A cell-and-plasma numerical model reveals hemodynamic stress and flow adaptation in zebrafish microvessels after morphological alteration.

The development of a functional cardiovascular system ensures a sustainable oxygen, nutrient and hormone delivery system for successful embryonic development and homeostasis in adulthood. While early vessels are formed by biochemical signaling and genetic programming, the onset of blood flow provides mechanical cues that participate in vascular remodeling of the embryonic vascular system. The zebrafish is a prolific animal model for studying the quantitative relationship between blood flow and vascular morphogenesis due to a combination of favorable factors including blood flow visualization in optically transparent larvae. In this study, we have developed a cell-and-plasma blood transport model using computational fluid dynamics (CFD) to understand how red blood cell (RBC) partitioning affect lumen wall shear stress (WSS) and blood pressure in zebrafish trunk blood vascular networks with altered rheology and morphology. By performing live imaging of embryos with reduced hematocrit, we discovered that cardiac output and caudal artery flow rates were maintained. These adaptation trends were recapitulated in our CFD models, which showed reduction in network WSS via viscosity reduction in the caudal artery/vein and via pressure gradient weakening in the intersegmental vessels (ISVs). Embryos with experimentally reduced lumen diameter showed reduced cardiac output and caudal artery flow rate. Factoring in this trend into our CFD models, simulations highlighted that lumen diameter reduction increased vessel WSS but this increase was mitigated by flow reduction due to the adaptive network pressure gradient weakening. Additionally, hypothetical network CFD models with different vessel lumen diameter distribution characteristics indicated the significance of axial variation in lumen diameter and cross-sectional shape for establishing physiological WSS gradients along ISVs. In summary, our work demonstrates how both experiment-driven and hypothetical CFD modeling can be employed for the study of blood flow physiology during vascular remodeling.

A mechanistic model of cross-bridge migration in RBC aggregation and disaggregation.

Red blood cells (RBCs) clump together under low flow conditions in a process called RBC aggregation, which can alter RBC perfusion in a microvascular network. As elevated RBC aggregation is commonly associated with cardiovascular and inflammatory diseases, a better understanding of aggregation is essential. Unlike RBC aggregation in polymer solutions which can be well explained by polymer depletion theory, plasma-mediated RBC aggregation has features that best match explanations with cross-bridging mechanisms. Previous studies have demonstrated the dominant role of fibrinogen (Fg) in promoting aggregate formation and recent cell-force spectroscopy (CFS) experiments on interacting RBC doublets in plasma have reported an inverse relationship between disaggregation force and the adhesive contact area between RBCs. This has led investigators to revisit the hypothesis of inter-RBC cross-bridging which involves cross-bridge migration under interfacial tension during the forced disaggregation of RBC aggregates. In this study, we developed the cross-bridge migration model (CBMM) in plasma that mechanistically represents the migrating cross-bridge hypothesis. Transport of mobile Fg cross-bridges (mFg) was calculated using a convection-diffusion transport equation with our novel introduction of convective cross-bridge drift that arises due to intercellular friction. By parametrically transforming the diffusivity of mFg in the CBMM, we were able to match experimental observations of both RBC doublet formation kinematics and RBC doublet disaggregation forces under optical tweezers tension. We found that non-specific cross-bridging promotes spontaneous growth of adhesion area between RBC doublets whereas specific cross-bridging tends to prevent adhesion area growth. Our CBMM was also able to correlate Fg concentration shifts from healthy population blood plasma to SLE (lupus) condition blood plasma with the observed increase in doublet disaggregation forces for the RBC doublets in SLE plasma.

High-Throughput Imaging of Blood Flow Reveals Developmental Changes in Distribution Patterns of Hemodynamic Quantities in Developing Zebrafish.

Mechanical forces from blood flow and pressure (hemodynamic forces) contribute to the formation and shaping of the blood vascular network during embryonic development. Previous studies have demonstrated that hemodynamic forces regulate signaling and gene expression in endothelial cells that line the inner surface of vascular tubes, thereby modifying their cellular state and behavior. Given its important role in vascular development, we still know very little about the quantitative aspects of hemodynamics that endothelial cells experience due to the difficulty in measuring forces in vivo. In this study, we sought to determine the magnitude of wall shear stress (WSS) exerted on ECs by blood flow in different vessel types and how it evolves during development. Utilizing the zebrafish as a vertebrate model system, we have established a semi-automated high-throughput fluorescent imaging system to capture the flow of red blood cells in an entire zebrafish between 2- and 6-day post-fertilization (dpf). This system is capable of imaging up to 50 zebrafish at a time. A semi-automated analysis method was developed to calculate WSS in zebrafish trunk vessels. This was achieved by measuring red blood cell flow using particle tracking velocimetry analysis, generating a custom-made script to measure lumen diameter, and measuring local tube hematocrit levels to calculate the effective blood viscosity at each developmental stage. With this methodology, we were able to determine WSS magnitude in different vessels at different stages of embryonic and larvae growth and identified developmental changes in WSS, with absolute levels of peak WSS in all vessel types falling to levels below 0.3 Pa at 6 dpf. Additionally, we discovered that zebrafish display an anterior-to-posterior trend in WSS at each developmental stage.