Browsing by Subject "endothelial cells"
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Item Differential HDAC6 Activity Modulates Ciliogenesis and Subsequent Mechanosensing of Endothelial Cells Derived from Pluripotent Stem Cells(Elsevier, 2018-07-24) Smith, Quinton; Macklin, Bria; Chan, Xin Yi; Jones, Hannah; Trempel, Michelle; Yoder, Mervin C.; Gerecht, Sharon; Pediatrics, School of MedicineSummary The role of primary cilia in mechanosensation is essential in endothelial cell (EC) shear responsiveness. Here, we find that venous, capillary, and progenitor ECs respond to shear stress in vitro in a cilia-dependent manner. We then demonstrate that primary cilia assembly in human induced pluripotent stem cell (hiPSC)-derived ECs varies between different cell lines with marginal influence of differentiation protocol. hiPSC-derived ECs lacking cilia do not align to shear stress, lack stress fiber assembly, have uncoordinated migration during wound closure in vitro, and have aberrant calcium influx upon shear exposure. Transcriptional analysis reveals variation in regulatory genes involved in ciliogenesis among different hiPSC-derived ECs. Moreover, inhibition of histone deacetylase 6 (HDAC6) activity in hiPSC-ECs lacking cilia rescues cilia formation and restores mechanical sensing. Taken together, these results show the importance of primary cilia in hiPSC-EC mechano-responsiveness and its modulation through HDAC6 activity varies among hiPSC-ECs.Item Differentiation, Evaluation, and Application of Human Induced Pluripotent Stem Cell–Derived Endothelial Cells(AHA, 2017-11) Lin, Yang; Gil, Chang-Hyun; Yoder, Mervin C.; Pediatrics, School of MedicineThe emergence of induced pluripotent stem cell (iPSC) technology paves the way to generate large numbers of patient-specific endothelial cells (ECs) that can be potentially delivered for regenerative medicine in patients with cardiovascular disease. In the last decade, numerous protocols that differentiate EC from iPSC have been developed by many groups. In this review, we will discuss several common strategies that have been optimized for human iPSC-EC differentiation and subsequent studies that have evaluated the potential of human iPSC-EC as a cell therapy or as a tool in disease modeling. In addition, we will emphasize the importance of using in vivo vessel-forming ability and in vitro clonogenic colony–forming potential as a gold standard with which to evaluate the quality of human iPSC-EC derived from various protocols.Item The Effects of High Fat Diet, Bone Healing, and BMP-2 Treatment on Endothelial Cell Growth and Function(Elsevier, 2021-05) Bhatti, Fazal Ur Rehman; Dadwal, Ushashi C.; Valuch, Conner R.; Tewari, Nikhil P.; Awosanya, Olatundun D.; Staut, Caio de Andrade; Sun, Seungyup; Mendenhall, Stephen K.; Perugini, Anthony J., III; Nagaraj, Rohit U.; Battini, Hanisha L.; Nazzal, Murad K.; Blosser, Rachel J.; Maupin, Kevin A.; Childress, Paul J.; Li, Jiliang; Kacena, Melissa A.; Orthopaedic Surgery, School of MedicineAngiogenesis is a vital process during the regeneration of bone tissue. The aim of this study was to investigate angiogenesis at the fracture site as well as at distal locations from obesity-induced type 2 diabetic mice that were treated with bone morphogenetic protein-2 (BMP-2, local administration at the time of surgery) to heal a femoral critical sized defect (CSD) or saline as a control. Mice were fed a high fat diet (HFD) to induce a type 2 diabetic-like phenotype while low fat diet (LFD) animals served as controls. Endothelial cells (ECs) were isolated from the lungs (LECs) and bone marrow (BMECs) 3 weeks post-surgery, and the fractured femurs were also examined. Our studies demonstrate that local administration of BMP-2 at the fracture site in a CSD model results in complete bone healing within 3 weeks for all HFD mice and 66.7% of LFD mice, whereas those treated with saline remain unhealed. At the fracture site, vessel parameters and adipocyte numbers were significantly increased in BMP-2 treated femurs, irrespective of diet. At distal sites, LEC and BMEC proliferation was not altered by diet or BMP-2 treatment. HFD increased the tube formation ability of both LECs and BMECs. Interestingly, BMP-2 treatment at the time of surgery reduced tube formation in LECs and humeri BMECs. However, migration of BMECs from HFD mice treated with BMP-2 was increased compared to BMECs from HFD mice treated with saline. BMP-2 treatment significantly increased the expression of CD31, FLT-1, and ANGPT2 in LECs and BMECs in LFD mice, but reduced the expression of these same genes in HFD mice. To date, this is the first study that depicts the systemic influence of fracture surgery and local BMP-2 treatment on the proliferation and angiogenic potential of ECs derived from the bone marrow and lungs.Item Endothelial actin depolymerization mediates NADPH oxidase-superoxide production during flow reversal(American Physiological Society (APS), 2014-01-01) Choy, Jenny S.; Lu, Xiao; Yang, Junrong; Zhang, Zhen-Du; Kassab, Ghassan S.; Department of Biomedical Engineering, Purdue School of Engineering and Technology, IUPUISlow moving blood flow and changes in flow direction, e.g., negative wall shear stress, can cause increased superoxide (O2·−) production in vascular endothelial cells. The mechanism by which shear stress increases O2·− production, however, is not well established. We tested the hypothesis that actin depolymerization, which occurs during flow reversal, mediates O2·− production in vascular endothelial cells via NADPH oxidase, and more specifically, the subunit p47phox. Using a swine model, we created complete blood flow reversal in one carotid artery, while the contralateral vessel maintained forward blood flow as control. We measured actin depolymerization, NADPH oxidase activity, and reactive oxygen species (ROS) production in the presence of various inhibitors. Flow reversal was found to induce actin depolymerization and a 3.9 ± 1.0-fold increase in ROS production as compared with forward flow. NADPH oxidase activity was 1.4 ± 0.2 times higher in vessel segments subjected to reversed blood flow when measured by a direct enzyme assay. The NADPH oxidase subunits gp91phox (Nox2) and p47phox content in the vessels remained unchanged after 4 h of flow reversal. In contrast, p47phox phosphorylation was increased in vessels with reversed flow. The response caused by reversed flow was reduced by in vivo treatment with jasplakinolide, an actin stabilizer (only a 1.7 ± 0.3-fold increase). Apocynin (an antioxidant) prevented reversed flow-induced ROS production when the animals were treated in vivo. Cytochalasin D mimicked actin depolymerization in vitro and caused a 5.2 ± 3.0-fold increase in ROS production. These findings suggest that actin filaments play an important role in negative shear stress-induced ROS production by potentiating NADPH oxidase activity, and more specifically, the p47phox subunit in vascular endothelium.Item Exploration of Endothelial Cell Invasion and Responses to Nicotine and Arginine by Streptococcus Mutans Serotype K Strains in a Sucrose-Induced Biofilm Lifestyle(2019-08) Wagenknecht, Dawn R.; Gregory, Richard L.; Windsor, L. Jack; Galli, Dominique M.; Lee, Chao-Hung; Ji, Julie; Childers, Noel K.Streptococcus mutans, an inhabitant of oral biofilm or dental plaque, adheres to the tooth surface via protein antigen I/II (PA I/II). Pathologic lesions of atherosclerosis (AT) and infective endocarditis (IE) harbor S. mutans. Serotypes f and k strains with collagen binding protein genes cbm and cnm are uncommon in the mouth, but these are the most prevalent S. mutans strains in AT and IE tissues and can invade endothelial cells (EC) in vitro. Tobacco use increases the risk for cardiovascular and oral diseases. Oral S. mutans encounter many substances including nicotine. Arginine is present in saliva and the EC glycocalyx that coats and protects ECs from shear forces of blood flow. Prior studies demonstrated arginine alters S. mutans biofilm. This work characterizes S. mutans serotype k strains and serotype c strains, the most prevalent in the mouth. The effects of nicotine and arginine on biofilm mass, metabolic activity and EC invasion were investigated. Biofilm production by serotypes c and k strains did not differ; there were no differences in responses to nicotine and arginine between these serotypes. Increased production of biofilm was associated with the cbm and cnm genes. Nicotine increased biofilm for all strains whereas arginine plus nicotine reduced bacteria and the extracellular polymeric substances. Previous EC invasion studies were performed with planktonic cultures of S. mutans; therefore, EC invasion by biofilm was evaluated. Significant factors for EC invasion by S. mutans are presence of the cbm gene and lack of PA I/II expression on the bacterial cell surface. Presence of the cnm gene increased EC invasion by biofilm but not planktonic cells. Planktonic cells of six strains invaded better than biofilm, whereas four strains showed increased invasion by biofilm cells. Neither nicotine nor arginine significantly altered the ability of S. mutans biofilm cells to invade ECs. Not all strains with cbm or cnm and no PA I/II expression invaded EC. A strain with PA I/II expression and without cbm and cnm genes invaded EC. While cbm, cnm and PA I/II expression are predictors of EC invasion, additional mechanisms for EC invasion by S. mutans remain to be revealed.Item Labeling of endothelial cells with magnetic microbeads by angiophagy(Springer, 2018-08) Thomas, Jessica; Jones, Desiree; Moldovan, Leni; Anghelina, Mirela; Gooch, Keith J.; Moldovan, Nicanor I.; Ophthalmology, School of MedicineObjectives Attachment of magnetic particles to cells is needed for a variety of applications but is not always possible or efficient. Simpler and more convenient methods are thus desirable. In this study, we tested the hypothesis that endothelial cells (EC) can be loaded with micron-size magnetic beads by the phagocytosis-like mechanism ‘angiophagy’. To this end, human umbilical vein EC (HUVEC) were incubated with magnetic beads conjugated or not (control) with an anti-VEGF receptor 2 antibody, either in suspension, or in culture followed by re-suspension using trypsinization. Results In all conditions tested, HUVEC incubation with beads induced their uptake by angiophagy, which was confirmed by (i) increased cell granularity assessed by flow cytometry, and (ii) the presence of an F-actin rich layer around many of the intracellular beads, visualized by confocal microscopy. For confluent cultures, the average number of beads per cell was 4.4 and 4.2, with and without the presence of the anti-VEGFR2 antibody, respectively. However, while the actively dividing cells took up 2.9 unconjugated beads on average, this number increased to 5.2 if binding was mediated by the antibody. Magnetic pulldown increased the cell density of beads-loaded cells in porous electrospun poly-capro-lactone scaffolds by a factor of 4.5 after 5 min, as compared to gravitational settling (p < 0.0001). Conclusion We demonstrated that EC can be readily loaded by angiophagy with micron-sized beads while attached in monolayer culture, then dispersed in single-cell suspensions for pulldown in porous scaffolds and for other applications.Item Mechanisms of Attachment of Tobacco-Treated Streptococcus mutans to Human Endothelial Cells(Office of the Vice Chancellor for Research, 2013-04-05) Miller, Alyssa R.Smoking has been proven to cause increased dental caries, which is an infectious disease caused by Streptococcus mutans, a gram-positive bacteria commonly found in the oral cavity. S. mutans is also known for its contribution to atherosclerosis, specifically the accumulation of plaque in the coronary arteries. This is facilitated by the interaction and binding of S. mutans to local endothelial cells (HUVEC). This study was conducted to explore the direct effects that tobacco has on the ability of S. mutans to affect endothelial cells that might lead to atherosclerosis. S. mutans were treated with different concentrations of nicotine and cigarette smoke condensate (CSC) to test if they affect the binding capabilities of S. mutans to endothelial cells. Blocking reagents, enolase antibody and purified DnaK, were also used to treat the HUVEC to observe the effects these reagents have on the ability of S. mutans to bind to the cells. Binding was measured by preforming a binding assay that incorporated these reagents and reading the absorbance using a spectrophotometer at 450 nm. To do this, sonicated HUVEC were added to a 96-well microtiter plate with 1% bovine serum albumin (BSA), followed by treated S. mutans, extra-avidin labeled horseradish peroxidase, and O-phenylenediamine (OPD). The experiment is still in progress; therefore, no results have been obtained thus far. However, it is expected that the nicotine/CSC treatment of S. mutans will increase binding to the endothelial cells thereby providing a possible mechanism of S. mutans contributing to atherosclerosis. The knowledge obtained from this experiment will be significant in developing treatment modalities to decrease the effects of smoking on cardiovascular disease.Item Mechano-sensitivity of nuclear lamin proteins in endothelial cells(2016-07-22) Jiang, Yizhi; Ji, Julie Ying Hui; Na, Sungsoo; Wallace, JosephAtherosclerosis is a chronic disease that happens mostly in aged people, and recently studies have showed many similarities between Hutchinson Gilford Progeria Syndrome (HGPS) cells and aging cells, implicating dysfunctions of lamin A/C in aging process and atherosclerosis, as HGPS is caused by a mutated form of lamin A/C. Blood flow in arteries is generating shear stress that is mostly applied on endothelial cells that align along inner blood vessel wall. At the same time, endothelial cells are also under stretch by periodic arterial pulses. Considering the fact that atherosclerosis is prone to developing at arterial branches with disturbed shear and increased stretch, it is highly possible that laminar flow and proper stretch force are regulating endothelium to function appropriately. In this thesis, the investigation of what effects laminar flow or cyclic stretch can bring to endothelial cells was conducted, and examination of lamin A/C expression under mechanical forces were elaborated and incorporated with cell senescence. Results showed that laminar shear stress and stretch force can regulate lamin A/C expression in different patterns, which were impaired in senescent cells.Item Mitochondrial Heme Synthesis Enzymes as Therapeutic Targets in Vascular Diseases(Frontiers, 2020-07-15) Shetty, Trupti; Corson, Timothy W.; Ophthalmology, School of MedicineItem Nicotine Effects Surface Bound Enolase on Streptococcus mutans and Its Binding to Human Plasminogen(Office of the Vice Chancellor for Research, 2013-04-05) Walters, Kamilah; Gregory, Richard L.Streptococcus mutans is the major bacterial agent responsible for dental caries. Previous research has shown that smokers have increased caries and that nicotine increases biofilm formation of S. mutans. S. mutans is also associated with atherosclerosis, another disease commonly found in smokers. However, little research has been done to investigate the direct effect of nicotine on the ability of S. mutans to bind to endothelial cells and lead to atherosclerosis. The two objectives of this study were to determine how nicotine affects the level of enolase, a glycolytic enzyme, on the surface of S. mutans, and next to determine its effect on binding of treated bacteria to human plasminogen, a protein present in the bloodstream. S. mutans strain UA159 was grown overnight in tryptic soy broth treated with 0, 0.5, 1, and 2 mg/mL nicotine at 37◦C in 5% CO2. These cells were used to coat a microtiter plate, and various levels of surface bound enolase and binding to plasminogen were determined using enzyme-linked immunosorbent assays (ELISA). A preliminary trial showed increase in both surface bound enolase and binding to plasminogen with increasing nicotine concentration. Similar results are to be expected with repetition of this procedure, indicating that nicotine up-regulates the bacterial expression of enolase and its binding to plasminogen, probably through plasminogen binding receptors, contributing to the virulence of S. mutans. Knowledge of the attachment mechanisms of S. mutans in the presence of tobacco may aid in prevention of tobacco-related atherosclerosis.