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Surface area-to-volume ratio, not cellular rigidity, determines red blood cell traversal through small capillaries

Arman Namvar, Adam J. Blanch, Matthew W. Dixon, Olivia M. S. Carmo, Boyin Liu, Snigdha Tiash, Oliver Looker, Dean Andrew, Li-Jin Chan, Wai-Hong Tham, Peter V. S. Lee, Vijay Rajagopal, Leann Tilley

Preprint posted on 11 July 2020 https://www.biorxiv.org/content/10.1101/2020.07.11.191494v1

Understanding red blood cell traversal through small capillaries

Selected by Mariana De Niz

Categories: biophysics, cell biology

Background

The mature human red blood cell (RBC) exhibits remarkable deformability and durability. Throughout their lifespan, RBCs circulate the body a vast number of times without repair, squeezing through capillaries and splenic inter-endothelial slits as small as 1-3µm. Therefore, the ability to undergo large deformations is key for RBC function. RBC deformability is determined by factors such as plasma membrane viscoelasticity, cytoplasmic viscosity, and cellular geometry- such as the surface area to volume (SA:V) ratio.Altogether, the individual effects of cell geometry and cellular rigidity on capillary traversal are not well understood.

The surface area of a healthy RBC is kept constant, and any increases in volume decrease the SA:V ratio, which adversely impacts RBC rheology. Upon infection with a malaria parasite, key biomechanical properties of the RBC are subverted. However, it remains unclear to what extent the higher rigidity of infected RBCs contributes directly to trapping of infected RBCs in small capillaries.

In their work, Namvar et al (1) monitored RBC traversal in an array of microchannels, with diameters such as those found in the smallest vessels, and examined the effects of independently manipulating cellular rigidity and SA:V ratio.

Figure 1. Surface area-to-volume ratio, not cellular rigidity, determines red blood cell traversal through small capillaries. Ektacytometry and HEMA analyses of parasitised RBCs. (From Figure 3h, Ref1.).

 

 

Key findings and developments

The authors began by calibrating measurements of RBC geometry. For this, they used a modified version of the microfluidic device called a Human Erythrocyte Microchannel Analyser (HEMA) to assess the ability of RBCs to traverse into wedge-shaped microchannels with a 5 µm diameter entry, and a 1.4 µm diameter exit. They then determined the minimum cylindrical diameter (MCD), which is the position where the RBC becomes lodged. Conforming the RBCs into the microchannels allows estimation of the cell surface area and volume. This analysis showed an average volume of 99 fL, an average surface area of 149 µm2, and a SA:V ratio of 1.50, with an average MCD of 3.03 µm.
The authors then examined the behaviour of RBCs under conditions in which the cellular rigidity was modified by chemical treatment with various concentrations of glutaraldehyde, but the SA:V ratio was held constant. They then used an ektacytometer to measure the elongation index of RBCs at shear stresses ranging from 0-20 Pa. At the physiologically relevant shear stress of 3 Pa, the ability of the RBCs treated with 0.004% glutaraldehyde to elongate was largely abrogated. No significant changes in SA:V ratio were observed between fixed and unfixed RBCs at glutaraldehyde concentrations of 0.001 and 0.004%, and the cells reach a similar MCD as unfixed RBCs. Altogether, these data show that cellular rigidity has little impact on RBC traversal into small capillaries.

As a next step, they examined the impact of altered cell volume on microchannel traversal and RBC elongation. Cell volume was altered by varying the osmolarity of the buffer solution. The authors found that at osmolarities close to the physiological range, the EI showed little dependence on osmolarity. Using the HEMA device, they showed that as cells over-hydrate at lower buffer osmolarities, there is a gradual increase in cell volume and they become lodged closer to the capillary entrance. Altogether, the work shows that moderate swelling of RBCs limits their traversal into microchannels but has less effect on their ability to undergo elongation.

The authors next explored the relevance of SA:V ratio changes in the context of Plasmodium infections. P. falciparum-infected RBCs(trophozoites) exhibited a rigidity profile similar to that of RBCs treated with 0.005% glutaraldehyde. In these cells, there was no significant difference in the mean SA:V ratio or MCD value compared to uninfected RBCs. Conversely, P. knowlesi-infected RBCs showed a decreased SA:V ratio and a higher MCD value compared to uninfected RBCs. Moreover, P. knowlesi-infected RBCs had reduced ability to elongate in flow, but appeared less rigid than P. falciparum-infected cells. These data confirm that SA:V ratio is a more important determinant than cell stiffness of the ability of RBCs to traverse small microchannels, with implications for the pathology of P. knowlesi infections.

Next, the authors compared the SA:V ratio of reticulocytes to mature RBCs, and their ability to traverse microchannels in the HEMA device. They found that two separate populations of reticulocytes (CD71+ and CD71-) exhibit the same SA:V ratio as mature RBCs and were found to reach the same MCD.

At any given point in the HEMA device, an RBC undergoes a complex distribution of stresses that cannot be measured experimentally. The authors developed a 3D computational simulation model of RBC traversal into the HEMA chip, to estimate the forces that RBCs would experience, and to probe the physical basis for the importance of SA:V ratio on RBC traversal through microchannels. This allowed them to find differences in pressure as RBCs traverse the microchannels, that best fit the experimental measurements. Using their 3D simulation model, the authors carried out two analyses of RBCs within the HEMA device, considering a) only membrane stiffness variation and b) only SA:V ratio variation. Simulations were performed on RBCs with different SA:V ratios. The authors concluded that passage of RBCs into the microchannels is highly dependent on the SA:V ratio.

The authors then performed simulations to predict whether high cell stiffness or low SA:V ratio would restrict the passage of a population of RBCs through the smallest capillaries encountered under physiological conditions, and found that the average MCD for healthy RBCs was between 2.73 and 3.29. Equally, they performed simulations to determine the minimum required pressure for cells with different rigidities and SA:V ratios to squeeze into small capillaries. They found that while shear modulus was not important, the minimum required pressure steeply increased as the SA:V ratio decreased. Altogether, the simulations showed that RBCs with a low SA:V ratio are more prone to trapping in small capillaries than cells with high cellular rigidity.

 

What I like about this preprint

I chose this preprint because I think while the focus in parasitology has been largely on the cell biology and molecular level, there is still a lot we don’t know from a biophysics point of view. I think this work addresses one important topic to better understand sequestration. I like the range of techniques approached, and I think this opens further questions for future exciting research.

 

References

  1. Namvar A., et al, Surface-to-volume ratio, not cellular rigidity, determines red blood cell traversal through small capillaries, bioRxiv, 2020.

 

Posted on: 20 July 2020

doi: https://doi.org/10.1242/prelights.23277

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Author's response

Leann Tilley shared

Open questions 

1.Congratulations on your exciting work. My first question to you is that you refer throughout the manuscript to small brain capillaries and how you tried to reproduce them in the HEMA device. Assumingly, there are small capillaries in all organs, and sequestration happens also in multiple organs. For instance, is there something specific to 3 µm brain capillaries, that you reproduced in HEMA devices- eg. geometry, pressure, morphology, circulation volume -that would differ from a 3 µm lung capillary (or of any other organ)?

You are correct. There are narrow capillaries in all organs, and sequestration of infected RBCs occurs at many different sites. Sequestration of infected RBCs in the capillaries of the brain is of particular interest from the point of view of malaria pathology. The inflammatory response to the trapped infected RBCs can result in the complication known as cerebral malaria, which is associated with coma and neurological sequalae. But our basic findings about the physical determinants of trapping apply equally well to capillaries at other sites. Our take home message is that rigidification of P. falciparum-infected RBCs is not, in and of itself, sufficient to hinder passage through microcapillaries.

2.Following from that question, if you reproduced in the HEMA device something specific to brain capillaries alone, do you expect different results if you reproduced characteristics of capillaries in other organs?

Ying Zheng (University of Washington) and Joseph Smith (Seattle Children’s Research Institute) have developed a beautiful 3D microvessel system, seeded with brain endothelial cells, that mimics the arteriole-capillary-venule (ACV) transition (doi: 10.1126/sciadv.aay7243). This system explores other important determinants of sequestration, namely the cytoadherence ligand and the vessel rheology. Such work will be critical to fully understanding sequestration and malaria pathology.

3.Do you think that from a biophysical point of view (ie SA:V ratio), further variables can be added to the HEMA device (such as ICAM1 expression), and would you expect this to affect your conclusions in any way? Can this be simulated?

The HEMA device is designed to give an accurate measure of surface area, volume and MCD and is excellent for measuring physical determinants of trapping. As mentioned in the answer to the question above, devices designed to measure adhesion have a larger diameter so that the effects of the cytoadhesion ligand and fluid flow parameters can be examined. Our current model could be extended to include biophysical properties related to the distribution of the cytoadhesion ligand and the vessel rheology. Such a model would enable us to integrate data from these disparate experimental assays to elucidate the fundamental biophysical properties that govern sequestration and malaria pathology.

4.A long-term discussion in the Plasmodium community, is the validity of rodent models for various specific pathologies. Have you tried your device with rodent uninfected and infected cells (with multiple Plasmodium strains with different capacities for sequestration)? Would a similar analysis to the one you show here, give further insights into why brain sequestration is a much more prominent finding in human malarias, but no so much rodent ones?

We have used the HEMA to look at normal mouse RBCs and RBCs with genetic defects to get information about SA:vol ratio (eg. doi: 10.1182/bloodadvances.2017009274). We have not looked at malaria parasite-infected mouse RBCs. It would be interesting to determine whether SA:vol is changed in malaria parasite-infected RBC as it would help to understand the determinants of pathology in rodent models.

 

5.The finding you did regarding the differences between P. falciparum and P. knowlesi is very interesting. Have you evaluated cells infected with other Plasmodium strains (eg. P. vivax, P. malariae, P. ovale) to determine precisely similar biophysical parameters? So far, mostly molecular explanations have been found regarding sequestration (or lack thereof) in some strains but not others.

We haven’t used the HEMA to look at RBCs infected with other species. We would like to do so, but access to such samples is difficult. Fortunately, the HEMA set-up is quite simple and would be amenable to taking into a field situation.

6.One of the findings you did is related to pressure. One theory is that upon sequestration of circulating cells, pressure would vary, affecting the variables for new cells entering a specific environment (eg. capillary). Can your computational model simulate this variable too?

Sequestration of infected RBCs will narrow that diameters of the capillaries to which they adhere – which will affect the ability of other RBCs to transit through that vessel. Our computer model predicts the pressures that need to be applied to force RBCs through capillaries of different diameters. The simulations confirm that RBC stiffening has surprisingly little effect, while RBC swelling has a dramatic effect.

7.Can you test in your device, infected and non-infected cells from humans with different bloodtypes, and even hemoglobinopathies? This would also be very useful to an understanding that has so far been purely molecular.

The HEMA device will be very useful for studies of RBCs with genetic defects, such as hereditary spherocytosis and hereditary elliptocytosis, that exhibit a decreased SA:V ratio. Circulation of RBCs with decreased SA:V ratio is predicted to impair blood flow through small capillaries, contributing to an enhanced risk of thrombosis.

8.Previous work in various disciplines has shown that nuclear deformations impact cell fate. If an infected cell (eg. trophozoite or schizont) undergoes the extreme deformability you present in this work, can your device be used to determine whether or not passage through small capillaries would influence the fate of the parasites either still during schizogony or following egress?

That’s an interesting idea. There are no studies to my knowledge of the downstream effect of cellular deformation on malaria parasite species, such as P. knowlesi, that can circulate as mature stage-infected RBCs or on Stage-V P. falciparum gametocyte-infected RBC, which also circulate. We know very little about the cytoskeletal meshworks that control nuclear positioning in these cells. The HEMA device could be used as part of a larger study of such phenomena.

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