A tool for the quantification of radial neo-vessels in chick chorioallantoic membrane angiogenic assays

Angiogenesis, the process of new blood vessels formation, plays a key role in different physiological and pathological conditions and it is considered a promising target for the development of new anti-inflammatory and anti-tumor therapies. Several assays have been developed to mimic the angiogenic process in vitro and in vivo. Here we propose a technique for the quantification of the pro-angiogenic or anti-angiogenic responses induced by different molecules when implanted in vivo on the chick embryo chorioallantoic membrane (CAM). At day 11 of development CAM is completely vascularized and neo-vessels induced by exogenous molecules converge radially to the implant. Our algorithm is an effective and rapid tool to characterize molecules endowed with proor anti-angiogenic effects by means of the quantification of the vessels present in the CAM macroscopic images. Based on conventional and dedicated image morphology tools, the proposed technique is able to discriminate radial from non-radial vessels, excluding the last ones from the count

Magnetic stimulation of the angiogenic potential of mesenchymal stromal cells in vascular tissue engineering

The authors acknowledge the financial support from Fundação para a Ciência e a Tecnologia (FCT-MEC), Portugal, through the dedicated project [PTDC/EDM-EDM/30828/2017] (BeLive) and PhD grant [SFRH/BD/114043/2015] and  through the project [EXPL/CTM-POL/1117/1135/2012] Moreover, the authors thanks POR Lisboa 2020 for the research project [PRECISE, Project N. 16394]. We acknowledge Dr. Marta Teixeira and the IPATIMUP facilities for the development of the ex vivo CAM experiments. The authors acknowledge Prof. Reyes Mallada (University of Zaragoza, Spain) for the use of the vibrating sample magnetometer (VSM) equipment and Dr. Pavel Strichovanec (University of Zaragoza, Spain) for the technical assistance provided during the experiments. We also acknowledge the Instituto de Medicina Molecular (IMM, Lisboa) for the services provided concerning the use of the Confocal Scanning Microscopy (Zeiss LSM 710). Publisher Copyright: © 2021 The Author(s). Published by National Institute for Materials Science in partnership with Taylor & Francis Group.The growing prevalence of vascular diseases worldwide has emphasized the need for novel tissue-engineered options concerning the development of vascularized 3D constructs. This study reports, for the first time, the use of external magnetic fields to stimulate mesenchymal stromal cells (MSCs) to increase the production of vascular endothelial growth factor-A (VEGF-A). Polyvinylalcohol and gelatin-based scaffolds, containing iron oxide nanoparticles, were designed for optimal cell magnetic stimulation. While the application of static magnetic fields over 24 h did not impact on MSCs proliferation, viability and phenotypic identity, it significantly increased the production of VEGF-A and guided MSCs morphology and alignment. The ability to enhance MSCs angiogenic potential was demonstrated by the increase in the number of new vessels formed in the presence of MSCs conditioned media through in vitro and in vivo models. Ultimately, this study uncovers the potential to manipulate cellular processes through short-term magnetic stimulation.publishersversionpublishe

Evidence for the use of ultrasound therapy for the management of mandibular osteoradionecrosis

Introduction: The treatment of mandibular osteoradionecrosis includes antibiotics and curettage, hyperbaric oxygen, surgery, and more recently, therapeutic ultrasound. The aim of this thesis was to establish the possible mechanisms of action of therapeutic ultrasound, that could explain its excellent clinical results. Material and Methods: Two ultrasound machines were evaluated, a 'traditional' (1 MHz and 3 MHz) and a 'long wave' machine (45 kHz). Ultrasound was applied to human mandibular osteoblasts, gingival fibroblasts, peripheral blood monocytes (PBMc) and mice calvaria. The following in vitro assays were performed: cell proliferation, collagen and non-collagenous protein (NCP) synthesis, bone resorption, cytokines and angiogenesis factors production using ELISA and RT-PCR techniques, and nitric oxide production. To evaluate the effects of ultrasound on angiogenesis in vivo, the chick chorioallanlbic membrane assay (CAM) was used. The use of near infrared spectroscopy (NIRS) for the measurement of radiotherapy effects in the mandible (deoxyhaemoglobin concentrations) was also evaluated. Results: Ultrasound stimulated bone formation in the mice calvaria. Cell proliferation assays showed an increase of DNA synthesis in fibroblasts and osteoblasts, up to 52%. Collagen/NCR synthesis was also enhanced, in fibroblasts up to 48%, and in osteoblasts up to 112%. Bone resorption, part of the bone turnover process, was promoted, and there is suggestion that the cyclo-oxygenase pathway is involved. In relation to cytokine production, a slight stimulation of IL-1beta was noted in all cell types. There was no difference in IL-6 and TNFalpha levels. The angiogenesis factors, IL-8 and bFGF, were significantly stimulated in osteoblasts, and VEGF was significantly stimulated in fibroblasts, osteoblasts and PBMc. RT-PCR showed that ultrasound induces mRNA transcription for several cytokines and bone related proteins, with the most evident effect being the induction of VEGF transcription in osteoblasts. The CAM assay showed that direct ultrasound application and insonated medium from fibroblasts induced angiogenesis in vivo. The best overall stimulatory intensities were 15 and 30 mW/cm2(SA) with 45 kHz ultrasound, and 0.1 and 0.4 W/cm2(SAPA) with 1 MHz ultrasound. The NIRS evaluation showed that it is very sensitive to measure deoxyhaemoglobin concentrations, however these measurements are not reproducible. No age correlations could be performed, and the differences between normal and radiotherapy mandibles was not significant because of the great variability in the measurements. Conclusions: These results show that ultrasound can correct hypocellularity, hypoxia and hypovascularity observed in osteoradionecrosis. It stimulates cell proliferation, bone formation, healing, and angiogenesis. Further in vivo experiments are recommended as well as prospective clinical trials using therapeutic ultrasound for the treatment and prevention of osteoradionecrosis, but NIRS cannot be used to measure the outcome of treatment. Therapeutic ultrasound is a viable option for the management of mandibular osteoradionecrosis, since it is effective, inexpensive and readily available

Microphysiological Systems for the Evaluation of Biomaterials in Regenerative Therapies

[eng] The design of bioresponsive materials capable of stimulating the body’s innate regenerative potential is opening unprecedented possibilities to treat tissue and organ failure, which is one of the most important burdens of healthcare systems worldwide. Unfortunately, their development is hampered by the lack of adequate preclinical models, which are essential in the successful transition of a biomaterial to the clinical trials phase. Most of the experiments rely on animal models, which usually fail to predict the material interactions with the human body, as they are unable to recapitulate the complexities of our physiology. During the last decades, the advancements in the field of microtechnology have allowed to create advanced cell culture systems capable of replicating tissue and organ-level physiology by mimicking relevant conditions such as cell organization or microenvironmental cues. These platforms, known as microphysiological systems (MPS), have shown in different studies their great potential in predicting mechanisms of action, safety, and efficacy of different drugs, attracting a lot of attention from the pharmaceutical industry and regulatory agencies. However, few studies have explored the possibility of using microphysiological systems for the preclinical testing of biomaterials. The goal of this thesis is to fill this knowledge gap by developing microfluidic cell culture systems that allow to reliably predict the actual in vivo response of different materials. One of the proposed platforms is aimed at assessing the potential of a biomaterial to stimulate endothelial progenitor cell recruitment in a bone tissue microenvironment. This is a critical step in the neovascularization and bone regeneration process that has not been properly studied due to the lack of adequate models. The proposed device allowed to identify the role of calcium ions in stimulating the recruitment of rat endothelial progenitor cells (rEPC) to the site of injury, which is mediated by an increase in the release of osteopontin, a chemotactic and mitogenic protein produced by rat bone-marrow mesenchymal stromal cells (BM-rMSC). The platform was also used to evaluate a calcium-releasing biomaterial based on electrospun polylactic acid (PLA) fibers with calcium-phosphate (CaP) nanoparticles. The results show a significant increase in terms of rEPC recruitment and the release of osteopontin and other pro-angiogenic and inflammatory proteins by BM-rMSC with respect to a regular PLA control, which is in close agreement with previous experiments performed in a murine in vivo model. The other platform proposed in this thesis is aimed at providing a physiologically relevant model of cardiac tissue to study a myocardial ischemia-reperfusion injury. There are currently no reliable in vitro models to mimic this disease, making these contributions extremely relevant for cardiac regeneration studies. A first prototype of the platform based on the combination of aligned electrospun PLA fibers with a user-friendly electrical stimulation setup in a microfluidic cell culture platform produced a biomimetic cardiac tissue in 2D. This was confirmed by the high anisotropy of the tissue constructs, based on the co- culture of neonatal mouse cardiomyocytes with cardiac fibroblasts, as well as the upregulation of several key cardiac markers such as contractile and structural proteins. In order to make the model more physiologically relevant, a second device was developed to obtain human-derived 3D tissues. This platform is based on the self-assembling of primary cardiac fibroblasts (hCF) co-cultured with human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) in a fibrin-based hydrogel around two microposts structures, which exert a passive mechanical tension that stimulates tissue maturation and cell alignment. We first performed a screening using 2D assays based on hPSC-CM monolayers to select the best environmental conditions to mimic an ischemia-reperfusion injury. We then characterized the response of the human- derived cardiac organoids to an ischemia-reperfusion injury, consisting of an 8 h culture period at 0 % oxygen in an ischemic solution that replicates the acidic and hyperkalemic conditions observed in vivo, followed by a refreshment with fully supplemented cell media and recovery of 21 % environmental oxygen concentrations. We observed a drastic increase in cell death by necrosis and apoptosis as well as a strong fibrotic response, characterized by an increase in hCF proliferation, differentiation towards myofibroblasts and collagen I deposition. Taken together, we believe that the platforms developed in this thesis constitute an extremely valuable and versatile tool to perform preclinical studies, offering a promising alternative to animal studies for the development of new biomaterials and drug discovery.[spa] El diseño de biomateriales capaces de estimular la capacidad innata del cuerpo de regenerarse está abriendo una oportunidad sin precedentes para el tratamiento y reemplazamiento de órganos y tejidos, una de las principales cargas en los sistemas de salud a nivel mundial. Desafortunadamente, el desarrollo de estas terapias se ve lastrado por la falta de modelos preclínicos adecuados, que son esenciales en la transición exitosa de un biomaterial a la aplicación clínica. La mayoría de estos experimentos se basan en el uso de modelos animales, que habitualmente fallan en la predicción de las interacciones que ocurren en el cuerpo humano, debido a las diferencias inherentes que existen en términos de fisiología. Durante las últimas décadas, los avances en el campo de la microtecnología han permitido crear plataformas de cultivo celular capaces de replicar elementos fisiológicos a nivel de tejidos y órganos denominados sistemas microfisiológicos. Estos sistemas han demostrado su gran utilidad en la predicción de mecanismos de acción, seguridad y eficacia de diferentes fármacos, atrayendo una gran atención por parte de las agencias regulatorias. Sin embargo, pocos estudios han explorado la posibilidad de usar este tipo de sistemas para la evaluación preclínica de biomateriales. El objetivo de esta tesis es realizar contribuciones en este campo mediante el desarrollo de sistemas microfluídicos de cultivo celular capaces de predecir la respuesta in vivo de diferentes materiales. En esta tesis se presentan principalmente dos modelos diferentes de sistemas microfisiológicos. El primer está relacionado con el reclutamiento de células progenitoras endoteliales en un entorno de regeneración ósea para el estudio de la vascularización de biomateriales, mientras que el segundo busca generar un modelo de tejido cardíaco fisiológicamente relevante para estudiar una lesión por isquemia-reperfusión y posibles terapias regenerativas

Exploration of quorum sensing peptides: the missing link between microbiome and cancer outcome?

The human microbiome was recently associated with diverse diseases. The explanation for this relationship is however not yet clarified. In this research, the interaction of quorum sensing peptides, produced by the bacteria, with human cancer cells was explored. First, the chemical characteristics of already known quorum sensing peptides (and analogues), their bacterial origin and quorum sensing related properties were summarized in the Quorumpeps database. The chemical diversity of these quorum sensing peptides was then analyzed, after which diverse model-peptides were selected for further research. After chemical synthesis of these peptides, the purity was investigated, next to the identity of the impurities. A thorough quality control is necessary to correctly interpret functional results. Eventually, the effect of the quorum sensing peptides on human cancer cell behaviour was investigated: we demonstrated that some quorum sensing peptides induced colon and breast cancer cell invasion and promoted angiogenesis; both processes can be linked to cancer metastasis. Some quorum sensing peptides were also found to pass the blood-brain barrier, indicating that these peptides can exert an effect on the brain tissue. Quorum sensing peptides can pass the intestinal barrier as well and thus reach the blood circulation; once they are present in the blood, they can interact with cells throughout the human body. The quorum sensing peptides were also found to remain sufficiently stable in human plasma. Moreover, the quorum sensing peptides demonstrated no haemolytic and no direct cell-killing effects. Finally, we reviewed the quality aspects of radiolabelled peptides as used not only in the biomedical research but also as diagnostics or therapeutics in the current shift towards personalized medicine

First-order statistical speckle models improve robustness and reproducibility of contrast-enhanced ultrasound perfusion estimates

Contrast-enhanced ultrasound (CEUS) permits the quantification and monitoring of adaptive tumor responses in the face of anti-angiogenic treatment, with the goal of informing targeted therapy. However, conventional CEUS image analysis relies on mean signal intensity as an estimate of tracer concentration in indicator-dilution modeling. This discounts additional information that may be available from the first-order speckle statistics in a CEUS image. Heterogeneous vascular networks, typical of tumor-induced angiogenesis, lead to heterogeneous contrast enhancement of the imaged tumor cross-section. To address this, a linear (B-mode) processing approach was developed to quantify the change in the first-order speckle statistics of B-mode cine loops due to the incursion of microbubbles. The technique, named the EDoF (effective degrees of freedom) method, was developed on tumor bearing mice (MDA-MB-231LN mammary fat pad inoculation) and evaluated using nonlinear (two-pulse amplitude modulated) contrast microbubble-specific images. To improve the potential clinical applicability of the technique, a second-generation compound probability density function for the statistics of two-pulse amplitude modulated contrast-enhanced ultrasound images was developed. The compound technique was tested in an antiangiogenic drug trial (bevacizumab) on tumor bearing mice (MDA-MB-231LN), and evaluated with gold-standard histology and contrast-enhanced X-ray computed tomography. The compound statistical model could more accurately discriminate anti-VEGF treated tumors from untreated tumors than conventional CEUS image. The technique was then applied to a rapid patient-derived xenograft (PDX) model of renal cell carcinoma (RCC) in the chorioallantoic membrane (CAM) of chicken embryos. The ultimate goal of the PDX model is to screen RCC patients for de novo sunitinib resistance. The analysis of the first-order speckle statistics of contrast-enhanced ultrasound cine loops provides more robust and reproducible estimates of tumor blood perfusion than conventional image analysis. Theoretically this form of analysis could quantify perfusion heterogeneity and provide estimates of vascular fractal dimension, but further work is required to determine what physiological features influence these measures. Treatment sensitivity matrices, which combine vascular measures from CEUS and power Doppler, may be suitable for screening of de novo sunitinib resistance in patients diagnosed with renal cell carcinoma. Further studies are required to assess whether this protocol can be predictive of patient outcome

Cerebrovascular development: mechanisms and experimental approaches

The cerebral vasculature plays a central role in human health and disease and possesses several unique anatomic, functional and molecular characteristics. Despite their importance, the mechanisms that determine cerebrovascular development are less well studied than other vascular territories. This is in part due to limitations of existing models and techniques for visualisation and manipulation of the cerebral vasculature. In this review we summarise the experimental approaches used to study the cerebral vessels and the mechanisms that contribute to their development

Collective locomotion of human cells, woundh healing and their control by extracts and isolated compounds from marine ivertebrates

The collective migration of cells is a complex integrated process that represents a common theme joining morphogenesis, tissue regeneration, and tumor biology. It is known that a remarkable amount of secondary metabolites produced by aquatic invertebrates displays active pharmacological properties against a variety of diseases. The aim of this review is to pick up selected studies that report the extraction and identification of crude extracts or isolated compounds that exert a modulatory effect on collective cell locomotion and/or skin tissue reconstitution and recapitulate the molecular, biochemical, and/or physiological aspects, where available, which are associated to the substances under examination, grouping the producing species according to their taxonomic hierarchy. Taken all of the collected data into account, marine invertebrates emerge as a still poorly-exploited valuable resource of natural products that may significantly improve the process of skin regeneration and restrain tumor cell migration, as documented by in vitro and in vivo studies. Therefore, the identification of the most promising invertebrate-derived extracts/molecules for the utilization as new targets for biomedical translation merits further and more detailed investigations

Focusing on metabolomic dysregulation and modulation of retinal metabolism to develop novel therapeutic strategies for diabetic retinopathy

Tese de doutoramento, Medicina (Oftalmologia), Universidade de Lisboa, Faculdade de Medicina, 2015Diabetic retinopathy (DR) is a neurovascular complication of diabetes mellitus and a leading cause of blindness in adults below the age of 65, in industrialized nations. Currently there are 126.6 million people with DR worldwide (34.6% of the total diabetic population) and it is estimated that this number will increase to 191.0 million by 2030. Generally, DR is divided into two stages: non-proliferative diabetic retinopathy (NPDR), an earlier phase characterized by appearance of microaneurysms, dot and blot hemorrhages, capillary occlusions and nerve fiber layer infarcts and; and proliferative diabetic retinopathy (PDR), the late-stage disease, which is diagnosed when pathological neovascular changes are identified on the retinal surface and/or vitreous. Diabetic macular edema (DME) can develop at any stage and reflects a pathological increase in retinal vascular permeability. Despite its high prevalence, current availability of preventive and therapeutic strategies is far from ideal. In fact, there are no reliable biomarkers to predict risk of developing DR and no effective, targeted and early-acting therapies to sustainably and safely prevent disease progression into its vision threatening stages: PDR and DME. The two main therapeutic options currently available for DR are laser photocoagulation and intravitreal injections of anti-vascular endothelial growth factor (VEGF) agents; these constitute “non-selective” and destructive (especially for laser therapy) approaches that are not only unable to effectively and sustainably arrest retinal disease progression in every case, but can also potentially induce a myriad of undesired off-target effects at the retinal level and even systemically, in the case of VEGF antagonists. Moreover, they act late in the disease course. The lack of reliable rodent models - there is no diabetic mouse model that spontaneously recapitulates the late stages of DR - has greatly hindered research progress and development of novel and effective drugs for PDR, further contributing to the present therapeutic scenario. In this dissertation I will introduce and present my experimental work in the context of the following concepts, which can potentially lead to development of targeted, earlier acting, less destructive and more effective future therapies for DR: (1) DR has long been regarded as a vascular disease and present-day DR management guidelines are still based on this assumption. However, a growing body of evidence shows that retinal neuronal function becomes impaired before vascular changes can be detected; these findings along with those showing that adequate retinal functioning depends on stable intercellular interactions within neurovascular units, suggest that disrupting retinal neurovascular crosstalk may play a critical role in promoting disease development and progression. (2) Metabolomic studies have been surprisingly neglected in the investigation of DR’s pathophysiology, and this is clearly reflected by the fact that the metabolome of human DR remains unknown. Furthermore, the neuroretina is one of the most metabolically demanding tissues in the body per unit weight, and diabetes is triggered by a metabolic defect that profoundly impairs cellular energy production. These features constitute a potentially disastrous combination in regard to retinal functioning and suggest that studying retinal energy metabolism in DR is critical. (3) Metabolic cycles of photoreceptors, interneurons and glial cells are still under debate and, even though it is known that intercellular communications within the neurovascular unit (NVU) are mediated by metabolites whose production becomes dysregulated under pathological conditions, the precise mechanisms underlying retinal neurovascular coupling are not fully identified. Gaining further insight into these interactions is pivotal because retinal NVUs are responsible for regulating blood flow for functionally dynamic retinal neuronal networks and, thus, for their proper functioning. Besides the points stated above, additional clinical clues were considered to guide the research plan presented in this dissertation. One the strongest came from a subset of long-term diabetic patients who appear to be protected from developing late-stage DR, by an unknown mechanism. Studying these patients provides an excellent opportunity to identify protective factors and to further understand the mechanisms involved in progression of DR. In order to better understand how metabolic dysregulation impacts development of DR, how neurovascular interactions become compromised in the diabetic retina, and to develop strategies to potentially restore homeostasis within the NVU, we decided to use metabolomic analyses. A highly sensitive metabolomics mass-spectrometry based approach was used in ocular and serum samples to identify the most prominent metabolic perturbations, to acquire a global overview of the metabolomic landscape in late-stage DR and to identify potentially protective circulating factors. At the ocular level, late-stage diabetic retinopathy was associated with severe dysregulation in amino acid levels; this was especially prominent in those generated during arginine metabolism, suggesting a preferential activity in the arginase pathway over the alternative Nitric Oxide Synthase (NOS) pathway; Studies in the Oxygen-Induced-Retinopathy (OIR) mouse, a non-diabetic model that develops features of ischemic retinopathy, revealed a very similar metabolic landscape and, in vivo global isotope analysis confirmed the presence of asymmetrical arginine metabolism by showing: (1) over-activity in the arginase pathway leading to enhanced proline production; and (2) reduced activity in the alternative NOS pathway, with potentially reduced NO production. Even though NO’s role in DR and other retinopathies is not clearly understood, NO is known to be an important modulator of cellular interactions within the NVU and its lower availability in specific locations and/or time-points in pathological conditions may significantly contribute to the disruption of retinal neurovascular crosstalk. The work presented in this dissertation has also described novel functions for interneurons (amacrine and horizontal cells) and photoreceptors within the NVU, by showing that these cells play an active role in regulating their primary vasculature and thus, their blood supply. Furthermore, it has also shown that dysfunction of retinal neurons in this capacity can directly alter their own blood supply, therefore providing additional clues for disrupted retinal neurovascular crosstalk. Metabolomic analyses comparing serum samples from diabetic patients with or without PDR (long-term diabetic patients “protected” from late-stage DR versus those who were non-protected) revealed that “protected” patients had higher circulating levels of a purine metabolite, inosine. To assess its therapeutic potential in conditions of retinal ischemia, inosine was delivered to the eye of the OIR mouse where it enhanced effective revascularization of ischemic retinal areas, thus significantly reducing pathological neovascularization. These effects were associated with a favorable modulation of the local pro-inflammatory response that could result from an improved overall retinal metabolic status. These beneficial effects on retinal metabolism induced by inosine injections were observed as: (1) a reduction in basal mitochondrial respiration in vaso-obliterated areas (i.e., ischemic areas), which can potentially increase retinal neuronal tolerance to hypoxia by reducing the metabolic mismatch created by scarce metabolic supply and high neuronal demand; and (2) a reduction in proline production, suggesting antagonism of the arginase pathway (which is hyperactive in oxygen-induced-retinopathy and potentially in human PDR). In summary, the work presented in this dissertation employed a metabolomic-focused approach with a strong focus on neurovascular crosstalk to answer two intriguing questions regarding DR: (1) What is the characteristic ocular metabolic landscape of severe DR? (2) Is the “protection” against severe DR (observed in some long-term diabetic patients) associated with differences in circulating metabolic factors? The answers to these questions, presented below, could serve as the basis of future targeted, more effective and earlier acting therapeutics that would revolutionize DR patient management. Identification of the most prominently affected metabolic pathways in eyes with severe DR has identified specific pathways of amino acid metabolism as potential targets for development of new drugs for DR. We have also identified a circulating protective factor, inosine, in “protected” patients and further investigated its ability to (1) prevent development of retinal ischemia and pathological neovascularization; (2) adjust retinal metabolism to the limited energy supplies in ischemic areas; and (3) counteract development of prominent metabolic dysregulation by potentially inhibiting the pathology-promoting arginase pathway. We believe that inosine can potentially become an effective, early-acting therapeutic agent to prevent progression of DR. In addition, these metabolites could potentially be used as reliable biomarkers for monitoring response to therapy and for predicting risk of developing or progressing DR. Finally, the work presented in this dissertation supports the concept that early intervention for treating DR will restore balance and stabilize cellular interactions within the NVU, thereby reversing the chronic stressors (e.g., extreme conditions of metabolic insufficiency in retinal ischemic areas) that ultimately drive development and progression of retinal pathology.Fundação para a Ciência e a Tecnologia (FCT