Highlighting Biophysics Research During Sickle Cell Awareness Month

September is National Sickle Cell Awareness Month in the United States. Sickle cell disease is an inherited blood disorder that affects approximately 100,000 Americans and millions worldwide. It is particularly common among people whose ancestors come from Sub-Saharan Africa, South America, Cuba, Central America, Saudi Arabia, India, and Mediterranean countries such as Turkey, Greece, and Italy.

To recognize the awareness month, we spoke with BPS member George Em Karniadakis, Brown University, and his collaborators Xuejin Li, Brown University, and Ming Dao, MIT, about their research related to sickle cell disease. Their research was also featured on the cover of the July 11, 2017, issue of Biophysical Journal.


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What is the connection between your research and sickle cell disease?

Sickle cell disease (SCD) is the first identified molecular disease affecting more than 270,000 new patients each year. Our interests are in modeling multiscale biological systems using new mathematical and computational tools that we develop in our teams at Brown University and MIT in conjunction with carefully selected microfluidic experiments at MIT. We have an ongoing NIH-funded joint project that focuses on developing such validated predictive models for the sickle cell disease (SCD). In this project, with close collaboration between clinicians, experimentalists, applied mathematicians and physical chemists, we have been  developing new predictive patient-specific models of SCD, linking sub-cellular, cellular, and vessel-level phenomena spanning across four orders of magnitude in spatio-temporal scales. So far we have developed a validated patient-specific and data-driven multiscale modeling approach to probe the biophysical mechanisms involved in SCD from hemoglobin polymerization to vaso-occlusion.

Why is your research important to those concerned about sickle cell disease?

SCD is one of the most common genetic blood disorders that can cause several types of chronic and acute complications such as vaso-occlusive crises (VOC), hemolytic anemia, and sequestration crisis. It is also the first identified molecular disease (as early as 1947 by Linus Pauling), and the underlying molecular cause of the disease has been understood for more than half a century. However, progress in developing treatments to prevent painful VOC and associated symptoms has been slow. Consequently, we have been developing a “first-principles” multiscale approach that can handle the disparity of molecular, mesoscopic and macroscopic phenomena involved in SCD simultaneously. Such simulations could potentially answer questions concerning the links among sickle hemoglobin (HbS) polymerization, cell sickling, blood flow alteration, and eventually VOC. We hope, in turn, that these models will help in assessing effective drug strategies to combat the clinical symptoms of this genetic blood disorder.

Karniadakis

Figure 1. Dynamic behavior of individual sickle RBCs flowing in microfluidic channel. Inside the yellow circles are trapped sickle RBCs at the microgates, and inside the white circles are deformable RBCs, which are capable of circumnavigating trapped cells ahead of them by choosing a serpentine path (indicated by the white arrows).

How did you get into this area of research?

We have been working on multiscale modeling of blood disorders for more than 10 years.  In the very beginning, we were interested in developing new computational paradigms in multiscale simulations, which would enable us to perform multiscale realistic simulation of blood flow in the brain of a patient with an aneurysm. We then realized that the mesoscopic modeling of red blood cells (RBCs) and hemorheology in general seems to be the most effective approach for modeling malaria and other hematologic disorders. Then, we shifted our attention to the particle-based modeling of blood flow by employing the dissipative particle dynamics (DPD) method, which can seamlessly represent the RBC membrane, cytoskeleton, cytosol, surrounding plasma, and even the parasite in the malaria-infected RBCs. We developed multiscale RBC models and employed them to predict mechanical and rheological properties of RBCs and quantify molecular-level mechanical forces involved in bilayer–cytoskeletal dissociation in blood disorder. In 2012, we started to work on SCD, after realizing that no multiscale simulation studies of SCD had been conducted before – our work is the first of its kind!

How long have you been working on it?

As we mentioned above, we have been working in this field for more than 10 years.

Do you receive public funding for this work? If so, from what agency?

Yes, we receive support from NHLBI, the institute within NIH focusing on blood disorders based on the interagency funding initiative pioneered by Dr. Grace Peng. For those who are interested in this multiscale consortium they can visit: https://www.imagwiki.nibib.nih.gov/

Have you had any surprise findings thus far?

Plenty! For example, at the vessel scale, using computer models, we have discovered that it is the soft and sticky type of RBCs that initiate the blockage process and lead to sickle cell crises and not the rigid sickle cells! This is the first study to identify a specific biophysical mechanism through which vaso-occlusion takes place. At the cellular scale, we have developed a tiny microfluidic device that can analyze the behavior of blood from SCD patients. Informed from the microfluidic experiments conducted by Dr. Ming Dao’s group at MIT, we have developed a unique patient-specific predictive model of sickle RBCs to characterize the complex behavior of sickle RBCs in narrow capillary-like microenvironment. At the sub-cellular (molecular) scale, we have developed a particle HbS model for studying the growth dynamics of polymer fibers (recent cover of Biophysical Journal). The simulations provide new details of how SCD manifests inside RBCs, which could help other medical researchers in developing new treatments.

What is particularly interesting about the work from the perspective of other researchers?

It is known that the primary cause of the clinical phenotype of SCD is the intracellular polymerization of sickle hemoglobin (HbS) resulting in sickling of RBCs in deoxygenated conditions. However, the clinical expression of SCD is heterogeneous, making it hard to predict the risk of VOC, and resulting in a serious challenge for disease management. Our data-driven stochastic multiscale models, based on particle methods, can be used to explore and understand the dynamics of collective processes associated with vaso-occlusion that links together sub-cellular, cellular, and vessel phenomena. A similar computational framework can be applied to study blood flow in other hematologic disorders, including malaria, hereditary spherocytosis and elliptocytosis, as well as other blood pathological conditions in patients with diabetes mellitus or AIDS.  For example, in ongoing work we have quantified the biophysical characteristics of RBCs in type-2 diabetes mellitus (T2DM), from which the simulation results and their comparison with currently available experimental data are helpful in identifying a specific parametric model that best describes the main hallmarks of T2DM RBCs.  Perhaps, the most important extension is to connect such multiscale models to all the “omics” technologies (genomics, proteomics, metabolomics, etc.) to implement the vision of precision medicine advocated both in the U.S. and around the world.

What is particularly interesting about the work from the perspective of the public?

Our studies provide new insight into what causes painful episodes in people with SCD. Using the computational models we could probe different mechanisms and validate diverse hypotheses regarding vaso-occlusion.  For example, we have shown that the rigid crescent-shaped RBCs —the hallmark of SCD — do not cause these blockages on their own. Instead, softer, deformable RBCs are known as cells that start the process by sticking to arteriolar and capillary walls. The rigid crescent-shaped cells then stack up behind these softer cells, creating a sort of a traffic jam.

Currently, hydroxyurea (HU) is the only approved medication in widespread use for the treatment of SCA, and it is thought to work by promoting the production of fetal hemoglobin, which can reduce sickling rate. Using the computational models, we can now run simulations that include fetal hemoglobin, which could help in establishing better dosage guidelines or in identifying a subgroup of patients who would benefit from this treatment or proposing a different type of treatment for others.

In addition, based on our own experience and knowledge, we also presented a short review in SIAM NEWs,   which provides the broader public with a general idea of computational modeling of blood disorders, including SCD. Here is the link to the review: https://sinews.siam.org/Details-Page/in-silico-medicine-multiscale-modeling-of-hematological-disorders.

Do you have a cool image you want to share with the blog post related to this research?

Yes, we have a cool image to share (figure 1). This image shows the different dynamic behavior between individual normal RBCs and sickle ones in microfluidic flow. Normal RBCs are round and flexible, and easily change shape to move through even the smallest blood vessels. Under deoxygenation, RBCs undergo sickling can be hard, sticky, and abnormally shaped, so they tend to get stuck at the microgates and block the blood flow. Once the adjacent microgates in the flow direction (from right to left) are blocked, the deformable RBCs (one is highlighted in white circle) appear to take a preferred path, i.e., they twist and turn along a serpentine path (as indicated by the white arrows) once they spot trapped sickle cells (one is highlighted in yellow circle) ahead of them.

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Biophysics on World Sickle Cell Day

June 19 is World Sickle Cell Awareness Day. Sickle cell disease refers to a group of inherited red blood cell disorders. The Center for Disease Control and Prevention estimates that sickle cell disease affects 90,000 to 100,000 people in the United States, a majority of whom are African-American. Worldwide, it is estimated that 300,000 children are born each year with sickle cell diseases, though many go undiagnosed in developing countries. We recently spoke with Biophysical Society member Frank Ferrone, Drexel University, about the research his lab conducts related to sickle cell disease. 

What is the connection between your research and sickle cell disease?

We have several projects underway that relate to sickle cell disease.   Let me give two.   In one, we are working to complete a device that can detect the presence of sickle cell disease, or sickle cell trait (and distinguish the two), using a drop of blood, in under a minute, at a cost of a few cents per test.   And we’re almost there!   In a more basic vein (sorry about that ) we are also exploring a radical hypothesis of ours that the structure of the sickle polymers that cause all the trouble in the disease is not understood properly—and if we are correct, our revision of this paradigm has implications for all pathological assembly diseases.

Why is your research important to those concerned about sickle cell disease?

In the US, children get tested, information gets shared (mostly), and emergency medicine is very good…and even here, sickle patients sometimes get misdiagnosed or fall through the cracks.  In certain nations, sickle cell disease afflicts as many as 1 in 7 newborn children, and the resources simply don’t exist to conduct expensive tests, or to track the families whose children are affected.   Thus an inexpensive and rapid test could be a godsend there.   At the same time in the US, the NCAA dictates that student athletes should have their status known, since sickle trait  poses a covert risk—kids can have no symptoms, and then under exertion experience tragic, even fatal, sickle-events.     Now, we supposedly have medical records of everyone’s test—but that’s if you did get tested, and if you have the records.   Imagine that instead of a kid having to wait for results of a test–an expensive one to boot–because his family can’t find his test results, that the school nurse could prick his finger and in a minute pronounce him good to go (or not).

As to the other project I mentioned, well there are currently no drugs that work by interfering with the polymeric structures that generate the pathology.   Maybe if we understood the polymers better, that could change.

How did you get into this area of research?

For my PhD I did a project on normal hemoglobin, using laser photolysis to trigger a structural change, and following it with kinetic CD (it was a first).   When looking around for a post-doc, I got invited to join Bill Eaton’s group at the NIH, using the photolysis trick to induce sickle cell hemoglobin polymerization.    I knew nothing about sickle cell at the time, but the project sounded interesting, the group was exciting, and, as Bill explained it, “eventually everybody comes to visit the NIH” so the environment was immensely stimulating.

How long have you been working on it?

It’s now on 40 years!   I carried the project with me to Drexel University’s Physics Department, where I’ve continued to work on this.

Do you receive public funding for this work? If so, from what agency?

While we did have many years of generous NIH support, at the moment we don’t have funding.

Have you had any surprise findings thus far?

Plenty!   When I was transitioning to Drexel from the NIH, we came up with the idea that there were two kinds of nucleation that generated the sickle fibers, not just one like everybody thought, mainly because we had these experiments that couldn’t be explained any other way.   That was a big surprise.   And now it’s turned out to be fairly common in pathological assemblies.

We found that the second type of nucleation—onto the surface of polymers—came about because intermolecular contacts that stabilize the interior of polymers also appear on the polymer exterior!   Anybody could have found this from the existing structures, but nobody thought to look.   Even we bumped into it by accident.

When we employed our models to understand the assembly process, we got another big surprise.   The ability of the hemoglobin molecules to oscillate about their equilibrium position in the polymer exercises a HUGE influence on the rate of the assembly, thanks to an effect known as vibrational entropy.   Weak, “sloppy” bonds generate much faster nucleation—which we demonstrated experimentally.

And one of our most recent surprises was that the polymer formation gets hung up in a metastable state because of the extreme crowding.   And in a red cell, that in turn leads to Brownian ratchet forces that can hold cells in narrow channels, like capillaries.

What is particularly interesting about the work from the perspective of other researchers?

Sickle cell is perhaps the “Granddaddy” of all the protein assembly diseases, which now include Alzheimer’s, prion diseases, Huntington’s, Parkinson’s… There is often a lot of hard biochemistry that goes into simply characterizing the assembly that is the root of these.   Sickle hemoglobin can be prepared in large quantities, purified simply, and even reconstructed by site-directed mutagenesis, with the result that much more sophisticated physical questions can be posed and tested without a ridiculous overhead of construction and purification.     Thus, as I mentioned, the double nucleation model, constructed for sickle cell polymerization, has been shown to operate on Aβ assembly.   In addition, to do the analysis, we needed to invent a new mathematical approach, adopting perturbation methods to deal with intractable differential equations.  This has been of great interest to others, too.   Finally, for years it was our “burden” that all the standard kinetic and equilibrium equations we used had to be modified to account for the high concentration of hemoglobin in the red cells.  I hated it!   Fortunately, we succeeded in working it out, but now with the burgeoning interest in molecular crowding, it turns out that our work has a lot of  applicability for another class of problems as well.

What is particularly interesting about the work from the perspective of the public?

Sickle cell disease has been known for over 100 years.   It’s known as the first molecular disease.   When people discuss gene therapy, sickle cell is invariably found in the list of targets.   And yet there is but one drug.   A solution to this long-standing problem generates interest.     Moreover, there are about 240,000 new cases (i.e. sickle cell births) yearly in Africa.   But the other part of the story, which maybe should be told to the public even more prominently, is that these assembly diseases are all connected in a fundamental way.    Our analytical methods led to a novel result in poly-Q assembly.   Our model was adopted for Aβ assembly.    Answering basic questions well generates a tide that indeed floats all boats.

Do you have a cool image you want to share related to this research?

We have a movie!   This was taken in our lab by Dr. Alexey Aprelev.   We have a narrow micro-fluidic channel, 4 µm wide and 1.5 µm deep, and a red cell.  In the movie the cell squeezes in and out just fine in response to externally oscillating the hydrostatic pressure.  The hemoglobin inside the cell has CO bound, which can be removed by light very efficiently.  Just as the cell is entering we turn on a laser, which you can see from the leaked light in the background.   Our oscillatory pressure now does nothing!  And most dramatically, note that the cell cannot exit either!   This trapping is due to Brownian ratchet forces, as we published in a letter in Biophysical Journal in 2012.   And once the laser is turned off, CO rebinds, and the cell once again can move in and out, recovering its deformability.  Click here to view the video.

Red Blood Cell Adhesion in Sickle Cell Disease

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Millions worldwide live with sickle cell disease, the most common inherited blood disorder. Sickle cell disease is due to a single-point mutation in the ÿ-globin gene resulting in the production of abnormal hemoglobin. In the deoxygenated state, hemoglobin polymerizes to form relatively stiff filaments forcing red blood cells to assume an irregular shape. It is these “sickled” red blood cells that are thought to significantly contribute to, if not initiate, occlusion of small blood vessels resulting in microvascular infarction, severe pain, widespread organ dysfunction, and early mortality.  The hallmark of the disease is the development of spontaneous, intermittent, disabling episodes of severe pain called vaso-occlusive episodes.

Our article discusses adhesion of normal and sickle cell disease human erythrocytes to endothelial laminin.  Erythrocyte adhesion to endothelium is thought to be a critical mediator of the complicated process of vaso-occlusion in sickle cell disease. This translational work is a collaboration between investigators at the schools of Engineering and Medicine at the University of Connecticut.  When our article was accepted for publication, we thought that an image on the journal cover would be a good way to attract attention to this devastating disease and to show, at least partially, the complexity of one of its major consequences in the circulatory system. The image was created by Kostyantyn Partola, who is a first-year Ph.D. candidate in our lab. It is a three-dimensional depiction of normally and abnormally shaped sickled red blood cells interacting with endothelial cells as well as white blood cells and platelets in a human blood vessel cross-section. Some of the sickled cells are adherent to the endothelium and partially obstruct blood flow, while other cells are shown flowing freely within the blood vessel. We tried to create an interesting picture by illustrating how the interaction of cells can mediate vasoocclusion.

Please visit the website of the Cellular Mechanics Laboratory at the University of Connecticut and the Comprehensive Sickle Cell Clinical and Research Center at the University of Connecticut Health Center for more information on our research.

–Jamie Maciaszek, Biree Andemariam, Krithika Abiraman, and George Lykotrafitis

Where Science Meets Art with BiophysJ Author George Lykotrafitis

Biophysical Journal author George Lykotrafitis, Assistant Professor of Mechanical Engineering at the University of Connecticut, discusses the image he created for the cover of the latest issue of BiophysJ.

In our lab, we develop computational models and perform experiments at the cellular level to study quantitatively how diseases affect the chemomechanical properties and structure of human cells. Our main focus is sickle cell disease, a hereditary blood disorder caused by a one point mutation in the gene that encodes adult hemoglobin. In the deoxygenated state, the defective hemoglobin (Hemoglobin S) polymerizes to form stiff hemoglobin fibers which interact with the erythrocyte membrane and alter its properties. As a result, the erythrocytes of patients with sickle cell disease are stiffer and more adherent than normal erythrocytes while they have an abnormal sickle shape in the deoxygenated state.

In this article, we present a solvent-free coarse-grain molecular dynamics model for the membrane of normal human erythrocytes. The model combines the lipid bilayer and the erythrocyte cytoskeleton, thus exhibiting both the fluidic behavior of the lipid bilayer and the elastic properties of the erythrocyte cytoskeleton. Three types of coarse-grained particles are introduced to represent clusters of lipid molecules, actin junctions and band-3 complexes. An in-house developed parallel molecular dynamics program is used for the work. The proposed model is part of a larger effort to simulate the biomechanical behavior of sickle red blood cells.

Visualization of molecular dynamics simulations is an essential part of the postprocessing of the numerical results. Here, we employ AtomEye as a visualization tool. The red particles represent actin junctions, the yellow particles represent band-3 complexes attached to the spectrin tetramer, and the light blue particles represent free band-3 complexes. The smaller particles correspond to the lipid bilayer. While the original file was created by using AtomEye, the image was enhanced by digital processing. The original length scales of the image were preserved. The artist Xiaohong (Lucia) Liao (PhD) helped us to create an artistically interesting composition.

All in the lab feel great pleasure that our image is selected for the cover of the Biophysical Journal. We believe that it will increase the visibility of this particular work and of our research in general. Please visit the website of the Cellular Mechanics Laboratory at the University of Connecticut for more information on our research and on our outreach activities.