Fibrin networks regulate protein transport during thrombus development.

Thromboembolic disease is a leading cause of morbidity and mortality worldwide. In the last several years there have been a number of studies attempting to identify mechanisms that stop thrombus growth. This paper identifies a novel mechanism related to formation of a fibrin cap. In particular, protein transport through a fibrin network, an important component of a thrombus, was studied by integrating experiments with model simulations. The network permeability and the protein diffusivity were shown to be important factors determining the transport of proteins through the fibrin network. Our previous in vivo studies in mice have shown that stabilized non-occluding thrombi are covered by a fibrin network ('fibrin cap'). Model simulations, calibrated using experiments in microfluidic devices and accounting for the permeable structure of the fibrin cap, demonstrated that thrombin generated inside the thrombus was washed downstream through the fibrin network, thus limiting exposure of platelets on the thrombus surface to thrombin. Moreover, by restricting the approach of resting platelets in the flowing blood to the thrombus core, the fibrin cap impaired platelets from reaching regions of high thrombin concentration necessary for platelet activation and limited thrombus growth. The formation of a fibrin cap prevents small thrombi that frequently develop in the absence of major injury in the 60000 km of vessels in the body from developing into life threatening events.

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.

Activated protein C blocks p53-mediated apoptosis in ischemic human brain endothelium and is neuroprotective.

Activated protein C (APC) is a systemic anti-coagulant and anti-inflammatory factor. It reduces organ damage in animal models of sepsis, ischemic injury and stroke and substantially reduces mortality in patients with severe sepsis. It was not known whether APC acts as a direct cell survival factor or whether its neuroprotective effect is secondary to its anti-coagulant and anti-inflammatory effects. We report that APC directly prevents apoptosis in hypoxic human brain endothelium through transcriptionally dependent inhibition of tumor suppressor protein p53, normalization of the pro-apoptotic Bax/Bcl-2 ratio and reduction of caspase-3 signaling. These mechanisms are distinct from those involving upregulation of the genes encoding the anti-apoptotic Bcl-2 homolog A1 and inhibitor of apoptosis protein-1 (IAP-1) by APC in umbilical vein endothelial cells. Cytoprotection of brain endothelium by APC in vitro required endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1), as did its in vivo neuroprotective activity in a stroke model of mice with a severe deficiency of EPCR. This is consistent with work showing the direct effects of APC on cultured cells via EPCR and PAR-1 (ref. 9). Moreover, the in vivo neuroprotective effects of low-dose mouse APC seemed to be independent of its anti-coagulant activity. Thus, APC protects the brain from ischemic injury by acting directly on brain cells.

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.

IFATS collection: Adipose stromal cell differentiation is reduced by endothelial cell contact and paracrine communication: role of canonical Wnt signaling.

Adipose stromal cells (ASC) are multipotential mesenchymal progenitor cells that are readily induced to undergo adipogenic differentiation, and we have recently demonstrated them to have functional and phenotypic overlap with pericytes lining microvessels in adipose tissues. In this study we addressed the hypothesis that modulation of ASC fate within this perivascular niche can occur via interaction with endothelial cells (EC), which serve to modulate the adipogenic potential of ASC. To this end, we investigated contact as well as paracrine effects of EC on ASC adipogenesis, in two-dimensional coculture and via conditioned medium and analyzed mutual gene expression changes by real-time reverse transcription polymerase chain reaction (PCR). A significant decrease in adipogenic differentiation was observed in ASC when they were cocultured with EC but not control fibroblasts. This endothelial cell-specific effect was accompanied by increased expression of factors involved in Wnt signaling, most prominently Wnt1, Wnt4, and Wnt10a, which are well-known inhibitors of adipogenesis. Suppression of Wnt1 but not Wnt 10a or scrambled control short interfering RNA in cocultures partially reversed the endothelial cell effect, thus increasing adipogenic differentiation, suggesting a plausible role of Wnt1 ligand in modulation of adipogenesis by the vasculature. Furthermore, addition of recombinant Wnt ligand or the Wnt signaling agonist inhibited adipogenic differentiation of ASC in the absence of EC. In conclusion, these data define the relationship in adipose tissue between ASC and EC in the perivascular niche, in which the latter act to repress adipogenesis, thereby stabilizing vasculature. It is tempting to speculate that abnormal endothelial function may be associated with pathologic derepression of adipogenesis. Disclosure of potential conflicts of interest is found at the end of this article.

Mice with a severe deficiency in protein C display prothrombotic and proinflammatory phenotypes and compromised maternal reproductive capabilities.

Anticoagulant protein C (PC) is important not only for maintenance of normal hemostasis, but also for regulating the host immune response during inflammation. Because mice with a designed total genetic deficiency in PC (PC-/- mice) die soon after birth, attempts to dissect PC function in various coagulation/inflammation-based pathologies through use of mice with less than 50% of normal PC levels have not been successful to date. In the current investigation, we have used a novel transgenic strategy to generate different mouse models expressing 1-18% of normal PC levels. In contrast to PC-/- mice, mice with only partial PC deficiency survived beyond birth and also developed thrombosis and inflammation. The onset and severity of these phenotypes vary significantly and are strongly dependent on plasma PC levels. Our findings additionally provide the first evidence that maternal PC is vital for sustaining pregnancy beyond 7.5 days postcoitum, likely by regulating the balance of coagulation and inflammation during trophoblast invasion. These low PC-expressing transgenic mouse lines provide novel animal models that can be used to elucidate the importance of PC in maintenance of the organism and in disease.

Suppression of hepatocyte growth factor production impairs the ability of adipose-derived stem cells to promote ischemic tissue revascularization.

The use of adipose-derived stem/stromal cells (ASCs) for promoting repair of tissues is a promising potential therapy, but the mechanisms of their action are not fully understood. We and others previously demonstrated accelerated reperfusion and tissue salvage by ASCs in peripheral ischemia models and have shown that ASCs secrete physiologically relevant levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor. The specific contribution of HGF to ASC potency was determined by silencing HGF expression. RNA interference was used to downregulate HGF expression. A dual-cassette lentiviral construct expressing green fluorescent protein (GFP) and either a small hairpin RNA specifically targeted to HGF mRNA (shHGF) or an inactive control sequence (shCtrl) were used to stably transduce ASCs (ASC-shHGF and ASC-shCtrl, respectively). Transduced ASC-shHGF secreted >80% less HGF, which led to a reduced ability to promote survival, proliferation, and migration of mature and progenitor endothelial cells in vitro. ASC-shHGF were also significantly impaired, compared with ASC-shCtrl, in their ability to promote reperfusion in a mouse hindlimb ischemia model. The diminished ability of ASCs with silenced HGF to promote reperfusion of ischemic tissues was reflected by reduced densities of capillaries in reperfused tissues. In addition, fewer GFP(+) cells were detected at 3 weeks in ischemic limbs of mice treated with ASC-shHGF compared with those treated with ASC-shCtrl. These results indicate that production of HGF is important for the potency of ASCs. This finding directly supports the emerging concept that local factor secretion by donor cells is a key element of cell-based therapies. Disclosure of potential conflicts of interest is found at the end of this article.

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.

Rat prostate tumors express cancer procoagulant, an activator of coagulation factor X.

Two common procoagulant activities associated with tumors are tissue factor and cancer procoagulant (CP), an activator of coagulation factor X. We have identified a convenient source of CP in transplanted Lobund-Wistar rat PA3 prostate tumors. CP activity was purified from 5 independent transplanted prostate tumors by column chromatography. The protein activated factor X in the absence of TF and factor VII. An antihuman CP antibody recognized rat CP in an ELISA and inactivated CP activity in a chromogenic assay. Lobund-Wistar prostate tumors may provide a convenient animal model useful in determining the role of CP in cancer development.

Generation of genetically-altered mice producing very low levels of coagulation factorVII.

It has been shown earlier that mice with a total targeted deletion of the factorVII gene (FVII(-/-)) die perinatally, thereby precluding study of adult animals with this total deficiency. Consequently, mice producing very low levels of FVII were developed by targeted replacement of the wild-type (WT) murine FYII gene with its corresponding cDNA, under control of the tetracycline transactivator (tTA) promoter. When backcrossed into the C57BI/6 strain, unchallenged mice containing two replaced FVII(tTA) alleles (FVII(tTA/tTA) produce approximately 0.7% of WT FVII levels, but yet live to adulthood despite displaying severely downregulated overall thrombin production and spontaneously developing cardiac fibrosis at a young adult age. This genetically-altered mouse line provides an excellent animal model to study consequences of a severe FVII deficiency in unchallenged mice and in mice subjected to a variety of experimental challenges.

Tissue distribution of fetal liver cells following in utero transplantation in mice.

Transplantation of hepatic stem cells in utero has been advanced as a potential clinical approach to a variety of diseases, including deficiencies of coagulation factors. Although syngeneic transplantation has met with some success, consideration needs to be given to the potential for transplanted cells to colonize nontarget tissues. Liver cells were harvested from Rosa26 embyros at embryonic age 12.5 days postconception (pc) and transplanted into the peritoneal cavity of syngeneic recipients in utero. Tissues were harvested from tissue recipients at various time points ranging from 1 to 328 days pc, and tissues were stained for beta-galactosidase to identify the existence of cells derived from Rosa26 donors. Beta-galactosidase-positive cells were found in the lung, liver, and brain as early as 20 days pc and through 328 days pc. Positive cells in these tissues existed as islands of cells that were morphologically similar to hepatocytes. In the spleen, individual beta-galactosidase-positive cells of both leukocytic and erythrocytic lineages were present, and suggest that hematopoietic cells were transferred to recipients along with hepatocytes. The lack of an inflammatory response to the beta-galactosidase-positive cells suggests that the donor cells were immunologically tolerated. In summary, the possibility that cells administered in utero may inadvertently colonize nontarget tissues suggests that clinical application of this method will need to be approached with diligence.

Gene targeting in hemostasis. Factor X.

Blood coagulation Factor X (FX) is a vitamin K-dependent serine protease that plays a central role in blood clotting. Additionally, FX may exert other functions beyond coagulation but the precise role of this protein in these processes and the in vivo relevance remains to be further delineated. The development of gene knockout technology has allowed for a direct means to determine the physiological relevance of various proteins in a number of normal and pathological processes. Mice with a total deficiency of FX have been generated by targeted deletion of all exons encoding the mature FX protein. The genotypic distribution indicated that homozygous deficiency results in partial embryonic lethality at embryonic day (E) 11.5-12.5 with signs of massive bleeding but no histologically evident defects in the vasculature of these embryos or their yolk sac. The majority of those that survive to term die within 5 days, most frequently from intraabdominal bleeding. The remainder die between postnatal day (P)5 and P20 with intraabdominal, subcutaneous, or intracranial bleeding or a combination thereof. These observations underline the importance of FX function in embryonic and postnatal survival and confirm that these mice serve as effective models of the bleeding disorders observed in human FX deficiency. While the early FX-/- lethality impedes investigation of potential morphogenetic functions of FX in vivo, such studies may however become feasible through the availability of mice expressing mutant FX proteins, e.g., EPR-1 binding site mutants, or of mice with conditional FX deficiency.

Mice with a severe deficiency of the endothelial protein C receptor gene develop, survive, and reproduce normally, and do not present with enhanced arterial thrombosis after challenge.

The endothelial cell Protein C receptor (EPCR) functions to enhance activation of anticoagulant Protein C (PC) by the thrombin/ thrombomodulin (Tm) complex on the surface of the endothelium. This overall system functions in anticoagulation, profibrinolytic, and antiinflammatory responses. Mice with a severe targeted deficiency of this receptor have been generated by integration of exogenous DNA elements into the 5'-untranslated region of the EPCR gene. Despite the retention of the entire endogenous EPCR coding sequence in the altered EPCR gene locus, only very low EPCR message contents were detected in mice by quantitative RT-PCR during embryogenesis and up to at least early adulthood. Immunohistochemical analysis of various regions of the arterial tree of mice up to 4 months of age, employing an anti-murine EPCR antibody, confirmed that undetectable levels of this protein were present in arterial regions during these periods. Despite this, these mice are not more prone to arterial thrombosis after challenge in a FeCl3 carotid artery thrombosis model. Small amounts (<10% of wild-type) of this protein were found in other tissues. Matings of mice homozygous for this deficiency led to normal births and survival of the offspring, in contrast to results by others demonstrating early embryonic lethality of a total EPCR deficiency. These data further show that minimal levels of EPCR are able to support male and female virility, as well as embryonic development, birth, and survival to adulthood.

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.

Mechano-rheological properties of the murine thrombus determined via nanoindentation and finite element modeling.

Deep vein thrombosis, pulmonary embolism, and abdominal aortic aneurysms are blood-related diseases that represent a major public health problem. These diseases are characterized by the formation of a thrombus (i.e., blood clot) that either blocks a major artery or causes an aortic rupture. Identifying the mechanical properties of thrombi can help determine when these incidents will occur. In this investigation, a murine thrombus, formed from platelet-rich plasma, calcium, and thrombin, was nanoindented and the elastic modulus was estimated via elastic contact theory. This information was used as input to an inverse finite element simulation, which determined optimal values for the elastic modulus and viscosity of the thrombus using a viscoelastic material model. A sensitivity analysis was also performed to determine which material parameters have the greatest affect on the simulation. Results from this investigation demonstrate the feasibility of the mechanical characterization of a murine thrombus using nanoindentation.

Multiscale model of fibrin accumulation on the blood clot surface and platelet dynamics.

A multiscale computational model of thrombus (blood clot) development is extended by incorporating a submodel describing formation of fibrin network through "fibrin elements" representing regions occupied by polymerized fibrin. Simulations demonstrate that fibrin accumulates on the surface of the thrombus and that fibrin network limits growth by reducing thrombin concentrations on the thrombus surface and decreasing adhesivity of resting platelets in blood near thrombus surface. These results suggest that fibrin accumulation may not only increase the structural integrity of the thrombus but also considerably contribute toward limiting its growth. Also, a fast Graphics Processing Unit implementation is described for a multiscale computational model of the platelet-blood flow interaction.

Modelling platelet-blood flow interaction using the subcellular element Langevin method.

In this paper, a new three-dimensional modelling approach is described for studying fluid-viscoelastic cell interaction, the subcellular element Langevin (SCEL) method, with cells modelled by subcellular elements (SCEs) and SCE cells coupled with fluid flow and substrate models by using the Langevin equation. It is demonstrated that: (i) the new method is computationally efficient, scaling as (N) for N SCEs; (ii) cell geometry, stiffness and adhesivity can be modelled by directly relating parameters to experimentally measured values; (iii) modelling the fluid-platelet interface as a surface leads to a very good correlation with experimentally observed platelet flow interactions. Using this method, the three-dimensional motion of a viscoelastic platelet in a shear blood flow was simulated and compared with experiments on tracking platelets in a blood chamber. It is shown that the complex platelet-flipping dynamics under linear shear flows can be accurately recovered with the SCEL model when compared with the experiments. All experimental details and electronic supplementary material are archived at http://biomath.math.nd.edu/scelsupplementaryinformation/.

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.

Identifying and genotyping transgene integration loci.

The random germline integration of genetically engineered transgenes has been a powerful technique to study the role of particular genes in variety of biological processes. Although the identification of the transgene insertion site is often not essential for functional analysis of the transgene, identifying the site can have practical benefit. Enabling one to distinguish between animals that are homozygous or hemizygous for the transgene locus could facilitate breeding strategies to produce animals with a large number of genetic markers. Furthermore, founder lines generated with the same transgene construct may exhibit different phenotypes and levels of transgene expression depending on the site of integration. The goal of this report was to develop a rapid protocol for the identification and verification of transgene insertion sites. To identify host genomic sequences at the coagulation Factor X transgene integration site, DNA from a tail snip of the transgenic mouse was digested with NcoI and circularized using T4 DNA ligase. Using appropriately positioned PCR primers annealing to a transgene fragment distal to a terminal transgene restriction site (NcoI), one could amplify a fragment containing the transgene terminal region and extending into the flanking genomic sequence at the insertion site. DNA sequence determination of the amplicon permitted identification of the insertion site using a BLASTN search. FISH analysis of a metaphase spread of primary fibroblasts derived from the transgenic mouse was consistent with the identification of insertion site near the end of mouse chromosome 14. Identification of transgene insertion sites will facilitate genotyping strategies useful for the construction of mice with multiple engineered genetic markers and to distinguish among different founder lines generated by the same transgene. Furthermore, identification of the insertion site is necessary to analyze unexpected phenotypes that might be caused by insertional inactivation of an endogenous gene.