Biomechanical effects of flow and coculture on human aortic and cord blood-derived endothelial cells.
ABSTRACT: Human endothelial cells derived from umbilical cord blood (hCB-ECs) represent a promising cell source for endothelialization of tissue engineered blood vessels. hCB-ECs cultured directly above human aortic smooth muscle cells (SMCs), which model native and tissue engineered blood vessels, produce a confluent endothelium that responds to flow like normal human aortic endothelial cells (HAECs). The objective of this study was to quantify the elastic modulus of hCB-ECs cocultured with SMCs under static and flow conditions using atomic force microscopy (AFM). Cytoskeleton structures were assessed by AFM cell surface imaging and immunofluorescence of F-actin. The elastic moduli of hCB-ECs and HAECs were similar and significantly smaller than the value for SMCs in monoculture under static conditions (p<0.05). In coculture, hCB-ECs and HAECs became significantly stiffer with moduli 160-180% larger than their corresponding values in monoculture. While the moduli of hCB-ECs and HAECs almost doubled in monoculture and flow condition, their corresponding values in coculture declined after exposure to flow. Both the number and diameter of cortical stress fiber per cell width increased in coculture and/or flow conditions, whereas the subcortical stress fiber density throughout the cell interior increased by a smaller amount. These findings indicate that changes to biomechanical properties in coculture and/or exposure to flow are correlated with changes in the cortical stress fiber density. For ECs, fluid shear stress appeared to have greater effect on the elastic modulus than the presence of SMCs and changes to the elastic modulus in coculture may be due to EC-SMC communication.
Project description:Coculture of endothelial cells (ECs) and smooth muscle cells (SMCs) in vitro can yield confluent monolayers or EC networks. Factors influencing this transition are not known. In this study, we examined whether the spatial arrangement of EC-SMC cocultures affected EC migration, network morphology, and angiogenic protein secretion. Human umbilical cord blood-derived ECs (hCB-ECs) were grown in coculture with human aortic SMCs in either a mixed or lamellar spatial geometry and analyzed over a culture period of 12 days. The hCB-ECs cultured on SMCs in a mixed system had higher cell speeds, shorter persistence times, and lower random motility coefficients than ECs in a lamellar system. By day 12 of coculture, mixed systems demonstrated greater anastomoses and capillary loop formation than lamellar systems as evidenced by a higher number of branch points, angle of curvature between branch points, and percentage of imaged area covered by networks. The network morphology was more uniform in the mixed systems than the lamellar systems with fewer EC clusters present after several days in culture. Proliferation of hCB-ECs was higher for mixed cocultures during the first 24?h of coculture, and then declined dramatically suggesting that proliferation only contributed to network formation during the early stages of coculture. Proteome assay results show reduced solution levels, but no change in intracellular levels of angiogenic proteins in lamellar systems compared to mixed systems. These data suggest that mixing ECs and SMCs together favors the formation of EC networks to a greater extent than a lamellar arrangement in which ECs form a cell layer above a confluent, quiescent layer of SMCs.
Project description:Atherosclerosis develops at regions of the arterial tree exposed to disturbed flow. The early stage of atherogenesis involves the adhesion of leukocytes (white blood cells [WBCs]) to and their transmigration across endothelial cells (ECs), which are located in close proximity to smooth muscle cells (SMCs). We investigated the effects of EC/SMC coculture and disturbed flow on the adhesion and transmigration of 3 types of WBCs (neutrophils, peripheral blood lymphocytes [PBLs], and monocytes) using our vertical-step flow (VSF) chamber, in which ECs were cocultured with SMCs in collagen gels. Such coculture significantly increased the adhesion and transmigration of neutrophils, PBLs, and monocytes under VSF, particularly in the reattachment area, where the rolling velocity of WBCs and their transmigration time were decreased, as compared with the other areas. Neutrophils, PBLs, and monocytes showed different subendothelial migration patterns under VSF. Their movements were more random and shorter in distance in the reattachment area. Coculture of ECs and SMCs induced their expressions of adhesion molecules and chemokines, which contributed to the increased WBC adhesion and transmigration. Our findings provide insights into the mechanisms of WBC interaction with the vessel wall (composed of ECs and SMCs) under the complex flow environments found in regions of prevalence for atherogenesis.
Project description:RATIONALE:Endothelial microRNA-126 (miR-126) modulates vascular development and angiogenesis. However, its role in the regulation of smooth muscle cell (SMC) function is unknown. OBJECTIVE:To elucidate the role of miR-126 secreted by endothelial cells (ECs) in regulating SMC turnover in vitro and in vivo, as well as the effects of shear stress on the regulation. METHODS AND RESULTS:Coculture of SMCs with ECs or treatment of SMCs with conditioned media from static EC monoculture (EC-CM) increased SMC miR-126 level and SMC turnover; these effects were abolished by inhibition of endothelial miR-126 and by the application of laminar shear stress to ECs. SMC miR-126 did not increase when treated with EC-CM from ECs subjected to inhibition of miR biogenesis, or with CM from sheared ECs. Depletion of extracellular/secreted vesicles in EC-CM did not affect the increase of SMC miR-126 by EC-CM. Biotinylated miR-126 or FLAG (DYKDDDDK epitope)-tagged Argonaute2 transfected into ECs was detected in the cocultured or EC-CM-treated SMCs, indicating a direct EC-to-SMC transmission of miR-126 and Argonaute2. Endothelial miR-126 represses forkhead box O3, B-cell lymphoma 2, and insulin receptor substrate 1 mRNAs in the cocultured SMCs, suggesting the functional roles of the transmitted miR-126. Systemic depletion of miR-126 in mice inhibited neointimal lesion formation of carotid arteries induced by cessation of blood flow. Administration of EC-CM or miR-126 mitigated the inhibitory effect. CONCLUSIONS:Endothelial miR-126 acts as a key intercellular mediator to increase SMC turnover, and its release is reduced by atheroprotective laminar shear stress.
Project description:Transplantation of ex vivo expanded human umbilical cord blood cells (hCB) only partially enhances the hematopoietic recovery after myelosuppressive therapy. Incubation of hCB with optimal combinations of cytokines and niche cells, such as endothelial cells (ECs), could augment the efficiency of hCB expansion. We have devised an approach to cultivate primary human ECs (hECs) in serum-free culture conditions. We demonstrate that coculture of CD34(+) hCB in direct cellular contact with hECs and minimal concentrations of thrombopoietin/Kit-ligand/Flt3-ligand resulted in a 400-fold expansion of total hematopoietic cells, 150-fold expansion of CD45(+)CD34(+) progenitor cells, and 23-fold expansion of CD45(+) Lin(-)CD34(hi+)CD45RA(-)CD49f(+) stem and progenitor cells over a 12-day period. Compared with cytokines alone, coculture of hCB with hECs permitted greater expansion of cells capable of multilineage engraftment and serial transplantation, hallmarks of long-term repopulating hematopoietic stem cells. Therefore, hECs establish a cellular platform for expansion of hematopoietic stem and progenitor cells and treatment of hematologic disorders.
Project description:RATIONALE:Elastin is an important ECM (extracellular matrix) protein in large and small arteries. Vascular smooth muscle cells (SMCs) produce the layered elastic laminae found in elastic arteries but synthesize little elastin in muscular arteries. However, muscular arteries have a well-defined internal elastic lamina (IEL) that separates endothelial cells (ECs) from SMCs. The extent to which ECs contribute elastin to the IEL is unknown. OBJECTIVE:To use targeted elastin (Eln) deletion in mice to explore the relative contributions of SMCs and ECs to elastic laminae formation in different arteries. METHODS AND RESULTS:We used SMC- and EC-specific Cre recombinase transgenes with a novel floxed Eln allele to focus gene inactivation in mice. Inactivation of Eln in SMCs using Sm22aCre resulted in depletion of elastic laminae in the arterial wall with the exception of the IEL and SMC clusters in the outer media near the adventitia. Inactivation of elastin in ECs using Tie2Cre or Cdh5Cre resulted in normal medial elastin and a typical IEL in elastic arteries. In contrast, the IEL was absent or severely disrupted in muscular arteries. Interruptions in the IEL resulted in neointimal formation in the ascending aorta but not in muscular arteries. CONCLUSIONS:Combined with lineage-specific fate mapping systems, our knockout results document an unexpected heterogeneity in vascular cells that produce the elastic laminae. SMCs and ECs can independently form an IEL in most elastic arteries, whereas ECs are the major source of elastin for the IEL in muscular and resistance arteries. Neointimal formation at IEL disruptions in the ascending aorta confirms that the IEL is a critical physical barrier between SMCs and ECs in the large elastic arteries. Our studies provide new information about how SMCs and ECs contribute elastin to the arterial wall and how local elastic laminae defects may contribute to cardiovascular disease.
Project description:Direct ultrasonic imaging of arterial and venous thrombi could aid in diagnosis and treatment planning by providing rapid and cost-effective measurements of thrombus volume and elastic modulus. Toward this end, it was demonstrated that open-air magnetomotive ultrasound (MMUS) provides specific contrast to superparamagnetic iron oxide-labeled model thrombi embedded in gelatin-based blood vessel-mimicking flow phantoms. MMUS was performed on model thrombi in the presence of pulsatile flow that mimics cardiac-induced motion found in real vasculature. The MMUS signal and contrast-to-noise ratio (CNR) were measured across a range of physiologically relevant thrombus volumes and elastic moduli. Model thrombus volumes as small as 0.5 ml were shown to be detectable (CNR > 1) over the entire range of elastic moduli tested (3.5-40 kPa). It was also found that MMUS signal and CNR are increased with increasing thrombus volume ( ) and decreasing elastic modulus ( ), while variations in pulsatile flow rate had little effect. These findings demonstrate that MMUS has promise as a direct in vivo thrombosis imaging modality for quantifying thrombus volume and stiffness.
Project description:Small-diameter synthetic vascular grafts have high failure rate and tissue-engineered blood vessels are limited by the scalability. Here we engineered bioactive materials for in situ vascular tissue engineering, which recruits two types of endogenous progenitor cells for the regeneration of blood vessels. Heparin was conjugated to microfibrous vascular grafts to suppress thrombogenic responses, and stromal cell-derived factor-1? (SDF-1?) was immobilized onto heparin to recruit endogenous progenitor cells. Heparin-bound SDF-1? was more stable than adsorbed SDF-1? under both static and flow conditions. Microfibrous grafts were implanted in rats by anastomosis to test the functional performance. Heparin coating improved the short-term patency, and immobilized SDF-1? further improved the long-term patency. SDF-1? effectively recruited endothelial progenitor cells (EPCs) to the luminal surface of the grafts, which differentiated into endothelial cells (ECs) and accelerated endothelialization. More interestingly, SDF-1? increased the recruitment of smooth muscle progenitor cells (SMPCs) to the grafts, and SMPCs differentiated into smooth muscle cells (SMCs) in vivo and in vitro. Consistently, SDF-1?-immobilized grafts had significantly higher elastic modulus. This work demonstrates the feasibility of simultaneously recruiting progenitor cells of ECs and SMCs for in situ blood vessel regeneration. This in situ tissue engineering approach will have broad applications in regenerative medicine.
Project description:To accelerate vein graft reendothelialization and reduce vein graft thrombosis by infusing human umbilical cord blood-derived endothelial cells (hCB-ECs) because loss of endothelium contributes to vein graft thrombosis and neointimal hyperplasia.Under steady flow conditions in vitro, hCB-ECs adhered to smooth muscle cells 2.5 to 13 times more than ECs derived from peripheral blood or human aorta (P<0.05). Compared with peripheral blood and human aorta ECs, hCB-ECs had 1.4-fold more cell surface ?(5)?(1) integrin heterodimers per cell (P<0.05) and proliferated on fibronectin 4- to 10-fold more rapidly (P<0.05). Therefore, we used hCB-ECs to enhance reendothelialization of carotid interposition vein grafts implanted in NOD.CB17-Prkdc(scid)/J mice. Two weeks postoperatively, vein grafts from hCB-EC-treated mice demonstrated approximately 55% reendothelialization and no luminal thrombosis. In contrast, vein grafts from sham-treated mice demonstrated luminal thrombosis in 75% of specimens (P<0.05) and only approximately 14% reendothelialization. In vein grafts from hCB-EC-treated mice, 33±10% of the endothelium was of human origin, as judged by human major histocompatibility class I expression.The hCB-ECs adhere to smooth muscle cells under flow conditions in vitro, accelerate vein graft reendothelialization in vivo, and prevent vein graft thrombosis. Thus, hCB-ECs offer novel therapeutic possibilities for vein graft disease.
Project description:Vascular endothelial cells (ECs) at arterial branches and curvatures experience disturbed blood flow and induce a quiescent-to-activated phenotypic transition of the adjacent smooth muscle cells (SMCs) and a subsequent smooth muscle hyperplasia. However, the mechanism underlying the flow pattern-specific initiation of EC-to-SMC signaling remains elusive. Our previous study demonstrated that endothelial microRNA-126-3p (miR-126-3p) acts as a key intercellular molecule to increase turnover of the recipient SMCs, and that its release is reduced by atheroprotective laminar shear (12 dynes/cm2) to ECs. Here we provide evidence that atherogenic oscillatory shear (0.5 ± 4 dynes/cm2), but not atheroprotective pulsatile shear (12 ± 4 dynes/cm2), increases the endothelial secretion of nonmembrane-bound miR-126-3p and other microRNAs (miRNAs) via the activation of SNAREs, vesicle-associated membrane protein 3 (VAMP3) and synaptosomal-associated protein 23 (SNAP23). Knockdown of VAMP3 and SNAP23 reduces endothelial secretion of miR-126-3p and miR-200a-3p, as well as the proliferation, migration, and suppression of contractile markers in SMCs caused by EC-coculture. Pharmacological intervention of mammalian target of rapamycin complex 1 in ECs blocks endothelial secretion and EC-to-SMC transfer of miR-126-3p through transcriptional inhibition of VAMP3 and SNAP23. Systemic inhibition of VAMP3 and SNAP23 by rapamycin or periadventitial application of the endocytosis inhibitor dynasore ameliorates the disturbed flow-induced neointimal formation, whereas intraluminal overexpression of SNAP23 aggravates it. Our findings demonstrate the flow-pattern-specificity of SNARE activation and its contribution to the miRNA-mediated EC-SMC communication.
Project description:The most promising alternatives to heart transplantation are left ventricular assist devices and artificial hearts; however, their use has been limited by thrombotic complications. To reduce these, sintered titanium (Ti) surfaces were developed, but thrombosis still occurs in approximately 7.5% of patients. We have invented a rapid-seeding technology to minimize the risk of thrombosis by rapid endothelialization of sintered Ti with human cord blood-derived endothelial cells (hCB-ECs). Human cord blood-derived endothelial cells were seeded within minutes onto sintered Ti and exposed to thrombosis-prone low fluid flow shear stresses. The hCB-ECs adhered and formed a confluent endothelial monolayer on sintered Ti. The exposure of sintered Ti to 4.4 dynes/cm for 20?hr immediately after rapid seeding resulted in approximately 70% cell adherence. The cell adherence was not significantly increased by additional ex vivo static culture of rapid-seeded sintered Ti before flow exposure. In addition, adherent hCB-ECs remained functional on sintered Ti, as indicated by flow-induced increase in nitric oxide secretion and reduction in platelet adhesion. After 15 day ex vivo static culture, the adherent hCB-ECs remained metabolically active, expressed endothelial cell functional marker thrombomodulin, and reduced platelet adhesion. In conclusion, our results demonstrate the feasibility of rapid-seeding sintered Ti with blood-derived hCB-ECs to generate a living antithrombotic surface.