Endothelial (dys)function: the target of physical exercise for prevention and treatment of cardiovascular disease.

AIM: Endothelial dysfunction has been recognized as the early event and the common feature of chronic disorders associated with increased risk for atherosclerotic heart diseases. While the beneficial effects of aerobic, moderate-intensity exercise on endothelial function are very well assessed, an intriguing doubt exists about the effects of long-term high-intensity physical activity. The aim of the present study was to compare recent findings of our group concerning homocysteine levels in athletes to available data in literature in order to clarify the meaning of such apparent metabolic paradox. METHODS: The studied population included 185 athletes: 180 healthy age and sex matched subjects served as control group. The assessed variables included homocysteine, folate, vitamin B12, total and HDL cholesterol, LDH, CPK and IL-6. Results were compared to available data in literature. RESULTS: The prevalence of hyperhomocysteinemia (>15 µmol/L) in athletes and controls was 55% and 15%, respectively. In the studied population, no correlation was found between homocysteine and all the other investigated variables. CONCLUSION: The present results suggest that intensive physical training could induce a pathological increase of homocysteine levels. With this regard, it has been suggested that the observed increases of cardio-vascular risk factors in athletes could represent an adaptative feature marker of muscle demand but would not actually lead to endothelial damage. This remains, however, a speculative hypothesis and further analysis are needed in order to clarify the clinical significance of those observations in order to better preserve the athletes immediate and future health.

Health risk screening in adolescents: a pilot study.

AIM: Even if youths are generally perceived to be healthy, adolescent years are associated with significant morbidity. Screening and counselling programmes seem to be cost-effective but adolescents prefer to rely on health care services for the treatment of diagnosed diseases or injuries rather than for preventive actions. Age oriented studies are needed for better understanding the health needs of adolescents in order to provide an adequate offer of preventive opportunities. METHODS: Eight hundred youths ranging from 13 to 18 years of age were recruited. Health status and risks were clustered into the following five categories: clinical assessment, substance use/abuse, nutritional habits, alcohol and tobacco consumption, physical status. Surprisingly, 33% of the youths were suggested to perform further clinical assessment and even more interestingly a significant number of them received a diagnosis of a symptomatic disorder for which he or she did not previously consider a medical visit to be necessary. RESULTS: As expected, alcohol consumption, tobacco smoking, drug use/abuse and sedentary habit represent the risky lifestyles commonly followed by adolescents. CONCLUSION: The present study confirms the importance of screening programs addressed to health issues and behavioural attitudes of adolescents even in light of the fact that they may underestimate even indicative symptoms.

Urinary and serum hormones profiles after testosterone enanthate administration in male hypogonadism: concerns on the detection of doping with testosterone in treated hypogonadal athletes.

OBJECTIVE: To describe serum and urinary hormones, androgens metabolites and testosterone/epitestosterone ratio profiles after testosterone administration in male hypogonadal volunteers, and to evaluate their possible usefulness in detecting doping with testosterone in treated hypogonadal athletes. DESIGN: Controlled open label design vs placebo; pharmacokinetic study. PARTICIPANTS: Ten male volunteers affected by severe hypogonadism (serum testosterone <2.31 ng/ml). INTERVENTIONS AND MAIN OUTCOME MEASURES: Serum and urinary parameters were evaluated, by radioimmunoassay and gas chromatography-mass spectrometry, before and at different time points for 7/3 weeks after a single administration of testosterone enanthate (250 mg) or placebo, respectively. RESULTS: As partially known, testosterone administration increased, with great individual variability, urinary concentrations of glucuronide testosterone, androsterone, etiocholanolone, 5alpha-androstane- 3alpha,17beta-diol, 5beta-androstane-3alpha,17beta-diol, testosterone/ epitestosterone and testosterone/LH ratios; and decreased epitestosterone and 5alpha-androstane-3beta,17beta-diol/5beta-androstane- 3alpha,17beta-diol ratio. Serum testosterone and dihydrotestosterone increased in all volunteers, and concentrations higher than the upper reference limits were observed in many volunteers until 2 weeks after testosterone administration. CONCLUSION: Whereas the observed prolonged hyperandrogenism partially limited data interpretation, the report ed characteristics of variation of urinary parameters might be used to suspect testosterone misuse in hypogonadal athletes treated with testosterone enanthate. In this sense, while the actual threshold for tes tos terone/epites tos ter one ratio was confirmed to be of reduced usefulness, we suggest a contemporary evaluation of whole urinary androgen metabolites profile and serum androgens, at specific time points after testosterone enanthate administration. Moreover, an adequate tailoring of treatment, to avoid transitory hyperandrogenism, is highly advisable. Further studies on strategies for detecting doping with testosterone in hypogonadal athletes are warranted.

Do aspartate and asparagine acute supplementation influence the onset of fatigue in intense exercise?

AIM: Oxaloacetic acid represents a fundamental intermediary in the metabolism of energy substrate. Asparagine and aspartate constitute precursor compounds of this substance. Therefore, they could affect tricarbossilic acids cycle. Besides, it was suggested that supplementation with aspartate and asparagine determines a muscular glycogen sparing during strenuous physical exercise, even if the real effectiveness remain controversial. The aim of the present pilot study was to evaluate the hypothesis that a supplementation with oxaloacetate precursors, precisely aspartate and asparagine, could improve sport performance during high intensity endurance exercise. METHODS: We recruited 15 male trained athletes, aged from 20 to 30 years (mean age: 24.13+/-3.87 years), practicing triathlon. We administered them placebo or aspartate (7 g) and asparagine (7 g) mixture, using a double blind technique, before performing an exhaustion stress test on cycloergometer carried out to 90% of each athlete's maximum oxygen consumption, previously determined. RESULTS: We evaluated lactatemia through earlobe punctures at the end of warming up, at the maximum effort and at recovery time (3 min, 5 min, 10 min, 15 min, 30 min). Furthermore, subjects were submitted to three blood samples from brachial artery in order to assess the glycemia (before the exercise, at the end of the exercise, and 30 min after the end of the exercise). CONCLUSION: The analysis of these parameters and the results of the ergometric tests after amino acids assumption indicate that acute supplementation with aspartate and asparagine do not significantly affect physical performance in athletes practicing high intensity exercises, and that acute administration of aspartate does not cause a sparing of muscle glycogen concentration.

Models of epithelial histogenesis.

Epithelial tissues emerge from coordinated sequences of cell renewal, specialization and assembly. Like corresponding immature tissues, adult epithelial tissues are provided by stem cells which are responsible for tissue homeostasis. Advances in epithelial histogenesis has permitted to clarify several aspects related to stem cell identification and dynamics and to understand how stem cells interact with their environment, the so-called stem cell niche. The development and maintenance of epithelial tissues involves epithelial-mesenchymal signalling pathways and cell-matrix interactions which control target nuclear factors and genes. The tooth germ is a prototype for such inductive tissue interactions and provides a powerful experimental system for the study of genetic pathways during development. Clonogenic epithelial cells isolated from developing as well mature epithelial tissues has been used to engineer epithelial tissue-equivalents, e.g. epidermal constructs, that are used in clinical practise and biomedical research. Information on molecular mechanisms which regulate epithelial histogenesis, including the role of specific growth/differentiation factors and cognate receptors, is essential to improve epithelial tissue engineering.

Stimulation of osteoblast growth by an electromagnetic field in a model of bone-like construct.

The histogenesis of bone tissue is strongly influenced by physical forces, including magnetic fields. Recent advances in tissue engineering has permitted the generation of three dimensional bone-like constructs. We have investigated the effects of electromagnetic stimulation on human osteoblast cells grown in a hydrophobic polyurethane scaffold. Bone-like constructs were stimulated by pulsed electromagnetic fields in a bioreactor. Proliferation, bone protein expression and calcified matrix production by osteoblasts were measured using histochemical methods. In stimulated cultures, the number of cells was significantly higher compared to static (control) cultures. In both stimulated and control cultures, cells were immunoreactive to osteoblast markers, including type-I collagen, osteocalcin and osteopontin, thus suggesting that the expression of bone-related markers was maintained throughout the in vitro experiments. Morphometric analysis of von Kossa-stained sections revealed that stimulation with electromagnetic field significantly increased matrix calcification. The data lend support to the view that the application of a magnetic field can be used to stimulate cell growth in bone-like constructs in vitro. This finding may be of interest for the production of biomaterials designed for clinical applications.

Differential distribution of elastic tissue in human natural skin and tissue-engineered skin.

Tissue-engineered skins (TES), manufactured by epidermal and dermal equivalents, are now being used in biological, pharmacotoxicological and clinical applications. It is thus interesting to know to what extent artificial organs are similar to natural counterparts. Elastic fibres are important constituents of the extracellular matrix of natural skin (NS). The aim of our study was to investigate the possible occurrence and distribution of elastic tissue in a model of human TES using different histochemical techniques, including classical Orcein and Fuchsin-Resorcin methods and immunohistochemistry, at both light and electron microscopical levels. Immunoperoxidase and high resolution immunogold methods were used. In NS, classical staining techniques and elastin-immunohistochemistry revealed a well-organized network of elastic fibres. High resolution immunocytochemistry revealed an intense labelling in the amorphous component of elastic fibres. Fibres of different diameters were immunostained. In TES, no stained elastic fibres were observed using classical staining techniques, and the interpretation of immunoperoxidase observations was not clear-cut. In contrast, immunogold staining at the electron microscopical level provided specific labelling of elastin-like immunoreactive material in the dermal equivalent. However, ultrastructural immunocytochemistry revealed that elastic tissue organization in TES was poor compared to that in NS. This study demonstrates that elastic fibres are a component of the extracellular matrix in this model of TES and suggests that fibroblasts of the dermal equivalent are engaged in matrix secretion. Nevertheless, the level of extracellular matrix organization in TES is low compared to NS. Moreover, this study also suggests that different models of bilayered TES may differ with respect to extracellular matrix organization. These aspects should be considered when TES is used in biological and pharmacotoxicological studies. A better understanding of the factors influencing extracellular matrix formation in TES is necessary to achieve further development of skin generation in vitro.

Cell proliferation and differentiation in a model of human skin equivalent.

Recent advances in culturing technology has permitted the production of organotypic models that may be referred to as human skin equivalents (HSE). We have studied histochemical, ultrastructural, and kinetic aspects of an HSE composed by an epidermal equivalent and a dermal equivalent separated by a basement membrane. Only keratinocytes and fibroblasts were present in the epidermal and dermal equivalents, respectively; cells of other lineages were lacking. Keratinocyte stratification and differentiation seemed similar to natural skin. Evidence is shown that such an HSE may also release growth factors such as vascular endothelial growth factor that are believed to play a role in skin grafting. The distribution of cycling cells as well as the values of the growth fraction are comparable to those observed in natural skin. Although the absence of several cells populations that reside in natural skin is a remarkable feature of this HSE, the high levels of tissue organization and cell differentiation lead us to believe that such an HSE may be considered a candidate substitute of human skin in biological, pharmacologic, and clinical applications.

Cell kinetics in a model of artificial skin. An immunohistochemical and flow cytometric analysis.

Bioengineered organs raised in vitro are candidate substitutes for natural organs in biological, pharmacological and clinical applications. We have studied cell kinetics in a human skin equivalent (HSE) using a combined immunohistochemical and flow cytometric approach. Morphological analysis has shown that, relative to unstimulated natural skin, cell proliferation mainly occurs in the basal layer of the epidermal equivalent. Immunohistochemical and flow cytometric measurements of the growth fraction suggested a cell turnover comparable to that of natural skin. Immunohistochemical labelling indices matched well with flow cytometric data. These observations are consistent with morphological and histochemical data demonstrating normal cell differentiation and tissue architecture in HSE and suggest that such HSE may be a usefull substitute for human skin.

Morphological aspects of an artificial skin.

Culture technology have permitted the generation of an artificial skin using human neonatal stem cells. A major advantage of this model is that epithelial-mesenchymal interactions are maintained. We have studied some morphological aspects concerning tissue organisation and cell differentiation using immunohistochemistry and electron microscopy. The epidermal equivalent was composed by a stratified and keratinized epithelium. The cells of this epithelium were cytokeratin-positive and disposed in different layers corresponding to natural skin, i.e. basal, spinous, granular and keratinized layers. The ultrastructural aspects concerning keratinocyte differentiation were comparable to natural epidermis. The dermal equivalent was composed by a loose connective tissue. The cells of the dermal equivalent were vimentin-positive and belonged to the fibroblast lineage. Although poorly developed, a basement membrane was detectable in the dermo-epidermal junction. The organ architecture and the high level of cell differentiation suggest that this bioengineered skin might be considered a useful substitute for natural skin in clinical, biological and pharmacological applications.

Immunohistochemical localization of lipoperoxidation products in normal human placenta.

4-Hydroxynonenal (4-HNE) is a major propagation product of lipid peroxidation that is supposed to be responsible for some of the effects associated with oxidative stress in tissues. We have investigated the possible occurrence and distribution of 4-HNE-immunoreactivity in human normal placenta using immunocytochemistry. Specific immunostaining was observed in cytotrophoblast cells, syncytiotrophoblast, some cells of the villous mesenchyme and some endothelial cells of first trimester and term placentae. The detection of 4-HNE-immunoreactivity in placenta raises the question whether lipoperoxidation products are produced locally in placental cells or represent exogenous products that derive from maternal blood flow. Since trophoblastic cells and villous macrophages are provided by a scavenger receptor, it is conceivable that these cells may play a protective role with regard to the diffusion of lipoperoxidation products from the mother to the embryo. However, since a significant degree of lipid oxidative modification does not take place in plasma, it is presumed that 4-HNE is a local product of placental metabolism. In line with this hypothesis, it is proposed that maternal low density lipoproteins, which are the major source of cholesterol for placental steroid synthesis, might be oxidized by villous cells during their traversal through the villous wall.

Cell proliferation in developing human dental pulp. A combined flow cytometric and immunohistochemical study.

Information concerning cell proliferation and differentiation in dental pulp may be important to understand tooth response to exogenous stimuli. Since few data concerning human dental pulp are available, we have investigated the growth fraction and the localization of proliferating cells in pulp tissue of third molars of young adult human males and females, using flow cytometry and immunohistochemistry. Flow cytometric analysis demonstrates a low proliferative activity of pulp tissue that appears to be confined to radicular pulp, as revealed by immunohistochemical detection of proliferating cells. No polyploid or aneuploid cell populations could be identified, and G2-blocked cells, if any, represented a negligible cell population. Odontoblasts, cells of the sub-odontoblastic layer, and cells of coronal pulp were found to be not proliferating under normal conditions. These data provide the basis for future investigations on proliferative activity and regenerative potentiality of human pulp cells in experimental and clinical situations.

Localization of proliferating cell nuclear antigen-immunoreactivity in human dental pulp and gingiva.

The proliferating cell nuclear antigen (PCNA) is regarded as an operational marker of proliferating cells. We have used PC10 monoclonal antibody to PCNA to reveal proliferation sites in human dental pulp and gingiva. Intense PCNA-immunoreactivity was observed in the basal layer of the gingiva lining epithelium and within some cells of the underlying connective tissues, including some endothelial and perivascular cells. PCNA-reactive cells were scattered throughout the pulp tissue, but were particularly numerous in the peripheral part. Since PCNA is an endogenous cell cycle-related molecule, we propose that PCNA-antibodies may represent useful for studying cell kinetics in human oral tissues in normal as well as pathological situations, such as tumours, wound healing and inflammation.

Ultrastructural Localization of elastin-like immunoreactivity in the extracellular matrix around human small lymphatic vessels.

The distribution of elastin-like immunoreactivity around small lymphatic vessels was investigated in three different human tissues (skin, heart and dental pulp) using high resolution immunocytochemistry. Quantitative assessment of the immunogold reaction was performed with an image analysis system. Intense and moderate elastin-immuno-reactivity was detected in the extracellular matrix around small lymphatic vessels of the skin and heart, respectively. By contrast, absence of immunostaining was observed around lymphatic vessels in the dental pulp. Although the staining was mostly detectable on the non-fibrillar amorphous component of the extracellular matrix, some microfibrils were also immunostained in close proximity to the lymphatic vessel wall. These findings support the concept that small lymphatic vessels may be heterogeneous with respect to the composition of the extracellular matrix around their wall. The observation that it is possible to observe small lymphatic vessels displaying low or no elastin-immunoreactivity in the adjoining matrix militates against the hypothesis that elastic fibers play a pivotal role in the mechanisms that regulate the function of small lymphatic vessels.

Catecholamines in human dental pulp. A combined immunohistochemical and chromatographic study.

Pharmacological studies have suggested that nerve-released catecholamines may play an important role in the regulation of vascular tone and in the modulation of sensory nerve activity in animal teeth. We have used tyrosine hydroxylase-immunohistochemistry to detect catecholamine-producing cells in human dental pulp and high performance liquid chromatography to identify and quantitate catecholamines in this tissue. Tyrosine hydroxylase-immunoreactivity was confined to a sub-population of nerve fibres that were mainly localized around blood vessels. Considerable concentrations of norepinephrine (17.8 +/- 3.75 pg/mg tissue) and much lower concentrations of dopamine and epinephrine (0.27 +/- 0.10 and 0.19 +/- 0.11 pg/mg, respectively) were measured in all samples examined. It is suggested that catecholamines in human dental pulp are exclusively contained in nervous structures that are mainly associated with blood vessels and that norepinephrine is the candidate neurotransmitter of these nerve fibres. These data provide the basis to further studies addressed to clarify the possible functions of catecholamines in human dental pulp during physiological as well as inflammatory situations.

Detection of bromo-deoxyuridine- and proliferating cell nuclear antigen-immunoreactivities in tooth germ.

The development of antibodies to cell cycle-related antigens provides the basis for immunochemical studies on cell kinetics. Bromo-deoxyuridine (BrdU) incorporated by S-phase traversing cells is an exogenous marker of replicating cells, whereas proliferating cell nuclear antigen (PCNA) is an endogenous marker of replicating cells. We have applied monoclonal antibodies to BrdU and PCNA to study cell kinetics in tooth germ by immunohistochemistry and flow cytometry. BrdU-antibody reacted only with S phase-traversing cells in pulse-labelling experiments, whereas PCNA-antibody reacted with G1, S and G2-M phases traversing cells. Although the number of PCNA-positive cells largely exceeded the number of BrdU-labelled cells, the pattern distribution of immunoreactive cells was similar using BrdU- and PCNA-antibodies as revealed by immunohistochemistry. The use of PCNA-antibody allowed the detection of proliferating cells also in human tooth germ. It is suggested that combined identification of BrdU and PCNA on one side and growth factors, oncoproteins or differentiation markers on the other side may constitute a useful approach to understand the mechanisms of cell differentiation in tooth germ.

Occurrence and distribution of matrix metalloproteinase-2-immunoreactivity in human embryonic tissues.

Tissue development and structure is controlled by dynamic and interactive relationships between cells and the extra-cellular matrix (ECM) which they secrete. We have investigated the occurrence and distribution of metalloproteinase-2 (MMP-2), an enzyme involved in the catabolism of ECM components, in human embryonic tissues by immunocytochemistry. Cells displaying MMP-2 immunoreactivity showed a widespread distribution in human embryonic tissues and organs. Cytoplasmic staining was detected in cells deriving from all three embryonic layers. Although further studies are needed to clarify the possible substrates of MMP-2 in developing tissues, these morphological data lend support to the hypothesis that ECM remodelling and degradation may represent a physiological counterpart of ECM deposition that occur during development.

Ultrastructural immunolocalization of enamel matrix proteins during early stages of ameloblast differentiation.

Enamel matrix proteins (EMP) represent specific molecular markers of ameloblast secretion. In order to study early differentiation stages of the cells of the inner enamel epithelium, we have investigated the ultrastructural localization of EMP-immunoreactivity in rat tooth germ. Pre-secretory stages of ameloblast differentiation were identified by the absence of EMP-immunoreactivity within epithelial cells as well as adjoining extra-cellular matrix. During subsequent secretory stages EMP-like immunoreactive material could be detected both within epithelial cells as well as within the adjoining extra-cellular matrix. The intensity of the immunoreactivity increased while advancing with the differentiation of epithelial cells. Intracellularly, EMP-immunoreactivity was detectable in cytoplasmic compartments involved in exocrine secretion pathway. During the early secretory stage, EMP-immunoreactive material was also detectable in the basement membrane of the epithelial-mesenchymal interface and within the pre-dentine, close to odontoblast plasma membranes and processes. It is thus suggested that EMP may cross the basement membrane between epithelial and mesenchymal cells. Our study suggests that this aspect might be important in molecular mechanisms that regulate epithelial-mesenchymal interactions during odontogenesis.

Expression of c-erbB-2 proto-oncogene-encoded protein (p185erbB-2) in functional rat ameloblasts.

The c-erbB-2 proto-oncogene encodes a transmembrane glycoprotein with tyrosine kinase activity, p185erbB-2, that has been previously localized in some developing and mature epithelia. The possible occurrence of p185 in the inner enamel epithelium of rat tooth germ was here investigated immunocytochemically. Postmitotic functional ameloblasts displayed intense p185-immunoreactivity, thus suggesting that c-erbB-2 proto-oncogene is active during odontogenesis. The expression of this gene in differentiated and functional cells of the enamel organ suggests that its role is not restricted to mitotic events but may also be important in signalling pathways related to other cell activities.

Endothelin-induced vasoconstriction in rabbit nasal mucosa.

Nasal blood flow is finely regulated by local release of neurotransmitters, neuropeptides and other bioactive molecules acting via paracrine mechanisms. We have investigated the effects of endothelin-1 (ET-1), a potent vasoconstrictor peptide, on the blood perfusion of rabbit nasal mucosa by laser Doppler flowmetry. After injection with ET-1, a potent and prolonged nasal vasoconstriction was observed. ET-immunoreactivity has previously been detected in nasal tissues and it is therefore suggested that ET-1 may participate in the regulation of nasal blood flow via paracrine mechanisms.