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1.
Methods Mol Biol ; 2206: 89-101, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754813

RESUMO

The rabbit corneal micropocket assay uses the avascular cornea as a substrate to study angiogenesis in vivo. The continuous monitoring of neovascular growth in the same animal allows for the evaluation of drugs acting as suppressors or stimulators of angiogenesis. Through the use of standardized slow-release pellets, a predictable angiogenic response can be quantified over the course of 1-2 weeks. Uniform slow-release pellets are prepared by mixing purified angiogenic growth factors such as basic fibroblast growth factor (FGF) or vascular endothelial growth factor (VEGF) and a synthetic polymer to allow for their slow release. A micropocket is surgically created in the cornea thickness under anesthesia and in sterile conditions. The angiogenesis stimulus (growth factor but also tissue fragment or cell suspension) is placed into the pocket in order to induce vascular outgrowth from the limbal capillaries where vessels are preexisting. On the following days, the neovascular development and progression are measured and qualified using a slit lamp, as well as the concomitant vascular phenotype or inflammatory features. The results of the assay allow to assess the ability of potential therapeutic molecules to modulate angiogenesis in vivo, both when released locally or given by ocular formulations or through systemic treatment. In this chapter the experimental details of the avascular rabbit cornea assay, the technical challenges, advantages, and limitations are discussed.

2.
Nutrients ; 12(6)2020 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-32471156

RESUMO

: Recently the use of food by-products as natural sources of biologically active substances has been extensively investigated especially for the development of functional foods fortified with natural antioxidants. Due to their content of bioactive compounds, such as carotenoids, flavonoids and limonoids, citrus peels could be suitable to formulate enriched olive oils able to boost healthy nutrition. The aim of this study was: (i) to determine the compositional and sensory profiles of citrus olive oil; and (ii) to evaluate its nutraceutical properties in rats with high fat diet-induced metabolic syndrome and oxidative stress. The results obtained show the potential of using citrus peels as a source of bioactive compounds to improve the sensory profile as well as the phytochemical composition of olive oil. We demonstrated that the production system of Citrus x aurantium olive oil and Citrus limon olive oil improves its organoleptic properties without altering its beneficial effects, which, like control extra virgin olive oil, showed protective effects relating to glucose and serum lipid levels, metabolic activity of adipocytes, myocardial tissue functionality, oxidative stress markers and endothelial function at blood vessel level.

3.
Int J Mol Sci ; 21(8)2020 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-32340282

RESUMO

Carbonic anhydrase IX (CA-IX) plays a pivotal role in regulation of pH in tumor milieu catalyzing carbonic acid formation by hydrating CO2. An acidification of tumor microenvironment contributes to tumor progression via multiple processes, including reduced cell-cell adhesion, increased migration and matrix invasion. We aimed to assess whether the pharmacological inhibition of CA-IX could impair tumor cell proliferation and invasion. Tumor epithelial cells from breast (MDA-MB-231) and lung (A549) cancer were used to evaluate the cytotoxic effect of sulfonamide CA-IX inhibitors. Two CA-IX enzyme blockers were tested, SLC-0111 (at present in phase Ib clinical trial) and AA-06-05. In these cells, the drugs inhibited cell proliferation, migration and invasion through shifting of the mesenchymal phenotype toward an epithelial one and by impairing matrix metalloprotease-2 (MMP-2) activity. The antitumor activity was elicited via apoptosis pathway activation. An upregulation of p53 was observed, which in turn regulated the activation of caspase-3. Inhibition of proteolytic activity was accompanied by upregulation of the endogenous tissue inhibitor TIMP-2. Collectively, these data confirm the potential use of CA-IX inhibitors, and in particular SLC-0111 and AA-06-05, as agents to be further developed, alone or in combination with other conventional anticancer drugs.

4.
Cancers (Basel) ; 11(12)2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31817719

RESUMO

Melanoma and non-small-cell lung carcinoma (NSCLC) cell lines are characterized by an intrinsic population of cancer stem-like cells (CSC), and high expression of detoxifying isozymes, the aldehyde dehydrogenases (ALDHs), regulating the redox state. In this study, using melanoma and NSCLC cells, we demonstrate that ALDH3A1 isozyme overexpression and activity is closely associated with a highly aggressive mesenchymal and immunosuppressive profile. The contribution of ALDH3A1 to the stemness and immunogenic status of melanoma and NSCLC cells was evaluated by their ability to grow in 3D forming tumorspheres, and by the expression of markers for stemness, epithelial to mesenchymal transition (EMT), and inflammation. Furthermore, in specimens from melanoma and NSCLC patients, we investigated the expression of ALDH3A1, PD-L1, and cyclooxygenase-2 (COX-2) by immunohistochemistry. We show that cells engineered to overexpress the ALDH3A1 enzyme enriched the CSCs population in melanoma and NSCLC cultures, changing their transcriptome. In fact, we found increased expression of EMT markers, such as vimentin, fibronectin, and Zeb1, and of pro-inflammatory and immunosuppressive mediators, such as NFkB, prostaglandin E2, and interleukin-6 and -13. ALDH3A1 overexpression enhanced PD-L1 output in tumor cells and resulted in reduced proliferation of peripheral blood mononuclear cells when co-cultured with tumor cells. Furthermore, in tumor specimens from melanoma and NSCLC patients, ALDH3A1 expression was invariably correlated with PD-L1 and the pro-inflammatory marker COX-2. These findings link ALDH3A1 expression to tumor stemness, EMT and PD-L1 expression, and suggest that aldehyde detoxification is a redox metabolic pathway that tunes the immunological output of tumors.

5.
Cells ; 8(12)2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31766389

RESUMO

Hyaluronic acid (HA) is used in substitutive and aesthetic medicine with various applications. Ultrapure absorbable HA (Bioregen®) and a mix of reticulated and free low molecular weight HA (Regenyal Idea Bioexpander®) (both provided by Regenyal Laboratories Srl, San Benedetto del Tronto (AP), Italy) represent a reliable hydrating device and skin filler, useful for skin blemishes, lines and wrinkles, and lip widening, respectively. The commercial products are known for their safety, but data on the molecular, cellular, and tissue responses are lacking. We aimed to evaluate the bioavailability and the pro-angiogenic features of the products Bioregen® and Bioexpander® in vitro on cultured endothelial cells (ECs) and dermal fibroblasts in vivo when injected into experimental animals. When added to fibroblasts and ECs, Bioexpander® induced cell migration. The two HA preparations were well tolerated, while a transient proangiogenic behavior of Bioexpander®, when implanted subcutaneously in mice, was found. The neovascular response was evident in the first week with higher levels of VEGF and FGF-2 before undergoing regression. In conclusion, our data strengthen the safety of HA synthetic preparations both in vitro and in vivo. Even if a proangiogenic response is documented, it is modest and transient, leading to tissue recovery and absence of an inflammatory infiltrate.

6.
Sci Rep ; 9(1): 9297, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243320

RESUMO

Neuropathic pain is characterized by an uncertain etiology and by a poor response to common therapies. The ineffectiveness and the frequent side effects of the drugs used to counteract neuropathic pain call for the discovery of new therapeutic strategies. Laser therapy proved to be effective for reducing pain sensitivity thus improving the quality of life. However, its application parameters and efficacy in chronic pain must be further analyzed. We investigated the pain relieving and protective effect of Photobiomodulation Therapy in a rat model of compressive mononeuropathy induced by Chronic Constriction Injury of the sciatic nerve (CCI). Laser (MLS-MiS) applications started 7 days after surgery and were performed ten times over a three week period showing a reduction in mechanical hypersensitivity and spontaneous pain that started from the first laser treatment until the end of the experiment. The ex vivo analysis highlighted the protective role of laser through the myelin sheath recovery in the sciatic nerve, inhibition of iNOS expression and enhancement of EAAT-2 levels in the spinal cord. In conclusion, this study supports laser treatment as a future therapeutic strategy in patients suffering from neuropathic pain induced by trauma.

7.
Prostaglandins Other Lipid Mediat ; 143: 106344, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31207300

RESUMO

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) as gefitinib are standard treatment of non-small cell lung cancer (NSCLC), but resistance often occurs. This study demonstrates that NSCLC cells resistant to gefitinib (GR cells) displayed a significantly higher microsomal prostaglandin E synthase-1 (mPGES-1) expression and activity than parental cells. Overexpression of mPGES-1/prostaglandin E-2 (PGE-2) signaling in GR cells was associated with acquisition of mesenchymal and stem-like cell properties, nuclear EGFR translocation and tolerance to cisplatin. mPGES-1 inhibition reduced mesenchymal and stem-like properties, and nuclear EGFR translocation in GR cells. Consistently, inhibition of mPGES-1 activity enhanced sensitivity to cisplatin and responsiveness to gefitinib in GR cells. We propose the mPGES-1/PGE-2 signaling as a potential target for treating aggressive and resistant lung cancers.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos , Gefitinibe/farmacologia , Neoplasias Pulmonares/patologia , Terapia de Alvo Molecular , Prostaglandina-E Sintases/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Dinoprostona/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores ErbB/metabolismo , Inativação Gênica , Humanos , Prostaglandina-E Sintases/deficiência , Prostaglandina-E Sintases/genética , Transdução de Sinais/efeitos dos fármacos
9.
J Exp Clin Cancer Res ; 37(1): 311, 2018 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-30541574

RESUMO

BACKGROUND: Aldehyde dehydrogenase 1A1 (ALDH1A1), a member of aldehyde dehydrogenase family, is a marker of stemness in breast cancer. During tumor progression cancer stem cells (CSCs) have been reported to secrete angiogenic factors to orchestrate the formation of pathological angiogenesis. This vasculature can represent the source of self-renewal of CSCs and the route for further tumor spreading. The aim of the present study has been to assess whether ALDH1A1 controls the output of angiogenic factors in breast cancer cells and regulates tumor angiogenesis in a panel of in vitro and in vivo models. METHODS: Stemness status of breast cancer cells was evaluated by the ability to form turmorspheres in vitro. A transwell system was used to assess the angiogenic features of human umbilical vein endothelial cells (HUVEC) when co-cultured with breast cancer cells MCF-7 harboring different levels of ALDH1A1. Under these conditions, we survey endothelial proliferation, migration, tube formation and permeability. Moreover, in vivo, MCF-7 xenografts in immunodeficient mice allow to evaluate blood flow, expression of angiogenic factors and microvascular density (MVD). RESULTS: In MCF-7 we observed that ALDH1A1 activity conferred stemness property and its expression correlated with an activation of angiogenic factors. In particular we observed a significant upregulation of hypoxia inducible factor-1α (HIF-1α) and proangiogenic factors, such as vascular endothelial growth factor (VEGF). High levels of ALDH1A1, through the retinoic acid pathway, were significantly associated with VEGF-mediated angiogenesis in vitro. Co-culture of HUVEC with ALDH1A1 expressing tumor cells promoted endothelial proliferation, migration, tube formation and permeability. Conversely, downregulation of ALDH1A1 in MCF-7 resulted in reduction of proangiogenic factor release/expression and impaired HUVEC angiogenic functions. In vivo, when subcutaneously implanted in immunodeficient mice, ALDH1A1 overexpressing breast tumor cells displayed a higher expression of VEGF and MVD. CONCLUSION: In breast tumors, ALDH1A1 expression primes a permissive microenvironment by promoting tumor angiogenesis via retinoic acid dependent mechanism. In conclusion, ALDH1A1 might be associated to progression and diffusion of breast cancer.


Assuntos
Aldeído Desidrogenase/metabolismo , Neoplasias da Mama/irrigação sanguínea , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Neoplásicas/metabolismo , Tretinoína/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Aldeído Desidrogenase/biossíntese , Aldeído Desidrogenase/genética , Aldeído Desidrogenase 1 , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Células Endoteliais da Veia Umbilical Humana , Humanos , Células MCF-7 , Camundongos , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retinal Desidrogenase , Transdução de Sinais , Transfecção
10.
Oncotarget ; 9(17): 13353-13365, 2018 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-29568362

RESUMO

Nitric oxide (NO) exerts conflicting effect on tumor growth and progression, depending on its concentration. We aimed to characterize the anti-cancer activity of a new NO donor, Ni(SalPipNONO) belonging to the class of metal-nonoates, in epithelial derived tumor cells, finally exploring its antiangiogenic properties. Tumor epithelial cells were screened to evaluate the cytotoxic effect of Ni(SalPipNONO), which was able to inhibit cell proliferation in a dose dependent manner, being more effective than the commercial DETA/NO. The human lung carcinoma cells A549 were chosen as model to study the anti-cancer mechanisms exerted by the compound. In these cells, Ni(SalPipNONO) inhibited clonogenicity and cell invasion, while promoting apoptosis. The antitumor activity was partly due to NO-cGMP dependent pathway, contributing to reduced cell number and apoptosis, and partly to the salicylaldehyde moiety and reactive oxygen species (ROS) activated ERK1/2 signaling converging on p53 dependent caspase-3 cleavage. An additional contribution by downstream cycloxygenase-2 (COX-2) derived cyclopentenones may explain the tumor inhibitory activities. As NO has been described to affect tumor angiogenesis, we checked this activity both on tumor and endothelial cell co-cultures and in Matrigel in vivo assay. Our data document that Ni(SalPipNONO) was able to both reduce angiogenic factor expression by tumor cells acting on hypoxia inducible factor-1α (HIF-1 α) level, and endothelial cell functions related to angiogenesis. Collectively, these data confirm the potential use of NO donors and in particular Ni(SalPipNONO) acting through multiple mechanisms, as an agent to be further developed to be used alone or in combination with conventional therapy.

11.
J Vasc Res ; 53(5-6): 255-268, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27923233

RESUMO

Lymphatic leakage can be seen as a detrimental phenomenon associated with fluid retention and deposition as well as gain of weight. Moreover, lymphatic dysfunction is associated with an inflammatory environment and can be a substrate for other health conditions. A number of treatments can ameliorate lymphatic vasculature: natural substances have been used as treatment options particularly suitable for their consolidated effectiveness and safety profile. Here we report the protective effect of AdipoDren®, an association of a series of plant-derived natural complexes, on lymphatic endothelium permeability promoted by interleukin-1 beta (IL-1ß) and the associated molecular mechanisms. AdipoDren® demonstrated a protective effect on dermal lymphatic endothelial cell permeability increased by IL-1ß. Reduced permeability was due to the maintenance of tight junctions and cell-cell localisation of occludin and zonula occludens-1 (ZO-1). Moreover, AdipoDren® reduced the expression of the inflammatory key element cyclooxygenase-2 (COX-2), while not altering the levels of endothelial and inducible nitric oxide synthases (eNOS and iNOS). The upregulation of antioxidant enzymatic systems (catalase and superoxide dismutase-1, SOD-1) and the downregulation of pro-oxidant markers (p22 phox subunit of NADPH oxidase) were also evident. In conclusion, AdipoDren® would be useful to ameliorate conditions of altered lymphatic vasculature and to support the physiological functionality of the lymphatic endothelium.


Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Endotélio Linfático/efeitos dos fármacos , Interleucina-1beta/farmacologia , Linfedema/tratamento farmacológico , Extratos Vegetais/farmacologia , Preparações de Plantas/farmacologia , Junções Íntimas/efeitos dos fármacos , Catalase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Endotélio Linfático/metabolismo , Endotélio Linfático/fisiopatologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Linfedema/metabolismo , Linfedema/fisiopatologia , NADPH Oxidases/metabolismo , Ocludina/metabolismo , Rutina/farmacologia , Superóxido Dismutase-1/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
12.
Pharmacol Res ; 107: 352-359, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27063892

RESUMO

The nickel-piperazine/NO donor compound, Ni(PipNONO)Cl, belonging to the family of compounds labelled as "metal-nonoates", due to its promising vasodilating activity, has been considered as a potential drug candidate in anti-hypertensive therapy. Drug efficacy has been evaluated in spontaneously hypertensive rats (SHR) in comparison with normotensive animals (C57BL/6 mice and WKY rats). In normotensive animals the metal-nonoate maintained blood pressure at basal level both following acute administration and after 30 days of treatment. In SHR, Ni(PipNONO)Cl reduced blood pressure in the dose range of 3-10mg/kg. When compared with a commercial NONOate, DETA/NO, used at the same doses, Ni(PipNONO)Cl was more active in reducing blood pressure in SHR than DETA/NO in the first two weeks, while the effect of the two molecules was similar in the third and fourth week. The degradation and control compound Ni(Pip)Cl2 had no effect on blood pressure and heart rate in same animal models. Remarkably, the blood pressure reduction induced by the new NO-donor Ni(PipNONO)Cl does not evoke changes in the heart rate and tolerance. Considering the mechanisms of vascular protection, 30 days of administration of Ni(PipNONO)Cl improved endothelial function in SHR by upregulating endothelial NO synthase (eNOS) through increased eNOS protein levels and downregulated Caveolin-1 (Cav-1), and by increasing superoxide dismutase 1 (SOD1) protein level in aortae. In cultured endothelial cells Ni(PipNONO)Cl restored the cell functions (cytoskeletal protein expression, migration and proliferation) altered by the inflammatory mediator interleukin-1ß (IL-1ß), impairing the endothelial to mesenchimal transition. In conclusion, Ni(PipNONO)Cl maintained unaltered blood pressure in normotensive mice and rats, and it exerted anti-hypertensive effect in SHR through the restoration of vascular endothelial protective functions.


Assuntos
Anti-Hipertensivos/uso terapêutico , Hipertensão/tratamento farmacológico , Níquel/uso terapêutico , Doadores de Óxido Nítrico/uso terapêutico , Piperazinas/uso terapêutico , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Frequência Cardíaca/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Hipertensão/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY
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