Biomedical subjects

Fabrizio Ledda

Publications and source records attributed to Fabrizio Ledda.

10 recordsLinked to original sources

PubMed2006

Role of conventional treatments on circulating and monocyte-derived dendritic cells in colorectal cancer.

We investigated the effects of metastasis, surgery and chemotherapy on both circulating dendritic cells (DCs) and monocyte-derived-DCs (MoDCs) in colorectal cancer (CRC) patients. Metastatic or nonmetastatic CRC patients had significantly reduced DC subsets compared to healthy subjects (p < 0.001). These cells were significantly higher in metastatic than in nonmetastatic patients. MoDCs were significantly lower in metastatic than healthy and nonmetastatic subjects (p < 0.001). Surgically treated patients had nearly one-half circulating DC subsets compared to healthy subjects (p < 0.001) while no difference was found between unoperated and healthy subjects. MoDCs obtained from tumor-bearing were significantly higher than in operated subjects. In both cases, MoDCs were significantly lower than in healthy subjects (p < 0.001). Circulating DCs and MoDCs were significantly lower in CRC patients, with or without chemotherapy, compared to healthy subjects (p < 0.001). Compared to untreated, chemotherapy-treated patients had 30% fewer DC subsets and lower MoDCs. Characterization of circulating DC subsets and MoDCs may elucidate CRC patients' immune system status.

Aged↗

PubMed2005

Oxytocin mediates the estrogen-dependent contractile activity of endothelin-1 in human and rabbit epididymis.

Epididymis is a sex steroid (androgen + estrogen)-sensitive duct provided with spontaneous motility, allowing sperm transport. We previously reported that the oxytocin (OT) receptor (OTR) mediates an estrogen-dependent increase in epididymal contractility. Because endothelin (ET)-1 also regulates epididymal motility, we tested its sex steroid dependence in a rabbit model. We demonstrated that estrogens up-regulate responsiveness to ET-1, which is reduced by blocking aromatase activity (letrozole, 2.5 mg/kg) or by triptorelin (2.9 mg/kg)-induced hypogonadism, whereas it is fully restored by estradiol valerate (3.3 mg/kg weekly) but not by testosterone enanthate (30 mg/kg weekly). However, changing sex steroid milieu did not affect either ET-1, its receptor gene, or protein expression. Two structurally distinct OTR-antagonists [(d(CH2)5(1), Tyr(Me)(2), Orn(8))-OT and atosiban] almost completely abolished ET-1 contractility, without competing for [125I]ET-1 binding, suggesting that OT/OTR partially mediates ET-1 action. Immunohistochemical studies in human and rabbit epididymis demonstrated that both OT and its synthesis-associated protein, neurophysin I, are expressed in the epithelial cells facing the muscular layer, suggesting local OT production. Quantitative RT-PCR demonstrated a high abundance of OT transcripts in human epididymis. OT transcript was also originally detected and partially sequenced in rabbit epididymis. To verify whether ET-1 regulates OT release, we used rabbit epididymal epithelial cell cultures. These cells expressed a high density of [125I]ET-1 binding sites and responded to ET-1 with a dose-dependent OT release. Hence, we propose that an ET-1-induced OT/OTR system activation underlies the estrogen-dependent hyperresponsiveness to ET-1. These local sources might promote the spontaneous motility necessary for sperm transport.

Animals↗

PubMed2005

Morphological and phenotypical characterization of human endothelial progenitor cells in an early stage of differentiation.

The exact phenotype and lineage of endothelial progenitor cells (EPCs) are still a matter of debate and different expansion protocols are used to obtain them. In this study, EPC expansion from peripheral blood mononuclear cells was analyzed within the first week of culture. Both the adherent and suspended cells, of which the latter usually discarded, were considered. We provide, for the first time, a systematic study of EPC phenotype and functional features within the first 3 days of culture. Moreover, within the 2nd day, both cellular fractions displayed a significant increase in endothelial marker expression which correlated with EPC properties.

Biomarkers↗

PubMed2005

Intracellular pathways triggered by the selective FLT-1-agonist placental growth factor in vascular smooth muscle cells exposed to hypoxia.

We have previously shown that hypoxia makes vascular smooth muscle cells (VSMCs) responsive to placental growth factor (PlGF) through the induction of functional fms-like tyrosine kinase (Flt-1) receptors. The aim of this study was to investigate the molecular mechanisms involved in the PlGF effects on proliferation and contraction of VSMCs previously exposed to hypoxia (3% O2). In cultured rat VSMCs exposed to hypoxia, PlGF increased the phosphorylation of protein kinase B (Akt), p38 and STAT3; activation of STAT3 was higher than that of other kinases. In agreement with this finding, the proliferation of hypoxia-treated VSMCs in response to PlGF was significantly impaired by the p38 and the phosphatidylinositol 3-kinase inhibitors SB202190 and LY294002, respectively, and was almost completely prevented by AG490, a janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor. Since hypoxia was able to reverse the vasorelaxant effect of PlGF into a vasoconstrictor response, the mechanism of this latter effect was also investigated. Significant Flt-1 activity was measured in isolated preparations from rat aorta exposed to hypoxia. Inhibitors of mitogen-activated protein kinase kinase, Akt and STAT3 induced a modest inhibition of the vasoconstrictor response to PlGF, while the p38 inhibitor SB202190 markedly impaired the PlGF-induced contractile response. These effects were selectively mediated by Flt-1 without any involvement of foetal liver kinase-1 receptors. These data are the first evidence that different intracellular pathways activated by Flt-1 receptor in VSMCs are involved in diverse biological effects of PlGF: while mitogen activated protein kinase kinase/extracellular signal regulated kinase(1/2) and JAK/STAT play a role in VSMC proliferation, p38 is involved in VSMC contraction. These findings may highlight the role of PlGF in vascular pathology.

Animals↗

PubMed2004

Androgens regulate phosphodiesterase type 5 expression and functional activity in corpora cavernosa.

By real-time RT-PCR and Western blot analysis, we found that phosphodiesterase type 5 (PDE5) mRNA and protein abundance was several fold higher in human male than in female reproductive tracts. The highest mRNA level (>1 x 10(7) molecules/microg total RNA) was detected in human corpora cavernosa (CC), where PDE5 protein was immunolocalized in both muscular and endothelial compartment. The possible role of androgens in regulating PDE5 expression was studied using a previously established rabbit model of hypogonadotropic hypogonadism. In this model, hypogonadism reduced, and testosterone (T) supplementation restored, CC PDE5 gene and protein expression. In addition, T supplementation completely rescued and even enhanced cyclic GMP conversion to metabolites, without changing IC(50) for sildenafil (IC(50) = 2.16 +/- 0.62 nm). In control CC strips, sildenafil dose-dependently increased relaxation induced by electrical field stimulation, with EC(50) = 3.42 +/- 1.7 nm. Hypogonadism reduced, and T increased, sildenafil effect on electrical field stimulation, again without changing their relative EC(50) values. CC sensitivity to the NO-donor NCX4040 was greater in hypogonadal rabbit strips than in control or T-treated counterparts. Moreover, sildenafil enhanced NCX4040 effect in eugonadal rabbit strips but not in hypogonadal ones. This suggests that androgens up-regulate PDE5 in rabbit penis. We also measured PDE5 gene expression and metabolic activity in human CC from male-to-female transsexual individuals, chronically treated with estrogens and cyproterone acetate. Comparing the observed values vs. eugonadal controls, PDE5 mRNA, protein, and functional activity were significantly reduced. In conclusion, we demonstrated, for the first time, that androgens positively regulate PDE5, thus providing a possible explanation about the highest abundance of this enzyme in male genital tract.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2003

Endogenous VEGF-A is responsible for mitogenic effects of MCP-1 on vascular smooth muscle cells.

Vessel wall remodeling is a complex phenomenon in which the loss of differentiation of vascular smooth muscle cells (VSMCs) occurs. We investigated the role of rat macrophage chemoattractant protein (MCP)-1 on rat VSMC proliferation and migration to identify the mechanism(s) involved in this kind of activity. Exposure to very low concentrations (1-100 pg/ml) of rat MCP-1 induced a significant proliferation of cultured rat VSMCs assessed as cell duplication by the counting of total cells after exposure to test substances. MCP-1 stimulated VSMC proliferation and migration in a two-dimensional lateral sheet migration of adherent cells in culture. Endogenous vascular endothelial growth factor-A (VEGF-A) was responsible for the mitogenic activity of MCP-1, because neutralizing anti-VEGF-A antibody inhibited cell proliferation in response to MCP-1. On the contrary, neutralizing anti-fibroblast growth factor-2 and anti-platelet-derived growth factor-bb antibodies did not affect VSMC proliferation induced by MCP-1. RT-PCR and Western blot analyses showed an increased expression of either mRNA or VEGF-A protein after MCP-1 activation (10-100 pg/ml), whereas no fms-like tyrosine kinase (Flt)-1 receptor upregulation was observed. Because we have previously demonstrated that hypoxia (3% O2) can enhance VSMC proliferation induced by VEGF-A through Flt-1 receptor upregulation, the effects of hypoxia on the response of VSMCs to MCP-1 were investigated. Severe hypoxia (3% O2) potentiated the growth-promoting effect of MCP-1, which was able to significantly induce cell proliferation even at a concentration as low as 0.1 pg/ml. These findings demonstrate that low concentrations of rat MCP-1 can directly promote rat VSMC proliferation and migration through the autocrine production of VEGF-A.

Animals↗

PubMed2003

Oxytocin receptor is expressed in the penis and mediates an estrogen-dependent smooth muscle contractility.

Oxytocin (OT) is released by the posterior pituitary during male orgasm and is supposed to participate in the ejaculatory process. We now report evidence demonstrating the presence of an OT receptor gene (real-time RT-PCR and Northern blot) and protein (immunohistochemistry, Western blot, and binding studies) expression in the rabbit and human corpus cavernosum (CC) and its possible involvement in postorgasmic penile detumescence. OT receptor is expressed in the penis at a concentration similar to that present in other portions of the male genital tract and mediates CC contractility. OT-induced CC contractility is clearly regulated by the changing sex steroid milieu. In fact, we found that in a rabbit model of hypogonadotropic hypogonadism (induced by a single administration of the long-acting GnRH agonist triptorelin pamoate, 2.9 mg/kg), OT responsiveness was strongly reduced and was completely restored by estradiol valerate (3.3 mg/kg weekly), but not by testosterone enanthate (30 mg/kg weekly). As we found that CC expresses both subtypes of estrogen receptors and P450 aromatase, we hypothesized a physiological role for endogenous estrogens in regulating OT responsiveness. We therefore treated adult rabbits with an aromatase inhibitor (letrozole, 2.5 mg/kg) or an antiestrogen (tamoxifen, 0.25 mg/kg) for 3 wk. Both treatments significantly reduced CC responsiveness to OT stimulation. In conclusion, these findings indicate that OT might participate in inducing postorgasmic penile flaccidity and suggest a new role for estrogens in the male: regulation of CC responsiveness to OT.

Animals↗

PubMed2002

Effect of hypoxia and endothelial loss on vascular smooth muscle cell responsiveness to VEGF-A: role of flt-1/VEGF-receptor-1.

OBJECTIVE: The influence of hypoxia and endothelial loss on the responsiveness of vascular smooth muscle cells (VSMCs) to vascular endothelial growth factor (VEGF-A) was tested. METHODS AND RESULTS: Exposure to hypoxia induced a potentiation of cultured cell proliferation in response to either the agonist for the VEGF receptor 1 (flt-1) placental growth factor (PlGF-1) or to VEGF-A. This effect was mediated by the mitogen activated protein kinase (MAPK) cascade, since it was inhibited by the MAPK kinase inhibitor PD98059 and by the farnesyl transferase inhibitor II. Accordingly, PlGF-1 activated extracellular signal-regulated kinase(1/2). In rat aortic rings deprived of endothelium and cultured in three-dimensional fibrin gels, an increased sprouting of tubular structures in response to VEGF-A was observed only under hypoxia. Studies on rat aorta preparations revealed an endothelium-dependent vasorelaxation in response to either VEGF-A or PlGF1, which was reversed to a contractile response in endothelium-deprived preparations exposed to hypoxia. Western blot and immunohistochemistry of endothelium-deprived preparations exposed to hypoxia showed flt-1 receptor expression in all medial cells. Conversely, flt-1 mRNA, of endothelium-deprived aortic preparations and of tubular structures, was unchanged by hypoxia. CONCLUSION: These findings demonstrate that experimental conditions mimicking pathological vascular injury can make VSMCs responsive to VEGF-A through the induction of functional flt-1 receptors.

Animals↗

PubMed2002

Inhibition of vascular smooth muscle cell growth by angiotensin type 2 receptor stimulation for in vitro organ culture model.

Recent data suggest that AT2 receptors in smooth muscle cells (VSMCs) of adult vessels may counterbalance AT1 receptor activity by inhibiting cell growth. The role of AT2 receptors in VSMC proliferation was investigated by using an in vitro organ culture model in which the sprouting of tubular structures was assessed. In preparations with endothelium, tubular growth was unaffected by angiotensin II (Ang II) but was inhibited by 50 +/- 9% by losartan and was increased by 110 +/- 15% by the AT2 antagonist PD123177. In endothelium-deprived preparations, growth inhibition (-49.1 +/- 0.5%) was observed when angiotensin II was added together with losartan 1 microM, whereas stimulation (59.8 +/- 14%) was induced by angiotensin II with 1 microM PD123177. In cultured VSMCs angiotensin II slightly promoted growth that was inhibited by losartan but was unaffected by PD123177. AT1a, AT1b, and AT2 mRNA expression was demonstrated in cells isolated from tubular structures grown from intact and endothelium-deprived rings, but only AT1a and AT1b mRNA was detected in cultured VSMCs. In conclusion, this paper proposes an in vitro organ culture model in which the expression of AT2 receptors in VSMCs is preserved and demonstrates AT2 receptor-mediated inhibition of VSMC proliferation in adult vessels.

Angiogenesis Inhibitors↗

PubMed2002

Estrogens, but not androgens, regulate expression and functional activity of oxytocin receptor in rabbit epididymis.

Previous binding and contractility studies indicate that oxytocin (OT) receptors are present in rabbit epididymis. To investigate the effect of changing endocrine milieu on OT responsiveness, we induced hypogonadism (hypo) in rabbits with a single administration of a long-acting GnRH analog, triptorelin, and we replaced hypogonadal rabbits with different sex steroids. After 2 months from triptorelin administration, testosterone (T) plasma levels were decreased and OT responsiveness abolished. Administration of T to hypo rabbits restored T plasma levels but not OT sensitivity. Because Western blot analysis indicated that both estrogen receptors and aromatase are expressed in the epididymis, we treated hypo rabbits with estradiol valerate (E2v). E2v not only completely restored OT responsiveness but also even amplified it. Accordingly, Northern and Western blot analysis indicated that both OT receptor gene and protein were strongly induced by E2v but not by T. Surprisingly, also the class I estrogen receptor antagonist, tamoxifen restored OT sensitivity in hypo rabbits. To verify whether endogenous estradiol is involved in the regulation of OT receptor responsiveness, we treated intact rabbits with an aromatase inhibitor, letrozole. Blocking aromatase activity almost completely abolished OT sensitivity. These findings suggest a new function of estrogens in the male: regulation of OT responsiveness in epididymis.

Androgens↗