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BACKGROUND AND AIMS: Atherosclerosis preferentially develops in arterial branches and curvatures where vascular endothelium is exposed to disturbed flow. In this study, the effects of disturbed flow on the regulation of vascular endothelial phosphoproteins and their contribution to therapeutic application in atherogenesis were elucidated. METHODS: Porcine models, large-scale phosphoproteomics, transgenic mice, and clinical specimens were used to discover novel site-specific phosphorylation alterations induced by disturbed flow in endothelial cells (ECs). RESULTS: A large-scale phosphoproteomics analysis of native endothelium from disturbed (athero-susceptible) vs. pulsatile flow (athero-resistant) regions of porcine aortas led to the identification of a novel atherosclerosis-related phosphoprotein vinculin (VCL) with disturbed flow-induced phosphorylation at serine 721 (VCLS721p). The induction of VCLS721p was mediated by G-protein-coupled receptor kinase 2 (GRK2)S29p and resulted in an inactive form of VCL with a closed conformation, leading to the VE-cadherin/catenin complex disruption to enhance endothelial permeability and atherogenesis. The generation of novel apolipoprotein E-deficient (ApoE-/-) mice overexpressing S721-non-phosphorylatable VCL mutant in ECs confirmed the critical role of VCLS721p in promoting atherosclerosis. The administration of a GRK2 inhibitor to ApoE-/- mice suppressed plaque formation by inhibiting endothelial VCLS721p. Studies on clinical specimens from patients with coronary artery disease (CAD) revealed that endothelial VCLS721p is a critical clinicopathological biomarker for atherosclerosis progression and that serum VCLS721p level is a promising biomarker for CAD diagnosis. CONCLUSIONS: The findings of this study indicate that endothelial VCLS721p is a valuable hemodynamic-based target for clinical assessment and treatment of vascular disorders resulting from atherosclerosis.
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Aterosclerosis , Células Endoteliales , Vinculina , Animales , Ratones , Aterosclerosis/patología , Células Endoteliales/patología , Endotelio Vascular/patología , Ratones Noqueados para ApoE , Fosforilación , Porcinos , HumanosRESUMEN
OBJECTIVES: Endothelial progenitor cells (EPCs) circulate with increased numbers in the peripheral blood of patients with highly-vascularised hepatocellular carcinoma (HCC) and contribute to angiogenesis and neovascularisation. We hypothesised that angiogenic EPCs, that is, colony forming unit-endothelial cells (CFU-ECs), and outgrowth EPCs, that is, endothelial colony-forming cells, may exert paracrine effects on the behaviours and metastatic capacities of human hepatoma cells. DESIGN: Various molecular and functional approaches ranging from in vitro cell culture studies on molecular signalling to in vivo investigations on cell invasion and orthotropic transplantation models in mice and clinical specimens from patients with HCC were used. RESULTS: Monocyte chemotactic protein-1 (MCP-1) was identified as a critical mediator released from CFU-ECs to contribute to the chemotaxis of Huh7 and Hep3B cells by inducing their microRNA-21 (miR-21) biogenesis through the C-C chemokine receptor-2/c-Jun N-terminal kinase/activator protein-1 signalling cascade. CFU-EC-induction of miR-21 in these cells activated their Rac1 and matrix metallopeptidase-9 by silencing Rho GTPase-activating protein-24 and tissue inhibitor of metalloproteinase-3, respectively, leading to increased cell mobility. MCP-1-induction of miR-21 induced epithelial-mesenchymal transformation of Huh7 cells in vitro and their intrahepatic metastatic capability in vivo. Moreover, increased numbers of MCP-1(+) EPCs and their positive correlations with miR-21 induction and metastatic stages in human HCC were found. CONCLUSIONS: Our results provide new insights into the complexity of EPC-HCC interactions and indicate that anticancer therapies targeting either the MCP-1 released from angiogenic EPCs or the miR-21 biogenesis in HCC cells may prevent the malignant progression of primary tumours.
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Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/fisiopatología , Quimiocina CCL2/fisiología , Células Progenitoras Endoteliales/fisiología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/fisiopatología , MicroARNs/fisiología , Línea Celular Tumoral , Quimiotaxis/fisiología , Técnicas de Cocultivo , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Metaloproteinasa 9 de la Matriz/metabolismo , Proteína de Unión al GTP rac1/fisiologíaRESUMEN
Engineering vascularized tissues remains a promising approach for treating ischemic cardiovascular diseases. The availability of 3D-bioprinted vascular grafts that induce therapeutic angiogenesis can help avoid necrosis and excision of ischemic tissues. Here, using a combination of living cells and biodegradable hydrogels, we fabricated 3D-printed biocompatible proangiogenic patches from endothelial cell-laden photo-crosslinked gelatin (EC-PCG) bioink and smooth muscle cell-encapsulated polyurethane (SMC-PU) bioink. Implantation of 3D-bioprinted proangiogenic patches in a mouse model showed that EC-PCG served as an angiogenic capillary bed, whereas patterned SMC-PU increased the density of microvessels. Moreover, the assembled patterns between EC-PCG and SMC-PU induced the geometrically guided generation of microvessels with blood perfusion. In a rodent model of hindlimb ischemia, the vascular patches rescued blood flow to distal tissues, prevented toe/foot necrosis, promoted muscle remodeling, and increased the capillary density, thereby improving the heat-escape behavior of ischemic animals. Thus, our 3D-printed vascular cell-laden bioinks constitute efficient and scalable biomaterials that facilitate the engineering of vascular patches capable of directing therapeutic angiogenesis for treating ischemic vascular diseases.
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Gelatina , Hidrogeles , Isquemia , Neovascularización Fisiológica , Poliuretanos , Impresión Tridimensional , Animales , Gelatina/química , Poliuretanos/química , Hidrogeles/química , Isquemia/terapia , Neovascularización Fisiológica/efectos de los fármacos , Ratones , Humanos , Miocitos del Músculo Liso/citología , Reactivos de Enlaces Cruzados/química , Células Endoteliales de la Vena Umbilical Humana , Miembro Posterior/irrigación sanguínea , Miembro Posterior/patología , Masculino , Ingeniería de Tejidos/métodos , Bioimpresión/métodosRESUMEN
MicroRNAs (miRs) and bone morphogenetic protein receptor-specific Smads are mechano-responsive molecules that play vital roles in modulating endothelial cell (EC) functions in response to blood flow. However, the roles of interplay between these molecules in modulating EC functions under flows remain unclear. We elucidated the regulatory roles of the interplay between miR-487a and Smad5 in EC proliferation in response to different flow patterns. Microarray and quantitative RT-PCR showed that disturbed flow with low and oscillatory shear stress (OS, 0.5 ± 4 dynes/cm2) upregulates EC miR-487a in comparison to static controls and pulsatile shear stress (12 ± 4 dynes/cm2). MiR-487a expression was higher in ECs in the inner curvature (OS region) than the outer curvature of the rat aortic arch and thoracic aorta and also elevated in diseased human coronary arteries. MiR-487a expression was promoted by nuclear phospho-Smad5, which bound to primary-miR-487a to facilitate miR-487a processing. Algorithm prediction and luciferase reporter and argonaute 2-immunoprecipitation assays demonstrated that miR-487a binds to 3'UTR of CREB binding protein (CBP) and p53. Knockdown and overexpression of miR-487a decreased and increased, respectively, phospho-Rb and cyclin A expressions through CBP and p53. A BrdU incorporation assay showed that miR-487a enhanced EC proliferation under OS in vitro and in disturbed flow regions of experimentally stenosed rat abdominal aorta in vivo. These results demonstrate that disturbed flow with OS induces EC expression of miR-487a through its enhanced processing by activated-Smad5. MiR-487 inhibits its direct targets CBP and p53 to induce EC cycle progression and proliferation. Our findings suggest that EC miR-487 may serve as an important molecular target for intervention against disturbed flow-associated vascular disorders resulting from atherosclerosis.
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Endothelial progenitor cells (EPCs) participate in the neovascularization processes in the development of hepatocellular carcinoma (HCC). We investigated whether interactions between EPCs and HCC cells affect chemotactic and pro-inflammatory activities of EPCs. Two distinct phenotypes of circulating EPCs, i.e., myeloid-derived EPCs (colony forming unit-endothelial cells, CFU-ECs) and outgrowth EPCs (endothelial-colony forming cells, ECFCs), were co-cultured with Huh7 and Hep3B cells by using transwell chamber and IBIDI(TM) Culture-Inserts and µ-slide plates. Transwell and horizontal migration/invasion assays and time-lapse microscopy were used to monitor and analyze the migration and invasion of EPCs induced by these HCC cells. A human cytokine antibody array was used to compare protein expression profiles in EPCs and HCC cells. Flow cytometry and electromobility shift analysis were used to detect nuclear factor-κB (NF-κB)-DNA binding activity and pro-inflammatory adhesion molecule expression in EPCs. Ectopic full-length CC chemokine receptor 6 (CCR6) plasmid was used to transfect into ECFCs to investigate the role of CCR6 in HCC-induced EPC migration and invasion. The results show that co-culture with Huh7 and Hep3B cells induces the expression of endothelial cell (EC) markers KDR, Flt1, CD31 and VE-cadherin in CFU-ECs, but down-regulates the expressions of CD31 and VE-cadherin in ECFCs. These HCC cells induce migration and invasion of CFU-ECs, but not ECFCs, and do not affect the cell cycle distribution in these EPCs. Cytokine protein array identifies macrophage inflammatory protein-3α (MIP-3α) produced by HCC cells as a critical factor responsible for the HCC-induced chemotaxis of CFU-ECs, which highly express the specific MIP-3α counterreceptor CCR6. Overexpressing CCR6 in ECFCs significantly increases their chemotaxis in response to HCC cells. Co-culturing EPCs with HCC cells results in decreases in NF-κB binding activity and hence intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expressions in EPCs. Our results indicate that HCC cells exert differential effects on CFU-ECs and ECFCs, with increased chemotaxis for CFU-ECs, but not ECFCs. This HCC-induced chemotaxis of CFU-ECs is mediated by MIP-3α produced by HCC cells, which targets to CCR6 on CFU-ECs. Tumors may provide a humoral microenvironment to attenuate the pro-inflammatory activity of EPCs, which might be associated with the tumor escape mechanism.
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Carcinoma Hepatocelular/fisiopatología , Quimiotaxis , Neoplasias Hepáticas/fisiopatología , Células Madre/citología , Antígenos CD/metabolismo , Cadherinas/metabolismo , Movimiento Celular , Células Cultivadas , Quimiocina CCL20/metabolismo , Técnicas de Cocultivo , Citocinas/metabolismo , Humanos , FN-kappa B/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Análisis por Matrices de Proteínas , Receptores CCR6/genética , Receptores CCR6/metabolismo , Células Madre/metabolismoRESUMEN
AIMS: The implication of circulating haematopoietic CD34(+) progenitors in the vasculature is unclear due to the lack of understanding of their characteristics and plasticity mediated by their cellular microenvironment. We investigated how vascular smooth muscle cells (SMCs) and their interactions with endothelial cells (ECs) affect the behaviour and plasticity of CD34(+)CD31(+) progenitors and the underlying mechanisms. METHODS AND RESULTS: Human peripheral blood-derived CD34(+)CD31(+) cells were directly transplanted into injured arteries in vivo and co-cultured with ECs and SMCs in vitro. CD34(+)CD31(+) progenitors injected into wire-injured mouse arteries differentiate into ECs and macrophages in the neoendothelial layer and neointima, respectively. SMC-co-culture increases CD34(+)CD31(+) cell mobility and adhesion to and transmigration across ECs. Sorted CD34(+)CD31(+) progenitors that adhered to ECs co-cultured with SMCs have the capacity to form capillary-like structures in Matrigel and chimeric blood vessels in vivo. Sorted transmigrated progenitors give rise to macrophages with increased pro-angiogenic activity. These differentiations of CD34(+)CD31(+) progenitors into ECs and macrophages are mediated by ß(2)-integrin and Notch-1, respectively. ß(2)-Integrin and Notch-1 are activated by their counterligands, intercellular adhesion molecule-1 (ICAM-1) and jagged-1, which are highly expressed in the neoendothelium and neointima in injured arteries. Intra-arterial injection of ß(2)-integrin-activated CD34(+)CD31(+) progenitors into wire-injured mouse arteries inhibits neointima formation. CONCLUSION: Our findings indicate that the peripheral vascular niches composed of ECs and SMCs may predispose haematopoietic CD34(+)CD31(+) progenitors to differentiate into ECs and macrophages through the activations of the ICAM-1/ß(2)-integrin and jagged-1/Notch-1 cascades, respectively.