<em>Fibroblast</em> <em>Growth</em> <em>Factors</em> (FGFs) are polypeptides with diverse activities in development and physiology. The mammalian Fgf family can be divided into the intracellular Fgf11/12/13/14 subfamily (iFGFs), the hormone-like Fgf15/21/23 subfamily (hFGFs), and the canonical Fgf subfamilies, including Fgf1/2/5, Fgf3/4/6, Fgf7/10/22, Fgf8/17/18, and Fgf9/16/<em>20</em>. However, all Fgfs are evolutionarily related. We propose that an Fgf13-like gene is the ancestor of the iFgf subfamily and the most likely evolutionary ancestor of the entire Fgf family. Potential ancestors of the canonical and hFgf subfamilies, Fgf4-, Fgf5-, Fgf8-, Fgf9-, Fgf10-, and Fgf15-like, appear to have derived from an Fgf13-like ancestral gene. Canonical FGFs function in a paracrine manner, while hFGFs function in an endocrine manner. We conclude that the ancestral Fgfs for these subfamilies acquired this functional diversity before the evolution of vertebrates. During the evolution of early vertebrates, the Fgf subfamilies further expanded to contain three or four members in each subfamily.

Cellular senescence irreversibly arrests cell proliferation in response to oncogenic stimuli. Human cells develop a senescence-associated secretory phenotype (SASP), which increases the secretion of cytokines and other <em>factors</em> that alter the behavior of neighboring cells. We show here that "senescent" mouse <em>fibroblasts</em>, which arrested <em>growth</em> after repeated passage under standard culture conditions (<em>20</em>% oxygen), do not express a human-like SASP, and differ from similarly cultured human cells in other respects. However, when cultured in physiological (3%) oxygen and induced to senesce by radiation, mouse cells more closely resemble human cells, including expression of a robust SASP. We describe two new aspects of the human and mouse SASPs. First, cells from both species upregulated the expression and secretion of several matrix metalloproteinases, which comprise a conserved genomic cluster. Second, for both species, the ability to promote the <em>growth</em> of premalignant epithelial cells was due primarily to the conserved SASP <em>factor</em> CXCL-1/KC/GRO-alpha. Further, mouse <em>fibroblasts</em> made senescent in 3%, but not <em>20</em>%, oxygen promoted epithelial tumorigenesis in mouse xenographs. Our findings underscore critical mouse-human differences in oxygen sensitivity, identify conditions to use mouse cells to model human cellular senescence, and reveal novel conserved features of the SASP.

Some propose using phosphate binders in the CKD population given the association between higher levels of phosphorus and mortality, but their safety and efficacy in this population are not well understood. Here, we aimed to determine the effects of phosphate binders on parameters of mineral metabolism and vascular calcification among patients with moderate to advanced CKD. We randomly assigned 148 patients with estimated GFR=<em>20</em>-45 ml/min per 1.73 m(2) to calcium acetate, lanthanum carbonate, sevelamer carbonate, or placebo. The primary endpoint was change in mean serum phosphorus from baseline to the average of months 3, 6, and 9. Serum phosphorus decreased from a baseline mean of 4.2 mg/dl in both active and placebo arms to 3.9 mg/dl with active therapy and 4.1 mg/dl with placebo (P=0.03). Phosphate binders, but not placebo, decreased mean 24-hour urine phosphorus by 22%. Median serum intact parathyroid hormone remained stable with active therapy and increased with placebo (P=0.002). Active therapy did not significantly affect plasma C-terminal <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 levels. Active therapy did, however, significantly increase calcification of the coronary arteries and abdominal aorta (coronary: median increases of 18.1% versus 0.6%, P=0.05; abdominal aorta: median increases of 15.4% versus 3.4%, P=0.03). In conclusion, phosphate binders significantly lower serum and urinary phosphorus and attenuate progression of secondary hyperparathyroidism among patients with CKD who have normal or near-normal levels of serum phosphorus; however, they also promote the progression of vascular calcification. The safety and efficacy of phosphate binders in CKD remain uncertain.

Human mesenchymal stem cells (hMSCs) expanded with and without <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) supplementation were compared with respect to their proliferation rate, ability to differentiate along the chondrogenic pathway in vitro, and their gene expression profiles. hMSCs expanded in FGF-supplemented medium were smaller and proliferated more rapidly than hMSCs expanded in control conditions. Chondrogenic cultures made with FGF-treated cells were larger and contain more proteoglycan than those made with control cells. Furthermore, aggregates of FGF-treated cells lacked the collagen type I-positive and collagen type II-negative outer layer characteristic of aggregates of control cells. A total of 358 unique transcripts were differentially expressed in FGF-treated hMSCs. Of these, 150 were upregulated and <em>20</em>8 downregulated. Seventeen percent of these genes affect proliferation. Known genes associated with cellular signaling functions comprised the largest percentage ( approximately <em>20</em>%) of differentially expressed transcripts. Eighty percent of differentially expressed extracellular matrix-related genes were downregulated. The present findings that FGF-2 enhances proliferation and differentiation of hMSCs adds to a <em>growing</em> body of evidence that cytokines modulate the differentiation potential and, perhaps, the multipotentiality of adult stem cells. With the generation of gene expression profiles of FGF-treated and control cells we have taken the first steps in the elucidation of the molecular mechanism(s) behind these phenomena.

Type I (alpha, beta) and type II (gamma) interferons (IFNs) can restrict the <em>growth</em> of many cell types. INF-stimulated gene transcription, a key early event in IFN response, acts through the Janus kinase-signal transducers and activators of transcription pathway, in which both IFN-alpha and IFN-gamma activate the transcription <em>factor</em> Stat1. A cell line lacking Stat1 (U3A) was not <em>growth</em>-arrested by IFN-alpha or IFN-gamma, and experiments were carried out with U3A cells permanently expressing normal or various mutant forms of Stat1 protein. Only cells in which complete Stat1 activity was available (Stat1alpha) were <em>growth</em>-inhibited by IFN-gamma. A mutant that supports <em>20</em>-30% normal transcription did not cause <em>growth</em> restraint. In contrast, IFN-alpha <em>growth</em> restraint was imposed by cells producing Stat1beta, which lacks transcriptional activation potential. This parallels earlier results showing the truncated Stat1 can function in IFN-alpha gene activation. In addition to experiments on long-term cultured cells, we also found that wild-type primary mouse embryonic <em>fibroblasts</em> were inhibited by IFNs, but <em>fibroblasts</em> from Stat1-deficient mouse embryos were not inhibited by IFNs.

Epithelial-to-mesenchymal transition (EMT) is a process that plays essential roles in development and wound healing that is characterized by loss of homotypic adhesion and cell polarity and increased invasion and migration. At the molecular level, EMT is characterized by loss of E-cadherin and increased expression of several transcriptional repressors of E-cadherin expression (Zeb-1, Zeb-2, Twist, Snail, and Slug). Early work established that loss of E-cadherin and increased expression of MMP-9 was associated with a poor clinical outcome in patients with urothelial tumors, suggesting that EMT might also be associated with bladder cancer progression and metastasis. More recently, we have used global gene expression profiling to characterize the molecular heterogeneity in human urothelial cancer cell lines (n = <em>20</em>) and primary patient tumors, and unsupervised clustering analyses revealed that the cells naturally segregate into two discrete "epithelial" and "mesenchymal" subsets, the latter consisting entirely of muscle-invasive tumors. Importantly, sensitivity to inhibitors of the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) or type-3 <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR3) was confined to the "epithelial" subset, and sensitivity to EGFR inhibitors could be reestablished by micro-RNA-mediated molecular reversal of EMT. The results suggest that EMT coordinately regulates drug resistance and muscle invasion/metastasis in urothelial cancer and is a dominant feature of overall cancer biology.

OBJECTIVE

Hydrogen sulfide (H(2)S) has been reported to be a gasotransmitter which regulates cardiovascular homeostasis. The present study aims to examine the hypothesis that hydrogen sulfide is able to promote angiogenesis.

METHODS

Angiogenesis was assessed using in vitro parameters (i.e. endothelial cell proliferation, adhesion, transwell migration assay, scratched wound healing and formation of tube-like structure) and in vivo by assessing neovascularization in mice. Phosphorylation of Akt was measured using Western blot analysis.

RESULTS

Exogenously administered NaHS (H(2)S donor) concentration-dependently (10-<em>20</em> micromol/l) increased cell <em>growth</em>, migration, scratched wound healing and tube-like structure formation in cultured endothelial cells. These effects of NaHS on endothelial wound healing and tube-like structure formation were prevented by either the phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294002 (5 micromol/l) or transfection of a dominant-negative mutant of Akt. NaHS increased Akt phosphorylation and this effect was also blocked by either LY 294002 or wortmannin (25 nmol/l). NaHS did not significantly alter the levels of vascular endothelial <em>growth</em> <em>factor</em>, mRNA expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> and angiopoietin-1, or nitric oxide metabolites. NaHS treatment (10 and 50 micromol kg(-1) day(-1)) significantly promoted neovascularization in vivo in mice.

CONCLUSIONS

The present study reports a novel proangiogenic role of H(2)S which is dependent on activation of Akt.

The mineralized matrix of osseous tissue harbors abundant mitogenic activity which is extractable by demineralizing solvents. In bovine bone powder free of blood and cartilage contamination, the volume concentration of mitogens is up to <em>20</em> times greater than in serum. <em>Growth</em> <em>factor</em> activity in bone extracts was quantitated on quiescent mouse BALB/c/3T3 <em>fibroblasts</em>, where [3H]thymidine incorporation for 48 h was stimulated up to <em>20</em>0-fold in a linear, dose-dependent manner. Six distinct bone-derived <em>growth</em> <em>factors</em> (BDGFs) have been resolved and partially purified (up to 44,000-fold) on heparin-Sepharose using NaCl gradient elution. Provisionally named by the NaCl molarity at which they elute, these BDGFs include BDGF-0.45 (25% of total activity). This <em>factor</em> is heat-stable and sensitive to dithiothreitol, and displaces 125I-labelled bovine platelet-derived <em>growth</em> <em>factor</em> in a radioreceptor assay. BDGF-0.45 (approximately 50 ng/g of bone) is closely related or identical to bovine platelet-derived <em>growth</em> <em>factor</em>. BDGF-1.1 (10%) has a pI of 5.2 and shows a 16,600-dalton doublet on sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blots stained with antiserum to bovine anionic <em>fibroblast</em> <em>growth</em> <em>factor</em>. Two activities with high heparin affinity resemble cationic forms of <em>fibroblast</em> <em>growth</em> <em>factor</em>. BDGF-1.5 is the dominant <em>factor</em> in fetal membranous bone (50%), but is less abundant in adult bone (<em>20</em>%). BDGF-1.7, a 17,500-18,400-dalton triplet, is virtually absent in fetal bone (7%) but abundant (30%) in adult bone and may be related to cartilage derived <em>growth</em> <em>factor</em>. Two minor activities, BDGF-0.1 (10%) and BDGF-2.0 (7%) have not been characterized. Proliferation of bovine capillary endothelial cells was strongly supported by BDGFs 1.1, 1.5, and 1.7, but not by 0.45. These four purified BDGFs and the crude bone extract were also strongly mitogenic for rat osteoblasts while depressing alkaline phosphatase specific activity by 2-3-fold. Bone exhibits the most complex spectrum of <em>growth</em> <em>factor</em> activities of any tissue yet described. Bone cells and other indigenous cell types must be considered as possible sources of the BDGFs, in addition to sequestration from blood. Mechanisms for unmasking or release of BDGFs from the mineralized matrix resulting in local action on target cells are undoubtedly important for the development and maintenance of bone tissue.

Neurogenesis, which may contribute to the ability of the adult brain to function normally and adapt to disease, nevertheless declines with advancing age. Adult neurogenesis can be enhanced by administration of <em>growth</em> <em>factors</em>, but whether the aged brain remains responsive to these <em>factors</em> is unknown. We compared the effects of intracerebroventricular <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2 and heparin-binding epidermal <em>growth</em> <em>factor</em>-like <em>growth</em> <em>factor</em> (HB-EGF) on neurogenesis in the hippocampal dentate subgranular zone (SGZ) and the subventricular zone (SVZ) of young adult (3-month) and aged (<em>20</em>-month) mice. Neurogenesis, measured by labelling with bromodeoxyuridine (BrdU) and by expression of doublecortin, was reduced by approximately 90% in SGZ and by approximately 50% in SVZ of aged mice. HB-EGF increased BrdU labelling in SGZ at 3 months by approximately 60% and at <em>20</em> months by approximately 450%, which increased the number of BrdU-labelled cells in SGZ of aged mice to approximately 25% of that in young adults. FGF-2 also stimulated BrdU labelling in SGZ, by approximately 25% at 3 months and by approximately 250% at <em>20</em> months, increasing the number of newborn neurones in older mice to approximately <em>20</em>% of that in younger mice. In SVZ, HB-EGF and FGF-2 increased BrdU incorporation by approximately 140% at 3 months and approximately 170% at <em>20</em> months, so the number of BrdU-labelled cells was comparable in untreated 3-month-old and <em>growth</em> <em>factor</em>-treated <em>20</em>-month-old mice. These results demonstrate that the aged brain retains the capacity to respond to exogenous <em>growth</em> <em>factors</em> with increased neurogenesis, which may have implications for the therapeutic potential of neurogenesis enhancement in age-associated neurological disorders.

BACKGROUND

There are several reports that engrafted mesenchymal stem cells (MSCs) stimulate angiogenesis in the ischemic heart, but the mechanism remains controversial. We hypothesize that transplantation of MSCs enhances vascular regeneration through a paracrine action.

METHODS

A transmural myocardial infarction was created by ligation of the left anterior descending coronary artery in rats. Those with an ejection fraction less than 0.70 1 week after myocardial infarction were included. Autologous MSCs (1 x 10(7); 0.2 mL) or culture medium (0.2 mL) was injected intramyocardially into the periinfarct zone (50 microL/injection at four sites; n = <em>20</em>/group). At 2 weeks after transplantation, Western blot analysis was used to assay the paracrine <em>factors</em> and proapoptotic proteins. Echocardiography to assess heart function was performed on additional groups at 8 weeks after implantation.

RESULTS

The angiogenic factors basic fibroblast growth factor, vascular endothelial growth factor, and stem cell homing factor (stromal cell-derived factor -1alpha) increased in the MSC-treated hearts compared with medium-treated hearts. This was accompanied by a downregulation of proapoptotic protein Bax in ischemic myocardium. Similarly, capillary density increased about 40% in MSC-treated hearts compared with medium-treated hearts (p = 0.001). Left ventricular contractility, indicated by fractional shortening, improved in MSC-treated hearts at 2 months after implantation (MSCs: 48.6% +/- 19.9%; medium: 18.7% +/- 6.4%; p = 0.004).

CONCLUSIONS

Autologous MSC transplantation attenuates left ventricular remodeling and improves cardiac performance. The major mechanism appears to be paracrine action of the engrafted cells, increasing angiogenesis and cytoprotection.

BACKGROUND

The pathophysiology of heart failure with normal ejection fraction (HFNEF) is still under discussion. Here we report the influence of cardiac inflammation on extracellular matrix (ECM) remodeling in patients with HFNEF.

RESULTS

We investigated left ventricular systolic and diastolic function in <em>20</em> patients with HFNEF and 8 control patients by conductance catheter methods and echocardiography. Endomyocardial biopsy samples were also obtained, and ECM proteins as well as cardiac inflammatory cells were investigated. Primary human cardiac <em>fibroblasts</em> were outgrown from the endomyocardial biopsy samples to investigate the gene expression of ECM proteins after stimulation with transforming <em>growth</em> <em>factor</em>-β. Diastolic dysfunction was present in the HFNEF patients compared with the control patients. In endomyocardial biopsy samples from HFNEF patients, we found an accumulation of cardiac collagen, which was accompanied by a decrease in the major collagenase system (matrix metalloproteinase-1) in the heart. Moreover, a subset of inflammatory cells, which expressed the profibrotic <em>growth</em> <em>factor</em> transforming <em>growth</em> <em>factor</em>-β, could be documented in the HFNEF patients. Stimulation of primary human cardiac <em>fibroblasts</em> from HFNEF patients with transforming <em>growth</em> <em>factor</em>-β resulted in transdifferentiation of <em>fibroblasts</em> to myo<em>fibroblasts</em>, which produced more collagen and decreased the amount of matrix metalloproteinase-1, the major collagenase in the human heart. A positive correlation between cardiac collagen, as well as the amount of inflammatory cells, and diastolic dysfunction was evident and suggests a direct influence of inflammation on fibrosis triggering diastolic dysfunction.

CONCLUSIONS

Cardiac inflammation contributes to diastolic dysfunction in HFNEF by triggering the accumulation of ECM.

We recently identified activating mutations of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) in bladder carcinoma. In this study we assessed the incidence of FGFR3 mutations in a series of 132 bladder carcinomas: <em>20</em> carcinoma in situ (CIS), 50 pTa, 19 pT1, and 43 pT2-4. All 48 mutations identified were identical to the germinal activating mutations that cause thanatophoric dysplasia, a lethal form of dwarfism. The S249C mutation, found in 33 of the 48 mutated tumors, was the most common. The frequency of mutations was higher in pTa tumors (37 of 50, 74%) than in CIS (0 of <em>20</em>, 0%; P < 0.0001), pT1 (4 of 19, 21%; P < 0.0001) and pT2-4 tumors (7 of 43, 16%; P < 0.0001). FGFR3 mutations were detected in 27 of 32 (84%) G1, 16 of 29 (55%) G2, and 5 of 71 (7%) G3 tumors. This association between FGFR3 mutations and low grade was highly significant (P < 0.0001). FGFR3 is the first gene found to be mutated at a high frequency in pTa tumors. The absence of FGFR3 mutations in CIS and the low frequency of FGFR3 mutations in pT1 and pT2-4 tumors are consistent with the model of bladder tumor progression in which the most common precursor of pT1 and pT2-4 tumors is CIS.

Scatchard analysis of binding of 125I-basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) to baby hamster kidney (BHK) cells revealed the presence of two binding sites: a high affinity site with KD of <em>20</em> pM and 80,000 sites per cell and a low affinity site with KD of about 2 nM and 600,000 sites per cell. The binding to the two sites could be separated by first washing the cells with 2 M NaCl at pH 7.5 which released the low affinity binding and then extracting the cells with 0.5% Triton X-100 to recover the 125I-basic FGF bound to high affinity sites. The binding to the high affinity site was acid sensitive, suggesting that it represented binding to the receptor. Binding to the low affinity site could be competed strongly by heparin and less strongly by heparan sulfate but not by chondroitin sulfate, dermatan sulfate, or keratan sulfate. Treatment of BHK cells with heparinase abolished 62% of the low affinity binding, suggesting that the low affinity binding represented binding to cell-associated, heparin-like molecules. A variety of other cell types, including bovine capillary endothelial (BCE) cells, also demonstrated both low and high affinity binding sites. To test whether the low affinity binding might play a role in the basic FGF stimulation of plasminogen activator (PA) production by BCE cells, heparin was added to BCE cultures at concentrations which totally blocked binding of 125I-basic FGF to the low affinity sites. Addition of the heparin did not diminish the increased PA production induced by basic FGF. This suggests that the low affinity binding has no direct role in the stimulation of PA production in BCE cells.

Platelet-derived <em>growth</em> <em>factor</em> (PDGF) and transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) markedly potentiate tissue repair in vivo. In the present experiments, both in vitro and in vivo responses to PDGF and TGF-beta were tested to identify mechanisms whereby these <em>growth</em> <em>factors</em> might each enhance the wound-healing response. Recombinant human PDGF B-chain homodimers (PDGF-BB) and TGF-beta 1 had identical dose-response curves in chemotactic assays with monocytes and <em>fibroblasts</em> as the natural proteins from platelets. Single applications of PDGF-BB (2 micrograms, 80 pmol) and TGF-beta 1 (<em>20</em> micrograms, 600 pmol) were next applied to linear incisions in rats and each enhanced the strength required to disrupt the wounds at 5 d up to 212% of paired control wounds. Histological analysis of treated wounds demonstrated an in vivo chemotactic response of macrophages and <em>fibroblasts</em> to both PDGF-BB and to TGF-beta 1 but the response to TGF-beta 1 was significantly less than that observed with PDGF-BB. Marked increases of procollagen type I were observed by immunohistochemical staining in <em>fibroblasts</em> in treated wounds during the first week. The augmented breaking strength of TGF-beta 1 was not observed 2 and 3 wk after wounding. However, the positive influence of PDGF-BB on wound breaking strength persisted through the 7 wk of testing. Furthermore, PDGF-BB-treated wounds had persistently increased numbers of <em>fibroblasts</em> and granulation tissue through day 21, whereas the enhanced cellular influx in TGF-beta 1-treated wounds was not detectable beyond day 7. Wound macrophages and <em>fibroblasts</em> from PDGF-BB-treated wounds contained sharply increased levels of immunohistochemically detectable intracellular TGF-beta. Furthermore, PDGF-BB in vitro induced a marked, time-dependent stimulation of TGF-beta mRNA levels in cultured normal rat kidney <em>fibroblasts</em>. The results suggest that TGF-beta transiently attracts <em>fibroblasts</em> into the wound and may stimulate collagen synthesis directly. In contrast, PDGF is a more potent chemoattractant for wound macrophages and <em>fibroblasts</em> and may stimulate these cells to express endogenous <em>growth</em> <em>factors</em>, including TGF-beta, which, in turn, directly stimulate new collagen synthesis and sustained enhancement of wound healing over a more prolonged period of time.

The platelet-derived <em>growth</em> <em>factor</em> (PDGF) is shown to be chemotactic for monocytes and neutrophils. Maximum monocyte chemotaxis to PDGF is fully equal to that achieved with C5a and occurs at congruent to <em>20</em> ng/ml (congruent to 0.7 nM). Maximum neutrophil chemotaxis is congruent to 60% that achieved with C5A but occurs at congruent to 1 ng/ml (congruent to 32 pM). The chemotactic activity of PDGF is blocked by specific antisera to PDGF and by protamine sulfate, a competitive inhibitor of PDGF binding to cell surfaces. In contrast to PDGF, epidermal <em>growth</em> <em>factor</em> shows no chemotactic activity for inflammatory cells at 0.5-100 ng/ml. The high level of chemotactic activity of PDGF suggests that in addition to its role as a mitogen for smooth muscle cells and <em>fibroblasts</em>, PDGF may be involved in attracting inflammatory cells to sites of platelet release.

To assess whether the progression of plasma cell tumors is accompanied by angiogenesis and secretion of matrix-degrading enzymes, bone marrow biopsy specimens from <em>20</em> patients with monoclonal gammopathy of undetermined significance (MGUS), 18 patients with nonactive multiple myeloma (MM), and 26 patients with active MM were evaluated for their angiogenic potential and matrix-metalloproteinase (MMP) production. A fivefold increase of the <em>factor</em> VIII+ microvessel area was measured by a planimetric method of point counting in the bone marrow of patients with active MM as compared with nonactive MM and MGUS patients (P <.01). When serum-free conditioned media (CM) of plasma cells isolated from the bone marrow of each patient were tested in vivo for their angiogenic activity in the chick embryo chorioallantoic membrane (CAM) assay, the incidence of angiogenic samples was significantly higher (P <. 01) in the active MM group (76%) compared with nonactive MM (33%) and MGUS (<em>20</em>%) groups. Moreover, a linear correlation (P <.01) was found between the extent of vascularization of the bone marrow of a given patient and the angiogenic activity exerted in the CAM assay by the plasma cells isolated from the same bone marrow. In vitro, a significantly higher fraction of the plasma cell CM samples from the active MM group stimulated human umbilical vein endothelial cell (HUVEC) proliferation (53%, P <.01), migration (42%, P <.05), and/or monocyte chemotaxis (38%, P <.05) when compared with nonactive MM and MGUS groups (ranging between 5% and 15% of the samples). Also, immunoassay of plasma cell extracts showed significantly higher (P <. 01) levels of the angiogenic basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2 in the active MM patients than in nonactive MM and MGUS patients (153 +/- 59, 23 +/- 17, and 31 +/- 18 pg FGF-2/100 micrograms of protein, respectively). Accordingly, neutralizing anti-FGF-2 antibody caused a significant inhibition (ranging from 54% to 68%) of the biological activity exerted on cultured endothelial cells and in the CAM assay by plasma cell CM samples from active MM patients. Finally, in situ hybridization of bone marrow plasma cells and gelatin-zymography of their CM showed that active MM patients express significantly higher (P <.01) levels of MMP-2 mRNA and protein when compared with nonactive MM and MGUS patients, whereas MMP-9 expression was similar in all groups. Taken together, these findings indicate that the progression of plasma cell tumors is accompanied by an increase of bone marrow neovascularization. This is paralleled by an increased angiogenic and invasive potential of bone marrow plasma cells, which is dependent, at least in part, by FGF-2 and MMP-2 production. Induction of angiogenesis and secretion of MMPs by plasma cells in active disease may play a role in their medullary and extramedullary dissemination, raising the hypothesis that angiostatic/anti-MMP agents may be used for therapy of MM.

Wound repair is initiated with the aggregation of platelets, formation of a fibrin clot, and release of <em>growth</em> <em>factors</em> from the activated coagulation pathways, injured cells, platelets, and extracellular matrix (ECM), followed by migration of inflammatory cells to the wound site. Thereafter, keratinocytes migrate over the wound, angiogenesis is initiated, and <em>fibroblasts</em> deposit and remodel the granulation tissue. Cell migration, angiogenesis, degradation of provisional matrix, and remodeling of newly formed granulation tissue, all require controlled degradation of the ECM. Disturbance in the balance between ECM production and degradation leads to formation of chronic ulcers with excessive ECM degradation, or to fibrosis, for example hypertrophic scars or keloids characterized by excessive accumulation of ECM components. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which as a group can degrade essentially all ECM components. So far, <em>20</em> members of the human MMP family have been identified. Based on their structure and substrate specificity, they can be divided into subgroups of collagenases, stromelysins, stromelysin-like MMPs, gelatinases, membrane-type MMPs (MT-MMPs), and other MMPs. In this review, the role of MMPs in normal wound repair as well as in chronic ulcers is discussed. In addition, the role of signaling pathways, in particular, mitogen-activated protein kinases (MAPKs) in regulating MMP expression is discussed as possible therapeutical targets for wound healing disorders.

Angiogenesis depends on <em>growth</em> <em>factors</em> and vascular cell adhesion events. Integrins and <em>growth</em> <em>factors</em> are capable of activating the ras/MAP kinase pathway in vitro, yet how these signals influence endothelial cells during angiogenesis is unknown. Upon initiation of angiogenesis with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) on the chick chorioallantoic membrane (CAM), endothelial cell mitogen-activated protein (MAP) kinase (ERK) activity was detected as early as 5 min yet was sustained for at least <em>20</em> h. The initial wave of ERK activity (5-1<em>20</em> min) was refractory to integrin antagonists, whereas the sustained activity (4-<em>20</em> h) depended on integrin alphavbeta3, but not beta1 integrins. Inhibition of MAP kinase kinase (MEK) during this sustained alphavbeta3-dependent ERK signal blocked the formation of new blood vessels while not influencing preexisting blood vessels on the CAM. Inhibition of MEK also blocked <em>growth</em> <em>factor</em> induced migration but not adhesion of endothelial cells in vitro. Therefore, angiogenesis depends on sustained ERK activity regulated by the ligation state of both a <em>growth</em> <em>factor</em> receptor and integrin alphavbeta3.

The human <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (hFGF23) and its autosomal dominant hypophosphatemic rickets (ADHR) mutant genes were incorporated into animals by naked DNA injection to investigate the action on phosphate homeostasis in vivo. The hFGF23 mutants (R176Q, R179Q, and R179W) markedly reduced serum phosphorus (6.2-6.9 mg/dl) compared with the plasmid MOCK (8.5 mg/dl). However, native hFGF23 did not affect serum phosphorus (8.6 mg/dl). Both hFGF23 and hFGF23R179Q mRNAs were expressed more than 100-fold in the liver 4 days after injection, however, the C-terminal portion of hFGF23 was detected only in the serum from hFGF23R179Q-injected animals (1109 pg/ml). hFGF23R179Q mutant was secreted as a 32-kDa protein, whereas, native hFGF23 was detected as a <em>20</em>-kDa protein in the cell-conditioned media. These results suggest the hFGF23R179Q protein is resistant to intracellular proteolytic processing. The hFGF23R179Q suppressed Na/P(i) co-transport activities both in kidney and in small intestine by 45 and 30%, respectively, as well as serum 1alpha,25-dihydroxyvitamin D(3) to less than 15 pg/ml. However, it had little effect on serum parathyroid hormone (PTH). Infusion of hFGF23R179Q protein normalized serum phosphorus in thyroparathyroidectomized rats without affecting serum calcium. Taken together, the FGF23 mutants reduce both phosphate uptake in intestine and phosphate reabsorption in kidney, independent of PTH action.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF-23) is critical to the pathogenesis of a distinct group of renal phosphate wasting disorders: tumor-induced osteomalacia, X-linked hypophosphatemia, and autosomal dominant and autosomal recessive hypophosphatemic rickets. Excess circulating FGF-23 is responsible for their major phenotypic features which include hypophosphatemia due to renal phosphate wasting and inappropriately low serum 1,25(OH)2D concentrations. To characterize the effects of FGF-23 on renal sodium-phosphate (Na/P(i)) cotransport and vitamin D metabolism, we administered FGF-23(R176Q) to normal mice. A single injection (0.33 microg/g body wt) induced significant hypophosphatemia, <em>20</em> and 29% decreases (P < 0.001) in brush-border membrane (BBM) Na/Pi cotransport at 5 and 17 h after injection, respectively, and comparable decreases in the abundance of type IIa Na/P(i) cotransporter protein in BBM. Multiple injections (6, 12, and 24 mug/day for 4 days) induced dose-dependent decreases (38, 63, and 75%, respectively) in renal abundance of 1alpha-hydroxylase mRNA (P < 0.05). To determine whether FGF-23(R176Q) exerts a direct action on 1alpha-hydroxylase gene expression, we examined its effects in cultured human (HKC-8) and mouse (MCT) renal proximal tubule cells. FGF-23(R176Q) (1 to 10 ng/ml) induced a dose-dependent decrease in 1alpha-hydroxylase mRNA with a maximum suppression of 37% (P < 0.05). Suppression was detectable after 6 h of exposure and maximal after 21 h. In MCT cells, FGF-23(R176Q) suppressed 1alpha-hydroxylase mRNA and activated the ERK1/2 signaling pathway. The MAPK inhibitor PD98059 effectively abolished FGF-23-induced suppression of 1alpha-hydroxylase mRNA by blocking signal transduction via ERK1/2. These novel findings provide evidence that FGF-23 directly regulates renal 1alpha-hydroxylase gene expression via activation of the ERK1/2 signaling pathway.

The effects of serum, transforming <em>growth</em> <em>factor</em> (TGF) beta 1, bFGF, and heparin on in vitro myo<em>fibroblast</em> transformation was studied. Primary rabbit corneal keratocytes were grown under serum-free conditions or in media supplemented with serum (10% fetal calf serum), TGF beta 1 (0.1-10 ng/ml), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) (0.1-10 ng/ml), or heparin (10 U/ml). Cells were analyzed for expression of alpha-smooth muscle actin (alpha-SM actin), alpha 5 beta 1 integrin (the high-affinity fibronectin receptor) and fibronectin by immunoprecipitation, Western blotting, and immunofluorescence. Corneal keratocytes grown in the presence of serum showed a typical <em>fibroblast</em> morphology with induction of alpha-SM actin expression in 1 to 10% of cells. Addition of bFGF blocked serum-induced alpha-SM actin expression, whereas addition of TGF beta 1 enhanced alpha-SM actin expression (100%), which in combination with heparin (10 U/ml), led to a pulling apart of the <em>fibroblast</em>ic sheet, simulating contraction. Under serum-free conditions, with or without bFGF and heparin, primary corneal <em>fibroblasts</em> appeared morphologically similar to in situ corneal keratocytes, demonstrating a broad, stellate morphology with interconnected processes and no alpha-SM actin expression. Addition of TGF beta 1 to serum-free cultures resulted in a dramatic transformation of corneal keratocytes to spindle-shaped, <em>fibroblast</em>-like cells that expressed alpha-SM actin in 100% of cells and exhibited a <em>20</em>-fold increase in fibronectin synthesis and a 13-fold increase in alpha 5 beta 1-integrin synthesis. These effects were blocked by the addition of neutralizing antibodies (16 micrograms/ml). Overall these data suggest that TGF beta 1 is a potent modulator of myo<em>fibroblast</em> transformation under serum-free conditions. In addition, the <em>growth</em> of keratocytes in serum appears to mimic, in part, in vivo activation and myo<em>fibroblast</em> transformation. We conclude that detailed study of TGF beta 1-induced myo<em>fibroblast</em> transformation under defined serum-free conditions will provide important insights into the myo<em>fibroblast</em> transformation process.

BACKGROUND

The antineoplastic compound paclitaxel (Taxol) causes an increased assembly of extraordinarily stable microtubules. The present study was designed to characterize the effects of paclitaxel on proliferation and migration of human arterial smooth muscle cells (haSMCs) in vitro and on neointima formation in an in vivo experimental rabbit model.

RESULTS

Both monocultures of haSMCs and cocultures with human arterial endothelial cells (haECs) were used. Cell <em>growth</em> after 4, 8, and 14 days was determined in the absence or presence of platelet-derived <em>growth</em> <em>factor</em>-AB (PDGF-AB), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), or thrombin. Nonstop paclitaxel exposure, as well as single-dose applications of paclitaxel for 24 hours or even <em>20</em> minutes (0.1 to 10.0 micromol/L), caused a complete and prolonged inhibition of haSMC <em>growth</em> up to day 14, with an IC50 of 2.0 nmol/L. Mitogens or cocultures with stimulating haECs did not significantly attenuate paclitaxel-induced effects. Immunohistochemistry showed characteristic cytoskeletal changes predominantly in the microtubule network. Additionally, in <em>20</em> male New Zealand White rabbits, intimal plaques were produced by electrical stimulation. In 10 animals, paclitaxel was locally applied by use of microporous balloons. Histologically, the intima wall area, wall thickness, and degree of stenosis were reduced significantly in paclitaxel-treated animals compared with controls.

CONCLUSIONS

Our data show that paclitaxel inhibits haSMC proliferation and migration in a dose-dependent manner in monocultures and cocultures even in the presence of mitogens. Furthermore, paclitaxel prevents neointima formation in rabbits after balloon angioplasty. The long-lasting effect after just several minutes' exposure time makes this lipophilic substance a promising candidate for local antiproliferative therapy of restenosis.

Midkine (MK) is a developmentally regulated, secreted <em>growth</em> <em>factor</em> homologous to pleiotrophin (PTN). To investigate the potential role of MK in tumor <em>growth</em>, we expressed MK in human SW-13 cells and studied receptor binding, signal transduction, and activity of MK. The MK protein stimulates soft agar colony formation in vitro and tumor <em>growth</em> of SW-13 cells in athymic nude mice, as well as proliferation of human endothelial cells from brain microvasculature and umbilical vein (HUVEC) in the low ng/ml range. MK binds to anaplastic lymphoma kinase (ALK), the receptor for PTN, with an apparent K(d) of 170 pm in intact cells, and this receptor binding of MK is competed by PTN with an apparent K(d) of approximately <em>20</em> pm. Monoclonal antibodies raised against the extracellular ligand-binding domain of ALK inhibit ALK receptor binding of MK as well as MK-stimulated colony formation of SW-13 cells. Furthermore, MK stimulates ALK phosphorylation in WI-38 human <em>fibroblasts</em> and activates PI3-kinase and MAP kinase signal transduction in WI-38, HUVEC, neuroblastoma (SH SY-5Y) and glioblastoma (U87MG) cells that express the ALK protein. We conclude that MK can act as a <em>growth</em>, survival, and angiogenic <em>factor</em> during tumorigenesis and signals through the ALK receptor.

We have investigated the effects of different <em>growth</em> <em>factors</em> on the proliferation and osteogenic potential of primary cultures of human bone marrow stromal cells (BMSC). <em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2 was the most effective in promoting <em>growth</em> of these cells in vitro. The size of colonies formed in clonal conditions was approximately 2.5 times larger in presence of FGF-2. Also the morphology of BMSC was affected: cells cultured in 10% FCS alone became flattened, whereas FGF-2 expanded cells maintained a <em>fibroblast</em>-like elongated phenotype. Levels of alkaline phosphatase activity in BMSC expanded with FGF-2 were significantly lower (56%) than control and, after stimulation with ascorbic acid, betaGlycerophosphate and dexamethasone, FGF-2 expanded BMSC deposited approximately 3-fold more mineralized matrix than control cells. We have assessed osteogenicity of BMSC on hydroxyapatite porous scaffolds (bioceramics) by an ectopic bone formation assay. FGF-2 expanded BMSC yielded a higher bone formation >><em>20</em>-fold) than control cells. We conclude that FGF-2, promoting BMSC proliferation, maintains cells in a more immature state allowing in vitro expansion of human osteo-progenitors which, associated with bioceramics, can differentiate in vivo and form bone tissue.