Patients with densely innervated tumors suffer with increased metastasis and decreased survival as compared to those with less innervated tumors. We hypothesize that in some tumors, nerves are acquired by a tumor-induced process, called axonogenesis. Here, we use PC12 cells as an in vitro neuronal model, human tumor samples and murine in vivo models to test this hypothesis. When appropriately stimulated, PC12 cells extend processes, called neurites. We show that patient tumors release vesicles, called exosomes, which induce PC12 neurite outgrowth. Using a cancer mouse model, we show that tumors compromised in exosome release are less innervated than controls. Moreover, in vivo pharmacological blockade of exosome release similarly attenuates tumor innervation. We characterize these nerves as sensory in nature and demonstrate that axonogenesis is potentiated by the exosome-packaged axonal guidance molecule, EphrinB1. These findings indicate that tumor released exosomes induce tumor innervation and exosomes containing EphrinB1 potentiate this activity.
Aims Cholesterol not only deposits in foam cells at the atherosclerotic plaque, but also plays an important role as a regulator of cell migration in atherogenesis. In addition, the progression of atherosclerosis leads to arterial wall stiffening, and thus altering the micromechanical environment of vascular smooth muscle cells (VSMCs) in vivo. Our studies aim to test the hypothesis that membrane cholesterol and substrate stiffness co-ordinate to regulate VSMCs biomechanics, and thus potentially regulate VSMCs migration and atherosclerotic plaque formation. Methods and results Methyl-β-cyclodextrin was used to manipulate membrane cholesterol content in VSMCs isolated from the descending thoracic aorta of male Sprague-Dawley rats and cultured on Type I collagen-coated polyacrylamide gel substrates with varying stiffness. Atomic force microscopy (AFM) was used to determine VSMCs stiffness and integrin-fibronectin (FN) adhesion. The alignment of submembranous actin filaments was visualized with AFM and confocal microscopy. The constriction force of rat aorta was measured ex vivo using a multi-wire myograph system. Our results demonstrated that cholesterol-depletion and substrate-softening induced a significant decrease in VSMCs stiffness and adhesion to FN, as well as cytoskeletal disorganization. In addition, the contractile force of rat aorta was reduced upon cholesterol-depletion. Cholesterol-enrichment resulted in an increase in stiffness, adhesion to FN, cytoskeletal organization of VSMCs compared with the cholesterol-depleted cells, and enhanced contractile force of rat aortas compared with the cholesterol-depleted vessel rings. Conclusion Cell membrane cholesterol and substrate stiffness synergistically affect VSMCs elastic modulus (E-modulus) by regulating the organization of the actin cytoskeleton. Except for the 3.5 kPa gel substrate, cholesterol-depletion decreased VSMCs-FN adhesion force, adhesion loading rate, cytoskeletal orientation, and E-modulus compared with the control VSMCs. Conversely, cholesterol-enrichment significantly increased cytoskeleton orientation, stiffness, and VSMCs-FN cell adhesion force compared with both control and cholesterol-depleted VSMCs on a soft substrate.
Due to the prevalence of cardiovascular diseases, there is a large need for small diameter vascular grafts that cannot be fulfilled using autologous vessels. Although medium to large diameter synthetic vessels are in use, no suitable small diameter vascular graft has been developed due to the unique dynamic environment that exists in small vessels. To achieve long term patency, a successful tissue engineered vascular graft would need to closely match the mechanical properties of native tissue, be non-thrombotic and non-immunogenic, and elicit the proper healing response and undergo remodeling to incorporate into the native vasculature. Electrospinning presents a promising approach to the development of a suitable tissue engineered vascular graft. This review provides a comprehensive overview of the different polymers, techniques, and functionalization approaches that have been used to develop an electrospun tissue engineered vascular graft.
Vascular smooth muscle cell (VSMC) migration is a critical step in the progression of cardiovascular disease and aging. Migrating VSMCs encounter a highly heterogeneous environment with the varying extracellular matrix (ECM) composition due to the differential synthesis of collagen and fibronectin (FN) in different regions and greatly changing stiffness, ranging from the soft necrotic core of plaques to hard calcifications within blood vessel walls. In this study, we demonstrate an application of a two-dimensional (2D) model consisting of an elastically tunable polyacrylamide gel of varying stiffness and ECM protein coating to study VSMC migration. This model mimics the in vivo microenvironment that VSMCs experience within a blood vessel wall, which may help identify potential therapeutic targets for the treatment of atherosclerosis. We found that substrate stiffness had differential effects on VSMC migration on type 1 collagen (COL1) and FN-coated substrates. VSMCs on COL1-coated substrates showed significantly diminished migration distance on stiffer substrates, while on FN-coated substrates VSMCs had significantly increased migration distance. In addition, cortical stress fiber orientation increased in VSMCs cultured on more rigid COL1-coated substrates, while decreasing on stiffer FN-coated substrates. On both proteins, a more disorganized cytoskeletal architecture was associated with faster migration. Overall, these results demonstrate that different ECM proteins can cause substrate stiffness to have differential effects on VSMC migration in the progression of cardiovascular diseases and aging.
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