The vascular network is arguably the most important organ in a human body. It connects all the other tissues and carries oxygen and nutrients to support their growth and maintenance. Vascular morphogenesis is a multi-step process that occurs in both embryogenesis and adults during pregnancy, wound healing and other pathological conditions. It is tightly regulated by a complex network of transcription factors and signaling pathways in endothelial cell (EC) intrinsic and extrinsic manners.

Slug (Snai2) is a member of the well-conserved Snail family of transcription factors and has roles in multiple developmental processes, including epithelial-to-mesenchymal transitions (EMT). Our lab has been previously demonstrated that Slug expression is up-regulated in angiogenic EC in vitro and is critical for sprouting, partly by controlling EC migration. Preliminary evidence suggests that Slug might play other roles in this process; however, a comprehensive investigation on the mechanisms of Slug-regulated vessel formation is lacking. In addition, although it has been suspected that Slug-mediated endothelial-to-mesenchymaltransition (EndoMT), a process similar to EMT, might have a role in angiogenesis, there is little direct experimental evidence supporting this hypothesis. Moreover, it is unclear whether the role of Slug in angiogenesis observed in our in vitro system also applies to in vivo models.

In this study, using various in vitro 3D culture models, as well as several in vivo models, we confirmed the critical role of Slug during both developmental and pathological vascular morphogenesis. Using RNA-sequencing analysis, we also demonstrated that Slug regulates an

EMT-like suite of target genes. The level of Slug expression determines whether EC perform normal sprouting angiogenesis or excessive pathological angiogenesis by directly suppressing Dll4-activated Notch signaling to upregulate VEGFR2. In vivo, reduced Notch signaling

completely rescues delayed retinal angiogenesis in SlugKO mice. Unlike Snail, Slug is absolutely required for pathological angiogenesis necessary for sustained tumor growth while it plays a transient role in developmental angiogenesis in mice. Slug, but not Snail, is activated in EC by the pro-angiogenic factor SDF-1 via its canonical receptor CXCR4 and subsequent ERK5 phosphorylation. These data are consistent with Slug regulating a partial endothelial-to mesenchymal transition in angiogenic EC.

Angiogenesis is a tightly regulated multi-step process in which new blood vessels form from the preexisting vasculature. Due to extensive research in the field of vascular biology, a plethora of knowledge exists about the growth factors, receptors, and signaling pathways that drive angiogenesis. However, our understanding of the transcription factors that bridge the gap between these signals and new gene expression remains incomplete.

Members of the Snail superfamily of zinc-finger transcription factors are expressed and required during both embryonic development and progression of malignant epithelial tumors. Specifically, this protein family drives developmental and pathological events by inducing epithelial to mesenchymal transitions (EMT). Snail (Snai1) and Slug (Snai2) are transcriptional repressors that belong to the Snail family of zinc-finger proteins. Importantly, expression of Snail or Slug in tumor cells is known to contribute to invasion and metastasis, and these genes have also been observed in angiogenic endothelial cells (EC) in vivo. Based on these observations, we asked whether angiogenic sprouting might share common attributes with EMT.

Using our in vitro angiogenesis model, we show that sprouting EC express and regulate Slug and Snail. To demonstrate a functional role for these genes, we utilized small interfering RNA (siRNA) to inhibit Slug or Snail expression and examine phenotypic alterations in EC undergoing angiogenesis. Interestingly, both Slug and Snail appear to be required for angiogenic sprouting as siRNA-mediated knockdown of either gene inhibits EC sprouting. In addition, we demonstrate that Slug regulates expression of membrane-type 1 matrix metalloproteinase (MT1-MMP; itself a regulator of vascular morphogenic events), but not vascular endothelial-cadherin (VE-Cadherin). Furthermore, lentiviral-mediated re-expression of MT1-MMP rescues the loss of sprouting induced by Slug knockdown, confirming that MT1-MMP is a crucial downstream target of Slug during sprouting angiogenesis. Importantly, we also observe enhanced Slug and Snail expression in tumor-associated blood vessels in multiple cancers. Taken together, these data identify a critical role for Slug expression in regulating pathological angiogenesis.

About 95% of anti-cancer drugs that show promise during preclinical study fail to gain FDA approval for clinical use. The gap between in vitro and in vivo preclinical study calls for physiologically relevant in vitro models that can better serve as a reliable drug-screening tool. The vascularized micro-tumor (VMT) is a novel three-dimensional model system that recapitulates the complex tumor microenvironment within a transparent microfluidic device that allows real-time study of drug response and tumor-stromal interactions. Here we validate our model system by showing that gene expression, tumor heterogeneity, and treatment response in the VMT more closely model tumor clinicopathology than current standard drug screening modalities.

The blood brain barrier (BBB) is a complex, multi-cellular structure that strictly regulates the passage of blood-borne molecules, ions, and cells from the periphery into the central nervous system (CNS). Its constituents include specialized endothelial cells (EC), pericytes, astrocytic end-feet, and the basal lamina. Due to this protective structure, hydrophilic drugs suboptimally accumulate in the brain after systemic administration contributing to the clinical failure of many CNS targeted therapies. Moreover, BBB dysfunction is observed in many neurodegenerative illnesses including Parkinson’s, Alzheimer’s, and Huntington’s disease. Thus, there is considerable interest in elucidating the underpinnings of BBB transport such that endogenous systems may be modulated or coopted to best treat CNS-associated diseases.

While maintained by exogenous factors, BBB properties are localized to and effectuated by the brain endothelium. Unlike ECs of the periphery, brain microvascular ECs (BMECs) maintain specialized tight junctions between single cells that restrict paracellular entry into the CNS, while a limited number of caveolae, reduced pinocytosis, and selective transporters limit transcellular entry. This specialization, induced by the developing neural milieu and maintained by non-endothelial brain constituents, is critical for BMEC identity. Consequently, there is a need for higher-order human-derived BBB in vitro models to replace conventional BMEC monolayer models that are better suited to complement in vivo (mouse) studies.

Based on our lab’s Vascularized Micro Organ (VMO) platform, this body of research aims to develop a novel BBB version – the Vascularized Micro Brain (VMB) – utilizing human-derived endothelial colony forming cells derived endothelial cells (ECFC-ECs), pericytes, astrocytes and iPSC-derived neural progenitor cells (iPSC-NPCs), that mimics the natural process of BBB induction and assembles into a perfused micro-vasculature. Herein, we demonstrate the ability of the VMB to mirror in vivo BBB gene expression and capture complex BBB-like vascular functions including reduced permeability and transcytosis. We believe that the VMB is optimally suited to study the BBB and its functional state during CNS-related pathologies, including neurodegenerative diseases and cancer, with the hope that it can better identify drugs likely to succeed in the clinic.