Extracellular vesicles (EVs) play important roles in tumor progression by altering immune surveillance, promoting vascular dysfunction, and priming distant sites for organotropic metastases. The miRNA expression patterns in circulating EVs are important diagnostic tools in cancer. However, multiple cell types within the tumor microenvironment (TME) including cancer cells and stromal cells (e.g. immune cells, fibroblasts, and endothelial cells, ECs) contribute to the pool of circulating EVs. Because EVs of different cellular origins have different functional properties, auditing the cargo derived from cell type-specific EVs in the TME is essential. Here, we demonstrate that a murine EC lineage-tracing model (Cdh5-CreERT2:ZSGreenl/s/l mice) can be used to isolate EC-derived extracellular vesicles (EC-EVs). We further show that purified ZSGreen+ EVs express expected EV markers, they are transferable to multiple recipient cells, and circulating EC-EVs from tumor-bearing mice harbor elevated levels of specific miRNAs (e.g. miR-30c, miR-126, miR-146a, and miR-125b) compared to non tumor-bearing counterparts. These results suggest that, in the tumor setting, ECs may systemically direct the function of heterotypic cell types either in the circulation or in different organ micro-environments via the cargo contained within their EVs.

hypertrophic (HCM) and dilated cardiomyopathy (DCM). In the current study we describe both computational and experimental investigations of 2 cardiac Troponin T (cTnT) mutations that occur in similar regions of the thin filament but result in divergent clinical phenotypes. Both experimental (Time resolved FRET (TR-FRET)) and computational results show that the D160E mutation repositions the flexible linker, moving it closer to the C-terminus of tropomyosin. This in turn alters weak electrostatic binding that decreases linker flexibility, ultimately leading to HCM. By contrast, Arg173Trp (R173W) also lies within the linker domain on cTnT but results in a dilated cardiomyopathy (DCM). Computation shows both average structure and time dependent dynamics are differentially modified in this mutational state. Specifically, the in silico average structure of the thin filament shows an increased distance between the linker region and the C-terminus of tropomyosin due to the R173W mutation. Differential scanning calorimetry on reconstituted thin filaments both with and without the R173W mutation suggest an impact on thermal stability and thus the thermodynamics of protein interaction. Our simulations and TR-FRET results illuminate the resultant perturbations to the WT structure and dynamics caused by the R173W mutation, and we contrast these effects to those found in the D160E mutation. Our findings provide a new framework for understanding the varying pathogenic mechanisms that drive clinical phenotypes in HCM and DCM in proximate thin filament mutations.

identity are plastic, and changes are found similar to those previously reported in human vein graft adaptation and animal models. These bioreactors adequately mimic the human arterial environment and enable testing of potential therapeutic agents directed against neointimal hyperplasia. Introduction and objectives:Understanding the processes that control vascular smooth muscle cell (VSMC) phenotypic modulation and determining which subpopulation of VSMCs migrate is critical for the design of treatment of intimal hyperplasia. Connective tissue growth factor (CTGF) has recently been reported to be upregulated in a variety of fibrotic disorders. We hypothesize that CTGF is a marker of a dedifferentiated VSMCs and therefore a target for treatment of intimal hyperplasia.Methods: Western blot analysis was used to determine protein expression. Bowden chambers were used for VSMC migration. Magnetic beads were labeled with CTGF antibody according to the manufacture's protocol. Porcine carotid arteries were microdissected into inner layer (intima/media) and outer layer (media/adventitia). VSMCs were transfected with myr-AKT1 using the nucleofection technique. Pig iliac arteries were injured and organ-cultured for 7 days.Results: Intimal hyperplastic VSMCs had higher baseline CTGF expression than normal VSMCs. VSMCs in the inner intimal/medial layer had increased differentiation markers. Outer medial/adventitial layer VSMCs had decreased contractile protein expression and increased CTGF expression. Balloon injury led to increased expression of CTGF in a subpopulation of VSMC as well as CTGF expression in cells within the intimal hyperplastic lesion. Intimal hyperplasia was recapitulated by addition of exogenous CTGF. High-expressing CTGF VSMCs (magnetic bead isolated) had decreased contractile protein expression and increased extracellular matrix expression. CTGF-positive VSMCs had 60% increased migration. Exogenous CTGF increased VSMC migration in a dose-dependent manner. Overexpression of myr-AKT1 increased CTGF and collagen IV expression in VSMCs.Conclusions: CTGF is an important regulator of VSMC migration and vascular remodeling through AKT1. CTGF is overexpressed in a subpopulation of VSMCs, which may be the promigratory cells that populate intimal hyperplasia lesions. These data will allow for more targeted therapies for intimal hyperplasia.