Using organ-on-a-chip technology to study haemorrhagic activities of snake venoms on endothelial tubules
Abstract
Snakebite envenomation is a major public health issue which causes severe morbidity and mortality, affecting millions of people annually. Of a diverse range of clinical manifestations, local and systemic haemorrhage are of particular relevance, as this may result in ischemia, organ failure and even cardiovascular shock. Thus far, in vitro studies have failed to recapitulate the haemorrhagic effects observed in vivo. Here, the authors present an organ-on-a-chip approach to investigate the effects of four different snake venoms on a perfused microfluidic blood vessel model. They assess the effect of the venoms of four snake species on epithelial barrier function, cell viability, and contraction/delamination. Our findings reveal two different mechanisms by which the microvasculature is being affected, either by disruption of the endothelial cell membrane or by delamination of the endothelial cell monolayer from its matrix. The use of our blood vessel model may shed light on the key mechanisms by which tissue-damaging venoms exert their effects on the capillary vessels, which could be helpful for the development of effective treatments against snakebites.
Introduction
Snakebite is one of the leading global health crises known to date, which claim between 81,000 and 138,000 lives annually, particularly in the (sub)tropical regions of this world1,2. Snake venom is comprised of a complex mixture of proteins and peptides, which poses a substantial threat to human life, causing significant morbidity and mortality. Snakebite morbidity, which is mainly caused by the toxins that directly or indirectly destroy cells, may cause permanent disability, which includes severe tissue loss (i.e., muscle and skin), chronic renal diseases and blindness1,3,4,5,6. Morbidity is estimated to occur in at least 400,000 snake bite victims annually1,2,5,7.
Many venoms have tissue-damaging activities that may result in a range of pathologies, including muscle and skin necrosis, acute kidney injury and haemorrhage1,7,8,9. The most medically relevant pathology caused by the tissue-damaging components in snake venoms is local and systemic bleeding1,10. Local and systemic haemorrhage may further promote ischemia, which may cause acute kidney injury and organ failure and could even lead to cardiovascular shock1,11,12. Haemorrhage is a rapid event in vivo with capillary endothelial cells showing drastic structural alterations within minutes13,14.
However, the same tissue-damaging components do not necessarily induce rapid toxicity to endothelial cells in vitro. The discrepancy between these two observations may arise from the fact that traditional in vitro systems do not take into account the mechanical action of haemodynamic forces acting on the endothelial cells, as is the case with in vivo systems12,15,16. To date, the study of tissue-damaging effects of snake venom toxins has been primarily based on two-dimensional cell culture models. These do not have the tubular morphology of vasculature found in vivo and lack important environmental cues from the cellular microenvironment, such as interaction with an extracellular matrix (ECM) and exposure to flow17,18. In an attempt to address this discrepancy, innovative approaches are required that advance our understanding of venom-induced haemorrhage and can be utilised to develop effective treatments. The development of snakebite treatments focusing on the neutralisation of the haemorrhagic effects of snake venom components relies on robust and quantitative in vitro models of blood vessels.
Organ-on-chip is an emerging technology that utilises microengineering to create three-dimensional models in microfluidic channel networks. In contrast to cells grown in 2D, blood vessels grown in microfluidic channels enable the inclusion of several important physiological parameters during cell culture, such as 3D tubular morphology, fluid perfusion, inclusion of ECM and exposure to biochemical gradients19. Organ-on-a-chip assays have been successfully used for the formation of perfused vascular models, including angiogenesis20,21, T-cell migration22, monocyte adhesion23, vascularisation of spheroids24 and real-time methods to study vascular barrier function25. In this latter study, organ-on-chip is combined with high-content imaging systems that enable direct monitoring of vascular barrier, viability and morphology.
In this study, we aimed to use a human blood vessel model grown in an organ-on-chip platform to simulate the hemorrhagic effect of different snake venoms while assessing their direct toxicity to endothelial cells. For this, we exposed microfluidic human blood vessels to a panel of four venoms from four different snake species. We used fluorescent tracer molecules and image analysis to assess endothelial barrier function and monitor vascular leakage following venom exposure. Furthermore, we assessed cellular viability and vessel morphology to evaluate the tissue-damaging activities of the snake venoms comprehensively. This allowed us to differentiate between different mechanisms of action by which venoms cause blood vessel disruption.
We found two distinct mechanisms by which the blood vessel is being affected: delamination of the endothelial cell monolayer from its matrix and disruption of the endothelial cell membrane. The workflow presented here could be utilised in the routine assessment of snake toxicity and its tissue-damaging and proteolytic effects and will be helpful for the development of effective snakebite treatments.