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Despite numerous molecular targeted therapies tested in glioblastoma (GBM), no significant progress in patient survival has been achieved in the last 20 years in the overall population of GBM patients except with TTfield setup associated with the standard of care chemoradiotherapy. Therapy resistance is associated with target expression heterogeneity and plasticity between tumors and in tumor niches. We focused on α5 integrin implicated in aggressive GBM in preclinical and clinical samples. To address the characteristics of α5 integrin heterogeneity we started with patient data indicating that elevated levels of its mRNA are related to hypoxia pathways. We turned on glioma stem cells which are considered at the apex of tumor formation and recurrence but also as they localize in hypoxic niches. We demonstrated that α5 integrin expression is stem cell line dependent and is modulated positively by hypoxia in vitro. Importantly, heterogeneity of expression is conserved in in vivo stem cell-derived mice xenografts. In hypoxic niches, HIF-2α is preferentially implicated in α5 integrin expression which confers migratory capacity to GBM stem cells. Hence combining HIF-2α and α5 integrin inhibitors resulted in proliferation and migration impairment of α5 integrin expressing cells. Stabilization of HIF-2α is however not sufficient to control integrin α5 expression. Our results show that AHR (aryl hydrocarbon receptor) expression is inversely related to HIF-2α and α5 integrin expressions suggesting a functional competition between the two transcription factors. Collectively, data confirm the high heterogeneity of a GBM therapeutic target, its induction in hypoxic niches by HIF-2α and suggest a new way to attack molecularly defined GBM stem cells.
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Heart failure remains one of the largest clinical burdens globally, with little to no improvement in the development of disease-eradicating therapeutics. Integrin targeting has been used in the treatment of ocular disease and cancer, but little is known about its utility in the treatment of heart failure. Here we sought to determine whether the second generation orally available, αvß3-specific RGD-mimetic, 29P , was cardioprotective. Male mice were subjected to transverse aortic constriction (TAC) and treated with 50 µg/kg 29P or volume-matched saline as Vehicle control. At 3 weeks post-TAC, echocardiography showed that 29P treatment significantly restored cardiac function and structure indicating the protective effect of 29P treatment in this model of heart failure. Importantly, 29P treatment improved cardiac function giving improved fractional shortening, ejection fraction, heart weight and lung weight to tibia length fractions, together with partial restoration of Ace and Mme levels, as markers of the TAC insult. At a tissue level, 29P reduced cardiomyocyte hypertrophy and interstitial fibrosis, both of which are major clinical features of heart failure. RNA sequencing identified that, mechanistically, this occurred with concomitant alterations to genes involved molecular pathways associated with these processes such as metabolism, hypertrophy and basement membrane formation. Overall, targeting αvß3 with 29P provides a novel strategy to attenuate pressure-overload induced cardiac hypertrophy and fibrosis, providing a possible new approach to heart failure treatment.
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Despite progress in the prevention and diagnosis of cancer, current technologies for tumor detection present several limitations including invasiveness, toxicity, inaccuracy, lengthy testing duration and high cost. Therefore, innovative diagnostic techniques that integrate knowledge from biology, oncology, medicinal and analytical chemistry are now quickly emerging in the attempt to address these issues. Following this approach, here we developed a paper-based electrochemical device for detecting cancer-derived Small Extracellular Vesicles (S-EVs) in fluids. S-EVs were obtained from cancer cell lines known to express, at a different level, the αvß6 integrin receptor, a well-established hallmark of numerous epithelial cancer types. The resulting biosensor turned out to recognize αvß6-containing S-EVs down to a limit of 0.7*103 S-EVs/mL with a linear range up to 105 S-EVs /mL, and a relative standard deviation of 11%, thus it may represent a novel opportunity for αvß6 expressing cancers detection.
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Tony Keller, a pioneer in the field of Nuclear Magnetic Resonance (NMR) spectroscopy, passed away on October 27, 2023, at the age of 86 in Spiez, Switzerland. His work and vision were essential to the development and commercialization of NMR spectrometers for many areas of scientific research.
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Receptor-selective peptides are widely used as smart carriers for specific tumor-targeted delivery. A remarkable example is the cyclic nonapeptide iRGD (CRGDKPGDC, 1) that couples intrinsic cytotoxic effects with striking tumor-homing properties. These peculiar features are based on a rather complex multistep mechanism of action, where the primary event is the recognition of RGD integrins. Despite the high number of preclinical studies and the recent success of a phase I trial for the treatment of pancreatic ductal adenocarcinoma (PDAC), there is little information available about the iRGD three-dimensional (3D) structure and integrin binding properties. Here, we re-evaluate the peptide's affinity for cancer-related integrins including not only the previously known targets αvß3 and αvß5 but also the αvß6 isoform, which is known to drive cell growth, migration, and invasion in many malignancies including PDAC. Furthermore, we use parallel tempering in the well-tempered ensemble (PT-WTE) metadynamics simulations to characterize the in-solution conformation of iRGD and extensive molecular dynamics calculations to fully investigate its binding mechanism to integrin partners. Finally, we provide clues for fine-tuning the peptide's potency and selectivity profile, which, in turn, may further improve its tumor-homing properties.
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Integrinas , Oligopéptidos , Línea Celular Tumoral , Oligopéptidos/química , Péptidos/química , Neoplasias PancreáticasRESUMEN
Serratia marcescens is an opportunistic human pathogen involved in antibiotic-resistant hospital acquired infections. Upon contact with the host epithelial cell and prior to internalization, Serratia induces an early autophagic response that is entirely dependent on the ShlA toxin. Once Serratia invades the eukaryotic cell and multiples inside an intracellular vacuole, ShlA expression also promotes an exocytic event that allows bacterial egress from the host cell without compromising its integrity. Several toxins, including ShlA, were shown to induce ATP efflux from eukaryotic cells. Here, we demonstrate that ShlA triggered a nonlytic release of ATP from Chinese hamster ovary (CHO) cells. Enzymatic removal of accumulated extracellular ATP (eATP) or pharmacological blockage of the eATP-P2Y2 purinergic receptor inhibited the ShlA-promoted autophagic response in CHO cells. Despite the intrinsic ecto-ATPase activity of CHO cells, the effective concentration and kinetic profile of eATP was consistent with the established affinity of the P2Y2 receptor and the known kinetics of autophagy induction. Moreover, eATP removal or P2Y2 receptor inhibition also suppressed the ShlA-induced exocytic expulsion of the bacteria from the host cell. Blocking α5ß1 integrin highly inhibited ShlA-dependent autophagy, a result consistent with α5ß1 transactivation by the P2Y2 receptor. In sum, eATP operates as the key signaling molecule that allows the eukaryotic cell to detect the challenge imposed by the contact with the ShlA toxin. Stimulation of P2Y2-dependent pathways evokes the activation of a defensive response to counteract cell damage and promotes the nonlytic clearance of the pathogen from the infected cell.
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Autofagia , Interacciones Huésped-Patógeno , Integrina alfa5beta1 , Receptores Purinérgicos P2Y2 , Serratia , Toxinas Biológicas , Animales , Cricetinae , Adenosina Trifosfato/metabolismo , Autofagia/efectos de los fármacos , Células CHO , Cricetulus , Exocitosis/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Integrina alfa5beta1/antagonistas & inhibidores , Integrina alfa5beta1/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Serratia/química , Serratia/efectos de los fármacos , Serratia/fisiología , Toxinas Biológicas/farmacología , HumanosRESUMEN
Human brain cells generated by in vitro cell programming provide exciting prospects for disease modeling, drug discovery and cell therapy. These applications frequently require efficient and clinically compliant tools for genetic modification of the cells. Recombinant adeno-associated viruses (AAVs) fulfill these prerequisites for a number of reasons, including the availability of a myriad of AAV capsid variants with distinct cell type specificity (also called tropism). Here, we harnessed a customizable parallel screening approach to assess a panel of natural or synthetic AAV capsid variants for their efficacy in lineage-related human neural cell types. We identified common lead candidates suited for the transduction of directly converted, early-stage induced neural stem cells (iNSCs), induced pluripotent stem cell (iPSC)-derived later-stage, radial glia-like neural progenitors, as well as differentiated astrocytic and mixed neuroglial cultures. We then selected a subset of these candidates for functional validation in iNSCs and iPSC-derived astrocytes, using shRNA-induced downregulation of the citrate transporter SLC25A1 and overexpression of the transcription factor NGN2 for proofs-of-concept. Our study provides a comparative overview of the susceptibility of different human cell programming-derived brain cell types to AAV transduction and a critical discussion of the assets and limitations of this specific AAV capsid screening approach.
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Dependovirus , Transportadores de Anión Orgánico , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Terapia Genética , Vectores Genéticos/genética , Humanos , Proteínas Mitocondriales/metabolismo , Transportadores de Anión Orgánico/metabolismo , Transducción GenéticaRESUMEN
Astrocytes have crucial functions in the central nervous system (CNS) and are major players in many CNS diseases. Research on astrocyte-centered diseases requires efficient and well-characterized gene transfer vectors. Vectors derived from the Adeno-associated virus serotype 9 (AAV9) target astrocytes in the brains of rodents and nonhuman primates. A recombinant (r) synthetic peptide-displaying AAV9 variant, rAAV9P1, that efficiently and selectively transduces cultured human astrocytes, has been described previously. Here, it is shown that rAAV9P1 retains astrocyte-targeting properties upon intravenous injection in mice. Detailed analysis of putative receptors on human astrocytes shows that rAAV9P1 utilizes integrin subunits αv, ß8, and either ß3 or ß5 as well as the AAV receptor AAVR. This receptor pattern is distinct from that of vectors derived from wildtype AAV2 or AAV9. Furthermore, a CRISPR/Cas9 genome-wide knockout screening revealed the involvement of several astrocyte-associated intracellular signaling pathways in the transduction of human astrocytes by rAAV9P1. This study delineates the unique receptor and intracellular pathway signatures utilized by rAAV9P1 for targeting human astrocytes. These results enhance the understanding of the transduction biology of synthetic rAAV vectors for astrocytes and can promote the development of advanced astrocyte-selective gene delivery vehicles for research and clinical applications.
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Astrocitos , Vectores Genéticos , Animales , Astrocitos/metabolismo , Dependovirus/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Ratones , Transducción GenéticaRESUMEN
Despite extensive treatment, glioblastoma inevitably recurs, leading to an overall survival of around 16 months. Understanding why and how tumours resist to radio/chemotherapies is crucial to overcome this unmet oncological challenge. Primary and acquired resistance to Temozolomide (TMZ), the standard-of-care chemotherapeutic drug, have been the subjects of several studies. This work aimed to evaluate molecular and phenotypic changes occurring during and after TMZ treatment in a glioblastoma cell model, the U87MG. These initially TMZ-sensitive cells acquire long-lasting resistance even after removal of the drug. Transcriptomic analysis revealed that profound changes occurred between parental and resistant cells, particularly at the level of the integrin repertoire. Focusing on α5ß1 integrin, which we proposed earlier as a glioblastoma therapeutic target, we demonstrated that its expression was decreased in the presence of TMZ but restored after removal of the drug. In this glioblastoma model of recurrence, α5ß1 integrin plays an important role in the proliferation and migration of tumoral cells. We also demonstrated that reactivating p53 by MDM2 inhibitors concomitantly with the inhibition of this integrin in recurrent cells may overcome the TMZ resistance. Our results may explain some integrin-based targeted therapy failure as integrin expressions are highly switchable during the time of treatment. We also propose an alternative way to alter the viability of recurrent glioblastoma cells expressing a high level of α5ß1 integrin.
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Nanometer-scale properties of the extracellular matrix influence many biological processes, including cell motility. While much information is available for single-cell migration, to date, no knowledge exists on how the nanoscale presentation of extracellular matrix receptors influences collective cell migration. In wound healing, basal keratinocytes collectively migrate on a fibronectin-rich provisional basement membrane to re-epithelialize the injured skin. Among other receptors, the fibronectin receptor integrin α5ß1 plays a pivotal role in this process. Using a highly specific integrin α5ß1 peptidomimetic combined with nanopatterned hydrogels, we show that keratinocyte sheets regulate their migration ability at an optimal integrin α5ß1 nanospacing. This efficiency relies on the effective propagation of stresses within the cell monolayer independent of substrate stiffness. For the first time, this work highlights the importance of extracellular matrix receptor nanoscale organization required for efficient tissue regeneration.
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Movimiento Celular , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Integrina alfa5beta1/metabolismo , Queratinocitos/metabolismo , Mecanotransducción Celular , Nanoestructuras , Cicatrización de Heridas , Adhesión Celular , Técnicas de Cultivo de Célula , Proliferación Celular , Uniones Célula-Matriz/metabolismo , Células HaCaT , Humanos , Hidrogeles , Propiedades de Superficie , Factores de TiempoRESUMEN
A major deficit in tissue engineering strategies is the lack of materials that promote angiogenesis, wherein endothelial cells from the host vasculature invade the implanted matrix to form new blood vessels. To determine the material properties that regulate angiogenesis, we have developed a microfluidic in vitro model in which chemokine-guided endothelial cell sprouting into a tunable hydrogel is followed by the formation of perfusable lumens. We show that long, perfusable tubes only develop if hydrogel adhesiveness and degradability are fine-tuned to support the initial collective invasion of endothelial cells and, at the same time, allow for matrix remodeling to permit the opening of lumens. These studies provide a better understanding of how cell-matrix interactions regulate angiogenesis and, therefore, constitute an important step towards optimal design criteria for tissue-engineered materials that require vascularization.
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Matriz Extracelular/química , Hidrogeles/química , Neovascularización Fisiológica , Ingeniería de Tejidos/métodos , Adhesividad , Dextranos/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Dispositivos Laboratorio en un Chip , Metacrilatos/química , Sulfonas/química , Ingeniería de Tejidos/instrumentaciónRESUMEN
Over recent years, αvß6 and αvß8 Arg-Gly-Asp (RGD) integrins have risen to prominence as interchangeable co-receptors for the cellular entry of herpes simplex virus-1 (HSV-1). In fact, the employment of subtype-specific integrin-neutralizing antibodies or gene-silencing siRNAs has emerged as a valuable strategy for impairing HSV infectivity. Here, we shift the focus to a more affordable pharmaceutical approach based on small RGD-containing cyclic pentapeptides. Starting from our recently developed αvß6-preferential peptide [RGD-Chg-E]-CONH2 (1), a small library of N-methylated derivatives (2-6) was indeed synthesized in the attempt to increase its affinity toward αvß8. Among the novel compounds, [RGD-Chg-(NMe)E]-CONH2 (6) turned out to be a potent αvß6/αvß8 binder and a promising inhibitor of HSV entry through an integrin-dependent mechanism. Furthermore, the renewed selectivity profile of 6 was fully rationalized by a NMR/molecular modeling combined approach, providing novel valuable hints for the design of RGD integrin ligands with the desired specificity profile.
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Antígenos de Neoplasias/química , Herpesvirus Humano 1/fisiología , Integrinas/química , Ligandos , Oligopéptidos/química , Péptidos Cíclicos/química , Antígenos de Neoplasias/metabolismo , Sitios de Unión , Células HEK293 , Humanos , Integrinas/metabolismo , Simulación del Acoplamiento Molecular , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/uso terapéutico , Unión Proteica , Internalización del Virus/efectos de los fármacosRESUMEN
Integrins have been extensively investigated as therapeutic targets over the last decades, which has been inspired by their multiple functions in cancer progression, metastasis, and angiogenesis as well as a continuously expanding number of other diseases, e.g., sepsis, fibrosis, and viral infections, possibly also Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2). Although integrin-targeted (cancer) therapy trials did not meet the high expectations yet, integrins are still valid and promising targets due to their elevated expression and surface accessibility on diseased cells. Thus, for the future successful clinical translation of integrin-targeted compounds, revisited and innovative treatment strategies have to be explored based on accumulated knowledge of integrin biology. For this, refined approaches are demanded aiming at alternative and improved preclinical models, optimized selectivity and pharmacological properties of integrin ligands, as well as more sophisticated treatment protocols considering dose fine-tuning of compounds. Moreover, integrin ligands exert high accuracy in disease monitoring as diagnostic molecular imaging tools, enabling patient selection for individualized integrin-targeted therapy. The present review comprehensively analyzes the state-of-the-art knowledge on the roles of RGD-binding integrin subtypes in cancer and non-cancerous diseases and outlines the latest achievements in the design and development of synthetic ligands and their application in biomedical, translational, and molecular imaging approaches. Indeed, substantial progress has already been made, including advanced ligand designs, numerous elaborated pre-clinical and first-in-human studies, while the discovery of novel applications for integrin ligands remains to be explored.
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High levels of reactive oxygen species (ROS) in tumors have been shown to exert anti-tumor activity, leading to the concept of ROS induction as therapeutic strategy. The organometallic compound ferrocene (Fc) generates ROS through a reversible one-electron oxidation. Incorporation of Fc into a tumor-targeting, bioactive molecule can enhance its therapeutic activity and enable tumor specific delivery. Therefore, we conjugated Fc to five synthetic, Arg-Gly-Asp (RGD)-based integrin binding ligands to enable targeting of the cell adhesion and signaling receptor integrin subtypes αvß3, α5ß1, or αvß6, which are overexpressed in various, distinct tumors. We designed and synthesized a library of integrin-ligand-ferrocene (ILF) derivatives and showed that ILF conjugates maintained the high integrin affinity and selectivity of their parent ligands. A thorough biological characterization allowed us to identify the two most promising ligands, an αvß3 (L2b) and an αvß6 (L3b) targeting ILF, which displayed selective integrin-dependent cell uptake and pronounced ferrocene-mediated anti-tumor effects in vitro, along with increased ROS production and DNA damage. Hence, ILFs are promising candidates for the selective, tumor-targeted delivery of ferrocene to maximize its anti-cancer efficacy and minimize systemic toxicity, thereby improving the therapeutic window of ferrocene compared to currently used non-selective anti-cancer drugs.
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Integrinas , Neoplasias , Humanos , Integrina alfa5beta1 , Integrina alfaVbeta3 , Ligandos , Metalocenos , Neoplasias/tratamiento farmacológicoRESUMEN
Over the last decades, several techniques have been developed to study cell adhesion; however, they present significant shortcomings. Such techniques mostly focus on strong adhesion related to specific protein-protein associations, such as ligand-receptor binding in focal adhesions. Therefore, weak adhesion, related to less specific or nonspecific cell-substrate interactions, are rarely addressed. Hence, we propose in this work a complete investigation of cell adhesion, from highly specific to nonspecific adhesiveness, using variable-angle total internal reflection fluorescence (vaTIRF) nanoscopy. This technique allows us to map in real time cell topography with a nanometric axial resolution, along with cell cortex refractive index. These two key parameters allow us to distinguish high and low adhesive cell-substrate contacts. Furthermore, vaTIRF provides cell-substrate binding energy, thus revealing a correlation between cell contractility and cell-substrate binding energy. Here, we highlight the quantitative measurements achieved by vaTIRF on U87MG glioma cells expressing different amounts of α 5 integrins and distinct motility on fibronectin. Regarding integrin expression level, data extracted from vaTIRF measurements, such as the number and size of high adhesive contacts per cell, corroborate the adhesiveness of U87MG cells as intended. Interestingly enough, we found that cells overexpressing α 5 integrins present a higher contractility and lower adhesion energy.
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PURPOSE: Increased angiogenesis after myocardial infarction is considered an important favorable prognostic parameter. The αvß3 integrin is a key mediator of cell-cell and cell-matrix interactions and an important molecular target for imaging of neovasculature and repair processes after MI. Thus, imaging of αvß3 expression might provide a novel biomarker for assessment of myocardial angiogenesis as a prognostic marker of left ventricular remodeling after MI. Currently, there is limited data available regarding the association of myocardial blood flow and αvß3 integrin expression after myocardial infarction in humans. METHODS: Twelve patients were examined 31 ± 14 days after MI with PET/CT using [18F]Galacto-RGD and [13N]NH3 and with cardiac MRI including late enhancement on the same day. Normal myocardium (remote) and areas of infarction (lesion) were identified on the [18F]Galacto-RGD PET/CT images by correlation with [13N]NH3 PET and cardiac MRI. Lesion/liver-, lesion/blood-, and lesion/remote ratios were calculated. Blood flow and [18F]Galacto-RGD uptake were quantified and correlated for each myocardial segment (AHA 17-segment model). RESULTS: In 5 patients, increased [18F]Galacto-RGD uptake was notable within or adjacent to the infarction areas with a lesion/remote ratio of 46% (26-83%; lesion/blood 1.15 ± 0.06; lesion/liver 0.61 ± 0.18). [18F]Galacto-RGD uptake correlated significantly with infarct size (R = 0.73; p = 0.016). Moreover, it correlated significantly with restricted blood flow for all myocardial segments (R = - 0.39; p < 0.0001) and even stronger in severely hypoperfused areas (R = - 0.75; p < 0.0001). CONCLUSION: [18F]Galacto-RGD PET/CT allows the visualization and quantification of myocardial αvß3 expression as a key player in angiogenesis in a subset of patients after MI. αvß3 expression was more pronounced in patients with larger infarcts and was generally more intense but not restricted to areas with more impaired blood flow, proving that tracer uptake was largely independent of unspecific perfusion effects. Based on these promising results, larger prospective studies are warranted to evaluate the potential of αvß3 imaging for assessment of myocardial angiogenesis and prediction of ventricular remodeling.
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Integrina alfaVbeta3 , Infarto del Miocardio , Humanos , Imagen por Resonancia Magnética , Infarto del Miocardio/diagnóstico por imagen , Miocardio , Tomografía Computarizada por Tomografía de Emisión de Positrones , Tomografía de Emisión de Positrones , Estudios ProspectivosRESUMEN
Here we present the use of surface nanopatterning of covalently immobilized BMP-2 and integrin selective ligands to determine the specificity of their interactions in regulating cell adhesion and focal adhesion assembly. Gold nanoparticle arrays carrying single BMP-2 dimers are prepared by block-copolymer micellar nanolithography and azide-functionalized integrin ligands (cyclic-RGD peptides or α5ß1 integrin peptidomimetics) are immobilized on the surrounding polyethylene glycol alkyne by click chemistry. Compared to BMP-2 added to the media, surface immobilized BMP-2 (iBMP-2) favors the spatial segregation of adhesion clusters and enhances focal adhesion (FA) size in cells adhering to α5ß1 integrin selective ligands. Moreover, iBMP-2 copresented with α5ß1 integrin ligands induces the recruitment of αvß3 integrins in FAs. When copresented with RGD, iBMP-2 induces the assembly of a higher number of FAs, which are not affected by α5ß1 integrin blocking. Our dual-functionalized platforms offer the possibility to study the crosstalk between integrins and BMP receptors, and more in general they could be used to address the spatial regulation of growth factors and adhesion receptors crosstalk on biomimetic surfaces.
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Oro , Nanopartículas del Metal , Adhesión Celular , Integrina alfa5beta1 , Integrina alfaVbeta3 , LigandosRESUMEN
Interactions between U2AF homology motifs (UHMs) and U2AF ligand motifs (ULMs) play a crucial role in early spliceosome assembly in eukaryotic gene regulation. UHM-ULM interactions mediate heterodimerization of the constitutive splicing factors U2AF65 and U2AF35 and between other splicing factors that regulate spliceosome assembly at the 3' splice site, where UHM domains of alternative splicing factors, such as SPF45 and PUF60, contribute to alternative splicing regulation. Here, we performed high-throughput screening using fluorescence polarization assays with hit validation by NMR and identified phenothiazines as general inhibitors of UHM-ULM interactions. NMR studies show that these compounds occupy the tryptophan binding pocket of UHM domains. Co-crystal structures of the inhibitors with the PUF60 UHM domain and medicinal chemistry provide structure-activity-relationships and reveal functional groups important for binding. These inhibitors inhibit early spliceosome assembly on pre-mRNA substrates in vitro. Our data show that spliceosome assembly can be inhibited by targeting UHM-ULM interactions by small molecules, thus extending the toolkit of splicing modulators for structural and biochemical studies of the spliceosome and splicing regulation.
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Fenotiazinas/química , Fenotiazinas/farmacología , Empalmosomas/efectos de los fármacos , Empalmosomas/metabolismo , Empalme Alternativo , Humanos , Unión Proteica/efectos de los fármacos , Dominios Proteicos , Precursores del ARN/genética , Precursores del ARN/metabolismo , Factores de Empalme de ARN/química , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Empalmosomas/genética , Factor de Empalme U2AF/química , Factor de Empalme U2AF/genética , Factor de Empalme U2AF/metabolismoRESUMEN
Intestinal transport and sensing processes and their interconnection to metabolism are relevant to pathologies such as malabsorption syndromes, inflammatory diseases, obesity and type 2 diabetes. Constituting a highly selective barrier, intestinal epithelial cells absorb, metabolize, and release nutrients into the circulation, hence serving as gatekeeper of nutrient availability and metabolic health for the whole organism. Next to nutrient transport and sensing functions, intestinal transporters including peptide transporter 1 (PEPT1) are involved in the absorption of drugs and prodrugs, including certain inhibitors of angiotensin-converting enzyme, protease inhibitors, antivirals, and peptidomimetics like ß-lactam antibiotics. Here, we verify the applicability of 3D organoids for in vitro investigation of intestinal biochemical processes related to transport and metabolism of nutrients and drugs. Establishing a variety of methodologies including illustration of transporter-mediated nutrient and drug uptake and metabolomics approaches, we highlight intestinal organoids as robust and reliable tool in this field of research. Currently used in vitro models to study intestinal nutrient absorption, drug transport and enterocyte metabolism, such as Caco-2 cells or rodent explant models are of limited value due to their cancer and non-human origin, respectively. Particularly species differences result in poorly correlative data and findings obtained in these models cannot be extrapolated reliably to humans, as indicated by high failure rates in drug development pipelines. In contrast, human intestinal organoids represent a superior model of the intestinal epithelium and might help to implement the 3Rs (Reduction, Refinement and Replacement) principle in basic science as well as the preclinical and regulatory setup.
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Neoatherosclerosis is defined as foamy macrophage infiltration into the peri-strut or neointimal area after stent implantation, potentially leading to late stent failure through progressive atherosclerotic changes including calcification, fibroatheroma, thin-cap fibroatheroma, and rupture with stent thrombosis (ST) in advanced stages. Human autopsy as well as intravascular imaging studies have led to the understanding of neoatherosclerosis formation as a similar but significantly accelerated pathophysiology as compared to native atherosclerosis. This acceleration is mainly based on disrupted endothelial integrity with insufficient barrier function and augmented transmigration of lipids following vascular injury after coronary intervention and especially after implantation of drug-eluting stents. In this review, we summarize translational insights into disease pathophysiology and discuss therapeutic approaches to tackle this novel disease entity. We introduce a novel animal model of neoatherosclerosis alongside accompanying in vitro experiments, which show impaired endothelial integrity causing increased permeability for low-density lipoprotein cholesterol resulting in foam cell transformation of human monocytes. In addition, we discuss novel intravascular imaging surrogates to improve reliable diagnosis of early stage neoatherosclerosis. Finally, a therapeutic approach to prevent in-stent neoatherosclerosis with magnesium-based bioresorbable scaffolds and systemic statin treatment demonstrated the potential to improve arterial healing and re-endothelialization, leading to significantly mitigated neoatherosclerosis formation in an animal model of neoatherosclerosis.
La neoateroesclerosis se define como infiltración de macrófagos espumosos en la zona periprotésica o de la neoíntima tras una implantación de stent, lo cual posiblemente derive en un fracaso tardío del stent mediante cambios ateroescleróticos progresivos, incluidos la calcificación, fibroateromas, fibroateromas de cápsula fina (FACF) y trombosis del stent (TS). Gracias a los estudios de autopsia humana y de imagen intravascular se ha podido comprender la formación de la neoateroesclerosis de una manera fisiopatológica similar a la ateroesclerosis nativa pero significativamente acelerada. Esta aceleración se basa principalmente en la alteración de la integridad endotelial con una función de barrera insuficiente y una mayor transmigración de lípidos a consecuencia de una lesión vascular tras una intervención coronaria y, especialmente, tras la implantación de stents farmacoactivos. En este artículo ofrecemos un resumen de las perspectivas translacionales sobre la fisiopatología de la enfermedad y analizamos los enfoques terapéuticos para abordar esta nueva enfermedad. Presentamos un modelo animal de neoateroesclerosis innovador junto con experimentos in vitro complementarios, en los cuales se pone de manifiesto que la integridad endotelial dañada causa una mayor permeabilidad para el colesterol de las LDL (LDL), lo que da lugar a que los monocitos se transformen en células espumosas. Asimismo, comentamos los criterios indirectos de valoración de imagen intravascular a fin de mejorar el diagnóstico fiable de la neoateroesclerosis en fase inicial. Por último, en un enfoque terapéutico para prevenir la neoateroesclerosis del stent con andamios de magnesio biorreabsorbibles (BRS) y un tratamiento sistémico con estatinas se demostró la posibilidad de mejorar la cicatrización y la reendotelización arteriales, lo que derivó en la formación de neoateroesclerosis significativamente más lenta en un modelo animal de neoateroesclerosis.