Fulvia Fabi

Neuropeptide Y‐induced potentiation of noradrenergic vasoconstriction in the human saphenous vein: involvement of endothelium generated thromboxane

We investigated the potentiating effect of low concentrations of neuropeptide Y (NPY) on the vasoconstriction induced by transmural nerve stimulation (TNS) and noradrenaline (NA) in human saphenous veins. The effects of (i) endothelium removal; (ii) the addition of the NO pathway precursor L‐arginine; (iii) the ETA/ETB endothelin receptor antagonist Ro 47‐0203; (iv) the cyclo‐oxygenase inhibitor, indomethacin; (v) the selective thromboxane A2 (TxA2) receptor antagonists Bay u3405 and ifetroban, and (vi) the TxA2 synthase inhibitor, UK 38485, were studied in order to gain information about the mechanisms of NPY‐induced potentiation. Contractile response curves for TNS (0.5–8 Hz) and for exogenously administered NA (0.1–3 μM) were obtained in superfused saphenous vein rings. The contractions induced by both TNS and NA at all tested frequencies and concentrations, respectively, were significantly potentiated by 50 nM NPY in endothelium intact veins. Conversely, in endothelium‐denuded vessel rings the contractile‐response curves to TNS and NA overlapped both in the absence and presence of NPY, thus suggesting that a release of vasoactive substances from endothelial cells could account for the noradrenergic NPY‐induced potentiation. In vessels with intact endothelium, the potentiating action of NPY on TNS and NA was unaffected by the presence of high concentrations of the NO precursor L‐arginine (3–10 mM) or the non‐selective ETA/ETB endothelin receptor antagonist, Ro 47‐0203 (10 μM). These data indicate that the NPY‐induced effect does not involve either the endothelium‐derived vasodilator nitric oxide or the vasoconstrictor endothelin. Conversely, in the presence of the cyclo‐oxygenase inhibitor, indomethacin (30 μM), NPY failed to potentiate the vasoconstrictions produced by either nerve stimulation or by exogenous NA, thus providing evidence that arachidonic acid metabolites through the cyclo‐oxygenase pathway are mainly responsible for the potentiation evoked by NPY. When the TxA2 receptor antagonists, Bay u 3405 (1 μM) and ifetroban (1 μM) were added to the superfusing medium, NPY did not alter either the frequency‐ or the concentration‐response curves for either TNS or NA. Accordingly, both TNS‐ and NA‐induced contractions were not potentiated by NPY in the presence of the TxA2 synthase inhibitor, UK 38485 (10 μM). This clearly demonstrates the pivotal role of TxA2 in NPY‐induced potentiation. In superfused vein rings with endothelium, a subthreshold concentration (0.2 nM) of the TxA2 mimetic U 46619 potentiated both TNS‐ and NA‐induced vasoconstrictions. This potentiation was higher at low stimulation frequencies and low NA concentrations, and resembled that produced by NPY. Our results indicate that in the human saphenous vein NPY potentiates the contractions produced by sympathetic nerve stimulation acting at the postjunctional level, primarily on endothelial cells. In particular, the NPY‐induced release of a cyclo‐oxygenase metabolite, namely TxA2, may have a synergistic effect on the vasoconstriction induced by the noradrenergic mediator. Thus, such a mechanism may play a key role in the maintenance of the sympathetic tone of large human capacitance vessels.

Evidence for sympathetic neurotransmission through presynaptic N-type calcium channels in human saphenous vein.

1 The specific type(s) of voltage‐sensitive calcium channels (VSCCs) involved in sympathetic neurotransmission have not yet been characterized in human vascular tissues. We therefore examined the functional role of the N‐ and L‐type VSCCs in human saphenous veins. 2 Contractile response curves for transmural nerve stimulation (TNS) and for exogenously administered noradrenaline (NA) were obtained in superfused saphenous vein rings. The contractions induced by TNS, but not by NA, were inhibited by 1 μm tetrodotoxin and by 10 μm guanethidine. Both responses were substantially reduced by 1 μm phentolamine, indicating that the contractions evoked by TNS were mediated by endogenous NA released from noradrenergic nerves. 3 In the presence of 2 μm ω‐conotoxin GVIA (omega Conus Geographus toxin, fraction VI A; ω‐CgTx), a polypeptide with specific inhibitory activity on N‐ and L‐type calcium channels, the neurally evoked contractions were almost completely abolished. In contrast, the responses induced by exogenous NA were not affected by the neurotoxin, thus providing evidence of the exclusive presynaptic action of ω‐CgTx. 4 In the presence of the calcium antagonist verapamil (10 μm), which selectively blocks L‐type VSCCs, the contractions induced by both TNS and NA were diminished to the same extent, suggesting that the organic calcium blocker is active only at the postjunctional level. 5 It is concluded that N‐type calcium channels are the main pathway of calcium entry controlling the functional responses induced by activating sympathetic nerves; the role of L‐type channels appears to be limited to the postjunctional level, modulating smooth muscle contractions.

Mechanisms of pulmonary vasoconstriction and bronchoconstriction produced by PAF in the guinea-pig: role of platelets and cyclo-oxygenase metabolites.

1 The mechanisms of action of platelet activating factor (PAF) in the bronchial and cardiovascular systems have not yet been fully elucidated. In order to characterize better and to ascertain whether the effects of PAF in both these systems may be ascribed to the same mechanisms, we examined the actions of PAF in the heart‐lung preparation of guinea‐pig (HLP). The role of platelets and of cyclo‐oxygenase metabolites was investigated. 2 In HLPs perfused with autologous blood, bolus injections of PAF (4–32 ng) produced major effects at the pulmonary vascular and bronchial levels. Both dose‐related pulmonary vascular hypertension and bronchoconstriction produced by PAF were diminished to the same extent (46% and, respectively, 47%) when HLPs were perfused with a medium consisting of homologous red blood cells suspended in physiological solution containing 3.5% dextran (RBC). This suggests that the effects of PAF partially depend on the presence of formed elements. 3 When indomethacin (30 μm) was added to the perfusing blood, the dose‐response curve for the pulmonary hypertensive responses produced by PAF was strongly reduced (90%) in comparison to control preparations, whereas the bronchoconstrictor effects of PAF were only partially diminished (23%). These data constitute direct evidence that products of the cyclo‐oxygenase pathway exert a major role in the vascular, rather than in the bronchial actions of PAF. 4 In HLPs perfused with RBC containing indomethacin (30 μm), the pulmonary vascular hypertensive responses produced by PAF were almost completely abolished, thus indicating that cyclo‐oxygenase products from tissues are involved in these effects. Conversely, PAF administration continued to cause dose‐related bronchoconstrictor responses that were reduced only partially in comparison with HLPs perfused with RBC in the absence of the cyclo‐oxygenase inhibitor. This implies that PAF also has direct action on the bronchoconstriction evoked. 5 At the cardiac level, administration of PAF in HLPs perfused with blood caused a dose‐related increase in right atrial pressure accompanied by a decrease in left atrial pressure and cardiac output, which were completely suppressed or attenuated by the absence of formed elements and the addition of indomethacin. This suggests that the progressive heart impairment is secondary to the severe pulmonary hypertension induced by PAF. 6 The results of this study performed in the heart‐lung preparation of the guinea‐pig, which made it possible to simultaneously record cardiovascular and bronchial parameters, indicate that various components are involved in the responses produced by PAF. It is suggested that different mechanisms depending on the relative contribution of these components may account for the PAF‐induced effects at the pulmonary vascular and airway levels.

Nitric oxide-dependent and -independent modulation of sympathetic vasoconstriction in the human saphenous vein

The possible modulation by the endothelium of the contractile responses to sympathetic nerve stimulation was examined in isolated superfused human saphenous vein. Contractile response curves for transmural nerve stimulation and noradrenaline were higher in endothelium-denuded than in intact human saphenous vein rings. In vessels with endothelium, transmural nerve stimulation- and noradrenaline-induced contractions were unaffected by the cyclooxygenase inhibitor, indomethacin (10 microM), but were potentiated by the nitric oxide (NO) synthase inhibitor, L-N omega-nitro-L-arginine (L-NNA, 3 microM) even when combined with D-arginine (0.3 mM), but not with L-arginine (0.3 mM). As in the case of noradrenaline, contractile responses to 5-HT, but not to KCI, were enhanced by endothelium removal, L-NNA or L-NNA plus D-arginine, but were unaffected by L-NNA plus L-arginine. The guanylyl cyclase inhibitor, methylene blue (10 microM), potentiated both transmural nerve stimulation- and noradrenaline-induced contractions in endothelium intact rings, whereas it enhanced, although to a lesser degree, only the neurally evoked contractions in endothelium-denuded human saphenous vein. In the vessels without endothelium L-NNA failed to affect the vasoconstriction induced by both transmural nerve stimulation and noradrenaline. Our results suggest that at least two inhibitory factors are involved in modulating the sympathetic vasoconstriction in the human saphenous vein: (1) at a postjunctional level, NO, the release of which from endothelial cells is probably stimulated by the activation of specific receptors, and (2) at a prejunctional level, an unidentified vasodilator agent, which is unmasked by the removal of the endothelium layer and which is probably co-released along with noradrenaline, and which acts through the guanylyl cyclase pathway.

Nitric oxide (NO) modulation of PAF‐induced cardiopulmonary action: interaction between NO synthase and cyclo‐oxygenase‐2 pathways

To further investigate into the mechanisms of PAF‐induced cardiopulmonary actions, we examined the effects of the nitric oxide synthase (NOS) inhibitor L‐Nω‐nitro‐L‐arginine (L‐NNA), of the specific cyclooxygenase‐2 (COX‐2) inhibitor NS 398, and of the combined presence of both COX and NOS inhibitors on the PAF responses in the heart lung preparation of guinea‐pig (HLP). In HLPs perfused with homologous blood, dose‐response curves for the haemodynamic and bronchial effects of PAF (1 – 32 ng) were carried out in the absence or presence of L‐NNA (200 μM). L‐NNA caused an increase in the resting pulmonary arterial pressure (PAP) without affecting the other basal values, and strongly potentiated the bronchoconstriction and pulmonary hypertension elicited by PAF. An enhancement of the PAF‐induced actions on right atrial pressure (RAP) and cardiac output (CO) was also observed. All the effects of L‐NNA were antagonized by L‐arginine (2 mM). The presence of L‐NNA in the perfusing blood of HLPs failed to affect the pulmonary hypertensive and bronchoconstrictor responses induced by the thromboxane A2 mimetic U46619 (0.05 – 1.6 μg), 5‐hydroxytryptamine (0.1 – 1.6 μg), and histamine (0.1 – 1.6 μg), thus suggesting that these PAF secondary mediators are not responsible for the hyper‐responsiveness to PAF induced by L‐NNA. Blocking COX‐2 pathway with NS 398 (15 – 30 μM) did not alter the cardiopulmonary resting variables. However, a reduction of the PAF‐mediated pulmonary hypertension, but not of bronchoconstriction, was observed. When L‐NNA was added to the perfusing medium of HLPs pre‐treated with NS 398 or with indomethacin (15 μM), the basal PAP values were enhanced. However, in the combined presence of COX and NOS inhibitors, only a slight increase in the hypertensive responses to the highest doses of PAF was observed, whereas the PAF mediated actions at bronchial and cardiac level were unaffected. This study indicates that (i) the cardiopulmonary actions induced by PAF are specifically modulated by endogenous NO through the NOS pathway, and (ii) COX‐2 isoform is involved in the pulmonary hypertensive, but not bronchoconstrictor, effects of PAF. Furthermore, an interaction between PAF stimulated COX, particularly COX‐2, and NOS pathways appears to take a functional role at both bronchial and cardiovascular level.

Antisense versus proopiomelanocortin mRNA reduces vascular risk in a murine model of type-2 diabetes following stress exposure in early post-natal life

Mechanisms of vascular complications in type-2 diabetes patients and animal models are matter of debate. We previously demonstrated that a double-stress model applied to male mice during nursing period produces enduring hyperfunction of endogenous opioid and adrenocorticotropin (ACTH)-corticosteroid systems, accompanied by type-2 diabetes-like alterations in adult animals. Administration of the opioid receptor antagonist naloxone, or of an antisense oligodeoxynucleotide versus proopiomelanocortin mRNA, capable to block the pro-opiomelanocortin-derived peptides β-endorphin and ACTH, selectively prevent these alterations. Here, we investigated alterations produced by our stress model on aorta endothelium-dependent relaxation and contractile responses. Mice, stressed during nursing period, showed in the adulthood hormonal and metabolic type-2 diabetes-like alterations, including hyperglycemia, increased body weight and increased plasma ACTH and corticosterone levels. Ex vivo isolated aorta rings, gathered from stressed mice, were less sensitive to noradrenaline-induced contractions versus controls. This effect was blocked by nitric-oxide synthase-inhibitor l-N(G)-nitroarginine added to bath organ solution. Aorta rings relaxation caused by acetylcholine was enhanced in stressed mice versus controls, but following treatment with the nitric-oxide donor sodium nitroprusside, concentration-relaxation curves in aorta from stressed groups were similar to controls. Therefore, vascular response alterations to physiologic-pharmacologic stimuli were apparently due to nitric-oxide hyperfunction-dependent mechanisms. Aorta functional alterations, and plasma stress hormones enhancement, were prevented in mice stressed and treated with antisense oligodeoxinucleotide, addressed to reduce ACTH- and corticosteroid-mediated hyperfunction. This study demonstrates the key role of ACTH-corticosteroid axis hyperfunction for the triggering of vascular conditions in male adult rodents following postnatal stress in a type-2 diabetes model.

The role of the Istituto Superiore di Sanità as the competent authority for Phase I trials in the translation of advanced therapies

Advanced therapy medicinal products (ATMP) can offer new, effective therapeutic options for the treatment of severe illnesses, including cancer, neurodegenerative and cardiovascular diseases. Translation of advanced therapies to the clinic has been slow despite significant academic research from academia and foundations. The implementation of 2001/20 Directive in Italy established that the development of an ATMP should follow the GXP rules - good manufacturing practice (GMP) for production, good laboratory practice (GLP) for non clinical safety studies and good clinical practice (GCP) for clinical trials. The high costs of GCP application and the needs for GMP facilities are perceived as the most important bottlenecks for the development of ATMP. Here it is pointed out that a strategic cooperation between different actors (academia, industry and experts in regulatory issues) is strongly needed. In particular, it is highlighted that the Istituto Superiore di Sanità, as the competent authority for the authorization of Phase I clinical trials, has a specific responsibility in fostering the translation of safe and effective therapies for human diseases.