Chiara Mioni

Recombinant human erythropoietin improves angiogenesis and wound healing in experimental burn wounds

Objective:Erythropoietin interacts with vascular endothelial growth factor (VEGF) and stimulates endothelial cell mitosis and motility; thus it may be of importance in the complex phenomenon of wound healing. The purpose of this study was to investigate the effect of recombinant human erythropoietin (rHuEPO) on experimental burn wounds. Design:Randomized experiment. Setting:Research laboratory. Subjects:C57BL/6 male mice weighing 25–30 g. Interventions:Mice were immersed in 80°C water for 10 secs to achieve a deep-dermal second degree burn. Animals were randomized to receive either rHuEPO (400 units/kg/day for 14 days in 100 &mgr;L subcutaneously) or its vehicle alone (100 &mgr;l/day distilled water for 14 days subcutaneously). On day 14 the animals were killed. Burn areas were used for histologic examination, evaluation of neoangiogenesis by immunohistochemistry, and expression (Western blot) of the specific endothelial marker CD31 as well as quantification of microvessel density, measurement of VEGF wound content (enzyme-linked immunosorbent assay), expression (Western blot) of endothelial and inducible nitric oxide synthases, and determination of wound nitric oxide (NO) products. Measurements and Main Results:rHuEPO increased burn wound reepithelialization and reduced the time to final wound closure. These effects were completely abated by a passive immunization with specific antibodies against erythropoietin. rHuEPO improved healing of burn wound through increased epithelial proliferation, maturation of the extracellular matrix, and angiogenesis. The hematopoietic factor augmented neoangiogenesis as suggested by the marked increase in microvessel density and by the robust expression of the specific endothelial marker CD31. Furthermore, rHuEPO enhanced the wound content of VEGF caused a marked expression of endothelial and inducible nitric oxide synthases and increased wound content of nitric oxide products. Conclusions:Our study suggests that rHuEPO may be an effective therapeutic approach to improve clinical outcomes after thermal injury.

Activation of the cholinergic anti-inflammatory pathway reduces NF-kappab activation, blunts TNF-alpha production, and protects againts splanchic artery occlusion shock.

ABSTRACT The cholinergic anti-inflammatory pathway has not yet been studied in splanchnic artery occlusion (SAO) shock. We investigated whether electrical stimulation (STIM) of efferent vagus nerves suppresses the inflammatory cascade in SAO shock. Animals were subjected to clamping of the splanchnic arteries for 45 min, followed by reperfusion. This surgical procedure resulted in an irreversible state of shock (SAO shock). Sham-operated animals were used as controls. Two minutes before the start of reperfusion, rats were subjected to bilateral cervical vagotomy (VGX) or sham surgical procedures. Application of constant voltage pulses to the caudal vagus ends (STIM: 5 V, 2 ms, 6 Hz for 15 min, 5 min after the beginning of reperfusion) increased survival rate (VGX + SAO + Sham STIM = 0% at 4 h of reperfusion; VGX + SAO + STIM = 90% at 4 h of reperfusion), reverted the marked hypotension, inhibited I&kgr;B&agr; liver loss, blunted the augmented nuclear factor-&kgr;B activity, decreased hepatic tumor necrosis factor (TNF)-&agr; mRNA (VGX + SAO + Sham STIM = 1.0 ± 1.9 TNF-&agr;/glyceraldehyde-3-phosphate dehydrogenase ratio; VGX + SAO + STIM = 0.3 ± 0.2 TNF-&agr;/glyceraldehyde-3-phosphate dehydrogenase ratio), reduced plasma TNF-&agr; (VGX + SAO + Sham STIM = 118 ± 19 pg/mL; VGX + SAO + STIM = 39 ± 8 pg/mL), ameliorated leukopenia, and decreased leukocyte accumulation, as revealed by means of myeloperoxidase activity in the ileum (VGX + SAO + Sham STIM = 7.9 ± 1 U/g tissue; VGX + SAO + STIM = 3.1 ± 0.7 U/g tissue) and in the lung (VGX + SAO + Sham STIM = 8.0 ± 1.0 U/g tissue; VGX + SAO + STIM = 3.2 ± 0.6 U/g tissue). Chlorisondamine, a nicotinic receptor antagonist, abated the effects of vagal stimulation. Our results show a parasympathetic inhibition of nuclear factor-&kgr;B and TNF-&agr; in SAO shock.

Vagus nerve mediates the protective effects of melanocortins against cerebral and systemic damage after ischemic stroke.

A vagus nerve-mediated, efferent cholinergic protective pathway activated by melanocortins is operative in circulatory shock and myocardial ischemia. Moreover, melanocortins have neuroprotective effects against brain damage after ischemic stroke. Here we investigated cerebral and systemic pathophysiologic reactions to focal cerebral ischemia in rats induced by intrastriatal microinjection of endothelin-1, and the possible protective role of the melanocortin-activated vagal cholinergic pathway. In the striatum and liver of saline-treated control rats, the activation of extracellular signal-regulated kinases, c-jun N-terminal kinases, and caspase-3, the increase in tumor necrosis factor-α (TNF-α) concentration and DNA fragmentation, as well as the increase in TNF-α plasma levels, occurred 10 and 20 h after the ischemic insult suggesting an activation of inflammatory and apoptotic responses. Treatment with [Nle4, D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH; 3 or 9 h after stroke) suppressed the inflammatory and apoptotic cascades at central and peripheral level. Bilateral vagotomy and pharmacologic blockade of peripheral nicotinic acetylcholine receptors blunted the protective effect of NDP-α-MSH. The present results show that focal brain ischemia in rats causes significant effects not only in the brain, but also in the liver. Moreover, our data support the hypothesis that a protective, melanocortin-activated, vagal cholinergic pathway is likely operative in conditions of ischemic stroke.

Selective melanocortin MC4 receptor agonists reverse haemorrhagic shock and prevent multiple organ damage

In circulatory shock, melanocortins have life‐saving effects likely to be mediated by MC4 receptors. To gain direct insight into the role of melanocortin MC4 receptors in haemorrhagic shock, we investigated the effects of two novel selective MC4 receptor agonists.

Further evidence that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors

In rats subjected to myocardial ischemia/reperfusion, melanocortin peptides, including gamma(1)-melanocyte-stimulating hormone (gamma(1)-MSH), are able to exert a protective effect by stimulating brain melanocortin MC(3) receptors. A non-melanocortin receptor belonging to a group of receptors for Phe-Met-Arg-Phe-NH(2) (FMRFamide)-like peptides may be involved in some of the cardiovascular effects of the gamma-MSHs. FMRFamide-like peptides and gamma(1)-/gamma(2)-MSH share, among other things, the C-terminal Arg-Phe sequence, which seems to be essential for cardiovascular effects in normal animals. So we aimed to further investigate which receptor and which structure are involved in the protective effects of melanocortins in anesthetized rats subjected to myocardial ischemia by ligature of the left anterior descending coronary artery (5 min), followed by reperfusion. In saline-treated rats, reperfusion induced, within a few seconds, a high incidence of ventricular tachycardia and ventricular fibrillation, and a high percentage of death within the 5 min of observation period. Reperfusion was associated with a massive increase in free radical blood levels and with an abrupt and marked fall in systemic arterial pressure. The i.v. treatment (162 nmol/kg) during the ischemic period with the adrenocorticotropin fragment 1-24 [ACTH-(1-24): the reference protective melanocortin which binds all melanocortin receptors], as well as with both the melanocortin MC(3) receptor agonists gamma(2)-MSH and [D-Trp(8)]gamma(2)-MSH, reduced the incidence of ventricular tachycardia, ventricular fibrillation and death, the increase in free radical blood levels and the fall in arterial pressure. On the contrary, gamma(2)-MSH-(6-12) (a fragment unable to bind melanocortin receptors) was ineffective. Such protective effect was prevented by the melanocortin MC(3)/MC(4) receptor antagonist SHU 9119. In normal (i.e., not subjected to myocardial ischemia/reperfusion) rats, the same i.v. dose (162 nmol/kg) of gamma(2)-MSH, [D-Trp(8)]gamma(2)-MSH and gamma(2)-MSH-(6-12) provoked a prompt and transient increase in arterial pressure; on the other hand, ACTH-(1-24), which lacks the C-terminal Arg-Phe sequence, decreased arterial pressure, but only at higher doses. Heart rate of normal rats was not affected by any of the assayed peptides. The present data confirm and extend our previous findings that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC(3) receptors. Moreover, they further support the notion that, in normal rats, cardiovascular effects of gamma-MSHs are mediated by receptors for FMRFamide-like peptides, for whose activation, but not for that of melanocortin MC(3) receptors, the C-terminal Arg-Phe structure being relevant.

Adrenocorticotropin reverses vascular dysfunction and protects against splanchnic artery occlusion shock

Tumour necrosis factor (TNF‐α) is involved in the pathogenesis of splanchnic artery occlusion (SAO) shock. On the other hand, inhibition of TNF‐α is an important component of the mechanism of action of melanocortins in reversing haemorrhagic shock. We therefore investigated the effects of the melanocortin peptide ACTH‐(1–24) (adrenocorticotropin fragment 1–24) on the vascular failure induced by SAO shock. SAO‐shocked rats had a decreased survival rate (0% at 4 h of reperfusion, while sham‐shocked rats survived for more than 4 h), enhanced serum TNF‐α concentrations (755±81 U ml−1), decreased mean arterial blood pressure, leukopenia, and increased ileal leukocyte accumulation, as revealed by means of myeloperoxidase activity (MPO=9.4±1 U g−1 tissue). Moreover, aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE, 1 nM–10 μM) (Emax and ED50 in shocked rats=7.16 mN mg−1 tissue and 120 nM, respectively; Emax and ED50 in sham‐shocked rats=16.31 mN mg−1 tissue and 100 nM, respectively), reduced responsiveness to acetylcholine (ACh, 10 nM‐10 μM) (Emax and ED50 in shocked rats=30% relaxation and 520 nM, respectively; Emax and ED50 in sham‐shocked rats=82% relaxation and 510 nM, respectively) and increased staining for intercellular adhesion molecule‐1 (ICAM‐1). ACTH‐(1–24) [160 μg kg−1 intravenously (i.v.), 5 min after SAO] increased survival rate [SAO+ACTH‐(1–24)=80% at 4 h of reperfusion], reversed hypotension, reduced serum TNF‐α (55±13 U ml−1), ameliorated leukopenia, reduced ileal MPO (1.2±0.2 U g−1 tissue), restored the reactivity to PE, improved the responsiveness to ACh and blunted the enhanced immunostaining for ICAM‐1 in the aorta. Adrenalectomy only in part–but not significantly–reduced the ACTH‐induced shock reversal, the survival rate of SAO+ACTH‐(1–24) adrenalectomized rats being 60% at 4 h of reperfusion; and methylprednisolone (80 mg−1 i.v., 5 min after SAO) had a non‐significant effect (10% survival) at 4 h of reperfusion. The present data show that melanocortins are effective also in SAO shock, their effect being, at least in part, mediated by reduced production of TNF‐α. Furthermore, they demonstrate, for the first time, that this inhibition is responsible for the adrenocorticotropin‐induced reversal of vascular failure and leukocyte accumulation.

Lipid peroxidation inhibition reduces NF-κB activation and attenuates cerulein-induced pancreatitis

Increased lipid peroxidation, enhanced nuclear factor kappa-B (NF- s B) activation and augmented tumor necrosis factor- f (TNF- f ) production have been implicated in cerulein-induced pancreatitis. We investigated whether lipid peroxidation inhibition might reduce NF- s B activation and the inflammatory response in cerulein-induced pancreatitis. Male Sprague-Dawley rats of 230-250 g body weight received administration of cerulein (80 w g/kg s.c. for each of four injections at hourly intervals). A control group received four s.c. injections of 0.9% saline at hourly intervals. Animals were randomized to receive either raxofelast, an inhibitor of lipid peroxidation (20 mg/kg i.p. administered with the first cerulein injection) or its vehicle (1 ml/kg of a 10% DMSO/NaCl solution). All these rats were sacrificed 2 h after the last injection of either cerulein or its vehicle. Raxofelast administration (20 mg/kg i.p. with the first cerulein) significantly reduced malondialdehyde (MDA) levels, an index of lipid peroxidation (CER+DMSO=3.075 - 0.54 w mol/g; CER+raxofelast= 0.693 - 0.18 w mol/g; p <0.001 ), decreased myeloperoxidase (MPO) activity ( CER+DMSO=22.2 - 3.54 mU/g; CER+raxofelast=9.07 - 2.05 mU/g; p <0.01 ), increased glutathione levels (GSH) (CER+DMSO= 5.21 - 1.79 w mol/g; CER+raxofelast=15.71 - 2.14 w mol/g; p <0.001 ), and reduced acinar cell damage evaluated by means of histology and serum levels of both amylase ( CER+DMSO=4063 - 707.9 U/l; CER+raxofelast=1198 - 214.4 U/l; p <0.001 ), and lipase (CER+DMSO=1654 - 330 U/l; CER+raxofelast= 386 - 118.2 U/l; p <0.001 ), Furthermore, raxofelast reduced pancreatic NF- s B activation and the TNF- f mRNA levels and tissue content of mature protein in the pancreas. Indeed, lipid peroxidation inhibition might be considered a potential therapeutic approach to prevent the severe damage in acute pancreatitis.

Involvement of the central nervous system in the protective effect of melanocortins in myocardial ischaemia/reperfusion injury

Melanocortin peptides exert, in rats, a protective effect in myocardial ischaemia followed by reperfusion, or permanent occlusion of a coronary artery. Moreover, melanocortins have an anti-shock effect. Since the mechanism of the life-saving effect of these peptides in haemorrhagic shock includes an essential brain loop, we aimed to determine whether the central nervous system (CNS) is also involved in the protective effect against the outcome of short-term myocardial ischaemia followed by reperfusion. Ischaemia was produced in anaesthetized rats by ligature of the left anterior descending coronary artery for 5 min. Reperfusion-induced ventricular tachycardia (VT), ventricular fibrillation (VF) and lethality, and the time-course of arterial blood pressure over 5 min following reperfusion were evaluated. Groups of 8-14 rats were used. Intravenous (i.v.) injection of ACTH-(1-24) (0.16-0.48 mg/kg) during the ischaemic period dose dependently reduced the incidence of VT, VF and of lethality. In saline-treated rats, coronary reperfusion caused VT in 100% animals, VF in 86%, and death in 86%. The highest dose of ACTH-(1-24) (0.48 mg/kg) completely prevented the occurrence of VT, VF and death in all rats (P<0.005). Moreover, the melanocortin peptide prevented the fall in mean arterial pressure (MAP) occurring during reperfusion. Treatment with ACTH-(1-24) by the intracerebroventricular (i.c.v.) route also reduced the incidence of VT, VF and lethality, and prevented the fall in MAP in a dose dependent manner. Complete (100%) protection occurred with an i.c.v. dose (0.048 mg/kg) 10 times less than that needed by the i.v. route. The present data show that in the protective effect of melanocortin peptides against the injury after myocardial ischaemia/reperfusion, the i.c.v. route of administration is more effective than the i.v. route. They suggest that a CNS mechanism, not yet identified, may be involved.

Lipid peroxidation inhibition by raxofelast improves angiogenesis and wound healing in experimental burn wounds.

We investigated the effects of raxofelast, a lipid peroxidation inhibitor, in an experimental model of burn wounds. C57BL/6 male mice of 25-30 g were immersed in 80°C water for 10 seconds to achieve a partial-thickness scald burn. Animals received intraperitoneally either raxofelast (20 mg/kg/day for 14 days in 100 μL) or its vehicle alone (100 μL/day for 14 days). On day 14, burn areas were used for measuring conjugated dienes, reduced glutathione levels, histological damage, neoangiogenesis by immunohistochemistry and expression (Western blot) of the specific endothelial marker CD31 as well as quantification of microvessel density, VEGF wound content, endothelial and inducible nitric oxide synthase (eNOS and iNOS) expression and wound nitrite content. Raxofelast decreased tissue conjugated dienes (vehicle 6.1 ± 1.4 ΔABS/mg protein; raxofelast 3.7 ± 0.8 ΔABS/mg protein), prevented tissue glutathione consumption (vehicle 3.2 ± 0.9 μmol/g protein; raxofelast 6.7 ± 1.8 μmol/g protein), increased epithelial proliferation, extracellular matrix maturation, and augmented neoangiogenesis as suggested by the marked increase in microvessel density and by the robust expression of the specific endothelial marker CD31 (vehicle 9.4 ± 1.1 integrated intensity; raxofelast 14.8 ± 1.8 integrated intensity). Furthermore, raxofelast enhanced VEGF wound content (vehicle 1.4 ± 0.4 pg/mg protein; raxofelast 2.4 ± 0.6 pg/mg protein), caused a marked expression of eNOS (vehicle 16.1 ± 3 integrated intensity; raxofelast 26.2 ± 4 integrated intensity) and iNOS (vehicle 9.1 ± 1.8 integrated intensity; raxofelast 16.2 ± 3.5 integrated intensity) and increased wound nitrite content. Lipid peroxidation inhibition by raxofelast may be an effective therapeutic approach to improve clinical outcomes after thermal injury.

Cannabinoid CB1 receptor blockade enhances the protective effect of melanocortins in hemorrhagic shock in the rat

Activation of peripheral cannabinoid CB(1) receptors contributes to hemorrhagic hypotension, and endocannabinoids produced by macrophages and platelets may be mediators of this effect. A number of studies have provided evidence that functional links exist in the mechanisms of action of cannabinoids and opioid peptides; and opioids too play an important role in the pathophysiology of hemorrhagic hypotension and shock. On the other hand, melanocortin peptides, which are the main endogenous functional antagonists of opioid peptides, have an antishock effect in animals and humans. Thus, we investigated whether an interaction exists between endocannabinoids and the endogenous opioid/antiopioid system also in a condition of hemorrhagic shock and, particularly, whether the blockade of cannabinoid CB(1) receptors potentiates the antishock effect of melanocortins. Urethane-anesthetized rats were stepwise bled until mean arterial pressure decreased to, and stabilized at, 21-23 mm Hg. In this model of hemorrhagic shock, which caused the death of all control rats within 30 min after vehicle (tween 80, 5% in saline) injection, the intravenous (i.v.) bolus injection of the cannabinoid CB(1) receptor antagonist N-piperidino-5-[4-chlorophenyl]-1-[2,4 dichlorophenyl]-4-methyl-3-pyrazolecarboxamide (SR141716A) increased mean arterial pressure, pulse pressure, respiratory rate and survival rate in a dose-related manner (0.1-3 mg/kg), an almost complete recovery of mean arterial pressure, pulse pressure and respiratory rate, and 100% survival at the end of the observation period (2 h), occurring with the dose of 3 mg/kg. The melanocortin ACTH-(1-24) (adrenocorticotropin) also produced in a dose-related manner (0.02-0.16 mg/kg i.v.) a restoration of cardiovascular and respiratory functions, and increased survival rate, an almost complete recovery and 100% survival at the end of the observation period (2 h) occurring with the dose of 0.16 mg/kg. When a subactive dose of SR141716A (0.2 mg/kg; 30% survival) was associated with a subactive dose of ACTH-(1-24) (0.02 mg/kg; 12% survival), a complete reversal of the shock condition was obtained with 100% survival at the end of the 2-h observation period. The present results show that the concurrent inhibition of both endogenous opioid and cannabinoid systems produces a reversal of hemorrhagic shock more effective than that produced by the inhibition of either of them. These data suggest that functional interactions between endocannabinoids and opioid/antiopioid are at work also in the pathophysiology of hemorrhagic shock.