Joseph P. Califano

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.

Cellular Traction Stresses Increase with Increasing Metastatic Potential

Cancer cells exist in a mechanically and chemically heterogeneous microenvironment which undergoes dynamic changes throughout neoplastic progression. During metastasis, cells from a primary tumor acquire characteristics that enable them to escape from the primary tumor and migrate through the heterogeneous stromal environment to establish secondary tumors. Despite being linked to poor prognosis, there are no direct clinical tests available to diagnose the likelihood of metastasis. Moreover, the physical mechanisms employed by metastatic cancer cells to migrate are poorly understood. Because metastasis of most solid tumors requires cells to exert force to reorganize and navigate through dense stroma, we investigated differences in cellular force generation between metastatic and non-metastatic cells. Using traction force microscopy, we found that in human metastatic breast, prostate and lung cancer cell lines, traction stresses were significantly increased compared to non-metastatic counterparts. This trend was recapitulated in the isogenic MCF10AT series of breast cancer cells. Our data also indicate that increased matrix stiffness and collagen density promote increased traction forces, and that metastatic cells generate higher forces than non-metastatic cells across all matrix properties studied. Additionally, we found that cell spreading for these cell lines has a direct relationship with collagen density, but a biphasic relationship with substrate stiffness, indicating that cell area alone does not dictate the magnitude of traction stress generation. Together, these data suggest that cellular contractile force may play an important role in metastasis, and that the physical properties of the stromal environment may regulate cellular force generation. These findings are critical for understanding the physical mechanisms of metastasis and the role of the extracellular microenvironment in metastatic progression.

The role of the cytoskeleton in cellular force generation in 2D and 3D environments

To adhere and migrate, cells generate forces through the cytoskeleton that are transmitted to the surrounding matrix. While cellular force generation has been studied on 2D substrates, less is known about cytoskeletal-mediated traction forces of cells embedded in more in vivo-like 3D matrices. Recent studies have revealed important differences between the cytoskeletal structure, adhesion, and migration of cells in 2D and 3D. Because the cytoskeleton mediates force, we sought to directly compare the role of the cytoskeleton in modulating cell force in 2D and 3D. MDA-MB-231 cells were treated with agents that perturbed actin, microtubules, or myosin, and analyzed for changes in cytoskeletal organization and force generation in both 2D and 3D. To quantify traction stresses in 2D, traction force microscopy was used; in 3D, force was assessed based on single cell-mediated collagen fibril reorganization imaged using confocal reflectance microscopy. Interestingly, even though previous studies have observed differences in cell behaviors like migration in 2D and 3D, our data indicate that forces generated on 2D substrates correlate with forces within 3D matrices. Disruption of actin, myosin or microtubules in either 2D or 3D microenvironments disrupts cell-generated force. These data suggest that despite differences in cytoskeletal organization in 2D and 3D, actin, microtubules and myosin contribute to contractility and matrix reorganization similarly in both microenvironments.

Implanted adipose progenitor cells as physicochemical regulators of breast cancer

Multipotent adipose-derived stem cells (ASCs) are increasingly used for regenerative purposes such as soft tissue reconstruction following mastectomy; however, the ability of tumors to commandeer ASC functions to advance tumor progression is not well understood. Through the integration of physical sciences and oncology approaches we investigated the capability of tumor-derived chemical and mechanical cues to enhance ASC-mediated contributions to tumor stroma formation. Our results indicate that soluble factors from breast cancer cells inhibit adipogenic differentiation while increasing proliferation, proangiogenic factor secretion, and myofibroblastic differentiation of ASCs. This altered ASC phenotype led to varied extracellular matrix (ECM) deposition and contraction thereby enhancing tissue stiffness, a characteristic feature of breast tumors. Increased stiffness, in turn, facilitated changes in ASC behavior similar to those observed with tumor-derived chemical cues. Orthotopic mouse studies further confirmed the pathological relevance of ASCs in tumor progression and stiffness in vivo. In summary, altered ASC behavior can promote tumorigenesis and, thus, their implementation for regenerative therapy should be carefully considered in patients previously treated for cancer.

Exogenous and endogenous force regulation of endothelial cell behavior.

Endothelial cells live in a dynamic environment where they are constantly exposed to external hemodynamic forces and generate cytoskeletal-based endogenous forces. These exogenous and endogenous forces are critical regulators of endothelial cell health and blood vessel maintenance at all generations of the vascular system, from large arteries to capillary beds. The first part of this review highlights the role of the primary exogenous hemodynamic forces of shear, cyclic strain, and pressure forces in mediating endothelial cell response. We then discuss the emergent role of the mechanical properties of the extracellular matrix and of cellular endogenous force generation on endothelial cell function, implicating substrate stiffness and cellular traction stresses as important mediators of endothelial cell health. The intersection of exogenous and endogenous forces on endothelial cell function is discussed, suggesting some of the many remaining questions in the field of endothelial mechanobiology.

Matrix Stiffness: A Regulator of Cellular Behavior and Tissue Formation

The extracellular environment is an essential mediator of cell health and provides both chemical and mechanical stimuli to influence single and collective cell behaviors. While historically there has been significant emphasis placed on chemical regulators within the extracellular matrix, the role of the mechanical environment is less well known. Here, we review the role of matrix mechanics on cell function and tissue integrity. Cellular responses to mechanical signals include differentiation, migration, proliferation, and alterations in cell–cell and cell–matrix adhesion. Interestingly, the mechanical properties of tissues are altered in many disease states, leading to cellular dysfunction and further disease progression. Successful regenerative medicine strategies must consider the native mechanical environment so that they are able to elicit a favorable cellular response and integrate into the native tissue structure.

Control of cell migration using a conducting polymer device

Control of cell migration is receiving a great deal of attention due to its relevance to the engineering of tissues. Here we report a device that contains a conducting polymer stripe and achieves a continuum of microenvironments for cell growth under the influence of an applied bias. Marked differences are observed in the migration behaviour of bovine aortic endothelial cells (ECs) as a function of location along the polymer stripe, and a 3-fold variation is achieved in EC migration speed and directional persistence time. Moreover, the device induces directional cell migration along the conducting polymer stripe. A gradient in adsorbed fibronectin indicates that a spatial variation in cell adhesion is at play. The ability to control cell migration behaviour using external electrical stimuli highlights the potential of using conducting polymers as “active” substrates for the non-invasive control of cell behaviour.

Matrix stiffening promotes a tumor vasculature phenotype

Significance Dysregulation of both vascular architecture and function is a hallmark of numerous diseases, including cancer. This dysregulation is currently largely attributed to up-regulated proangiogenic growth factors. Here, we show that the stiffness of the underlying extracellular matrix also plays a central role in promoting angiogenesis and a characteristic tumor-like vasculature both in vitro and in vivo. The matrix stiffness-mediated angiogenesis is dependent on increased matrix metalloprotease activity. In addition, increased matrix cross-linking disrupts endothelial cell–cell junctional integrity and results in leakier vasculature. These results suggest that altered tissue mechanics, which are characteristic of solid tumors, directly influence vascular phenotype and, subsequently, may impair therapeutic delivery and efficacy. Tumor microvasculature tends to be malformed, more permeable, and more tortuous than vessels in healthy tissue, effects that have been largely attributed to up-regulated VEGF expression. However, tumor tissue tends to stiffen during solid tumor progression, and tissue stiffness is known to alter cell behaviors including proliferation, migration, and cell–cell adhesion, which are all requisite for angiogenesis. Using in vitro, in vivo, and ex ovo models, we investigated the effects of matrix stiffness on vessel growth and integrity during angiogenesis. Our data indicate that angiogenic outgrowth, invasion, and neovessel branching increase with matrix cross-linking. These effects are caused by increased matrix stiffness independent of matrix density, because increased matrix density results in decreased angiogenesis. Notably, matrix stiffness up-regulates matrix metalloproteinase (MMP) activity, and inhibiting MMPs significantly reduces angiogenic outgrowth in stiffer cross-linked gels. To investigate the functional significance of altered endothelial cell behavior in response to matrix stiffness, we measured endothelial cell barrier function on substrates mimicking the stiffness of healthy and tumor tissue. Our data indicate that barrier function is impaired and the localization of vascular endothelial cadherin is altered as function of matrix stiffness. These results demonstrate that matrix stiffness, separately from matrix density, can alter vascular growth and integrity, mimicking the changes that exist in tumor vasculature. These data suggest that therapeutically targeting tumor stiffness or the endothelial cell response to tumor stiffening may help restore vessel structure, minimize metastasis, and aid in drug delivery.

Tissue stiffness regulates serine/arginine-rich protein-mediated splicing of the extra domain B-fibronectin isoform in tumors

Significance Alternative splicing is the main mechanism that drives protein diversity; however, little is known about the physiological cues that control splicing. Here, we show that the stiffness of the extracellular matrix mediates protein splicing in cells both in vitro and in vivo. Alternative splicing mediated by matrix stiffness occurs through the phosphorylation of splicing regulatory factors, serine/arginine rich (SR) proteins, and depends on the PI3K signaling pathway. Because the SR family of proteins are conserved among both vertebrates and invertebrates and are known to also be involved in genome stabilization, translation, and mRNA export, these results suggest a previously unidentified mechanism by which cells can respond and adapt to their mechanical microenvironment in both healthy and diseased states. Alternative splicing of proteins gives rise to different isoforms that play a crucial role in regulating several cellular processes. Notably, splicing profiles are altered in several cancer types, and these profiles are believed to be involved in driving the oncogenic process. Although the importance of alternative splicing alterations occurring during cancer is increasingly appreciated, the underlying regulatory mechanisms remain poorly understood. In this study, we use both biochemical and physical tools coupled with engineered models, patient samples, and a murine model to investigate the role of the mechanical properties of the tumor microenvironment in regulating the production of the extra domain-B (EDB) splice variant of fibronectin (FN), a hallmark of tumor angiogenesis. Specifically, we show that the amount of EDB-FN produced by endothelial cells increases with matrix stiffness both in vitro and within mouse mammary tumors. Matrix stiffness regulates splicing through the activation of serine/arginine rich (SR) proteins, the splicing factors involved in the production of FN isoforms. Activation of the SR proteins by matrix stiffness and the subsequent production of EDB-FN are dependent on intracellular contractility and PI3K-AKT signaling. Notably, matrix stiffness-mediated splicing is not limited to EDB-FN, but also affects splicing in the production of PKC βII and the VEGF 165b splice variant. Together, these results demonstrate that the mechanical properties of the microenvironment regulate alternative splicing and establish a previously unidentified mechanism by which cells can adapt to their microenvironment.

Fabrication of substrates with defined mechanical properties and topographical features for the study of cell migration.

Both substrate topography and substrate mechanical properties are known to influence cell behavior, but little is known about how they act in concert. Here, a method is presented to introduce topographical features into PA hydrogel substrates that span a wide range of physiological E values. Gel swelling plays a significant role in the fidelity of protruding micromolded features, with the most efficient pattern transfer occurring at a crosslinking concentration equal to or greater than ≈5%. In contrast, swelling does not influence the spacing fidelity of microcontact printed islands of collagen on 2D PA substrates. BAECs cultured on micromolded PA substrates exhibit contact guidance along ridges patterned for all E tested.