Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro

Abstract

Angiogenesis is a complex morphogenetic process whereby endothelial cells from existing vessels invade as multicellular sprouts to form new vessels. Here, we have engineered a unique organotypic model of angiogenic sprouting and neovessel formation that originates from preformed artificial vessels fully encapsulated within a 3D extracellular matrix. Using this model, we screened the effects of angiogenic factors and identified two distinct cocktails that promoted robust multicellular endothelial sprouting. The angiogenic sprouts in our system exhibited hallmark structural features of in vivo angiogenesis, including directed invasion of leading cells that developed filopodia-like protrusions characteristic of tip cells, following stalk cells exhibiting apical-basal polarity, and lumens and branches connecting back to the parent vessels. Ultimately, sprouts bridged between preformed channels and formed perfusable neovessels. Using this model, we investigated the effects of angiogenic inhibitors on sprouting morphogenesis. Interestingly, the ability of VEGF receptor 2 inhibition to antagonize filopodia formation in tip cells was context-dependent, suggesting a mechanism by which vessels might be able to toggle between VEGF-dependent and VEGFindependent modes of angiogenesis. Like VEGF, sphingosine-1-phosphate also seemed to exert its proangiogenic effects by stimulating directional filopodial extension, whereas matrix metalloproteinase inhibitors prevented sprout extension but had no impact on filopodial formation. Together, these results demonstrate an in vitro 3D biomimetic model that reconstitutes the morphogenetic steps of angiogenic sprouting and highlight the potential utility of the model to elucidate the molecular mechanisms that coordinate the complex series of events involved in neovascularization.A ngiogenesis, the process by which new capillary vessels sprout from existing vasculature, plays a critical role in embryonic development and wound healing, and its dysregulation can contribute to cancer progression as well as numerous inflammatory and ischemic diseases (1, 2). Consequently, therapeutic strategies to suppress, enhance, or normalize angiogenesis are widely sought to treat a broad spectrum of diseases (1, 2). The most mature among these approaches targets the activity of angiogenic growth factors, such as vascular endothelial growth factor (VEGF), to modulate relevant signaling pathways and control the angiogenesis process. Indeed, inhibitors of such pathways have emerged as a mainstay therapy for some cancers and diabetic retinopathy (3-5). However, it is still unclear how the endothelial cells (ECs) lining blood vessels form new vessels, or how angiogenic factors regulate such a dynamic, multicellular process.Examining the physical process of angiogenesis requires experimental systems in which the formation of new capillary vessels can be easily observed and manipulated. Commonly used in vivo models such as the mouse dorsal window chamber, chick chorioallantoic membrane, and mouse corneal micro...

Valve Interstitial Cells Act in a Pericyte Manner Promoting Angiogensis and Invasion by Valve Endothelial Cells

“…Additionally, the question of whether the VEVIS invasive sprouts are indicative of functional in vivo angiogenesis complete with lumen formation and blood carrying capacity remains unclear. Future studies should include an in vitro angiogenesis assay that incorporates flow to better demonstrate an EC lumen 22 and an in vivo model to test VIC ability to support vessel formation 19 . Nonetheless, this work expands our understanding of valve cell-cell organization and communication with implications for valve homeostasis, heart valve tissue engineering, and the pathology of CAVD.…”

Development and Applications of Microfluidic Devices for Cell Culture in Cell Biology

“…The interstial flow is the extracellular fluid that exists in the interstitial spaces between tissue spaces, can direct the migration of cancer cells [79] (Figure 4). [80]. (b) Schematic for microfluidic cell migration assay enabling direct comparison of cell migration behavior between the condition and control sides [81].…”

“…Angiogenesis is crucial to include in cancer models, because it is a prerequisite for tumor growth, invasion, progression, and metastasis. One strategy to build vascular models is to separate two parallel microfluidic channels by a hydrogel filled channel in the middle [80]. The other way is the vascular structures fully embedded within 3D ECM [83].…”

“…Development of these micro engineering approaches has opened entirely new possibilities to create in vitro models that reconstitute more complex 3D organ level structures and to integrate crucial dynamic mechanical cues as well as chemical signals. There have been different kinds of tissue or organ chip models for specific research (Figure 6), such as liver-on-a-chip, [96] kidney-on-a-chip, [97,98] gut-on-a-chip, [99,100] lung-on-a-chip, [101,102] heart-on-a-chip [103] and vessel-on-a-chip [104].…”

Force Measurement Tools to Explore Cadherin Mechanotransduction

“…Microtissues of aligned cardiac cells suspended in ECM between fl exible cantilevers allow whole tissue force measurements while cells engage in cell -cell and cell -ECM adhesions in 3D (Figure 4c) (Boudou et al, 2012;Legant et al, 2009). Three dimensional in vitro angiogenesis models including the bead sprouting assay (Nakatsu et al, 2003), lumen formation assay (Koh et al, 2008), and more complex models such as those recently developed by Zheng et al (2012) and Nguyen et al (2013) are currently limited in their ability to examine mechanical regulation of vessel homeostasis and the angiogenic process. Another widely used 3D model, the in vitro skin model, has provided an excellent platform to study homophilic and heterophilic cell interactions within a multilayer, structured 3D environment that recapitulates the native setting (Figure 3d) (Boelsma et al, 2000;Alt-Holland et al, 2008).…”