Modifiable risk factors for erectile dysfunction: an assessment of the awareness of such factors in patients suffering from ischaemic heart disease.

Up to 40% of cases of erectile dysfunction (ED) originate from vascular disturbances associated with atherosclerotic disease, leading to the previously proven concomitance between ischaemic heart disease (IHD) and ED. The aim of this study was to evaluate patients' knowledge about modifiable risk factors for ED. The evaluated group of patients was composed of 502 male patients undergoing cardiac rehabilitation and receiving treatment for IHD. The patients' knowledge of risk factors for ED linked to IHD was assessed with an original survey. The presence of ED was assessed using an abridged version of the International Index of Erectile Function-5 questionnaire. Increase in leisure-time physical activity was estimated using a leaflet based on the Framingham questionnaire. In all, 189 participants were unable to name any modifiable ED risk factors, and only 31 patients knew all 6 of them. The most frequently mentioned ED risk factor was smoking, whereas the least frequently mentioned was sedentary lifestyle. Awareness of smoking as an ED risk factor was closely related to the patients' level of education, place of residence, smoking and underlying ED in the individual patient. The ability to classify diabetes as a risk factor for ED was significantly related to the patients' level of education, place of residence, and the prevalence of diabetes in the evaluated group of respondents. The same relations were observed regarding hyperlipidaemia. Awareness of the negative impact a sedentary lifestyle has on the erectile process was found to be closely related to the patients' age, as well as their level of education. The performed study demonstrates the poor knowledge of IHD patients about the modifiable risk factors for ED. The factor that patients are the least aware of is sedentary lifestyle, which, simultaneously, is the risk factor that most frequently affects the respondents.

A hypoxic response induced in MatLyLu cells by cobalt chloride results in an enhanced angiogenic response by the chick chorioallantoic membrane.

Hypoxia is an important regulator of hypoxia inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) expression. Using Dunning rat prostate tumour cells (MatLyLu) we examined the induction of HIF-1alpha and VEGF by hypoxia and if the induction of these proteins would lead to angiogenesis in the chick chorioallantoic membrane (CAM). MatLyLu cells were exposed to 1% O2, in 5.25% CO2 and 94.75% N2, or treated with 100 mM CoCl2 to simulate hypoxia. Conditioned medium was analyzed using Western blots for HIF-1alpha or VEGF. VEGF levels were quantified using ELISA. Hypoxia significantly increased both HIF-1alpha and VEGF protein production. MatLyLu cells (1x10(6) viable cells in 50 microl of medium) grown under normoxic conditions induced in angiogenesis in the CAM, evaluated by the formation of a spoke wheel, and in cross sections by the number of blood vessels, following 5-day culture. This response was significantly increased using CoCl2-treated cells. Culture medium alone with or without CoCl2 was not angiogenic. These results provide direct evidence for the role of hypoxia in the induction of HIF-1alpha and VEGF which act as key angiogenic signals.

Nerve growth factor and neuroimmune interactions in inflammatory diseases.

Discovered almost 50 years ago, nerve growth factor (NGF) has been extensively studied in various biological systems. NGF has recently been suggested to play an important role in mediating and/or regulating immune response, in addition to its trophic and tropic effects on nerve growth and regeneration It is clear that in complex interactions between immune cells and nervous system NGF plays a central role. We have only just begun to identify and understand the direct mechanisms by which NGF activates target cells, the precise identity of the target cells, and the particular factors released from target cells. Nerve growth factor together with possibly other neurotrophins such as BDNF (brain-derived nerve growth factor), GDNF (glial-derived nerve growth factor) or NT3 are important modulators of immunity. More detailed studies are needed at the receptor, mediator and cellular levels to better understand the neuroimmunomodulatory properties of neurothrophins and NGF. The nature of the involvement of NGF in inflammation and inflammatory diseases remains a particularly interesting question. By blocking NGF or mediators released upon NGF activation, we are able to control the progress of inflammation, thereby opening many therapeutic opportunities for the future.

Contact guidance of chick embryo neurons on single scratches in glass and on underlying aligned human skin fibroblasts.

The influence of substratum topography on the morphology and orientation of neurites of chick embryo neurons was studied. Two series of experiments are reported. One concerned the behaviour of growth cones when the axons become contact-guided by the surface texture. The second studied contact guidance of neurites extending on a compact layer of fixed aligned human skin fibroblasts (HSF). It was observed that when the growth cones of sensory neurons isolated from dorsal root ganglions encountered a single scratch in a glass surface (0.1-2 microm in depth and diameter) they turned and continued movement following the axis of the scratch. These neurons became contact-guided as a result of the sequence of events. The growth cone filopodia recognized the irregularity in the substratum surface, whereas the growth cone lamella stabilized contact with the scratch and moved forward along the scratch axis. Scanning electron microscope revealed that the single scratches 150 nm in width and ca. 100 nm deep growth cone filopodia less than 200 nm in diameter could detect and react by turning into them. These filopodia extensions followed the edge of scratches. However, phase contrast and Nomarski's differential interference contrast appeared insufficient for analysis of primary contact guidance of fine growth cone filopodia which themselves are often less than 200 nm. In neuron cultures on fixed aligned HSF, the neuron aggregates assumed spindle-like shapes, and sparsely seeded individual neurons extended axons along the long axes of the fibroblasts. The axons extended significantly further on the fixed underlying fibroblasts than on collagen-covered glass. In crowded cultures of neurons, the cells extended neurites ignoring both the surface anisotropy (the scratches) and the orientation of the aligned fibroblasts. Immunofluorescence staining of neurons with antibodies against neurofilaments made it possible to analyse their shape and orientation on the fibroblasts. Computer-assisted image analysis permitted the observed alignment of the neurites to be characterized quantitatively.

A nerve growth factor peptide retards seizure development and inhibits neuronal sprouting in a rat model of epilepsy.

Kindling, an animal model of epilepsy wherein seizures are induced by subcortical electrical stimulation, results in the upregulation of neurotrophin mRNA and protein in the adult rat forebrain and causes mossy fiber sprouting in the hippocampus. Intraventricular infusion of a synthetic peptide mimic of a nerve growth factor domain that interferes with the binding of neurotrophins to their receptors resulted in significant retardation of kindling and inhibition of mossy fiber sprouting. These findings suggest a critical role for neurotrophins in both kindling and kindling-induced synaptic reorganization.

Intraventricular administration of antibodies to nerve growth factor retards kindling and blocks mossy fiber sprouting in adult rats.

Repeated subconvulsive electrical stimulation of certain areas of the forebrain leads to kindling, a progressive and permanent amplification of evoked epileptiform activity, which is a model for human temporal lobe epilepsy. Recent studies have shown that kindling induces synthesis of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) but not neurotrophin-3 (NT-3) in the hippocampus and cortex. Kindling also elicits mossy fiber sprouting and functional synaptogenesis in the supragranular layer, the hilus, and the CA3 region of the hippocampus. Intraventricular administration of antibodies to NGF has been shown to effectively block septohippocampal sprouting in the adult rat, and has been reported to retard amygdaloid kindling. In the present study, we have investigated the possible role of NGF in both kindling and kindling-associated sprouting. We have confirmed a kindling-induced sprouting of the mossy fibers into the stratum oriens of the CA3 region of the hippocampus, utilizing a new semiquantitative method of analysis based on Timm staining. Previous studies found no overt signs of hippocampal damage with this kindling paradigm, indicating that the increased Timm staining likely reflects a purely activity-induced sprouting. Intraventricular infusion of affinity-purified anti-NGF IgGs (which cross-react with NT-3 but not BDNF) resulted in both significant retardation of kindling and inhibition of the kindling-induced mossy fiber sprouting. The findings suggest a role for NGF in both these phenomena.

NGF deprivation of adult rat brain results in cholinergic hypofunction and selective impairments in spatial learning.

Cholinergic hypofunction has often been correlated with a variety of behavioural impairments. In the present study, adult Wistar rats were intraventricularly infused with antibodies to nerve growth factor (anti-NGF) to examine the effects on cholinergic neurons of the basal forebrain, and on behavioural performance. Immunocytochemical techniques indicated that chronically infused anti-NGF penetrates into the basal forebrain, cortex, striatum, corpus callosum and hippocampus, confirming previous findings after a single injection. Treatment with anti-NGF for 1 or 2 weeks resulted in a significant decrease of 27-33% in density of choline acetyltransferase immunostaining of the cholinergic cell bodies in the medial septum and vertical diagonal band, and a 26% reduction in choline acetyltransferase enzyme activity in the septal area. An array of spatial learning Morris water maze tasks was used to distinguish between acquisition skills and the flexible use of learned information in novel tests. Rats subjected to the spatial learning paradigm received anti-NGF infusion for 2 weeks prior to and for another 2 weeks during the behavioural testing. The anti-NGF-treated animals were found to be no different from those receiving control serum in the Morris water maze acquisition task, either in the latency to find the platform or in the time spent searching in the training quadrant when the platform was removed. However, in consecutive extinction trials, anti-NGF rats continued to search in the empty training quadrant, suggesting the occurrence of perseveration; control rats expanded their search over other areas of the pool.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and modulation of nerve growth factor in murine keratinocytes (PAM 212).

Nerve growth factor (NGF) is a polypeptide that is required for normal development and maintenance of the sympathetic and sensory nervous systems. Skin has been shown to contain relatively high amounts of NGF, which is in keeping with the finding that the quantity of NGF in a tissue is proportional to the extent of sympathetic innervation of that organ. Since the keratinocyte, a major cellular constituent of the skin, is known to produce other growth factors and cytokines, our experiments were designed to determine whether keratinocytes are a source of NGF. Keratinocyte-conditioned media from the keratinocyte cell line PAM 212 contained NGF-like activity, approximately 2-3 ng/ml, as detected by the neurite outgrowth assay. Freshly isolated BALB/c keratinocytes contained approximately 0.1 ng/ml. Using a cDNA probe directed against NGF, we demonstrated the presence of a 1.3-kb NGF mRNA in both PAM 212 and BALB/c keratinocytes. Since ultraviolet radiation (UV) is a potentially important modulating factor for cytokines in skin, we examined the effect of UV on NGF mRNA expression. Although UV initially inhibited the expression of keratinocyte NGF mRNA (4 h), by 24 h an induction of NGF mRNA was seen. The NGF signal could also be induced by phorbol esters. Thus, keratinocytes synthesize and express NGF, and its expression is modulated by UVB and phorbol esters.

Serum factors that stimulate fatty acid oxidation: physiological specificity.

In the accompanying paper (Wice et al., 1986) we reported that serum from chickens contains small molecular weight compounds that stimulate long-chain fatty acid oxidation ten fold or more in HeLa cells. Here we show that this response is not limited to specific sera or to specific target cells. The specificity of the metabolic response to these factors was also investigated. They had no effect on the following major pathways of HeLa cell metabolism: 1) the oxidation of the medium-chain fatty acid, octanoic acid, 2) the rate of glycolysis of glucose, 3) the flux of glucose carbon through the oxidative arm of the pentose cycle, 4) the entry of pyruvate into the citrate cycle, 5) the oxidation of glutamine carbon, 6) the utilization rate of oxygen or 7) the rate of fatty acid synthesis. Furthermore, the increased oxidation of long-chain fatty acids was not a result of an increased uptake into the cells. Thus, the serum factors appear to be very specific for the oxidation of long-chain fatty acids for energy. Since carnitine also stimulates long-chain fatty acid oxidation in these cells, it seems likely that these compounds either facilitate the activity of carnitine or provide the same function--presumably the transport of long-chain fatty acid into and out of the mitochondria.

Serum factors that stimulate fatty acid oxidation: properties of factors.

Cultured heart muscle cells, but not HeLa cells, oxidize long-chain fatty acids in medium containing dialyzed serum. Addition of chicken serum dialysate (or non-dialized serum) stimulated palmitic acid oxidation by HeLa cells 10 to 20 fold. This serum activity was not eliminated by lipid extraction, ethanol or acid precipitation, alkaline phosphatase treatment, or autoclaving. About 80% was lost after any one of the following treatments: 6N HCl at 110 degrees C for 16 hr, pepsin, Dowex cation exchange at pH 3, or 1N KOH at 100 degrees C for 1 hr. Serum activity was separated into five or more peaks by gel filtration with Sephadex G-10. Each of these peak fractions was further purified by HPLC using a cyanopropyl-bonded resin. Carnitine, which is important for the transport of long-chain fatty acids into mitochondria for oxidation, also stimulated the oxidation of palmitate. However, these serum factors are not known precursors to carnitine since its immediate precursor 4-n-trimethylaminobutyrate, did not stimulate palmitate oxidation. Total carnitine, including that in acylcarnitine compounds, was approximately 15 microM in the chicken sera to give approximately 0.7 microM in the medium. Based on the fraction of total activity accountable by carnitine and fractional stability to acid, alkali, and pepsin, about 75% of the activity is from non-carnitine compounds. Only one of the factors appears to be carnitine or an acylcarnitine derivative. Several lines of evidence suggest that the other factors are peptide compounds.

Comparative energy metabolism in cultured heart muscle and HeLa cells.

The yields of energy from oxidation of fatty acids, glucose, and glutamine were compared in cultures of chick embryo heart muscle (heart) and HeLa cells. Aerobic energy production, as measured by oxygen utilization, was comparable in the two cell types. In media containing dialyzed sera, the rates of incorporation of fatty acids directly into lipids were similar in both cells and accounted for greater than 97% of fatty acid metabolism in HeLa cells. However, in heart cells only 45% ended in lipid, 42% in protein, and 13% was released as CO2; the latter two products probably reflect the oxidation of fatty acids to acetyl-coenzyme A (-CoA) and its subsequent metabolism in the citrate cycle. Increased serum concentration in the medium did not affect fatty acid metabolism in HeLa cultures, but resulted in greater oxidation by heart cells (greater than 100 times that by HeLa cells). The metabolisms of both glucose and glutamine were similar in heart and HeLa cells with greater than or equal to 60% of glucose carbon ending as medium lactate and only 3-5% converted to acetyl-CoA. About 25% of glutamine carbon ended as CO2 and increased utilizations with increasing serum concentrations was accountable in both cells by increased lactate from glucose and glutamate from glutamine. CO2 production (and energy) from glutamine was independent of glutamine concentration within a tenfold range of physiological concentrations. The yields of energy have been calculated. In 10% dialyzed calf serum, oxidation of glutamine carbon provided about half of the total energy in heart cells; glucose about 35-45%, with most coming from glycolysis; oxidation of fatty acid carbon provided only 5-10%. That greater than 90% of the aerobic energy comes from glutamine in both cells can account for the comparable rates of oxygen utilization. HeLa cells derived little or no energy from fatty acids.

Induction of an attachment and spreading on glass of Ehrlich ascites tumour cells by alpha-saturated polyprenols.

It has been observed that a dolichol, alpha dihydro-undecaprenol, induces phenotypic changes in Ehrlich ascites tumour [EAT] cells. In the presence of this dolichol in the medium. EAT cells attach to glass and spread on it but grow in an overlapping pattern. This spreading takes place after a lag of between 72-96 hours at continuous exposure to the dolichol. When cells, once induced to spread, are trypsinized, they can spread once more within 3-6 hours in the dolichol-free medium. It is suggested that dolichols, which act as lipid intermediates in protein glycosylation, may represent a class of compounds which by interference with the biosynthesis of plasma membrane constituents influence surface properties of EAT cells and induce spreading. The results presented in this paper support the view that sugar moieties of plasma membrane constituents play a role in controlling cell attachment, spreading, and intercellular communication.