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...

Gradients of diffusive molecules within 3D extracellular matrix (ECM) are essential in guiding many processes such as development, angiogenesis, and cancer. The spatial distribution of factors that guide these processes is complex, dictated by the distribution and architecture of vasculature and presence of surrounding cells, which can serve as sources or sinks of factors. To generate temporally and spatially defined soluble gradients within a 3D cell culture environment, we developed an approach to patterning microfluidically ported microchannels that pass through a 3D ECM. Micromolded networks of sacrificial conduits ensconced within an ECM gel precursor solution are dissolved following ECM gelation to yield functional microfluidic channels. The dimensions and spatial layout of channels are readily dictated using photolithographic methods, and channels are connected to external flow via a gasket that also serves to house the 3D ECM. We demonstrated sustained spatial patterning of diffusive gradients dependent on the architecture of the microfluidic network, as well as the ability to independently populate cells in either the channels or surrounding ECM, enabling the study of 3D morphogenetic processes. To highlight the utility of this approach, we generated model vascular networks by lining the channels with endothelial cells and examined how channel architecture, through its effects on diffusion patterns, can guide the location and morphology of endothelial sprouting out from the channels. We show that locations of strongest gradients define positions of angiogenic sprouting, suggesting a mechanism by which angiogenesis is regulated in vivo and a potential means to spatially defining vasculature in tissue engineering applications. This flexible 3D microfluidic approach should have utility in modeling simple tissues and will aid in the screening and identification of soluble factor conditions that drive morphogenetic events such as angiogenesis.

The ultimate design of functionally therapeutic engineered tissues and organs will rely on our ability to engineer vasculature that can meet tissue-specific metabolic needs. We recently introduced an approach for patterning the formation of functional spatially organized vascular architectures within engineered tissues in vivo. Here, we now explore the design parameters of this approach and how they impact the vascularization of an engineered tissue construct after implantation. We used micropatterning techniques to organize endothelial cells (ECs) into geometrically defined "cords," which in turn acted as a template after implantation for the guided formation of patterned capillaries integrated with the host tissue. We demonstrated that the diameter of the cords before implantation impacts the location and density of the resultant capillary network. Inclusion of mural cells to the vascularization response appears primarily to impact the dynamics of vascularization. We established that clinically relevant endothelial sources such as induced pluripotent stem cell-derived ECs and human microvascular endothelial cells can drive vascularization within this system. Finally, we demonstrated the ability to control the juxtaposition of parenchyma with perfused vasculature by implanting cords containing a mixture of both a parenchymal cell type (hepatocytes) and ECs. These findings define important characteristics that will ultimately impact the design of vasculature structures that meet tissue-specific needs.

Angiogenic sprouting is a critical process involved in vascular network formation within tissues. During sprouting, tip cells and ensuing stalk cells migrate collectively into the extracellular matrix while preserving cell–cell junctions, forming patent structures that support blood flow. Although several signaling pathways have been identified as controlling sprouting, it remains unclear to what extent this process is mechanoregulated. To address this question, we investigated the role of cellular contractility in sprout morphogenesis, using a biomimetic model of angiogenesis. Three-dimensional maps of mechanical deformations generated by sprouts revealed that mainly leader cells, not stalk cells, exert contractile forces on the surrounding matrix. Surprisingly, inhibiting cellular contractility with blebbistatin did not affect the extent of cellular invasion but resulted in cell–cell dissociation primarily between tip and stalk cells. Closer examination of cell–cell junctions revealed that blebbistatin impaired adherens-junction organization, particularly between tip and stalk cells. Using CRISPR/Cas9-mediated gene editing, we further identified NMIIA as the major isoform responsible for regulating multicellularity and cell contractility during sprouting. Together, these studies reveal a critical role for NMIIA-mediated contractile forces in maintaining multicellularity during sprouting and highlight the central role of forces in regulating cell–cell adhesions during collective motility.

Cell -cell adhesions serve to mechanically couple cells, allowing for long-range transmission of forces across cells in development, disease, and homeostasis. Recent work has shown that such contacts also play a role in transducing mechanical cues into a wide variety of cellular behaviors important to tissue function. As such, understanding the mechanical regulation of cells through their adhesion molecules has become a point of intense focus. This review will highlight the existing and emerging technologies and models that allow for exploration of cadherin-based adhesions as sites of mechanotransduction.