OBJECTIVE

We examine the effect of a Chinese herbal medicine mixture on erectile function in a rat model of hypercholesterolemic erectile dysfunction.

METHODS

In this study 32, 3-month-old Sprague-Dawley rats were used. The 8 control animals were fed a normal diet and the remaining 24 were fed 1% cholesterol diet for 4 months. After 2 months herbal medicine was added to the drinking water of the treatment group of <em>16</em> rats but not the cholesterol only group of 8. Of the <em>16</em> rats 8 received 25 mg./kg. per day (group 1) and 8 received 50 mg./kg. per day (group 2) of Chinese herbal medicine mixture. Serum cholesterol levels were measured at 2 and 4 months. At 4 months erectile function was evaluated with cavernous nerve electrostimulation in all animals. Penile tissues were collected for electron microscopy, and to perform Western blot for endothelial nitric oxide synthase, neuronal nitric oxide synthase, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and caveolin-1.

RESULTS

Serum cholesterol levels were significantly higher in animals fed the 1% cholesterol diet compared to controls at 2 and 4 months. Nevertheless, there was no significant difference among group 1 (145 +/- 30 mg./dl.), group 2 (157 +/- 20) and the cholesterol only group (143 +/- 15). Systemic arterial pressure was not significantly different between the animals that were fed the 1% cholesterol diet and the controls. During electrostimulation of the cavernous nerve peak sustained intracavernous pressure was significantly lower in the cholesterol only group (50 +/- 23 cm. H2O) compared to the control group. Conversely erectile function was not impaired in the herbal medicine treated rats. Electron microscopy showed many caveolae with fingerlike processes in the cavernous smooth muscle and endothelial cell membranes in control and treated rats but not in the cholesterol only group of rats. Western blot did not show a difference among groups in protein expression for endothelial nitric oxide synthase and neuronal nitric oxide synthase in penile tissue but caveolin-1 and bFGF protein expression was significantly higher in groups 1 and 2 than in the cholesterol only and control groups.

CONCLUSIONS

Rats developed erectile dysfunction after being fed a 1% cholesterol diet for 4 months. Although serum cholesterol levels were similar in the cholesterol only rats and those treated with Chinese herbal medicine mixture, erectile response was significantly better in the treated group. The mechanism of the herbal medicine is unknown. High levels of bFGF and caveolin-1 expression in the treated group may protect the cavernous smooth muscle and endothelial cells from the harmful effect of high serum cholesterol.

OBJECTIVE

Although there are a few reports on GH therapy in achondroplasia, these were based on a small sample and/or short-term observation. To clarify the effectiveness of GH treatment on short stature in achondroplasia and hypochondroplasia, a long-term treatment study in a larger number of patients was performed.

METHODS

Forty-two children (<em>16</em> males and 26 females, age 3-14 years) with achondroplasia were examined in this study. Initially, we evaluated hypothalamic-pituitary function and point mutation analysis as previously reported. After the evaluation, the children were treated with GH for more than 2 years; then post-treatment <em>growth</em> velocity and body proportion parameters were determined.

RESULTS

The 35 typical variants of our achondroplasia patients showed previously reported point mutation in the fibroblast growth factor receptor 3 gene. The annual height gain during GH therapy was significantly greater than that before therapy (3.9 +/- 1.0 cm/year before treatment vs 6.5 +/- 1.8 cm/year for the first year and 4.6 +/- 1.6 cm/year for the second year of treatment). The body disproportion had not been aggravated during the treatment period.

CONCLUSIONS

We conclude that GH might be beneficial in the treatment of short stature in children with achondroplasia in the first 2 years of treatment.

In the absence of descending spinal and supraspinal afferent inputs, neurons in the developing lumbar spinal cord of the chick embryo undergo regressive changes including cellular atrophy and degeneration between embryonic days 10 and <em>16</em>. There are significant decreases in the number of motoneurons, interneurons, and sensory (dorsal root ganglion) neurons. Although there are several possible explanations for how afferents might regulate the maintenance of neuronal viability, we have focused attention on the putative role of neurotrophic agents in these events. Previous studies have shown that specific tissue extracts (e.g., muscle, brain), soluble proteins, <em>growth</em> <em>factors</em>, and trophic agents can promote the in vitro and in vivo survival of avian motoneurons during the period of natural cell death (embryonic days 6-10). Several of these agents were also effective following deafferentation. These included brain extract (BEX), muscle extract (MEX), conditioned medium from astrocyte cultures (ACM), as well as the following neurotrophic agents: nerve <em>growth</em> <em>factor</em> (NGF), brain-derived neurotrophic <em>factor</em> (BDNF), neurotrophin-3 (NT-3), S-100, insulin-like <em>growth</em> <em>factor</em>-I (IGF-I), ciliary neurotrophic <em>factor</em> (CNTF), platelet-derived <em>growth</em> <em>factor</em> (PDGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and leukemia inhibitory <em>factor</em> (CDF/LIF). Both transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) and acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) were ineffective. Although considerable more work is needed to determine which (and how) specific CNS-derived trophic agents regulate motoneuron survival, the present results are consistent with the notion that neurotrophic agents released from or modulated by synaptic inputs to target neurons promote neuronal differentiation and survival in the CNS.

Neural stem cells play an important role in neurogenesis of the adult central nervous system (CNS). Inhibition of neurogenesis has been suggested to be an underlying mechanism of radiation-induced CNS damage. Here we developed an in vivo/ in vitro clonogenic assay to characterize the survival of neural stem cells after exposure to ionizing radiation. Cells were isolated from the rat cervical spinal cord and plated as single cell suspensions in defined medium containing epidermal <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. The survival of the proliferating cells was determined by their ability to form neurosphere colonies. The number and size of neurospheres were analyzed quantitatively at day 10, 12, 14 and <em>16</em> after plating. Plating cells from 5, 10 and 15 mm of the cervical spinal cord resulted in a linear increase in the number of neurospheres from day 10-<em>16</em>. Compared to the nonirradiated spinal cord, there was a significant decrease in the number and size of neurosphere colonies cultured from a 10-mm length of the rat spinal cord after a single dose of 5 Gy. When dissociated neurospheres derived from a spinal cord that had been irradiated with 5 Gy were allowed to differentiate, the percentages of neurons, oligodendrocytes and astrocytes as determined by immunocytohistochemistry were not altered compared to those from the nonirradiated spinal cord. Secondary neurospheres could be obtained from cells dissociated from primary neurospheres that had been cultured from the irradiated spinal cord. In conclusion, exposure to ionizing radiation reduces the clonogenic survival of neural stem cells cultured from the rat spinal cord. However, neural stem cells retain their pluripotent and self-renewing properties after irradiation. A neurosphere-based assay may provide a quantitative measure of the clonogenic survival of neural stem cells in the adult CNS after irradiation.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF2) is a potent neurotrophic <em>factor</em> that is involved in brain development and the formation of long-term memory. It has recently been shown that acute FGF2, administered at the time of learning, enhances long-term memory for contextual fear conditioning as well as extinction of conditioned fear in developing rats. As other research has shown that administering FGF2 on the first day of life leads to long-term morphological changes in the hippocampus, in the present study we investigated whether early life exposure to FGF2 affects contextual fear conditioning, and renewal following extinction, later in life. Experiment 1 demonstrated that a single injection of FGF2 on Postnatal Day (PND) 1 did not lead to any detectable changes in contextual fear conditioning in PND <em>16</em> or PND 23 rats. Experiments 2 and 3 demonstrated that 5 days of injections of FGF2 (from PND 1-5) facilitated contextual fear conditioning in PND <em>16</em> and PND 23 rats. Experiment 4 demonstrated that the observed facilitation of memory was not due to FGF2 increasing rats' sensitivity to foot shock. Experiment 5 showed that early life exposure to FGF2 did not affect learning about a discrete conditioned stimulus, but did allow PND <em>16</em> rats to use contextual information in more complex ways, leading to context-dependent extinction of conditioned fear. These results further implicate FGF2 as a critical signal involved in the development of learning and memory.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> binding protein (FGF-BP) is a secreted protein that binds FGF-1 and FGF-2 and is involved in mobilization and activation of FGFs from the extracellular matrix. FGF-BP overexpression as well as ribozyme-mediated reduction of endogenous FGF-BP revealed that FGF-BP can be rate-limiting for tumor <em>growth</em> and angiogenesis. Recent studies showed that FGF-BP expression is up-regulated during early phases of tumorigenesis, indicating that the role of FGF-BP in angiogenesis is a critical early step in the development and progression of tumors. Human papillomavirus type <em>16</em> (HPV <em>16</em>) is highly associated with the development of anogenital cancers. Here we demonstrate that the stable expression of the E6 oncogene of HPV <em>16</em> leads to an activation of the FGF-BP promoter in primary human foreskin keratinocytes (one of the natural host cells of these viruses). This is associated with an increase in the steady state levels of FGF-BP mRNA and FGF-BP protein in cells stably expressing E6. Transient E6 expression revealed that the observed activation of the FGF-BP promoter by the viral oncogene is an early process which is independent from immortalization/transformation events in the cells.

We examined the potential neurotrophic effects of bone morphogenetic protein (BMP)-2 on the survival and differentiation of neurons cultured from the rat developing striatum at embryonic day <em>16</em>, a period during which the mRNAs for BMP-2 and its receptor subunits (types IA, IB, and II) were detected. BMP-2 exerted potent activity to promote the survival of striatal neurons and increased the number of surviving microtubule-associated protein-2-positive cells by 2.4-fold as compared with the control cultures after 4 days in vitro. Although basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) also showed relatively high activity to promote the survival of striatal neurons, transforming <em>growth</em> <em>factor</em>-beta1, -beta2, and -beta3, glial cell line-derived neurotrophic <em>factor</em>, or brain-derived neurotrophic <em>factor</em> promoted their survival weakly. Striatal neurons cultured in the presence of BMP-2 or bFGF possessed extensive neurite out<em>growth</em>s, the majority of which were GABA-immunoreactive. Inhibition of glial cell proliferation by 5-fluorodeoxyuridine did not affect the capacity of BMP-2 to promote the survival of striatal GABAergic neurons. In contrast, the ability of bFGF to promote the survival of striatal neurons was inhibited significantly by the treatment of cells with 5-fluorodeoxyuridine. All these results suggest that BMP-2 exerts potent neurotrophic effects on the striatal GABAergic neurons in a glial cell-independent manner.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-1 (FGF-1) and FGF-2 are heparin-binding polype ptides which express potent mitogenic properties in neoplastic cells. In the present study, we have examined the contribution of endogenous FGF-1 and FGF-2 to the autocrine <em>growth</em> of HSY human salivary-gland adenocarcinoma cells in vitro. Using specific monoclonal antibodies against FGF-1 and FGF-2, immunohistochemical analysis of HSY cells revealed strong expression of both FGF-1 and FGF-2 in the cytoplasm and nucleus. Consistent with these data, 2 molecular mass species of FGF-1 (<em>16</em> and 18 kDa) and 3 FGF-2 (18, 24 and 27 kDa) were identified in HSY cells by Western-blot analysis. Scatchard analysis of FGF binding sites on HSY cells indicated the presence of 23,000 [125I]FGF-1 binding sites/cells with a dissociation constant (KD) of 178 pM and 13,000 [125I]FGF-2 binding sites/cell with a KD of 102 pM. In addition, HSY cells were shown to express the mRNA for FGF receptor-1 (FGFR-1) by reverse transcription-polymerase chain reaction (RT-PCR), confirming the existence of high-affinity FGF binding sites. The influence of endogenous FGF-1 and FGF-2 on HSY cell <em>growth</em> was evaluated by suppressing the expression and activity of FGF by using anti-sense oligonucleotides and neutralizing antibodies. The addition of 50 micron FGF-1-specific anti-sense oligonucleotides to HSY cells resulted in a 61% inhibition of cell <em>growth</em>, while 50 microM FGF-2-specific anti-sense oligonucleotides resulted in a 76% inhibition. These effects were dose-dependent and specific, since sense oligonucleotides were ineffective in inhibiting HSY cell <em>growth</em> at the same concentration. Furthermore, HSY cell <em>growth</em> was suppressed in the presence of anti-FGF-1 or anti-FGF-2 neutralizing antibody, resulting in a 58% inhibition at 8 micromilligrams/ml. Our observations suggest that FGF-1 and FGF-2 may act as autocrine regulators by interacting with FGF receptors on HSY cells.

In a previous study, the authors demonstrated that meningioma cells secrete platelet-derived <em>growth</em> <em>factor</em> (PDGF)-like molecules that stimulate their own <em>growth</em> in an autocrine manner. Based on that finding, a study was undertaken to examine the effect of trapidil, a drug known to have an antagonistic action against PDGF, on cell proliferation of human meningiomas in culture. Trapidil showed a dose-dependent inhibition of meningioma cell proliferation in the absence of any exogenous mitogenic stimulation. The maximum effect was observed at a concentration of 100 micrograms/ml, with the decrease in cell <em>growth</em> ranging from <em>16</em>% to 54% compared to control samples. Trapidil similarly inhibited the basal deoxyribonucleic acid (DNA) synthesis assessed by [3H]-thymidine incorporation in three of seven meningiomas. While the conditioned medium generated from meningioma cells remarkably stimulated the proliferation of meningioma cells (<em>16</em>6% to 277% of control), this effect was strikingly inhibited by the addition of trapidil. Trapidil also inhibited conditioned medium-stimulated DNA synthesis, even when there was no effect on basal DNA synthesis. Furthermore, trapidil significantly inhibited the epidermal <em>growth</em> <em>factor</em> (EGF)-stimulated proliferation of meningioma cells. This inhibitory effect on EGF-stimulated cell proliferation was also observed in nontumorous <em>fibroblasts</em>, demonstrating that trapidil is not an antagonist specific to PDGF. The addition of trapidil (30 micrograms/ml) in combination with bromocriptine (1 microM) showed an additive inhibitory effect on the meningioma cell <em>growth</em> compared to trapidil or bromocriptine alone. The overall results suggest that trapidil exhibits an inhibitory effect on meningioma cell proliferation through blocking the mitogenic stimulation induced by autocrine or exogenous <em>growth</em> <em>factors</em>, and may be considered as a possible new approach to the medical treatment of meningiomas.

To determine whether neural precursor cells have region-specific <em>growth</em> properties, we compared the proliferation, mitogenicity, and differentiation of these cells isolated from the embryonic day <em>16</em> rat forebrain and spinal cord. Neural precursor cells isolated from both regions were cultured in <em>growth</em> medium supplemented with epidermal <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, or epidermal <em>growth</em> <em>factor</em>+basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. Under all three conditions, both neural precursor cell populations proliferated for multiple passages. While spinal cord-derived neural precursor cells proliferated moderately faster in epidermal <em>growth</em> <em>factor</em>-enriched <em>growth</em> medium, brain-derived cells proliferated much faster in basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-enriched <em>growth</em> medium. When exposed to both epidermal <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, the two neural precursor cell populations expanded and proliferated more rapidly than when exposed to a single <em>factor</em>, with brain-derived neural precursor cells expanding significantly faster than spinal cord-derived ones (P<0.0001). Differentiation studies showed that both neural precursor cell populations were multi-potent giving rise to neurons, astrocytes, and oligodendrocytes. However, neuronal differentiation from brain-derived neural precursor cells was greater than spinal cord-derived ones (11.95+/-5.00% vs 1.92+/-1.13%; passage 2). Further, the two neural precursor cell populations differentiated into a similar percentage of oligodendrocytes (brain: 8.66+/-5.85%; spinal cord: 7.69+/-3.91%; passage 2). Immunofluorescence and Western blot studies showed that neural precursor cells derived from both regions expressed receptors for basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and epidermal <em>growth</em> <em>factor</em>. However, brain-derived neural precursor cells expressed higher levels of the two receptors than spinal cord-derived ones in <em>growth</em> medium containing epidermal <em>growth</em> <em>factor</em>+basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. Thus, our results showed that neural precursor cells isolated from the two regions of the CNS have distinct properties and <em>growth</em> requirements. Identifying phenotypic differences between these neural precursor cell populations and their <em>growth</em> requirements should provide new insights into the development of cell therapies for region-specific neurological degenerative diseases.

OBJECTIVE

Laminar shear stress is an important stimulus in the endothelium-dependent control of vascular tone and of vascular remodeling processes. Based on previous studies demonstrating integrin-mediated release of fibroblast growth factor 2 (FGF-2), we investigated whether shear stress-induced integrin activation requires the involvement of an extracellular protease.

METHODS

Cultured porcine aortic endothelial cells (PAEC) were exposed to laminar shear stress (16 dyn/cm(2)), whereas static cells served as controls.

RESULTS

Exposure of PAEC to shear stress led to an increased activity of a protease in supernatants. This protease could be characterized as elastase but was different from neutrophil and pancreatic elastases. The enhanced activity was accompanied by the activation of integrin α(v)β(3) and p38 MAPK, and followed by an increased FGF-2 concentration in the supernatant. Pretreatment with inhibitors of either elastase or integrin α(v)β(3) resulted in a reduction of FGF-2 release. The observed effects of shear stress on integrin α(v)β(3) and p38 MAPK activation, as well as on FGF-2 release could be mimicked by application of pancreatic elastase to static endothelial cells.

CONCLUSIONS

By inducing the release of an endothelial elastase, shear stress induces an integrin-dependent release of FGF-2 from endothelial cells.

Alcohol use disorders (AUDs), including alcohol abuse and dependence, have been linked to the development of acute lung injury (ALI). Prior clinical investigations suggested an association between AUDs and abnormal alveolar epithelial permeability mediated through pulmonary oxidative stress that may partially explain this relationship. We sought to determine if correcting pulmonary oxidative stress in the setting of AUDs would normalize alveolar epithelial permeability in a double-blinded, randomized, placebo-controlled trial of Protandim, a nutraceutical reported to enhance antioxidant activity. We randomized 30 otherwise healthy AUD subjects to receive directly observed inpatient oral therapy with either Protandim (1,350 mg/day) or placebo. Subjects underwent bronchoalveolar lavage (BAL) and blood sampling before study drug administration and after 7 days of therapy; all AUD subjects completed the study protocol without adverse events. BAL total protein was measured at each timepoint as an indicator of alveolar epithelial permeability. In subjects with AUDs, before study drug initiation, BAL total protein values were not significantly higher than in 11 concurrently enrolled controls (P = 0.07). Over the 7-day study period, AUD subjects did not exhibit a significant change in BAL total protein, regardless of their randomization to Protandim {n = 14, -2% [intraquartile range (IQR), -56-146%]} or to placebo [n = <em>16</em>, 77% (IQR -20-290%); P = 0.19]. Additionally, among those with AUDs, no significant changes in BAL oxidative stress indexes, epithelial <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em>, interleukin-1β, or interleukin-10 were observed regardless of drug type received. Plasma thiobarbituric acid reactive substances, a marker of lipid peroxidation, decreased significantly over time among AUD subjects randomized to placebo (P < 0.01). These results suggest that Protandim for 7 days in individuals with AUDs who are newly abstinent does not alter alveolar epithelial permeability. However, our work demonstrates the feasibility of safely conducting clinical trials that include serial bronchoscopies in a vulnerable population at risk for acute lung injury.

Extracts from bovine pituitaries and other tissues contained basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-like peptides of 22-26 kda, co-fractionating with smaller, <em>16</em>-20 kda bFGFs. Heparin-bound, 22-26 kda bFGFs were converted to smaller, heparin-binding forms by tryptic proteolysis. In solution, 22-26 kda bFGFs were converted to smaller, heparin-binding forms by an activity present in pituitary extracts. Calcium protected higher molecular weight pituitary bFGFs from truncation by the endogenous activity, which was not acid-activated, co-purified with bFGF during heparin-sepharose chromatography, remained operant at high salt concentrations and was inhibited by phenylmethan-sulfonyl fluoride.

A procedure for the isolation and cultivation of endothelium from the marginal vessels of the rabbit ear is described. Endothelial cells, isolated by slow perfusion with a trypsin solution, are cultured in minimal essential medium supplemented with 10% fresh rabbit serum for up to 6 mo. In primary culture, marginal vessel endothelial cells grow in an expanding circular pattern with closely apposed cell membranes. Weibel-Palade bodies, subcellular organelles unique to endothelial cells in situ, are present in both primary and in serially cultivated cells (12 passages). In intact skin, Weibel-Palade (W-P) bodies are observed in the perinuclear cytoplasm in close proximity to the cell membrane facing the vascular lumen. 8-<em>16</em> tubules of 200 A diameter are present in each body. In primary and subcultured cells, W-P bodies of identical size are seen in the vicinity of the Golgi apparatus and in close proximity to the outer cell membrane. At the optimum serum concentration (10%), a cell doubling time of 72-96 h is observed. When <em>growth</em> in normal rabbit serum and in platelet-poor serum is compared, a slower <em>growth</em> rate is observed in the absence of platelets, suggesting that <em>factors</em> released by platelets affect endothelial cell proliferation. However, addition of crude platelet <em>factor</em> does not substitute for complete serum. <em>Fibroblast</em> <em>growth</em> <em>factor</em> is not mitogenic for rabbit marginal vessel endothelium in vitro.

Retinoids are effective <em>growth</em> modulators of human ovarian carcinoma cell lines. Their effects are mediated by nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which are transcriptional <em>factors</em> and members of the steroid/thyroid receptor superfamily. To our knowledge, until now, the cellular distribution of RAR proteins in human ovarian tumor specimens is unknown. This study provides new data on the differential cellular localization of RAR alpha protein in <em>16</em> serous adenocarcinomas originating from the ovaries, fallopian tubes, and the peritoneum. Using an affinity-purified antiserum specific for RAR alpha and a monoclonal antibody recognizing the full-length estrogen receptor molecule (clone 6F11), we performed immunohistochemistry on frozen tissue sections and examined the relationship between RAR alpha and estrogen receptor protein expression by comparing the percentage of immunostained tumor cells for either receptor. Our findings indicate a strong linear relationship between the percentages of RAR alpha- and estrogen receptor-labeled tumor cells as determined by linear regression analysis (P < 0.005, r = 0.825). A modest inverse relationship was found between the percentage of RAR alpha-positive tumor cells and histological grade, attesting to a differentiation-dependent trend (P < 0.04). No significant relationship was found between RAR alpha-labeled cells and clinical stage (P = 0.139), site of tumor origin (ovaries versus fallopian tubes versus peritoneum) (P = 0.170), and primary versus metastatic lesion (P = 0.561). Thus, serous adenocarcinomas are capable of expressing RAR alpha and estrogen receptor despite high histological grade and advanced stage of neoplastic disease. Compared with the heterogeneous localization of RAR alpha in cancer cells, there was widespread RAR alpha immunoreactivity in tumor-infiltrating lymphocytes, vascular endothelial cells, and stromal <em>fibroblasts</em>, underscoring the value of immunohistochemistry in the accurate determination of RAR/(RXR) content in tumor specimens.

The purpose of this study was to evaluate the long-term effectiveness of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in achieving neovascularization following ischemia from arterial ligation and to determine an optimal dosage level. We used an Ameroid constrictor to produce progressive occlusion of the left femoral artery of rabbits. At 2 weeks, the rabbits were randomized to receive intravenous injection of vehicle (group A, n = 15); 3 microg/kg/day bFGF (group B, n = 12); 10 microg/kg/day bFGF (group C, n = 12); or <em>16</em> microg/kg/day bFGF (group D, n = 15) for 3 days. At 1 to 37 days after surgery, we assessed limb neovascularization by transcutaneous oximetry (TCPO(2)), angiography, heart rate, arterial pressure, peripheral vascular resistance (PRU), and muscle blood flow (MBF) during steady-state intra-arterial infusion of saline (basal), acetylcholine, papaverine, or serotonin under anesthesia and capillary density (cap/mm(2)) and capillary per muscle fiber ratio (cap/F). Groups B and C showed significantly greater change in TCPO(2) over time than groups A and D (P < 0.0001). Group D showed the lowest TCPO(2) values from days 14 to 37 and group C the highest. Groups B and C showed a higher number of vessels filled with contrast agent than groups A and D (P < 0.0001). Calf cap/mm(2) and cap/F were significantly higher in groups B and C than groups A and D (P < 0.0001). Calf basal MBF values were higher in groups B and C than in groups A and D, but were not statistically significant. Group D showed the highest level in basal PRU. There were no significant differences in heart rate or blood pressure among the groups. These results show (1) treatment with bFGF has no adverse hemodynamic effects, (2) bFGF enhances angiogenesis and circulation at moderate doses, and these effects persist at least several weeks, and (3) high doses of bFGF may inhibit angiogenesis and collateral circulation.

Stimulated human blood monocytes and alveolar macrophages are known to elaborate a soluble <em>factor</em>(s) that inhibits <em>fibroblast</em> proliferation by stimulating <em>fibroblast</em> prostaglandin (PG) production. However, the <em>factor</em>(s) mediating these effects has never been completely characterized and its relationship to known cytokines has never been fully defined. In this study, we demonstrate that this <em>factor</em>(s) is partially trypsin-sensitive, is between <em>16</em> and 24 kilodaltons molecular weight, and elutes between 0.13 and 0.25 M NaCl and 24 and 38% 1-propanol from anion exchange and reverse phase columns, respectively. In addition, we demonstrate that interleukin-1 (IL-1) and tumor necrosis <em>factor</em> (TNF) have similar patterns of elution and that the <em>fibroblast</em> <em>growth</em>-inhibiting and PGE-stimulating activities in monocyte and alveolar macrophage supernatants are partially reversed with neutralizing antibodies against IL-1-beta or TNF. However, this inhibition is not due to IL-1-beta or TNF, alone, because each has a mild stimulatory effect on <em>fibroblast</em> proliferation. In contrast, a dose-dependent inhibition of <em>fibroblast</em> proliferation is noted when <em>fibroblasts</em> are simultaneously exposed to recombinant IL-1 and TNF. This inhibition is reversed when <em>fibroblast</em> PG production is blocked and appears to result from IL-1 and TNF synergistically stimulating <em>fibroblast</em> PGE production. Thus, the PG-mediated inhibition of <em>fibroblast</em> proliferation caused by stimulated human mononuclear phagocytes is not the result of a single cytokine but is instead at least partly mediated by an interaction of IL-1 and TNF.

Except rare instances of allogeneic stem cell transplantation, treatment of idiopathic myelofibrosis (IMF) is only palliative and based on cytostatic treatment (hydroxyurea and anagrelide), androgen therapy, steroids and splenectomy. Thalidomide is an anti-angiogenic and immunomodulatory drug with a wide spectrum of activities, which are not clearly understood. Current data suggest that the action of thalidomide is related to several different mechanisms, including suppression of tumor necrosis <em>factor</em>, effects on basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, vascular endothelial <em>growth</em> <em>factor</em>, interleukins and interferons, downregulation of selected cell surface adhesion molecules, and changes in the lymphocyte subsets. We administered thalidomide to <em>16</em> patients with IMF (15 men, one women) who had transfusion-dependent anemia, thrombocytopenia or symptomatic splenomegaly. Median age was 59 yr (range: 52-78). Patients received thalidomide at an escalating dose from 100 to 400 mg/d (median 300 mg). The drug was discontinued in four patients because of progressive disease (two) or polyneuropathy (two). Other adverse effects were obstipation (10), fatigue (eight) and edema (two). Clinical response has now been observed for a median duration of 9 months (range: 3-20). Fifteen patients are evaluable. Anemia improved in six of 10 patients who were anemic. Platelet counts improved in five of seven patients with thrombocytopenia. Splenomegaly regressed in three of 13 patients. Lactate dehydrogenase (LDH) decreased in seven of 12 patients, but increased in four patients. LDH levels were not correlated with clinical response. In summary, thalidomide appears useful in the treatment of IMF.

OBJECTIVE

Restenosis after stent implantation is caused by endothelial cell damage and subsequent neointimal formation. The objective of this study is to elucidate the relevance of endothelial progenitor cells (EPCs) in the development of in-stent restenosis in patients undergoing stent implantation.

METHODS

The subjects were 46 patients who underwent coronary stenting. Blood samples were collected at the time of follow-up coronary angiography after coronary stenting. EPCs were isolated from blood samples and cultured. Their phenotypes were confirmed by uptake of acetylated low-density lipoprotein and binding of fluorescein isothiocyanate-labeled Ulex europaeus agglutinin 1 lectin. The number of colony-forming units (CFUs) and the senescent cells, determined by acidic beta-galactosidase staining, was counted. Angiogenic growth factors secreted by EPCs, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), hepatocyte growth factor (HGF), and macrophage chemoattractant protein (MCP-1) from the culture medium were also measured by enzyme-linked immunosorbent assay.

RESULTS

Patients with in-stent restenosis (defined as >40% stenosis, n=16) had a decreased number of CFUs (p<0.05), and increased senescent cells (p<0.05), compared to patients without restenosis (n=30). There was no significant difference of angiogenic growth factors (VEGF, HGF, b-FGF, and MCP-1) secreted by EPCs between the two groups. On multivariate analysis, an increased number of senescent EPCs was the indepen-dent factor associated with in-stent restenosis (OR 1.10, 95% CI 1.01 to 1.20).

CONCLUSIONS

These data suggested that EPCs might be involved in the development of in-stent restenosis.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterised by the clonal proliferation of the haematopoietic precursors together with the progressive development of bone marrow fibrosis. This stromal alteration is an important clinical issue and specific prognostic markers are not currently available. In bone marrow biopsies from 58 PMF patients, we explored the methylation pattern of genes encoding cytokines involved in the stromal reaction, namely platelet-derived <em>growth</em> <em>factor</em>-beta (PDGFB), transforming <em>growth</em> <em>factor</em>-beta (TGFB) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF2). We also evaluated the methylation profile of the Long Interspersed Nucleotide Element 1 (LINE-1). PDGFB, FGF2 and LINE-1, but not TGFB, were significantly differently methylated in PMF compared to controls. Significantly, PDGFB hypomethylation ((<em>16</em>%) was correlated with a favourable PMF prognosis (grade of marrow fibrosis, p=0.03; International Prognostic Scoring Systems p=0.01 and Dynamic International Prognostic Scoring Systems, p=0.02). Although the basis of the association of PDGFB hypomethylation with favourable prognosis remains to be clarified, we speculate that hypomethylation in PMF could represent the effect of acquired somatic mutations in genes involved in epigenetic regulation of the genome.

BACKGROUND

Reabsorption of bile acids from the intestine by ileal bile acid transporter is pivotal for the enterohepatic circulation of BAs and sterol homoeostasis.

OBJECTIVE

To assess tolerability and study, bile acid metabolism in a phase 1 trial with the selective ileal bile acid transporter inhibitor A4250.

METHODS

A randomised double-blind, single-ascending dose (SAD) and multiple-ascending-dose study consisting of five cohorts comprising 40 individuals with a single administration of A4250 (0.1, 0.3, 1, 3, or 10 mg) or placebo and three cohorts comprising 24 individuals with a 1-week administration of A4250 (1 or 3 mg once daily or 1.5 mg twice daily) or placebo. For the multiple-ascending-dose study, bile acids were measured by HPLC-MS in plasma and faeces, and fibroblast growth factor 19 (FGF19) and 7α-hydroxy-4-cholesten-3-one (C4) were measured in plasma.

RESULTS

No serious adverse events occurred and all participants finished the trial per protocol. At the end of the multiple-ascending-dose study, plasma total bile acids and FGF19 decreased by 47% and 76%, respectively, at 3 mg/day (P < 0.01), and by 15% and 16%, respectively, at 1.5 mg twice daily (P < 0.05). Plasma C4 and faecal bile acids increased at all dose regimens, by 555%, 664%, 292% and 338%, 421%, 420%, respectively (P < 0.01-0.05). The primary bile acids cholic and chenodeoxycholic acids constituted the majority of faecal bile acids in the A4250-treated groups.

CONCLUSIONS

A4250 is well tolerated. By blocking ileal bile acid transporter in the terminal ileum, it highly efficiently interrupts the enterohepatic circulation of BAs, and should be of benefit to patients with cholestatic liver diseases. Clinical Trial registration EudraCT 2013-001175-21.

BACKGROUND

In recent years, several genes were found to be involved in the process of epididymo-testicular descent, the most frequently cited ones include INSL3, HOXA10, GNRHR, and KAL1. In this study, we analyzed the differences in gene expression profiles between cryptorchid and descended testes. In particular, we analyzed expression of all recently published genes known to be associated with undescended testis.

METHODS

Twenty-two testicular biopsies from 18 boys were analyzed. We analyzed gene expression in <em>16</em> cryptorchid and 6 descended testes using Affymetrix Human Genome U133 Plus 2.0 GeneChips, and validated the results with qPCR.

RESULTS

3,688 transcripts were differentially expressed with an adjusted p value of <0.05 and a change of at least 1.5-fold. The list contained 1,866 downregulated and 1,822 upregulated transcripts in the cryptorchid testes. A novel observation in our study was that the fibroblast growth factor receptor 1 gene (FGFR1) and its mediators SOS1 and RAF1 were expressed less in undescended testes.

CONCLUSIONS

Based on our results, it is possible that a subtle dysfunction (expression) of the FGFR1, SOS1 and RAF1 genes is involved in the development of the most common male reproductive tract disorder - unilateral or bilateral cryptorchidism.

Several members of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family have the potential to protect the intestine against the side effects of radiation therapy. FGF1 is capable of signaling through all subtypes of FGF receptors (FGFRs), whereas FGF7 and FGF10 activate only the epithelial-specific subtype, FGFR2IIIb (FGFR2b). The present study compared the protective activity of FGF1, FGF7 and FGF10 and examined the profiles of FGFR expression in the jejunum of BALB/c mice given total-body irradiation (TBI) with gamma rays. TBI caused drastic increases in FGFR1-4 transcript levels in the jejunum. However, FGFR2b protein temporarily decreased at 12 and 24 h after irradiation. FGF1 pretreatment minimized the number of apoptotic cells in jejunal crypts at <em>16</em> and 24 h after irradiation and increased crypt survival most effectively. In addition, pretreatment with FGF7 or FGF10 decreased FGFR1 transcript levels. The greater effectiveness of FGF1 to enhance crypt survival was also observed even when each FGF was administered 1 h after irradiation. These findings indicate that FGF1 is more potent than FGF7 or FGF10 for protection of the intestine against radiation exposure and suggest that the profiles of FGFR expression in the intestine favor the FGF1 signaling pathway before and during the initial period after irradiation.

The present study was carried out to determine if an insulin-like <em>growth</em> <em>factor</em> (IGF) type activity might be produced by embryonal carcinoma-derived cells. The cell line used to condition <em>growth</em> medium for the isolation of secreted <em>growth</em> <em>factors</em> was a newly established Dif 5 cell type. Dif 5 cells are a differentiated endoderm-like cell type derived from F9 embryonal carcinoma cells (which possess properties similar to mouse embryonic stem cells) following extensive exposure to retinoic acid. When <em>growth</em> medium conditioned by Dif 5 cells is chromatographed on Sephadex G-75 in 1 M acetic acid two peaks of activity are observed which compete for specific [125I]iodo multiplication stimulating activity (MSA) binding to PYS cells. MSA is the rat homologue of human IGF-II. The high molecular weight fraction (Mr approximately 60K) apparently corresponds to IGF-binding protein as determined by its ability to bind [125I]iodo-MSA. The low molecular weight fraction (Mr approximately 8K) is biologically active as this fraction stimulates [3H]thymidine incorporation into serum-starved chick embryo <em>fibroblasts</em>. Radioimmunoassay data indicate that the IGF-like activity produced by Dif 5 cells is more closely related to IGF-II than to IGF-I. Undifferentiated embryonal carcinoma stem cell lines (F9, Nulli, and PCC4) produced little of this MSA-like activity, while PYS-2 (parietal endoderm-like) cells produced about <em>16</em> ng MSA/10(6) cells/24 hr as determined by radioimmunoassay. Dif 5 and PSA-5E (visceral endoderm-like) cells, are found to secrete significant amounts of MSA into the <em>growth</em> medium (30-50 ng MSA/10(6) cells/24 hr). These findings offer further support to a proposal that MSA (IGF-II) produced by endoderm cells, particularly visceral endoderm, may serve as an early embryonic <em>growth</em> <em>factor</em>.