Michael R. King

Age-Related Intimal Stiffening Enhances Endothelial Permeability and Leukocyte Transmigration

Inhibiting endothelial cell contractility reverses the deleterious effects of age-related matrix stiffening on normal cell function, which could help prevent the development of atherosclerosis. Rock Your Heart Out According to novelist Thomas Bailey Aldrich, “To keep the heart unwrinkled, to be hopeful, kindly, cheerful, reverent, is to triumph over old age” (from Ponkapoag Papers). Unfortunately, despite a positive attitude, aging is accompanied by several changes of heart, at least at the cellular level. One age-related “wrinkle” is stiffening of the extracellular matrix that lines the blood vessels, a change that has been linked to atherosclerosis; yet, the cellular and mechanical features that couple the two conditions have remained elusive. Now, using a clever combination of biomaterials, cells, aortas, and mice, Huynh and colleagues have demystified the correlation between aging and atherosclerosis, showing that cell contractility is at the heart of it all. The authors first developed an in vitro system that mimicked the basic structures of both young and old blood vessels. Synthetic hydrogel matrices of varying stiffnesses were seeded with bovine aortic endothelial cells. By administering a solution of fluorescently labeled molecules to the cell-gel system and watching how the dye moved across the cell layer, Huynh et al. determined that permeability increased as a function of matrix stiffness, suggesting that age alone was a disruptive factor. These results were confirmed ex vivo by performing atomic force microscopy with decellularized thoracic aortas from both young (~10 weeks) and old (~92 weeks) mice. In both of these systems, the enhanced vessel permeability resulted from an increase in the distance—or junction—between neighboring cells. This increase in the so-called gap junction width also permitted the passage of leukocytes through the endothelial cell monolayer; along with leaky vasculature, cellular transmigration is a hallmark of atherosclerosis progression. Because the Rho signaling pathway is linked to the cellular cytoskeleton and, in turn, contractility, Huynh et al. hypothesized that they could reverse the effects of age-related intimal stiffening by inhibiting Rho-associated kinase (ROCK). By administering a pharmacological ROCK inhibitor (Y-27632) to their in vitro setup and to old mice, the authors showed that gap junction widths and endothelial cellular forces decreased. In vitro, the inhibitor also prevented leukocyte transmigration. These observations suggest that directly interfering with Rho signaling is a viable treatment option for age-related atherosclerosis. And because inhibitors of Rho signaling, such as fasudil, are already available in the clinic, one might say that physicians and researchers are ready to rock. Age is the most significant risk factor for atherosclerosis; however, the link between age and atherosclerosis is poorly understood. During both aging and atherosclerosis progression, the blood vessel wall stiffens owing to alterations in the extracellular matrix. Using in vitro and ex vivo models of vessel wall stiffness and aging, we show that stiffening of extracellular matrix within the intima promotes endothelial cell permeability—a hallmark of atherogenesis. When cultured on hydrogels fabricated to match the elasticity of young and aging intima, endothelial monolayers exhibit increased permeability and disrupted cell-cell junctions on stiffer matrices. In parallel experiments, we showed a corresponding increase in cell-cell junction width with age in ex vivo aortas from young (10 weeks) and old (21 to 25 months) healthy mice. To investigate the mechanism by which matrix stiffening alters monolayer integrity, we found that cell contractility increases with increased matrix stiffness, mechanically destabilizing cell-cell junctions. This increase in endothelial permeability results in increased leukocyte extravasation, which is a critical step in atherosclerotic plaque formation. Mild inhibition of Rho-dependent cell contractility using Y-27632, an inhibitor of Rho-associated kinase, or small interfering RNA restored monolayer integrity in vitro and in vivo. Our results suggest that extracellular matrix stiffening alone, which occurs during aging, can lead to endothelial monolayer disruption and atherosclerosis pathogenesis. Because previous therapeutics designed to decrease vascular stiffness have been met with limited success, our findings could be the basis for the design of therapeutics that target the Rho-dependent cellular contractile response to matrix stiffening, rather than stiffness itself, to more effectively prevent atherosclerosis progression.

Multiparticle adhesive dynamics: Hydrodynamic recruitment of rolling leukocytes

The slow rolling motion of leukocytes along the walls of blood vessels mediated by specific receptor-ligand adhesion is important in inflammation and occurs in postcapillary venules over a wide range of wall shear stresses and vessel diameters. The ability of hydrodynamic collisions between cells to induce capture of free-stream leukocytes to a selectin-bearing surface under shear flow was studied experimentally by using a cell-free assay. It was found that carbohydrate-coated spherical beads, representing model leukocytes, tend to attach to the adhesive wall 4–5 cell diameters up- or downstream of a slowly rolling or stationary adhesive bead. A key feature of such “hydrodynamic recruitment” is that only glancing, indirect collisions occurring close to the plane will result in downstream attachment. A direct numerical simulation of cell capture and rolling that includes multiparticle hydrodynamic interactions is shown to reproduce the observed behavior accurately. The theory predicts that hydrodynamic recruitment will occur in the absence of buoyancy effects and over a range of shear rates, suggesting that the mechanism may be important in vivo. This theory is supported by measurements of leukocyte capture in vivo using the hamster cheek pouch model.

Use of naturally occurring halloysite nanotubes for enhanced capture of flowing cells.

The development of individualized treatments for cancer can be facilitated by more efficient methods for separating cancer cells from patient blood in such a way that they remain viable for live cell assays. We have previously shown that immobilized P-selectin protein can be used on the inner surface of a microscale flow system to induce leukemic cells and leukocytes to roll at different velocities and relative fluxes, thereby creating a means for rapid cell fractionation without inflicting cellular damage. In this study, we explore a method to more efficiently capture leukemic and epithelial cancer cells from flow by altering the nanoscale topography of the inner surface of P-selectin-coated microtubes. This functionalized topography is achieved by attaching naturally occurring halloysite nanotubes to the microtube surface via a monolayer of poly-L-lysine), followed by functionalization with recombinant human selectin protein. We have found that halloysite nanotube coatings promote increased capture of leukemic cells and have determined the key parameters for controlling cell capture under flow: halloysite content and selectin density. Ultimately, selectin-functionalized nanotube coatings should provide a means for enhanced cancer cell isolation from whole blood and other mixtures of cells.

Toxoplasma gondii Triggers Release of Human and Mouse Neutrophil Extracellular Traps

ABSTRACT Neutrophils have recently been shown to release DNA-based extracellular traps that contribute to microbicidal killing and have also been implicated in autoimmunity. The role of neutrophil extracellular trap (NET) formation in the host response to nonbacterial pathogens has received much less attention. Here, we show that the protozoan pathogen Toxoplasma gondii elicits the production of NETs from human and mouse neutrophils. Tachyzoites of each of the three major parasite strain types were efficiently entrapped within NETs, resulting in decreased parasite viability. We also show that Toxoplasma activates a MEK-extracellular signal-regulated kinase (ERK) pathway in neutrophils and that the inhibition of this pathway leads to decreased NET formation. To determine if Toxoplasma induced NET formation in vivo, we employed a mouse intranasal infection model. We found that the administration of tachyzoites by this route induced a rapid tissue recruitment of neutrophils with evidence of extracellular DNA release. Taken together, these data indicate a role for NETs in the host innate response to protozoan infection. We propose that NET formation limits infection by direct microbicidal effects on Toxoplasma as well as by interfering with the ability of the parasite to invade target host cells.

A physical sciences network characterization of non-tumorigenic and metastatic cells

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences–Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis.

TRAIL-coated leukocytes that kill cancer cells in the circulation

Significance This paper describes a unique approach to target and kill cancer cells in the bloodstream, in which the extensive surface area of circulating leukocytes is used to display the cancer-specific TNF-related apoptosis inducing ligand (TRAIL) and E-selectin adhesion receptor to the surrounding fluid. The approach is inspired by the cytotoxic activity of natural killer cells and is quite effective at killing cancer cells both in vitro with human blood samples and in mouse blood circulation. The mechanism is surprising and unexpected in that this repurposing of leukocytes in flowing blood is more effective than directly targeting the cancer cells with liposomes or soluble protein. Metastasis through the bloodstream contributes to poor prognosis in many types of cancer. Mounting evidence implicates selectin-based adhesive interactions between cancer cells and the blood vessel wall as facilitating this process, in a manner similar to leukocyte trafficking during inflammation. Here, we describe a unique approach to target and kill colon and prostate cancer cells in the blood that causes circulating leukocytes to present the cancer-specific TNF-related apoptosis inducing ligand (TRAIL) on their surface along with E-selectin adhesion receptor. This approach, demonstrated in vitro with human blood and also in mice, mimics the cytotoxic activity of natural killer cells and increases the surface area available for delivery of the receptor-mediated signal. The resulting “unnatural killer cells” hold promise as an effective means to neutralize circulating tumor cells that enter blood with the potential to form new metastases.

Computational and Experimental Models of Cancer Cell Response to Fluid Shear Stress

It has become evident that mechanical forces play a key role in cancer metastasis, a complex series of steps that is responsible for the majority of cancer-related deaths. One such force is fluid shear stress, exerted on circulating tumor cells by blood flow in the vascular microenvironment, and also on tumor cells exposed to slow interstitial flows in the tumor microenvironment. Computational and experimental models have the potential to elucidate metastatic behavior of cells exposed to such forces. Here, we review the fluid-generated forces that tumor cells are exposed to in the vascular and tumor microenvironments, and discuss recent computational and experimental models that have revealed mechanotransduction phenomena that may play a role in the metastatic process.

Targeted drug delivery to circulating tumor cells via platelet membrane-functionalized particles

Circulating tumor cells (CTCs) are responsible for metastases in distant organs via hematogenous dissemination. Fundamental studies in the past decade have suggested that neutralization of CTCs in circulation could represent an effective strategy to prevent metastasis. Current paradigms of targeted drug delivery into a solid tumor largely fall into two main categories: unique cancer markers (e.g. overexpression of surface receptors) and tumor-specific microenvironment (e.g. low pH, hypoxia, etc.). While relying on a surface receptor to target CTCs can be greatly challenged by cancer heterogeneity, targeting of tumor microenvironments has the advantage of recognizing a broader spectrum of cancer cells regardless of genetic differences or tumor types. The blood circulation, however, where CTCs transit through, lacks the same tumor microenvironment as that found in a solid tumor. In this study, a unique microenvironment was confirmed upon introduction of cancer cells of different types into circulation where activated platelets and fibrin were physically associated with blood-borne cancer cells. Inspired by this observation, synthetic silica particles were functionalized with activated platelet membrane along with surface conjugation of tumor-specific apoptosis-inducing ligand cytokine, TRAIL. Biomimetic synthetic particles incorporated into CTC-associated micro-thrombi in lung vasculature and dramatically decreased lung metastases in a mouse breast cancer metastasis model. Our results demonstrate a Trojan Horse strategy of neutralizing CTCs to attenuate metastasis.

E-selectin liposomal and nanotube-targeted delivery of doxorubicin to circulating tumor cells

The presence of circulating tumor cells (CTCs) is believed to lead to the formation of secondary tumors via an adhesion cascade involving interaction between adhesion receptors of endothelial cells and ligands on CTCs. Many CTCs express sialylated carbohydrate ligands on their surfaces that adhere to selectin protein found on inflamed endothelial cells. We have investigated the feasibility of using immobilized selectin proteins as a targeting mechanism for CTCs under flow. Herein, targeted liposomal doxorubicin (L-DXR) was functionalized with recombinant human E-selectin (ES) and polyethylene glycol (PEG) to target and kill cancer cells under shear flow, both when immobilized along a microtube device or sheared in a cone-and-plate viscometer in a dilute suspension. Healthy circulating cells such as red blood cells were not targeted by this mechanism and were left to freely circulate, and minimal leukocyte death was observed. Halloysite nanotube (HNT)-coated microtube devices immobilized with nanoscale liposomes significantly enhanced the targeting, capture, and killing of cancer cells. This work demonstrates that E-selectin functionalized L-DXR, sheared in suspension or immobilized onto microtube devices, provides a novel approach to selectively target and deliver chemotherapeutics to CTCs in the bloodstream.

Mechanical Shedding of L-selectin from the Neutrophil Surface during Rolling on Sialyl Lewis x under Flow

The interaction of L-selectin expressed on leukocytes with endothelial cells leads to capture and rolling and is critical for the recruitment of leukocytes into sites of inflammation. It is known that leukocyte activation by chemoattractants, the change of osmotic pressure in cell media, or cross-linking of L-selectin all result in rapid shedding of L-selectin. Here we present a novel mechanism for surface cleavage of L-selectin on neutrophils during rolling on a sialyl Lewis x-coated surface that involves mechanical force. Flow cytometry and rolling of neutrophils labeled with Qdot®-L-selectin antibodies in an in vitro flow chamber showed that the mechanical shedding of L-selectin occurs during rolling and depends on the amount of shear applied. In addition, the mechanical L-selectin shedding causes an increase in cell rolling velocity with rolling duration, suggesting a gradual loss of L-selectin and is mediated by p38 mitogen-activated protein kinase activation. Thus, these data show that mechanical force induces the cleavage of L-selectin from the neutrophil surface during rolling and therefore decreases the adhesion of cells to a ligand-presenting surface in flow.