We have investigated gene amplification of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor-4 (FGFR4) gene in 30 primary breast tumor samples and <em>15</em> gynecological tumor samples. Ten percent of the breast tumors showed 2- to 4-fold amplification. Amplification was found more frequently in estrogen- and progesterone-receptor-positive tumors and in tumors with high lymph-node involvement. Breast tumor samples were also analyzed for the amplification of fgfr3 and erbB2 genes and the chromosome 11q13 located genes hst1/int2/bcl1/sea. erbB2 gene was amplified 2- to 13-fold in 13% of the cases, but no amplification of int2/hst1/bcl1/sea amplicon was found. Gynecological tumors were also analyzed for the amplification of fgfr4 and fgfr3 genes and for int2 and hst1 oncogenes. Eleven of the <em>15</em> gynecological tumors were ovarian neoplasms including 2 benign tumors; the remainder comprised 1 ovarian metastasis of breast cancer; 1 endometrial cancer; 1 uterine leiomyosarcoma and 1 carcinosarcoma of the fallopian tube. In gynecological tumors, fgfr4 gene was found to be amplified in 2 ovarian tumors. Amplification of hst1 was found in 1 benign ovarian tumor. Thus, the fgfr4 gene may be involved in breast and ovarian tumorigenesis.

The development of high myopia is associated with altered scleral extracellular matrix biochemistry. Previous studies highlight the importance of collagen turnover in this process, yet it is unclear which <em>factors</em> control scleral remodeling. This study used a mammalian model of myopia to investigate the capacity of TGF (transforming <em>growth</em> <em>factor</em>)-beta1, -beta2, and -beta3 to influence scleral remodeling in myopia. RT-PCR confirmed the presence of all mammalian TGF-beta isoforms in scleral tissue and scleral <em>fibroblasts</em>. Myopia was experimentally induced via monocular deprivation of pattern vision, and animals were allocated to two groups depending on the duration of treatment (1 or 5 days). Down-regulation of each isoform was apparent after only 1 day of myopia development (TGF-beta1, -32%; TGF-beta2, -27%; TGF-beta3, -42%). Whereas the decrease in TGF-beta1 and -beta3 expression was relatively constant between the two time points, differential down-regulation of TGF-beta2 was found between days 1 (-27%) and 5 (-50%). In vitro experiments, using primary scleral <em>fibroblasts</em>, demonstrated the capacity of all isoforms to increase collagen production in a dose-dependent manner. Changes in TGF-beta levels, which mimicked those during myopia induction, caused an approximately <em>15</em>% reduction in collagen synthesis, which is qualitatively similar to those previously reported in vivo. These data represent the first demonstration of TGF-beta3 expression in the sclera and implicate all three TGF-beta isoforms in the control of scleral remodeling during myopia development. In addition, the early alterations in TGF-beta expression levels may reflect a role for these cytokines in mediating the retinoscleral signal that controls myopic eye <em>growth</em>.

Interleukin-6 is a pleiotropic cytokine with a wide range of effects, including induction of B-cell and cytotoxic T-cell differentiation, and induction of acute phase reactant production by hepatocytes. Interleukin-6 also can act as an autocrine <em>growth</em> <em>factor</em> in malignancy. Various cell types produce interleukin-6, including T and B cells, monocytes, <em>fibroblasts</em>, and some solid tumor cells. In previous work we detected the production of substantial amounts of interleukin-6 by human ovarian cancer cells, including the ovarian cancer cell lines CAOV-3, OVCAR-3, and SKOV-3, and several primary ovarian tumor cultures. In this study we retrospectively examined 90 separate serum specimens for interleukin-6 in 36 patients with epithelial ovarian cancer. The mean serum interleukin-6 concentration of those ovarian cancer patients with macroscopic disease (n = 57) was 0.26 +/- 0.04 U/ml (mean +/- SEM). Healthy adult donors have interleukin-6 serum levels of 0.12 +/- 0.03 U/ml. Sixteen of 21 ovarian cancer patients with macroscopic disease (76%) had elevated (greater than 0.20 U/ml) levels of serum interleukin-6, with levels approaching 1 U/ml in some patients (p less than 0.01). Of those nine patients with bulky tumor (residual greater than 2 cm), eight (89%) had an elevated interleukin-6 level (mean, 0.31 +/- 0.05), while eight of 12 (66%) with minimal residual disease (less than 2 cm) had elevated levels. Only two of <em>15</em> (13%) patients who were in clinical remission and who had microscopic disease had elevated values. Of the 36 patients, 22 were CA 125 negative (less than 35 U/ml), and of these, four had elevated interleukin-6 levels. Of the 14 patients with an elevated CA 125 level, 12 (86%) had elevated interleukin-6 levels. In those 16 patients in whom serial levels of interleukin-6 were measured, rising levels were found over a 3 to 4 month interval in nine (56%); this correlated with tumor progression. Furthermore, the subsequent survival of patients was shown to correlate with the level of interleukin-6, such that patients whose levels were elevated greater than 0.20 U/ml interleukin-6 survived a mean of 12.5 months, compared with 27.2 months for patients with normal levels (p less than 0.001). These data support the concept that interleukin-6 may be a useful tumor marker in some patients with epithelial ovarian cancer, as it correlates with the tumor burden, clinical disease status, and survival.

With only a fraction of patients responding to cancer immunotherapy, a better understanding of the entire tumor microenvironment is needed. Using single-cell transcriptomics we chart the fibroblastic landscape during pancreatic ductal adenocarcinoma (PDAC) progression in animal models. We identify a population of carcinoma-associated <em>fibroblasts</em> (CAFs) programmed by transforming <em>growth</em> <em>factor</em> beta and expressing the leucine-rich repeat containing <em>15</em> (LRRC<em>15</em>) protein. These LRRC<em>15</em>+ CAFs surround tumor islets and are absent from normal pancreatic tissue. The presence of LRRC<em>15</em>+ CAFs in human patients was confirmed in >80,000 single-cells from 22 PDAC patients as well as immunohistochemistry on samples from 70 patients. Furthermore, immunotherapy clinical trials comprising over 600 patients across 6 cancer types revealed elevated levels of the LRRC<em>15</em>+ CAF signature correlated with poor response to anti-PD-L1 therapy. This work has important implications for targeting non-immune elements of the tumor microenvironment to boost responses of cancer patients to immune checkpoint blockade therapy.

Keratinocytes and dermal endothelial cells, excluding leukocytes that infiltrate wounds, are the main source of soluble <em>factors</em> regulating healing of skin ulcers. We used immunohistochemistry to analyze the expression of various chemotactic and <em>growth</em> <em>factors</em> and their receptors in the margin of diabetic foot ulcers and in normal nondiabetic foot skin. Our study found significantly elevated expression of transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1) and type I TGF-beta receptors (TGFbetaR1), granulocyte macrophage colony-stimulating <em>factor</em> (GM-CSF), and epidermal <em>growth</em> <em>factor</em> (EGF) in keratinocytes in the ulcer margin (p < 0.05). Significantly increased expression of monocyte chemotactic protein-1, GM-CSF, CXCR1, and TGFbetaRI and decreased expression of interleukin (IL)-10, IL-<em>15</em>, and TGF-beta1 were observed in ulcer dermal endothelial cells (p < 0.05). There was a lack of up-regulation of IL-8, CCR2A, IL-10 receptor, GM-CSF receptor, platelet-derived <em>growth</em> <em>factors</em> and their receptors, vascular endothelial <em>growth</em> <em>factor</em> and its type II receptor, EGF receptor, insulin-like <em>growth</em> <em>factor</em>-1, and nitric oxide synthase-2 in both KCs and endothelial cells in the ulcer. Finally, there was a lack of up-regulation of IL-10 and IL-<em>15</em> in keratinocytes and of EGF, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and nitric oxide synthase-3 in endothelial cells in the ulcer margins. The enhanced expression of some <em>factors</em> responsible for KC behavior could suggest an unimpaired capacity of keratinocytes to reepithelialize the margin of diabetic foot ulcers. However, lack of up-regulation of some angiogenic and leukocyte chemotactic <em>factors</em>, associated with the reduced influx of immune cells, may account for a poor formation of granulation tissue and chronicity of ulcer epithelialization.

As bones are levers for skeletal muscle to exert forces, both are complementary and essential for locomotion and individual autonomy. In the past decades, the idea of a bone-muscle unit has emerged. Numerous studies have confirmed this hypothesis from in utero to aging works. Space flight, bed rest as well as osteoporosis and sarcopenia experimentations have allowed to accumulate considerable evidence. Mechanical loading is a key mechanism linking both tissues with a central promoting role of physical activity. Moreover, the skeletal muscle secretome accounts various molecules that affect bone including insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2), interleukin-6 (IL-6), IL-<em>15</em>, myostatin, osteoglycin (OGN), FAM5C, Tmem119 and osteoactivin. Even though studies on the potential effects of bone on muscle metabolism are sparse, few osteokines have been identified. Prostaglandin E2 (PGE2) and Wnt3a, which are secreted by osteocytes, osteocalcin (OCN) and IGF-1, which are produced by osteoblasts and sclerostin which is secreted by both cell types, might impact skeletal muscle cells. Cartilage and adipose tissue are also likely to participate to this control loop and should not be set aside. Indeed, chondrocytes are known to secrete Dickkopf-1 (DKK-1) and Indian hedgehog (Ihh) and adipocytes produce leptin, adiponectin and IL-6, which potentially modulate bone and muscle metabolisms. The understanding of this system will enable to define new levers to prevent/treat sarcopenia and osteoporosis at the same time. These strategies might include nutritional interventions and physical exercise.

Stimulation of <em>fibroblasts</em> with serum <em>growth</em> <em>factors</em> results in the rapid activation of a set of immediate-early genes, among them 3CH134. We have purified a bacterially expressed form of the 3CH134-encoded polypeptide and demonstrated that it has intrinsic protein-tyrosine-phosphatase (PTPase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) activity in vitro. This activity is optimal at pH 7.5, is sensitive to vanadate and cysteinyl modifying agents, and is insensitive to a panel of serine/threonine phosphatase inhibitors. Purified 3CH134 protein displays a high degree of selectivity among the tyrosine-phosphorylated polypeptide substrates tested. Under our assay conditions, the rates of dephosphorylation are in the order EDNDYINASL peptide < myelin basic protein < reduced, carboxyamidomethylated, and maleylated lysozyme (RCML) < p42mapk. There is a 200-fold range in rates for these substrates, with p42mapk dephosphorylated <em>15</em>-fold more rapidly than RCML. Although 3CH134 is most closely related to the tyrosine/serine dual-specificity phosphatase VH1, we failed to detect any 3CH134-directed activity on casein or RCML phosphorylated on serine/threonine residues by cAMP-dependent protein kinase. Since 3CH134 expression is controlled transcriptionally and posttranscriptionally, it may represent a class of PTPases whose activity is regulated at the level of protein synthesis and degradation.

Collagenase-3 (MMP-13) is a novel matrix metalloproteinase, the expression of which has so far only been documented in human breast carcinomas and osteoarthritic cartilage. In this study we have examined the expression of MMP-13 during human fetal development. Northern blot hybridizations revealed abundant expression of MMP-13 mRNAs in total RNA from fetal cartilage and calvaria at gestational age of <em>15</em> weeks. By in situ hybridization MMP-13 transcripts were detected in chondrocytes of hypertrophic cartilage in vertebrae of the spinal column and in the dorsal end of ribs undergoing ossification, as well as in osteoblasts and periosteal cells below the inner periosteal region of ossified ribs. In contrast, no expression of MMP-13 could be detected in osteoclasts. Furthermore, expression of MMP-13 mRNA was detected in osteoblasts and <em>fibroblasts</em> primarily on the inner side of calvarial bone of the skull at 16 weeks of gestation. Expression of MMP-13 mRNA by primary human fetal chondrocytes in culture was enhanced by transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) and inhibited by bone morphogenetic protein-2 (BMP-2). No expression of MMP-13 mRNA could be noted in other fetal tissues, including the skin, lungs, neural tissue, muscle, and liver. These results suggest that MMP-13 plays an important role in the extracellular matrix remodeling during fetal bone development both via endochondral and intramembranous ossification.

BACKGROUND

The loss of alveolar walls is a hallmark of emphysema. As fibroblasts play an important role in the maintenance of alveolar structure, a change in fibroblast phenotype could be involved in the pathogenesis of this disease. In a previous study we found a reduced in vitro proliferation rate and number of population doublings of parenchymal lung fibroblasts from patients with emphysema and we hypothesized that these findings could be related to a premature cellular aging of these cells. In this study, we therefore compared cellular senescence markers and expression of respective genes between lung fibroblasts from patients with emphysema and control patients without COPD.

METHODS

Primary lung fibroblasts were obtained from 13 patients with moderate to severe lung emphysema (E) and 15 controls (C) undergoing surgery for lung tumor resection or volume reduction (n = 2). Fibroblasts (8E/9C) were stained for senescence-associated beta-galactosidase (SA-beta-Gal). In independent cultures, DNA from lung fibroblasts (7E/8C) was assessed for mean telomere length. Two exploratory 12 k cDNA microarrays were used to assess gene expression in pooled fibroblasts (3E/3C). Subsequently, expression of selected genes was evaluated by quantitative PCR (qPCR) in fibroblasts of individual patients (10E/9C) and protein concentration was analyzed in the cell culture supernatant.

RESULTS

The median (quartiles) percentage of fibroblasts positive for SA-beta-Gal was 4.4 (3.2;4.7) % in controls and 16.0 (10.0;24.8) % in emphysema (p = 0.001), while telomere length was not different. Among the candidates for differentially expressed genes in the array (factor>> or = 3), 15 were upregulated and 121 downregulated in emphysema. qPCR confirmed the upregulation of insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-rP1 (p = 0.029, p = 0.0002), while expression of IGFBP-5, -rP2 (CTGF), -rP4 (Cyr61), FOSL1, LOXL2, OAZ1 and CDK4 was not different between groups. In line with the gene expression we found increased cell culture supernatant concentrations of IGFBP-3 (p = 0.006) in emphysema.

CONCLUSIONS

These data support the hypothesis that premature aging of lung fibroblasts occurs in emphysema, via a telomere-independent mechanism. The upregulation of the senescence-associated IGFBP-3 and -rP1 in emphysema suggests that inhibition of the action of insulin and insulin-like growth factors could be involved in the reduced in vitro-proliferation rate.

Ligands of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) abrogate the stimulation of collagen gene transcription induced by transforming <em>growth</em> <em>factor</em>-beta (TGF-beta). Here, we delineate the mechanisms underlying this important novel physiological function for PPAR-gamma in connective tissue homeostasis. First, we demonstrated that antagonistic regulation of TGF-beta activity by PPAR-gamma ligands involves cellular PPAR-gamma, since <em>15</em>-deoxy-Delta12,14-prostaglandin J(2) (<em>15</em>d-PGJ(2)) failed to block TGF-beta-induced responses in either primary cultures of PPAR-gamma-null murine embryonic <em>fibroblasts</em>, or in normal human skin <em>fibroblasts</em> with RNAi-mediated knockdown of PPAR-gamma. Next, we examined the molecular basis underlying the abrogation of TGF-beta signaling by PPAR-gamma in normal human <em>fibroblasts</em> in culture. The results demonstrated that Smad-dependent transcriptional responses were blocked by PPAR-gamma without preventing Smad2/3 activation. In contrast, the interaction between activated Smad2/3 and the transcriptional coactivator and histone acetyltransferase p300 induced by TGF-beta, and the accumulation of p300 on consensus Smad-binding DNA sequences and histone H4 hyperacetylation at the COL1A2 locus, were all prevented by PPAR-gamma. Wild-type p300, but not a mutant form of p300 lacking functional histone acetyltransferase, was able to restore TGF-beta-induced stimulation of COL1A2 in the presence of PPAR-gamma ligands. Collectively, these results indicate that PPAR-gamma blocked Smad-mediated transcriptional responses by preventing p300 recruitment and histone H4 hyperacetylation, resulting in the inhibition of TGF-beta-induced collagen gene expression. Pharmacological activation of PPAR-gamma thus may represent a novel therapeutic approach to target p300-dependent TGF-beta profibrotic responses such as stimulation of collagen gene expression.

OBJECTIVE

This study aimed to quantitate and compare the concentration of vascular endothelial growth factor (VEGF) in aqueous humor samples from patients with neovascular glaucoma (NVG), primary open-angle glaucoma (POAG), and cataract, as well as in serum samples of healthy human subjects.

METHODS

The authors collected aqueous humor samples by using their previously published technique of limbal paracentesis. The authors determined the concentration of VEGF by using a competitive enzyme immunoassay system and four-parameter logistic curve fitting and performed statistical analysis by using the Mann-Whitney-Wilcoxon test.

RESULTS

The authors detected VEGF in 12 of 12 samples from patients with NVG (mean +/- standard error of the mean, 29.267 +/- 7.350 ng/ml), 15 of 28 samples from patients with POAG (0.726 +/- 0.204 ng/ml), 4 of 20 aqueous humor samples from patients with cataract (0.257 +/- 0.043 ng/ml), and 16 of 16 human serum samples (20.246 +/- 1.568 ng/ml). The mean concentration of VEGF in aqueous humor of patients with NVG was 40- and 113-fold higher than that in patients with POAG and cataract, respectively, and the difference was statistically significant (P < 0.01). The VEGF level in patients with POAG was elevated compared with that in patients with cataract (P < 0.05). Although the mean concentration of VEGF in aqueous humor of patients with NVG was approximately 1.45-fold higher than that in serum, the difference was not significant (P>> 0.05).

CONCLUSIONS

The authors' findings show that patients with NVG had a significantly increased level of VEGF in the aqueous humor and implicate VEGF as an important factor in the pathogenesis of intraocular neovascularization in these patients. The authors discuss the possible role of the ciliary epithelium, in addition to retina, in the production of VEGF and the complementary function of basic fibroblast growth factor and other growth factors.

Levels of mRNA for IFN-beta 2/B cell differentiation <em>factor</em>2/hepatocyte-stimulating <em>factor</em> (IFN-beta 2) in confluent quiescent cultures of human diploid <em>fibroblasts</em> (FS-4 strain) are enhanced by TNF, IL-1 alpha and beta, platelet-derived <em>growth</em> <em>factor</em> (PDGF) and IFN-beta 1. Of these cytokines, IL-1 alpha and beta cause a particularly strong increase in the accumulation of IFN-beta 2 mRNA in <em>fibroblasts</em>. We have evaluated whether the IFN-beta 2 gene is regulated at the transcriptional level by using nuclear run-on transcription assays. We observed that the IFN-beta 2 gene is transcribed at a low level in uninduced FS-4 cells and that this transcriptional activity is increased 2- to 3-fold in cycloheximide-treated cells, 20- to 35-fold in IL-1 alpha-treated cells, and 5- to <em>15</em>-fold in TNF-treated cells. PDGF and IFN-beta 1 enhance transcription across the IFN-beta 2 gene 2- to 3-fold. The enhancing effect of IL-1 alpha on IFN-beta 2 gene transcription, but not that of TNF, PDGF, or IFN-beta 1, is inhibited by cycloheximide, suggesting that newly-synthesized protein is involved in the increase in IFN-beta 2 transcription in response to IL-1 alpha but not in the response to the other stimuli. Furthermore, the enhancement of IFN-beta 2 transcription is sustained for up to 14 h after IL-1 alpha induction but is transient and declines to base line levels within 6 h after TNF addition. These observations suggest that there are important differences in the mechanisms by which IL-1 alpha and TNF increase IFN-beta 2 gene transcription in <em>fibroblasts</em>.

This study analyzes the expression of monocyte chemoattractant protein-1 (MCP-1) by inflamed synovial tissue and defines its regulation in cultured synoviocytes. Synoviocytes from patients with rheumatoid arthritis and osteoarthritis express the 0.7-kb MCP-1 mRNA. Stimulation of synoviocytes with IL-1, TNF-alpha, LPS, platelet-derived <em>growth</em> <em>factor</em>, and transforming <em>growth</em> <em>factor</em>-beta-1, but not with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> causes a marked increase in MCP-1 mRNA levels. Expression of the MCP-1 gene is inducible by activators of the protein kinase A (cAMP) and C (PMA) signal transduction pathways and is differentially regulated by the steroids dexamethasone and retinoic acid. Cultured synoviocytes de novo synthesize 12-, <em>15</em>-, and <em>15</em>.2-kDa MCP-1 proteins, which increase after stimulation with IL-1. Synovial tissues from donors without joint disease and from patients with rheumatoid or osteoarthritis were analyzed for MCP-1 mRNA expression by in situ hybridization. In these samples MCP-1 mRNA expressing cells were predominantly found in the sublining cell layers, whereas specimens of normal synovial tissue contained only few positive cells. These results identify synoviocytes as a source of MCP-1. Its expression is controlled by peptide regulatory <em>factors</em> that are known to be present in arthritic joints. Detection of cells producing MCP-1 mRNA in synovial tissues from patients with arthritis shows that this gene is expressed in vivo and suggests that MCP-1 can play a role in recruiting monocytes in joint inflammation.

Progesterone is the hormone of pregnancy and unequivocally required in all mammals for maternal support of conceptus (embryo/fetus and associated membranes) survival and development. The actions of progesterone are mediated by the progesterone receptor (PR). However, the endometrial lumenal (LE) and glandular epithelia (GE) of a number of species exhibit a loss of PR expression prior to the stages of uterine receptivity and implantation. In sheep, PR expression becomes undetectable in the endometrial LE after Day 11 and then in the GE after Day 13. Loss of PR in the GE appears to be required for onset of differentiated functions in terms of production of secretory proteins, such as uterine milk proteins (UTMP) and osteopontin (OPN). Therefore, the actions of progesterone on endometrial epithelia during most of gestation appear to be mediated by the endometrial stroma that remains PR-positive throughout pregnancy. Stromal cells produce several <em>growth</em> <em>factors</em>, such as hepatocyte <em>growth</em> <em>factor</em> (HGF) and <em>fibroblast</em> <em>growth</em> <em>factors</em>-7 and -10 (FGF-7, FGF-10), that have receptors expressed specifically in the endometrial epithelia. These <em>factors</em> may be progesterone-responsive and mediate epithelial-mesenchymal interactions that are crucial for support of pregnancy. Studies of the uterine gland knockout (UGKO) ewe indicate that uterine glands and, by default, their secretions are required for peri-implantation conceptus survival and <em>growth</em>. A complex servomechanism, involving hormones from the ovary and conceptus as well as endogenous betaretroviruses expressed in the endometrial LE and GE, is proposed to regulate endometrial gland differentiation and function during gestation. At estrus, estrogen increases PR expression in the endometrial epithelia. High levels of endogenous Jaagsiekte sheep retroviruses (enJSRVs) are expressed in the PR-positive endometrial LE and GE in response to increasing progesterone and are hypothesized to stimulate trophoblast proliferation and production of interferon (IFN) tau. IFN tau, the pregnancy recognition hormone produced by the trophoblast from Days 10 to 21, acts in a paracrine manner on the PR-negative endometrial LE and superficial GE to inhibit transcription of estrogen receptor alpha (ER) and oxytocin receptor (OTR) genes. These actions of IFN tau maintain progesterone production from the corpus luteum by abrogating release of luteolytic pulses of prostaglandin F2 alpha (PGF) from the endometrial epithelium. The antiluteolytic effects of IFN tau are dependent on progesterone. Progesterone stimulation over 8-10 days suppresses expression of the PR gene in the LE and then GE. Loss of the PR in the LE is concomitant with decreases in mucin glycoprotein one (MUC-1), an inhibitor of blastocyst implantation. As the conceptus begins implantation on Day <em>15</em>, the binucleate trophectodermal cells then differentiate and produce placental lactogen (PL), a member of the prolactin (PRL) and <em>growth</em> hormone (GH) family. PL stimulates GE proliferation and production of secretory proteins, such as UTMP and OPN. Interestingly, the effects of PL on the GE appear to require the absence of PR and prior exposure to IFN tau. During mid-pregnancy, the mononuclear trophectodermal cells produce GH that can also act on a progestinized uterus to stimulate GE hypertrophy and secretory function. The actions of this servomechanism are proposed to stimulate GE hyperplasia from Days 20 to 50 and then GE hypertrophy and maximal differentiated function after Day 50 when the majority of fetal <em>growth</em> and development occurs during gestation.

Endostatin is a Mr 20,000 COOH-terminal fragment of collagen XVIII that inhibits the <em>growth</em> of several primary tumors. We report here the cloning and expression of mouse endostatin in both prokaryotic and eukaryotic expression systems. Soluble recombinant protein expressed in yeast (<em>15</em>-20 mg/L) inhibited the proliferation and migration of endothelial cells in response to stimulation by basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. A rabbit polyclonal antibody was raised that showed positive immunoreactivity to the recombinant protein expressed from both systems. Importantly, the biological activity of the mouse recombinant protein could be neutralized by this antiserum in both endothelial proliferation and chorioallantoic membrane assays. Systemic administration of endostatin at 10 mg/kg suppressed the <em>growth</em> of renal cell cancer in a nude mouse model. The inhibition of tumor <em>growth</em> with soluble yeast-produced protein was comparable to that obtained with non-refolded precipitated protein expressed from bacteria. In addition, two closely related COOH-terminal deletion mutants of endostatin were also tested and showed strikingly differing activity. Collectively, these findings demonstrate the expression of a biologically active form of mouse endostatin in yeast, define a role for the molecule in inhibiting endothelial cell migration, extend its antitumor effects to renal cell carcinoma, and provide a formal proof (via the neutralizing antiserum experiments and the mutant data) that endostatin (and not a possible contaminant) acts as an antiangiogenic agent. Finally, the high level expression of mouse endostatin in yeast serves as an endotoxin free, soluble source of protein for fundamental studies on the mechanisms of tumor <em>growth</em> suppression by angiogenesis inhibitors.

Synovial membrane biopsy specimens from <em>15</em> rheumatoid arthritis patients were examined using routine histologic stains and monoclonal antibodies directed against cell surface antigens. Three patterns of lymphoid cell infiltrates were recognized: 1) diffuse infiltration of T cells that surrounded clusters of germinal center B cells (3 patients); 2) diffuse T cell infiltration, lacking germinal centers (8 patients); and 3) proliferation of subsynovial <em>fibroblasts</em>, with relatively few lymphoid cells (4 patients). The synovial, subsynovial, and perivascular tissues in each of the patterns exhibited a high frequency of HLA-DR antigen, HLA-DS antigen, transferrin receptor, and/or epidermal <em>growth</em> <em>factor</em> receptor. In contrast, normal or osteoarthritic synovial tissues did not display a marked increase of these antigens or receptors. Cells bearing natural killer antigen were infrequent in each of these patterns. Active synovitis, synovial effusions, anemia, and elevated sedimentation rate were present in rheumatoid arthritis patients with each of the three histologic patterns. Immunohistologic characterization of synovial membrane infiltrates by these monoclonal antibodies provides additional information about pathogenesis of rheumatoid arthritis and may help in predicting responses to different therapeutic modalities.

This study determined the presence of interleukin 1 (IL-1), interleukin 6 (IL-6), tumour necrosis <em>factor</em> alpha (TNF alpha), tumour necrosis <em>factor</em> beta (TNF beta), interferon gamma (IFN gamma), transforming <em>growth</em> <em>factor</em> beta 2 (TGF beta 2) and <em>fibroblast</em> proliferation activity (FPA) in vitreous aspirates from eyes undergoing vitrectomy for the treatment of retinal detachment complicated by proliferative vitreoretinopathy (PVR) or uncomplicated retinal detachment (RD). Cadaveric vitreous from normal subjects were used as controls. The results showed that IL-1 and IL-6 predominated in vitreous from eyes with PVR or RD, and that concentrations of IL-6 greater than 20 pg/ml were more frequently found in PVR than in RD (p = 0.031) or control specimens (p = 0.006). Low levels of TNF alpha were observed in 4/18 eyes with PVR, 1/<em>15</em> eyes with RD and 1/<em>15</em> control vitreous, and small concentrations of TNF alpha were seen in 3/18 eyes with PVR, 1/<em>15</em> eyes with RD and 2/<em>15</em> control vitreous. IFN gamma was detected in 12/18 eyes with PVR, but only in 5/<em>15</em> eyes with RD (p = 0.048) and 6/<em>15</em> control specimens. TGF beta 2 was present in all vitreous samples at concentrations ranging from 100 to 4,500 pg/ml with no significant differences among the three groups. Control vitreous possessed the greatest FPA when compared with vitreous from eyes with PVR (p = 0.031) or RD (p = 0.048). These observations provide further evidence that cytokine-mediated pathways of inflammation are involved in the pathogenesis of PVR and point to the possible involvement of IL-1, IL-6 and IFN gamma in cellular interactions leading to chronicity.

Activation of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) receptors elicits diverse cellular responses including <em>growth</em>, mitogenesis, migration, and differentiation. The intracellular signaling pathways that mediate these important processes are not well understood. In Caenorhabditis elegans, suppressors of clr-1 identify genes, termed soc genes, that potentially mediate or activate signaling through the EGL-<em>15</em> FGF receptor. We demonstrate that three soc genes, soc-1, soc-2, and sem-5, suppress the activity of an activated form of the EGL-<em>15</em> FGF receptor, consistent with the soc genes functioning downstream of EGL-<em>15</em>. We show that soc-2 encodes a protein composed almost entirely of leucine-rich repeats, a domain implicated in protein-protein interactions. We identified a putative human homolog, SHOC-2, which is 54% identical to SOC-2. We find that shoc-2 maps to 10q25, shoc-2 mRNA is expressed in all tissues assayed, and SHOC-2 protein is cytoplasmically localized. Within the leucine-rich repeats of both SOC-2 and SHOC-2 are two YXNX motifs that are potential tyrosine-phosphorylated docking sites for the SEM-5/GRB2 Src homology 2 domain. However, phosphorylation of these residues is not required for SOC-2 function in vivo, and SHOC-2 is not observed to be tyrosine phosphorylated in response to FGF stimulation. We conclude that this genetic system has allowed for the identification of a conserved gene implicated in mediating FGF receptor signaling in C. elegans.

OBJECTIVE

To perform a phase I trial of recombinant human endostatin (rhEndostatin; EntreMed, Rockville, MD) given as a daily 20-minute intravenous (IV) injection in adult patients with refractory solid tumors.

METHODS

The daily dose was increased from <em>15</em> to 240 mg/m(2) by a <em>factor</em> of 100% in cohorts of three patients. In the absence of dose-limiting toxicity, uninterrupted treatment was continued until the tumor burden increased by more than 50% from baseline. Correlative studies included dynamic contrast-enhanced magnetic resonance imaging of tumor blood flow, urinary vascular endothelial <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> levels, rhEndostatin serum pharmacokinetics, and monitoring of circulating antibodies to rhEndostatin.

RESULTS

There were no notable treatment related toxicities among <em>15</em> patients receiving a total of 50 monthly cycles of rhEndostatin. One patient with a pancreatic neuroendocrine tumor had a minor response and two patients showed disease stabilization. Linearity in the pharmacokinetics of rhEndostatin was indicated by dose-proportionate increases in the area under the curve for the first dose and the peak serum concentration at steady state. Daily systemic exposure to rhEndostatin in patients receiving 240 mg/m(2)/d was approximately 50% lower than that provided by the therapeutically optimal dose in preclinical studies.

CONCLUSIONS

rhEndostatin administered as a 20-minute daily IV injection at doses up to 240 mg/m(2) showed no significant toxicities. Evidence of clinical benefit was observed in three patients. Due to high variability between the peak and trough serum concentrations associated with the repeated short IV infusion schedule, daily serum drug levels only briefly exceeded concentrations necessary for in vitro antiangiogenic effects.

OBJECTIVE

The objective of this study was to evaluate the safety and tolerance of increasing single and repeated (n = 2) doses of intramuscular naked plasmid DNA encoding for fibroblast growth factor (FGF) type 1 (NV1FGF) administered to patients with unreconstructible end-stage peripheral arterial occlusive disease (PAD). The secondary objectives were to determine the biologic activity of NV1FGF on hemodynamic and clinical parameters associated with improved perfusion.

METHODS

Fifty-one patients with unreconstructible peripheral arterial occlusive disease with rest pain or tissue necrosis underwent treatment with intramuscular NV1FGF. Increasing single (500, 1000, 2000, 4000, 8000, and 16,000 microg) and repeated (2 x 500, 2 x 1000, 2 x 2000, 2 x 4000, and 2 x 8000 microg) doses of NV1FGF were injected into the ischemic thigh and calf. Arteriography was performed before treatment and was repeated 12 weeks after treatment. Side effects and serious adverse events were monitored. Measurements of plasma and urine levels were performed to evaluate NV1FGF plasmid distribution. Serum FGF-1 was measured as an analysis of gene expression at the protein level. Transcutaneous oxygen pressure, ankle brachial index, toe brachial index, pain assessment with visual analog scale, and ulcer healing also were assessed. The safety results are presented for 51 patients, and the clinical outcomes are presented for the first 15 patients (500 to 4000 microg) who completed the 6-month follow-up study.

RESULTS

NV1FGF was well tolerated. Sixty-six serious adverse events were reported; however, none were considered to be related to NV1FGF. Four patients had adverse events that were possibly or probably related to the study treatment: injection site pain, pain, peripheral edema, myasthenia, and paresthesia. No laboratory adverse events were related to the study treatment. Two deaths remote from the treatment were considered not related. Biodistribution of plasmid was limited and transient in plasma and absent in urine. No increase in the FGF-1 serum level was detected. A significant reduction in pain (P <.001) and aggregate ulcer size (P <.01) was associated with an increased transcutaneous oxygen pressure (P <.01) as compared with baseline pretreatment values. A significant increase in ankle brachial index (P <.01) was seen.

CONCLUSIONS

NV1FGF is well tolerated and potentially could be effective for the treatment of patients with end-stage limb ischemia. Biologic parameters indicate improved perfusion after NV1FGF administration. Dose response is not yet evident. The safety of NV1FGF and the magnitude of improvement observed in this study encourage further investigation with a placebo-controlled, double-blind clinical trial.

During wound healing, <em>fibroblasts</em> are recruited from the surrounding tissue to accomplish repair. The requisite migration and proliferation of the <em>fibroblasts</em> is promoted by <em>growth</em> <em>factors</em> including those that activate the epidermal <em>growth</em> <em>factor</em> receptor (EGFR). Counterstimulatory <em>factors</em> in wound fluid are postulated to limit this response; among these <em>factors</em> is the ELR-negative CXC chemokine, interferon inducible protein-10 (IP-10). We report here that IP-10 inhibited EGF- and heparin-binding EGF-like <em>growth</em> <em>factor</em>-induced Hs68 human dermal <em>fibroblast</em> motility in a dose-dependent manner (to 52% and 44%, respectively, at 50 ng/ml IP-10), whereas IP-10 had no effect on either basal or EGFR-mediated mitogenesis (96 +/- <em>15</em>% at 50 ng/ml). These data demonstrate for the first time a counterstimulatory effect of IP-10 on a specific induced <em>fibroblast</em> response, EGFR-mediated motility. To define the molecular basis of this negative transmodulation of EGFR signaling, we found that IP-10 did not adversely impact receptor or immediate postreceptor signaling as determined by tyrosyl phosphorylation of EGFR and two major downstream effectors phospholipase C-gamma and erk mitogen-activated protein kinases. Morphological studies suggested which biophysical steps may be affected by demonstrating that IP-10 treatment resulted in an elongated cell morphology reminiscent of failure to detach the uropod; in support of this, IP-10 pretreatment inhibited EGF-induced cell detachment. These data suggested that calpain activity may be involved. The cell permeant agent, calpain inhibitor I, limited EGF-induced motility and de-adhesion similarly to IP-10. IP-10 also prevented EGF- induced calpain activation (reduced by 71 +/- 7%). That this inhibition of EGF-induced calpain activity was secondary to IP-10 initiating a cAMP-protein kinase A-calpain cascade is supported by the following evidence: (a) the cell permeant analogue 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) prevented EGF-induced calpain activity and motility; (b) other ELR-negative CXC chemokines, monokine induced by IFN-gamma and platelet <em>factor</em> 4 that also generate cAMP, inhibited EGF-induced cell migration and calpain activation; and (c) the protein kinase A inhibitor Rp-8-Br-cAMPS abrogated IP-10 inhibition of cell migration, cell detachment, and calpain activation. Our findings provide a model by which IP-10 suppresses EGF-induced cell motility by inhibiting EGF-induced detachment of the trailing edges of motile cells.

In this report, we have investigated the secretion and synthesis of transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1) by human dermal <em>fibroblast</em> cultures in response to hypoxia (2% oxygen), and have compared it to standard oxygen culture conditions (<em>15</em>% oxygen at the cell surface). Sandwich enzyme-linked immunosorbent assay (SELISA) showed a selective and progressive increase in secretion of the TGF-beta 1 isoform in response to hypoxia, up to ninefold after cultures were exposed to low oxygen for 72 h; TGF-beta 2 peptide levels were not increased. We then investigated the transcriptional regulation of the TGF-beta 1 gene in response to low and standard oxygen tensions. In the first 24-48 h, TGF-beta 1 mRNA levels decreased steadily in both oxygen environments. This mRNA decline continued for up to 72 h in standard oxygen but not in cultures exposed to low oxygen tension. At 72 h, steady-state TGF-beta 1 mRNA levels were 8 times greater in low compared to standard oxygen, and this increase was reversible upon re-exposure of <em>fibroblast</em> cultures to standard oxygen tension for 24 h. Elevated TGF-beta 1 m-RNA levels in both low and standard oxygen declined steadily and with the same half-life after the addition of actinomycin D, suggesting that hypoxia increased TGF-beta 1 transcription rather than mRNA stability. We conclude that low oxygen tension upregulates the synthesis of TGF-beta 1 by human dermal <em>fibroblasts</em>, and leads to increased secretion of this peptide.

The binding interactions for the three primary reactants of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) system, basic FGF (bFGF), an FGF receptor, FGFR1, and the co<em>factor</em> heparin/heparan sulfate (HS), were explored by isothermal titrating calorimetry, ultracentrifugation, and molecular modeling. The binding reactions were first dissected into three binary reactions: (1) FGFR1 + bFGF<=>>FGFR1/bFGF, K1 = 41 (+/- 12) nM; (2) FGFR1 + HS<=>>FGFR1/HS, K2 = 104 (+/- 17) microM; and (3) bFGF + HS<=>>bFGF/HS, K3 = 470 (+/- 20) nM, where HS = low MW heparin, approximately 3 kDa. The first, binding of bFGF to FGFR1 in the absence of HS, was found to be a simple binary binding reaction that is enthalpy dominated and characterized by a single equilibrium constant, K1. The conditional reactions of bFGF and FGFR1 in the presence of heparin were then examined under conditions that saturate only the bFGF heparin site (1.5 equiv of HS/bFGF) or saturate the HS binding sites of both bFGF and FGFR1 (1.0 mM HS). Both 3-and 5-kDa low MW heparins increased the affinity for FGFR1 binding to bFGF by approximately 10-fold (Kd = 4.9 +/- 2.0 nM), relative to the reaction with no HS. In addition, HS, at a minimum of 1.5 equiv/bFGF, induced a second FGFR1 molecule to bind to another lower affinity secondary site on bFGF (K4 = 1.9 +/- 0.7 microM) in an entropy-dominated reaction to yield a quaternary complex containing two FGFR1, one bFGF, and at least one HS. Molecular weight estimates by analytical ultracentrifugation of such fully bound complexes were consistent with this proposed composition. To understand these binding reactions in terms of structural components of FGFR1, a three-dimensional model of FGFR1 was constructed using segment match modeling. Electrostatic potential calculations confirmed that an elongated cluster, approximately <em>15</em> x 35 A, of nine cationic residues focused positive potential (+2kBT) to the solvent-exposed beta-sheet A, B, E, C' surface of the D(II) domain model, strongly implicating this locus as the HS binding region of FGFR1. Structural models for HS binding to FGFR1, and HS binding to bFGF, were built individually and then assembled to juxtapose adjacent binding sites for receptor and HS on bFGF, against matching proposed <em>growth</em> <em>factor</em> and HS binding sites on FGFR1. The calorimetric binding results and the molecular modeling exercises suggest that bFGF and HS participate in a concerted bridge mechanism for the dimerization of FGFR1 in vitro and presumably for mitogenic signal transduction in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

BACKGROUND

Vascular endothelial growth factor (VEGF) is a proangiogenic molecule produced by melanoma cells. We hypothesized that administration of bevacizumab (Bev), a monoclonal antibody that neutralizes VEGF, with low-dose interferon alfa-2b (IFN-alpha2b), an inhibitor of basic fibroblast growth factor (FGF), would lead to the regression of metastatic melanoma.

METHODS

Patients with metastatic melanoma were randomized to receive Bev (15 mg/kg intravenously every 2 weeks) with or without low-dose IFN-alpha2b (1 MU/m2 subcutaneously daily). Patients exhibiting a clinical response or stable disease after 12 weeks were treated until disease progression.

RESULTS

Thirty-two patients (16 per arm) were accrued (18 male, 14 female; mean age 57.5 years). Both regimens were well tolerated. Six patients developed easily managed exacerbations of preexisting hypertension. Two patients developed grade 3 proteinuria that resolved after a treatment break. IFN-alpha2b therapy was associated with grade 1 to 2 constitutional symptoms. Arterial thromboembolic complications were observed in three patients (two mild myocardial infarctions, one transient ischemic attack), all of whom had risk factors. One patient (Bev plus IFN-alpha2b arm) had locally recurrent scalp disease that partially responded to therapy. Eight patients (five Bev, three Bev plus IFN-alpha2b) had prolonged disease stabilization (24 to 146 weeks). Plasma levels of VEGF and FGF did not correlate with any clinical parameter. The patient with the longest period of stable disease had the highest baseline VEGF and FGF.

CONCLUSIONS

Bev was well tolerated at this dose and prolonged disease stabilization was achieved in one-quarter of metastatic melanoma patients. Low-dose IFN-alpha2b did not augment the activity of Bev.