The sleeping brain differs from the waking brain in its electrophysiological and molecular properties, including the expression of <em>growth</em> <em>factors</em> and immediate early genes (IEG). Sleep architecture and homeostatic regulation of sleep in neonates is distinct from that of adults. Hence, the present study addressed the question whether the unique homeostatic response to sleep deprivation in neonates is reflected in mRNA expression of the IEG cFos, brain-derived nerve <em>growth</em> <em>factor</em> (BDNF), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF2) in the cortex. As sleep deprivation is stressful to developing rats, we also investigated whether the increased levels of corticosterone would affect the expression of <em>growth</em> <em>factors</em> in the hippocampus, known to be sensitive to glucocorticoid levels. At postnatal days 16, <em>20</em>, and 24, rats were subjected to sleep deprivation, maternal separation without sleep deprivation, sleep deprivation with 2 h recovery sleep, or no intervention. mRNA expression was quantified in the cortex and hippocampus. cFos was increased after sleep deprivation and was similar to control level after 2 h recovery sleep irrespective of age or brain region. BDNF was increased by sleep deprivation in the cortex at P<em>20</em> and P24 and only at P24 in the hippocampus. FGF2 increased during recovery sleep at all ages in both brain regions. We conclude that cortical BDNF expression reflects the onset of adult sleep-homeostatic response, whereas the profile of expression of both <em>growth</em> <em>factors</em> suggests a trophic effect of mild sleep deprivation.

OBJECTIVE

Fibroblast growth factor 23 (FGF23) levels are elevated in patients with autosomal dominant polycystic kidney disease (ADPKD) and X-linked hypophosphatemia (XLH), but only the latter is characterized by a renal phosphate wasting phenotype. This study explored potential mechanisms underlying resistance to FGF23 in ADPKD.

METHODS

FGF23 and klotho levels were measured, and renal phosphate transport was evaluated by calculating the ratio of the maximum rate of tubular phosphate reabsorption to GFR (TmP/GFR) in 99 ADPKD patients, 32 CKD patients, 12 XLH patients, and 20 healthy volunteers. ADPKD and CKD patients were classified by estimated GFR (CKD stage 1, ≥90 ml/min per 1.73 m(2); CKD stage 2, 60-89 ml/min per 1.73 m(2)).

RESULTS

ADPKD patients had 50% higher FGF23 levels than did XLH patients; TmP/GFR was near normal in most ADPKD patients and very low in XLH patients. Serum klotho levels were lowest in the ADPKD group, whereas the CKD and XLH groups and volunteers had similar levels. ADPKD patients with an apparent renal phosphate leak had two-fold higher klotho levels than those without. Serum klotho values correlated inversely with cyst volume and kidney growth.

CONCLUSIONS

Loss of klotho might be a consequence of cyst growth and constrain the phosphaturic effect of FGF23 in most patients with ADPKD. Normal serum klotho levels were associated with normal FGF23 biologic activity in all XLH patients and a minority of ADPKD patients. Loss of klotho and FGF23 increase appear to exceed and precede the changes that can be explained by loss of GFR in patients with ADPKD.

Thrombopoietin (TPO), the specific cytokine that regulates platelet production, is expressed in human bone marrow (BM), kidney, and liver. There appears to be no regulation of TPO in the kidney and liver, but TPO messenger RNA (mRNA) expression can be modulated in the stromal cells of the BM. In this study, we used primary human BM stromal cells as a model to study the regulation of TPO mRNA expression in response to various platelet alpha-granular proteins. We showed that platelet-derived <em>growth</em> <em>factor</em> (PDGF) BB and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) 2 stimulated TPO mRNA expression in both a dose-dependent and time-dependent manner. The addition of 50 ng/mL of PDGF and <em>20</em> ng/mL of FGF resulted in maximal induction of TPO mRNA expression in 4 hours. We also found that platelet <em>factor</em> 4 (PF4), thrombospondin (TSP), and transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) are negative modulators of megakaryocytopoiesis. We observed suppression in TPO mRNA expression with 1 microg/mL of both PF4 and TSP and 50 ng/mL of TGF-beta, with maximal suppression occurring 4 hours after the addition of these proteins. Finally, the addition of whole-platelet lysate produced a dose-dependent inhibition of TPO expression. On the basis of these findings, we propose that the platelet alpha-granular proteins studied may regulate TPO gene expression in BM stromal cells by means of a feedback mechanism.

The ras gene product (p21) is thought to transduce signals from various <em>growth</em> and differentiation <em>factors</em>. p21 is a GTP-binding protein, and its activity is regulated by the bound GDP/GTP ratio. We analysed p21-bound nucleotides in cell lysates of rat pheochromocytoma cell line PC12 cells stimulated with various <em>factors</em>. Nerve <em>growth</em> <em>factors</em> (NGF) rapidly increased the relative amount of active p21-GTP complex to as much as <em>20</em>% of the total amount of p21 within 2 min. The amount of p21-GTP then declined to 8% after 10 min, and this level was sustained for at least 2 h. Epidermal <em>growth</em> <em>factor</em> (EGF) also stimulated a rapid accumulation of p21-GTP to the same extent as seen with NGF, but the amount of p21-GTP declined to 5% after 10 min and gradually returned to the basal level within 60 min. In contrast, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, interleukin 6 and dibutyryl cAMP, which induce neuronal differentiation of PC12 cells, did not stimulate the accumulation of p21-GTP at any time point examined. Phorbol 12-myristate 13-acetate also had no effect. Interestingly, the protein kinase inhibitor K-252a specifically suppressed the NGF-induced accumulation of p21-GTP, but did not suppress the EGF-induced response. These results strongly suggest that an active p21-GTP complex transduces the differentiation signal from NGF. It may also be suggested that the process of activating p21 is mediated by a K-252a-sensitive protein kinase(s).

BACKGROUND

The purpose of the phase I dose-escalation study was to evaluate the maximum tolerated dose (MTD) of BIBF 1120, an oral triple angiokinase inhibitor of vascular endothelial growth factor, platelet derived growth factor and fibroblast growth factor receptors, combined with paclitaxel and carboplatin.

METHODS

Patients with advanced gynecological malignancies received BIBF 1120 twice-daily (b.i.d.) continuously at 100, 150, 200 or 250 mg, combined with paclitaxel (175 mg/m(2)) and carboplatin (area under the curve 5 min.mg/ml) every 3 weeks. The MTD, safety, pharmacokinetics (PK) and clinical activity were evaluated.

RESULTS

Twenty-two patients were treated. Three experienced dose-limiting toxic effects in the first treatment cycle: 1 of 13 at 200 mg b.i.d. BIBF 1120 [diarrhea, common terminology criteria for adverse events (CTCAE) grade 3]; two of two at 250 mg b.i.d. BIBF 1120 (elevated alanine aminotransferase and aspartate aminotransferase, CTCAE grade 3/4). The MTD was defined as 200 mg b.i.d. Principal adverse events were gastrointestinal disorders. No clinically relevant drug-drug interaction was observed after 20 days treatment with 200 mg b.i.d. BIBF 1120 on the PK of paclitaxel or carboplatin and vice versa.

CONCLUSIONS

The MTD of BIBF 1120 in a 20-day continuous dosing regimen with standard-dose paclitaxel and carboplatin was 200 mg b.i.d. This combination had an acceptable safety profile and no clinically relevant drug-drug interactions. Further evaluation of this combination is warranted in this indication.

Endothelial injury is the primary pathogenic event leading to the renal thrombotic microangiopathic lesions typical of the hemolytic uremic syndrome (HUS). Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is an angiogenic <em>growth</em> <em>factor</em> released by injured endothelial cells. In a previous study we have found a significant accumulation of bFGF in human immunodeficiency virus (HIV)-transgenic mice with renal disease. Here we investigated whether bFGF was accumulated in the circulation and kidneys of two children with HIV-associated HUS (HIV-HUS), and studied the mechanisms involved in this process. The plasma levels of bFGF in children with HIV-HUS (124+/-<em>20</em> pg/ml) were increased compared with five children with HIV nephropathy (49+/-6 pg/ml) and twenty HIV-infected children without renal disease (26+/-4 pg/ml, P<0.001). Immunohistochemistry and receptor binding studies showed that bFGF was accumulated bound to heparan sulfate proteoglycans in renal glomeruli and interstitium surrounding renal tubules in HIV-HUS kidneys. Basic FGF stimulated the proliferation of mesangial and urinary renal tubular epithelial cells isolated from both patients. These findings support the hypothesis that bFGF and its low-affinity binding sites may play a relevant role in modulating the process of glomerular and renal tubular regeneration during the acute stages of HIV-HUS. A follow-up study in a larger sample population is required to confirm these results.

On six human hepatocellular carcinoma (HCC) cell lines (KIM-1, KYN-1, KYN-2, KYN-3, HAK-1A, and HAK- 1B), we examined expressions and functions of the proteins and messenger RNAs (mRNAs) of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and its receptor, i.e., <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor-1 (FGFR-1), as well as mRNA expressions of FGFR-2 approximately 4. All six cell lines expressed the proteins and mRNAs of bFGF and FGFR-1, and at least one of FGFR-2 approximately 4 mRNAs. Two of the six cell lines (KYN-1 and KYN-3) presented significant release of bFGF in culture supernatant, while the release in the remaining four cell lines was quite small. Addition of anti-bFGF neutralizing antibody (1, 10, or <em>20</em> microg/mL) to culture medium resulted in marked suppression of cell proliferation in all cell lines except HAK-1A. On the other hand, addition of exogenous bFGF (0.1, 1, or 5 ng/mL) to culture medium stimulated cell proliferation except in KIM-1 and KYN-2. When KIM-1 was transplanted to nude mice and anti-bFGF antibody was injected subcutaneously to a space surrounding the developed tumor, tumor proliferation was significantly suppressed in nude mice that received anti-bFGF antibody than in control mice, but there were no histological differences between the groups, including blood space formation in the stroma. In conclusion, hepatocellular carcinoma (HCC) cells may possess a proliferation mechanism regulated by an autocrine mechanism, a paracrine mechanism, or both, which are mediated by bFGF/FGFR.

BACKGROUND

Preimplantation cross-talk between a functional blastocyst and the endometrium is critical for successful blastocyst implantation. This interaction is mediated in part by endometrial cytokines/growth factors secreted by glandular epithelium into the uterine cavity. Recent evidence suggests that blastocyst-derived hCG may influence the endometrial milieu in conception cycles thereby enhancing receptivity and implantation success. This study investigated the effect of hCG on the secretory profile of a select cohort of 44 cytokines/growth factors from primary human endometrial epithelial cells (hEECs). These factors included those with both known and unknown roles during receptivity and implantation. The expression of one previously unknown hCG-regulated factor, fibroblast growth factor 2 (FGF2), in human endometrium and its effects on hEEC function were further examined.

METHODS

hEECs isolated from endometrial biopsies collected from fertile cycling women (n = 15) were treated ± recombinant hCG (0.2-20 IU/ml) for 48 h and conditioned media was quantitatively analysed using Luminex™ multiplex technology. FGF2 was further investigated by immunohistochemistry, western blot and cell-adhesion assays.

RESULTS

Of 44 cytokines/growth factors examined, 39 were produced by hEECs with a distinct profile. hCG (2 IU/ml) significantly increased the production of six factors, including those with known roles in receptivity and trophoblast function (interleukin-11), blastocyst migration and adhesion (CXCL10), blastocyst development (granulocyte macrophage colony-stimulating factor) and one unknown with respect to receptivity and implantation (FGF2). Up-regulation of known hCG-regulated proteins, vascular endothelial growth factor and leukaemia inhibitory factor, validated this study. Immunoreactive epithelial FGF2 increased across the menstrual cycle, being highest in secretory and first trimester pregnancy endometrium in vivo. FGF2 (100 ng/ml) stimulated phosphorylation of ERK1/2 in hEEC with no effect on ERK1/2 abundance and stimulated hEEC adhesion to fibronectin and collagen IV (components of blastocyst/trophectoderm extracellular matrix).

CONCLUSIONS

These findings clearly support roles for hCG and FGF2 in the blastocyst-endometrial cross-talk important for endometrial receptivity and blastocyst implantation.

Suggestions exist that, in addition to traditional <em>growth</em> <em>factors</em>, the extracellular matrix (ECM) of a cell can regulate its proliferation. This hypothesis was investigated with normal and transformed <em>fibroblasts</em> because they exhibit specific intracellular responses after adherence to ECM and produce large quantities of ECM proteins. Although cells cultured on different ECM proteins grew more rapidly than those on plastic, adherence and cell <em>growth</em> on an individual ECM protein were not correlated. To test if ECM can stimulate cell <em>growth</em>, soluble ECM proteins were given to cells after plating. In this culture system only collagen VI (CVI), at a concentration of <em>20</em> microg/ml in medium, increased 3T3 cell number to 402% of control by 72 h. Similar increases of human <em>fibroblasts</em> and HT 1080 cell numbers were noted. DNA synthesis of all three cell types increased 24 h after addition of soluble CVI. A mixture of CVI single chains, yielded by reduction and alkylation, was not stimulatory. However, this mixture efficiently inhibited the DNA synthesis induced by native CVI. Antibody inhibition studies showed that the region of CVI stimulating proliferation differs from the site bound by the integrin receptor alpha2beta1, which mediates cell adhesion to immobilized CVI. Heparin inhibited a portion of CVI-induced proliferation. These data demonstrate that CVI can stimulate mesenchymal cell <em>growth</em> via a pathway that is independent of the integrin alpha2beta1 and that the stimulatory region appears to be within the native helical portion of the collagen.

A gly(388)arg polymorphism (rs351855) in the transmembrane domain of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR4) is associated with increased risk, staging, and metastasis in several different types of cancer. To specifically assess the impact of the polymorphic FGFR4 in colorectal cancer (CRC), we engineered CRC cell lines with distinct endogenous expression patterns to overexpress either the FGFR4(gly) or FGFR4(arg) alleles. The biologic analyses revealed an oncogenic importance for both polymorphic alleles, but FGFR4(gly) was the stronger inducer of tumor <em>growth</em>, whereas FGFR4(arg) was the stronger inducer of migration. An evaluation of clinical specimens revealed that FGFR4 was upregulated in <em>20</em>/71 patients independent of gly(388)arg status. There was no correlation between the presence of an FGFR4(arg) allele and CRC or polyp risk in 3,471 participants of the CORSA study. However, among 182 patients with CRC, FGFR4(arg)-carriers had a fivefold higher risk of tumors that were stage II or greater. Together, our results established that both allelic forms of FGFR4 exert an oncogenic impact and may serve equally well as therapeutic targets in CRC. One important implication of our findings is that FGFR4(arg)-carriers are at a higher risk for more aggressive tumors and therefore may profit from early detection measures.

The effects of fluoride (<em>20</em> mumol/L) and bovine bone extract (17 micrograms/ml) were determined on cultures of human bone cells, embryonic chick bone cells, and human skin <em>fibroblasts</em>. The incorporation of [3H]thymidine into DNA was measured 16 hours after the addition of <em>factors</em>. After three to five days treatment, Triton X-100 extracts of the cells were assayed for acid phosphatase activity, in the presence and absence of tartrate, and for alkaline phosphatase activity. Fluoride stimulated [3H]thymidine incorporation and specific activity of alkaline phosphatase in human bone cells and chick bone cells but not in human skin cells. Fluoride also stimulated the cell population doubling rate of the human bone cells with an optimum of approximately <em>20</em> mumol/L. Bovine bone extract stimulated thymidine uptake into DNA several-fold and decreased alkaline phosphatase activity in all three types of cultured cells. The specific activity of tartrate-resistant acid phosphatase was increased in bone cells but not in skin <em>fibroblasts</em>. These results suggest that fluoride specifically stimulates the proliferation and differentiation of osteoblasts, while the <em>growth</em> <em>factors</em> in bovine bone extract primarily stimulate proliferation of bone cells. Cultures of human bone cells respond similarly to chick calvarial cells when treated with fluoride or bovine bone extract.

Relationships between mevalonate and DNA synthesis were explored in quiescent human <em>fibroblasts</em> stimulated with human platelet-derived <em>growth</em> <em>factor</em> (PDGF). Studies of others have indicated that mevalonate, or a product of mevalonate other than cholesterol, is essential for DNA replication. The present studies were designed to determine whether there was a critical time in the cell cycle when mevalonate was necessary for later DNA synthesis to occur. Compactin and mevinolin, inhibitors of hydroxymethylglutaryl CoA reductase, were employed to block both the synthesis of mevalonate and of DNA. Compactin inhibited the sharp peak of DNA synthesis which occurs in cells 24 h after PDGF addition in a concentration-dependent manner. This suppression of DNA synthesis was not prevented by low density lipoprotein but was fully reversed by mevalonate. Compactin inhibited DNA synthesis when the inhibitor was present during the time interval 10-<em>20</em> h after PDGF addition. Its presence only in the interval before 10 h or after <em>20</em> h had no effect. Conversely, mevalonate could fully overcome the compactin block in DNA synthesis when present during the period of from 10-<em>20</em> h after PDGF addition. Mevalonate present only before 10 h or after <em>20</em> h had no effect. When mevalonate was added to mevinolin-blocked cells for the interval 10-15 h after PDGF, the mevinolin block of DNA synthesis was 68% overcome; in contrast, only <em>20</em>% of the reversal of the mevinolin block was seen when mevalonate was added from 15-<em>20</em> h. Addition of mevalonate for only the 2-h interval of from 10-12 h after PDGF overcame the mevinolin block of DNA synthesis (assayed at 24 h) by 50%. The results show that there is a critical time period, several h before S phase, when PDGF-stimulated cells require mevalonate in order for DNA synthesis to proceed at 24 h. This critical period comprised the interval of approximately 10-<em>20</em> h after PDGF addition and especially the early part of this interval.

1. Metformin is one of the most commonly used drugs for the treatment of Type 2 diabetes. Accumulating evidence suggests that metformin also has cardioprotective effects. In the present study, we investigated the cardioprotective effects of metformin and the mechanisms involved. 2. A rat model of chronic heart failure was established by permanent left coronary artery occlusion. Heart failure rats were randomly divided into four groups: (i) a saline-treated group given 4 mL/kg day via intragastric gavage; (ii) a metformin-treated group, given 100 mg/kg metformin once daily via intragastric gavage; (iii) a group treated with 5 mg/kg 5'-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR), an AMP-activated protein kinase (AMPK) agonist, every second day; and (iv) a group treated with 100 mg/kg per day metformin + <em>20</em> mg/kg, i.p., compound C (an AMPK antagonist). After 4 weeks treatment, echocardiography was used to assess left ventricular (LV) dimensions and function. Expression of AMPK, endothelial nitric oxide synthase (eNOS) and transforming <em>growth</em> <em>factor</em> (TGF)-β1 was determined by reverse transcription-polymerase chain reaction and western blot analysis. 3. Metformin administration significantly improved cardiac function and LV remodelling, as evidenced by increases in LV systolic pressure and LV ejection fraction and decreases in LV end-diastolic diameter and LV end-systolic diameter. These beneficial effects of metformin were associated with increased AMPK and eNOS phosphorylation, as well as reductions in insulin, TGF-β1, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and tumour necrosis <em>factor</em>-α levels in the circulation and/or myocardium. 4. The results indicate that chronic low-dose metformin confers significant cardioprotective effects against chronic heart failure by activating the AMPK-eNOS pathway.

OBJECTIVE

Fibroblast growth factor 2 (FGF-2) protects the heart from ischaemia- and reperfusion-induced cell death by a mechanism linked to protein kinase C (PKC)ε-mediated connexin 43 (Cx43) phosphorylation. Cx43 localizes predominantly to gap junctions, but has also been detected at subsarcolemmal (SSM), but not interfibrillar (IFM), mitochondria, where it is considered important for cardioprotection. We have now examined the effect of FGF-2 administration to the heart on resistance to calcium-induced permeability transition (mPTP) of isolated SSM vs. IFM suspensions, in relation to mitochondrial PKCε/Cx43 levels, phosphorylation, and the presence of peptide Gap27, a Cx43 channel blocker.

RESULTS

FGF-2 perfusion increased resistance to calcium-induced mPTP in SSM and IFM suspensions by 2.9- and 1.7-fold, respectively, compared with their counterparts from vehicle-perfused hearts, assessed spectrophotometrically as cyclosporine A-inhibitable swelling. The salutary effect of FGF-2 was lost in SSM, but not in IFM, in the presence of Gap27. FGF-2 perfusion increased relative levels of PKCε, phospho(p) PKCε, and Tom-20 translocase in SSM and IFM, and of Cx43 in SSM. Phospho-serine (pS) 262- and pS368-Cx43 showed a 30- and 8-fold increase, respectively, in SSM from FGF-2-treated, compared with untreated, hearts. Stimulation of control SSM with phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased both calcium tolerance and mitochondrial Cx43 phosphorylation at S262 and S368. The PMA-induced phosphorylation of mitochondrial Cx43 was prevented by εV1-2, a PKCε-inhibiting peptide.

CONCLUSIONS

SSM are more responsive than IFM to FGF-2-triggered protection from calcium-induced mPTP, by a mitochondrial Cx43 channel-mediated pathway, associated with mitochondrial Cx43 phosphorylation at PKCε target sites.

OBJECTIVE

Many common neurosurgical procedures, including anterior temporal lobectomy and endoscopic ventricular puncture, allow neurosurgeons to retrieve portions of the germinal subventricular zone (SVZ). Isolation and maintenance of precursor cells from this zone can be used for hypothesis-driven experiments with a goal of improving our understanding of the basic mechanisms of central nervous system injury or disease and the potential of cell-based therapies to treat them. This article details our ability to reliably harvest, isolate, characterize, and maintain normal adult human brain SVZ precursor cells.

METHODS

Normal SVZ specimens were retrieved as part of anterior temporal lobe resections during planned epilepsy surgery. Dissociated SVZ specimens were plated and incubated in epidermal growth factor and basic fibroblast growth factor for more than 1 year to select for and expand normal neural precursor cells.

RESULTS

Self-renewal and immunocytochemical experiments proved the feasibility of long-term expansion of a slowly dividing nestin+, vimentin+, and glial fibrillary acidic protein-positive astrocyte capable of generating new neurons and glia. These mitotically active bipotent human precursors generated a large number of progeny and possessed significant self-renewal capacity, demonstrated by their ability to generate neurospheres. Cryopreservation was reliable with no loss of the precursor phenotype.

CONCLUSIONS

We have adapted techniques to allow for the isolation and long-term propagation of human adult neural precursors that are capable of generating both neurons and astrocytes in vitro. We have exploited the cell's self-renewal capacity to significantly and consistently expand human neural precursor cells for as long as 20 months. These findings suggest that cells derived from the SVZ during routine surgery may provide a renewable source of human neural precursor cells to study the biological mechanism of central nervous system disease or for application in cell-based human transplantation paradigms.

OBJECTIVE

To determine the expression of vascular endothelial growth factor-C (VEGF-C) in the synovial fluid of patients with rheumatoid arthritis (RA) and to investigate the regulation of VEGF-C production by major proinflammatory cytokines in fibroblast-like synoviocytes (FLS).

METHODS

The concentrations of VEGF-C, tumor necrosis factor-alpha (TNF-alpha), and interleukin 1beta (IL-1beta) were measured using an ELISA method in synovial fluids obtained from 20 patients with RA and 20 with osteoarthritis (OA). Primary cultured RA FLS were stimulated with TNF-alpha or IL-1beta, and the expression levels of VEGF-C mRNA and protein were assessed by quantitative real-time polymerase chain reaction and ELISA.

RESULTS

Significantly higher levels of VEGF-C were found in RA synovial fluids compared to OA synovial fluids. VEGF-C levels showed a highly significant correlation with the levels of both TNF-alpha and IL-1beta in the synovial fluid of patients with RA. TNF-alpha stimulation significantly increased VEGF-C mRNA and protein expression in RA FLS in a dose-dependent manner. A tendency to increased expression of VEGF-C was also observed after IL-1beta stimulation in FLS.

CONCLUSIONS

Overexpression of VEGF-C in FLS by stimulation with TNF-alpha may play an important role in the progression of synovial inflammation and hyperplasia in RA by contributing to local lymphangiogenesis and angiogenesis.

BACKGROUND

In previous experiments, we showed that heparin oligosaccharides inhibit the angiogenic cytokine fibroblast growth factor-2. Here, we present the first in vivo study of size-fractionated heparin oligosaccharides in four models of angiogenesis that are progressively less dependent on fibroblast growth factor-2.

METHODS

Heparin oligosaccharides were prepared using size-exclusion gel filtration chromatography and characterized through depolymerization and strong anion exchange high-performance liquid chromatography. Size-defined oligosaccharides (20 mg/kg/d) were given to mice bearing s.c. sponges that were injected with fibroblast growth factor-2 (100 ng/d). After 14 days, octasaccharides and decasaccharides reduced the microvessel density to levels below control. In a second experiment, HEC-FGF2 human endometrial cancer cells that overexpress fibroblast growth factor-2 were implanted in a hollow fiber placed s.c. in vivo. Oligosaccharides were given at 20 mg/kg/d for 2 weeks and the data again showed that octasaccharides significantly reduced microvessel density around the fiber (P = 0.03). In a more complex model, where angiogenesis was induced by a broad spectrum of growth factors, including vascular endothelial growth factor, we implanted H460 lung carcinoma cells in hollow fibers and treated the animals with oligosaccharides at 20 mg/kg/d over 3 weeks. Octasaccharides reduced the microvessel density to that of control. Preliminary investigation of 6-O-desulfated heparins showed that these also had antiangiogenic activity.

RESULTS

Finally, we examined the inhibitory potential of hexasaccharides and octasaccharides given at 20 mg/kg/d and these inhibited the growth of H460 lung carcinoma in vivo. At clinically attainable concentrations, significant anticoagulation (activated partial thromboplastin time, anti-factor Xa, and anti-factor IIa) was not observed in vitro unless species containing>> or =16 saccharide residues were investigated.

CONCLUSIONS

Thus, our preclinical data show that heparin octasaccharides represent novel antiangiogenic compounds that can be given without the anticoagulant effects of low molecular weight heparin.

An optimal culture system for human pluripotent stem cells should be fully defined and free of animal components. To date, most xeno-free culture systems require human feeder cells and/or highly complicated culture media that contain activators of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) and transforming <em>growth</em> <em>factor</em>-β (TGFβ) signaling pathways, and none provide for replacement of FGF/TGFβ ligands with chemical compounds. The Wnt/β-catenin signaling pathway plays an important role in mouse embryonic stem cells in leukemia inhibitory <em>factor</em>-independent culture; however, the role of Wnt/β-catenin signaling in human pluripotent stem cell is still poorly understood and controversial because of the dual role of Wnts in proliferation and differentiation. Building on our previous investigations of small molecules modulating Wnt/β-catenin signaling in mouse embryonic stem cells, we identified a compound, ID-8, that could support Wnt-induced human embryonic stem cell proliferation and survival without differentiation. Dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) is the target of the small molecule ID-8. Its role in human pluripotent cell renewal was confirmed by DYRK knockdown in human embryonic stem cells. Using Wnt and the DYRK inhibitor ID-8, we have developed a novel and simple chemically defined xeno-free culture system that allows for long-term expansion of human pluripotent stem cells without FGF or TGFβ activation. These culture conditions do not include xenobiotic supplements, serum, serum replacement, or albumin. Using this culture system, we have shown that several human pluripotent cell lines maintained pluripotency >><em>20</em> passages) and a normal karyotype and still retained the ability to differentiate into derivatives of all three germ layers. This Wnt-dependent culture system should provide a platform for complete replacement of <em>growth</em> <em>factors</em> with chemical compounds.

We sought to evaluate safety, tolerability, pharmacokinetics, and pharmacodynamics of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), administered as a single intracoronary injection, to subjects with stable angina pectoris secondary to coronary artery disease. bFGF, an angiogenic <em>growth</em> <em>factor</em>, has been shown to enhance collateral development in animal models of progressive coronary occlusion. To our knowledge, this study represents the initial introduction of parenteral bFGF into humans. This was a phase 1, randomized, dose-escalation trial of bFGF in 25 subjects with coronary artery disease and stable angina. Subjects were randomized 2:1 to a single dose of bFGF or placebo, injected into the left main coronary artery. bFGF doses ranged from 3 to 100 microg/kg, increasing in half-log increments. bFGF was generally well tolerated at doses of 3 to 30 microg/kg. Plasma clearance was <em>20</em> +/- 2 ml/kg/min, with an elimination half-life of 85 +/- 11 minutes. bFGF caused acute hypotension ( approximately 10%) that did not appear to be dose-related through the dose range studied. Of the 9 subjects who received 30 to 100 microg/kg bFGF, 2 had sustained hypotension, mild to moderate in severity, lasting 1 to 3 days, and 3 subjects developed bradycardia hours to days after bFGF administration. bFGF dilated epicardial coronary arteries (7.4 +/- 2.5% mean diameter increase, p <0.02). Transient mild thrombocytopenia and proteinuria were observed in some subjects in the 30-microg/kg cohort. No subject had signs suggesting systemic angiogenesis. Thus, intracoronary bFGF, at doses of 3 to 30 microg/kg, was generally well tolerated in subjects with stable angina.

Previous findings suggest that both the Tat polypeptide encoded by HIV-1 and Tat-derived peptides can induce angiogenesis via activation of the KDR receptor for Vascular Endothelial <em>Growth</em> <em>Factor</em> (VEGF). We identified <em>20</em> amino acids and 12 amino acid peptides corresponding to the cysteine-rich and basic domains of HIV-1 Tat which inhibited (125)I-VEGF(165) binding to KDR and neuropilin-1 (NP-1) receptors in endothelial cells. Cysteine-rich and basic Tat peptides inhibited VEGF-induced ERK activation and mitogenesis in endothelial cells, and inhibited angiogenesis in vitro at concentrations similar to those which inhibited VEGF receptor binding. These peptides also inhibited proliferation, angiogenesis, and ERK activation induced by basic <em>fibroblast</em> <em>growth</em> <em>factor</em> with similar potency and efficacy. Surprisingly, we found that both cysteine-rich and basic domain Tat peptides strikingly induced apoptosis in endothelial cells, independent of their effects on VEGF and bFGF. Furthermore, we found no evidence for direct biological effects of recombinant Tat on VEGF receptor binding, ERK activation, endothelial cell survival, or mitogenesis. These findings demonstrate novel properties of Tat-derived peptides and indicate that their major effect in endothelial cells is apoptosis independent of specific inhibition of VEGF receptor activation.

The expression of SPARC (secreted protein acidic and rich in cysteine/osteonectin/BM-40) is elevated in endothelial cells participating in angiogenesis in vitro and in vivo. SPARC acts on endothelial cells to elicit changes in cell shape and to inhibit cell cycle progression. In addition, SPARC binds to and diminishes the mitotic activity of vascular endothelial <em>growth</em> <em>factor</em>. To determine the effect(s) of SPARC on angiogenic responses in vivo, we implanted polyvinyl alcohol sponges subcutaneously into wild-type and SPARC-null mice. On days 12 and <em>20</em> following implantation, SPARC-null mice showed increased cellular invasion of the sponges in comparison to wild-type mice. Areas of the sponge with the highest cell density exhibited the highest numbers of vascular profiles in both wild-type and SPARC-null animals. The endothelial component of the vessels was substantiated by immunoreactivity with three different markers specific for endothelial cells. Although sponges from SPARC-null relative to wild-type mice were populated by significantly more cells and blood vessels, an increase in the ratio of vascular to nonvascular cells was not apparent. No differences in the percentage of proliferating cells within the sponge were detected between wild-type and SPARC-null sections. However, elevated levels of vascular endothelial <em>growth</em> <em>factor</em> were associated with sponges from SPARC-null versus wild-type mice. An increase in vascular endothelial <em>growth</em> <em>factor</em> production was also observed in SPARC-null primary dermal <em>fibroblasts</em> relative to those of wild-type cells. In conclusion, we have shown that the fibrovascular invasion of polyvinyl alcohol sponges is enhanced in mice lacking SPARC, and we propose that increased levels of vascular endothelial <em>growth</em> <em>factor</em> account, at least in part, for this response.

A primary receptor-binding region of mouse epidermal <em>growth</em> <em>factor</em> (EGF) was identified by comparing the relative affinities of selected synthetic fragments with overlapping sequences in the EGF receptor-binding assay, using human foreskin <em>fibroblasts</em>. Only synthetic peptides containing the amino acid residues <em>20</em>-31 in the mouse EGF sequence showed the ability to compete with 125I-labeled EGF in binding to EGF receptors. The affinities of the cyclic EGF fragment [Ala<em>20</em>]EGF-(14-31) and the linear [(S-acetamidomethyl)-Cys<em>20</em>,31]-EGF-(<em>20</em>-31) were approximately 1/10(4) of the affinity of EGF. Despite their reduced receptor affinities, these two peptides exhibited the in vitro biological activities of native EGF, while fragments from other regions of the EGF molecule were devoid of these biological properties. The peptides induced DNA synthesis in human foreskin <em>fibroblasts</em> as measured by [3H]thymidine incorporation into DNA. They also induced EGF receptor clustering and activated the EGF-sensitive kinase, enhancing the autophosphorylation of EGF receptors in a dose-related manner. Moreover, a major antigenic determinant of EGF for rabbit anti-EGF antibodies was identified within this same localized region of the EGF molecule by competition experiments utilizing the synthetic EGF fragments. The predominant EGF antigenic determinant(s) was also found within the fragment [(S-acetamidomethyl)Cys<em>20</em>,31]-EGF-(<em>20</em>-31). The accessibility of the residues in positions <em>20</em>-31 for antibody recognition is consistent with the conclusion that these residues constitute or contain a major receptor-binding region for EGF.

We have modified a method (Gilles et al: Anal. Biochem., 159:109-113, 1986) for measuring cell number, that is based on the binding of crystal violet to cell nuclei and used it to assay effects of various glycosaminoglycans on <em>growth</em> of human lung <em>fibroblasts</em>. The procedure was modified by <em>growing</em> cells in microcultures (96 well microplates) and by measuring the amount of adsorbed dye using a microplate photometer after solubilisation of the cells with detergent. There was a linear relationship between absorbance and cell number measured by a Coulter Counter. The method is rapid and sensitive and can be used for screening many samples as well as measuring <em>growth</em> rates at low initial cell densities. Even the low <em>growth</em> rates obtained in the absence of serum can be detected. Human lung <em>fibroblasts</em> were <em>growth</em> arrested by serum deprivation and then grown for periods of up to 4 days in the presence of serum and exogenously added glycosaminoglycans (range, 0.1-100 micrograms/ml). Hyaluronan, chondroitin sulfate, and dextran sulfate were without effects, whereas dermatan sulfate, certain heparan sulfates, and heparin suppressed <em>growth</em> (<em>20</em>%-50% inhibition). The antiproliferative activity of dermatan sulfate increased with increasing iduronate content. Certain heparan sulfates, with moderately high sulfate and L-iduronate contents were better inhibitors than heparin despite the fact that the latter glycan has even higher sulfate and L-iduronate contents. The antiproliferative effect of exogenous glycans appeared after a lag period of 3-4 days, suggesting that they interfered with <em>factors</em> produced by the cells. Furthermore, the degree of inhibition was greater when the initial cell density was low. Above a certain level of seeded cells (approx. 10,000 cells/well), there was no inhibition by any of the glycosaminoglycans. It is possible that exogenous glycans cannot overcome endogenous <em>growth</em>-promoting effects in densely seeded cultures.

A431 cells have been used as an immunogen for generating monoclonal antibodies against the epidermal <em>growth</em> <em>factor</em> (EGF) receptor. Two immunoglobulin M and eight immunoglobulin G3 anti-EGF receptor antibodies were cloned. All ten antibodies immunoprecipitated biosynthetically labeled mature A431 cell EGF receptor and were able to recognize the receptor in Western blotting. However, none of the antibodies immunoprecipitated precursor polypeptides of the A431 cell EGF receptor, neither did they recognize EGF receptors from human foreskin <em>fibroblasts</em>, human placenta, nor a human-mouse hybrid cell expressing EGF receptor. The antibodies were found to bind to glycolipids from A431 cells and it was shown that the determinant involved was the blood group A antigen. It appears that this determinant is present on both the EGF receptor and glycolipids of A431 cells but is not expressed on EGF receptors from other human cells tested. One of the monoclonal antibodies raised was used for immunoaffinity purification of the EGF receptor. The procedure took advantage of the carbohydrate nature of the antigenic determinant by employing sugar-specific elution. The mild conditions permitted the purification of A431 cell EGF receptor (70-80% pure) that possessed an intrinsic EGF-stimulated tyrosine kinase activity with a specific activity of about <em>20</em> nmol/min/mg.