markers pdx1, ptf1a, and hlxb9la in different zebrafish models of BMP loss of function, we demonstrate that the BMP pathway is required between <em>20</em> and 24 h postfertilization to specify the ventral pancreatic bud. Knockdown experiments show that bmp2a, expressed in the lateral plate mesoderm at these stages, is essential for ventral pancreas specification. Bmp2a action is not restricted to the pancreatic domain and is also required for the proper expression of hepatic markers. By contrast, through the analysis of fgf10(-/-); fgf24(-/-) embryos, we reveal the specific role of these two FGF ligands in the induction of the ventral pancreas and in the repression of the hepatic fate. These mutants display ventral pancreas agenesis and ectopic masses of hepatocytes. Overall, these data highlight the dynamic role of BMP and FGF in the patterning of the hepatopancreatic region.

The <em>fibroblast</em> is a prominent cellular component of the periodontal ligament. It is believed to play an important role in collagen metabolism in health and disease. The turnover of collagen in the periodontal ligament is believed to be controlled by the balance between collagen synthesis and degradation. The family of matrix metalloproteinases and their inhibitors is one of the mechanisms which regulates this balance. The <em>factors</em> that regulate the synthesis of collagenase and its inhibitor, TIMP-1, by the periodontal ligament cell are poorly understood. The present study was undertaken to assess the effect of interleukin-1 beta (IL-1 beta), platelet-derived <em>growth</em> <em>factor</em> (PDGF), and transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta) on the expression of collagenase (MMP-1) and TIMP-1 mRNA in periodontal derived <em>fibroblasts</em> using reverse transcription polymerase chain reaction (RT-PCR). Early passage periodontal ligament derived <em>fibroblasts</em> were treated with IL-1 beta (10 and 100 pg/ml), two isoforms of PDGF, -AA and -BB (4 and <em>20</em> ng/ml) and TGF-beta (1 and 10 ng/ml). Treatment with <em>growth</em> <em>factors</em> from 2 to 24 hours revealed that the largest effects on MMP-1 mRNA occurred after 24 hours. IL-1 beta induced a 5 to 9 fold increase in MMP-1 mRNA. The two isoforms of PDGF had less of an effect (3 to 5 fold) on MMP-1 mRNA whereas TGF-beta induced a 25 to 50% decrease in the expression of this message. None of the <em>growth</em> <em>factors</em> had an effect on TIMP-1 mRNA expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoids have been shown to accelerate fetal lung type II cell maturation, and this effect appears, in part, to be mediated via <em>fibroblasts</em>. To identify glucocorticoid induced genes in fetal lung <em>fibroblasts</em>, we screened a cDNA library from cortisol-treated fetal lung <em>fibroblasts</em> with a subtracted cDNA probe which was enriched for sequences specific for cortisol-treated fetal lung <em>fibroblasts</em>. Fifty-seven clones were isolated from the cDNA library. One cDNA represented approximately 30% of the 57 clones. Analysis of DNA sequence homology suggested that this cDNA encodes the rat transforming <em>growth</em> <em>factor</em>-beta 3 (TGF beta 3). We found that TGF beta 3 mRNA was expressed in fetal lung <em>fibroblasts</em> but not epithelial cells. Expression of message in fetal lung <em>fibroblasts</em> was developmentally regulated. TGF beta 3 mRNA levels were low during the pseudoglandular stage (day 18), peaked during the early canalicular stage of lung development (day 19), then fell again at days <em>20</em> and 21 (term = 22 days). Exposure of fetal lung <em>fibroblasts</em> to cortisol increased TGF beta 3 mRNA expression in a time- and dose-dependent manner. Maternal administration of dexamethasone also enhanced mRNA expression of TGF beta 3 in fetal lung <em>fibroblasts</em>. These data suggest that glucocorticoids may mediate their stimulatory effect on lung maturation by inducing TGF beta 3 expression in fetal lung <em>fibroblasts</em>.

BACKGROUND

Fibroblast growth factor 9 (FGF9) enhances cell proliferation and invasiveness in several malignant diseases. The aim of the present study is to investigate the role of FGF9 in postoperative recurrence after radical prostatectomy.

METHODS

Cell viability and invasion of LNCaP cells were assessed using MTT assay and Matrigel invasion assay, respectively, in the presence or absence of treatment with recombinant FGF9. Tissues obtained during a radical prostatectomy in 133 male patients were immunohistochemically stained using anti-FGF9 antibody.

RESULTS

Cell viability and invasion of LNCaP was significantly enhanced by treatment with recombinant FGF9. Immunohistochemical staining detected FGF9-positive cells in 20 samples. The prevalence of FGF9-positive cells in cases with a Gleason score of 8 or higher was 34.2%, which was significantly higher than that in those with Gleason scores of 7 or lower (7.3%, P=0.0003), respectively. The 3-year biochemical relapse-free survival rate was 17.5% in cases with FGF9-positive cells, which was significantly lower than that in cases in which FGF9-positive cells were not detectable (75.5%, P < 0.0001).

CONCLUSIONS

These results indicate that FGF9 can stimulate proliferation and invasion in prostate cancer cells, thus FGF9 could be a candidate of a predictive factor for recurrence after radical prostatectomy.

BACKGROUND

High expression of vascular endothelial growth factor (VEGF) induces subepithelial fibrosis associated with angiogenesis in patients with asthma. Thrombin is recognized as a new candidate mediating airway remodeling. Therefore, this study was designed to determine the role of up-regulated thrombin activity induced by VEGF on airway remodeling in patients with asthma.

METHODS

Levels of biochemical parameters in induced sputum were examined in 21 asthmatic patients and 11 normal control subjects.

RESULTS

Thrombin activity in induced sputum was significantly higher in asthmatic patients than in normal control subjects: median, 3.67 U/mL; range, 1.15 to 10.2 U/mL; vs median, 1.26 U/mL; range, 0.93 to 2.42 U/mL (p < 0.0001). In contrast, protein C activity in induced sputum was lower in asthmatic patients than in normal control subjects: median, 20%; range, 5 to 30%; vs 41%; range, 30 to 59% (p < 0.0001). VEGF level in induced sputum was positively correlated with thrombin activity in asthmatic patients (r = 0.55, p = 0.02), while inversely correlated with protein C activity (r = - 0.57, p = 0.01). Levels of basic fibroblast growth factor (bFGF), a major profibrotic factor, were also significantly higher in asthmatic patients than in normal control subjects. Moreover, thrombin activity was significantly correlated with bFGF level in asthmatic patients (r = 0.67, p = 0.003).

CONCLUSIONS

Increase in VEGF level leads to up-regulation of thrombin activity in asthmatic airways, and this elevated thrombin activity induces elevation of bFGF level. It will become to be a new strategy of asthma therapy to attenuate thrombin activity for the regulation of airway remodeling.

Generation of contractile forces as <em>fibroblasts</em> attach and migrate through collagenous substrates is a fundamental behavior, yet its regulation and consequences are obscure. Although the transforming <em>growth</em> <em>factor</em>-betas (TGF-beta) are similarly important in fibrosis and tissue repair, their role in contraction is controversial. Using a quantitative, 3D collagen culture model we have measured the effects of TGF-beta1 and -beta3 on contractile forces generated by human dermal <em>fibroblasts</em>. Maximal stimulation was between 7.5 and 15 ng/ml of TGF-beta1. Higher doses were inhibitory (30 ng/ml), giving a bell-shaped dose response. The initial rate of force generation was increased sevenfold (15 ng/ml). A similar response pattern was seen with TGF-beta3 alone. However, the addition of both isoforms together stimulated a biphasic increase in force generation, suggesting that there was a distinct temporal cooperativity between the two isforms. This very early onset (10-<em>20</em> min) of stimulation suggested that TGF-beta might act through cell attachment and integrin function and the effect of TFG-beta on expression of fibronectin (FnR) and vitronectin (VnR) integrin receptors was monitored over the same time scale. TGF-beta1 dramatically up-regulated VnR expression, relative to FnR, over time but the optimal time for this was 2-4 h later than that of force stimulation. It is concluded that TGF-beta1 and -beta3 behave here primarily as mechanoregulatory <em>growth</em> <em>factors</em> and that stimulation of integrin expression may be a consequence of the altered cell stress.

BACKGROUND

Ethanol exposure during development leads to various forms of neuronal damage. Because neural stem cells (NSCs) play a pivotal role in the development and maturation of the central nervous system, it is important to understand the effect of ethanol on NSC differentiation. In this study, we investigated the effect of ethanol on differentiation of cultured NSCs in the presence and absence of neurotrophic factors.

METHODS

NSCs were derived from rat embryos on embryonic day 14. Cells were exposed to ethanol with or without neurotrophic factors, insulin-like growth factor-1 (IGF-1), or brain-derived neurotrophic factor (BDNF). The effect of ethanol on differentiation was quantified by measurement of optical density of each sample following to microtubule-associated protein 2 enzyme-linked immunosorbent assay and counting of the number of microtubule-associated protein 2-positive cells microscopically. In addition, cell viability of cultured cortical neurons that were exposed to similar concentrations of ethanol was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay.

RESULTS

Ethanol (20-100 mM) inhibited NSC differentiation induced by basic fibroblast growth factor removal, whereas those concentrations of ethanol did not affect neuronal survival. Both IGF-1 and BDNF promoted generation of neurons in the absence of ethanol. Moreover, they suppressed the inhibitory effect of ethanol on NSC differentiation.

CONCLUSIONS

Ethanol inhibited NSC differentiation at concentrations much lower than what compromised neuronal survival. Ethanol-induced differential inhibition was reduced by both IGF-1 and BDNF. These results suggest that ethanol inhibits stem cell differentiation through alteration of cellular pathways related to neurotrophic factor signaling.

CD34 is a transmembrane glycoprotein constitutively expressed on endothelial cells and hematopoietic stem cells. Use of CD34-recognizing antibodies has helped in the identification and isolation of CD34+ endothelial precursors from embryonic and adult tissues. However, CD34-null mice display no vascular abnormalities, demonstrating that CD34 antigen expression is not required for normal vascular development. Here we show that a CD34- cell population that includes endothelial cell precursors can be isolated from cord blood. In the presence of angiogenic <em>factors</em>, these cells mature to express the endothelial cell markers vascular endothelial-cadherin, vascular endothelial <em>growth</em> <em>factor</em> receptor-1 and -2, Tie-1 and -2 (tyrosine kinase with immunoglobulin and epidermal <em>growth</em> <em>factor</em> homology domains), von Willebrand <em>factor</em>, and CD31 while maintaining their CD34- status, and can be expanded in vitro for over <em>20</em> passages. Moreover, in functional studies, these cells can undergo extracellular matrix-dependent morphogenic changes into capillary-like tubular structures. When transplanted into immunodeficient mice in conjunction with tumor cells or with the proangiogenic <em>factor</em> basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, these cells can form functional microvessels arising along with host blood cells. These studies provide strong evidence for the existence of CD34- endothelial cell precursors in cord blood and suggest the use of ex vivo-expanded cord blood CD34- cells as a unique tool for the investigation of postnatal lineage diversification.

Reactive gliosis was revealed by immunocytochemistry using antibodies against the glial fibrillary acidic protein (GFAP) after a stab or an electrolytic lesion administered to the cerebral cortex, corpus callosum, striatum, or hippocampus of a 6-day-old rat. The intensity of the gliosis was about the same in the various structures injured and did not change with the delay of 3, 7, or <em>20</em> days between the injury and the sacrifice of the animals. When basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was injected in the lesion locus just after the lesion was performed, it resulted (as soon as 3 days after injury) in a strong astrogliosis that was enhanced after a delay of 7 days, the astrocytes in the lesion area exhibiting enlarged cell processes and intense GFAP-positive immunoreactivity. After a delay of <em>20</em> days, the astrocytes were not dispersed any more but packed in three or four layers along the borders of the lesion, thus reducing its extension. This suggests a possible role for bFGF in promoting scar formation following brain injury.

We examined the time course of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) immunoreactivity and its mRNA level mainly in the hippocampus after transient forebrain ischemia using immunohistochemistry, enzyme immunoassay (EIA), Western blot analysis and in situ hybridization. Neuronal death in the hippocampal CA1 subfield was observed 72 h after <em>20</em> min of ischemia. The number of bFGF-immunoreactive(IR) cells increased 48 h-5 days after ischemia in all hippocampal regions. At 10 and 30 days, the bFGF-IR cells in the CA1 subfield had further increased in numbers and altered their morphology, enlarging and turning into typical reactive astrocytes with the advancing neuronal death in that area. In contrast, the number of bFGF-IR cells in other hippocampal regions had decreased 30 days after ischemia. The EIA study showed a drastic increase in bFGF levels in the hippocampus 48 h after ischemia (150% of that in normal rat) which was followed by further increases. In Western blot analysis, three immunoreactive bands whose molecular weights correspond to 18, 22 and 24 kDa were observed in normal rat and ischemia increased all their immunoreactivities. In the in situ hybridization study of the hippocampus, bFGF mRNA positive cells were observed in the CA1 subfield in which many bFGF-IR cells existed after ischemia. These data demonstrate that transient forebrain ischemia leads to an early and strong induction of bFGF synthesis in astrocytes, suggesting that the role of bFGF is related to the function of the reactive astrocytes which appear following brain injury.

With diabetes affecting 5% to 10% of the US population, development of a more effective treatment for chronic diabetic wounds is imperative. Clinically, the current treatment in topical wound management includes debridement, topical antibiotics, and a state-of-the-art topical dressing. State-of-the-art dressings are a multi-layer system that can include a collagen cellulose substrate, neonatal foreskin <em>fibroblasts</em>, <em>growth</em> <em>factor</em> containing cream, and a silicone sheet covering for moisture control. Wound healing time can be up to <em>20</em> weeks. The future of diabetic wound healing lies in the development of more effective artificial "smart" matrix skin substitutes. This review article will highlight the need for novel smart matrix therapies. These smart matrices will release a multitude of <em>growth</em> <em>factors</em>, cytokines, and bioactive peptide fragments in a temporally and spatially specific, event-driven manner. This timed and focal release of cytokines, enzymes, and pharmacological agents should promote optimal tissue regeneration and repair of full-thickness wounds. Development of these kinds of therapies will require multidisciplinary translational research teams. This review article outlines how current advances in proteomics and genomics can be incorporated into a multidisciplinary translational research approach for developing novel smart matrix dressings for ulcer treatment. With the recognition that the research approach will require both time and money, the best treatment approach is the prevention of diabetic ulcers through better foot care, education, and glycemic control.

The effect of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) on the <em>growth</em> of chondrocytes in soft agar was examined. FGF induced colony formation by chick embryo and rabbit chondrocytes. The colony-forming efficiency of FGF-exposed chondrocytes was similar to that of Rous sarcoma virus-transformed chondrocytes (15-<em>20</em>%). Other mitogenic agents tested, such as epidermal <em>growth</em> <em>factor</em>, insulin, insulin-like <em>growth</em> <em>factor</em>-l, and platelet-derived <em>growth</em> <em>factor</em>, induced very low levels of colony formation. The induction of <em>growth</em> in soft agar of chondrocytes by FGF was not due to cells' phenotypic transformation, because chondrocytes grown in soft agar with FGF retained the ability to synthesize cartilage-characteristic proteoglycan. FGF did not induce <em>growth</em> in soft agar of chondrocytes whose phenotypic expression was suppressed by retinoic acid or 5-bromodeoxyuridine. In addition, FGF did not induce <em>growth</em> in soft agar of primary <em>fibroblasts</em> and normal rat kidney (NRK) cells. These results suggest that FGF selectively stimulates <em>growth</em> of differentiated chondrocytes in soft agar.

BACKGROUND

Fibroblast growth factor (FGF-23) is a novel phosphaturic factor. Current data suggest that serum phosphate, dietary phosphate and 1,25 dihydroxyvitamin D regulate circulating FGF-23 levels in vivo. We examined if hypogonadism-induced increases in serum phosphate are associated with increases in circulating FGF-23 in healthy men in the absence of dietary manipulation.

METHODS

25 healthy men were administered goserelin acetate (GnRH analog) 3.6 mg subcutaneously every 4 weeks for 12 weeks to induce acute testosterone and estrogen deficiency. Subjects consumed an ad libitum diet. Morning fasting blood and urine samples were collected to measure serum phosphate, serum intact FGF-23, PTH, and the maximum tubular reabsorption of phosphate (T(m)P/GFR) at baseline, weeks 4 and 12. The changes in serum FGF-23 and phosphate at weeks 4 and 12 were compared to baseline using paired t-tests.

RESULTS

Goserelin therapy decreased mean serum testosterone levels from 543+/-160 ng/dL to 33+/-15 ng/dL at week 4 (p<0.001), and to 20+/-10 ng/dL at week 12 (p<0.001). Serum phosphate increased significantly from 3.4+/-0.6 mg/dL to 3.9+/-0.4 mg/dL at week 4 (p=0.002), and to 4.3+/-0.4 mg/dL at week 12 (p<0.001). T(m)P/GFR increased significantly from 3.2+/-0.6 mg/dL to 3.6+/-0.5 mg/dL at week 4 (p<0.004), and to 4.1+/-0.6 mg/dL at week 12 (p<0.001). FGF-23 levels, however, did not change during the 12-week study.

CONCLUSIONS

Gonadal steroid deprivation increased serum phosphate levels in men but did not affect serum FGF-23 concentrations. The absence of any change in circulating FGF-23 suggests that supraphysiologic levels of serum phosphate may be required to stimulate circulating FGF-23 or that FGF-23 production is primarily sensitive to changes in dietary phosphate or 1,25 dihydroxyvitamin D within this physiologic serum phosphate range.

OBJECTIVE

To develop a method for grafting endothelial cells isolated from organ-cultured adult human corneas onto the denuded Descemet's membrane of human recipients.

METHODS

Adult human or porcine corneal endothelial cells were isolated and maintained in monolayer cultures before seeding. Recipient corneas were stripped of their own endothelium by one of three different methods (mechanical, chemical, or physical) and the completeness of removal assessed after vital staining. The utility of each method was evaluated by monitoring the quality of attachment of the seeded-cell population. The seeding density of transplanted cells required for optimal results also was determined and the final numeric cell density achieved on recipient corneas after culturing for 7-<em>20</em> days ascertained. The influence of incubating source cells with <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), both on this latter parameter and on cell morphology, also was evaluated. The functional integrity of regrafted endothelium was assessed in 24-h perfusion experiments.

RESULTS

The seeding of between 150,000 and 700,000 cells onto recipient corneas, followed by gentle centrifugation to improve attachment, yielded maximal final numeric cell densities of 3,450/mm2 and 1,850/mm2 in porcine and human lines, respectively. Recipient corneas were most effectively denuded of their own endothelium by freezing-and-thawing. The newly established endothelial monolayer remained stable for up to <em>20</em> days in organ culture (longest period monitored). FGF treatment did not enhance the final numeric density of cells attained on recipient corneas, but it did have a beneficial effect on their morphology. Only those recipient corneas that exhibited a well-differentiated monolayer of seeded endothelial cells underwent stromal deswelling near to physiologic levels.

CONCLUSIONS

A practical working model has been developed, whereby recipient corneas stripped of their own endothelium can be furnished with a "new," near-normal endothelium by appropriate manipulations of the seeded-cell population. This now paves the way for a realistic tackling of the problem of endothelial cell paucity in donor corneas destined for transplantation.

Insulin-like <em>growth</em> <em>factor</em> (IGF)-binding protein-5 (IGFBP-5) is cleaved by a serine protease that is secreted by <em>fibroblasts</em> and porcine smooth muscle cells (pSMC) in culture. To investigate whether other serine proteases could cleave this substrate at physiologically relevant concentrations, we determined the proteolytic effects of thrombin on IGFBP-5. Human alpha-thrombin (0.0008 NIH U/ml) cleaved IGFBP-5 into 24-, 23-, and <em>20</em>-kDa non-IGF-I-binding fragments. Cleavage occurred at a physiologically relevant thrombin concentration. The effect was specific for IGFBP-5, as other forms of IGFBPs, e.g. IGFBP-1, IGFBP-2, and IGFBP-4 were not cleaved by thrombin. Although IGFBP-3 was cleaved by thrombin, this effect required a 50-fold greater thrombin concentration. [35S]Methionine labeling followed by immunoprecipitation confirmed that IGFBP-5 that was constitutively synthesized by pSMC cultures was also degraded by thrombin into 24-, 23-, and <em>20</em>-kDa fragments. The binding of IGF-I to IGFBP-5 partially inhibited IGFBP-5 degradation by thrombin, and an IGF analog that does not bind to IGFBP-5 had no effect. Thrombin did not account for the serine protease activity that had been shown previously to be present in pSMC-conditioned medium. This was proven by showing that 1) no immunoreactive thrombin could be detected in the pSMC-conditioned medium; 2) the IGFBP-5 fragments that were generated by thrombin showed three cleavage sites (Arg192-Ala193, Arg156-Ile157, and Lys1<em>20</em>-His121), whereas the serine protease in conditioned medium cleaves IGFBP-5 at a different site; and 3) hirudin had no effect on IGFBP-5 cleavage by the protease in pSMC medium; however, it inhibited IGFBP-5 degradation by thrombin. To determine the physiological significance of IGFBP-5 cleavage, the effect of an IGFBP-5 mutant that is resistant to cleavage by the pSMC protease and has been shown to inhibit IGF-I actions in pSMC was determined. This mutant inhibited IGF-I-stimulated DNA synthesis, but if thrombin was added simultaneously, IGF-I was fully active. In summary, physiological concentrations of thrombin degrade IGFBP-5. Degradation can be blocked by hirudin and is partially inhibited by IGF-I binding. Generation of active thrombin in vessel walls may be a physiologically relevant mechanism for controlling IGF-I bioactivity.

We have previously reported the presence of proteolytic activity in conditioned medium from human <em>fibroblast</em> cultures that cleaves insulin-like <em>growth</em> <em>factor</em>-binding protein-5 (IGFBP-5) into non-IGF-I-binding fragments. Coincubation of IGF-I or IGF-II and IGFBP-5 with <em>fibroblast</em> cultures decreased proteolysis. The protease was purified by heparin-Sepharose affinity chromatography. The purified protease cleaved IGFBP-5 into 22-, <em>20</em>-, and 17-kilodalton non-IGF-I-binding fragments. Protease inhibitor profiles obtained using partially purified enzyme showed that it was a calcium-dependent serine protease. After chelation with EDTA, the activity could only be partially restored with zinc, indicating that it was probably not a metalloprotease. The protease was specific for IGFBP-5 and did not cleave pure IGFBP-1, -2, -3, or -4. IGF-I and IGF-II caused minimal inhibition of proteolysis in vitro. This suggests that the IGF-I-induced increase in IGFBP-5 in <em>fibroblast</em> medium is only partially due to direct protease inhibition. Heparin, antithrombin-III (AT-III), and heparin co<em>factor</em>-II had inhibitory activity, and heparin potentiated the activity of AT-III. Synthetic peptides, that contained the active sites of AT-III and alpha 1-antichymotrypsin, were also inhibitory. Peptides containing sequences found in two basic regions of IGFBP-5 were tested, and one had inhibitory activity. In summary, <em>fibroblasts</em> secrete a serine protease that cleaves IGFBP-5 and is specific for this form of IGFBP. The protease has properties that are similar to kallikreins, a family of serine proteases that is known to cleave epidermal and nerve <em>growth</em> <em>factor</em>-binding proteins.

A biodegradable non-woven hyaluronic acid polymer scaffold (Hyaff 11) was analysed in vitro as a carrier vehicle for differentiation and mineralization of rat bone marrow stromal cells (BMSC). BMSC were grown on Hyaff 11 in a mineralizing medium in the presence/absence of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). Osteoblastic differentiation was investigated by light and electron microscopy analysing the expression of osteogenic markers: calcium, alkaline phosphatase (AP), osteopontin (OP), bone sialoprotein (BSP) and collagen type 1. We also measured proliferation, AP activity and mRNA expression of AP and osteocalcin (OC). Electron microscopy and Toluidine-blue staining demonstrated that bFGF accelerated (day <em>20</em> vs. day 40) and increased mineralization. With bFGF, calcium, OP and BSP were strongly enhanced at day 40, whereas AP decreased. Our in vitro results demonstrate that Hyaff 11 is a useful vehicle for <em>growth</em>, differentiation and mineralization of rat BMSC, and that it permits bone development.

Hepatocyte <em>growth</em> <em>factor</em> (HGF), a potent mitogen for mature hepatocytes, has been considered to act as a hepatotropic <em>factor</em> for liver regeneration. We examined the effect of HGF on albumin synthesis and DNA synthesis of adult rat hepatocytes cultured at various cell densities. HGF stimulated albumin synthesis of hepatocytes by 40-60% when they were cultured at higher cell densities such that there was tight cell-cell contact. But at lower cell densities HGF failed to stimulate albumin synthesis. In contrast, the stimulatory effect of HGF on DNA synthesis of hepatocytes was more potent at lower than at higher cell densities: HGF did not stimulate DNA synthesis of hepatocytes cultured at confluent cell density. Thus, HGF seems to stimulate both albumin synthesis and DNA synthesis of hepatocytes, in a reciprocal relationship depending on cell density. When the effects of various cytokines were examined, epidermal <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em>-alpha, and acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> also stimulated albumin synthesis by <em>20</em>-30%. However, transforming <em>growth</em> <em>factor</em>-beta 1, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and interleukin-1 beta had no effect on albumin synthesis, while interleukin-6 inhibited it by 42%. Thus HGF was the most potent in stimulating albumin synthesis in these cytokines. Since HGF is markedly increased in the liver or plasma following various liver insults, HGF may be involved in liver regeneration through the potential to stimulate both cell <em>growth</em> and liver-specific functions such as albumin synthesis in a cell density-dependent manner.