Abstract
BackgroundPericytes, surrounding the endothelium, fulfill diverse functions that are crucial for vascular homeostasis. The loss of pericytes is associated with pathologies, such as diabetic retinopathy and Alzheimer’s disease. Thus, there exists a need for an experimental system that combines pharmacologic manipulation and quantification of pericyte coverage during sprouting angiogenesis. Here, we describe an in vitro angiogenesis assay that develops lumenized vascular sprouts composed of endothelial cells enveloped by pericytes, with the additional ability to comparatively screen the effect of multiple small molecules simultaneously. For automated analysis, we also present an ImageJ plugin tool we developed to quantify sprout morphology and pericyte coverage.MethodsHuman umbilical vein endothelial cells and human brain vascular pericytes were coated on microcarrier beads and embedded in fibrin gels in a 96-well plate to form lumenized vascular sprouts. After treatment with pharmacologic compounds, sprouts were fixed, stained, and imaged via optical z-sections over the area of each well. The maximum intensity projections of these images were stitched together to form montages of the wells, and those montages were processed and analyzed.ResultsVascular sprouts formed within 4–12 days and contained a patent lumen surrounded by a layer of human endothelial cells and pericytes. Using our workflow and image analysis, pericyte coverage after treatment with various compounds was successfully quantified.ConclusionsHere we present a robust in vitro assay using primary human vascular cells that allows researchers to analyze the effects of multiple compounds on sprouting angiogenesis and pericyte coverage. Our ImageJ plugin offers automated evaluation across multiple different vascular parameters, such as sprout length, cell density, branch points, and pericyte coverage.
Highlights
Pericytes, surrounding the endothelium, fulfill diverse functions that are crucial for vascular homeostasis
Lumen formation and pericyte coverage in multi-cellular sprouts To assess vascular lumen formation within endothelial cell (EC)-derived vascular sprouts with and without pericytes, we seeded Human umbilical vein endothelial cells (HUVEC) in the presence or absence of Human brain vascular pericytes (HBVP) onto microcarrier beads that subsequently were embedded into fibrin gels
Our methodology allows for quantitative assessment of morphology and pericyte coverage in vascular sprouts developed from human endothelial cells and human pericytes in vitro
Summary
Pericytes, surrounding the endothelium, fulfill diverse functions that are crucial for vascular homeostasis. The loss of pericytes is associated with pathologies, such as diabetic retinopathy and Alzheimer’s disease. Sprouting is induced by activation of endothelial cells (ECs) via growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), Eglinger et al Inflammation and Regeneration (2017) 37:2 the endothelium is frequently the cause or consequence of many diseases [8,9,10], resulting in increased vascular permeability and defective vessel maturation, which promote vessel leakage and inflammation [11]. For instance, can induce detachment of pericytes from quiescent vasculature, thereby activating ECs to sprout into the surrounding tissue [12]. After stroke, pericytes constrict around capillaries to decrease blood flow. The blood– brain barrier is disrupted, leading to progressive neuronal damage [15]
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