Material-driven fibronectin fibrillogenesis to engineer cell function

This thesis ventures with the extracellular matrix protein (ECM) fibronectin (FN) as an interface protein in the interaction between cells and materials to design microenvironment for future use in tissue engineering. It is studied the FN adsorption and conformations, cell behaviour to different FN conformation, cell adhesion, reorganisation and remodelling of FN at the material interface, the role of growth factors (GF) and their interactions with components of the extracellular matrix (ECM), the immunology cell response, and the stem cell fate influenced by the extrinsic signals coming from the engineered microenvironments using ECM's proteins. To investigate the FN response, in terms of adsorbed amount and conformation to different chemical properties of the material, model surfaces were used. Self assembled monolayers (SAM) with different percentages of two different chemical groups were used: CH3 and OH. FN adsorption, initial cell adhesion and signalling (focal adhesions, integrin expression and phosphorylation of FAK) is related with the reorganisation and secretion of FN and matrix degradation. It is shown that matrix degradation at the cell material interface depends on surface chemistry in metalloproteinase-dependent way. A direct relationship between FN activity at the cell-material interface and metalloproteinase 9 (MMP9) expression was found, being the product of a sequence of events that include integrin expression, focal adhesion formation, matrix reorganisation and focal adhesion kinase (FAK) phosphorylation. Two different materials with subtle variations in their chemical composition were employed as a drastically different FN conformation: from a globular conformation on PMA (poly (methyl acrylate)) to the formation of a well-interconnected FN network (similar to the FN physiological fibrillar network) triggered by PEA (poly (ethyl acrylate)). The formation of focal adhesions (vinculin), FAK expression and phosphorylation, specific integrin binding, protein and gene expression for ¿5 and ¿v was studied, seeking to correlate cell adhesion with matrix degradation. It is demonstrated that the material-driven FN fibrillogenesis on PEA triggers proteolytic activity: MMP activity is higher as a compensatory mechanism to the inability of cells to reorganise this FN network. Looking into the role of protein-material interactions and stem cell fate, and with the knowledge on PEA, we engineer different synergistic microenvironments to direct cell and stem cell fate. FN has a growth factor (GF) binding domain on its molecule (FNIII12-14) and has been demonstrated to produce a synergistic response when occurs at the same time the recognition of the cell binding domain (FNIII9-10). It is demonstrated that this domain is available on the FN coated PEA, and exploiting these interactions between PEA, FN and GF, it is developed a microenvironment to control cell behaviour and tissue repair. It is studied the BMP2 binding and presentation, the effect of BMP2 presentation on MSC proliferation and differentiation. These systems allow not only enhanced activity of GF compared to soluble administration, but also reduce GF doses, improving safety and cost effectiveness. Finally, the immunological reaction of the microenvironment developed is studied using dendritic cells, beside the conformational structure of ECM protein importance in DC integrin-based activation it is studied, helping to establish the field of adhesion-based modulation of DC as a general mechanism that has previously not been defined. The microenvironment didn't induce any maturation in DC, while different FN conformation shows differences in DC morphology and citokine level production (IL-10 and IL-12).En esta tesis se estudia la interacción de una proteina de la matriz extracelular, fibronectina (FN) como interfase en la interacción entre células y materiales, para diseñar microambientes con el propósito de ser usados en el futuro en ingeniería tisular. Se estudia la adsorción y conformación de FN y la relación con el diferente comportamiento celular: la adhesión celular, la reorganización y remodelado de la FN en la interfase célula-material, el papel que juegan los factores de crecimiento y sus interacciones con los componentes de la matriz extracelular, la respuesta immunológica y el destino celular de células madre influenciadas por las señales extrínsecas provenientes de microambientes elaborados a partir de proteínas de la matriz extracelular. Con el objetivo de investigar la respuesta a la FN en términos de conformación y cantidad absorbida a diferentes propiedades químicas del material, se usaron materiales modelo: monocapas autoensambladas (self-assembled monolayers, SAM). Las químicas estudiadas fueron CH3 and OH. La adsorption de FN, adhesion y señalización (adhesiones focales, expresión de interinas y fosforilación de quinasas de adhesiones focales (FAK)) se estudiaron en relación a la reorganización y secreción de FN y degradación de la matriz extracelular. Se demuestra que la degradación de la matriz extracelular en la interfase célula-material depende de la química de la superficie, a través de las metaloproteinasas. Se ha descubierto una relación directa entre la actividad de la FN que se encuentra en el material y la expresión de metaloproteinasa 9 (MMP9), a través de la expresión de integrinas, formación de adhesiones focales, reorganización de la matriz extracelular y fosforilación de FAK En el siguiente capítulo se emplean materiales poliméricos con una sutil diferencia en la composición química, provocando una diferencia drástica en la conformación de la FN: se pasa de una conformación globular en PMA (polimetil acrilato) a una conformación en forma de red interconectada en PEA (polietil acrilato). Con el propósito de relacionar la adhesión celular con la degradación de la matriz extracelular, se estudia la formación de adhesiones focales (vinculina), la expresión y fosforilación de FAK, la unión específica de integrinas y la expresión de las integrinas ¿5 and ¿v. Se demuestra que la formación de una red de FN sobre PEA induce la actividad proteolítica: la actividad de las MMPs es mayor, actuando como mecanismo compensatorio a la incapacidad de reorganización de la red de FN. Haciendo uso de la conformación de la FN sobre PEA, se estudiaron las interacciones entre la proteína-material y el destino celular de células madres. La FN posee un dominio de unión de factores de crecimiento (FNIII12-14) y se ha demostrado que se produce una respuesta sinérgica cuando el reconocimiento ocurre junto con el dominio de unión celular (FNIII9-10). En esta tesis se demuestra que el dominio de unión de factores de crecimiento está disponible en la conformación que adquiere sobre PEA y se diseñan microambientes para controlar el comportamiento celular y regeneración de tejido. Se estudia la unión y presentación de BMP2 y su efecto en la diferenciación de células madre mesenquimales. Los microambientes desarrollados, ademas de mejorar la actividad de los factores de crecimiento comparado con la administración soluble, también reduce la cantidad de factores de crecimiento que se tendría que administrar, mejorando la seguridad y efectividad. Finalmente se estudió la reacción inmunológica a los microambientes desarrollados usando células dendríticas, estudiando además la influencia de la estructura de la conformación de las proteínas en la activación de las células dendríticas a través de las integrinas. Los microambientes no indujeron ninguna maduración de células dendríticas, mientras que la conformación de la FN muestra controlEn aquesta tesi s'estudia la interacció entre una proteïna de la matriu extracel.lular, fibronectina (FN) com interfase en la interaccio entre cèl·lules i materials, per a dissenyar microambients amb el propòsit d'utilitzar-se al futur en enginyeria tissular. S'estudia l'adsorció i conformació de la FN i la relació amb el diferent comportament cel·lular: l'adhesió cel·lular, la reorganització i remodelat de la FN a la interfase cèl·lula-material, el paper que juguen els factors de creixement i les seus interaccions amb els components de la matriu extracel·lular, la resposta immunològica i el destí cel·lular de cèl·lules mare influenciades pels senyals extrínseques provinents de microambients elaborats a partir de proteïnes de la matriu extracel·lular. Amb l'objectiu d'investigar la respostar a la FN en termes de conformació i quantitat absorbida a diferents propietats químiques del material, s'utilitzaren materials model: monocapes autoacoblades (self-assembled monolayers, SAM). Les químiques estudiades van ser CH3 and OH. L'absorció de FN, adhesió i senyalització (adhesions focals, expressió d'integrines i fosforilació de quinases d'adhesions focals (FAK)) es van estudiar en relació a al reorganització i secreció de la FN i degradació de la matriu extracel·lular. Es demostra que la degradació de la matriu extracelular en la interfase cèl·lula-material depèn de la química de la superficie, a través de les metal·loproteïnases. S'ha descobert una relació directa entra l'activitat de la FN que es troba en el material i l'expressió de metaloproteinasa 9, a través de l'expressió d'integrines, formació d'adhesions focals, reorganització de la matriu extracel·lular i fosforilació de FAK. Al següent capítol es fan servir materials polimèrics amb una subtil diferència en la composició química, provocant una diferència dràstica en la conformació de la FN: es passa d'una conformació globular en PMA (polimetil acrilat) a una conformació en forma de xarxa interconnectada en PEA (polietil acrilat). Amb el propòsit de relacionar l'adhesió cel·lular amb la degradació de la matriu extracel·lular, s'estudia la formació d'adhesions focals (vinculina), l'expressió i fosforilació de FAK, la unió específica d'integrines i l'expressió de les integrines ¿5 and ¿v. Es demostra que la formació d'una xarxa de FN sobre PEA indueix l'activitat proteolítica: l'activitat de les MMPs és més gran, actuant com a mecanisme compensatori a la incapacitat de reorganització de la xarxa de FN. Fent ús de la conformació de la FN sobre PEA, es van estudiar les interaccions entre la proteïna-material i el destí cel·lular de cèl·lules mares. La FN posseeix un domini d'unió de factors de creixement (FNIII12-14) i s'ha demostrat que es produeix una resposta sinèrgica quan el reconeixement ocurreix juntament amb el domini d'unió cel·lular (FNIII9- 10). En aquesta tesi es demostra que el domini d'unió de factors de creixement està disponible a la conformació que adquireix sobre PEA i es dissenyen microambients per controlar el comportament cel·lular i regeneració de teixit. S'estudia la unió i presentació de BMP2 i el seu efecte en la diferenciació de cèl·lules mare mesenquimals. Els microambientes desenvolupats, a més de millorar l'activitat dels factors de creixement comparat amb l'administració soluble, també redueix la quantitat de factors de creixement que s'hauria d'administrar, millorant la seguretat i efectivitat. Finalment es va estudiar la reacció immunològica als microambients desenvolupats usant cèl·lules dendrítiques, estudiant a més la influència de l'estructura de la conformació de les proteïnes en l'activació de les cèl·lules dendrítiques a través de les integrines. Els microambients no van induir cap maduració de cèl·lules dendrítiques, mentre que la conformació de la FN mostra controlar la morfologia de les cèl·lules dendrítiques iLlopis Hernández, V. (2017). Material-driven fibronectin fibrillogenesis to engineer cell function [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90412TESI

Role of Surface Chemistry in Protein Remodeling at the Cell-Material Interface

Background: The cell-material interaction is a complex bi-directional and dynamic process that mimics to a certain extent the natural interactions of cells with the extracellular matrix. Cells tend to adhere and rearrange adsorbed extracellular matrix (ECM) proteins on the material surface in a fibril-like pattern. Afterwards, the ECM undergoes proteolytic degradation, which is a mechanism for the removal of the excess ECM usually approximated with remodeling. ECM remodeling is a dynamic process that consists of two opposite events: assembly and degradation. Methodology/Principal Findings: This work investigates matrix protein dynamics on mixed self-assembled monolayers (SAMs) of –OH and –CH3 terminated alkanethiols. SAMs assembled on gold are highly ordered organic surfaces able to provide different chemical functionalities and well-controlled surface properties. Fibronectin (FN) was adsorbed on the different surfaces and quantified in terms of the adsorbed surface density, distribution and conformation. Initial cell adhesion and signaling on FN-coated SAMs were characterized via the formation of focal adhesions, integrin expression and phosphorylation of FAKs. Afterwards, the reorganization and secretion of FN was assessed. Finally, matrix degradation was followed via the expression of matrix metalloproteinases MMP2 and MMP9 and correlated with Runx2 levels. We show that matrix degradation at the cell material interface depends on surface chemistry in MMP-dependent way. Conclusions/Significance: This work provides a broad overview of matrix remodeling at the cell-material interface, establishing correlations between surface chemistry, FN adsorption, cell adhesion and signaling, matrix reorganization and degradation. The reported findings improve our understanding of the role of surface chemistry as a key parameter in the design of new biomaterials. It demonstrates the ability of surface chemistry to direct proteolytic routes at the cell-material interface, which gains a distinct bioengineering interest as a new tool to trigger matrix degradation in different biomedical applications

Material-driven fibronectin assembly for high-efficiency presentation of growth factors

Growth factors (GFs) are powerful signaling molecules with the potential to drive regenerative strategies, including bone repair and vascularization. However, GFs are typically delivered in soluble format at supraphysiological doses because of rapid clearance and limited therapeutic impact. These high doses have serious side effects and are expensive. Although it is well established that GF interactions with extracellular matrix proteins such as fibronectin control GF presentation and activity, a translation-ready approach to unlocking GF potential has not been realized. We demonstrate a simple, robust, and controlled material-based approach to enhance the activity of GFs during tissue healing. The underlying mechanism is based on spontaneous fibrillar organization of fibronectin driven by adsorption onto the polymer poly(ethyl acrylate). Fibrillar fibronectin on this polymer, but not a globular conformation obtained on control polymers, promotes synergistic presentation of integrin-binding sites and bound bone morphogenetic protein 2 (BMP-2), which enhances mesenchymal stem cell osteogenesis in vitro and drives full regeneration of a nonhealing bone defect in vivo at low GF concentrations. This simple and translatable technology could unlock the full regenerative potential of GF therapies while improving safety and cost-effectiveness

Neutrophil gelatinase-associated lipocalin is a biomarker of acute-on-chronic liver failure and prognosis in cirrhosis

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is a syndrome that occurs in cirrhosis characterized by organ failure(s) and high mortality rate. There are no biomarkers of ACLF. The LCN2 gene and its product, neutrophil gelatinase-associated lipocalin (NGAL), are upregulated in experimental models of liver injury and cultured hepatocytes as a result of injury by toxins or proinflammatory cytokines, particularly Interleukin-6. The aim of this study was to investigate whether NGAL could be a biomarker of ACLF and whether LCN2 gene may be upregulated in the liver in ACLF. METHODS: We analyzed urine and plasma NGAL levels in 716 patients hospitalized for complications of cirrhosis, 148 with ACLF. LCN2 expression was assessed in liver biopsies from 29 additional patients with decompensated cirrhosis with and without ACLF. RESULTS: Urine NGAL was markedly increased in ACLF vs. no ACLF patients (108(35-400) vs. 29(12-73)μg/g creatinine; p<0.001) and was an independent predictive factor of ACLF; the independent association persisted after adjustment for kidney function or exclusion of variables present in ACLF definition. Urine NGAL was also an independent predictive factor of 28day transplant-free mortality together with MELD score and leukocyte count (AUROC 0.88(0.83-0.92)). Urine NGAL improved significantly the accuracy of MELD in predicting prognosis. The LCN2 gene was markedly upregulated in the liver of patients with ACLF. Gene expression correlated directly with serum bilirubin and INR (r=0.79; p<0.001 and r=0.67; p<0.001), MELD (r=0.68; p<0.001) and Interleukin-6 (r=0.65; p<0.001). CONCLUSIONS: NGAL is a biomarker of ACLF and prognosis and correlates with liver failure and systemic inflammation. There is remarkable overexpression of LCN2 gene in the liver in ACLF syndrome. LAY SUMMARY: Urine NGAL is a biomarker of acute-on-chronic liver failure (ACLF). NGAL is a protein that may be expressed in several tissues in response to injury. The protein is filtered by the kidneys due to its small size and can be measured in the urine. Ariza, Graupera and colleagues found in a series of 716 patients with cirrhosis that urine NGAL was markedly increased in patients with ACLF and correlated with prognosis. Moreover, gene coding NGAL was markedly overexpressed in the liver tissue in ACLF

Remodelado de la matriz extracelular en la interfase célula--¿proteína--¿material

[ES] La respuesta celular a los sustratos sintéticos puede ser controlada a través de los fenómenos que ocurren en la interfase célula-material. Uno de los fenómenos menos estudiados es el remodelado de las proteínas de la matriz en dicha interfase. La adhesión de las células sobre los diferentes sustratos sintéticos no se produce de forma directa entre las células y los materiales, tienen que estar presentes proteínas de la matriz extracelular (ECM) como lo son fibronectina (FN), vitronectina, laminina, etc. La conformación de dichas proteínas es muy importante y va a influir en la posterior respuesta celular, ya que dependiendo de ella unos dominios u otros de estas proteínas van a quedar o no expuestos, fomentando la adhesión celular y permitiendo la interacción con los receptores celulares, como las integrinas dando lugar a la formación de complejos de adhesión focal que anclan las células a la superficie y dan lugar a la respuesta celular. De esta interacción inicial depende la respuesta celular que haya posteriormente y de ella la capacidad que tengan las células para reorganizar la capa de proteínas adsorbidas en la superficie de un material. El trabajo consistirá en comprender el remodelado de la matriz extracelular en la interfase célula-material sobre materiales de química superficial ligeramente diferente (poliacrilato de etilo y poliacrilato de metilo), los cuales dan lugar a una conformación muy diferente de la FN causando diferentes respuestas celulares iniciales. Para esto se ha estudiado la adsorción de la FN sobre los diferentes sustratos a dos concentraciones diferentes (2 y 20 ¿g/ml) a las cuales se ha visto que la distribución y conformación es también dependiente de ésta. Se cuantificó la cantidad de FN adsorbida mediante western blot (WB) y la conformación de dicha proteína haciendo uso de un anticuerpo (cuyo epítopo es uno de los dominio de unión celular) mediante ELISA. Dado que la adsorción de la FN depende mucho en la respuesta celular inicial se estudió la formación de adhesiones focales mediante inmunofluorescencia de vinculina y la señalización celular ocurrida tras esta mediante la expresión proteica de FAK (kinasa de adhesión focal) y pFAK. Como estudios más detallados, los que dependen de los ya comentados, se investigaron las diferencias en la expresión de integrina ¿5 mediante la técnica de entrecruzamiento y extracción, seguida de inmuofluorescencia, así como su expresión proteica (WB) y génica (RT-PCR), al igual que la integrina ¿v. Finalmente, se correlacionó estos resultados con la expresión génica y actividad de las metaloproteinasas de la matriz (analizadas mediante PCR y zimografía respectivamente). Todos los resultados nos hacen ver como conclusión general que la adsorción de proteínas es un proceso muy importante que va a tener implicaciones en la respuesta celular y por lo tanto en la actividad celular en cuanto a degradación de la matriz extracelular.[EN] Matrix remodeling at the cell-material interface is an important issue to direct cell behaviour on biomaterials for regenetive medicine and tissue engineering. Cell adhesion on different synthetic substrates does not occur directly between cells and materials, but it is mediated by extracellular matrix proteins (ECM) such as fibronectin (FN), vitronectin, laminin, etc. somehow immobilized on the materials surface Conformation attained by these proteins after adsorption is of outmost importance to trigger subsequent cellular responses. So, protein conformation will define which proteins domains are available for cell adhesion, promoting the activation of cell receptors, integrins, and leading to the formation of focal adhesion complexes which anchor the cells to the material surface. Afterwards, cells tend to rearrange the adsorbed protein layer on the surface of a material, secrete new matrix and degrade it, a process called remodeling. This work includes cell remodeling of the extracellular matrix at the cell-material interface using substrates of slightly different surface chemistry (poly(ethyl acrylate) and poly(methyl acrylate)), which give rise to a very different conformation of FN causing different initial cell responses. To do so we have studied FN adsorption on different substrates at two different concentrations (2 and 20 ¿g/ml), which results in different distribution and conformation of the protein at the material interface. The amount of adsorbed FN was analysed by western blot (WB) and the conformation of the protein by ELISA using an antibody whose epitope is one of the cell-binding domain. As FN adsorption determines the initial cellular response, we have studied the formation of focal adhesions (vinculin) by immunofluorescence and subsequent cell signalling by FAK expression (focal adhesion kinase) and its phosphorylation pFAK. More detailed studies were performed to get further insights into integrin expression by using integrin crosslinking and extraction followed by immunofluorescence, as well as protein expression (WB), and gene expression (RT-PCR) for ¿5 and ¿v, to correlate cell adhesion with matrix degradation, characterized by gene expression and the activity of matrix metalloproteinases (analysed by PCR and zymography, respectively). All results conclude that protein adsorption is a very important process that will have implications in theLlopis Hernández, V. (2012). Remodelado de la matriz extracelular en la interfase célula--¿proteína--¿material. http://hdl.handle.net/10251/27431Archivo delegad

Role of material-driven fibronectin fibrillogenesis in protein remodeling

Protein remodeling at the cell–material interface is an important phenomenon that should be incorporated into the design of advanced biomaterials for tissue engineering. In this work, we address the relationship between fibronectin (FN) activity at the material interface and remodeling, including proteolytic cascades. To do so, we studied FN adsorption on two chemically similar substrates, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA), which resulted in different distribution and conformation of the protein at the material interface: FN organized spontaneously upon adsorption on PEA into physiological-like fibrils, through a process called material-driven FN fibrillogenesis. The amount of adsorbed FN and its conformation were investigated in two different coating concentrations (2 and 20 μg/mL). Since FN activity at the material interface determines the initial cellular response, we followed the formation of focal adhesions (vinculin) and subsequent cell signaling by focal adhesion kinase (FAK) expression and its phosphorylation (pFAK). More detailed studies were performed to get further insights into integrin binding by crosslinking and extraction followed by immunofluorescence, as well as protein and gene expression for α&lt;sub&gt;5&lt;/sub&gt; and α&lt;sub&gt;v&lt;/sub&gt;. To correlate cell adhesion with matrix degradation, gene expression and activity (zymography) of matrix metalloproteinases (MMPs) were measured. Overall, we demonstrated that the material-driven FN fibrillogenesis triggers proteolytic activity: MMP activity was higher on the material-driven FN fibrils, as a compensatory mechanism to the inability of cells to reorganize this FN network

Adiciones y correcciones a la orquidoflora valenciana, IX

Some data about rare orchid taxa at the Valencian Community (Spain) or expansions of their distribution area to new shires are shown; The confirmed presence of Dactylorhiza maculata for the Valencian Community must be emphasized. KSe aportan datos sobre algunos táxones escasos de la familia Orchidaceae en la Comunidad Valenciana o ampliaciones de área a nuevas comarcas; a destacar la confirmación de la presencia de Dactylorhiza maculata en la Comunidad Valencian

Prevalence of Depression and Fish Consumption among First Year Spanish University Students: UniHcos Project

The World Health Organization estimates that one fifth of university students have experienced major depressive disorder at some point in their lives. Nutrition may be one of the modifiable factors that influence the development of depression. Specifically, low omega-3 fatty acid and vitamin D levels, both nutrients found in high quantities in fish, have been linked to depressive disorders. The main objective of this study was to evaluate the prevalence of depression among young Spanish university students, in addition to the pattern of fish consumption among students and the possible relationship between fish consumption and the presence of depression. Data were collected retrospectively from a nationally representative sample of 11,485 Spanish university students aged 18 years or older in 11 Spanish universities, from 2012 to 2022. The respondents were analyzed according to frequency of consumption and compliance with weekly recommendations for fish intake and the presence of depression. Regression models were also performed to determine students’ odds of depression as a function of compliance with recommendations according to selected sociodemographic variables. The prevalence of depression was 10.5%; it was more prevalent in women, older students and in those with both high and low BMIs. In addition, it was also more prevalent in those that lived outside the family home, with roommates and those who were employed. Sixty-seven percent of the students met the fish intake recommendations. The most common frequency of fish consumption was 1–2 times/week (44.2%), and the least frequent was 2.3% daily fish consumption. Students from northern universities were more likely to consume fish (68.4%) than those from southern universities (66.4%). Non-consumption of fish was found to increase the risk of depression (ORa = 1.45 (1.28–1.64); AF = 31.0% (21.9–39.0)), but it was the student’s own conditions that had the greatest influence on the development of the disorder. In summary, a lower consumption of fish seems to be associated with a higher incidence of depression in Spanish university students; however, other social factors of the student may influence the development of the disorder, and all of this should be taken into account for the development of prevention strategies

Subtle variations in polymer chemistry modulate substrate stiffness and fibronectin activity

A family of polymer substrates which consists of a vinyl backbone chain with the side groups –COO(CH&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;x&lt;/sub&gt;CH&lt;sub&gt;3&lt;/sub&gt;, with x = 0, 1, 3, 5 was prepared. Substrates with decreasing stiffness, characterised by the elastic modulus at 37 °C, and similar chemical groups were obtained. Firstly, we have investigated whether these minute variations in polymer chemistry lead to differences in fibronectin (FN) adsorption: the same FN density was obtained on every substrate (450 ng cm−&lt;sup&gt;2&lt;/sup&gt;) but the supramolecular organisation of the protein at the material interface, as obtained with AFM, was different for x = 0 and the other surfaces (x = 1, 3, 5). Consequently, this allows one to use a set of substrates (x = 1, 3, 5) to investigate the effect of substrate stiffness on cell behavior as the unique physical parameter, i.e. after ruling out any influence of the length of the side group on protein conformation. Moreover, the importance of investigating the intermediate layer of proteins at the cell-material interface is stressed: the effect of x = 0 and x = 1 on cell behavior cannot be ascribed to the different stiffness of the substrate anymore, since the biological activity of the protein on the material surface was also different. Afterwards, initial cellular interaction was investigated using MC3T3-E1 osteoblasts-like cells and focusing on actin cytoskeleton development, focal adhesion formation and the ability of cells to reorganize the adsorbed FN layer on the different substrates. Image analysis was used to quantify the frequency distribution of the focal plaques, which revealed broader distributions on the stiffer substrates, with formation of larger focal plaques revealing that cells exert higher forces on stiffer substrates

Neutrophil gelatinase-associated lipocalin is a biomarker of acute-on-chronic liver failure and prognosis in cirrhosis

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is a syndrome that occurs in cirrhosis characterized by organ failure(s) and high mortality rate. There are no biomarkers of ACLF. The LCN2 gene and its product, neutrophil gelatinase-associated lipocalin (NGAL), are upregulated in experimental models of liver injury and cultured hepatocytes as a result of injury by toxins or proinflammatory cytokines, particularly Interleukin-6. The aim of this study was to investigate whether NGAL could be a biomarker of ACLF and whether LCN2 gene may be upregulated in the liver in ACLF. METHODS: We analyzed urine and plasma NGAL levels in 716 patients hospitalized for complications of cirrhosis, 148 with ACLF. LCN2 expression was assessed in liver biopsies from 29 additional patients with decompensated cirrhosis with and without ACLF. RESULTS: Urine NGAL was markedly increased in ACLF vs. no ACLF patients (108(35-400) vs. 29(12-73)μg/g creatinine; p<0.001) and was an independent predictive factor of ACLF; the independent association persisted after adjustment for kidney function or exclusion of variables present in ACLF definition. Urine NGAL was also an independent predictive factor of 28day transplant-free mortality together with MELD score and leukocyte count (AUROC 0.88(0.83-0.92)). Urine NGAL improved significantly the accuracy of MELD in predicting prognosis. The LCN2 gene was markedly upregulated in the liver of patients with ACLF. Gene expression correlated directly with serum bilirubin and INR (r=0.79; p<0.001 and r=0.67; p<0.001), MELD (r=0.68; p<0.001) and Interleukin-6 (r=0.65; p<0.001). CONCLUSIONS: NGAL is a biomarker of ACLF and prognosis and correlates with liver failure and systemic inflammation. There is remarkable overexpression of LCN2 gene in the liver in ACLF syndrome. LAY SUMMARY: Urine NGAL is a biomarker of acute-on-chronic liver failure (ACLF). NGAL is a protein that may be expressed in several tissues in response to injury. The protein is filtered by the kidneys due to its small size and can be measured in the urine. Ariza, Graupera and colleagues found in a series of 716 patients with cirrhosis that urine NGAL was markedly increased in patients with ACLF and correlated with prognosis. Moreover, gene coding NGAL was markedly overexpressed in the liver tissue in ACLF