Lesioned axons within the dorsal roots fail to regenerate through the peripheral nerve transition zone and into the spinal cord. This regenerative failure leads to a persistent loss of sensory function. To induce axonal <em>growth</em> across this barrier, we used recombinant adenovirus to express <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF2), nerve <em>growth</em> <em>factor</em> (NGF), L1 cell adhesion molecule (L1), or beta-galactosidase (LacZ) within the endogenous glia of the dorsal spinal cord <em>16</em> d after injury. Expression of either FGF2 or NGF, but not L1 or LacZ, induced robust axonal regeneration into normal as well as ectopic locations within the dorsal spinal cord. This regeneration led to near-normal recovery of thermal sensory function. Functional recovery and the majority of regenerating axons within the dorsal horn disappeared with recutting of the sensory roots. Injections of adenovirus encoding NGF, but not FGF2, also resulted in extensive sprouting of noninjured sensory axons, which we previously demonstrated could cause hyperalgesia and chronic pain. Thus, neurotrophic <em>factor</em> gene therapy administered as late as <em>16</em> d after injury may serve as a useful treatment to elicit recovery after dorsal root avulsion; however, the choice of neurotrophin is important to induce selective regeneration of damaged axons.

Epidermal <em>growth</em> <em>factor</em> (EGF) receptors extracted with Triton X-100 from human skin <em>fibroblasts</em> and A431 epidermoid carcinoma cells rapidly lose EGF-binding activity precipitable with polyethylene glycol. The presence of concanavalin A which can cross-link and, thereby, aggregate the receptors, allowed quantitative recovery of the lost EGF-binding activity. Scatchard analysis of EGF binding of Triton X-100-solubilized receptors showed that A431 cells and skin <em>fibroblasts</em> possess approximately 1.5 X 10(6) and 7 X 10(4) EGF-binding sites/cell, respectively, which exhibit similar affinities for the ligand. The heavy isotope density-shift method was employed to determine whether differences in rates of receptor synthesis or decay account for the large difference in number of receptors/cell between the two cell types. After shifting cells to medium containing heavy (15N, 13C, and 2H) amino acids, light and heavy receptors, solubilized from total cellular membranes, were resolved by isopycnic banding on density gradients and then quantitated. It was demonstrated that A431 cells synthesize EGF receptors at a rate 12 times faster than skin <em>fibroblasts</em> and that the half-life for receptor decay of A431 cells is somewhat longer (t1/2 = <em>16</em> h) than that (t1/2 = 9 h) of <em>fibroblasts</em>. Down-regulation of cell surface and total cellular EGF-binding capacity in A431 cells occurs with a t1/2 of 2-3 h and results in a 70-83% decrease in receptor level in 12 h. Scatchard analysis revealed that these changes in EGF binding were due to an alteration of receptor number and not EGF-binding affinity. Rates of EGF receptor synthesis and inactivation/decay were determined by the heavy isotope density-shift method. No change in the rate of receptor synthesis occurred as a consequence of EGF receptor down-regulation. Down-regulation, however, caused a decrease in receptor half-life from <em>16</em> to 4.5 h. These results indicate that EGF-dependent regulation of EGF receptor level in A431 cells involves an alteration of the rate of receptor inactivation.

METHODS

This study analyzed immunohistological features of the extruded or sequestrated intervertebral disc of the lumbar spine. To clarify the pathogenesis of neovascularization, cells isolated from herniated disc were cultured and examined biologically.

OBJECTIVE

The objectives of this study were to characterize the histologic features of extruded or sequestrated discs and inflammatory cells that infiltrate along the margins of the disc tissue and to clarify the pathogenesis of neovascularization observed at the edge of the disc tissue.

BACKGROUND

When some of the contents of the disc extrudes into the epidural space and is considered "foreign," an autoimmune response develops, which can lead to a chronic inflammatory response. However, the pathogenesis of inflammatory cell infiltrations and neovascularization are not clearly defined.

METHODS

The herniated discs were obtained during surgery and were stained with anti-interleukin-1, intercellular adhesion molecule-1, lymphocyte function-associated antigen, and basic fibroblast growth factor antibodies by using an indirect immunoperoxidase method. Cells isolated from herniated disc were cocultured with human endothelial cells and basic fibroblast growth factor contained by cultured disc cells were measured by radioimmunoassay.

RESULTS

The ingrowth of granulation tissue with vascularization, occurring at the edge of fibrocartilage fragment, was present at 11 of 16 of extruded and 3 of 5 of sequestrated discs. Anti-interleukin-1, intracellular adhesion molecule-1, lymphocyte function-associated antigen, and basic fibroblast growth factor were expressed on most of mononuclear cells infiltrating into the extruded or sequestrated disc. Cells from the extruded or sequestrated disc demonstrated significantly greater levels of basic fibroblast growth factor than those from the protruded disc, and they enhanced the proliferation of endothelial cells.

CONCLUSIONS

This study showed that mononuclear cells infiltrating along the margins of extruded discs expressed inflammatory mediators and might induce neovascularization and persistence of inflammation.

Two <em>growth</em> <em>factors</em> have been purified to homogeneity from either bovine hypothalamus or brain by heparin affinity chromatography. Both stimulate the <em>growth</em> of murine 3T3 <em>fibroblasts</em> and bovine capillary endothelial cells. One heparin-binding <em>growth</em> <em>factor</em> (HGF alpha), purified from either tissue by elution from heparin with 1.0 M sodium chloride, is obtained in a yield of 0.4 mg/kg of tissue. Its apparent molecular weight is <em>16</em> 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and its amino acid composition is identical with that of the acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> recently isolated from bovine brain by a multistep chromatographic procedure [Thomas, K. A., Rios-Candelore, M., & Fitzpatrick M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 357-361]. A second <em>growth</em> <em>factor</em> (HGF beta), isolated from either tissue by elution from heparin with 2.0 M sodium chloride, is obtained in a yield of 0.02 mg/kg of tissue. Its apparent molecular weight is 18 000 by SDS-PAGE, and its amino acid composition differs from that of HGF alpha. These results confirm the existence of two distinct <em>growth</em> <em>factors</em> in bovine neural tissue and establish that the acidic endothelial cell <em>growth</em> <em>factor</em> from hypothalamus and the acidic brain <em>fibroblast</em> <em>growth</em> <em>factor</em> are identical.

Graves' disease (GD), an autoimmune process involving thyroid and orbital tissue, is associated with lymphocyte abnormalities including expansion of memory T cells. Insulin-like <em>growth</em> <em>factor</em> receptor-1 (IGF-1R)-bearing <em>fibroblasts</em> overpopulate connective tissues in GD. IGF-1R on <em>fibroblasts</em>, when ligated with IgGs from these patients, results in the expression of the T cell chemoattractants, IL-<em>16</em> and RANTES. We now report that a disproportionately large fraction of peripheral blood T cells express IGF-1R (CD3+IGF-R+). CD3+IGF-1R+ T cells comprise 48 +/- 4% (mean +/- SE; n = 33) in patients with GD compared with 15 +/- 3% (n = 21; p < 10(-8)) in controls. This increased population of IGF-1R+ T cells results, at least in part, from an expansion of CD45RO+ T cells expressing the receptor. In contrast, the fraction of CD45RA+IGF-1R+ T cells is similar in GD and controls. T cells harvested from affected orbital tissues in GD reflect similar differences in the proportion of IGF-1R+CD3+ and IGF-1R+CD4+CD3+ cells as those found in the peripheral circulation. GD-derived peripheral T cells express durable, constitutive IGF-1R expression in culture and receptor levels are further up-regulated following CD3 complex activation. IGF-1 enhanced GD-derived T cell incorporation of BrdU (p < 0.02) and inhibited Fas-mediated apoptosis (p < 0.02). These findings suggest a potential role for IGF-1R displayed by lymphocytes in supporting the expansion of memory T cells in GD.

<em>Fibroblast</em>-3D collagen matrix culture provides a model system to analyze cell physiology under conditions that more closely resemble tissue than conventional 2D cell culture. Previous work has focused primarily on remodeling and contraction of collagen matrices by <em>fibroblasts</em>, and there has been little research on migration of cell populations within the matrix. Here, we introduce a nested collagen matrix model to analyze migration of <em>fibroblasts</em> in 3D collagen matrices. Nested collagen matrices were prepared by embedding contracted cell-containing matrices (also called dermal equivalents) inside cell-free matrices; migration occurred from the former to the latter. Control experiments with human dermal fragments in place of dermal equivalents confirmed the reliability of the model. Human <em>fibroblast</em> migration in nested collagen matrices occurred after a lag phase of 8-<em>16</em> h, and cells migrating out of the inner matrices were bipolar with leading dendritic extensions. Migration was myosin II, Rho kinase and metalloproteinase-dependent but did not require plasma fibronectin. Platelet-derived <em>growth</em> <em>factor</em> but not lysophosphatidic acid or serum stimulated cell migration, although all three of these physiological agonists promote matrix remodeling and contraction. The nested collagen matrix model is a relatively easy, rapid and quantitative method to measure migration of cell populations. Our studies using this model demonstrate important differences between regulation of <em>fibroblast</em> migration and remodeling in collagen matrices.

Addition of cholera toxin (100 ng/ml) to quiescent cultures of Swiss 3T3 cells acts synergistically with serum (2-4%), insulin, phorbol esters, epidermal <em>growth</em> <em>factor</em>, and <em>fibroblast</em>-derived <em>growth</em> <em>factor</em> to stimulate DNA synthesis. In the presence of insulin, cholera toxin caused a dose-dependent increase in cumulative [3H]thymidine incorporation into acid-insoluble material and in the intracellular cyclic AMP (cAMP) level. The dose--response curves for the two processes were similar. Furthermore, addition of 1-methyl-3-isobutylxanthine (15--500 microM) or of 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (5--100 microM), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase which are potent inhibitors of cyclic nucleotide phosphodiesterase activity, stimulated DNA synthesis and increased cAMP levels in Swiss 3T3 cells. These compounds strikingly potentiated the effect of cholera toxin on DNA synthesis and on cAMP levels. When quiescent Swiss 3T3 cells were exposed to cholera toxin (100 ng/ml) and insulin at 10 micrograms/ml (4- to 7-fold increase in cAMP level) or to these agents and 1-methyl-3-isobutyl xanthine at 50 microM (35-fold increase in cAMP level), DNA synthesis began after a lag of <em>16</em> hr. These results indicate that cAMP acts as a mitogenic signal for Swiss 3T3 cells and differ from the widely held view that cyclic AMP inhibits the proliferation of <em>fibroblast</em> cells.

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)(1) can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial. The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium(2). Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs(3). Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)(4). Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions(5-7). Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established(8). To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells(9), adult skin cells(10), foreskin <em>fibroblasts</em>(11-12), amniotic mesenchymal cells(13)). However, the efficiency of maintaining undifferentiated hESCs using human foreskin <em>fibroblast</em>-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A(14). Obviously, there is an evident difference in <em>growth</em> <em>factor</em> production by mouse and human feeder cells. Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key <em>factor</em> regulating the expression of Tgfβ1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs(15-<em>16</em>). For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the <em>growth</em> of hESCs. Here, we present an optimized method for the isolation and culture of mouse embryonic <em>fibroblasts</em> (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

In a previous study, we showed that heparitinase releases a 14-saccharide sequence (Oligo-H) from heparan sulfate (HS) with the structure delta GlcUA beta 1,4GlcNSO3-alpha 1,4[IdceA(2S)alpha 1,4GlcNSO3]5 alpha 1,4IdceA alpha 1,4GlcNAc (where IdceA(2S) represents iduronic acid 2-sulfate), which binds to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) with high affinity (Turnbull, J. E., Fernig, D., Ke, Y., Wilkinson, M. C. & Gallagher, J. T. (1992) J. Biol. Chem. 267, 10337-10341). This paper describes further work on the binding properties of HS saccharides and their capacity to mediate bFGF activity in a mitogenesis assay in which responsiveness is dependent on the addition of HS or heparin. Saccharides prepared by heparinase or nitrous acid digestion and heparitinase-resistant fragments five disaccharide units (degree of polymerization (dp) = 10) or less in size were unable to activate bFGF. However, heparitinase-resistant saccharides of dp12-<em>16</em> were active in the assay; the dp14 and dp<em>16</em> fractions were equivalent in activity to heparin and more active than the parent HS. Saccharides of the same size and basic structure as the active fractions >> or = dp12) bound to bFGF with high relative affinity. Active saccharides were composed mainly of N-sulfated disaccharides, the predominant unit being IdceA(2S)-GlcNSO3. This was enriched at least 5-fold in the active saccharides by comparison with the original HS. In addition, the dp12 and dp14 active fractions had a notably low content of trisulfated disaccharides (IdceA(2S)-GlcNSO3(6S)) (where GlcNSO3(6S) represents N-sulfated glucosamine 6-sulfate), which are the major repeat units of heparin. The data show that sequences similar in size and basic structure to Oligo-H can mediate the mitogenic activity of bFGF. Overall, the results provide further evidence that specific HS sequences are generated biosynthetically in order to fulfill particular biological functions such as activation of bFGF.

Circulating levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21), a metabolic regulator of glucose, lipid, and energy homeostasis, are elevated in obese diabetic subjects, raising questions about potential FGF21 resistance. Here we report tissue expression changes in FGF21 and its receptor components, and we describe the target-organ and whole-body responses to FGF21 in ob/ob and diet-induced obese (DIO) mice. Plasma FGF21 concentrations were elevated 8- and <em>16</em>-fold in DIO and ob/ob mice, respectively, paralleling a dramatic increase in hepatic FGF21 mRNA expression. Concurrently, expression levels of βKlotho, FGF receptor (FGFR)-1c, and FGFR2c were markedly down-regulated in the white adipose tissues (WAT) of ob/ob and DIO mice. However, dose-response curves of recombinant human FGF21 (rhFGF21) stimulation of ERK phosphorylation in the liver and WAT were not right shifted in disease models, although the magnitude of induction in ERK phosphorylation was partially attenuated in DIO mice. Whole-body metabolic responses were preserved in ob/ob and DIO mice, with disease models being more sensitive and responsive than lean mice to the glucose-lowering and weight-loss effects of rhFGF21. Endogenous FGF21 levels, although elevated in diseased mice, were below the half-maximal effective concentrations of rhFGF21, suggesting a state of relative deficiency. Hepatic and WAT FGF21 mRNA expression levels declined after rhFGF21 treatment in the absence of the increased expression levels of βKlotho and FGFR. We conclude that overt FGF21 resistance was not evident in the disease models, and increased hepatic FGF21 expression as a result of local metabolic changes is likely a major cause of elevated circulating FGF21 levels.

The contribution of specific type I collagen remodeling in angiogenesis was studied in vivo using a quantitative chick embryo assay that measures new blood vessel <em>growth</em> into well-defined fibrillar collagen implants. In response to a combination of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF), a strong angiogenic response was observed, coincident with invasion into the collagen implants of activated <em>fibroblasts</em>, monocytes, heterophils, and endothelial cells. The angiogenic effect was highly dependent on matrix metalloproteinase (MMP) activity, because new vessel <em>growth</em> was inhibited by both a synthetic MMP inhibitor, BB3103, and a natural MMP inhibitor, TIMP-1. Multiple MMPs were detected in the angiogenic tissue including MMP-2, MMP-13, MMP-<em>16</em>, and a recently cloned MMP-9-like gelatinase. Using this assay system, wild-type collagen was compared to a unique collagenase-resistant collagen (r/r), with regard to the ability of the respective collagen implants to support cell invasion and angiogenesis. It was found that collagenase-resistant collagen constitutes a defective substratum for angiogenesis. In implants made with r/r collagen there was a substantial reduction in the number of endothelial cells and newly formed vessels. The presence of the r/r collagen, however, did not reduce the entry into the implants of other cell types, that is, activated <em>fibroblasts</em> and leukocytes. These results indicate that fibrillar collagen cleavage at collagenase-specific sites is a rate-limiting event in <em>growth</em> <em>factor</em>-stimulated angiogenesis in vivo.

<em>Growth</em> <em>factors</em> play an important role in supraspinatus tendon-to-bone healing. The objective of this study was to evaluate the temporal expression of 8 different <em>growth</em> <em>factors</em> in tendon-to-bone healing in an animal model. We hypothesize that <em>growth</em> <em>factors</em> exhibit unique temporal profiles that correlate to specific stages in the acute process of the supraspinatus tendon. To test this hypothesis, rats underwent bilateral supraspinatus tendon detachment and repair. Animals were euthanized at 1, 2, 4, 8, and <em>16</em> weeks. Immunohistochemical staining was done using antibodies for basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), bone morphogenetic protein 12 (BMP-12), BMP-13, BMP-14, cartilage oligomeric matrix protein (COMP), connective tissue <em>growth</em> <em>factor</em> (CTGF), platelet-derived <em>growth</em> <em>factor</em>-B (PDGF-B), and transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1). Immunoassays showed an increase in the expression of all <em>growth</em> <em>factors</em> at 1 week, followed by a return to control or undetectable levels by <em>16</em> weeks in both the insertion and midsubstance. Future studies will investigate the different impacts of <em>growth</em> <em>factor</em> expression in tendon to bone healing.

The P97 promoter upstream of the oncogenic early genes of human papillomavirus (HPV)-<em>16</em> is active in keratinocytes and in cervical carcinoma cells due to a 5' keratinocyte-dependent cis enhancer. In this study, we have mapped the main enhancer activity to an 88-nucleotide (nt) fragment composed of multiple cis elements. A 63-nt promoter-proximal enhancer core was sufficient for P97 activation in a human keratinocytic cell line, HaCaT, and in cervical carcinoma cells. Although the enhancer functioned poorly in hepatoma cells or in <em>fibroblasts</em>, nuclear extracts from different cells protected similar cis elements from DNase I digestion. Two protected half-palindromic NF-I/CTF sites within the 63-nt core were necessary for its function; one represents a "cytokeratin element" (CK), a previously described 8-nt sequence shared with cytokeratin gene promoters. Both sites formed complexes of the same apparent size and relative binding affinity with NF-I/CTF-like <em>factor</em>(s) present in all cells tested. Although cell-dependent P97 activation could be determined by similar, yet distinct NF-I/CTF-like proteins, adjacent cis elements in the enhancer core were also required for function, and may thus interact with additional transcription <em>factors</em>. A 25-nt distal module with two AP-1 sites increased enhancer activity and cooperated with cis elements of the proximal core. Each AP-1 site as well as a third AP-1 site near the promoter bound c-Jun and Jun/Fos in vitro, and was activated by c-Jun and c-Fos in transfections. In addition to cell type-dependent activation, HPV-<em>16</em> P97 transcription may therefore respond to <em>growth</em> <em>factors</em> and oncogene products via the AP-1 pathway.

During angiogenesis, endothelial cells react to stimulation with finely tuned signaling responses. The role of calcium-regulated signaling in angiogenesis has not been defined. This study investigated the calcium dependency of endothelial cell proliferation and invasion by using an inhibitor of ligand-stimulated calcium influx, CAI (carboxy-amidotriazole). Incubation with CAI significantly inhibited proliferation of human umbilical vein endothelial cells (HUVECs) in response to serum (IC50 = 1 microM) or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF2; P2 < 0.005 at 10 microM). Statistically significant inhibition of HUVEC adhesion and motility to basement membrane proteins laminin, fibronectin, and type IV collagen was demonstrated (adhesion, P2 < 0.004-0.01; motility, P2 < 0.009-0.018). Marked inhibition of native and FGF2-induced gelatinase activity was shown by zymogram analysis and was confirmed by Northern blot analysis. CAI inhibited HUVEC tube formation on Matrigel and inhibited in vivo angiogenesis in the chicken chorioallantoic membrane assay, 67% at 20 microM and 56% at 10 microM compared with <em>16</em>% for an inactive CAI analog or 9% for 0.1% dimethyl sulfoxide control. Incubation of HUVECs with CAI and/or FGF2 followed by immunoprecipitation with anti-phosphotyrosine antibody showed inhibition of FGF2-induced tyrosine phosphorylation of proteins in the range 110-150 kDa. These results suggest that calcium-regulated events are important in native and FGF2-stimulated HUVEC proliferation and invasion, perhaps through regulation of FGF2-induced phosphorylation events, and indicate a role for calcium in the regulation of angiogenesis in vivo.

The present study was performed to compare serum concentrations of maternal and fetal angiogenic <em>growth</em> <em>factors</em> in IUGR (intrauterine <em>growth</em> restriction) and normal pregnancy at the time of delivery. VEGF (vascular endothelial <em>growth</em> <em>factor</em>), PlGF (placental <em>growth</em> <em>factor</em>), sFlt-1 (soluble fms-like tyrosine kinase 1), sKDR (soluble kinase domain receptor) and bFGF (basic <em>fibroblast</em> <em>growth</em> <em>factor</em>) were measured by ELISA in serum from a maternal peripheral vein, the umbilical vein and the umbilical arteries in 15 women with pregnancies complicated by IUGR and <em>16</em> controls (women with normal pregnancies). In IUGR, sFlt-1 was increased, and PlGF and sKDR were decreased, in both maternal serum and serum from the umbilical vein. Additionally, bFGF was increased in serum from the umbilical vein of women with pregnancies complicated by IUGR. No significant differences in <em>growth</em> <em>factor</em> concentrations between the groups were found in serum from the umbilical artery. In both groups, levels of VEGF were higher and levels of sFlt-1 were lower in serum from the umbilical vein and umbilical artery compared with maternal serum. PlGF levels were found to be lower in serum from the umbilical vein compared with maternal serum in both groups, whereas PlGF levels in serum from the umbilical artery were significantly lower only in the control group. These findings suggest an imbalance of angiogenic and anti-angiogenic <em>factors</em> in IUGR, with formation of an anti-angiogenic state in maternal and, to a lesser extent, umbilical vein blood. The placenta appears to play a central role in the release of sFlt-1 into maternal and umbilical blood. Umbilical artery blood was unaffected in IUGR, indicating that the fetus does not contribute to changes in angiogenic <em>growth</em> <em>factor</em> concentrations.

OBJECTIVE

The overall goal was to determine whether chronic ischemia and hypercholesterolemia interfere with bladder function and structure. The roles of atherosclerosis-induced chronic ischemia and hypercholesterolemia in bladder fibrosis and non-compliance were studied in the rabbit. The relationship between ischemia-induced changes in the expression of transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) and the severity of bladder fibrosis was also investigated.

METHODS

Male New Zealand White rabbits were divided into chronic bladder ischemia (CBI, n = 11), hypercholesterolemia (Hch, n = 8) and control (n = 8) groups. The CBI group underwent balloon endothelial injury of the iliac arteries and received a 0.5% cholesterol diet. The Hch group received a 0.5% cholesterol diet alone. The control group was placed on a regular diet. After 16 weeks, iliac artery and bladder wall blood flow measurements, cystometrograms (CMG) and aorto-iliac arteriograms were obtained in all animals. Iliac arteries and bladder tissues were processed for histological staining and computer-assisted histomorphometric image analysis. The expressions of TGF-beta1 and bFGF in bladder tissue were determined by immunohistochemical staining utilizing monoclonal antibodies.

RESULTS

At 16 weeks, arteriography and histology showed significant diffuse atherosclerotic occlusive disease of the aorto-iliac arteries in the CBI group. Iliac artery and bladder wall blood flows were significantly decreased in the CBI group compared with the Hch and control groups. Atherosclerosis-induced CBI shifted the volume-pressure curve to the left and caused severe bladder fibrosis. Hypercholesterolemia also caused fibrosis and non-compliance but to a much lesser extent compared with those caused by CBI. In histomorphometry, the percentage of detrusor smooth muscle was moderately decreased in the Hch group and severely decreased in the CBI group compared with the control group. In immunohistochemical stains of bladder tissues, bFGF expression was similar in the three groups of animals. TGF-beta1 expression was significantly greater in bladder tissues from the CBI group compared with the Hch and control groups.

CONCLUSIONS

Our studies show that atherosclerosis-induced chronic ischemia increases TGF-beta1 expression in the bladder leading to fibrosis, smooth muscle atrophy and non-compliance. Hypercholesterolemia also interferes with bladder structure and compliance but to a significantly lesser extent compared with CBI. Our studies suggest that arterial insufficiency and hypercholesterolemia, common aging-associated disorders, may play important roles in the pathophysiology of voiding dysfunction in the elderly.

Human induced pluripotent stem cells (iPSCs) represent a scalable source of potentially any cell type for disease modeling and therapeutic screening. We have a particular interest in modeling skeletal muscle from various genetic backgrounds; however, efficient and reproducible methods for the myogenic differentiation of iPSCs have not previously been demonstrated. Ectopic myogenic differentiation 1 (MyoD) expression has been shown to induce myogenesis in primary cell types, but the same effect has been unexpectedly challenging to reproduce in human iPSCs. In this study, we report that optimization of culture conditions enabled direct MyoD-mediated differentiation of iPSCs into myoblasts without the need for an intermediate step or cell sorting. MyoD induction mediated efficient cell fusion of mature myocytes yielding multinucleated myosin heavy chain-positive myotubes. We applied the same approach to dystrophic iPSCs, generating <em>16</em> iPSC lines from <em>fibroblasts</em> of four patients with Duchenne and Becker muscular dystrophies. As seen with iPSCs from healthy donors, within 36 hours from MyoD induction there was a clear commitment toward the myogenic identity by the majority of iPSCs in culture (50%-70%). The patient iPSC-derived myotubes successfully adopted the skeletal muscle program, as determined by global gene expression profiling, and were functionally responsive to treatment with hypertrophic proteins insulin-like <em>growth</em> <em>factor</em> 1 (IGF-1) and wingless-type MMTV integration site family, member 7A (Wnt7a), which are being investigated as potential treatments for muscular dystrophy in clinical and preclinical studies, respectively. Our results demonstrate that iPSCs have no intrinsic barriers preventing MyoD from inducing efficient and rapid myogenesis and thus providing a scalable source of normal and dystrophic myoblasts for use in disease modeling and drug discovery.

Recent studies indicate that insulin-like <em>growth</em> <em>factor</em>-II (IGF-II) acts as an autocrine differentiation <em>factor</em> for skeletal myoblasts in culture. IGF-II mRNA and protein are induced as early events in muscle differentiation, and the rate and extent of IGF-II secretion correlate with both biochemical and morphological differentiation. Here we show that IGF-II also functions as an essential survival <em>factor</em> during the transition from proliferating to differentiating myoblasts. Stably transfected C2 muscle cell lines were established in which a mouse IGF-II cDNA was expressed in the antisense orientation relative to the constitutively active Moloney sarcoma virus promoter. IGF-II antisense cells proliferated normally in <em>growth</em> medium containing 20% serum but underwent rapid death when placed in low serum differentiation medium. Death was accompanied by characteristic markers of apoptosis with more than 90% of cells showing DNA fragmentation within 12-<em>16</em> h. Myoblast death was prevented by IGF-I, des [1-3] IGF-I, IGF-II, and insulin with a dose potency consistent with activation of the IGF-I receptor; death also could be blocked by the protein synthesis inhibitor, cycloheximide. Exogenous IGFs additionally stimulated passage through a single cell cycle and subsequently induced terminal differentiation. Cell survival and cell cycle progression also were enhanced by <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 and platelet-derived <em>growth</em> <em>factor</em>-bb, but these peptides did not promote differentiation. Our results define a novel system for studying apoptotic cell death and its prevention by <em>growth</em> <em>factors</em>, underscore the importance of IGF action in minimizing inappropriate cell death, and indicate that shared signal transduction pathways may mediate myoblast survival in vitro.

OBJECTIVE

To evaluate the efficacy of low-level laser therapy (LLLT) on collateral circulation and microcirculation if a blood vessel is occluded.

BACKGROUND

Investigators have attempted prostaglandin and ultrasound therapy to promote improvements in the vascular bed of deprived tissue after an injury, which may lead to occlusion of the blood vessels.

METHODS

Thirty-four adult rabbits were used in this study, two of them considered 0-h reading group, while the rest were divided into two equal groups, with <em>16</em> rabbits each: control and those treated with LLLT. Each rabbit underwent two surgical operations; the medial aspect of each thigh was slit, the skin incised and the femoral artery exposed and ligated. The site of the operation in the treated group was irradiated directly following the operation and for 3 d after, one session daily for 10 min/session. The laser system used was a gallium-aluminum-arsenide (Ga-Al-As) diode laser with a wavelength of 904 nm and power of 10 mW. Blood samples collected from the femoral artery above the site of the ligation were sent for examination with high-performance liquid chromatography (HPLC) to determine the levels of adenosine, <em>growth</em> hormone (GH) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF). Tissue specimens collected from the site of the operation, consisting of the artery and its surrounding muscle fibers, were sent for histopathological examination to determine the fiber/capillary (F/C) ratio and capillary diameter. Blood samples and tissue specimens were collected at 4, 8, 12, <em>16</em>, 20, 24, 48 and 72 h postoperatively from the animals of both groups, control and treated.

RESULTS

Rapid increases in the level of adenosine, GH, and FGF occurred. The F/C ratio and capillary diameter peaked at 12-<em>16</em> h; their levels declined gradually, reaching normal values 72 h after irradiation in the treated group. Numerous collateral blood vessels proliferated the area, with marked increases in the diameters of the original blood vessels.

CONCLUSIONS

The results indicated that LLLT accelerated collateral circulation and enhanced microcirculation and seemed to be unique in the normalization of the functional features of the injured area, which could lead to occlusion of the regional blood vessels.

OBJECTIVE

To explore new strategies for effective isolation, preservation, and expansion of human corneal endothelial cells (HCECs).

METHODS

Human corneal Descemet's membrane and corneal endothelial cells were digested with collagenase A or Dispase II in supplemented hormonal epithelial medium (SHEM) for 1.5 to <em>16</em> hours. HCEC aggregates derived from collagenase A digestion were preserved in serum-free medium with low or high calcium for up to 3 weeks. Cryosections of HCEC aggregates were subjected to immunostaining with ZO-1, connexin 43, type IV collagen, laminin-5, and perlecan, and apoptosis was determined by TUNEL or cell-viability assay. For expansion, HCEC aggregates were seeded directly or after brief treatment with trypsin/EDTA in SHEM, with or without additional bovine pituitary extract (BPE), nerve <em>growth</em> <em>factor</em> (NGF), or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). The resultant HCECs were immunostained with ZO-1, connexin 43, and Ki67.

RESULTS

Digestion with collagenase A, but not Dispase, of the stripped Descemet's membrane generated HCEC aggregates, which preserved cell-cell junctions and basement membrane components. High cell viability of HCEC aggregates was preservable in a serum-free, high-calcium, but not low-calcium, medium for at least 3 weeks. Brief treatment of HCEC aggregates with trypsin/EDTA resulted in a higher proliferation rate than without, when cultured in SHEM, and the resultant confluent monolayer of hexagonal cells retained cell-cell junctions. However, additional BPE, NGF, or bFGF did not increase cell proliferation, whereas additional BPE or bFGF disrupted cell-cell junctions.

CONCLUSIONS

Collagenase A digestion successfully harvested aggregates with viable HCECs that were preservable for at least 3 weeks in a serum-free, high-calcium medium and, with brief trypsin/EDTA treatment, expanded in the SHEM into a monolayer with hexagonal cells that exhibited characteristic cell junctions.

OBJECTIVE

In children with CKD, information is limited regarding the prevalence and determinants of fibroblast growth factor 23 excess and 1,25-dihyroxyvitamin D deficiency across the spectrum of predialysis CKD. This study characterized circulating concentrations of fibroblast growth factor 23 and 1,25-dihyroxyvitamin D, and investigated their interrelationships and associations with GFR and secondary hyperparathyroidism in children with CKD who were enrolled in the Chronic Kidney Disease in Children observational cohort study.

METHODS

Plasma fibroblast growth factor 23 concentrations and determinants of mineral metabolism were measured in 464 children ages 1-16 years with predialysis CKD. GFR was measured by plasma disappearance of iohexol in 70% of participants and estimated by the Chronic Kidney Disease in Children estimating equation using serum creatinine and cystatin C concentrations in the remainder of the participants. Participants were grouped according to CKD stage and by 10-ml/min categories of GFR.

RESULTS

Median GFR for the cohort was 45 ml/min per 1.73 m(2) (interquartile range=33-57; range=15-109). Plasma fibroblast growth factor 23 concentration was above the normal range in 67% of participants (with higher levels observed among participants with lower GFR) before higher levels of serum parathyroid hormone and phosphorus were observed. Plasma fibroblast growth factor 23 levels were 34% higher in participants with glomerular disease than in participants with nonglomerular disease, despite similar GFR. Serum phosphorus levels, adjusted for age, were significantly lower at GFR of 60-69 ml/min per 1.73 m(2) than higher GFR, but thereafter they became higher in parallel with fibroblast growth factor 23 as GFR declined. Serum 1,25-dihyroxyvitamin D concentrations were lower in those participants with low GFR values, high fibroblast growth factor 23 levels, 25-hydroxyvitamin D deficiency, and proteinuria. Secondary hyperparathyroidism was present in 55% of participants with GFR<50 ml/min per 1.73 m(2).

CONCLUSIONS

In children with predialysis CKD, high plasma fibroblast growth factor 23 is the earliest detectable abnormality in mineral metabolism, and levels are highest in glomerular diseases.

OBJECTIVE

To determine whether it is possible to induce proliferation in the endothelium of older donor corneas by treatment of the intact monolayer with EDTA.

METHODS

Corneas from donors 52 to 75 years of age were obtained from an eye bank and were usually cut in quarters to increase sample size. The effect of EDTA dose (0.02-2.0 mg/ml) and incubation time (6, 30, and 60 minutes) on endothelial cell-cell contacts was evaluated by staining for ZO-1, a cell junction marker. Cell death was tested by a commercial live-dead assay. Corneal pieces were incubated for 0, 24, 48, or 60 hours in culture medium (M-199, 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 20 ng/ml fibroblast growth factor) before EDTA treatment. After treatment, pieces were incubated in the same medium for 24, 48, 72, or 96 hours to permit cell cycle entry. Tissue was fixed, stained for Ki67 (a marker for late G1-phase through the M-phase), and mounted in medium containing propidium iodide to visualize all nuclei. Confocal images were evaluated by computer (Image software; NIH, Bethesda, MD) to count Ki67-positive and propidium iodide-stained cells.

RESULTS

EDTA released corneal endothelial cell-cell contacts in a dose- and time-dependent manner. At doses and incubation times tested, EDTA did not induce significant cell death. Preincubation in culture medium for 24 hours was needed for endothelial cells to efficiently initiate proliferation in response to EDTA. The endothelium of corneas incubated in mitogen-containing medium for up to 108 hours without EDTA treatment did not stain for Ki67. EDTA at 2.0 mg/ml for 60 minutes appeared optimal and stimulated 16% to 18% of the cells to proliferate. Ki67-positive mitotic figures were visible 48 hours after exposure to EDTA. Formation of daughter cells was visible after double-staining for Ki67 and ZO-1.

CONCLUSIONS

EDTA released cells from contact inhibition and promoted proliferation in corneal endothelium from older donors. The authors hypothesize that corneal endothelium from older individuals divide in situ when exposed to positive growth factors under conditions in which cells have been transiently released from contact inhibition.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGF) and their high-affinity receptors (FGFR) represent an extensive cellular <em>growth</em> and survival system. Aim of this study was to evaluate the contribution of FGF/FGFR-mediated signals to the malignant <em>growth</em> of non-small cell lung cancer (NSCLC) and to assess their potential as targets for therapeutic interventions. Multiple FGFR mRNA splice variants were coexpressed in NSCLC cells (n = <em>16</em>) with predominance of FGFR1. Accordingly, both expression of a dominant-negative FGFR1 (dnFGFR1) IIIc-green fluorescent protein fusion protein and application of FGFR small-molecule inhibitors (SU5402 and PD<em>16</em>6866) significantly reduced <em>growth</em>, survival, clonogenicity, and migratory potential of the majority of NSCLC cell lines. Moreover, dnFGFR1 expression completely blocked or at least significantly attenuated s.c. tumor formation of NSCLC cells in severe combined immunodeficient mice. Xenograft tumors expressing dnFGFR1 exhibited significantly reduced size and mitosis rate, enhanced cell death, and decreased tissue invasion. When FGFR inhibitors were combined with chemotherapy, antagonistic to synergistic in vitro anticancer activities were obtained depending on the application schedule. In contrast, simultaneous blockage of FGFR- and epidermal <em>growth</em> <em>factor</em> receptor-mediated signals exerted synergistic effects. In summary, FGFR-mediated signals in cooperation with those transmitted by epidermal <em>growth</em> <em>factor</em> receptor are involved in <em>growth</em> and survival of human NSCLC cells and should be considered as targets for combined therapeutic approaches.

Galactosyl beta-1,3-N-acetyl galactosamine (Gal beta-1,3-GalNAc) (Thomsen Friedenreich antigen), the Class I core sequence in O-linked oligosaccharide chains, behaves as an oncofetal antigen showing increased expression in many epithelial malignancies. Previous work has shown that peanut agglutinin (PNA), a lectin that binds Gal beta-1,3-GalNAc, stimulates proliferation in HT-29 (human colon cancer) cells and normal human colonic epithelium and this implies that cell surface glycoproteins which express Gal beta-1,3-GalNAc may play an important role in the regulation of epithelial cell proliferation. We have now studied the effect on epithelial cells of another dietary Gal beta-1,3-GalNAc-binding lectin, the edible mushroom Agaricus bisporus lectin (ABL). This differs from PNA in its ability to bind also to sialylated Gal beta-1,3-GalNAc. In contrast to PNA, ABL (25 micrograms/ml) inhibited incorporation of [3H]-thymidine into DNA of HT29 colon cancer cells by 87% (95% confidence limit, 85-89%), Caco-2 colon cancer cells by <em>16</em>% (95% confidence limit, 12-20%), MCF-7 breast cancer cells by 50% (95% confidence limit, 47-52%), and Rama-27 rat mammary <em>fibroblasts</em> by 55% (95% confidence limit, 51-60%) when these cells were grown for 24 h in serum-free medium. When assessed by cell count, similar inhibition of proliferation of HT29 cells by ABL was found. In the presence of 2% fetal calf serum (which contains the ABL-binding glycoprotein fetuin), the inhibitory effect of ABL on cell proliferation was still demonstrable but at increased ABL concentration (60 micrograms/ml for 49% inhibition). Ten micrograms/ml ABL completely abolished the stimulatory effect on [3H]thymidine incorporation of epidermal <em>growth</em> <em>factor</em> (100 pg/ml) and PNA (25 micrograms/ml) and markedly inhibited the stimulatory effect of insulin (50 ng/ml). ABL (0.2 mg/ml) caused no cytotoxicity to HT29, MCF-7, and Rama-27 cells as measured by trypan blue exclusion, and inhibition of proliferation in HT29 cells caused by 50 micrograms/ml ABL was reversible after removal of the lectin. Binding studies with 125I-labeled ABL suggested a single class of binding site with an apparent Kd value of (4.12 +/- 0.29) x 10(-7) M with (3.6 +/- 0.3) x 10(7) binding sites/cell. A. bisporus lectin is a reversible noncytotoxic inhibitor of epithelial cell proliferation which deserves study as a potential agent for cancer therapy.