Differentiation and characterization of human iPSC-derived vascular endothelial cells under physiological shear stress.
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ABSTRACT:
SUBMITTER: Abutaleb NO
PROVIDER: S-EPMC7995664 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe endothelial cells that form the lumen of capillaries and microvessels are an important component of the blood-brain barrier. Cell phenotype is regulated by transducing a range of biomechanical and biochemical signals in the local microenvironment. Here we report on the role of shear stress in modulating the morphology, motility, proliferation, apoptosis, and protein and gene expression, of confluent monolayers of human brain microvascular endothelial cells derived from induced pluripotent stem cells.MethodsTo assess the response of derived human brain microvascular endothelial cells (dhBMECs) to shear stress, confluent monolayers were formed in a microfluidic device. Monolayers were subjected to a shear stress of 4 or 12 dyne cm-2 for 40 h. Static conditions were used as the control. Live cell imaging was used to assess cell morphology, cell speed, persistence, and the rates of proliferation and apoptosis as a function of time. In addition, immunofluorescence imaging and protein and gene expression analysis of key markers of the blood-brain barrier were performed.ResultsHuman brain microvascular endothelial cells exhibit a unique phenotype in response to shear stress compared to static conditions: (1) they do not elongate and align, (2) the rates of proliferation and apoptosis decrease significantly, (3) the mean displacement of individual cells within the monolayer over time is significantly decreased, (4) there is no cytoskeletal reorganization or formation of stress fibers within the cell, and (5) there is no change in expression levels of key blood-brain barrier markers.ConclusionsThe characteristic response of dhBMECs to shear stress is significantly different from human and animal-derived endothelial cells from other tissues, suggesting that this unique phenotype that may be important in maintenance of the blood-brain barrier. The implications of this work are that: (1) in confluent monolayers of dhBMECs, tight junctions are formed under static conditions, (2) the formation of tight junctions decreases cell motility and prevents any morphological transitions, (3) flow serves to increase the contact area between cells, resulting in very low cell displacement in the monolayer, (4) since tight junctions are already formed under static conditions, increasing the contact area between cells does not cause upregulation in protein and gene expression of BBB markers, and (5) the increase in contact area induced by flow makes barrier function more robust.
| S-EPMC5543552 | biostudies-literature
Project description:Objective: The vulnerability of atherosclerotic plaques is among the leading cause of ischemic stroke. High wall shear stress (WSS) promotes the instability of atherosclerotic plaques by directly imparting mechanical stimuli, but the specific mechanisms remain unclear. We speculate that modulation of mechanosensitive genes may play a vital role in accelerating the development of plaques. The purpose of this study was to find mechanosensitive genes in vascular endothelial cells (ECs) through combining microarray data with bioinformatics technology and further explore the underlying dynamics-related mechanisms that cause the progression and destabilization of atherosclerotic plaques. Methods: Microarray data sets for human vascular ECs under high and normal WSS were retrieved from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified through the R language. The performance of enrichment analysis and protein-protein interaction (PPI) network presented the biological function and signaling pathways of the DEGs. Hub genes were identified based on the PPI network and validated by GEO data sets. Predicted transcription factor (TF) genes and miRNAs interaction with potential mechanosensitive genes were identified by NetworkAnalyst. Results: A total of 260 DEGs, 121 upregulated and 139 downregulated genes, were screened between high and normal WSS from GSE23289. A total of 10 hub genes and four cluster modules were filtered out based on the PPI network. The enrichment analysis showed that the biological functions of the hub genes were mainly involved in responses to unfolded protein and topologically incorrect protein, and t to endoplasmic reticulum stress. The significant pathways associated with the hub genes were those of protein processing in the endoplasmic reticulum, antigen processing, and presentation. Three out of the 10 hub genes, namely, activated transcription factor 3 (ATF3), heat shock protein family A (Hsp70) member 6 (HSPA6), and dual specificity phosphatase 1 (DUSP1, also known as CL100, HVH1, MKP-1, PTPN10), were verified in GSE13712. The expression of DUSP1 was higher in the senescent cell under high WSS than that of the young cell. The TF-miRNA-mechanosensitive gene coregulatory network was constructed. Conclusion: In this work, we identified three hub genes, ATF3, HSPA6, and DUSP1, as the potential mechanosensitive genes in the human blood vessels. DUSP1 was confirmed to be associated with the senescence of vascular ECs. Therefore, these three mechanosensitive genes may have emerged as potential novel targets for the prediction and prevention of ischemic stroke. Furthermore, the TF-miRNA-mechanosensitive genes coregulatory network reveals an underlying regulatory mechanism and the pathways to control disease progression.
| S-EPMC8787366 | biostudies-literature
Project description:Extracellular vesicles (EVs) are increasingly used as delivery vehicles for drugs and bioactive molecules, which usually require intravascular administration. The endothelial cells covering the inner surface of blood vessels are susceptible to the shear stress of blood flow. Few studies demonstrate the interplay of red blood cell-derived EVs (RBCEVs) and endothelial cells. Thus, the phagocytosis of EVs by vascular endothelial cells during blood flow needs to be elucidated. In this study, red blood cell-derived extracellular vesicles (RBCEVs) were constructed to investigate endothelial cell phagocytosis in vitro and animal models. Results showed that low magnitude shear stress including low shear stress (LSS) and oscillatory shear stress (OSS) could promote the uptake of RBCEVs by endothelial cells in vitro. In addition, in zebrafish and mouse models, RBCEVs tend to be internalized by endothelial cells under LSS or OSS. Moreover, RBCEVs are easily engulfed by endothelial cells in atherosclerotic plaques exposed to LSS or OSS. In terms of mechanism, oxidative stress induced by LSS is part of the reason for the increased uptake of endothelial cells. Overall, this study shows that vascular endothelial cells can easily engulf EVs in areas of low magnitude shear stress, which will provide a theoretical basis for the development and utilization of EVs-based nano-drug delivery systems in vivo. Graphical abstract Image 1 Highlights • We recently reported that endothelial cells were amateur phagocytic cells for RBCEVs engulfment.• Low magnitude shear stress (LSS and OSS) can increase the uptake of RBCEVs by endothelial cells in vitro and in vivo.• ROS induced by low magnitude shear stress acts as an accelerator to enhance endothelial cells uptake of RBCEVs.
| S-EPMC8586717 | biostudies-literature
Project description:BackgroundEndothelial cells play a pivotal role in cardiovascular physiology and pathology by providing a barrier to the bloodstream. In the current study, we investigated the phenotype and barrier function of endothelial cells in response to shear stress under pro-atherogenic conditions.MethodsEndothelial cells were exposed to laminar shear stress (LSS) in a parallel-plate flow chamber containing oxidized low-density lipoprotein (oxLDL) in the perfusion solution, or remained static. We quantified the response of endothelial monolayers to LSS and oxLDL in terms of cell viability, barrier integrity, vascular endothelial cadherin (VE-cadherin) availability, focal adhesion (FA) remodeling, and monocyte-endothelial interactions.ResultsOur results showed that oxLDL stimulation and static conditions synergized to enhance endothelial barrier disruption. Under the same oxLDL challenge, the application of 25 dynes/cm2 LSS on the endothelial monolayer decreased the passage of fluorescein isothiocyanate (FITC)-dextran by 37.79%, increased transendothelial electrical resistance (TEER) by 24.97% compared with static cells (P<0.05), which was accompanied by reduced intercellular gap formation, relatively solid cell-substrate adhesion. Compared with static cells, endothelial cells exposed to both laminar flow and oxLDL had less small FAs, less monocyte adhesion and transmigration, and alleviated overexpression of VCAM-1 and MCP-1. Meanwhile, the oxLDL-induced internalization of VE-cadherin and vinculin were also attenuated by laminar flow, and this change was more pronounced at LSS of 25 dynes/cm2 than 5 dynes/cm2.ConclusionsStatic conditions favor, whereas physiologically higher levels of LSS ameliorate endothelial barrier disruption under pro-atherogenic stress, which is related to the improved availability of VE-cadherin and vinculin on the cell surface.
| S-EPMC7723531 | biostudies-literature
Project description:Bacterial dissemination via the cardiovascular system is the most common cause of infection mortality. A key step in dissemination is bacterial interaction with endothelia lining blood vessels, which is physically challenging because of the shear stress generated by blood flow. Association of host cells such as leukocytes and platelets with endothelia under vascular shear stress requires mechanically specialized interaction mechanisms, including force-strengthened catch bonds. However, the biomechanical mechanisms supporting vascular interactions of most bacterial pathogens are undefined. Fibronectin (Fn), a ubiquitous host molecule targeted by many pathogens, promotes vascular interactions of the Lyme disease spirochete Borrelia burgdorferi Here, we investigated how B. burgdorferi exploits Fn to interact with endothelia under physiological shear stress, using recently developed live cell imaging and particle-tracking methods for studying bacterial-endothelial interaction biomechanics. We found that B. burgdorferi does not primarily target insoluble matrix Fn deposited on endothelial surfaces but, instead, recruits and induces polymerization of soluble plasma Fn (pFn), an abundant protein in blood plasma that is normally soluble and nonadhesive. Under physiological shear stress, caps of polymerized pFn at bacterial poles formed part of mechanically loaded adhesion complexes, and pFn strengthened and stabilized interactions by a catch-bond mechanism. These results show that B. burgdorferi can transform a ubiquitous but normally nonadhesive blood constituent to increase the efficiency, strength, and stability of bacterial interactions with vascular surfaces. Similar mechanisms may promote dissemination of other Fn-binding pathogens.
| S-EPMC5410840 | biostudies-other
Project description:The role of nanotopographical extracellular matrix (ECM) cues in vascular endothelial cell (EC) organization and function is not well-understood, despite the composition of nano- to microscale fibrillar ECMs within blood vessels. Instead, the predominant modulator of EC organization and function is traditionally thought to be hemodynamic shear stress, in which uniform shear stress induces parallel-alignment of ECs with anti-inflammatory function, whereas disturbed flow induces a disorganized configuration with pro-inflammatory function. Since shear stress acts on ECs by applying a mechanical force concomitant with inducing spatial patterning of the cells, we sought to decouple the effects of shear stress using parallel-aligned nanofibrillar collagen films that induce parallel EC alignment prior to stimulation with disturbed flow resulting from spatial wall shear stress gradients. Using real time live-cell imaging, we tracked the alignment, migration trajectories, proliferation, and anti-inflammatory behavior of ECs when they were cultured on parallel-aligned or randomly oriented nanofibrillar films. Intriguingly, ECs cultured on aligned nanofibrillar films remained well-aligned and migrated predominantly along the direction of aligned nanofibrils, despite exposure to shear stress orthogonal to the direction of the aligned nanofibrils. Furthermore, in stark contrast to ECs cultured on randomly oriented films, ECs on aligned nanofibrillar films exposed to disturbed flow had significantly reduced inflammation and proliferation, while maintaining intact intercellular junctions. This work reveals fundamental insights into the importance of nanoscale ECM interactions in the maintenance of endothelial function. Importantly, it provides new insight into how ECs respond to opposing cues derived from nanotopography and mechanical shear force and has strong implications in the design of polymeric conduits and bioengineered tissues.
| S-EPMC4758680 | biostudies-literature
Project description:We identified primary cilia and centrosomes in cultured human umbilical vein endothelial cells (HUVEC) by antibodies to acetyl-alpha-tubulin and capillary morphogenesis gene-1 product (CMG-1), a human homologue of the intraflagellar transport (IFT) protein IFT-71 in Chlamydomonas. CMG-1 was present in particles along primary cilia of HUVEC at interphase and around the oldest basal body/centriole at interphase and mitosis. To study the response of primary cilia and centrosomes to mechanical stimuli, we exposed cultured HUVEC to laminar shear stress (LSS). Under LSS, all primary cilia disassembled, and centrosomes were deprived of CMG-1. We conclude that the exposure to LSS ends the IFT in cultured endothelial cells.
| S-EPMC2172280 | biostudies-literature
Project description:The current work reports the functional characterization of human induced pluripotent stem cells (iPSCs)- arterial and venous-like endothelial cells (ECs), derived in chemically defined conditions, either in monoculture or seeded in a scaffold with mechanical properties similar to blood vessels. iPSC-derived arterial- and venous-like endothelial cells were obtained in two steps: differentiation of iPSCs into endothelial precursor cells (CD31pos/KDRpos/VE-Cadmed/EphB2neg/COUP-TFneg) followed by their differentiation into arterial and venous-like ECs using a high and low vascular endothelial growth factor (VEGF) concentration. Cells were characterized at gene, protein and functional levels. Functionally, both arterial and venous-like iPSC-derived ECs responded to vasoactive agonists such as thrombin and prostaglandin E2 (PGE2), similar to somatic ECs; however, arterial-like iPSC-derived ECs produced higher nitric oxide (NO) and elongation to shear stress than venous-like iPSC-derived ECs. Both cells adhered, proliferated and prevented platelet activation when seeded in poly(caprolactone) scaffolds. Interestingly, both iPSC-derived ECs cultured in monoculture or in a scaffold showed a different inflammatory profile than somatic ECs. Although both somatic and iPSC-derived ECs responded to tumor necrosis factor-α (TNF-α) by an increase in the expression of intercellular adhesion molecule 1 (ICAM-1), only somatic ECs showed an upregulation in the expression of E-selectin or vascular cell adhesion molecule 1 (VCAM-1).
| S-EPMC6405900 | biostudies-other
Project description:Quiescence is commonly used to describe the inactive state of endothelial cells (ECs) in monolayers that have reached homeostasis. Experimentally quiescence is usually described in terms of the relative change in cell activity (e.g. turnover, speed, etc.) in response to a perturbation (e.g. solute, shear stress, etc.). The objective of this study is to provide new insight into EC quiescence by quantitatively defining the morphology and activity of confluent cell monolayers in response to shear stress and vascular modulators. Confluent monolayers of human umbilical vein ECs (HUVECs) were subjected to a range of shear stresses (4-16 dyne cm-2) under steady flow. Using phase contrast, time-lapse microscopy and image analysis, we quantified EC morphology, speed, proliferation, and apoptosis rates over time and detected differences in monolayer responses under various media conditions: basal media supplemented with growth factors, interleukin-8, or cyclic AMP. In all conditions, we observed a transition from cobblestone to spindle-like morphology in a dose-dependent manner due to shear stress. Cyclic AMP enhanced the elongation and alignment of HUVECs due to shear stress and reduced steady state cell speed. We observed the lowest proliferation rates below 8 dyne cm-2 and found that growth factors and cyclic AMP reduced proliferation and apoptosis; interleukin-8 similarly decreased proliferation, but increased apoptosis. We have quantified the response of ECs in confluent monolayers to shear stress and vascular modulators in terms of morphology, speed, proliferation and apoptosis and have established quantifiable metrics of cell activity to define vascular quiescence under shear stress.
| S-EPMC5490251 | biostudies-literature
Project description:Hypertension is a primary risk factor for cardiovascular diseases including myocardial infarction and stroke. Major determinants of blood pressure are vasodilatory factors such as nitric oxide (NO) released from the endothelium under the influence of fluid shear stress exerted by the flowing blood. Several endothelial signaling processes mediating fluid shear stress-induced formation and release of vasodilatory factors have been described. It is, however, still poorly understood how fluid shear stress induces these endothelial responses. Here we show that the endothelial mechanosensitive cation channel PIEZO1 mediated fluid shear stress-induced release of adrenomedullin, which in turn activated its Gs-coupled receptor. The subsequent increase in cAMP levels promoted the phosphorylation of endothelial NO synthase (eNOS) at serine 633 through protein kinase A (PKA), leading to the activation of the enzyme. This Gs/PKA-mediated pathway synergized with the AKT-mediated pathways leading to eNOS phosphorylation at serine 1177. Mice with endothelium-specific deficiency of adrenomedullin, the adrenomedullin receptor, or Gαs showed reduced flow-induced eNOS activation and vasodilation and developed hypertension. Our data identify fluid shear stress-induced PIEZO1 activation as a central regulator of endothelial adrenomedullin release and establish the adrenomedullin receptor and subsequent Gs-mediated formation of cAMP as a critical endothelial mechanosignaling pathway regulating basal endothelial NO formation, vascular tone, and blood pressure.
| S-EPMC6597232 | biostudies-literature