Cysteine cathepsins are altered by flow within an engineered in vitro microvascular niche.

Simone A Douglas,Manu O Platt,Kristina Haase,Roger D Kamm

doi:10.1063/5.0023342

Abstract

Throughout the process of vascular growth and remodeling, the extracellular matrix (ECM) concurrently undergoes significant changes due to proteolytic activity—regulated by both endothelial and surrounding stromal cells. The role of matrix metalloproteinases has been well-studied in the context of vascular remodeling, but other proteases, such as cysteine cathepsins, could also facilitate ECM remodeling. To investigate cathepsin-mediated proteolysis in vascular ECM remodeling, and to understand the role of shear flow in this process, in vitro microvessels were cultured in previously designed microfluidic chips and assessed by immunostaining, zymography, and western blotting. Primary human vessels (HUVECs and fibroblasts) were conditioned by continuous fluid flow and/or small molecule inhibitors to probe cathepsin expression and activity. Luminal flow (in contrast to static culture) decreases the activity of cathepsins in microvessel systems, despite a total protein increase, due to a concurrent increase in the endogenous inhibitor cystatin C. Observations also demonstrate that cathepsins mostly co-localize with fibroblasts, and that fibrin (the hydrogel substrate) may stabilize cathepsin activity in the system. Inhibitor studies suggest that control over cathepsin-mediated ECM remodeling could contribute to improved maintenance of in vitro microvascular networks; however, further investigation is required. Understanding the role of cathepsin activity in in vitro microvessels and other engineered tissues will be important for future regenerative medicine applications.

Highlights

Vasculogenesis is the formation of de novo microvessels usually observed in embryogenesis, tumor growth, and after extensive vascular damage;[1,2] this differs from angiogenesis, where microvessels are formed from pre-existing vessels, usually after injury.[3]
Microvascular networks were developed in a large-scale version of a singlegel channel PDMS device using a coculture of human umbilical vein endothelial cells (HUVECs) and normal human lung fibroblasts in a fibrin gel, as previously described.[32,33]
Proteolytic activity is required for matrix and vascular remodeling to occur—and we proposed and demonstrated that cathepsins actively contribute to this process

Summary

Introduction

Vasculogenesis is the formation of de novo microvessels usually observed in embryogenesis, tumor growth, and after extensive vascular damage;[1,2] this differs from angiogenesis, where microvessels are formed from pre-existing vessels, usually after injury.[3] These growth processes are complex but well-coordinated, involving ECM remodeling, endothelial cell migration, cytokine secretion, lumen formation, and mural cell (i.e., pericyte) recruitment.[1,2,3] The complexities involved in vascular growth, when disrupted, can lead to vascular regression While many of these growth processes have been characterized using in vitro vessel systems, less research has focused on vessel regression. The mechanisms driving these changes require clear investigations to aid in the generation and long-term maintenance of vascularized tissueengineered substrates

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