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.

Despite significant progress over the past few decades, creating a tissue-engineered vascular graft with replicated functions of native blood vessels remains a challenge due to the mismatch in mechanical properties, low biological function, and rapid occlusion caused by restenosis of small diameter vessel grafts (<6 mm diameter). A scaffold with similar mechanical properties and biocompatibility to the host tissue is ideally needed for the attachment and proliferation of cells to support the building of engineered tissue. In this study, pectin hydrogel nanofiber scaffolds with two different oxidation degrees (25 and 50%) were prepared by a multistep methodology including periodate oxidation, electrospinning, and adipic acid dihydrazide crosslinking. Scanning electron microscopy (SEM) images showed that the obtained pectin nanofiber mats have a nano-sized fibrous structure with 300–400 nm fiber diameter. Physicochemical property testing using Fourier transform infrared (FTIR) spectra, atomic force microscopy (AFM) nanoindentations, and contact angle measurements demonstrated that the stiffness and hydrophobicity of the fiber mat could be manipulated by adjusting the oxidation and crosslinking levels of the pectin hydrogels. Live/Dead staining showed high viability of the mesenchymal stem cells (MSCs) cultured on the pectin hydrogel fiber scaffold for 14 days. In addition, the potential application of pectin hydrogel nanofiber scaffolds of different stiffness in stem cell differentiation into vascular cells was assessed by gene expression analysis. Real-time polymerase chain reaction (RT-PCR) results showed that the stiffer scaffold facilitated the differentiation of MSCs into vascular smooth muscle cells, while the softer fiber mat promoted MSC differentiation into endothelial cells. Altogether, our results indicate that the pectin hydrogel nanofibers have the capability of providing mechanical cues that induce MSC differentiation into vascular cells and can be potentially applied in stem cell-based tissue engineering.

Key points This study demonstrates and evaluates the changes in rat vascular smooth muscle cell biomechanics following statin‐mediated cholesterol depletion. Evidence is presented to show correlated changes in migration and adhesion of vascular smooth muscle cells to extracellular matrix proteins fibronectin and collagen. Concurrently, integrin α5 expression was enhanced but not integrin α2. Atomic force microscopy analysis provides compelling evidence of coordinated reduction in vascular smooth muscle cell stiffness and actin cytoskeletal orientation in response to statin‐mediated cholesterol depletion. Proof is provided that statin‐mediated cholesterol depletion remodels total vascular smooth muscle cell cytoskeletal orientation that may additionally participate in altering ex vivo aortic vessel function. It is concluded that statin‐mediated cholesterol depletion may coordinate vascular smooth muscle cell migration and adhesion to different extracellular matrix proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell. Abstract Not only does cholesterol induce an inflammatory response and deposits in foam cells at the atherosclerotic plaque, it also regulates cellular mechanics, proliferation and migration in atherosclerosis progression. Statins are HMG‐CoA reductase inhibitors that are known to inhibit cellular cholesterol biosynthesis and are clinically prescribed to patients with hypercholesterolemia or related cardiovascular conditions. Nonetheless, the effect of statin‐mediated cholesterol management on cellular biomechanics is not fully understood. In this study, we aimed to assess the effect of fluvastatin‐mediated cholesterol management on primary rat vascular smooth muscle cell (VSMC) biomechanics. Real‐time measurement of cell adhesion, stiffness, and imaging were performed using atomic force microscopy (AFM). Cellular migration on extra cellular matrix (ECM) protein surfaces was studied by time‐lapse imaging. The effect of changes in VSMC biomechanics on aortic function was assessed using an ex vivo myograph system. Fluvastatin‐mediated cholesterol depletion (‐27.8%) lowered VSMC migration distance on a fibronectin (FN)‐coated surface (‐14.8%) but not on a type 1 collagen (COL1)‐coated surface. VSMC adhesion force to FN (+33%) and integrin α5 expression were enhanced but COL1 adhesion and integrin α2 expression were unchanged upon cholesterol depletion. In addition, VSMC stiffness (‐46.6%) and ex vivo aortic ring contraction force (‐40.1%) were lowered and VSMC actin cytoskeletal orientation was reduced (‐24.5%) following statin‐mediated cholesterol depletion. Altogether, it is concluded that statin‐mediated cholesterol depletion may coordinate VSMC migration and adhesion to different ECM proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell and aortic function.

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Stem cell differentiation on a decellularized native blood vessel scaffold under mechanical stimulation for vascular tissue engineering.