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Periodically, the European Space Agency (ESA) updates scientific roadmaps in consultation with the scientific community. The ESA SciSpacE Science Community White Paper (SSCWP) 9, "Biology in Space and Analogue Environments", focusses in 5 main topic areas, aiming to address key community-identified knowledge gaps in Space Biology. Here we present one of the identified topic areas, which is also an unanswered question of life science research in Space: "How to Obtain an Integrated Picture of the Molecular Networks Involved in Adaptation to Microgravity in Different Biological Systems?" The manuscript reports the main gaps of knowledge which have been identified by the community in the above topic area as well as the approach the community indicates to address the gaps not yet bridged. Moreover, the relevance that these research activities might have for the space exploration programs and also for application in industrial and technological fields on Earth is briefly discussed.
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The mitogen-activated protein kinase (MAPK/ERK) pathway is pivotal in controlling the proliferation and survival of melanoma cells. Several mutations, including those in BRAF, exhibit an oncogenic effect leading to increased cellular proliferation. As a result, the combination therapy of a MEK inhibitor with a BRAF inhibitor demonstrated higher efficacy and lower toxicity than BRAF inhibitor alone. This combination has become the preferred standard of care for tumors driven by BRAF mutations. Aldehyde dehydrogenase 1A1 (ALDH1A1) is a known marker of stemness involved in drug resistance in several type of tumors, including melanoma. This study demonstrates that melanoma cells overexpressing ALDH1A1 displayed resistance to vemurafenib and trametinib through the activation of PI3K/AKT signaling instead of MAPK axis. Inhibition of PI3K/AKT signaling partially rescued sensitivity to the drugs. Consistently, pharmacological inhibition of ALDH1A1 activity downregulated the activation of AKT and partially recovered responsiveness to vemurafenib and trametinib. We propose ALDH1A1 as a new potential target for treating melanoma resistant to MAPK/ERK inhibitors.
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Família Aldeído Desidrogenase 1 , Resistencia a Medicamentos Antineoplásicos , Melanoma , Células-Tronco Neoplásicas , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-akt , Retinal Desidrogenase , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Melanoma/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Linhagem Celular Tumoral , Família Aldeído Desidrogenase 1/metabolismo , Família Aldeído Desidrogenase 1/genética , Retinal Desidrogenase/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Pirimidinonas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Piridonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Vemurafenib/farmacologia , Aldeído Desidrogenase/metabolismo , Aldeído Desidrogenase/antagonistas & inibidores , Aldeído Desidrogenase/genética , Antineoplásicos/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , FenótipoRESUMO
Insufficient vessel maintenance adversely impacts patients in terms of tissue reperfusion following stroke or myocardial infarction, as well as during wound healing. Angiogenesis impairment is a feature typical of metabolic disorders acting at the cardiovascular level, such as diabetes. Therapeutic angiogenesis regulation offers promising clinical implications, and natural compounds as pro-angiogenic nutraceuticals hold valuable applications in regenerative medicine. By using cultured endothelial cells from human umbilical veins (HUVEC) we studied functional and molecular responses following exposure to erucin, a natural isothiocyanate derived from Brassicaceae plants and extracted from the seeds of rocket. Erucin (at nanomolar concentrations) promotes cell migration and tube formation, similar to vascular endothelial growth factor (VEGF), through mobilizing paxillin at endothelial edges. At the molecular level, erucin induces signaling pathways typical of angiogenesis activation, namely Ras, PI3K/AKT, and ERK1/2, leading to VEGF expression and triggering its autocrine production, as pharmacological inhibition of soluble VEGF and VEGFR2 dampens endothelial functions. Furthermore, erucin, alone and together with VEGF, preserves endothelial angiogenic functions under pathological conditions, such as those induced in HUVEC by high glucose (HG) exposure. Erucin emerges as a compelling candidate for therapeutic revascularization applications, showcasing promising prospects for natural compounds in regenerative medicine, particularly in addressing angiogenesis-related disorders.
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Movimento Celular , Glucose , Células Endoteliais da Veia Umbilical Humana , Isotiocianatos , Fator A de Crescimento do Endotélio Vascular , Humanos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Isotiocianatos/farmacologia , Movimento Celular/efeitos dos fármacos , Paxilina/metabolismo , Indutores da Angiogênese/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Brassicaceae/química , Neovascularização Fisiológica/efeitos dos fármacos , Sulfetos , TiocianatosRESUMO
Progress in mechanobiology allowed us to better understand the important role of mechanical forces in the regulation of biological processes. Space research in the field of life sciences clearly showed that gravity plays a crucial role in biological processes. The space environment offers the unique opportunity to carry out experiments without gravity, helping us not only to understand the effects of gravitational alterations on biological systems but also the mechanisms underlying mechanoperception and cell/tissue response to mechanical and gravitational stresses. Despite the progress made so far, for future space exploration programs it is necessary to increase our knowledge on the mechanotransduction processes as well as on the molecular mechanisms underlying microgravity-induced cell and tissue alterations. This white paper reports the suggestions and recommendations of the SciSpacE Science Community for the elaboration of the section of the European Space Agency roadmap "Biology in Space and Analogue Environments" focusing on "How are cells and tissues influenced by gravity and what are the gravity perception mechanisms?" The knowledge gaps that prevent the Science Community from fully answering this question and the activities proposed to fill them are discussed.
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The present white paper concerns the indications and recommendations of the SciSpacE Science Community to make progress in filling the gaps of knowledge that prevent us from answering the question: "How Do Gravity Alterations Affect Animal and Human Systems at a Cellular/Tissue Level?" This is one of the five major scientific issues of the ESA roadmap "Biology in Space and Analogue Environments". Despite the many studies conducted so far on spaceflight adaptation mechanisms and related pathophysiological alterations observed in astronauts, we are not yet able to elaborate a synthetic integrated model of the many changes occurring at different system and functional levels. Consequently, it is difficult to develop credible models for predicting long-term consequences of human adaptation to the space environment, as well as to implement medical support plans for long-term missions and a strategy for preventing the possible health risks due to prolonged exposure to spaceflight beyond the low Earth orbit (LEO). The research activities suggested by the scientific community have the aim to overcome these problems by striving to connect biological and physiological aspects in a more holistic view of space adaptation effects.
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Vascular inflammation (VI) represents a pathological condition that progressively affects the integrity and functionality of the vascular wall, thus leading to endothelial dysfunction and the onset of several cardiovascular diseases. Therefore, the research of novel compounds able to prevent VI represents a compelling need. In this study, we tested erucin, the natural isothiocyanate H2S-donor derived from Eruca sativa Mill. (Brassicaceae), in an in vivo mouse model of lipopolysaccharide (LPS)-induced peritonitis, where it significantly reduced the amount of emigrated CD11b positive neutrophils. We then evaluated the anti-inflammatory effects of erucin in LPS-challenged human umbilical vein endothelial cells (HUVECs). The pre-incubation of erucin, before LPS treatment (1, 6, 24 h), significantly preserved cell viability and prevented the increase of reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-α) levels. Moreover, erucin downregulated endothelial hyperpermeability and reduced the loss of vascular endothelial (VE)-Cadherin levels. In addition, erucin decreased vascular cell adhesion molecule 1 (VCAM-1), cyclooxygenase-2 (COX-2) and microsomal prostaglandin E-synthase 1 (mPGES-1) expression. Of note, erucin induced eNOS phosphorylation and counteracted LPS-mediated NF-κB nuclear translocation, an effect that was partially abolished in the presence of the eNOS inhibitor L-NAME. Therefore, erucin can control endothelial function through biochemical and genomic positive effects against VI.
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Endotélio Vascular , Transdução de Sinais , Humanos , Camundongos , Animais , Endotélio Vascular/metabolismo , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/metabolismoRESUMO
This study is preliminary to an experiment to be performed onboard the International Space Station (ISS) and on Earth to investigate how low gravity influences the healing of sutured human skin and vein wounds. Its objective was to ascertain whether these tissue explants could be maintained to be viable ex vivo for long periods of time, mimicking the experimental conditions onboard the ISS. We developed an automated tissue culture chamber, reproducing and monitoring the physiological tensile forces over time, and a culture medium enriched with serelaxin (60 ng/mL) and (Zn(PipNONO)Cl) (28 ng/mL), known to extend viability of explanted organs for transplantation. The results show that the human skin and vein specimens remained viable for more than 4 weeks, with no substantial signs of damage in their tissues and cells. As a further clue about cell viability, some typical events associated with wound repair were observed in the tissue areas close to the wound, namely remodeling of collagen fibers in the papillary dermis and of elastic fibers in the vein wall, proliferation of keratinocyte stem cells, and expression of the endothelial functional markers eNOS and FGF-2. These findings validate the suitability of this new ex vivo organ culture system for wound healing studies, not only for the scheduled space experiment but also for applications on Earth, such as drug discovery purposes.
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Pele , Cicatrização , Humanos , Pele/metabolismo , Suturas , Queratinócitos/fisiologia , Procedimentos NeurocirúrgicosRESUMO
Exogenous nitric oxide appears a promising therapeutic approach to control cancer progression. Previously, a nickel-based nonoate, [Ni(SalPipNONO)], inhibited lung cancer cells, along with impairment of angiogenesis. The Zn(II) containing derivatives [Zn(PipNONO)Cl] exhibited a protective effect on vascular endothelium. Here, we have evaluated the antitumor properties of [Zn(PipNONO)Cl] in human lung cancer (A549) and melanoma (A375) cells. Metastasis initiates with the epithelial-mesenchymal transition (EMT) process, consisting of the acquisition of invasive and migratory properties by tumor cells. At not cytotoxic levels, the nonoate significantly impaired A549 and A375 EMT induced by transforming growth factor-ß1 (TGF-ß1). Reduction of the mesenchymal marker vimentin, upregulated by TGF-ß1, and restoration of the epithelial marker E-cadherin, reduced by TGF-ß1, were detected in both tumor cell lines in the presence of Zn-nonoate. Further, the endothelial-mesenchymal transition achieved in a tumor-endothelial cell co-culture was assessed. Endothelial cells co-cultured with A549 or A375 acquired a mesenchymal phenotype with increased vimentin, alpha smooth muscle actin and Smad2/3, and reduced VE-cadherin. The presence of [Zn(PipNONO)Cl] maintained a typical endothelial phenotype. In conclusion, [Zn(PipNONO)Cl] appears a promising therapeutic tool to control tumor growth and metastasis, by acting on both tumor and endothelial cells, reprogramming the cells toward their physiologic phenotypes.
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ALDH1A1 is a cytosolic enzyme upregulated in tumor cells, involved in detoxifying cells from reactive aldehydes and in acquiring resistance to chemotherapeutic drugs. Its expression correlates with poor clinical outcomes in a number of cancers, including melanoma. The present study hypothesized that the increased ALDH1A1 expression and activity upregulated the release of proangiogenic factors from melanoma cells, which regulate angiogenic features in endothelial cells (ECs) through a rearrangement of the Notch pathway. In vivo, when subcutaneously implanted in immunodeficient mice, ALDH1A1 overexpressing melanoma cells displayed a higher microvessel density. In a 3D multicellular system, obtained coculturing melanoma cancer cells with stromal cells, including ECs, melanoma ALDH1A1 overexpression induced the recruitment of ECs into the core of the tumorspheres. By using a genes array, overexpression of ALDH1A1 in tumor cells also promoted modulation of Notch cascade gene expression in ECs, suggesting an interaction between tumor cells and ECs mediated by enrichment of angiogenic factors in the tumor microenvironment. To confirm this hypothesis, inactivation of ALDH1A1 by the pharmacological inhibitor CM037 significantly affected the release of angiogenic factors, including IL8, from melanoma cells. High levels of ALDH1A1, through the retinoic acid pathway, regulated the activation of NFkBp65 and IL8. Further, in a 2D coculture system, the addition of an IL8 neutralizing antibody to ECs cocultured with melanoma cells forced to express ALDH1A1 dampened endothelial angiogenic features, both at the molecular (in terms of gene and protein expression of mediators of the Notch pathway) and at the functional level (proliferation, scratch assay, tube formation and permeability). In conclusion, these findings demonstrated the existence of a link between melanoma ALDH1A1 expression and EC Notch signaling modification that results in a proangiogenic phenotype. Based on the crucial role of ALDH1A1 in melanoma control of the tumor microenvironment, the enzyme seems a promising target for the development of novel drugs able to interrupt the crosstalk between cancer (stem) cells and endothelial cells.
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Família Aldeído Desidrogenase 1 , Células Endoteliais , Melanoma , Retinal Desidrogenase , Família Aldeído Desidrogenase 1/genética , Animais , Células Endoteliais/metabolismo , Interleucina-8/genética , Melanoma/genética , Melanoma/patologia , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Receptores Notch , Retinal Desidrogenase/genética , Transdução de Sinais , Microambiente TumoralRESUMO
As human spaceflight progresses with extended mission durations, the demand for effective and safe drugs will necessarily increase. To date, the accepted medications used during missions (for space motion sickness, sleep disturbances, allergies, pain, and sinus congestion) are administered under the assumption that they act as safely and efficaciously as on Earth. However, physiological changes have been documented in human subjects in spaceflight involving fluid shifts, muscle and bone loss, immune system dysregulation, and adjustments in the gastrointestinal tract and metabolism. These alterations may change the pharmacokinetics (PK) and pharmacodynamics of commonly used medications. Frustratingly, the information gained from bed rest studies and from in-flight observations is incomplete and also demonstrates a high variability in drug PK. Therefore, the objectives of this review are to report (i) the impact of the space environmental stressors on human physiology in relation to PK; (ii) the state-of-the-art on experimental data in space and/or in ground-based models; (iii) the validation of ground-based models for PK studies; and (iv) the identification of research gaps.
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Voo Espacial , Ausência de Peso , Adaptação Fisiológica , Repouso em Cama , HumanosRESUMO
The aim of personalized medicine is to detach from a "one-size fits all approach" and improve patient health by individualization to achieve the best outcomes in disease prevention, diagnosis and treatment. Technological advances in sequencing, improved knowledge of omics, integration with bioinformatics and new in vitro testing formats, have enabled personalized medicine to become a reality. Individual variation in response to environmental factors can affect susceptibility to disease and response to treatments. Space travel exposes humans to environmental stressors that lead to physiological adaptations, from altered cell behavior to abnormal tissue responses, including immune system impairment. In the context of human space flight research, human health studies have shown a significant inter-individual variability in response to space analogue conditions. A substantial degree of variability has been noticed in response to medications (from both an efficacy and toxicity perspective) as well as in susceptibility to damage from radiation exposure and in physiological changes such as loss of bone mineral density and muscle mass in response to deconditioning. At present, personalized medicine for astronauts is limited. With the advent of longer duration missions beyond low Earth orbit, it is imperative that space agencies adopt a personalized strategy for each astronaut, starting from pre-emptive personalized pre-clinical approaches through to individualized countermeasures to minimize harmful physiological changes and find targeted treatment for disease. Advances in space medicine can also be translated to terrestrial applications, and vice versa. This review places the astronaut at the center of personalized medicine, will appraise existing evidence and future preclinical tools as well as clinical, ethical and legal considerations for future space travel.
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Wound healing is a complex phenomenon that involves different cell types with various functions, i.e., keratinocytes, fibroblasts, and endothelial cells, all influenced by the action of soluble mediators and rearrangement of the extracellular matrix (ECM). Physiological angiogenesis occurs in the granulation tissue during wound healing to allow oxygen and nutrient supply and waste product removal. Angiogenesis output comes from a balance between pro- and antiangiogenic factors, which is finely regulated in a spatial and time-dependent manner, in order to avoid insufficient or excessive nonreparative neovascularization. The understanding of the factors and mechanisms that control angiogenesis and their change following unloading conditions (in a real or simulated space environment) will allow to optimize the tissue response in case of traumatic injury or medical intervention. The potential countermeasures under development to optimize the reparative angiogenesis that contributes to tissue healing on Earth will be discussed in relation to their exploitability in space.
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The problem of drug resistance in cancer patients has been well in mind from the beginning of modern medicine and oncology treatments with the so called conventional cytotoxic therapy. With the advent of target therapy against tumor angiogenesis and in particular against the vascular endothelial growth factor (VEGF)/VEGF receptor system, researchers thought that resistance could be no more a problem, since the low pattern of proliferation displayed by endothelial cells. However, beside the efficacy demonstrated by antiangiogenic drugs, resistance during prolonged drug treatments appears as a limiting feature. Nowadays, various mechanisms of resistance to antiangiogenic therapeutics have been discovered, either innate and depending on the host, or acquired by the tumor cells, especially as a consequence of induced hypoxia by antiangiogenic drugs and the redundancy of proangiogenic factors in the tumor microenvironment, and other forms of tumor neovascularization, than sprouting angiogenesis. Here, we have reviewed the preclinical and clinical evidence for mechanisms of resistance to antiangiogenic drugs reported so far. The knowledge of the mechanisms underneath antiangiogenic drug resistance could be of help in the choice of the more appropriate drug, the development of novel therapeutic strategies, the design of proper drug combination protocols or new formulations of antiangiogenic strategies.
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Inibidores da Angiogênese , Resistencia a Medicamentos Antineoplásicos , Neoplasias , Inibidores da Angiogênese/farmacologia , Células Endoteliais , Humanos , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Preservation of vascular wall integrity against degenerative processes associated with ageing, fat-rich diet and metabolic diseases is a timely therapeutical challenge. The loss of endothelial function and integrity leads to cardiovascular diseases and multiorgan inflammation. The protective effects of the H2S-donor erucin, an isothiocyanate purified by Eruca sativa Mill. seeds, were evaluated on human endothelial and vascular smooth muscle cells. In particular, erucin actions were evaluated on cell viability, ROS, caspase 3/7, inflammatory markers levels and the endothelial hyperpermeability in an inflammatory model associated with high glucose concentrations (25 mM, HG). Erucin significantly prevented the HG-induced decrease in cell viability as well as the increase in ROS, caspase 3/7 activation, and TNF-α and IL-6 levels. Similarly, erucin suppressed COX-2 and NF-κB upregulation associated with HG exposure. Erucin also caused a significant inhibition of p22phox subunit expression in endothelial cells. In addition, erucin significantly prevented the HG-induced increase in endothelial permeability as also confirmed by the quantification of the specific markers VE-Cadherin and ZO-1. In conclusion, our results assess anti-inflammatory and antioxidant effects by erucin in vascular cells undergoing HG-induced inflammation and this protection parallels the preservation of endothelial barrier properties.
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Microgravity-induced bone loss is currently a significant and unresolved health risk for space travelers, as it raises the likelihood for irreversible changes that weaken skeletal integrity and the incremental onset of fracture injuries and renal stone formation. Another issue related to bone tissue homeostasis in microgravity is its capacity to regenerate following fractures due to weakening of the tissue and accidental events during the accomplishment of particularly dangerous tasks. Today, several pharmacological and non-pharmacological countermeasures to this problem have been proposed, including physical exercise, diet supplements and administration of antiresorptive or anabolic drugs. However, each class of pharmacological agents presents several limitations as their prolonged and repeated employment is not exempt from the onset of serious side effects, which limit their use within a well-defined range of time. In this review, we will focus on the various countermeasures currently in place or proposed to address bone loss in conditions of microgravity, analyzing in detail the advantages and disadvantages of each option from a pharmacological point of view. Finally, we take stock of the situation in the currently available literature concerning bone loss and fracture healing processes. We try to understand which are the critical points and challenges that need to be addressed to reach innovative and targeted therapies to be used both in space missions and on Earth.
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Osso e Ossos/metabolismo , Ausência de Peso/efeitos adversos , Doenças Ósseas Metabólicas/metabolismo , Cálcio , Exercício Físico/fisiologia , Humanos , Voo EspacialRESUMO
The vascular endothelium consists of a single layer of squamous endothelial cells (ECs) lining the inner surface of blood vessels. Nowadays, it is no longer considered as a simple barrier between the blood and vessel wall, but a central hub to control blood flow homeostasis and fulfill tissue metabolic demands by furnishing oxygen and nutrients. The endothelium regulates the proper functioning of vessels and microcirculation, in terms of tone control, blood fluidity, and fine tuning of inflammatory and redox reactions within the vessel wall and in surrounding tissues. This multiplicity of effects is due to the ability of ECs to produce, process, and release key modulators. Among these, gasotransmitters such as nitric oxide (NO) and hydrogen sulfide (H2S) are very active molecules constitutively produced by endotheliocytes for the maintenance and control of vascular physiological functions, while their impairment is responsible for endothelial dysfunction and cardiovascular disorders such as hypertension, atherosclerosis, and impaired wound healing and vascularization due to diabetes, infections, and ischemia. Upregulation of H2S producing enzymes and administration of H2S donors can be considered as innovative therapeutic approaches to improve EC biology and function, to revert endothelial dysfunction or to prevent cardiovascular disease progression. This review will focus on the beneficial autocrine/paracrine properties of H2S on ECs and the state of the art on H2S potentiating drugs and tools.
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The fine control of inflammation following injury avoids fibrotic scars or impaired wounds. Due to side effects by anti-inflammatory drugs, the research is continuously active to define alternative therapies. Among them, physical countermeasures such as photobiomodulation therapy (PBMT) are considered effective and safe. To study the cellular and molecular events associated with the anti-inflammatory activity of PBMT by a dual-wavelength NIR laser source, human dermal fibroblasts were exposed to a mix of inflammatory cytokines (IL-1ß and TNF-α) followed by laser treatment once a day for three days. Inducible inflammatory key enzymatic pathways, as iNOS and COX-2/mPGES-1/PGE2, were upregulated by the cytokine mix while PBMT reverted their levels and activities. The same behavior was observed with the proangiogenic factor vascular endothelial growth factor (VEGF), involved in neovascularization of granulation tissue. From a molecular point of view, PBMT retained NF-kB cytoplasmatic localization. According to a change in cell morphology, differences in expression and distribution of fundamental cytoskeletal proteins were observed following treatments. Tubulin, F-actin, and α-SMA changed their organization upon cytokine stimulation, while PBMT reestablished the basal localization. Cytoskeletal rearrangements occurring after inflammatory stimuli were correlated with reorganization of membrane α5ß1 and fibronectin network as well as with their upregulation, while PBMT induced significant downregulation. Similar changes were observed for collagen I and the gelatinolytic enzyme MMP-1. In conclusion, the present study demonstrates that the proposed NIR laser therapy is effective in controlling fibroblast activation induced by IL-1ß and TNF-α, likely responsible for a deleterious effect of persistent inflammation.
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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.
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Bioensaio/métodos , Córnea/patologia , Neovascularização da Córnea/patologia , Neovascularização Patológica/patologia , Animais , Córnea/metabolismo , Neovascularização da Córnea/metabolismo , Neovascularização Patológica/metabolismo , Coelhos , Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The vascular endothelium is one of the first barriers encountered by drugs and xenobiotics, which, once administered, enter the blood stream and diffuse to all organs through blood vessels. The continuous exposure of endothelial cells to drugs and chemical compounds turns out to be a huge risk for the cardiovascular system, as these substances could compromise endothelial vitality and function and create irreparable, localized or systemic damages. For this reason, a special attention should be paid to the safety of developing drugs on the cardiovascular system. In this study we focused our attention on carbonic anhydrase (CA)-IX inhibitors. CA-IX is an enzyme over-expressed in tumor cells in response to hypoxia, which is involved in pH control of the neoplastic mass microenvironment and in tumor progression. Specifically, we evaluated the safety on human umbilical vein endothelial cells (HUVEC) of CA-IX inhibitor AA-06-05, compared to its lead compound SLC-0111, for which the efficacy on tumor cells has already been proven. In this analysis we detected an impairment in viability and mitochondrial metabolism of HUVECs treated with AA-06-05 (but not with SLC-0111) in the concentration range 1-10 µM. These data were accompanied by an increase in the expression of the cell cycle negative regulator, p21, and a down-regulation of the pro-survival proteins ERK1/2 and AKT, both in their phosphorylated and total forms. The data obtained document the likelihood for CA-IX inhibitor AA-06-05 to be developed as new anticancer drug, but a particular attention should be paid to its potential side effects on endothelial cells due to its targeting on other CA isoforms as CA-I, with ubiquitous localization and physiological significance.
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Antineoplásicos/farmacologia , Anidrase Carbônica IX/antagonistas & inibidores , Inibidores da Anidrase Carbônica/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Sulfonamidas/farmacologia , Antígenos de Neoplasias/metabolismo , Antineoplásicos/toxicidade , Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/toxicidade , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Compostos de Fenilureia/toxicidade , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sulfonamidas/toxicidade , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
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.