<em>Growth</em> of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed of a 1:1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing <em>15</em> mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 micrograms/ml glutathione, 10 micrograms/ml insulin, 10 micrograms/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 micrograms/ml ethanolamine, 20 ng/ml epidermal <em>growth</em> <em>factor</em>, 2.0 nM 17 beta-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate. Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 micrograms/ml Tf, and 200 micrograms/ml BSA, which sustained MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA, insulin and insulin-like <em>growth</em> <em>factor</em> I (IGF-I) were mitogenic with ED50 values of 2 to 3 ng/ml and 30 to <em>15</em>0 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these <em>factors</em> in Tf-BSA but required 10-fold higher concentrations for ED50. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At less than or equal to ng/ml concentrations, epidermal <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em> II, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> were mitogenic for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA. This new method offers a convenient means of comparing the potencies of <em>growth</em>-promoting <em>factors</em> on human breast cancer cells without interfering activities known to be present in serum.

Both keloids (KLs) and hypertrophic scars (HSs) are considered as dermal fibroproliferative diseases that differ clinically and histopathologically. Although several <em>factors</em> have been postulated in the etiopathogenesis of these conditions, there has been <em>growing</em> evidence to suggest the role of COXs in the pathogenesis of abnormal wound healing because of the reduction of formation of KL and HS in patients using nonsteroidal anti-inflammatory drugs and a COX-2 inhibitor. The aim of the present work is to evaluate the pattern and localization of COX-1 and COX-2 expression in KL and HS compared with surgical scars. COX-1 and COX-2 were analyzed on skin biopsies of 30 patients who presented with KL (<em>15</em>) and HS (<em>15</em>) and 10 normal surgical scars (controls). Both COX-1 and COX-2 were expressed not only in dermal components (<em>fibroblasts</em>, inflammatory cells, and endothelial cells) but also in keratinocytes of the overlying epidermis in the different studied scar lesions. The percentage of COX-1 expression increased progressively from surgical scar (40%) to HS (53.3%) to KL (100%) with a statistically significant difference (P = 0.002). COX-2 was expressed in 100% of surgical scars, 73.3% of HS and 86.7% of KL with the absence of significant differences (P>> 0.05). The significant difference in COX-1 expression between HS and KL may refer to the presence of different pathways for the emergence of these diseases. The expression of COX-2 in all scars (normal or abnormal) indicates its active role as an inflammatory mediator. Keratinocytes play an active role in induction of scarring by up-regulation of inflammatory mediators, such as COX-1 and COX-2.

A nonselective cation channel that we characterized in the mouse L-cell membrane becomes quiescent with serum deprivation (arrested cell <em>growth</em>) and rapidly active upon readdition of serum or, specifically, platelet-derived <em>growth</em> <em>factor</em> (PDGF). Using the patch-clamp technique, we find that the predominant channel in the LMTK- cell line is a bursting nonselective cation channel (the NS channel). In cell-attached and inside-out patches, the channel has a conductance of 28 pS; equal selectivity for Na+, K+, and Cs+; and no anion or divalent cation permeability. The channel open probability is voltage insensitive and in inside-out patches does not correlate with intracellular calcium (0.5 nM to 50 microM). When cultures are rendered quiescent by incubation in serum-free medium, channel open probability is virtually 0 as compared to 0.26 (+/- 0.17) in exponentially <em>growing</em> cultures. If mitogenesis is initiated by readdition of serum to quiescent cells while maintaining cell-attached recording, there is a rapid (<em>15</em>-30 s) activation of the channel (n = 12). The open probability of the patch increases (greater than 0.75) for 2-3 min and then decreases. We have attempted applications of several <em>growth</em> <em>factors</em> (<em>fibroblast</em>-derived <em>growth</em> <em>factor</em>, epidermal <em>growth</em> <em>factor</em>, insulin, bombesin, alpha-thrombin, and vasopressin, individually or in combination) but find that only PDGF (5-100 ng/ml; n = 9) produces channel activation. This activation should provide a Na+ entry pathway parallel to that of the Na/H exchanger.

A hepatic invasive human colorectal xenograft model was derived in nude mice by selection through the liver of the parental cell line, C170. Following intraperitoneal injection, tumours selectively grew on the liver in>> 80% of the animals within <em>15</em>-20 days. The liver-invading xenograft line, renamed C170HM2, had a significantly greater expression of the Lewisx antigen compared to C170 (mean linear fluorescence per cell>> 1000 compared with 500 for C170, P < 0.02). C170HM2 had significantly elevated proliferation (when compared with C170) in the presence of epidermal (P < 0.001) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (P < 0.001). C170HM2 also mitogenically responded to type I collagen (derived from rat tails), unlike C170. C170HM2 tumours when invading the liver expressed both interstitial collagenase and gelatinase activity at the invading edge.

T lymphocytes bearing the gammadelta-TCR accumulate during wound healing and inflammation. However, the role of gammadelta-T lymphocytes in fibrogenic tissue reactions is not well understood. Therefore, we addressed the question of whether human gammadelta-T cells express and synthesize connective tissue <em>growth</em> <em>factor</em> (CTGF), a <em>factor</em> known to regulate fibrogenesis and wound healing. In addition, the lymphoblastic leukemia T cell line (Loucy) that possesses characteristics typical of gammadelta-T cells was used as a model to evaluate the regulation of CTGF gene expression. Blood gammadelta-T cells isolated from healthy donors were grown in the presence of IL-<em>15</em>/TGF-beta1 for 48 h and assessed for the expression and synthesis of CTGF. Nonstimulated human blood gammadelta-T cells and Loucy gammadelta-T cells expressed low levels of CTGF mRNA. Costimulation of the cells with IL-<em>15</em> and TGF-beta1 resulted in a substantially increased level of CTGF mRNA expression within 4-8 h, and it remained elevated for at least 48 h. In contrast, no CTGF mRNA was detected when nonstimulated and stimulated human CD4+ alphabeta-T cells were analyzed. In addition, Western blot analysis of human gammadelta-T cell lysates prepared 4 days following stimulation with IL-<em>15</em> and TGF-beta1 revealed a 38-kDa CTGF protein in cell lysates of human gammadelta-T cells. Detection was confirmed using Colo 849 <em>fibroblasts</em>, which can constitutively express high levels of CTGF. In conclusion, we herein present novel evidence that in contrast to CD4+ alphabeta-T cells human gammadelta-T cells are capable of expressing CTGF mRNA and synthesizing its corresponding protein, which supports the concept that gammadelta-T cells may contribute to wound healing or tissue fibrotic processes.

A retroviral vector (N2-SV-GC) was constructed by inserting a normal human glucocerebrosidase (GC) cDNA under control of the SV40 early region promoter into the Moloney murine leukemia virus-derived N2 vector. N2-SV-GC produced human GC in murine 3T3 <em>fibroblasts</em> at levels in the range of the endogenous murine GC as determined by enzymatic assay and Western blot analysis. The N2-SV-GC retroviral vector was used for studies of gene transduction of murine hematopoietic progenitor cells (HPC). Infection of bone marrow cultured for 2 to 10 days in medium containing hematopoietic <em>growth</em> <em>factors</em> was significantly more efficient than infection of freshly isolated marrow cells (24% to 32% G418-resistant CFU-GM v <em>15</em>%, respectively). The marrow infected by N2-SV-GC was maintained in long-term bone marrow culture (LTBMC) and had a stable level of G418-resistant HPC over 2 months of serial assays. The human GC gene of the vector was persistently expressed in the nonadherent cell fraction of the murine LTBMC as determined by Northern blotting, Western blotting, and immunohistochemical staining using a monoclonal antibody specific for human GC. N2-SV-GC also expressed the human GC gene in day 12 CFU-S. LTBMC represents a novel system for retroviral vector-mediated gene transduction of HPC and may accurately predict the activities of vectors in vivo.

It has recently been postulated that a variety of <em>growth</em> <em>factors</em> may be released from cancellous bone after an acromioplasty. The aim of this study was to demonstrate the presence of <em>growth</em> <em>factors</em> in the subacromial space after acromioplasty. Between October 2006 and March 2007, 23 patients underwent arthroscopic acromioplasty. A sample of at least 3 ml of fluid from the shoulder was obtained <em>15</em> min after the end of the procedure. At the same time another sample of 3 ml of the patient's venous blood was obtained as a control. The concentrations of <em>growth</em> <em>factors</em> in the fluids collected were determined using enzyme-linked immunosorbent assay (ELISA). The <em>growth</em> <em>factors</em> assayed were platelet-derived <em>growth</em> <em>factor</em>-AB (PDGF-AB), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> basic (bFGF) and transforming <em>growth</em> <em>factor</em> beta 1 (TGF-beta1). The concentrations of TGF-beta1 (p = 0.0001), PDGF-AB (p = 0.02), and bFGF (p < 0.0001) were significantly higher in the fluid from the subacromial space than in the blood sample. There are high concentrations of several <em>growth</em> <em>factors</em> in the subacromial space after acromioplasty.

BACKGROUND

In kidney transplant recipients, persistent hyperparathyroidism leads to hypercalcemia and increased urinary phosphorus excretion. The calcimimetic drug cinacalcet effectively decreases parathyroid hormone (PTH) levels and corrects hypercalcemia in these patients. The purpose of the present study is to examine the effect of cinacalcet treatment on determinants of renal phosphorus reabsorption under steady-state conditions.

METHODS

Open-label prospective uncontrolled trial.

METHODS

10 stable kidney transplant recipients with persistent hyperparathyroidism.

METHODS

Cinacalcet, 30 and 60 mg/d, for 2 weeks.

METHODS

Changes in urinary phosphorus excretion in timed urine samples, intact and carboxy-terminal (C-term) fibroblast growth factor 23 (FGF-23), intact PTH, venous pH, and bicarbonate values at defined intervals over 24 hours.

RESULTS

Cinacalcet decreased renal phosphorus excretion in the first 8 hours by 30% to 40%, but not from 8 to 24 hours after drug administration. Serum phosphorus levels normalized in all patients. Cinacalcet markedly decreased plasma intact PTH levels (60%; P < 0.001). Cinacalcet also decreased mean intact FGF-23 levels from 67 +/- 8 (SE) to 51 +/- 5 and to 54 +/- 6 pg/mL (P < 0.001) and mean C-term FGF-23 levels from 108 +/- 15 to 87 +/- 9 and to 101 +/- 9 RU/mL (P < 0.01), respectively. There was high correlation between intact FGF-23 and C-term FGF-23 levels (r = 0.598; P < 0.001). Acid-base status was unchanged.

CONCLUSIONS

This is a small study and does not examine the long-term effect of cinacalcet treatment.

CONCLUSIONS

Cinacalcet effectively corrected urinary phosphate wasting in kidney transplant recipients, resulting in normalization of serum phosphorus levels. The phosphatemic effects of cinacalcet correlated with a marked decrease in the phosphaturic hormone PTH, rather than with a change in FGF-23 levels or acid-base status, highlighting the importance of PTH in posttransplantation hypophosphatemia.

The present study sought to determine the interaction between the novelty-seeking trait and cocaine treatment on gene expression in the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) system. Specifically, we assessed the regulation of FGFR1 in response to cocaine in animals that were selectively bred on the basis of their locomotor response to a novel environment. High-responder (HR) rats are those that exhibit increased locomotor response and exploratory behavior in a novel environment and low-responder (LR) rats are those that exhibit lower levels of exploratory behavior and are less active. Both phenotypes received daily injections of either cocaine (<em>15</em> mg/kg, i.p.) or saline for 7 consecutive days. Animals were sacrificed 45 min following their last injection and FGFR1 gene expression was assessed in the hippocampus and prefrontal cortex by mRNA in situ hybridization. HR-bred rats exhibited increased FGFR1 mRNA in the hippocampus compared to LR-bred rats. Furthermore, cocaine decreased FGFR1 mRNA in the hippocampus and increased FGFR1 mRNA in the prefrontal cortex. Finally, HR and LR rats differed in their response to cocaine between brain regions. In the hippocampus, cocaine decreased gene expression in HR-bred rats without affecting LR-bred rats, whereas in the prefrontal cortex cocaine increased gene expression in LR-bred rats without affecting HR-bred rats. These results suggest that cocaine interacts with the novelty-seeking trait to alter gene expression. Thus, the FGF system may contribute to individual differences in the response to drugs of abuse.

The sulfonic acid polymers poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS), poly(4-styrenesulfonic acid) (PSS), and poly(anetholesulfonic acid) (PAS) proved to be highly potent inhibitors of angiogenesis in the chick chorioallantoic membrane (CAM) assay. PAMPS was found to achieve a dose-dependent inhibition of microvessel formation in the CAM assay ranging from 57 +/- 16% inhibition at 10 micrograms/disc to 72 +/- <em>15</em>% at <em>15</em>0 micrograms/ disc. Also, PSS and PAS caused a strong inhibition of angiogenesis (55 +/- 19% and 48 +/- 16%, respectively, at 50 micrograms/disc), whereas poly(vinylsulfonic acid) (PVS) was found to be inactive at this dose. The compounds proved to be nontoxic for the developing chick embryo at these doses. Suramin, which was included as a reference compound, caused only a slight inhibition of vascular density, at a dose of <em>15</em>0 micrograms/disc, whereas pentosan polysulfate (PPS) was found to be toxic. PAMPS, PAS, and PSS, but not PVS, inhibited microvessel formation in the rat aorta-ring assay. In addition, the increased [3H-methyl]dThd uptake in endothelial cells in vitro upon stimulation with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was inhibited by PAMPS, PAS, and PSS at 20 micrograms/ml. A strong correlation (r = 0.95) was found between the antiangiogenic effect of the sulfonic acid polymers in the CAM assay and their inhibition of the bFGF-induced mitogenic response, indicating that bFGF is the target for these sulfonic acid polymers. These results suggest that sulfonic acid polymers, and in particular PAMPS, may be considered as specific, nontoxic angiogenesis inhibitors.

The Trk family of tyrosine protein kinase receptors plays a significant role in the development and maintenance of neural tissues. It has been recently shown that Trk receptors are also expressed by a wide range of normal non-neuronal tissues in humans in a cell type-specific manner. In the present study, the expression patterns of TrkA in 337 non-neuronal invasive carcinomas of <em>15</em> different human tissues were investigated immunohistochemically. Overall, 133 (39%), 101 (30%) and 103 (31%) tumors exhibited strong, moderate and no TrkA immunoreactivity, respectively. Esophageal and thyroid carcinomas expressed high levels of TrkA, whereas the levels in gastric and colon cancers were low. TrkA expression was detected not only in carcinomas originating from TrkA-positive normal counterpart tissues, including the esophagus, breast, lung and uterus, but also in those from TrkA-negative tissues/cells of the thyroid, liver and ovary. Immunostaining for nerve <em>growth</em> <em>factor</em>-beta, the specific ligand for TrkA, in esophageal and breast carcinomas demonstrated its immunoreactivity in stromal <em>fibroblasts</em> and some TrkA-expressing tumor cells. These results suggest that paracrine/autocrine regulation via stromal/tumoral NGF-tumoral TrkA interaction may be involved in the <em>growth</em> of certain non-neuronal carcinomas.

OBJECTIVE

Fibroblast growth factor (FGF)-2 is one of the most powerful angiogenic growth factors to be evaluated as an agent for the promotion of angiogenesis. The aim of this study is to investigate whether intratracheal administration of controlled-release FGF-2 microspheres restores pulmonary function in beagle dogs with emphysema.

METHODS

Randomized, controlled, experimental animal study.

METHODS

Eighteen Wister rats and 15 adult beagle dogs.

METHODS

In the rat study, we compared the time profiles of the radioactivity remaining after intratracheal injection of 125I-labeled FGF-2, either incorporated with the controlled-release microspheres or as an aqueous solution. In the dog study, elastase-induced emphysema models were developed in 10 animals, classified into the following three groups: control group (n = 5), emphysema model with empty microspheres-treated group (FGF - group, n = 5), and emphysema model with FGF-2 containing microspheres-treated group (FGF + group, n = 5).

RESULTS

In the rat study, controlled-release microspheres maintained higher whole-lung FGF-2 concentrations after intratracheal administration. In the dog study, Pa(O2) in the FGF + group was significantly higher than in the FGF - group after treatment. Pulmonary perfusion dynamic MRI revealed significant improvement in the signal intensity of damaged lung with the FGF + group. Linear intercept of the FGF + group was significantly reduced than the FGF - group.

CONCLUSIONS

Results indicate that intratracheal administration of FGF-2 induced an increase in pulmonary blood flow in the damaged lung and led to recovery of pulmonary function. The controlled-release microsphere system increased the effectiveness of FGF-2.

Three cell lines derived from fin (WSF), head soft tissue (WSHST) and body muscle (WSBM) were established from white sturgeon (Acipenser transmontanus). Characterization included determination of optimal <em>growth</em> kinetics, karyotyping, and mitochondrial ribosomal RNA (rRNA) genotyping. The primary cultures of these cells were generated by the explant technique using the L-<em>15</em> medium supplemented with 20% fetal bovine serum and epidermal/<em>fibroblast</em> <em>growth</em> <em>factors</em>. The cells grew between <em>15</em>-30 degrees C, but optimal <em>growth</em> occurred at 25 degrees C with a doubling time of 48 hours. The cell lines can be readily maintained in-vitro and have been subcultured over 35 times. Following cryopreservation in liquid nitrogen, thawed cells exhibit a viability of>> 90% after a 16-month storage period. Chromosomal typing of these cell lines at their 17th passage revealed a chromosomal distribution of 242 to 278 with an apparent peak ranging from 250 to 260. Polymerase chain reaction amplification of mitochondrial 16S ribosomal RNA and sequence analysis indicated 100% identity of the sequences found in the cell lines with those found in the source animal, confirming that the cell lines were of A. transmontanus origin.

Monitoring bile acids as signal molecules in combination with a bile acid synthesis marker and the FXR regulator <em>fibroblast</em> <em>growth</em> <em>factor</em> 19 (FGF19), this study addresses significant postprandial changes. The efficacy of the different pathways to regulate bile acid synthesis through short heterodimer partner (SHP) dependent FXR modulation in liver, and SHP independent activation via FGF19 is demonstrated. Characteristic changes of the bile acid profile during an oral glucose tolerance test (oGTT) were investigated in 73 individuals. <em>15</em> bile acid species including conjugated and unconjugated forms were quantitatively determined with LC-MS/MS in serum samples collected at three time points during the oGTT. All conjugated bile acid species showed the same time course, a significant increase at 60 min after the glucose intake and an incline at 120 min. In contrast, a consistent decline of all unconjugated bile acids was monitored. 7α-Hydroxy-4-cholesten-3-one, an early bile acid synthesis marker, showed an inverse response with a significant decrease at 60 min which proves the efficient and rapid downregulation of CYP7A1 via FXR activation through bile acid signaling. Significantly higher levels of FGF19 were observed 120 min after glucose intake and 60 min after bile acid excursion.

The influence of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), a central nervous system (CNS)-derived molecule, on survival of trunk neural crest cells was investigated. As previously shown (C. Kalcheim and N. M. Le Douarin, 1986, Dev. Biol. 116, 451-466), the interposition of untreated silastic membranes between neural tube and neural crest cells of the dorsal root ganglion (DRG) anlage led to selective death of neural crest cells that remained distally located with respect to the implants. Membranes were then treated with laminin and bFGF (100 ng/ml) and implanted. Under these conditions, rescued cells were observed for over 30 hr after grafting in <em>15</em> of 19 embryos. In contrast, no surviving cells could be found in any of 10 control embryos implanted with laminin-treated silastic membranes. We have also investigated the effects of bFGF on survival of identified subpopulations of trunk neural crest cells cultured with somite cells in a serum-free, chemically defined medium. bFGF promoted a dose-dependent increase in the number of HNK-1-positive nonneuronal cells in 1- to 4-day-old cultures (1.8- to 8.2-fold over controls using FGF at concentrations of 10 pg/ml to 1 ng/ml, respectively). FGF had no mitogenic effect on the neural crest-derived nonneuronal cells since the number of HNK-1-immunoreactive nonneuronal cells having incorporated [3H]thymidine into their nuclei remained unchanged in control as compared to treated cultures. However, the same concentrations of FGF were found to stimulate the incorporation of [3H]thymidine into acid-insoluble material in somite cultures devoid of neural crest. Moreover, bFGF significantly enhanced survival of nonneuronal cells in pure neural crest cultures established from neural crest clusters, thus demonstrating a direct effect of bFGF on survival and/or differentiation of neural crest-derived nonneuronal cells. These data support the hypothesis that CNS-derived molecules influence early development of selective subsets of neural crest cells developing into sensory ganglia.

BACKGROUND

Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial sutures and the factors regulating its expression in vitro.

METHODS

Microarray analysis was performed on rat posterior frontal and sagittal cranial sutures at 5, 10, 15, 20, and 30 days of life (n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor beta1, or cultured with primary rat nonsuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction.

RESULTS

BMP3 expression in the posterior frontal suture decreased over the time course analyzed, whereas it increased in the sagittal suture. Notably, BMP3 expression was higher in the patent sagittal suture during the window of posterior frontal suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01).

CONCLUSIONS

BMP3 is expressed in rat cranial sutures in a temporal pattern suggesting involvement in cranial suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial suture biology.

This review highlights the huge advances made in the understanding of Crohn's disease in the last <em>15</em> years. The pathogenic immune response in the gut wall is a highly polarised T helper cell type 1 response, probably directed against antigens of the commensal flora. There is marked over-expression of pro-inflammatory cytokines such as tumor necrosis <em>factor</em> (TNF)-alpha and increased production of matrix degrading enzymes by <em>fibroblasts</em> and macrophages, which are probably responsible for ulceration and fistula formation. Crohn's disease runs in families and the susceptibility genes identified so far are associated with innate recognition of microbial products (Nod2) or epithelial barrier function (OCTN cation transporter genes and DLG5). Endogenous healing pathways mediated by transforming <em>growth</em> <em>factor</em> (TGF)-beta1 are inhibited because mucosal inflammatory cells express Smad7, the endogenous intracellular inhibitor of TGF-beta signalling. This makes it unlikely that enteral feeds containing TFG-beta are therapeutic by means of direct anti-inflammatory effects, however TGF-beta may still be involved because it is a well known epithelial motogen and may promote mucosal healing, in synergy with changes in mucosal bacterial populations as a result of the change in the diet.

Successful approaches to tissue engineering smooth muscle tissues utilize biodegradable scaffolds seeded with autologous cells. One common problem in using biological scaffolds specifically is the difficulty of inducing cellular penetration and controlling de novo extracellular matrix deposition/remodeling in vitro. Our hypothesis was that small intestinal submucosa (SIS) exposed to specific mechanical stimulation regimes would modulate the synthesis of de novo collagen and elastin by bladder smooth muscle cells (BSMC) within the SIS matrix. We further hypothesized that the cytokines vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2), two key <em>growth</em> <em>factors</em> involved in epithelial mesenchymal signaling, will promote the cellular penetration into SIS necessary for mechanical stimulation. BSMC were seeded at 0.5 x 10(6) cells/cm(2) onto the luminal side of SIS specimens. VEGF (10 ng/mL) and FGF-2 (5 ng/mL) were added to each insert in the media every other day for up to 7 days in static culture. Following static culture, specimens were stretched strip-biaxially under <em>15</em>% peak strain at either 0.5 or 0.1 Hz for an additional 7 days. Following the culture period, specimens were assayed histologically and biochemically for cellular penetration, proliferation, elastin, collagen, and protease activity. Histological analyses demonstrated that in standard culture media, BSMC remained on the surface of the SIS while both FGF-2 and VEGF profoundly promoted in<em>growth</em> of the BSMC into the SIS. The penetration of the cells in response to these cytokines was confirmed using a Transwell assay. Following cellular penetration, BSMC produced significant amounts of elastic fibers under cyclic mechanical stretching at 0.1 Hz under <em>15</em>% stretch, as evidenced by colorimetric assay and histology using a Verhoeff-Van Gieson stain. Protease activity was assessed in the media and found to be statistically increased in static culture following FGF-2 treatment. These findings demonstrate, for the first time, the capability of BSMC to produce histologically apparent elastin fibers in vitro. Moreover, our results suggest that a strategy involving <em>growth</em> <em>factors</em> and controlled mechanical stimulation may be used to engineer functional, elastin-rich tissue replacements using decellularized biologically derived scaffolds.

Formation of bone-like tissue in culture by stromal bone marrow cells (SBMC) derived from young <em>growing</em> rats is dependent on dexamethasone (Dex) (Cell Tissue Res 254:317; 1988) and is significantly enhanced by basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) (J Bone Miner Res 8:919; 1993). The aim of this study was to examine the effect of maturation on the osteogenic potential and the response to Dex and bFGF of SBMC by using cultures derived from young <em>growing</em> (6 weeks old) and adult (9 months old) rats. SBMC cultures were grown in the presence of Dex (10(-8) or 10(-7) mol/L) at both P(0) and P(1) and either in the presence or absence of bFGF. The effect of Dex and bFGF on mineralized bone-like tissue (MBT) formation was assessed at P(1). The highest levels of mineralized tissue formation in P(1) subcultures in the absence of bFGF were obtained when cultures derived from young rats (6 weeks old) were treated with Dex 10(-7) and 10(-8) mol/L at P(0) and P(1), respectively, and when cultures derived from adult rats were exposed to Dex 10(-8) mol/L both at P(0) and P(1). Under these optimal Dex concentrations, the amount of MBT formed by adult rat-derived cultures was <em>15</em>-fold lower than that of young rat-derived ones. The addition of bFGF to P(0) cultures or to P(1) cultures grown under optimal Dex conditions enhanced MBT formation in P(1) cultures derived from both young and adult rats, but this effect was considerably more pronounced in the adult rat-derived cultures. The maximal levels of MBT formation were produced by cultures derived from adult rats treated with bFGF at both P(0) and P(1), whereas in cultures derived from young rats, the addition of bFGF at P(0) was not necessary for maximal MBT production. This stimulating effect of bFGF on MBT formation by adult rat-derived cultures was accompanied by a 2.2-, 1.8-, and 4.3-fold increase in proliferation, alkaline phosphatase activity, and Ca(2+) deposition rate, respectively. bFGF increased the level of glucocorticoid receptor by approximately 2. 3-fold in Dex-treated cultures derived from young animals. These results indicate that maturation is associated with a decrease in the proportion of osteoprogenitor cells in the stromal bone marrow and in their capacity to express the osteogenic phenotype. They further point to the significant role of bFGF in stimulating proliferation and osteogenic expression of stromal bone marrow osteoprogenitors derived from adult rats.

Unilateral pneumonectomy in rats causes compensatory <em>growth</em> of the remaining lung. During this <em>growth</em>, there are large increases in the cell numbers and in the rates of collagen and non-collagen protein production. We examined possible mechanisms by which these changes might occur. Assessment of the effect of bronchoalveolar lavage (BAL) fluid on <em>fibroblasts</em> in vitro demonstrated the presence of stimulatory activity for <em>fibroblast</em> replication in control animals. This activity was greatly increased two and six days postpneumonectomy (1<em>15</em> +/- 26% and 75 +/- 18% above control values, respectively), but had returned to normal by 14 days. Preliminary characterization suggests that the activity is heat labile and consists of at least two moieties with apparent molecular weights of 5-<em>15</em> kD and 70-220 kD. The activity was partially blocked by antibodies to insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1), and levels of IGF-1 were increased by about 100% (p less than 0.001) two days after pneumonectomy compared with control values. Examination of BAL cells demonstrated an early influx of leucocytes into the remaining lung of pneumonectomized rats. At two days, about 25% of the lavageable cells were neutrophils, but macrophages were the predominant cell type at all times. The extravascular albumin space of the lung increased by about 65% (p less than 0.01), six days after pneumonectomy. The influx of circulatory proteins and cells are potential sources of the increased mitogenic activity observed in the lung.

Tumor necrosis <em>factor</em>-like weak inducer of apoptosis (TWEAK) could play a role in the regulation of interleukin (IL)-<em>15</em> and IL-18, cytokines crucial for angiogenesis and uterine natural killer (uNK) cell recruitment during embryo implantation. We therefore confirmed the endometrial presence of TWEAK/<em>fibroblast</em> <em>growth</em> <em>factor</em> inducible-14 (Fn-14) and documented simultaneously the cytotoxic KIR receptor (NKp46) of uNK cells in the human endometrium while TWEAK, Fn-14, IL-<em>15</em>, and IL-18 mRNA were quantified by real-time polymerase chain reaction in relation to the recruitment of CD56+ cells among fertile control women and patients who had failed to implant after assisted reproduction treatment.

alpha-Macroglobulins derived from plasma or secreted by macrophages are platelet-derived <em>growth</em> <em>factor</em> (PDGF) binding proteins that compete with cell-surface receptors on <em>fibroblasts</em> for PDGF binding. alpha 2-Macroglobulin (alpha 2M) derived from bovine plasma was tested for its ability to modulate the PDGF-induced proliferation of primary passage rat lung <em>fibroblasts</em> (RLFs) and a human skin <em>fibroblast</em> cell line (CRL <em>15</em>08). <em>Fibroblasts</em> were grown in 10% fetal bovine serum (FBS) for 24 hr, then washed with serum-free medium before adding serum-free defined medium (SFDM) containing insulin and transferrin. To this medium were added varying concentrations of human plasma-derived AB-PDGF and alpha 2 M, alone or in combination. Receptor-recognized alpha 2M was prepared by treatment with methylamine. Both native alpha 2M and the alpha 2M-methylamine (alpha 2M-MA) were tested for <em>growth</em> promoting activity in the absence or presence of PDGF. After 3 days, a concentration-dependent <em>growth</em> curve of <em>fibroblast</em> proliferation was demonstrated for PDGF alone, with near maximal stimulation reached at <em>15</em>-20 ng/ml PDGF. alpha 2M and alpha 2M-MA alone had no effect on cell proliferation. However, alpha 2M-MA concentrations above 32 micrograms/ml synergistically enhanced PDGF-stimulated proliferation greater than 100% in the presence of <em>15</em> ng/ml PDGF. Native alpha 2M enhanced PDGF-stimulated <em>growth</em> 80-100% above PDGF controls only at low concentrations (32-64 micrograms/ml alpha 2M). High concentrations of native alpha 2M (128-256 micrograms/ml) either had no effect on <em>growth</em> or were inhibitory to PDGF-stimulated <em>growth</em>, depending on the cell type tested. Rat lung <em>fibroblasts</em> were shown to secrete a <em>factor</em>(s) that inhibited the trypsin-binding capacity of native alpha 2M. We further demonstrated that early passage RLFs possess specific cell-surface receptors for [125I]-PDGF and [125I]-alpha 2M-MA, and preincubation of RLFs with alpha 2M-MA increased the specific binding of [125I]-PDGF to the cell surface of these <em>fibroblasts</em>. Considered together, these data support the view that receptor-recognized alpha 2M synergistically enhances the proliferative capacity of PDGF. We postulate that receptor-recognized alpha Ms enhance PDGF-stimulated <em>growth</em> by increasing the local concentration of PDGF at the cell surface, where the PDGF could be released in close proximity to its own receptors.

OBJECTIVE

The growth of ocular neovascularization is regulated by a balance between stimulating and inhibiting growth factors. Somatostatin affects angiogenesis by inhibiting the growth hormone-insulin-like growth factor axis and also has a direct antiproliferative effect on human retinal endothelial cells. The purpose of our study is to investigate the expression of somatostatin receptor (sst) subtypes and particularly sst subtype 2A (sst2A) in normal human macula, and to study sst2A in different stages of age-related maculopathy (ARM), because of the potential anti-angiogenic effect of somatostatin analogues.

METHODS

Sixteen eyes (10 enucleated eyes, 4 donor eyes, and 2 surgically removed choroidal neovascular [CNV] membranes) of 15 patients with eyes at different stages of ARM were used for immunohistochemistry. Formaldehyde-fixed paraffin-embedded slides were incubated with a polyclonal anti-human sst2A antibody. mRNA expression of five ssts and somatostatin was determined in the posterior pole of three normal human eyes by reverse transcriptase-polymerase chain reaction.

RESULTS

The immunohistochemical expression of sstA in newly formed endothelial cells and fibroblast-like cells was strong in fibrovascular CNV membranes. mRNA of sst subtypes 1, 2A, and 3, as well as somatostatin, was present in the normal posterior pole; sst subtypes 4 and 5 were not detectable.

CONCLUSIONS

Most early-formed CNV in ARM express sst2A. The presence of mRNA of sst subtype 2A was observed in normal human macula, and subtypes 1 and 3 and somatostatin are also present. sst2A receptors bind potential anti-angiogenic somatostatin analogues such as octreotide. Therefore, somatostatin analogues may be an effective therapy in early stages of CNV in ARM.

OBJECTIVE

Fibroadenomas are benign tumours composed of both glandular and fibrous tissue. The mechanisms regulating the growth of these tumours and the relation between the stromal and epithelial cells are poorly understood. Acidic fibroblast growth factor (aFGF) is a well known fibroblast activator, which acts through four specific cell surface receptors, among which, fibroblast growth factor receptor 4 (FGFR4) is highly specific. The aim of this study was to evaluate the distribution of aFGF and FGFR4 in specific cell types of fibroadenomas to understand their possible role in the growth of these breast lesions.

METHODS

Formalin fixed and paraffin wax embedded tissues from 15 fibroadenomas and peritumoral normal breasts were investigated for the expression of aFGF and FGFR4 using immunohistochemistry. The presence of aFGF mRNA was also investigated using in situ hybridisation.

RESULTS

Immunoreactivity for aFGF and FGFR4 was seen in epithelial cells, but it was lacking in myoepithelial cells of both normal tissues and fibroadenomas. Strong FGFR4 immunoreactivity was found in stromal fibroblasts, which were also weakly positive for aFGF. aFGF mRNA was detected in epithelial cells and in some stromal fibroblasts.

CONCLUSIONS

These results suggest a paracrine/autocrine modulation of epithelial and stromal cells of fibroadenomas through an aFGF-FGFR4 interaction. This interaction might regulate various cell functions and the growth of fibroadenomas.