Trends in the design and use of elastin-like recombinamers as biomaterials

Producción CientíficaElastin-like recombinamers (ELRs), which derive from one of the repetitive domains found in natural elastin, have been intensively studied in the last few years from several points of view. In this mini review, we discuss all the recent works related to the investigation of ELRs, starting with those that define these polypeptides as model intrinsically disordered proteins or regions (IDPs or IDRs) and its relevance for some biomedical applications. Furthermore, we summarize the current knowledge on the development of drug, vaccine and gene delivery systems based on ELRs, while also emphasizing the use of ELR-based hydrogels in tissue engineering and regenerative medicine (TERM). Finally, we show different studies that explore applications in other fields, and several examples that describe biomaterial blends in which ELRs have a key role. This review aims to give an overview of the recent advances regarding ELRs and to encourage further investigation of their properties and applications.Comisión Europea (project NMP-2014-646075)Ministerio de Economía, Industria y Competitividad (projects PCIN-2015-010 / MAT2016-78903-R / BES-2014-069763)Junta de Castilla y León (project VA317P18

Bioengineered dynamic systems based on elastin-like recombinamers

La ingeniería tisular y la medicina regenerativa son campos interdisciplinares que tienen como objetivo desarrollar sustitutos biológicos que restauren, mantengan o mejoren la función de un tejido o de un órgano perdida por lesión o enfermedad. Los biomateriales utilizados en aplicaciones biomédicas persiguen alcanzar características esenciales que promuevan la nueva morfogénesis tisular creando un entorno favorable para el crecimiento celular, así como las propiedades físicas y químicas adecuadas requeridas para un tejido determinado. En décadas pasadas, se han encontrado varios obstáculos para seleccionar biomateriales funcionales que imiten la matriz extracelular natural (MEC). A la inversa, los polímeros naturales derivados de proteínas de la matriz extracelular ofrecen la ventaja de la señalización biológica, pero pueden ser antigénicas y sus propiedades físicas son más difíciles de modular que en los polímeros sintéticos. La biodegradación es una característica crucial en los implantes biomédicos y su modulación ha sido ampliamente estudiada. Presentar un tiempo de degradación sincronizado con la formación de los nuevos tejidos es un objetivo altamente perseguido. La tasa de degradación está influenciada por muchas características de los polímeros, incluida la morfología y la orientación de la cadena, la cristalinidad inicial, el peso molecular, la presencia de monómeros y oligómeros residuales y la pureza. Existen varios métodos para cambiar y controlar la velocidad de degradación de los polímeros sintéticos y naturales, como la copolimerización, la mezcla, los aditivos y la irradiación, pero estos métodos presentan diferentes inconvenientes, como el cambio en las propiedades mecánicas, la pobre biocompatibilidad y el retraso en la bioabsorción. Debido a estos inconvenientes, puede ser útil producir biomateriales que sean sensibles a ciertos enzimas que se valgan de la respuesta inflamatoria que se produce por la propia lesión o introducción del implante. Los avances en ingeniería genética han facilitado la producción de proteínas modificadas que abarcan una amplia gama de propiedades y funcionalidades, como la adhesión celular, la señalización celular, la elasticidad y la biodegradabilidad. Actualmente, los recombinamers de tipo elastina (ELR) muestran propiedades sobresalientes para su aplicación en regeneración de tejidos. Los ELR son polipéptidos artificiales con un alto contenido en elastina que los convirtió en materiales capaces de imitar la ECM. Tienen interesantes propiedades mecánicas que van desde los elastómeros ideales hasta el plástico que permiten, por lo tanto, un esfuerzo mecánico repetitivo y confieren resistencia mecánica a través de un mecanismo separado que soporta varias funciones del tejido. Por último, son extremadamente biocompatibles debido a su composición aminoácidica que implica la repetición de secuencias basadas en las secuencias encontradas en la elastina de los mamíferos. En el primer estudio de esta tesis se emplearon dos ELRs con una bioactividad complementaria (CC-RGD y CCC-REDV), las cisteínas están incluidas en la región amino terminal, para desarrollar un sistema 2D en el que la adhesión celular selectiva está controlada espacialmente para su posterior aplicación en la tecnología de biosensores como dispositivos de diagnóstico o en la aplicación de ingeniería de tejidos y medicina regenerativa. Específicamente, en este estudio, las superficies modelo de oro se funcionalizaron con diferentes porcentajes de dos ELRs (CC-RGD y CCC-REDV), lo que demuestra que una composición óptima de 75% CCC-REDV y 25% CC-RGD conduce a una adhesión selectiva de células endoteliales en un sistema de co-cultivo. Luego, una vez que se funcionalizó toda la superficie con 100% de CC-RGD, se limpiaron áreas específicas mediante ablación con láser sin interferir en las propiedades topográficas, hidrófobas y mecánicas de las superficies. Estas áreas se funcionalizaron con un 75% de CCC-REDV y un 25% de CC-RGD y después de los estudios in vitro, se observó una distribución bien definida de células HUVEC y HFF1, es decir, un control espacial selectivo sobre la adhesión de las células HUVEC. Estos hallazgos sugieren un enfoque innovador para la generación de biosensores y / o andamios que requieren la organización espacial y temporal de las células. A continuación, el segundo estudio de esta tesis consiste en la mejora de la actividad angiogénica de los hidrogeles basados en ELR cuando se pega químicamente un pequeño péptido llamado QK que mima el factor de acrecimiento VEGF. Además de las secuencias de adhesión celular (RGD y REDV), un ELR utilizado en este trabajo presenta un sitio proteolítico (VGVAPG) que pertenece a la enzima elastasa. La actividad angiogénica de estos hidrogeles se estudió in vitro, demostrando que no altera las propiedades mecánicas y morfológicas de los ELR, sino la actividad biológica, como la adhesión y proliferación celular. Cuando se inyectó in vivo, se observó una mejor formación de microvasculatura que facilita la perfusión y la conexión con el tejido circundante. Estos resultados mostraron que los hidrogeles basados en ELR-QK son construcciones diseñadas óptimas con una potencial actividad angiogénica que podría emplearse en ingeniería de tejidos y medicina regenerativa para el tratamiento de enfermedades cardiovasculares. El tercer estudio de esta tesis tiene como objetivo lo de controlar en tiempo y espacio la infiltración célular y la biodegradación en un sistema estructurado tridimensional (3D). Los ELR empleados en este trabajo, además de una secuencia de adhesión celular universal (RGD), contienen sitios proteolíticos que se degradan en respuesta al activador de plasminógeno uroquinasa (uPA) con diferente tasa de degradación (rápida y lenta). Aprovechando estas características, en este estudio se desarrolló un sistema 3D que consiste en discos de hidrogeles de tres capas, con una capa interna que contiene el componente de degradación rápida y las capas externas que contienen el ELR de degradación lenta. Esos discos se han implantado por vía subcutánea en ratones y su evolución se siguió a lo largo del tiempo. Como resultado del diseño y de la secuencia de infiltración celular programada, la capa interna se colonizó primero siguiendo un patrón de adentro hacia afuera. Estos resultados demostraron que el sistema basado en 3D-ELR podría aplicarse como un sustituto tisular para la aplicación biomédica que requiere una regeneración controlada y vascularización a lo largo del tiempo. En resumen, los estudios mostrados en esta Tesis brindan nuevos conocimientos sobre los hidrogeles basados en ELR que se utilizarán en ingeniería de tejidos y medicina regenerativa.Departamento de Bioquímica y Biología Molecular y FisiologíaDoctorado en Investigación Biomédic

Use of proteolytic sequences with different cleavage kinetics as a way to generate hydrogels with preprogrammed cell-infiltration patterns imparted over their given 3D spatial structure

Producción CientíficaControl over biodegradation processes is crucial to generate advanced functional structures with a more interactive and efficient role for biomedical applications. Herein, a simple, high-throughput approach is developed based on a 3D-structured system that allows a preprogramed spatial-temporal control over cell infiltration and biodegradation. The 3D-structured system is based on elastin-like recombinamers (ELRs) characterized by differences in the kinetics of their peptide cleavage and consists of a three-layer hydrogel disk comprising an internal layer containing a rapidly degrading component, with the external layers containing a slow-degrading ELR. This structure is intended to invert the conventional pattern of cell infiltration, which goes from the outside to the inside of the implant, to allow an anti-natural process in which infiltration takes place first in the internal layer and later progresses to the outer layers. Time-course in vivo studies proved this hypothesis, i.e. that it is possible to drive the infiltration of cells over time in a given 3D-structured implant in a controlled and predesigned way that is able to overcome the natural tendency of conventional cell infiltration. The results obtained herein open up the possibility of applying this concept to more complex systems with multiple biological functions.European Commission (NMP-2014-646075, PITNGA-2012-317306)Ministerio de Economía, Industria y Competitividad ( grants PCIN-2015-010, MAT2015-68901-R, MAT2016-78903-R, MAT2016-79435-R)Junta de Castilla y León (VA015U16)Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó

Tethering QK peptide to enhance angiogenesis in elastin-like recombinamer (ELR) hydrogels

Producción CientíficaThe development of new capillary networks in engineered constructs is essential for their survival and their integration with the host tissue. It has recently been demonstrated that ELR-based hydrogels encoding different bioactivities are able to modulate their interaction with the host after injection or implantation, as indicated by an increase in cell adhesion and the ability to trigger vascularization processes. Accordingly, the aim of this study was to increase their angiogenic ability both in vitro and in vivo using a small VEGF mimetic peptide named QK, which was tethered chemically to ELR-based hydrogels containing cell-adhesion sequences in their backbone, such as REDV and RGD, as well as a proteolytic site (VGVAPG). In vitro studies were performed using a co-culture of endothelial and fibroblast cells encapsulated into the ELR-based hydrogels in order to determine cell proliferation after 21 days of culture, as well as the number of cell-cell interactions. It was found that although the presence of this peptide does not influence the morphological and rheological properties of these hydrogels, it has an effect on cell behaviour, inducing an increase in cell proliferation and the formation of endothelial cell clusters. In vivo studies demonstrate that the QK peptide enhances the formation of prominent functional capillaries at three weeks post-injection, as confirmed by H&E staining and CD31 immunohistochemistry. The newly formed functional microvasculature ensures perfusion and connection with surrounding tissues. These results show that ELR-QK hydrogels increase capillary network formation and are therefore attractive candidates for application in tissue regeneration, for example for the treatment of cardiovascular diseases such as myocardial infarction or ischemia.European Commission (NMP-2014-646075, PITN-GA-2012-317306)Ministerio de Economía, Industria y Competitividad (Projects PCIN-2015-010, MAT2015- 68901-R, MAT2016-78903-R)Junta de Castilla y León (programa de apoyo a proyectos de investigación - Ref. VA015U16)Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó

Spatial control and cell adhesion selectivity on model gold surfaces grafted with elastin-like recombinamers

Producción CientíficaA simple system for cell selectivity and spatially controlled cell adhesion has been developed using model gold surfaces grafted with a combination of two ELRs containing into their backbone cell-adhesion domains such as RGD and REDV. Grafting onto gold was achieved via redox reaction through thiol groups present in amino terminal cysteine tails of the ELRs. The correlation among contact angle, SEM micrographs, AFM, XPS and QCM-D have been carried out. After in-depth adhesion studies, a mixture of 75% ELR-REDV and 25% ELR-RGD was found to exhibit high selectivity for endothelial cells, promoting strong adhesion thereof. Consequently, certain areas of gold surfaces (strips) were cleaned by laser ablation and functionalized with the mixture 75% ELR-REDV - 25% ELR-RGD leading to a spatial segregation of the co-culture made of HUVEC and HFF1 cells. This platform therefore exhibits selective spatial control over cell adhesion associated with the bioactive epitopes (RGD and REDV) contained in the ELR sequence, since each functionalized surface (including strips) have similar topographic, hydrophobic and mechanical properties.2020-092020-09Comisión Europea (NMP-2014-646075, MSCA-ITN-2014-642687)Ministerio de Economía, Industria y Competitividad (PCIN-2015-010, MAT2015-68901-R, MAT2016-78903-R and MAT2016-79435-R)Junta de Castilla y León (programa de apoyo a proyectos de investigación - Ref. Project VA015U16)Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó

ELASTIN-LIKE RECOMBINAMERS FOR MULTI-MODAL DRUG DELIVERY SYSTEMS

Elastin is a protein with a key role in most of mammalian tissues and it is widely expressed in the extracellular matrix present over myocardium, cartilage and skin. Its elastogenic activity relies on the main cellular components of the tissue network, for instance, endothelial cells, fibroblasts, chondrocytes, and keratinocytes [1]. Nevertheless, the human elastin is naturally synthesized in early age, leading to a drawback based on the low availability, due to the stop in the natural synthetic mechanism with ageing. A clever strategy to overcome such an issue is based on the development of genetically-engineered elastin-mimicking peptides fabrication, so-called elastin-like recombinamers (ELRs), thus balancing the low availability of natural elastin and tuning the biomaterial structuring and behaviour. Relevant advances in the field can derive from the investigation of the morphological, mechanical, in-vitro and delivery-related properties of ELRs-based systems, fabricated in the form of either hydrogel or microspheres. Different scaffold constructs are studied herein, i.e., microspheres, hydrogel and microsphere integrated hydrogel in order to assess their delivery suitability and thoroughly understand the hierarchical complex structuring of the elastin-like recombinamer self- assembly mechanisms. We used two ELRs (1-HRGD6-cyclooctyne, 2-REDV-N3) modified with the two different reactive groups needed to form hydrogels via a click reaction and functionalized with two different bioactive sequences RGD and REDV that would promote cell adhesion. In this study the most stable and optimal concentration ratio of ELRs based hollow spheres exhibited no reduction in cellular metabolic activity. The sacrificial template-based method allowed us to engineer hollow spheres with a first layer of the ELRs HRGD6-component followed by a second layer of the ELRs REDV-component, by means of copper free click-chemistry reaction. The ELRs hollow spheres-tethered ELRs hydrogel was prepared by adding the pre-fabricated ELRs hollow spheres. The hydrogel construct was characterized by rheology, NMR, and Synchrotron Radiation SAXS (SRSAXS). Hollow spheres were characterized by TEM, SEM, DLS and FT-IR. Drug upload and release were assessed by means of ELISA, confocal microscopy and all constructs were successfully tested for cell metabolic activity, revealing no cytotoxicity. ELR-based hollow microspheres were fabricated and successfully entrapped into an ELR- hydrogel matrix. Release studies have been performed, determining the ELRs platform suitability as drug delivery system

Identification of additional risk loci for stroke and small vessel disease: a meta-analysis of genome-wide association studies

BACKGROUND: Genetic determinants of stroke, the leading neurological cause of death and disability, are poorly understood and have seldom been explored in the general population. Our aim was to identify additional loci for stroke by doing a meta-analysis of genome-wide association studies. METHODS: For the discovery sample, we did a genome-wide analysis of common genetic variants associated with incident stroke risk in 18 population-based cohorts comprising 84 961 participants, of whom 4348 had stroke. Stroke diagnosis was ascertained and validated by the study investigators. Mean age at stroke ranged from 45·8 years to 76·4 years, and data collection in the studies took place between 1948 and 2013. We did validation analyses for variants yielding a significant association (at p<5 × 10(-6)) with all-stroke, ischaemic stroke, cardioembolic ischaemic stroke, or non-cardioembolic ischaemic stroke in the largest available cross-sectional studies (70 804 participants, of whom 19 816 had stroke). Summary-level results of discovery and follow-up stages were combined using inverse-variance weighted fixed-effects meta-analysis, and in-silico lookups were done in stroke subtypes. For genome-wide significant findings (at p<5 × 10(-8)), we explored associations with additional cerebrovascular phenotypes and did functional experiments using conditional (inducible) deletion of the probable causal gene in mice. We also studied the expression of orthologs of this probable causal gene and its effects on cerebral vasculature in zebrafish mutants. FINDINGS: We replicated seven of eight known loci associated with risk for ischaemic stroke, and identified a novel locus at chromosome 6p25 (rs12204590, near FOXF2) associated with risk of all-stroke (odds ratio [OR] 1·08, 95% CI 1·05-1·12, p=1·48 × 10(-8); minor allele frequency 21%). The rs12204590 stroke risk allele was also associated with increased MRI-defined burden of white matter hyperintensity-a marker of cerebral small vessel disease-in stroke-free adults (n=21 079; p=0·0025). Consistently, young patients (aged 2-32 years) with segmental deletions of FOXF2 showed an extensive burden of white matter hyperintensity. Deletion of Foxf2 in adult mice resulted in cerebral infarction, reactive gliosis, and microhaemorrhage. The orthologs of FOXF2 in zebrafish (foxf2b and foxf2a) are expressed in brain pericytes and mutant foxf2b(-/-) cerebral vessels show decreased smooth muscle cell and pericyte coverage. INTERPRETATION: We identified common variants near FOXF2 that are associated with increased stroke susceptibility. Epidemiological and experimental data suggest that FOXF2 mediates this association, potentially via differentiation defects of cerebral vascular mural cells. Further expression studies in appropriate human tissues, and further functional experiments with long follow-up periods are needed to fully understand the underlying mechanisms

Long-term outcomes of the global tuberculosis and COVID-19 co-infection cohort

Background: Longitudinal cohort data of patients with tuberculosis (TB) and coronavirus disease 2019 (COVID-19) are lacking. In our global study, we describe long-term outcomes of patients affected by TB and COVID-19. Methods: We collected data from 174 centres in 31 countries on all patients affected by COVID-19 and TB between 1 March 2020 and 30 September 2022. Patients were followed-up until cure, death or end of cohort time. All patients had TB and COVID-19; for analysis purposes, deaths were attributed to TB, COVID-19 or both. Survival analysis was performed using Cox proportional risk-regression models, and the log-rank test was used to compare survival and mortality attributed to TB, COVID-19 or both. Results: Overall, 788 patients with COVID-19 and TB (active or sequelae) were recruited from 31 countries, and 10.8% (n=85) died during the observation period. Survival was significantly lower among patients whose death was attributed to TB and COVID-19 versus those dying because of either TB or COVID-19 alone (p&lt;0.001). Significant adjusted risk factors for TB mortality were higher age (hazard ratio (HR) 1.05, 95% CI 1.03-1.07), HIV infection (HR 2.29, 95% CI 1.02-5.16) and invasive ventilation (HR 4.28, 95% CI 2.34-7.83). For COVID-19 mortality, the adjusted risks were higher age (HR 1.03, 95% CI 1.02-1.04), male sex (HR 2.21, 95% CI 1.24-3.91), oxygen requirement (HR 7.93, 95% CI 3.44-18.26) and invasive ventilation (HR 2.19, 95% CI 1.36-3.53). Conclusions: In our global cohort, death was the outcome in &gt;10% of patients with TB and COVID-19. A range of demographic and clinical predictors are associated with adverse outcomes

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2

Worldwide trends in underweight and obesity from 1990 to 2022: a pooled analysis of 3663 population-representative studies with 222 million children, adolescents, and adults

Background Underweight and obesity are associated with adverse health outcomes throughout the life course. We estimated the individual and combined prevalence of underweight or thinness and obesity, and their changes, from 1990 to 2022 for adults and school-aged children and adolescents in 200 countries and territories. Methods We used data from 3663 population-based studies with 222 million participants that measured height and weight in representative samples of the general population. We used a Bayesian hierarchical model to estimate trends in the prevalence of different BMI categories, separately for adults (age ≥20 years) and school-aged children and adolescents (age 5–19 years), from 1990 to 2022 for 200 countries and territories. For adults, we report the individual and combined prevalence of underweight (BMI &lt;18·5 kg/m2) and obesity (BMI ≥30 kg/m2). For school&#x2;aged children and adolescents, we report thinness (BMI &lt;2 SD below the median of the WHO growth reference) and obesity (BMI &gt;2 SD above the median). Findings From 1990 to 2022, the combined prevalence of underweight and obesity in adults decreased in 11 countries (6%) for women and 17 (9%) for men with a posterior probability of at least 0·80 that the observed changes were true decreases. The combined prevalence increased in 162 countries (81%) for women and 140 countries (70%) for men with a posterior probability of at least 0·80. In 2022, the combined prevalence of underweight and obesity was highest in island nations in the Caribbean and Polynesia and Micronesia, and countries in the Middle East and north Africa. Obesity prevalence was higher than underweight with posterior probability of at least 0·80 in 177 countries (89%) for women and 145 (73%) for men in 2022, whereas the converse was true in 16 countries (8%) for women, and 39 (20%) for men. From 1990 to 2022, the combined prevalence of thinness and obesity decreased among girls in five countries (3%) and among boys in 15 countries (8%) with a posterior probability of at least 0·80, and increased among girls in 140 countries (70%) and boys in 137 countries (69%) with a posterior probability of at least 0·80. The countries with highest combined prevalence of thinness and obesity in school-aged children and adolescents in 2022 were in Polynesia and Micronesia and the Caribbean for both sexes, and Chile and Qatar for boys. Combined prevalence was also high in some countries in south Asia, such as India and Pakistan, where thinness remained prevalent despite having declined. In 2022, obesity in school-aged children and adolescents was more prevalent than thinness with a posterior probability of at least 0·80 among girls in 133 countries (67%) and boys in 125 countries (63%), whereas the converse was true in 35 countries (18%) and 42 countries (21%), respectively. In almost all countries for both adults and school-aged children and adolescents, the increases in double burden were driven by increases in obesity, and decreases in double burden by declining underweight or thinness. Interpretation The combined burden of underweight and obesity has increased in most countries, driven by an increase in obesity, while underweight and thinness remain prevalent in south Asia and parts of Africa. A healthy nutrition transition that enhances access to nutritious foods is needed to address the remaining burden of underweight while curbing and reversing the increase in obesit