A compartment model of VEGF distribution in blood, healthy and diseased tissues

<p>Abstract</p> <p>Background</p> <p>Angiogenesis is a process by which new capillaries are formed from pre-existing blood vessels in physiological (e.g., exercise, wound healing) or pathological (e.g., ischemic limb as in peripheral arterial disease, cancer) contexts. This neovascular mechanism is mediated by the vascular endothelial growth factor (VEGF) family of cytokines. Although VEGF is often targeted in anti-angiogenic therapies, there is little knowledge about how its concentration may vary between tissues and the vascular system. A compartment model is constructed to study the VEGF distribution in the tissue (including matrix-bound, cell surface receptor-bound and free VEGF isoforms) and in the blood. We analyze the sensitivity of this distribution to the secretion rate, clearance rate and vascular permeability of VEGF.</p> <p>Results</p> <p>We find that, in a physiological context, VEGF concentration varies approximately linearly with the VEGF secretion rate. VEGF concentration in blood but not in tissue is dependent on the vascular permeability of healthy tissue. Model simulations suggest that relative VEGF increases are similar in blood and tissue during exercise and return to baseline within several hours. In a pathological context (tumor), we find that blood VEGF concentration is relatively insensitive to increased vascular permeability in tumors, to the secretion rate of VEGF by tumors and to the clearance. However, it is sensitive to the vascular permeability in the healthy tissue. Finally, the VEGF distribution profile in healthy tissue reveals that about half of the VEGF is complexed with the receptor tyrosine kinase VEGFR2 and the co-receptor Neuropilin-1. In diseased tissues, this binding can be reduced to 15% while VEGF bound to the extracellular matrix and basement membranes increases.</p> <p>Conclusion</p> <p>The results are of importance for physiological conditions (e.g., exercise) and pathological conditions (e.g., peripheral arterial disease, coronary artery disease, cancer). This mathematical model can serve as a tool for understanding the VEGF distribution in physiological and pathological contexts as well as a foundation to investigate pro- or anti-angiogenic strategies.</p

Pharmacokinetics and pharmacodynamics of VEGF-neutralizing antibodies

<p>Abstract</p> <p>Background</p> <p>Vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis, and its role in cancer biology has been widely studied. Many cancer therapies target angiogenesis, with a focus being on VEGF-mediated signaling such as antibodies to VEGF. However, it is difficult to predict the effects of VEGF-neutralizing agents. We have developed a whole-body model of VEGF kinetics and transport under pathological conditions (in the presence of breast tumor). The model includes two major VEGF isoforms VEGF₁₂₁and VEGF₁₆₅, receptors VEGFR1, VEGFR2 and co-receptors Neuropilin-1 and Neuropilin-2. We have added receptors on parenchymal cells (muscle fibers and tumor cells), and incorporated experimental data for the cell surface density of receptors on the endothelial cells, myocytes, and tumor cells. The model is applied to investigate the action of VEGF-neutralizing agents (called "anti-VEGF") in the treatment of cancer.</p> <p>Results</p> <p>Through a sensitivity study, we examine how model parameters influence the level of free VEGF in the tumor, a measure of the response to VEGF-neutralizing drugs. We investigate the effects of systemic properties such as microvascular permeability and lymphatic flow, and of drug characteristics such as the clearance rate and binding affinity. We predict that increasing microvascular permeability in the tumor above 10^-5cm/s elicits the undesired effect of increasing tumor interstitial VEGF concentration beyond even the baseline level. We also examine the impact of the tumor microenvironment, including receptor expression and internalization, as well as VEGF secretion. We find that following anti-VEGF treatment, the concentration of free VEGF in the tumor can vary between 7 and 233 pM, with a dependence on both the density of VEGF receptors and co-receptors and the rate of neuropilin internalization on tumor cells. Finally, we predict that free VEGF in the tumor is reduced following anti-VEGF treatment when VEGF₁₂₁comprises at least 25% of the VEGF secreted by tumor cells.</p> <p>Conclusions</p> <p>This study explores the optimal drug characteristics required for an anti-VEGF agent to have a therapeutic effect and the tumor-specific properties that influence the response to therapy. Our model provides a framework for investigating the use of VEGF-neutralizing drugs for personalized medicine treatment strategies.</p

The Presence of VEGF Receptors on the Luminal Surface of Endothelial Cells Affects VEGF Distribution and VEGF Signaling

Vascular endothelial growth factor (VEGF) is a potent cytokine that binds to specific receptors on the endothelial cells lining blood vessels. The signaling cascade triggered eventually leads to the formation of new capillaries, a process called angiogenesis. Distributions of VEGF receptors and VEGF ligands are therefore crucial determinants of angiogenic events and, to our knowledge, no quantification of abluminal vs. luminal receptors has been performed. We formulate a molecular-based compartment model to investigate the VEGF distribution in blood and tissue in humans and show that such quantification would lead to new insights on angiogenesis and VEGF-dependent diseases. Our multiscale model includes two major isoforms of VEGF (VEGF121 and VEGF165), as well as their receptors (VEGFR1 and VEGFR2) and the non-signaling co-receptor neuropilin-1 (NRP1). VEGF can be transported between tissue and blood via transendothelial permeability and the lymphatics. VEGF receptors are located on both the luminal and abluminal sides of the endothelial cells. In this study, we analyze the effects of the VEGF receptor localization on the endothelial cells as well as of the lymphatic transport. We show that the VEGF distribution is affected by the luminal receptor density. We predict that the receptor signaling occurs mostly on the abluminal endothelial surface, assuming that VEGF is secreted by parenchymal cells. However, for a low abluminal but high luminal receptor density, VEGF binds predominantly to VEGFR1 on the abluminal surface and VEGFR2 on the luminal surface. Such findings would be pertinent to pathological conditions and therapies related to VEGF receptor imbalance and overexpression on the endothelial cells and will hopefully encourage experimental receptor quantification for both luminal and abluminal surfaces on endothelial cells

VEGF immobilization and VEGFR2 trafficking and phosphorylation: in vitro and in vivo implications

Modern drug development is marked by high failure rates in translation to the clinic. Further, many drugs that succeed in clinical trials work for only a fraction of patients. Systems pharmacology attempts to address these challenges by improving our understanding of the disease-therapy system, integrating detailed molecular interactions, cellular signaling, tissue architecture, and whole body physiology. I built cutting-edge, molecularly-detailed, multi-scale computational models to study the effects of immobilization of growth factors on signaling in angiogenesis, focusing in particular on the binding of vascular endothelial growth factor (VEGF) family members to the ECM. While most studies of VEGF signaling use only VEGF presented in solution, there is evidence that a large potion of VEGF may be ECM-bound in vivo, and relative expression of isoforms binding to ECM vs. found only in solution varies by tissue and changes in disease, motivating further study of this question. Starting at the in vitro level, we showed that differential signaling of VEGF-receptor 2 (VEGFR2) in response to soluble vs. immobilized VEGF can be explained by reduced internalization of ECM-VEGF-VEGFR2 complexes. Moving in vivo, we predicted differences in both growth factor distribution and receptor activation by VEGF family ligands, as a function of their ECM-binding properties. These predictions are consistent with observed vascular phenotypes in mice expressing single VEGF isoforms. Next, we explored how VEGF splicing changes in peripheral artery disease lead to impaired angiogenic responses to ischemia. Our model showed that the VEGF165b isoform, which does not bind to ECM or to the coreceptor NRP1, is a weak activator of VEGFR2 in vivo, and competes for binding to VEGF-receptor 1, but not VEGF-receptor 2. Finally, we used this model to screen potential therapeutic strategies designed to promote VEGF-mediated revascularization in ischemic disease and tissue engineering applications. Within a single system, we compared failed and promising biomaterial-based VEGF delivery systems, antibody-based therapeutics, and gene therapy strategies to identify key rules for design, optimization, and translation of these pro-angiogenic therapies

PET study of intravitreal adalimumab pharmacokinetics in a uveitis rat model

X. García-Otero is grateful to the IDIS (Health Research Institute of Santiago de Compostela) for financing his predoctoral research fellowship. C. Mondelo-García, E. Bandín-Vilar and A. Fernández-Ferreiro are grateful to the Carlos III Health Institute for financing their personnel contracts: JR20/00026, CM20/00135 and JR18/00014.S

Writing 3D in vitro models of human tendon within a biomimetic fibrillar support platform

Dissertação de mestrado em Engenharia Biomédica (especialização em Biomateriais, Reabilitação e Biomecânica)As patologias do tendão são doenças altamente debilitantes, para as quais os tratamentos atuais permanecem desafiadores e têm resultados de recuperação pouco relevantes. Deste modo, modelos in vitro relevantes, que permitam o estudo da tendinopatia e a testagem de novas abordagens regenerativas para desenvolver melhores tratamentos são altamente necessários. Neste trabalho, propomos o fabrico automatizado de sistemas microfisiológicos bioimpressos em 3D (MPS), incorporados numa plataforma de suporte fibrilar biomimética baseada na automontagem de nanocristais de celulose (CNCs). A matriz extracelular descelularizada do tendão (dECM) foi usada para produzir biotinta que recapitula de perto as pistas biofísicas e bioquímicas do nicho de células do tendão e, assim, autoinduz a diferenciação tenogénica de células-estaminais derivadas do tecido adiposo humano (hASCs). Dois MPS foram desenvolvidos: um sistema de monocultura que recria os padrões celulares e o fenótipo do tendão; e um sistema multicelular, com a incorporação de células endoteliais para estudar a comunicação entre o tendão e o sistema vascular, que desempenha papéis críticos na tendinopatia e no desenvolvimento do tendão. Ambos os MPS mostraram alta viabilidade celular, proliferação e alinhamento durante a cultura até 21 dias, e o hidrogel de dECM induziu a diferenciação de células-estaminais em direção à linhagem tenogénica, mostrado pela expressão de marcadores relacionados com o tendão, como Scleraxis (SCX) e Tenomodulin (TNMD). Notavelmente, as células endoteliais migram em direção ao compartimento do tendão, mostrando a atração química existente entre os dois compartimentos, mas não o invadiram. A comunicação com células endoteliais parece aumentar a diferenciação tenogénica das hASCs. No geral, o sistema proposto pode ser promissor para o fabrico automatizado de modelos organotípicos de tendão num-chip que será uma nova ferramenta valiosa para estudar a fisiologia e as patologias do tendão, bem como o efeito de medicamentos para o tratamento de tendinopatias.Tendon pathologies are highly debilitating diseases, for which current treatments remains challenging, and has poor recovery outcomes. Therefore, relevant in vitro models allowing to study tendinopathies and test new regenerative approaches to develop better treatments are highly needed. Here we propose the automated fabrication of 3D bioprinted microphysiological systems (MPS) embedded into a biomimetic fibrillar support platform based on self-assembling of cellulose nanocrystals (CNCs). Tendon decellularized extracellular matrix (dECM) was used to produce bioink that closely recapitulate the biophysical and biochemical cues of tendon cell niche, and thus self-induce the tenogenic differentiation of human adipose derived stem cells (hASCs). Two MPS were developed: a monoculture system that recreates the cellular patterns and phenotype of tendon core; and a multicellular system, incorporating endothelial cells to study the crosstalk between the tendon and the vascular compartments, which plays critical roles in tendinopathy and tendon development. Both MPS showed high cell viability, proliferation, and alignment during culture up to 21 days, and the dECM hydrogel induced stem cell differentiation towards tenogenic lineage, as shown by the expression of tendon-related markers such as Scleraxis (SCX) and Tenomodulin (TNMD). Remarkably, endothelial cells migrate towards tendon compartment, showing the existing chemoattraction between the two compartments, but did not invade it. The crosstalk with endothelial cells seem to boost hASCs tenogenesis. Overall, the proposed system might be promising for the automated fabrication of organotypic tendon-on-chip models that will be a valuable new tool to study tendon physiology and pathologies, as well as the effect of drugs for the treatment of tendinopathy

Neuroinflammation, Mast Cells, and Glia: Dangerous Liaisons

The perspective of neuroinflammation as an epiphenomenon following neuron damage is being replaced by the awareness of glia and their importance in neural functions and disorders. Systemic inflammation generates signals that communicate with the brain and leads to changes in metabolism and behavior, with microglia assuming a pro-inflammatory phenotype. Identification of potential peripheral-to-central cellular links is thus a critical step in designing effective therapeutics. Mast cells may fulfill such a role. These resident immune cells are found close to and within peripheral nerves and in brain parenchyma/meninges, where they exercise a key role in orchestrating the inflammatory process from initiation through chronic activation. Mast cells and glia engage in crosstalk that contributes to accelerate disease progression; such interactions become exaggerated with aging and increased cell sensitivity to stress. Emerging evidence for oligodendrocytes, independent of myelin and support of axonal integrity, points to their having strong immune functions, innate immune receptor expression, and production/response to chemokines and cytokines that modulate immune responses in the central nervous system while engaging in crosstalk with microglia and astrocytes. In this review, we summarize the findings related to our understanding of the biology and cellular signaling mechanisms of neuroinflammation, with emphasis on mast cell-glia interactions

Tendon Immune Regeneration: Insights on the Synergetic Role of Stem and Immune Cells during Tendon Regeneration

Tendon disorders represent a very common pathology in today’s population, and tendinopathies that account 30% of tendon-related injuries, affect yearly millions of people which in turn cause huge socioeconomic and health repercussions worldwide. Inflammation plays a prominent role in the development of tendon pathologies, and advances in understanding the underlying mechanisms during the inflammatory state have provided additional insights into its potential role in tendon dis-orders. Different cell compartments, in combination with secreted immune modulators, have shown to control and modulate the inflammatory response during tendinopathies. Stromal compartment represented by tenocytes has shown to display an important role in orchestrating the inflammatory response during tendon injuries due to the interplay they exhibit with the immune-sensing and infiltrating compartments, which belong to resident and recruited immune cells. The use of stem cells or their derived secretomes within the regenerative medicine field might represent synergic new therapeutical approaches that can be used to tune the reaction of immune cells within the damaged tissues. To this end, promising opportunities are headed to the stimulation of macrophages polarization towards anti-inflammatory phenotype together with the recruitment of stem cells, that possess immunomodulatory properties, able to infiltrate within the damaged tissues and improve tendinopathies resolution. Indeed, the comprehension of the interactions between tenocytes or stem cells with the immune cells might considerably modulate the immune reaction solving hence the inflammatory response and preventing fibrotic tissue formation. The purpose of this review is to compare the roles of distinct cell compartments during tendon homeostasis and injury. Furthermore, the role of immune cells in this field, as well as their interactions with stem cells and tenocytes during tendon regeneration, will be discussed to gain insights into new ways for dealing with tendinopathies

Local origins impart conserved bone type-related differences in human osteoblast behaviour

Osteogenic behaviour of osteoblasts from trabecular, cortical and subchondral bone were examined to determine any bone type-selective differences in samples from both osteoarthritic (OA) and osteoporotic (OP) patients. Cell growth, differentiation; alkaline phosphatase (TNAP) mRNA and activity, Runt-related transcription factor-2 (RUNX2), SP7-transcription factor (SP7), bone sialoprotein-II (BSP-II), osteocalcin/bone gamma-carboxyglutamate (BGLAP), osteoprotegerin (OPG, TNFRSF11B), receptor activator of nuclear factor-κβ ligand (RANKL, TNFSF11) mRNA levels and proangiogenic vascular endothelial growth factor-A (VEGF-A) mRNA and protein release were assessed in osteoblasts from paired humeral head samples from age-matched, human OA/OP (n = 5/4) patients. Initial outgrowth and increase in cell number were significantly faster (p < 0.01) in subchondral and cortical than trabecular osteoblasts, in OA and OP, and this bone type-related differences were conserved despite consistently faster growth in OA. RUNX2/SP7 levels and TNAP mRNA and protein activity were, however, greater in trabecular than subchondral and cortical osteoblasts in OA and OP. BSP-II levels were significantly greater in trabecular and lowest in cortical osteoblasts in both OA and OP. In contrast, BGLAP levels showed divergent bone type-selective behaviour; highest in osteoblasts from subchondral origins in OA and trabecular origins in OP. We found virtually identical bone type-related differences, however, in TNFRSF11B:TNFSF11 in OA and OP, consistent with greater potential for paracrine effects on osteoclasts in trabecular osteoblasts. Subchondral osteoblasts (OA) exhibited highest VEGF-A mRNA levels and release. Our data indicate that human osteoblasts in trabecular, subchondral and cortical bone have inherent, programmed diversity, with specific bone type-related differences in growth, differentiation and pro-angiogenic potential in vitro

Microfluidic organ-on-chip for assessing the transport of therapeutic molecules and polymeric nanoconstructs

openThe selective permeation of molecules and nanomedicines across the diseased vasculature dictates the success of a therapeutic intervention. Yet, in vitro assays cannot recapitulate relevant differences between the physiological and pathological microvasculature. Here, a double-channel microfluidic device was engineered to comprise vascular and extravascular compartments connected through a micropillar membrane with tunable permeability. The vascular compartment was coated by endothelial cells to achieve permeability values ranging from 0.1 um/sec, following a cyclic adenosine monophosphate (cAMP) pre-treatment (25 ug/mL), up to 2 um/sec, upon exposure to Mannitol, Lexiscan or in the absence of cells. Fluorescent microscopy was used to monitor the vascular behavior of 250 kDa Dextran molecules, 200 nm polystyrene nanoparticles (PB), and 1,000-400 nm discoidal polymeric nanoconstructs (DPN), under different permeability and flow conditions. In the proposed on-chip microvasculature, it was confirmed that permeation enhancers could favor the perivascular accumulation of ~ 200 nm, in a dose and time dependent fashion, while have no effect on larger particles. Moreover, the microfluidic device was used to interrogate the role of particle deformability in vascular dynamics. In the presence of a continuous endothelium, soft DPN attached to the vasculature more avidly at sub-physiological flows (100 um/sec) than rigid DPN, whose deposition was larger at higher flow rates (1 mm/sec). The proposed double-channel microfluidic device can be efficiently used to systematically analyze the vascular behavior of drug delivery systems to enhance their tissue specific accumulation.openXXXIII CICLO - BIOINGEGNERIA E ROBOTICA - BIOENGINEERING AND ROBOTICSPAOLO DECUZZIBarbato, MARIA GRAZI