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Denser matrix that slows sprout invasion leads to smaller sprout diameters. (A) Representative images (max intensity projections) of invading endothelial cells (ECs) in response to varying collagen density. All conditions were cultured for 3 days with 250 nM sphingosine 1-phosphate (S1P) in endothelial cell growth medium 2 [EGM2; supplemented with 25 ng ml–1 phorbol 12-myristate 13-acetate (PMA)] added to the chemokine channel within collagen hydrogels of the indicated density. Nuclei (magenta), 5-ethynyl-2′-deoxyuridine (EdU; cyan), Ulex Europaeus Agglutinin-1 (UEA; white), and yellow dashed lines indicate parent vessel edge. (B–D) Quantifications of proliferation, invasion speed, and sprout diameter as a function of collagen hydrogel density. For proliferation: n ≥ 6 per condition, for invasion speed: n ≥ 60 per condition, and for sprout diameter: n ≥ 100 per condition. (E,F) Relationships between proliferation and sprout diameter (E) and invasion speed and sprout diameter (F), with red dashed lines indicating a linear regression and statistical analyses performed by Pearson’s correlation. Sample size for each mean is identical to those of panels (B–D). All data presented as mean ± SD; *indicates a statistically significant comparison with P < 0.05 (one-way ANOVA).
Source publication
Angiogenesis is a complex, multicellular process that involves bidirectional interactions between extracellular matrix (ECM) and collectively invading endothelial cell (EC) sprouts that extend the microvasculature during development, wound healing, and disease processes. While many aspects of angiogenesis have been well studied, the relationship be...
Citations
... Mechanistically, cancer cells increase production and secretion of various angiogenic factors, including growth hormones and cytokines, into extracellular matrix (ECM) to activate endothelial cells (ECs) [15,16]. The activated ECs can degrade endothelial basement membrane to allow them to migrate into the ECM [17]. The migrated ECs then proliferate, further migrate toward the source of stimulants, form the hollow tubes, and finally create new vascular meshes [17]. ...
... The activated ECs can degrade endothelial basement membrane to allow them to migrate into the ECM [17]. The migrated ECs then proliferate, further migrate toward the source of stimulants, form the hollow tubes, and finally create new vascular meshes [17]. Recent evidence has demonstrated the involvement of ARID1A in regulating angiogenesis [18,19]. ...
Background
Defects and deficiency of AT-rich interactive domain-containing protein 1A (ARID1A) encoded by a tumor suppressor gene ARID1A have recently been suggested to get involved in angiogenesis, a crucial process in carcinogenesis. However, molecular mechanisms of ARID1A deficiency to induce angiogenesis in kidney cancer remain underinvestigated.
Methods
We performed large-scale identification of ARID1A protein interactors in renal tubular epithelial cells (RTECs) using immunoprecipitation (IP) followed by nanoLC-ESI-LTQ-Orbitrap tandem mass spectrometry (MS/MS). Their roles in angiogenesis were investigated using various assays.
Results
A total of 74 ARID1A-interacting proteins were identified. Protein–protein interactions analysis revealed that these identified proteins interacted directly or indirectly with ARID1A. Among them, the direct interaction between ARID1A and β-actin was validated by IP and reciprocal IP followed by Western blotting. Small interfering RNA (siRNA) was used for single and double knockdowns of ARID1A and ACTB. Semi-quantitative RT-PCR demonstrated that deficiency of ARID1A, but not ACTB, significantly affected expression of angiogenesis-related genes in RTECs (VEGF and FGF2 were increased, whereas PDGF and EGF were decreased). However, the knockdowns did not affect TGFB1 and FGF1 levels. The quantitative mRNA expression data of VEGF and TGFB1 were consistent with the secreted levels of their protein products as measured by ELISA. Only secreted products derived from ARID1A-deficient RTECs significantly increased endothelial cells (ECs) migration and tube formation. Some of the other carcinogenic features could also be confirmed in the ARID1A-deficient RTECs, including increased cell migration and chemoresistance. Double knockdowns of both ARID1A and ACTB did not enhance the effects of single ARID1A knockdown in all assays.
Conclusions
We report herein a large dataset of the ARID1A-interacting proteins in RTECs using an IP-MS/MS approach and confirm the direct interaction between ARID1A and β-actin. However, the role of ARID1A deficiency in angiogenesis is independent of β-actin.
... We observed that PMA is a strong inducer of sprouting angiogenesis [28,33,38]. PMA disrupts the integrity of the endothelial cell layer and upregulates the synthesis of proteinases, such as matrix metalloproteinases, that are essential for cell invasion in the early stages of angiogenesis [30,39]. PMA significantly accelerated migration, as well as vascular network formation and stabilization. ...
Human induced pluripotent stem cell (hiPSC)-derived brain spheroids can recapitulate the complex cytoarchitecture of the brain, as well as the genetic/epigenetic footprint of human brain development. However, hiPSC-derived 3D models such as spheroid and organoids does not have a perfusable microvascular network, which plays a vital role in maintaining homeostasis in vivo . With the critical balance of positive and negative angiogenic modulators, 3D microvascular network can be achieved by angiogenesis. This paper reports on a microfluidic-based three-dimensional, cortical spheroid grafted on the vascular-network. Vascular network was formed by inducing angiogenic sprouting using concentration gradient-driven angiogenic factors in the microfluidic device. We investigate critical factors for angiogenic vascular network formation with spheroid placement, including 1) a PKCα activator, phorbol-12-myristate-13-acetate (PMA); 2) orientation of endothelial cells under perfusion and permeability of vascular network; 3) effect of extracellular matrix (ECM) types and their densities on angiogenesis; and 4) integration with cortical spheroid on vascular network. This paper demonstrates proof of concept for the potential utility of a membrane-free in vitro cortical spheroid tissue construct with perfusable microvascular network that can be scaled up to a high throughput platform. It can provide a cost-effective alternative platform to animal testing by modeling brain diseases and disorders, and screening drugs.
... Therefore, even though the directional persistence of EC migration is high during angiogenesis in ageing-affected organisms, EC migration is diminished under the combined influence of the mechanisms mentioned above, resulting in reduced vascular sprouting. At the same time, the increased tension allows stem cells (proliferating endothelial cells) to implant into the developing bud and proliferate indefinitely, thereby extending the vascular bud [76,77]. FN undergoes a non-enzymatic glycation reaction during ageing, and glycated FN readily binds to RAGE, competing with the receptor VEGFR2 for c-Src binding and thereby directly inhibiting the formation of the VEGFR2c-Src complex and affecting the activation of downstream angiogenic signals, such as those of Rac and MEK, required for EC proliferation. ...
Each step in angiogenesis is regulated by the extracellular matrix (ECM). Accumulating evidence indicates that ageing-related changes in the ECM driven by cellular senescence lead to a reduction in neovascularisation, reduced microvascular density, and an increased risk of tissue ischaemic injury. These changes can lead to health events that have major negative impacts on quality of life and place a significant financial burden on the healthcare system. Elucidating interactions between the ECM and cells during angiogenesis in the context of ageing is neceary to clarify the mechanisms underlying reduced angiogenesis in older adults. In this review, we summarize ageing-related changes in the composition, structure, and function of the ECM and their relevance for angiogenesis. Then, we explore in detail the mechanisms of interaction between the aged ECM and cells during impaired angiogenesis in the older population for the first time, discussing diseases caused by restricted angiogenesis. We also outline several novel pro-angiogenic therapeutic strategies targeting the ECM that can provide new insights into the choice of appropriate treatments for a variety of age-related diseases. Based on the knowledge gathered from recent reports and journal articles, we provide a better understanding of the mechanisms underlying impaired angiogenesis with age and contribute to the development of effective treatments that will enhance quality of life.
... This system, Figure 5, also explicitly shows how cells can advance over the irregular hydrogel pores slower than through the open trail. This tip cell advancement is comparable not only to Figure 1D but also to the results obtained by Wang et al. (2021) where ECM-cell interactions regulate the sprout diameter. We illustrate the dynamics of our model in Figure 6, where we changed the initial condition to an initial sphere, which will also act as our cell reservoir, to solely focus on the tip advancement that is the region of interest in our model. ...
Sprouting angiogenesis is a core biological process critical to vascular development. Its accurate simulation, relevant to multiple facets of human health, is of broad, interdisciplinary appeal. This study presents an in-silico model replicating a microfluidic assay where endothelial cells sprout into a biomimetic extracellular matrix, specifically, a large-pore, low-concentration fibrin-based porous hydrogel, influenced by chemotactic factors. We introduce a novel approach by incorporating the extracellular matrix and chemotactic factor effects into a unified term using a single parameter, primarily focusing on modelling sprouting dynamics and morphology. This continuous model naturally describes chemotactic-induced sprouting with no need for additional rules. In addition, we extended our base model to account for matrix sensing and degradation, crucial aspects of angiogenesis. We validate our model via a hybrid in-silico experimental method, comparing the model predictions with experimental results derived from the microfluidic setup. Our results underscore the intricate relationship between the extracellular matrix structure and angiogenic sprouting, proposing a promising method for predicting the influence of the extracellular matrix on angiogenesis.
... 26 In addition, collagen fibers that define the pore size are sufficiently deformable to support angiogenic sprouting of endothelial cells. 56,57 Thus, our findings suggest that the significant increases in collagen matrix pore size due to HA or DS addition may facilitate the initial extension and sustained elongation of multicellular processes into matrix void spaces during cancer invasion or endothelial sprouting. ...
Fibrillar collagens and glycosaminoglycans (GAGs) are structural biomolecules that are natively abundant to the extracellular matrix (ECM). Prior studies have quantified the effects of GAGs on the bulk mechanical properties of the ECM. However, there remains a lack of experimental studies on how GAGs alter other biophysical properties of the ECM, including ones that operate at the length scales of individual cells such as mass transport efficiency and matrix microstructure. Here we characterized and decoupled the effects of the GAG molecules chondroitin sulfate (CS) dermatan sulfate (DS) and hyaluronic acid (HA) on the stiffness (indentation modulus), transport (hydraulic permeability), and matrix microarchitecture (pore size and fiber radius) properties of collagen-based hydrogels. We complement these biophysical measurements of collagen hydrogels with turbidity assays to profile collagen aggregate formation. Here we show that CS, DS, and HA differentially regulate the biophysical properties of hydrogels due to their alterations to the kinetics of collagen self-assembly. In addition to providing information on how GAGs play significant roles in defining key physical properties of the ECM, this work shows new ways in which stiffness measurements, microscopy, microfluidics, and turbidity kinetics can be used complementary to reveal details of collagen self-assembly and structure.
... Recently, the Baker group showed that dynamic interactions between stalk cells and the neighbouring ECM were at the core of sprouting angiogenesis. Applying combined forces and proteolysis, sprout stalk cells indeed compact and degrade the ECM, opening a space for three-dimensional expansion that depends on the matrix density and the forces at play [294]. This matrix-mediated cell-cell mechanical communication was found to be critical to direct cell migration and organize the vascular network, guaranteeing viable function [65,292]. ...
... These interactions can even lead to positive feedback loops that may prevent endothelial homeostasis [316]. Moreover, the mechanical response of ECs is bidirectional: EC response is dictated by the surrounding environment, which in turn is impacted by EC behaviour [65,292,294], adding another level of coupling. ...
Favouring or thwarting the development of a vascular network is essential in fields as diverse as oncology, cardiovascular disease or tissue engineering. As a result, understanding and controlling angiogenesis has become a major scientific challenge. Mechanical factors play a fundamental role in angiogenesis and can potentially be exploited for optimizing the architecture of the resulting vascular network. Largely focusing on in vitro systems but also supported by some in vivo evidence, the aim of this Highlight Review is dual. First, we describe the current knowledge with particular focus on the effects of fluid and solid mechanical stimuli on the early stages of the angiogenic process, most notably the destabilization of existing vessels and the initiation and elongation of new vessels. Second, we explore inherent difficulties in the field and propose future perspectives on the use of in vitro and physics-based modelling to overcome these difficulties.
... Furthermore, it is proposed that collagen is important for EC invasion and lumen formation during angiogenesis, while laminin is crucial during the vessel maturation and stabilization phase. Interestingly, and relevant to our studies, recent works have demonstrated the importance of ECM during the sprouting angiogenesis process (68)(69)(70). Thus, the direct effect of ECM proteins on EC function during angiogenesis, neovascularization, and blood vessel maturation highlights the need for investigation into their regulation. ...
The (Pro)renin receptor ((P)RR), also known as ATP6AP2, is a single-transmembrane protein that is implicated in a multitude of biological processes. However, the exact role of ATP6AP2 during blood vessel development remains largely undefined. Here, we use an inducible endothelial cell (EC)-specific Atp6ap2 knockout mouse model to investigate the role of ATP6AP2 during both physiological and pathological angiogenesis in vivo. We observed that postnatal deletion of Atp6ap2 in ECs results in cell migration defects, loss of tip cell polarity and subsequent impairment of retinal angiogenesis. In vitro, Atp6ap2 deficient ECs similarly displayed reduced cell migration, impaired sprouting, and defective cell polarity. Transcriptional profiling of ECs isolated from Atp6ap2 mutant mice further indicated regulatory roles in angiogenesis, cell migration and extracellular matrix composition. Mechanistically, we provided evidence that expression of various extracellular matrix components is controlled by ATP6AP2 via the extracellular-signal-regulated kinase (ERK) pathway. Furthermore, Atp6ap2 deficient retinas exhibited reduced revascularization in an oxygen induced retinopathy model. Collectively, our results demonstrated a critical role of ATP6AP2 as a regulator of developmental and pathological angiogenesis.
... This system, Fig.5, also explicitly shows how cells can advance over the irregular hydrogel pores slower than through the open trail. This tip cell advancement is comparable not only to Fig.1.D but also to the results obtained in 56 where ECM-cell interactions regulate the sprout diameter. We illustrate the dynamics of our model in Fig.6, where we changed the initial condition to an initial sphere, which will also act as our cell reservoir, to solely focus on the tip advancement that is the region of interest in our model. ...
Migration is a fundamental cellular behaviour that plays an essential role in vascular development and angiogenesis. Due to its relevance to many aspects of human health, the ability to accurately reproduce cell migration is of broad and multidisciplinary interest. This work presents a model to reproduce a microfluidic assay in which endothelial cells chemotactically migrate into a fibrin-based porous hydrogel. Endothelial cells emanate from a parent vessel through the extracellular matrix towards the increasing chemotactic factor concentration. We couple into the same parameter the extracellular matrix and the chemotactic factor distribution. We focus our efforts on modelling sprouting dynamics and morphology, providing a new framework to understand cell migration and the influence of the extracellular matrix. The model naturally describes chemotactic cell behaviour in response to the extracellular matrix structure. We further extend our model to allow extracellular matrix sensing and degradation. We validated the model based on a hybrid in silico-experimental approach by comparing it against the experimental results obtained in the microfluidic assay. Together, our findings highlight the nontrivial role of the extracellular matrix structure in angiogenic sprouting and offer an approach to predicting the effect of the extracellular matrix.
... Neovascularization is an imperative phase of wound healing that occurs through angiogenesis and vasculogenesis [57]. Angiogenesis is hallmarked with the formation of new blood capillaries via sprouting, which requires dynamic and spatially regulated interaction between endothelial cells, angiogenesis factors, and surrounding ECM proteins [58,59]. The formation of new sprouts after treatment with the nanofibrous scaffolds at day 7 is depicted in Fig. 6. ...
Burn wound care always remains a herculean task owing to its myriad of complications following injury. The current work describes the development of a biodegradable 3D framework fabricated from PVA, gelatin, chitosan polymers embedded with ferulic acid and resveratrol with direct influence on cellular recruitment and activation, without altering the temporal sequence of the healing process. The structural analysis of the scaffold revealed randomly oriented highly interconnected porous structured fibers which favored cell adhesion, migration and proliferation. Furthermore, the physico-chemical characterisation elucidated good compatibility of the drugs with the scaffolds and in-vitro release profile exhibited the sustained release of bioactive components up to 96 h endorsing the fabricated nanofibrous scaffold as a controlled drug delivery system. The drug incorporated nanofibrous construct exhibited an extensive potential in augmenting angiogenesis and cell migration (82% wound closure at 8 h) in in-vitro studies. The biotherapeutic nanofibrous scaffold enhanced the restoration of wounded tissue in in-vivo models. Thus, the present study strides way for developing, biomimetic nanofibrous scaffolds with sequential drug delivery for chronic wound management.
... Angiogenic neovessels remodel the ECM through traction, proteolytic degradation, and cell-matrix adhesion, and the ECM properties influence vascular growth and alignment. The cell-ECM mechanical interaction was also found to regulate endothelial sprouting speed and proliferation, thereby regulate sprout stalk diameter [94]. ...
The tumor ecosystem with heterogeneous cellular compositions and the tumor microenvironment has increasingly become the focus of cancer research in recent years. The extracellular matrix (ECM), the major component of the tumor microenvironment, and its interactions with the tumor cells and stromal cells have also enjoyed tremendously increased attention. Like the other components of the tumor microenvironment, the ECM in solid tumors differs significantly from that in normal organs and tissues. We review recent studies of the complex roles the tumor ECM plays in cancer progression, from tumor initiation, growth to angiogenesis and invasion. We highlight that the biomolecular, biophysical, and mechanochemical interactions between the ECM and cells not only regulate the steps of cancer progression, but also affect the efficacy of systemic cancer treatment. We further discuss the strategies to target and modify the tumor ECM to improve cancer therapy.
