Malgorzata M. Kamocka

A multiscale model of thrombus development

A two-dimensional multiscale model is introduced for studying formation of a thrombus (clot) in a blood vessel. It involves components for modelling viscous, incompressible blood plasma; non-activated and activated platelets; blood cells; activating chemicals; fibrinogen; and vessel walls and their interactions. The macroscale dynamics of the blood flow is described by the continuum Navier–Stokes equations. The microscale interactions between the activated platelets, the platelets and fibrinogen and the platelets and vessel wall are described through an extended stochastic discrete cellular Potts model. The model is tested for robustness with respect to fluctuations of basic parameters. Simulation results demonstrate the development of an inhomogeneous internal structure of the thrombus, which is confirmed by the preliminary experimental data. We also make predictions about different stages in thrombus development, which can be tested experimentally and suggest specific experiments. Lastly, we demonstrate that the dependence of the thrombus size on the blood flow rate in simulations is close to the one observed experimentally.

A Multiscale Model of Venous Thrombus Formation with Surface-Mediated Control of Blood Coagulation Cascade

A combination of the extended multiscale model, new image processing algorithms, and biological experiments is used for studying the role of Factor VII (FVII) in venous thrombus formation. A detailed submodel of the tissue factor pathway of blood coagulation is introduced within the framework of the multiscale model to provide a detailed description of coagulation cascade. Surface reactions of the extrinsic coagulation pathway on membranes of platelets are studied under different flow conditions. It is shown that low levels of FVII in blood result in a significant delay in thrombin production, demonstrating that FVII plays an active role in promoting thrombus development at an early stage.

Study of blood flow impact on growth of thrombi using a multiscale model

An extended multiscale model is introduced for studying the formation of platelet thrombi in blood vessels. The model describes the interplay between viscous, incompressible blood plasma, activated and non-activated platelets, as well as other blood cells, activating chemicals, fibrinogen and vessel walls. The macroscale dynamics of the blood flow is represented by the continuous submodel in the form of the Navier–Stokes equations. The microscale cell-cell interactions are described by the stochastic Cellular Potts Model (CPM). Simulations indicate that increase in flow rates leads to greater structural heterogeneity of the clot. As heterogeneous structural domains within the clot affect thrombus stability, understanding the factors influencing thrombus structure is of significant biomedical importance.

Two-photon intravital imaging of thrombus development

Thrombus development in mouse mesenteric vessels following laser-induced injury was monitored by high-resolution, near-real-time, two-photon, intravital microscopy. In addition to the use of fluorescently tagged fibrin(ogen) and platelets, plasma was labeled with fluorescently tagged dextran. Because blood cells exclude the dextran in the single plane, blood cells appear as black silhouettes. Thus, in addition to monitoring the accumulation of platelets and fibrin in the thrombus, the protocol detects the movement and incorporation of unlabeled cells in and around it. The developing thrombus perturbs the blood flow near the thrombus surface, which affects the incorporation of platelets and blood cells into the structure. The hemodynamic effects and incorporation of blood cells lead to the development of thrombi with heterogeneous domain structures. Additionally, image processing algorithms and simulations were used to quantify structural features of developing thrombi. This analysis suggests a novel mechanism to stop the growth of developing thrombus.

Computational Approaches to Studying Thrombus Development

In addition to descriptive biological models, many computational models have been developed for hemostasis/thrombosis that provide quantitative characterization of thrombus development. Simulations using computational models that have been developed for coagulation reactions, platelet activation, and fibrinogen assembly have been shown to be in close agreement with experimental data. Models of processes involved in hemostasis/thrombosis are being integrated to simulate the development of the thrombus simultaneously in time and space. Further development of computational approaches can provide quantitative insights leading to predictions that are not obvious from qualitative biological models.

Pro-inflammatory angiogenesis is mediated by p38 MAP kinase

Chronic inflammation is tightly linked to diseases associated with endothelial dysfunction including aberrant angiogenesis. To better understand the endothelial role in pro‐inflammatory angiogenesis, we analyzed signaling pathways in continuously activated endothelial cells, which were either chronically exposed to soluble TNF or the reactive oxygen species (ROS) generating H2O2, or express active transmembrane TNF. Testing in an in vitro capillary sprout formation assay, continuous endothelial activation increased angiogenesis dependent on activation of p38 MAP kinase, NADPH oxidase, and matrix metalloproteinases (MMP). p38 MAP kinase‐ and MMP‐9‐dependent angiogenesis in our assay system may be part of a positive feed forward autocrine loop because continuously activated endothelial cells displayed up‐regulated ROS production and subsequent endothelial TNF expression. The pro‐angiogenic role of the p38 MAP kinase in continuously activated endothelial cells was in stark contrast to the anti‐angiogenic activity of the p38 MAP kinase in unstimulated control endothelial cells. In vivo, using an experimental prostate tumor, pharmacological inhibition of p38 MAP kinase demonstrated a significant reduction in tumor growth and in vessel density, suggesting a pro‐angiogenic role of the p38 MAP kinase in pathological angiogenesis in vivo. In conclusion, our results suggest that continuous activation of endothelial cells can cause a switch of the p38 MAP kinase from anti‐angiogenic to pro‐angiogenic activities in conditions which link oxidative stress and autocrine TNF production. J. Cell. Physiol. 226: 800–808, 2011.

Multiscale models of thrombogenesis.

To restrict the loss of blood follow from the rupture of blood vessels, the human body rapidly forms a clot consisting of platelets and fibrin. However, to prevent pathological clotting within vessels as a result of vessel damage, the response must be regulated. Clots forming within vessels (thrombi) can restrict the flow of blood causing damage to tissues in the flow field. Additionally, fragments dissociating from the primary thrombus (emboli) may lodge and clog vessels in the brain (causing ischemic stroke) or lungs (resulting in pulmonary embolism). Pathologies related to the obstruction of blood flow through the vasculature are the major cause of mortality in the United States. Venous thromboembolic disease alone accounts for 900,000 hospitalizations and 300,000 deaths per year and the incidence will increase as the population ages (Wakefield et al. J Vasc Surg 2009, 49:1620–1623). Thus, understanding the interplay between the many processes involved in thrombus development is of significant biomedical value. In this article, we first review computational models of important subprocesses of hemostasis/thrombosis including coagulation reactions, platelet activation, and fibrin assembly, respectively. We then describe several multiscale models integrating these subprocesses to simulate temporal and spatial development of thrombi. The development of validated computational models and predictive simulations will enable one to explore how the variation of multiple hemostatic factors affects thrombotic risk providing an important new tool for thrombosis research. WIREs Syst Biol Med 2012 doi: 10.1002/wsbm.1160

Applying Small Molecule Signal Transducer and Activator of Transcription-3 (STAT3) Protein Inhibitors as Pancreatic Cancer Therapeutics

Constitutively activated STAT3 protein has been found to be a key regulator of pancreatic cancer and a target for molecular therapeutic intervention. In this study, PG-S3-001, a small molecule derived from the SH-4-54 class of STAT3 inhibitors, was found to inhibit patient-derived pancreatic cancer cell proliferation in vitro and in vivo in the low micromolar range. PG-S3-001 binds the STAT3 protein potently, Kd = 324 nmol/L by surface plasmon resonance, and showed no effect in a kinome screen (>100 cancer-relevant kinases). In vitro studies demonstrated potent cell killing as well as inhibition of STAT3 activation in pancreatic cancer cells. To better model the tumor and its microenvironment, we utilized three-dimensional (3D) cultures of patient-derived pancreatic cancer cells in the absence and presence of cancer-associated fibroblasts (CAF). In this coculture model, inhibition of tumor growth is maintained following STAT3 inhibition in the presence of CAFs. Confocal microscopy was used to verify tumor cell death following treatment of 3D cocultures with PG-S3-001. The 3D model was predictive of in vivo efficacy as significant tumor growth inhibition was observed upon administration of PG-S3-001. These studies showed that the inhibition of STAT3 was able to impact the survival of tumor cells in a relevant 3D model, as well as in a xenograft model using patient-derived cells. Mol Cancer Ther; 15(5); 794–805. ©2016 AACR.

Segmentation, reconstruction, and analysis of blood thrombus formation in 3D 2-photonmicroscopy images

We study the problem of segmenting, reconstructing, and analyzing the structure growth of thrombi (clots) in blood vessels in vivo based on 2-photon microscopic image data. First, we develop an algorithm for segmenting clots in 3D microscopic images based on density-based clustering and methods for dealing with imaging artifacts. Next, we apply the union-of-balls (or alpha-shape) algorithm to reconstruct the boundary of clots in 3D. Finally, we perform experimental studies and analysis on the reconstructed clots and obtain quantitative data of thrombus growth and structures. We conduct experiments on laser-induced injuries in vessels of two types of mice (the wild type and the type with low levels of coagulation factor VII) and analyze and compare the developing clot structures based on their reconstructed clots from image data. The results we obtain are of biomedical significance. Our quantitative analysis of the clot composition leads to better understanding of the thrombus development, and is valuable to the modeling and verification of computational simulation of thrombogenesis.

Combined experimental and simulation study of blood clot formation

A multiscale model of blood clot (thrombus) formation is extended by adding a sub-model of the tissue factor (TF) pathway of blood coagulation to provide more biologically relevant description of coagulation process. A combination of experimental approach, image analysis and a multiscale modeling is used for studying role of factor VII and fibrin in limiting growth of a blood clot. The simulations obtained using new extended model, generated a hypothesis that formation of a fiber cap is capable of stopping blood clot growth by blocking release of thrombin, causing activation of platelets, into a blood stream. This was confirmed by comparison with reconstructed three dimensional experimental images of clots formed in mice with normal and limited levels of factor VII.