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Scientific community have gained lots of new insights in the genetic and biochemical background of different conditions, rare diseases included, settling the basis for preclinical models that are helping to identify new biomarkers and therapeutic targets. Translational Medicine (TM) is an interdisciplinary area of biomedicine with an essential role in bench-to-bedside transition enhancement, generating a circular flow of knowledge transference between research environment and clinical setting, always centered in patient needs. Here, we present different tools used in TM and an overview of what is being done related to hereditary hemorrhagic telangiectasia (HHT), as a disease's model. This work is focused on how this combination of basic and clinical research impacts in HHT patient's daily clinical management and also looking into the future. Further randomized clinical trials with HHT patients should assess the findings of this bench-to-bedside transition. The benefits of this basic and clinical research combination, may not only be important for HHT patients but for patients with other vascular diseases sharing angiogenic disturbances.

Human basophils, first identified over 140 years ago, account for just 0.5-1% of circulating leukocytes. While this scarcity long hampered basophil studies, innovations during the past 30 years, beginning with their isolation and more recently in the development of mouse models, have markedly advanced our understanding of these cells. Although dissimilarities between human and mouse basophils persist, the overall findings highlight the growing importance of these cells in health and disease. Indeed, studies continue to support basophils as key participants in IgE-mediated reactions, where they infiltrate inflammatory lesions, release pro-inflammatory mediators (histamine, leukotriene C4: LTC4) and regulatory cytokines (IL-4, IL-13) central to the pathogenesis of allergic diseases. Studies now report basophils infiltrating various human cancers where they play diverse roles, either promoting or hampering tumorigenesis. Likewise, this activity bears remarkable similarity to the mounting evidence that basophils facilitate wound healing. In fact, both activities appear linked to the capacity of basophils to secrete IL-4/IL-13, with these cytokines polarizing macrophages toward the M2 phenotype. Basophils also secrete several angiogenic factors (vascular endothelial growth factor: VEGF-A, amphiregulin) consistent with these activities. In this review, we feature these newfound properties with the goal of unraveling the increasing importance of basophils in these diverse pathobiological processes.

Simple Summary Glioblastoma is the most aggressive primary brain tumor, with the highest incidence and the worst prognosis. Life expectancy from diagnosis remains dismal, at around 15 months, despite surgical resection and treatment with radiotherapy and chemotherapy. Given the aggressiveness of the tumor and the inefficiency of the treatments adopted to date, the scientific research investigates innovative therapeutic approaches. Importantly, angiogenesis represents one of the main features of glioblastoma, becoming in the last few years a major candidate for target therapy. Metformin, a well-established therapy for type 2 diabetes, offered excellent results in preventing and fighting tumor progression, particularly against angiogenic mechanisms. Therefore, the purpose of this review is to summarize and discuss experimental evidence of metformin anti-cancer efficacy, with the aim of proposing this totally safe and tolerable drug as add-on therapy against glioblastoma. Abstract Glioblastoma is the most common primitive tumor in adult central nervous system (CNS), classified as grade IV according to WHO 2016 classification. Glioblastoma shows a poor prognosis with an average survival of approximately 15 months, representing an extreme therapeutic challenge. One of its distinctive and aggressive features is aberrant angiogenesis, which drives tumor neovascularization, representing a promising candidate for molecular target therapy. Although several pre-clinical studies and clinical trials have shown promising results, anti-angiogenic drugs have not led to a significant improvement in overall survival (OS), suggesting the necessity of identifying novel therapeutic strategies. Metformin, an anti-hyperglycemic drug of the Biguanides family, used as first line treatment in Type 2 Diabetes Mellitus (T2DM), has demonstrated in vitro and in vivo antitumoral efficacy in many different tumors, including glioblastoma. From this evidence, a process of repurposing of the drug has begun, leading to the demonstration of inhibition of various oncopromoter mechanisms and, consequently, to the identification of the molecular pathways involved. Here, we review and discuss metformin’s potential antitumoral effects on glioblastoma, inspecting if it could properly act as an anti-angiogenic compound to be considered as a safely add-on therapy in the treatment and management of glioblastoma patients.

High-dose standard-of-care chemotherapy is the only option for triple-negative breast cancer (TNBC) patients, which eventually die due to metastatic tumors. Recently, metronomic chemotherapy (mCHT) showed advantages in treating TNBCs leading us to investigate the anti-metastatic and anti-angiogenic potential of metronomic 5-Fluorouracil plus Vinorelbine (5-FU+VNR) on endothelial cells (ECs) and TNBCs in comparison to standard treatment (STD). We found that 10-fold lower doses of 5-FU+VNR given mCHT vs. STD inhibits cell proliferation and survival of ECs and TNBC cells. Both schedules strongly affect ECs migration and invasion, but in TNBC cells mCHT is significantly more effective than STD in impairing cell migration and invasion. The two treatments disrupt FAK/VEGFR/VEGF signaling in both ECs and TNBC cells. mCHT, and to a much lesser extent STD treatment, induces apoptosis in ECs, whereas it switches the route of cell death from apoptosis (as induced by STD) to autophagy in TNBC cells. mCHT-treated TNBCs-derived conditioned medium also strongly affects ECs’ migration, modulates different angiogenesis-associated proteins, and hampers angiogenesis in matrix sponge in vivo. In conclusion, mCHT administration of 5-FU+VNR is more effective than STD schedule in controlling cell proliferation/survival and migration/invasion of both ECs and TNBC cells and has a strong anti-angiogenic effect. Our data suggest that the stabilization of tumor growth observed in TNBC patients treated with mCHT therapy schedule is likely due not only to direct cytotoxic effects but also to anti-metastatic and anti-angiogenic effects.

Osteoarthritis (OA) is a joint disease characterized by inflammation of the synovium, angiogenesis, cartilage degradation, and osteophyte formation. Harpagophytum Procumbens DC. ex Meisn., Boswellia Serrata Roxb., Curcuma longa L., Bromelain and Escin (Aesculus hippocastanum) are plants which extracts, together to Bromelain and Escin (Aesculus hippocastanum) are traditionally used in OA. However, their mechanistic role remains unclear. We aimed to investigate whether these bioactives alone or in combination (as in Flonat Fast®) can suppress TNF-α-induced inflammation, angiogenesis, and osteophyte formation using two cell models involved in OA: endothelial cells and monocytes. Each plant extract was evaluated for its polyphenol content, antioxidant activity, and toxicity. In endothelial cells and monocytes, expression of genes involved in OA was assessed, functional assays for inflammation and angiogenesis were performed, and impairment of reactive oxygen species production (ROS) was evaluated. Exposure of cells to the bioactives alone and in combination before cytokine stimulation resulted in differential counterregulation of several gene and protein expressions, including those for cyclooxygenases-2, metalloproteinase-9, transforming growth factor β1, and bone morphogenic protein-2. We demonstrated that these bioactives modulated monocyte adhesion to endothelial cells as well as cell migration and endothelial angiogenesis. Consistent with radical scavenging activity in the cell-free system, the bioactives curbed TNF-α-stimulated intracellular ROS production. We confirmed the potential anti-inflammatory and antiangiogenic effects of the combination of Harpagophytum procumbens, Boswellia, Curcuma, Bromelain, and Escin and provided new mechanistic evidence for their use in OA. However, further clinical studies are needed to evaluate the true clinical utility of these bioactives as supportive, preventive, and therapeutic agents.

Colorectal cancer; Hepatobiliary tumour; Neoangiogenesis Cáncer colorrectal; Tumores hepatobiliares; Neoangiogénesis Càncer colorectal; Tumors hepatobiliars; Neoangiogènesi Gastrointestinal tumours are a heterogeneous group of neoplasms that arise in the gastrointestinal tract and hepatobiliary system. Their incidence is rising globally and they currently represent the leading cause of cancer-related mortality worldwide. Anti-angiogenic agents have been incorporated into the treatment armamentarium of most of these malignancies and have improved survival outcomes, most notably in colorectal cancer and hepatocellular carcinoma. New treatment combinations with immunotherapies and other agents have led to unprecedented benefits and are revolutionising patient care. In this review, we detail the mechanisms of action of anti-angiogenic agents and the preclinical rationale underlying their combinations with immunotherapies. We review the clinical evidence supporting their use across all gastrointestinal tumours, with a particular emphasis on colorectal cancer and hepatocellular carcinoma. We discuss available biomarkers of response to these therapies and their utility in routine clinical practice. Finally, we summarise ongoing clinical trials in distinct settings and highlight the preclinical rationale supporting novel combinations.

Angiogenesis, a process characterized by the formation of new blood vessels from pre-existing ones, is a crucial step in tumor growth and dissemination. Given the ability of tumors to interfere with multiple or different molecular pathways to promote angiogenesis, there is an increasing need to therapeutically block tumor progression by targeting multiple antiangiogenic pathways. Natural polyphenols present health-protective properties, which are likely attributed to their ability to activate multiple pathways involved in inflammation, carcinogenesis, and angiogenesis. Recently, increased attention has been addressed to the ability of flavonoids, the most abundant polyphenols in the diet, to prevent cancer by suppressing angiogenesis. Here we investigate the mechanisms by which xanthohumol (the major prenylated flavonoid of the hop plant Humulus lupulus L.) and nobiletin (flavonoid from red-orange Citrus sinensis) can modulate the effects of Tumor Necrosis Factor-alpha (TNF-alpha) on human umbilical vein endothelial cells (HUVEC). The results reported in this paper show that xanthohumol and nobiletin pretreatment of HUVEC inhibits the effects induced by TNF-alpha on cell migration, invasion capability, and colon cancer cell adhesion on the endothelial monolayer. Moreover, the pretreatment reduces metalloproteinases and adhesion molecules' expression. Finally, our results highlight that xanthohumol and nobiletin can counteract the effects of TNF-alpha on angiogenesis and invasiveness, mainly through Vascular Endothelial Growth Factor and NF-kappa B pathways. Since angiogenesis plays an important pathological role in the progression of several diseases, our findings may provide clues for developing xanthohumol and nobiletin as therapeutic agents against angiogenesis-associated diseases.

Background: The type 2 deiodinase (DIO2) converts thyroxine to 3,3′,5-triiodothyronine (T3), modulating intracellular T3. An increase in DIO2 within muscle stem cells during skeletal muscle regeneration leads to T3-dependent potentiation of differentiation. The muscle stem cell niche comprises numerous cell types, which coordinate the regeneration process. For example, muscle stem cells provide secretory signals stimulating endothelial cell-mediated vascular repair, and, in turn, endothelial cells promote muscle stem differentiation. We hypothesized that Dio2 loss in muscle stem cells directly impairs muscle stem cell-endothelial cell communication, leading to downstream disruption of endothelial cell function. Methods: We assessed the production of proangiogenic factors in differentiated C2C12 cells and in a C2C12 cell line without Dio2 (D2KO C2C12) by real-time quantitative-polymerase chain reaction and enzyme-linked immunosorbent assay. Conditioned medium (CM) was collected daily in parallel to evaluate its effects on human umbilical vein endothelial cell (HUVEC) proliferation, migration and chemotaxis, and vascular network formation. The effects of T3-treatment on vascular endothelial growth factor (Vegfa) mRNA expression in C2C12 cells and mouse muscle were assessed. Chromatin immunoprecipitation (ChIP) identified thyroid hormone receptor (TR) binding to the Vegfa gene. Using mice with a targeted disruption of Dio2 (D2KO mice), we determined endothelial cell number by immunohistochemistry/flow cytometry and evaluated related gene expression in both uninjured and injured skeletal muscle. Results: In differentiated D2KO C2C12 cells, Vegfa expression was 46% of wildtype (WT) C2C12 cells, while secreted VEGF was 45%. D2KO C2C12 CM exhibited significantly less proangiogenic effects on HUVECs. In vitro and in vivo T3 treatment of C2C12 cells and WT mice, and ChIP using antibodies against TRα, indicated that Vegfa is a direct genomic T3 target. In uninjured D2KO soleus muscle, Vegfa expression was decreased by 28% compared with WT mice, while endothelial cell numbers were decreased by 48%. Seven days after skeletal muscle injury, D2KO mice had 36% fewer endothelial cells, coinciding with an 83% decrease in Vegfa expression in fluorescence-activated cell sorting purified muscle stem cells. Conclusion: Dio2 loss in the muscle stem cell impairs muscle stem cell-endothelial cell crosstalk via changes in the T3-responsive gene Vegfa, leading to downstream impairment of endothelial cell function both in vitro and in vivo.

Interactions of malignant multiple myeloma (MM) plasma cells with the microenvironment control MM plasma-cell growth, survival, drug-resistance and dissemination. As microvascular density increases in the bone marrow in MM, we investigated whether bone marrow MM endothelial cells control disease progression via the junctional adhesion molecule-A (JAM-A). Membrane and cytoplasmic JAM-A levels were upregulated in MM endothelial cells in 111 patients with newly diagnosed MM and in 201 with relapsed/refractory MM compared to the levels in patients with monoclonal gammopathy of undetermined significance and healthy controls. Elevated membrane expression of JAM-A on MM endothelial cells predicted poor clinical outcome. Mechanistically, addition of recombinant JAM-A to MM endothelial cells increased angiogenesis, whereas inhibition of this adhesion molecule impaired angiogenesis and MM growth in two-dimensional and three-dimensional in vitro cell cultures and chorioallantoic membrane assays. To corroborate these findings, we treated MM-bearing mice with a JAM-A-blocking monoclonal antibody and demonstrated impaired MM progression, corresponding to decreased MM-related vascularity. These findings support the concept that JAM-A is an important mediator of MM progression through facilitating MM-associated angiogenesis. Elevated JAM-A expression on bone marrow endothelial cells is an independent prognostic factor for the survival of both patients with newly diagnosed MM and those with relapsed/refractory MM. Blocking JAM-A restricts angiogenesis in vitro, in utero and in vivo and represents a suitable druggable molecule to halt neo-angiogenesis and MM progression.

[eng] Tumours grow in a self-imposed hypoglycaemic microenvironment due to their exacerbated metabolic needs. To thrive in these harsh conditions, malignant cells must adapt their own metabolic routes and hijack the host nutrient supply infrastructure through extracellular signalling. This study aimed at characterising the secretory response of tumoral cells in response to glucose deprivation, both the molecular signalling leading to this response and the consequences of this response for the tumour. In particular, this study focused on a cytokine never before linked to glucose deprivation: LIF. Through use of in silico, in vitro, and in vivo approaches we made two novel findings: firstly, we determined that LIF induction happens through non-canonical N-glycosylation dependent signalling, in the context of glucose deprivation. Furthermore, we have determined that the signalling mechanism leading to LIF production encompasses a complex combination of transcriptional and post-transcriptional signals that involve PERK but none of its canonical transcription factors. Secondly, we discovered a novel pro-tumorigenic role for this cytokine as an angiogenic. Our findings addressed the gap in literature regarding the mechanism for LIF production in tumours, providing insight relevant to future development of therapeutic approaches. And most importantly, we provide an integrative view of not only how glucose deprivation leads to production of LIF, but also how this cytokine could lead to the resolution of the local hypoglycaemia via angiogenesis., [spa] Los tumores imponen un microambiente de constante hipoglicemia debido a su rápida proliferación y al exacerbado consumo de metabolitos que esto conlleva. Para prosperar en estas circunstancias, las células cancerosas alteran su metabolismo y sus rutas de señalización de forma que puedan abusar de la infraestructura de aporte de nutrientes del organismo. El objetivo de este estudio es caracterizar la respuesta secretora del tumor en respuesta a la falta de glucosa, tanto al nivel de la señalización molecular que integra esta respuesta como al nivel de las consecuencias externas que estas señales tienen para el tumor. En particular, este estudio se centra en una citoquina que nunca antes se había relacionado con la falta de glucosa: LIF (por sus siglas en inglés: Factor Inhibidor de la Leucemia). A través del uso de técnicas in silico, in vitro e in vivo hemos alcanzado dos descubrimientos. En primer lugar, hemos determinado que la producción de LIF está inducida a través de una ruta no-canónica de detección de la falta de glucosa basada en la falta de N-glicosilación. Además, hemos determinado que el mecanismo que sigue a este fallo en la glicosilación incluye una compleja combinación de señales transcripcionales y postranscripcionales dependientes de PERK, pero no de los factores de transcripción tradicionalmente asociados a esta quinasa. En segundo lugar, hemos descubierto un nuevo rol pro-tumorigénico de esta citoquina como factor pro-angiogénico. Nuestros hallazgos cubren un vacío en la literatura en lo relativo al mecanismo que causa la producción de LIF por parte del tumor, lo cual podría servir de base para nuevas aproximaciones terapéuticas. Además, hemos desarrollado una visión unificada que explica cómo la falta de glucosa resulta en la producción de esta citoquina, y cómo LIF a su vez puede actuar para resolver la hipoglicemia local restaurando el riego sanguíneo mediante la angiogénesis.

Multiple sclerosis is a chronic disease of the central nervous system characterized by demyelination and destruction of axons. The most common form of the disease is the relapsing-remitting multiple sclerosis in which episodic attacks with typical neurological symptoms are followed by episodes of partial or complete recovery. One of the underestimated factors that contribute to the pathogenesis of multiple sclerosis is excessive angiogenesis. Here, we review the role of angiogenesis in the onset and in the development of the disease, the molecular mechanisms underlying angiogenesis, the current therapeutic approaches, and the potential therapeutic strategies with a look at natural compounds as multi-target drugs with both neuroprotective and anti-angiogenic properties.

Background Despite the increasing number of treatment options, reliable prognostic/predictive biomarkers are still missing for patients affected by metastatic clear cell renal cell carcinoma (mccRCC). Methods Patients with mccRCC undergoing standard first line treatment were enrolled. Blood (12 ml) was drawn at treatment baseline and circulating free DNA (cfDNA) was extracted from plasma. Next-generation sequencing (NGS) was performed on cfDNA using the Oncomine Pan-Cancer Cell-Free Assay and clinical outcomes were correlated with liquid biopsy findings. Results A total of 48 patients were enrolled, 12 received immunotherapy and 36 received a vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor (TKI). A cfDNA cut-off of 0.883 ng/μl stratified patients based on progression-free survival (PFS) and overall survival (OS) (p = 0.001 and p = 0.008, respectively). cfDNA amount was also correlated with best response (p = 0.006). Additional cfDNA cut-points divided patients into short, intermediate and long responders, with PFS of 4.87 vs 9.13 vs 23.1 months, respectively (p Conclusions The present study demonstrates that cfDNA and TP53 are potential predictive biomarkers of response in mccRCC to be further explored in larger and/or prospective studies.

L’angiogènesi, la formació de nous vasos sanguinis a partir dels preexistents, és essencial per al desenvolupament normal i juga un paper crucial en patologies com el càncer, la diabetis i l’aterosclerosi. Tot i una extensa investigació, molts dels aspectes de l’angiogènesi segueixen sense comprendre’s. Resultats experimentals recents indiquen que les cèl·lules endotelials, que recobreixen les parets internes dels vasos sanguinis, es reorganitzen dins dels vasos en creixement i que l’elongació dels brots està dominada per aquesta barreja de cèl·lules durant les primeres etapes del procés. S’ha demostrat que els reordenaments cel·lulars estan regulats per l’adaptació dinàmica de l’expressió gènica o el fenotip cel·lular. Tot i això, la majoria dels models teòrics d’angiogènesi no tenen en compte aquests fenòmens i, en canvi, assumeixen que les posicions de les cèl·lules són fixes i que el fenotip cel·lular és irreversible durant la germinació. En aquesta tesi, formulem un model multiescala de brotació angiogènica impulsada per reordenaments cel·lulars dinàmics. El nostre model explica la barreja cel·lular, que està regulada per un model estocàstic de senyalització subcel·lular vinculada al canvi de fenotip. Inicialment ens enfoquem en la brotació angiogènica primerenca, quan els efectes de la proliferació cel·lular són insignificants. Validem el nostre model contra les dades experimentals disponibles. A continuació, quantifiquem el reordenament cel·lular que ocorre durant la germinació i investiguem com canvia l’estructura de ramificació vascular en funció del nivell de mescla cel·lular. Els nostres resultats suggereixen que la barreja de cèl·lules afecta directament la morfologia de les vasculatures en creixement. En particular, els reordenaments de cèl·lules endotelials amb fenotips diferents poden generar canvis a l’estructura de la xarxa, ja que l’adaptació del fenotip cel·lular és més lenta que la migració cel·lular. La barreja cel·lular també contribueix a la remodelació de la matriu extracel·lular que, alhora, guia el creixement vascular. Per investigar els efectes de la proliferació cel·lular, que opera en escales temporals més llargues que la migració cel·lular, primer desenvolupem un mètode, basat en la teoria de grans desviacions, que ens permet reduir la complexitat computacional del nostre model multiescala híbrid fent coarse-graining de la dinàmica interna de les seves cèl·lules-agents. El mètode de coarse-graining (CG) és aplicable a sistemes en què el comportament dels agents es descriu amb sistemes estocàstics amb múltiples estats estacionaris estables. La tècnica CG redueix el sistema estocàstic original a un procés Markovià a l’espai dels seus estats estacionaris estables. El nostre mètode CG conserva la descripció original dels estats dels agents (en lloc de convertir-los en discrets) i les transicions estocàstiques entre ells, alhora que redueix considerablement la complexitat computacional de les simulacions de models. Després de formular el mètode CG per a una classe general de models híbrids, il·lustrem el seu potencial aplicant-ho al nostre model d’angiogènesi. Fem un CG del model subcel·lular, que determina l’especificació del fenotip cel·lular. Això redueix substancialment el cost computacional de les simulacions. Després, ampliem el nostre model per tenir en compte la proliferació cel·lular i el validem utilitzant les dades experimentals disponibles. Aquest marc ens permet estudiar el creixement de la xarxa en escales de temps associades amb l’angiogènesi in vivo i investigar com la variació de la taxa de proliferació cel·lular afecta el creixement de la xarxa. En resum, aquest treball proporciona una nova visió dels comportaments cel·lulars complexos que impulsen la germinació angiogènica. Alhora, avança en el camp de la modelització teòrica mitjançant la formulació del mètode CG, que aplana el camí per a una reducció sistemàtica dels models multiescala híbrids. La angiogénesis, la formación de nuevos vasos sanguíneos a partir de los preexistentes, es esencial para el desarrollo normal y juega un papel crucial en patologías como el cáncer, la diabetes y la aterosclerosis. A pesar de una extensa investigación, muchos de los aspectos de la angiogénesis siguen sin comprenderse. Resultados experimentales recientes indican que las células endoteliales, que recubren las paredes internas de los vasos sanguíneos, se reorganizan dentro de los vasos en crecimiento y que la elongación de los brotes está dominada por esta mezcla de células durante las primeras etapas del proceso. Se ha demostrado que los reordenamientos celulares están regulados por la adaptación dinámica de la expresión génica o el fenotipo celular. Sin embargo, la mayoría de los modelos teóricos de angiogénesis no tienen en cuenta estos fenómenos y, en cambio, asumen que las posiciones de las células son fijas y que el fenotipo celular es irreversible durante la germinación. En esta tesis, formulamos un modelo multiescala de brotación angiogénica impulsada por reordenamientos celulares dinámicos. Nuestro modelo explica la mezcla celular, que está regulada por un modelo estocástico de señalización subcelular vinculada al cambio de fenotipo. Inicialmente nos enfocamos en la brotación angiogénica temprana, cuando los efectos de la proliferación celular son insignificantes. Validamos nuestro modelo contra los datos experimentales disponibles. A continuación cuantificamos el reordenamiento celular que ocurre durante la germinación e investigamos cómo cambia la estructura de ramificación vascular en función del nivel de mezcla celular. Nuestros resultados sugieren que la mezcla de células afecta directamente la morfología de las vasculaturas en crecimiento. En particular, los reordenamientos de células endoteliales con fenotipos distintos pueden generar cambios en la estructura de la red, ya que la adaptación del fenotipo celular es más lenta que la migración celular. La mezcla celular también contribuye a la remodelación de la matriz extracelular que, a su vez, guía el crecimiento vascular. Con el fin de investigar los efectos de la proliferación celular, que opera en escalas temporales más largas que la migración celular, primero desarrollamos un método, basado en la teoría de las grandes desviaciones, que nos permite reducir la complejidad computacional de nuestro modelo multiescala híbrido mediante coarse-graining de la dinámica interna de sus células-agentes. El método de coarse-graining (CG) es aplicable a sistemas en los que el comportamiento de los agentes se describe mediante sistemas estocásticos con múltiples estados estacionarios estables. La técnica CG reduce el sistema estocástico original a un proceso Markoviano en el espacio de sus estados estacionarios estables. Nuestro método CG conserva la descripción original de los estados de los agentes (en lugar de convertirlos en discretos) y las transiciones estocásticas entre ellos, al tiempo que reduce considerablemente la complejidad computacional de las simulaciones de modelos. Después de formular el método CG para una clase general de modelos híbridos, ilustramos su potencial aplicándolo a nuestro modelo de angiogénesis. Hacemos un CG del modelo subcelular, que determina la especificación del fenotipo celular. Esto reduce sustancialmente el coste computacional de las simulaciones. Luego, ampliamos nuestro modelo para tener en cuenta la proliferación celular y lo validamos utilizando los datos experimentales disponibles. Este marco nos permite estudiar el crecimiento de la red en escalas de tiempo asociadas con la angiogénesis in vivo e investigar cómo la variación de la tasa de proliferación celular afecta el crecimiento de la red. En resumen, este trabajo proporciona una nueva visión de los comportamientos celulares complejos que impulsan la germinación angiogénica. Al mismo tiempo, avanza en el campo de la modelización teórica mediante la formulación del método CG, que allana el camino para una reducción sistemática de los modelos multiescala híbridos. Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for normal development and plays a crucial role in such pathologies as cancer, diabetes and atherosclerosis. In spite of extensive research, many aspects of how new vessels sprout from existing vasculature remain unclear. Recent experimental results indicate that endothelial cells, lining the inner walls of blood vessels, rearrange within growing vessels and that sprout elongation is dominated by cell mixing during the early stages of angiogenesis. Cell rearrangements have been shown to be regulated by dynamic adaptation of cell gene expression, or cell phenotype. However, most theoretical models of angiogenesis do not account for these phenomena and instead assume that cell positions are fixed and cell phenotype is irreversible during sprouting. In this thesis, we formulate a multiscale model of angiogenic sprouting driven by dynamic cell rearrangements. Our model accounts for cell mixing which is regulated by a stochastic model of subcellular signalling linked to phenotype switching. We initially focus on early angiogenic sprouting when the effects of cell proliferation are negligible. We validate our model against available experimental data. We then use it to develop a measure to quantify the amount of cell rearrangement that occurs during sprouting and investigate how the branching structure of vascular networks changes as the level of cell mixing varies. Our results suggest that cell shuffling directly affects the morphology of growing vasculatures. In particular, rearrangements of endothelial cells with distinct phenotypes can drive changes in the network structure since cell phenotype adaptation is slower than cell migration. Cell mixing also contributes to remodelling of the extracellular matrix which, in turn, guides vascular growth. In order to investigate the effects of cell proliferation, which operates on longer timescales than cell migration, we first develop a method, based on large deviation theory, which allows us to reduce the computational complexity of our hybrid multiscale model by coarse-graining the internal dynamics of its cell-agents. The coarse-graining (CG) method is applicable to systems in which agent behaviour is described by stochastic systems with multiple stable steady states. The CG technique reduces the original stochastic system to a Markov jump process on the space of its stable steady states. Our CG method preserves the original description of agent states (instead of converting them to discrete ones) and stochastic transitions between them, while considerably reducing the computational complexity of model simulations. After formulating the CG method for a general class of hybrid models, we illustrate its potential by applying it to our model of angiogenesis. We coarse-grain the subcellular model, which determines cell phenotype specification. This substantially reduces the computational cost of simulations. We then extend our model to account for cell proliferation and validate it using available experimental data. This framework allows us to study network growth on timescales associated with angiogenesis in vivo and to investigate how varying the cell proliferation rate affects network growth. Summarising, this work provides new insight into the complex cell behaviours that drive angiogenic sprouting. At the same time, it advances the field of theoretical modelling by formulating a coarse-graining method, which paves the way for a systematic reduction of hybrid multiscale models. Universitat Autònoma de Barcelona. Programa de Doctorat en Matemàtiques

ANTECEDENTS: L'endometriosi empitjora la qualitat de vida i causa dolor pelvià crònic, especialment en dones joves. El retard en el diagnòstic pot trigar de mitjana uns 7 anys a causa de la manca d'eines de diagnòstic precoç, cosa que negativament impacta en el benestar de les pacients i en la fertilitat, tenint a més, alts costos econòmics i socials. La recurrència és una altra prioritat en la investigació en endometriosi, ja que fins a un 40-50% de pacients intervingudes recorren durant els 5 anys posteriors a la cirurgia. Consegüentment, la detecció de pacients en risc de recurrència podria permetre adequar el seguiment postquirúrgic al risc de recurrència de cada pacient i individualitzar-ne els tractaments. Per tant, hi ha una necessitat d'identificació de noves tècniques que puguin proporcionar un diagnòstic precoç i eines pronòstiques per disminuir la comorbilitat associada. En aquest context, la nostra hipòtesi va ser que l'angiogènesi té un paper important en la patogènesi d'endometriosi. OBJECTIUS: Analitzar l'expressió de factors angiogènics, en concret, endoglina (Eng), tirosina quinasa 1 tipus fms (sFlt-1) i factor de creixement placentari (PlGF) en plasma, aspirat endometrial i teixit endometriòsic per identificar potencials biomarcadors diagnòstics i pronòstics de la malaltia. METODOLOGIA: Per a aquest estudi prospectiu de casos i controls se'n van reclutar 77 amb endometriosi (amb confirmació histològica de la malaltia) i 103 dones sanes sense endometriosi. L'estudi es va desenvolupar a l'Hospital de la Vall d'Hebron (Barcelona, Espanya) entre els anys 2014 i 2020. Les mostres de plasma van ser analitzades mitjançant kits ELISA comercialitzats per a l'expressió de sEng, sFlt-1 i PlGF. Les mostres de teixit d'aspirat endometrial i de teixit endometriòsic es van analitzar i l'expressió de sEng, sFlt-1 i PlGF va ser mesurada mitjançant reacció en cadena de polimerasa en temps real (RTqPCR). RESULTATS: En plasma, sFlt-1 va ser augmentat en endometriosi i va presentar valors sota la corba ROC de 0.78 com a biomarcador diagnòstic. No hi va haver diferències per la sEng i el PlGF. En aspirat endometrial, tots els factors analitzats van ser significativament més baixos en endometriosi en comparació del grup control i no hi va haver diferències en l'expressió de factors en funció del tipus de la malaltia (adenomiosi, endometriosi ovàrica-profunda, afectació orgànica). Tot i que no hi va haver diferències estadísticament significatives entre l'endometriosi recurrent i la no recurrent, hi va haver una tendència cap a una menor expressió de Eng, sFlt-1 i PlGF al grup d'endometriosi recurrent. En teixit endometriòsic, tots els factors angiogènics van ser elevats en endometriosi, incloent els diferents subtipus (ovàrica i profunda). L'anàlisi de la recurrència en endometriosi primària va mostrar un increment en els nivells de factors angiogènics en aquelles pacients amb recurrència i va proporcionar valors AUC en rang de 0.75-0.87 com a biomarcadors pronòstics. CONLUSIÓ: sFlt-1 podria ser un biomarcador diagnòstic en plasma. Tots els biomarcadors estudiats a nivell d'aspirat endometrial han demostrat una alta capacitat diagnòstica de l'endometriosi. Eng, sFlt-1 i PlGF podrien ser biomarcadors pronòstics amb capacitat de predir la recurrència a nivell de teixit endometrial ectòpic. A totes les mostres analitzades, no vam poder predir la severitat o el tipus de la malaltia mitjançant l'anàlisi de Eng, sFlt-1 i PlGF. ANTECEDENTES: La endometriosis empeora la calidad de vida y causa dolor pélvico crónico, especialmente en mujeres jóvenes. El retraso en el diagnóstico puede tardar de media unos 7 años debido a la falta de herramientas de diagnóstico tempranas, lo que negativamente impacta en el bienestar de las pacientes y en la fertilidad, teniendo, además, altos costes económicos y sociales. La recurrencia es otra prioridad en la investigación en endometriosis, ya que hasta un 40-50% de pacientes intervenidas recurren en los 5 años posteriores a la cirugía. Consecuentemente, la detección de pacientes en riesgo de recurrencia podría permitir adecuar el seguimiento postquirúrgico al riesgo de recurrencia de cada paciente e individualizar los tratamientos. Por tanto, existe una necesidad de identificación de nuevas técnicas que puedan proporcionar un diagnóstico temprano y herramientas pronósticas para disminuir la comorbilidad asociada. En este contexto, nuestra hipótesis fue que la angiogenesis desarrolla un papel importante en la patogénesis de endometriosis. OBJETIVOS: Analizar la expresión de factores angiogénicos, en concreto, endoglina (Eng), tirosina quinasa 1 tipo fms (sFlt-1) y factor de crecimiento placentario (PlGF) en plasma, aspirado endometrial y tejido endometriósico para identificar potenciales biomarcadores diagnósticos y pronósticos de la enfermedad. METODOLOGÍA: Para este estudio prospectivo de casos y controles se reclutaron 77 con endometriosis (con confirmación histológica de la enfermedad) y 103 mujeres sanas sin endometriosis. El estudio fue desarrollado en el Hospital de la Vall d'Hebron (Barcelona, España) entre los años 2014 y 2020. Las muestras de plasma fueron analizadas mediante kits ELISA comercializados para la expresión de Eng, sFlt-1 and PlGF. Las muestras de tejido de aspirado endometrial y de tejido endometriósico se analizaron y la expresión de Eng, sFlt-1 y PlGF fue medida mediante reacción en cadena de polimerasa en tiempo real (RTqPCR). RESULTADOS: En plasma, sFlt-1 fue aumentado en endometriosis y presentó valores bajo la curva ROC de 0.78 como biomarcador diagnóstico. No hubo diferencias para la Eng y PlGF. En aspirado endometrial, todos los factores analizados fueron significativamente más bajos en endometriosis en comparación con grupo control y no hubo diferencias en la expresión de factores en función del tipo de la enfermedad (adenomiosis, endometriosis ovárica-profunda, afectación orgánica). Aunque no hubo diferencias estadísticamente significativas entre la endometriosis recurrente y la no recurrente, hubo una tendencia hacia una menor expresión de Eng, sFlt-1 y PlGF en el grupo de endometriosis recurrente. En tejido endometriósico, todos los factores angiogénicos fueron elevados en endometriosis, incluyendo los diferentes subtipos (ovárica y profunda). El análisis de la recurrencia en endometriosis primaria mostró un incremento en los niveles de factores angiogénicos en aquellas pacientes con recurrencia y proporcionó valores AUC en rango de 0.75-0.87 como biomarcadores pronósticos. CONCLUSIONES: sFlt-1 podría ser un biomarcador diagnóstico en plasma. Todos los biomarcadores estudiados a nivel de aspirado endometrial han demostrado alta capacidad diagnóstica de la endometriosis. Eng, sFlt-1 y PlGF podrían ser biomarcadores pronósticos con capacidad de predecir la recurrencia a nivel de tejido endometrial ectópico. En todas las muestras analizadas, no pudimos predecir la severidad o el tipo de la enfermedad mediante el análisis de Eng, sFlt-1 y PlGF. BACKGROUND: Endometriosis causes impaired quality of life and chronic pelvic pain, especially in young women. The diagnosis delay is around 7 years due to the lack of diagnostic tools, which causes negative impact in patient's welfare and fertility, and has high economic and social costs. Recurrence is also the other priority in endometriosis research because up to 40-50% of patients recurs in the following 5 years after the surgery. Consequently, the detection of these patients could let an appropriate follow-up that would be beneficial for them. There is an unmet clinical need in the identification of novel techniques that provide earlier diagnosis and prognostic tools to decrease associated comorbidity. In this context, we hypothesized that angiogenesis may play an important role in endometriosis pathogenesis. OBJECTIVE: Analyze the expression of angiogenic factors in plasma, endometrial biopsy and endometriotic tissue to identify potential biomarkers for endometriosis diagnosis and prognosis. STUDY DESIGN: In this prospective case-control study, 77 women suffering from endometriosis (with histological confirmation of the disease) and 103 healthy controls without endometriosis were recruited in the Vall d'Hebron Hospital (Barcelona, Spain) between 2014 and 2020. Plasma samples were analyzed for protein expression levels of endoglin, sFlt-1 and PlGF using commercially available ELISA kits. Tissue samples from endometrial biopsy and endometriotic tissue were analyzed and RNA expression of endoglin (Eng), soluble fms-like tyrosine kinase-1 (sFlt-1), and placental growth factor (PlGF) was assessed by real-time polymerase chain reaction. RESULTS: In plasma samples, sFlt-1 was upregulated in endometriosis and presented an AUC value of 0.78 as a diagnostic biomarker. No differences were observed in endoglin and PlGF. In endometrial biopsy, all assessed biomarkers were significantly lower in endometriosis in comparison to the control group. The angiogenic activity in uterine aspirate was similar among adenomyosis, deep-ovarian endometriosis, and organ involvement. Although there were no statistical differences between recurrent and non-recurrent endometriosis, there was a tendency for downregulation of Eng, sFlt-1 and PlGF in the recurrence endometriosis group. In tissue samples, all angiogenic factors were upregulated in endometriosis, including different subtypes (deep and ovarian endometriosis). The analysis of recurrence in the primary endometriosis showed an increased levels of angiogenic factors in those patients with recurrence and showed AUC values ranging from 0.75 to 0.87 as a prognostic biomarkers. CONCLUSIONS: sFlt-1 could be a diagnostic biomarker in plasma samples. All studied biomarkers have demonstrated high accuracy for endometriosis diagnosis in endometrial biopsy. Hence, there was no correlation with the severity of the disease. Endoglin, sFlt-1 and PlGF might be prognostic biomarkers predicting recurrence in ectopic endometrial tissues. In all simples, we could not predict the severity or subtypes of the disease by analyzing expression of Eng, sFlt-1 y PlGF.

Formyl peptide receptors (FPR1, FPR2 and FPR3) are innate immune sensors of pathogen and commensal bacteria and have a role in colonic mucosa homeostasis. We identified FPR1 as a tumour suppressor in gastric cancer cells due to its ability to sustain an inflammation resolution response with antiangiogenic potential. Here, we investigate whether FPR1 exerts similar functions in colorectal carcinoma (CRC) cells. Since it has been shown that the commensal bacterium Lactobacillus rhamnosus GG (LGG) can promote intestinal epithelial homeostasis through FPR1, we explored the possibility that it could induce proresolving and antiangiogenic effects in CRC cells. We demonstrated that pharmacologic inhibition or genetic deletion of FPR1 in CRC cells caused a reduction of proresolving mediators and a consequent upregulation of angiogenic factors. The activation of FPR1 mediates opposite effects. Proresolving, antiangiogenic and homeostatic functions were also observed upon treatment of CRC cells with supernatant of LGG culture, but not of other lactic acid or nonprobiotic bacteria (i.e. Bifidobacterium bifidum or Escherichia coli). These activities of LGG are dependent on FPR1 expression and on the subsequent MAPK signalling activation. Thus, the innate immune receptor FPR1 could be a regulator of the balance between microbiota, inflammation and cancer in CRC models.

Beyond their well-known functions in the acute phases of the immune response, neutrophils play important roles in the various phases of tumor initiation and progression, through the release of their stored or newly synthesized mediators. In addition to reactive oxygen species, cytokines, chemokines, granule proteins and lipid mediators, neutrophil extracellular traps (NETs) can also be released upon neutrophil activation. NET formation can be achieved through a cell-death process or in association with the release of mitochondrial DNA from viable neutrophils. NETs are described as extracellular fibers of DNA and decorating proteins responsible for trapping and killing extracellular pathogens, playing a protective role in the antimicrobial defense. There is increasing evidence, however, that NETs play multiple roles in the scenario of cancer-related inflammation. For instance, NETs directly or indirectly promote tumor growth and progression, fostering tumor spread at distant sites and shielding cancer cells thus preventing the effects of cytotoxic lymphocytes. NETs can also promote tumor angiogenesis and cancer-associated thrombosis. On the other hand, there is some evidence that NETs may play anti-inflammatory and anti-tumorigenic roles. In this review, we focus on the main mechanisms underlying the emerging effects of NETs in cancer initiation and progression.

Angiogenesis, the active formation of new blood vessels from pre-existing ones, is a complex and demanding biological process that plays an important role in physiological as well as pathological settings. Recent evidence supports cell metabolism as a critical regulator of angiogenesis. However, whether and how cell metabolism regulates endothelial growth factor receptor levels and nucleotide synthesis remains elusive. We here shown in both human cell lines and mouse models that during developmental and pathological angiogenesis, endothelial cells (ECs) use glutaminolysis-derived glutamate to produce aspartate (Asp) via aspartate aminotransferase (AST/GOT). Asp leads to mTORC1 activation which, in turn, regulates endothelial translation machinery for VEGFR2 and FGFR1 synthesis. Asp-dependent mTORC1 pathway activation also regulates de novo pyrimidine synthesis in angiogenic ECs. These findings identify glutaminolysis-derived Asp as a regulator of mTORC1-dependent endothelial translation and pyrimidine synthesis. Our studies may help overcome anti-VEGF therapy resistance by targeting endothelial growth factor receptor translation.

Lay abstract Extended-stage small-cell lung cancer (ES-SCLC) is a highly aggressive lung cancer subtype, accounting for 13-15% of all lung cancers. For several years, the standard treatment for this disease was based on polychemotherapy, with a rapid disease response but with an equally rapid disease progression. The new standard treatment has recently been changed, based on the results of two large clinical trials, which showed the efficacy and safety of the combination of chemotherapy with immunotherapy compared to chemotherapy alone. Nevertheless, prognosis of ES-SCLC remains poor, and new treatment strategies are urgently needed. Therefore, we designed the CeLEBrATE trial to investigate whether the combination of chemotherapy with antiangiogenetic therapy and immunotherapy is safe and could improve survival in patients with ES-SCLC.Based on improved survival from the addition of PD-L1 inhibitors in phase III trials, the combination of immunotherapy and platinum-doublet chemotherapy has become the new standard treatment for extended-stage small-cell lung cancer (ES-SCLC). Furthermore, the antiangiogenetic agent bevacizumab showed a longer progression-free survival by targeting VEGF that has pleiotropic effects, including immunosuppressive ones. We, therefore, hypothesized that targeting angiogenesis would improve the efficacy of chemoimmunotherapy. The CeLEBrATE trial is an open-label, multicenter, phase II study designed to assess the efficacy and safety of the combination of carboplatin and etoposide plus bevacizumab and atezolizumab in treatment-naive patients with ES-SCLC. The primary end point is overall survival rate at 1 year, while secondary end points include overall response rate, progression-free survival and toxicity.

Lung carcinoids are neuroendocrine tumors that comprise well-differentiated typical (TCs) and atypical carcinoids (ACs). Preclinical models are indispensable for cancer drug screening since current therapies for advanced carcinoids are not curative. We aimed to develop a novel in vivo model of lung carcinoids based on the xenograft of lung TC (NCI-H835, UMC-11, and NCI-H727) and AC (NCI-H720) cell lines and patient-derived cell cultures inTg(fli1a:EGFP)y1zebrafish embryos. We exploited this platform to test the anti-tumor activity of sulfatinib. The tumorigenic potential of TC and AC implanted cells was evaluated by the quantification of tumor-induced angiogenesis and tumor cell migration as early as 24 h post-injection (hpi). The characterization of tumor-induced angiogenesis was performed in vivo and in real time, coupling the tumor xenograft with selective plane illumination microscopy on implanted zebrafish embryos. TC-implanted cells displayed a higher pro-angiogenic potential compared to AC cells, which inversely showed a relevant migratory behavior within 48 hpi. Sulfatinib inhibited tumor-induced angiogenesis, without affecting tumor cell spread in both TC and AC implanted embryos. In conclusion, zebrafish embryos implanted with TC and AC cells faithfully recapitulate the tumor behavior of human lung carcinoids and appear to be a promising platform for drug screening.

Angiogenin (Ang) is a potent angiogenic protein that is overexpressed in many types of cancer at concentration values correlated to the tumor aggressiveness. Here, by means of an integrated multi-technique approach based on crystallographic, spectrometric and spectroscopic analyses, we demonstrate that the anti-cancer drug oxaliplatin efficiently binds angiogenin. Microscopy cellular studies, carried out on the prostate cancer cell (PC-3) line , show that oxaliplatin inhibits the angiogenin prompting effect on cell proliferation and migration, which are typical features of angiogenesis process. Overall, our findings point to angiogenin as a possible target of oxaliplatin, thus suggesting a potential novel mechanism for the antineoplastic activity of this platinum drug and opening the avenue to novel approaches in the combined anti-cancer anti-angiogenic therapy.

Reciprocal interactions between endothelial cells (ECs) and adipocytes are fundamental to maintain white adipose tissue (WAT) homeostasis, as illustrated by the activation of angiogenesis upon WAT expansion, a process that is impaired in obesity. However, the molecular mechanisms underlying the crosstalk between ECs and adipocytes remain poorly understood. Here, we show that local production of polyamines in ECs stimulates adipocyte lipolysis and regulates WAT homeostasis in mice. We promote enhanced cell-autonomous angiogenesis by deleting Pten in the murine endothelium. Endothelial Pten loss leads to a WAT-selective phenotype, characterized by reduced body weight and adiposity in pathophysiological conditions. This phenotype stems from enhanced fatty acid β-oxidation in ECs concomitant with a paracrine lipolytic action on adipocytes, accounting for reduced adiposity. Combined analysis of murine models, isolated ECs and human specimens reveals that WAT lipolysis is mediated by mTORC1-dependent production of polyamines by ECs. Our results indicate that angiocrine metabolic signals are important for WAT homeostasis and organismal metabolism.

We aimed to overcome intratumoral heterogeneity in clear cell renal cell carcinoma (clearRCC). One hundred cases of clearRCC were sampled. First, usual standard sampling was applied (1 block/cm of tumor); second, the whole tumor was sampled, and 0.6 mm cores were taken from each block to construct a tissue microarray; third, the residual tissue, mapped by taking pieces 0.5 × 0.5 cm, reconstructed the entire tumor mass. Precisely, six randomly derived pieces of tissues were placed in each cassette, with the number of cassettes being based on the diameter of the tumor (called multisite 3D fusion). Angiogenic and immune markers were tested. Routine 5231 tissue blocks were obtained. Multisite 3D fusion sections showed pattern A, homogeneous high vascular density (10%), pattern B, homogeneous low vascular density (8%) and pattern C, heterogeneous angiogenic signatures (82%). PD-L1 expression was seen as diffuse (7%), low (33%) and absent (60%). Tumor-infiltrating CD8 scored high in 25% (pattern hot), low in 65% (pattern weak) and zero in 10% of cases (pattern desert). Grading was upgraded in 26% of cases (G3–G4), necrosis and sarcomatoid/rhabdoid characters were observed in, respectively, 11 and 7% of cases after 3D fusion (p = 0.03). CD8 and PD-L1 immune expressions were higher in the undifferentiated G4/rhabdoid/sarcomatoid clearRCC subtypes (p = 0.03). Again, 22% of cases were set to intermediate to high risk of clinical recurrence due to new morphological findings of all aggressive G4, sarcomatoid/rhabdoid features by using 3D fusion compared to standard methods (p = 0.04). In conclusion, we propose an easy-to-apply multisite 3D fusion sampling that negates bias due to tumor heterogeneity.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation, predominantly affecting the lung parenchyma and peripheral airways, that results in progressive and irreversible airflow obstruction. COPD development is promoted by persistent pulmonary inflammation in response to several stimuli (e.g., cigarette smoke, bacterial and viral infections, air pollution, etc.). Angiogenesis, the formation of new blood vessels, and lymphangiogenesis, the formation of new lymphatic vessels, are features of airway inflammation in COPD. There is compelling evidence that effector cells of inflammation (lung-resident macrophages and mast cells and infiltrating neutrophils, eosinophils, basophils, lymphocytes, etc.) are major sources of a vast array of angiogenic (e.g., vascular endothelial growth factor-A (VEGF-A), angiopoietins) and/or lymphangiogenic factors (VEGF-C, -D). Further, structural cells, including bronchial and alveolar epithelial cells, endothelial cells, fibroblasts/myofibroblasts, and airway smooth muscle cells, can contribute to inflammation and angiogenesis in COPD. Although there is evidence that alterations of angiogenesis and, to a lesser extent, lymphangiogenesis, are associated with COPD, there are still many unanswered questions.

Blood vessels form vast networks in all vertebrate organs to sustain tissue growth, repair and homeostatic metabolism, but they also contribute to a range of diseases with neovascularisation. It is, therefore, important to define the molecular mechanisms that underpin blood vessel growth. The receptor tyrosine kinase KIT is required for the normal expansion of hematopoietic progenitors that arise during embryogenesis from hemogenic endothelium in the yolk sac and dorsal aorta. Additionally, KIT has been reported to be expressed in endothelial cells during embryonic brain vascularisation and has been implicated in pathological angiogenesis. However, it is neither known whether KIT expression is widespread in normal organ endothelium nor whether it promotes blood vessel growth in developing organs. Here, we have used single-cell analyses to show that KIT is expressed in endothelial cell subsets of several organs, both in the adult and in the developing embryo. Knockout mouse analyses revealed that KIT is dispensable for vascularisation of growing organs in the midgestation embryo, including the lung, liver and brain. By contrast, vascular changes emerged during late-stage embryogenesis in these organs from KIT-deficient embryos, concurrent with severe erythrocyte deficiency and growth retardation. These findings suggest that KIT is not required for developmental tissue vascularisation in physiological conditions, but that KIT deficiency causes foetal anaemia at late gestation and thereby pathological vascular remodelling.

ObjectivePrimary sclerosing cholangitis (PSC) is in 70% of cases associated with inflammatory bowel disease. The hypermorphic T108M variant of the orphan G protein-coupled receptor GPR35 increases risk for PSC and ulcerative colitis (UC), conditions strongly predisposing for inflammation-associated liver and colon cancer. Lack of GPR35 reduces tumour numbers in mouse models of spontaneous and colitis associated cancer. The tumour microenvironment substantially determines tumour growth, and tumour-associated macrophages are crucial for neovascularisation. We aim to understand the role of the GPR35 pathway in the tumour microenvironment of spontaneous and colitis-associated colon cancers.DesignMice lacking GPR35 on their macrophages underwent models of spontaneous colon cancer or colitis-associated cancer. The role of tumour-associated macrophages was then assessed in biochemical and functional assays.ResultsHere, we show that GPR35 on macrophages is a potent amplifier of tumour growth by stimulating neoangiogenesis and tumour tissue remodelling. Deletion of Gpr35 in macrophages profoundly reduces tumour growth in inflammation-associated and spontaneous tumour models caused by mutant tumour suppressor adenomatous polyposis coli. Neoangiogenesis and matrix metalloproteinase activity is promoted by GPR35 via Na/K-ATPase-dependent ion pumping and Src activation, and is selectively inhibited by a GPR35-specific pepducin. Supernatants from human inducible-pluripotent-stem-cell derived macrophages carrying the UC and PSC risk variant stimulate tube formation by enhancing the release of angiogenic factors.ConclusionsActivation of the GPR35 pathway promotes tumour growth via two separate routes, by directly augmenting proliferation in epithelial cells that express the receptor, and by coordinating macrophages’ ability to create a tumour-permissive environment.

[eng] The pleiotropic WT1 transcription factor (WT1) was first discovered in renal tumours. It contains four zinc-finger motifs at the C-terminus which are important for DNA binding to activate gene expression and is also involved in post-transcriptional processes. The expression of WT1 is essential during development of multiple organs, among them the cardiovascular structures. In the developing heart, WT1 is strongly expressed in the epicardium, and in cardiac endothelial cells. After cardiac injury, WT1 is temporally upregulated in both lineages in the infarcted area and border zone, which points to a potential role for WT1 in cardiac repair and regeneration. In this thesis, we focused on the role of Wt1 in endothelial cells, particularly in cardiac endothelial cells during coronary vessel formation, and in the adult heart after myocardial infarction. To support some of these findings, the mouse retinal vasculature was used as a model for two-dimensional angiogenic studies. We have demonstrated that Wt1 is present in CECs during coronary development, and that Wt1 deletion abrogates for endothelial proliferation, migration, and maturation during both retinal and coronary vasculature formation in vivo. Also, RNAseq analysis of embryonic Wt1-KOΔEC CECs suggests that Wt1 controls several downstream pathways. However, the importance of Wt1 functions in neovascularization in the adult heart after injury, specifically after 14 days of MI, has proven to be limited. The so-called recapitulation of embryonic programs in the neovascularization processes arising after injury was not sufficient for Wt1 to exert a differential effect on the adult heart after myocardial infarction. We observed no significant functional changes in cardiac output, assessed by ECG imaging, nor in tissue remodelling, visualized by analysis of the fibrotic scar in the infarcted region. However, we did observe changes in Wt1-KOΔEC CECs at the molecular and transcriptomic level, suggesting that WT1 may activate some genetic programmes in these cells, even if its effects are not able to alter evident cardiac functions.

Bladder cancer is the most common tumor of the urinary system, with a high incidence in the male population. Surgery and intravesical instillations can eradicate it, although recurrences are very common, with possible progression. For this reason, adjuvant therapy should be considered in all patients. Resveratrol displays a biphasic dose response both in vitro and in vivo (intravesical application) with an antiproliferative effect at high concentrations and antiangiogenic action in vivo (intraperitoneal application) at a low concentration, suggesting a potential role for it in clinical management as an adjuvant to conventional therapy. In this review, we examine the standard therapeutical approach to bladder cancer and the preclinical studies that have investigated resveratrol in xenotransplantation models of bladder cancer. Molecular signals are also discussed, with a particular focus on the STAT3 pathway and angiogenic growth factor modulation.

The high output heart failure is a clinical condition in which the systemic congestion is associated to a high output state, and it can be observed in a non-negligible percentage of hematological diseases, particularly in multiple myeloma, a condition in which the risk of adverse cardiovascular events may increase, with a worse prognosis for patients. For this reason, though an accurate literature search, we provided in this review a complete overview of different pathogenetic mechanisms responsible for high output heart failure in multiple myeloma. Indeed, this clinical finding is present in the 8% of multiple myeloma patients, and it may be caused by artero-venous shunts, enhanced angiogenesis, glutamminolysis, hyperammonemia and hemorheological alterations with increase in plasma viscosity. The high output heart failure in multiple myeloma is associated with significant morbidity and mortality, emphasizing the need for a multidisciplinary approach.

Angiogenic sprouting relies on collective migration and coordinated rearrangements of endothelial leader and follower cells. VE-cadherin-based adherens junctions have emerged as key cell-cell contacts that transmit forces between cells and trigger signals during collective cell migration in angiogenesis. However, the underlying molecular mechanisms that govern these processes and their functional importance for vascular development still remain unknown. We previously showed that the F-BAR protein PACSIN2 is recruited to tensile asymmetric adherens junctions between leader and follower cells. Here we report that PACSIN2 mediates the formation of endothelial sprouts during angiogenesis by coordinating collective migration. We show that PACSIN2 recruits the trafficking regulators EHD4 and MICAL-L1 to the rear end of asymmetric adherens junctions to form a recycling endosome-like tubular structure. The junctional PACSIN2/EHD4/MICAL-L1 complex controls local VE-cadherin trafficking and thereby coordinates polarized endothelial migration and angiogenesis. Our findings reveal a molecular event at force-dependent asymmetric adherens junctions that occurs during the tug-of-war between endothelial leader and follower cells, and allows for junction-based guidance during collective migration in angiogenesis.

Diabetic retinopathy; Endostatin; Intravitreal vessels Retinopatia diabètica; Endostatina; Vasos intravítres Retinopatía diabética; Endostatina; Vasos intravítreos Endostatin, a naturally cleaved fragment of type XVIII collagen with antiangiogenic activity, has been involved in the regulation of neovascularization during diabetic retinopathy. Here, the intracellular distribution of endostatin in healthy mouse and human neuroretinas has been analyzed. In addition, to study the effect of experimental hyperglycemia on retinal endostatin, the db/db mouse model has been used. Endostatin protein expression in mouse and human retinas was studied by immunofluorescence and Western blot, and compared with db/db mice. Eye fundus angiography, histology, and immunofluorescence were used to visualize mouse retinal and intravitreal vessels. For the first time, our results revealed the presence of endostatin in neurons of mouse and human retinas. Endostatin was mainly expressed in bipolar cells and photoreceptors, in contrast to the optic disc, where endostatin expression was undetectable. Diabetic mice showed a reduction of endostatin in their retinas associated with the appearance of intravitreal vessels at the optic disc in 50% of db/db mice. Intravitreal vessels showed GFAP positive neuroglia sheath, basement membrane thickening by collagen IV deposition, and presence of MMP-2 and MMP-9 in the vascular wall. All together, these results point that decreased retinal endostatin during experimental diabetes is associated with optic disc intravitreal vascularization. Based on their phenotype, these intravitreal vessels could be neovessels. However, it cannot be ruled out the possibility that they may also represent persistent hyaloid vessels. This work was supported by the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Spain (grant number PI16/00719); the Fundação para a Ciência e a Tecnologia, Ministerio da Educação e Ciência, Portugal (grant numbers SFRH/BD/95330/2013 and SFRH/BPD/102573/2014); and Fondo Europeo de Desarrollo Regional (FEDER).

The use of copper (Cu2+) and cobalt (Co2+) has been described to stimulate blood vessel formation, a key process for the success of tissue regeneration. However, understanding how different concentrations of these ions affect cellular response is important to design scaffolds for their delivery to better fine tune the angiogenic response. On the one hand, gene expression analysis and the assessment of tubular formation structures with human umbilical vein endothelial cells (HUVEC) revealed that high concentrations (10μM) of Cu2+in early times and lower concentrations (0.1 and 1μM) at later times (day 7) enhanced angiogenic response. On the other hand, higher concentrations (25μM) of Co2+during all time course increased the angiogenic gene expression and 0.5, 5 and 25μM enhanced the ability to form tubular structures. To further explore synergistic effects combining both ions, the non-toxic concentrations were used simultaneously, although results showed an increased cell toxicity and no improvement of angiogenic response. These results provide useful information for the design of Cu2+or Co2+delivery scaffolds in order to release the appropriate concentration during time course for blood vessel stimulation.

The loss of skin integrity has always represented a major challenge for clinicians dealing with dermal defects, such as ulcers (diabetic, vascular and chronic), postoncologic resections (i.e., radical vulvectomy) or dermatologic disorders. The introduction in recent decades of acellular dermal matrices (ADMs) supporting the repair and restoration of skin functionality represented a significant step toward achieving clean wound repair before performing skin grafts. Hard-to-heal ulcers generally depend on local ischemia and nonadequate vascularization. In this context, one possible innovative approach could be the prevascularization of matrices with vessel-forming cells (inosculation). This paper presents a comparative analysis of the most widely used dermal templates, i.e., Integra® Bilayer Matrix Wound Dressing, PELNAC®, PriMatrix® Dermal Repair Scaffold, Endoform® Natural Dermal Template, and Myriad Matrix®, testing their ability to be colonized by human adult dermal microvascular endothelial cells (ADMECs) and to induce and support angiogenesis in vitro and in vivo. By in vitro studies, we demonstrated that Integra® and PELNAC® possess superior pro-adhesive and pro-angiogenetic properties. Animal models allowed us to demonstrate the ability of preseeded ADMECs on Integra® to promote the engraftment, integration and vascularization of ADMs at the site of application.

Nerve growth factor (NGF) is recognized as a pleiotropic molecule, exerting a variety of biological effects on different cell types and pathophysiological conditions, and its role in tissue wound healing has been recently highlighted. However, the preferential cellular target of NGF is still elusive in the complex cellular and molecular cross talk that accompanies wound healing. Thus, to explore possible NGF cellular targets in skin wound healing, we investigated the in vitro NGF responsiveness of keratinocytes (cell line HEKa), fibroblasts (cell line BJ), and endothelial cells (cell line HUVEC), also in the presence of adverse microenvironmental conditions, e.g., hyperglycemia. The main results are summarized as follows: 1) NGF stimulates keratinocyte proliferation and HUVEC proliferation and angiogenesis in a dose-dependent manner although it has no effect on fibroblast proliferation; 2) NGF stimulates keratinocyte but not fibroblast migration in the wound healing assay; and 3) NGF completely reverts the proliferation impairment of keratinocytes and the angiogenesis impairment of HUVECs induced by high d-glucose concentration in the culture medium. These results contribute to better understanding possible targets for the therapeutic use of NGF in skin repair.

The efficacy of metformin in treating cancer has been extensively investigated since epidemiologic studies associated this anti-diabetic drug with a lower risk of cancer incidence. Since tumors are complex systems, in which cancer cells coexist and interact with several different types of non-malignant cells, it is not surprising that anti-cancer drugs affect not only cancer cells, but also the abundance and functions of cells of the tumor microenvironment. Recent years have seen a wide collection of reports showing how metformin, as well as other complex I inhibitors, may influence cancer progression by modulating the phenotype of non-transformed cells in a tumor. In this review, we particularly focus on the effect of metformin on angiogenesis, cancer-associated fibroblasts, tumor-associated macrophages and cancer immunosuppression.

Aims Cardiac ischaemia does not elicit an efficient angiogenic response. Indeed, lack of surgical revascularization upon myocardial infarction results in cardiomyocyte death, scarring, and loss of contractile function. Clinical trials aimed at inducing therapeutic revascularization through the delivery of pro-angiogenic molecules after cardiac ischaemia have invariably failed, suggesting that endothelial cells in the heart cannot mount an efficient angiogenic response. To understand why the heart is a poorly angiogenic environment, here we compare the angiogenic response of the cardiac and skeletal muscle using a lineage tracing approach to genetically label sprouting endothelial cells. Methods and results We observed that overexpression of the vascular endothelial growth factor in the skeletal muscle potently stimulated angiogenesis, resulting in the formation of a massive number of new capillaries and arterioles. In contrast, response to the same dose of the same factor in the heart was blunted and consisted in a modest increase in the number of new arterioles. By using Apelin-CreER mice to genetically label sprouting endothelial cells we observed that different pro-angiogenic stimuli activated Apelin expression in both muscle types to a similar extent, however, only in the skeletal muscle, these cells were able to sprout, form elongated vascular tubes activating Notch signalling, and became incorporated into arteries. In the heart, Apelin-positive cells transiently persisted and failed to give rise to new vessels. When we implanted cancer cells in different organs, the abortive angiogenic response in the heart resulted in a reduced expansion of the tumour mass. Conclusion Our genetic lineage tracing indicates that cardiac endothelial cells activate Apelin expression in response to pro-angiogenic stimuli but, different from those of the skeletal muscle, fail to proliferate and form mature and structured vessels. The poor angiogenic potential of the heart is associated with reduced tumour angiogenesis and growth of cancer cells.

Well-differentiated lung neuroendocrine tumours (Lu-NETs), classified as typical (TC) and atypical (AC) carcinoids, represent 30% of NETs. Angiogenesis plays an essential role in NET development and progression. A higher vascular network is a marker of differentiation, with positive prognostic implications. Materials and Methods: We retrospectively evaluated microvessel density (MVD) by CD34 immunohistochemical (IHC) staining and hypoxia by IHC staining for Hypoxia-inducible factor 1α (HIF-1α), comparing right- and left-lung parenchyma in 53 lung NETs. Results: The median age was 66 years (39–81), 56.6% males, 24.5% AC, 40.5% left-sided tumours and 69.8% TNM stage I. The mitotic count was