Lorenzo Cinci

Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodelling.

In the post‐infarcted heart, grafting of precursor cells may partially restore heart function but the improvement is modest and the mechanisms involved remain to be elucidated. Here, we explored this issue by transplanting C2C12 myoblasts, genetically engineered to express enhanced green fluorescent protein (eGFP) or eGFP and the cardiotropic hormone relaxin (RLX) through coronary venous route to swine with experimental chronic myocardial infarction. The rationale was to deliver constant, biologically effective levels of RLX at the site of cell engraftment. One month after engraftment, histological analysis showed that C2C12 myoblasts selectively settled in the ischaemic scar and were located around blood vessels showing an activated endothelium (ICAM‐1‐,VCAM‐positive). C2C12 myoblasts did not trans‐differentiate towards a cardiac phenotype, but did induce extracellular matrix remodelling by the secretion of matrix metalloproteases (MMP) and increase microvessel density through the expression of vascular endothelial growth factor (VEGF). Relaxin‐producing C2C12 myoblasts displayed greater efficacy to engraft the post‐ischaemic scar and to induce extracellular matrix re‐modelling and angiogenesis as compared with the control cells. By echocardio‐graphy, C2C12‐engrafted swine showed improved heart contractility compared with the ungrafted controls, especially those producing RLX. We suggest that the beneficial effects of myoblast grafting on cardiac function are primarily dependent on the paracrine effects of transplanted cells on extracellular matrix remodelling and vascularization. The combined treatment with myoblast transplantation and local RLX production may be helpful in preventing deleterious cardiac remodelling and may hold therapeutic possibility for post‐infarcted patients.

Ceramide: A Key Signaling Molecule in a Guinea Pig Model of Allergic Asthmatic Response and Airway Inflammation

Although mechanisms involved in the pathogenesis of asthma remain unclear, roles for oxidative/nitrosative stress, epithelial cell apoptosis, and airway inflammation have been documented. Ceramide is a sphingolipid with potent proinflammatory and proapoptotic properties. This study aimed at determining whether increased formation of ceramide contributes to the development of airway inflammation and hyper-responsiveness, using a well characterized in vivo model of allergic asthmatic response and airway inflammation in ovalbumin-sensitized guinea pigs. Aerosol administration of ovalbumin increased ceramide levels and ceramide synthase activity in the airway epithelium associated with respiratory abnormalities, such as cough, dyspnea, and severe bronchoconstriction. These abnormalities correlated with nitrotyrosine formation in the airway epithelium and oxidative/nitrosative stress, epithelial cell apoptosis, and airway inflammation evident by the infiltration of neutrophils and eosinophils in lung tissues, mast cell degranulation, and release of prostaglandin D2 and proinflammatory cytokines. Inhibition of de novo ceramide synthesis with the competitive and reversible inhibitor of ceramide synthase fumonisin B1 (0.25, 0.5 and 1 mg/kg b.wt.), given i.p. daily for 4 days before allergen challenge, attenuated nitrotyrosine formation and oxidative/nitrosative stress, epithelial cell apoptosis, and airway inflammation while improving the respiratory and histopathological abnormalities. These results implicate ceramide in the development of allergic asthmatic response and airway inflammation. Strategies aimed at reducing the levels of ceramide and downstream events should yield promising novel anti-asthmatic agents.

3‐Iodothyronamine: a modulator of the hypothalamus‐pancreas‐thyroid axes in mice

BACKGROUND AND PURPOSE Preclinical pharmacology of 3‐iodothyronamine (T1AM), an endogenous derivative of thyroid hormones, indicates that it is a rapid modulator of rodent metabolism and behaviour. Since T1AM undergoes rapid enzymatic degradation, particularly by MAO, we hypothesized that the effects of T1AM might be altered by inhibition of MAO.

α-Conotoxin RgIA protects against the development of nerve injury-induced chronic pain and prevents both neuronal and glial derangement

&NA; &agr;‐Conotoxin RgIA prevents the development of chronic pain, halts progression of damage in injured nerves, and limits downstream pathological alterations in dorsal root ganglion and spinal cord. &NA; Neuropathic pain affects millions of people worldwide, causing substantial disability and greatly impairing quality of life. Commonly used analgesics or antihyperalgesic compounds are generally characterized by limited therapeutic outcomes. Thus, there is a compelling need for novel therapeutic strategies able to prevent nervous tissue alterations responsible for chronic pain. The &agr;9&agr;10 nicotinic acetylcholine receptor antagonist &agr;‐conotoxin RgIA (RgIA), a peptide isolated from the venom of a carnivorous cone snail, induces relief in both acute and chronic pain models. To evaluate potential disease‐modifying effects of RgIA, the compound was given to rats following chronic constriction injury (CCI) of the sciatic nerve. Two or 10 nmol RgIA injected intramuscularly once a day for 14 days reduced the painful response to suprathreshold stimulation, increased pain threshold to nonnoxious stimuli, and normalized alterations in hind limb weight bearing. Histological analysis of the sciatic nerve revealed that RgIA prevented CCI‐induced decreases of axonal compactness and diameter, loss of myelin sheath, and decreases in the fiber number. Moreover, RgIA significantly reduced edema and inflammatory infiltrate, including a decrease of CD86+ macrophages. In L4‐L5 dorsal root ganglia, RgIA prevented morphometric changes and reduced the inflammatory infiltrate consistent with a disease‐modifying effect. In the dorsal horn of the spinal cord, RgIA prevented CCI‐induced activation of microglia and astrocytes. These data suggest that RgIA‐like compounds may represent a novel class of therapeutics for neuropathic pain that protects peripheral nervous tissues as well as prevents central maladaptive plasticity by inhibiting glial cell activation.

Functional and histopathological improvement of the post-infarcted rat heart upon myoblast cell grafting and relaxin therapy

Although the myocardium contains progenitor cells potentially capable of regenerating tissue upon lethal ischaemic injury, their actual role in post‐infarction heart healing is negligible. Therefore, transplantation of extra‐cardiac stem cells is a promising therapeutic approach for post‐infarction heart dysfunction. Paracrine cardiotropic factors released by the grafted cells, such as the cardiotropic hormone relaxin (RLX), may beneficially influence remodelling of recipient hearts. The current study was designed to address whether grafting of mouse C2C12 myoblasts, genetically engineered to express green fluorescent protein (C2C12/GFP) or GFP and RLX (C2C12/RLX), are capable of improving long‐term heart remodelling in a rat model of surgically induced chronic myocardial infarction. One month after myocardial infarction, rats were treated with either culture medium (controls), or C2C12/GFP cells, or C2C12/RLX cells plus exogenous RLX, or exogenous RLX alone. The therapeutic effects were monitored for 2 further months. Cell transplantation and exogenous RLX improved the main echocardiographic parameters of cardiac function, increased myocardial viability (assessed by positron emission tomography), decreased cardiac sclerosis and myocardial cell apoptosis and increased microvascular density in the post‐infarction scar tissue. These effects were maximal upon treatment with C2C12/RLX plus exogenous RLX. These functional and histopathological findings provide further experimental evidence that myoblast cell grafting can improve myocardial performance and survival during post‐infarction heart remodelling and dysfunction. Further, this study provides a proof‐of‐principle to the novel concept that genetically engineered grafted cells can be effectively employed as cell‐based vehicles for the local delivery of therapeutic cardiotropic substances, such as RLX, capable of improving adverse heart remodelling.

Oxaliplatin Neurotoxicity Involves Peroxisome Alterations. PPARγ Agonism as Preventive Pharmacological Approach

The development of neuropathic syndromes is an important, dose limiting side effect of anticancer agents like platinum derivates, taxanes and vinca alkaloids. The causes of neurotoxicity are still unclear but the impairment of the oxidative equilibrium is strictly related to pain. Two intracellular organelles, mitochondria and peroxisomes cooperate to the maintaining of the redox cellular state. Whereas a relationship between chemotherapy-dependent mitochondrial alteration and neuropathy has been established, the role of peroxisome is poor explored. In order to study the mechanisms of oxaliplatin-induced neurotoxicity, peroxisomal involvement was evaluated in vitro and in vivo. In primary rat astrocyte cell culture, oxaliplatin (10 µM for 48 h or 1 µM for 5 days) increased the number of peroxisomes, nevertheless expression and functionality of catalase, the most important antioxidant defense enzyme in mammalian peroxisomes, were significantly reduced. Five day incubation with the selective Peroxisome Proliferator Activated Receptor-γ (PPAR-γ) antagonist G3335 (30 µM) induced a similar peroxisomal impairment suggesting a relationship between PPARγ signaling and oxaliplatin neurotoxicity. The PPARγ agonist rosiglitazone (10 µM) reduced the harmful effects induced both by G3335 and oxaliplatin. In vivo, in a rat model of oxaliplatin induced neuropathy, a repeated treatment with rosiglitazone (3 and 10 mg kg−1 per os) significantly reduced neuropathic pain evoked by noxious (Paw pressure test) and non-noxious (Cold plate test) stimuli. The behavioral effect paralleled with the prevention of catalase impairment induced by oxaliplatin in dorsal root ganglia. In the spinal cord, catalase protection was showed by the lower rosiglitazone dosage without effect on the astrocyte density increase induced by oxaliplatin. Rosiglitazone did not alter the oxaliplatin-induced mortality of the human colon cancer cell line HT-29. These results highlight the role of peroxisomes in oxaliplatin-dependent nervous damage and suggest PPARγ stimulation as a candidate to counteract oxaliplatin neurotoxicity.

A selective antagonist of histamine H4 receptors prevents antigen‐induced airway inflammation and bronchoconstriction in guinea pigs: involvement of lipocortin‐1

Among the pathogenic mechanisms of asthma, a role for oxidative/nitrosative stress has been well documented. Recent evidence suggests that histamine H4 receptors play a modulatory role in allergic inflammation. Here we report the effects of compound JNJ 7777120 (JNJ), a selective H4 receptor antagonist, on antigen‐induced airway inflammation, paying special attention to its effects on lipocortin‐1 (LC‐1/annexin‐A1), a 37 kDA anti‐inflammatory protein that plays a key role in the production of inflammatory mediators.

Activation of cannabinoid receptors prevents antigen-induced asthma-like reaction in guinea pigs

In this study we evaluated the effects of the CB1/CB2 cannabinoid receptor agonist CP55, 940 (CP) on antigen‐induced asthma‐like reaction in sensitized guinea pigs and we tested the ability of the specific CB2 receptor antagonist SR144528 (SR) and CB1 receptor antagonist AM251 (AM) to interfere with the effects of CP. Ovalbumin‐sensitized guinea pigs placed in a respiratory chamber were challenged with the antigen given by aerosol. CP (0.4 mg/kg b.wt.) was given i.p. 3 hrs before ovalbumin challenge. Sixty minutes before CP administration, some animals were treated i.p. with either AM, or SR, or both (0.1 mg/kg b.wt.). Respiratory parameters were recorded and quantified. Lung tissue specimens were then taken for histopathological and morphometric analyses and for eosinophilic major basic protein immunohistochemistry. Moreover, myeloperoxidase activity, 8‐hydroxy‐2‐deoxyguanosine, cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP) levels, and CB1 and CB2 receptor protein expression by Western blotting were evaluated in lung tissue extracts. In the bronchoalveolar lavage fluid, the levels of prostaglandin D2 and tumour necrosis factor‐α TNF‐α were measured. Ovalbumin challenge caused marked abnormalities in the respiratory, morphological and biochemical parameters assayed. Treatment with CP significantly reduced these abnormalities. Pre‐treatment with SR, AM or both reverted the protective effects of CP, indicating that both CB1 and CB2 receptors are involved in lung protection. The noted treatments did not change the expression of cannabinoid receptor proteins, as shown by Western blotting. These findings suggest that targeting cannabinoid receptors could be a novel preventative therapeutic strategy in asthmatic patients.

Anti-inflammatory effects of low molecular weight heparin derivative in a rat model of carrageenan-induced pleurisy.

Low molecular weight heparin derivatives are characterized by low anti‐coagulant activity and marked anti‐inflammatory effects that allow for these molecules to be viewed as a new class of non‐steroidal anti‐inflammatory drugs (NSAIDs). We show here that K5NOSepiLMW, an O‐sulphated heparin‐like semi‐synthetic polymer of the D‐glucuronic acid–N‐acetyleparoson disaccharide unit with low molecular weight, has marked anti‐inflammatory effects in a rat model of acute inflammation, the carrageenan‐induced pleurisy, commonly used to test NSAID efficacy. A 30‐min. pre‐treatment with K5NOSepiLMW (0.1, 0.5 and 1 mg/kg b.wt., given intrapleurally) attenuated the recruitment of leucocytes in the lung tissue and the pleural exudate, inhibited the induction of inducible nitric oxide synthase and cyclooxygenase‐2 (COX‐2), thereby abating the generation of nitric oxide and pro‐inflammatory prostaglandins such as PgE2 and PGF1α, reduced the inflammation‐induced nitroxidative lung tissue injury, as shown by tissue thiobarbituric acid‐reactive substances and nitrotyrosine, and blunted the local generation of cytokines such as interleukin‐1β and tumour necrosis factor‐α. All these parameters were markedly increased by intrapleural carrageenan in the absence of any pre‐treatment. The anti‐inflammatory action of K5NOSepiLMW is specific, as judged by the lack of therapeutic effects of B4/110, a biologically inactive cognate polysaccharide, given in the place of the authentic molecule. Moreover, K5NOSepiLMW showed similar effects as celecoxib (1 mg/kg b.wt), a COX‐2 inhibitor and well‐known NSAID. This study provides further insight into the mechanisms underlying the beneficial effects of heparin derivatives in inflammation and identifies K5NOSepiLMW as a novel, promising anti‐inflammatory drug.

Exosomes secreted from human colon cancer cells influence the adhesion of neighboring metastatic cells: Role of microRNA-210

ABSTRACT Cancer-secreted exosomes influence tumor microenvironment and support cancer growth and metastasis. MiR-210 is frequently up-regulated in colorectal cancer tissues and correlates with metastatic disease. We investigated whether exosomes are actively released by HCT-8 colon cancer cells, the role of exosomal miR-210 in the cross-talk between primary cancer cells and neighboring metastatic cells and its contribution in regulating epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). After 7 d of culture, a subpopulation of viable HCT-8 cells detached the monolayer and started to grow in suspension, suggesting anoikis resistance and a metastatic potential. The expression of key proteins of EMT revealed that these cells were E-cadherin negative and vimentin positive further confirming their metastatic phenotype and the acquisition of anoikis resistance. Metastatic cells, in the presence of adherently growing HCT-8, continued to grow in suspension whereas only if seeded in cell-free wells, were able to adhere again and to form E-cadherin positive and vimentin negative new colonies, suggesting the occurrence of MET. The chemosensitivity to 5 fluorouracil and to FOLFOX-like treatment of metastatic cells was significantly diminished compared to adherent HCT-8 cells. Of note, adherent new colonies undergoing MET, were insensitive to both chemotherapeutic strategies. Electron microscopy analysis demonstrated that adherently growing HCT-8, actually secreted exosomes and that exosomes in turn were taken up by metastatic cells. When exosomes secreted by adherently growing HCT-8 were administered to metastatic cells, MET was significantly inhibited. miR-210 was significantly upregulated in exosomes compared to its intracellular levels in adherently growing HCT-8 cells and correlated to anoikis resistance and EMT markers. Exosomes containing miR-210 might be considered as EMT promoting signals that preserve the local cancer-growth permissive milieu and also guide metastatic cells to free, new sites of dissemination.