Confluent Endothelial Cell Monolayers Research Articles

Electrospun polyhedral oligomeric silsequioxane-poly(carbonate-urea) urethane for fabrication of hemocompatible small-diameter vascular grafts with angiogenesis capacity

The clinical utility of small-diameter vascular grafts (SDVGs) is limited due to the possibility of thrombosis and intimal hyperplasia. These features can delay the development of a functional endothelial cell (EC) monolayer on the luminal surface of grafts. Therefore, the development and fabrication of vascular grafts (VGs) with comparable extracellular matrix (ECM) functions are mandatory to elicit hemocompatible confluent EC monolayers, and angiogenesis behavior inside the body. To promote the interactions between ECs and the surface of electrospun polyacrylic acid-grafted polyhedral oligomeric silsesquioxane-poly(carbonate-urea)-urethane (PAAc-POSS-PCUU), in this research, the surface of nanofibers was modified by covalently immobilizing extracted soluble proteins from aorta (ESPA) using EDC/NHS chemistry. The ATR-FTIR spectroscopy, WCA, and SEM microscopy confirmed the binding of acrylic acid and soluble vascular proteins on the surface of electrospun fibers. The PAAc-POSS-PCUU nanofibers and engineered biomimetic Pro-PAAc-POSS-PCUU nanofibers exhibited excellent biocompatibility indicated by increased survival rate (p < 0.05). Western blotting revealed the increase of VE-cadherin, Tie-2, vWF, and VEGFR-2 in HUVECs after being plated on PAAc-POSS-PCUU and Pro-PAAc-POSS-PCUU scaffolds, indicating appropriate angiogenesis behavior (p < 0.05). Besides, the antioxidant capacity was induced by the increase of SOD and GPx activity (p < 0.05). Additionally, blood compatibility tests revealed that Pro-PAAc-POSS-PCUU nanofibers accelerate the formation of a single EC layer without hemolysis and platelet adhesion. Taken together, Pro-PAAc-POSS-PCUU nanofibers exhibited excellent blood compatibility, and angiogenesis behavior, making them a promising candidate for clinical applications.

Anti-PF4/Heparin Monoclonal Antibodies Induce Thrombus Formation in an Ex Vivo Endothelialized Microfluidic System

Introduction: Heparin-induced thrombocytopenia (HIT) is a serious adverse reaction to heparin. Antibodies against platelet factor 4 (PF4) or a heparin/PF4 complex have been reported to activate platelets, neutrophils and possibly endothelial cells, resulting in thrombocytopenia, hypercoagulability and thromboembolic complications. While the impact of anti-PF4/heparin antibodies on platelet, neutrophil and monocyte activation has been extensively studied, the role of endothelial cells in HIT-associated thrombosis remains underexplored. In particular, the interactions between HIT-induced procoagulancy and endothelial cells under dynamic conditions is not completely analyzed. In this report, we investigated how HIT antibodies induce thrombosis in an endothelialized microfluidic system. Methods: Fibrinogen microfluidic channels were coated with confluent monolayer of human umbilical vein endothelial cells (HUVECs). Cells were primed with low-dose TNF-α to induce a sub-thrombotic endothelial activation, prior to perfusion of whole blood. Recalcified unstimulated or Thrombin Receptor Activator Peptide 6 (TRAP-6) activated whole blood was perfused at a venous flow rate. HIT-thrombosis model was established and tested with monoclonal anti-PF4/heparin antibodies in whole blood. In brief, whole blood was pre-incubated with unfractionated heparin (UFH, 0.2 IU/mL and 100 IU/mL). Next, anti-PF4/heparin antibodies were introduced to the whole blood mixture and incubated for 30 minutes. Whole blood was recalcified and perfused over resting or primed endothelial cells at a venous shear stress. Thrombus formation was recorded over time. Results: The endothelialized microfluidic model successfully captures sub-thrombotic conditions under venous shear. Activated platelets induced a procoagulant shift and three-dimensional thrombi. We applied our thrombosis model to investigate thrombus formation induced by a HIT mimicking monoclonal antibody. We show that the heparin-dependent platelet activation predicts the thrombotic response in the microfluidic system: HIT antibodies in absence of heparin did not exert a pro-thrombotic effect on activated endothelial cells. Co-incubation with 0.2 IU/mL UFH induced thrombosis, embolization of thrombus material and channel occlusion only when endothelial cells were primed with TNF-α. The pro-thrombotic effect of HIT antibodies was fully reversed at the super-therapeutic dose of 100 IU/mL UFH. Conclusions: HIT antibodies induce thrombosis and embolization in a system emulating physiological environment. For the first time, we present a comprehensive thrombosis model that incorporates the established thrombogenicity of HIT-mimicking antibodies. We show that our thrombosis model incorporates both endothelial- and blood-based control of thrombosis. Primed endothelial cells and antibody-induced procoagulancy trigger thrombosis in a concerted fashion, allowing investigation of the underlying mechanisms and possible preclinical evaluation of potential inhibitors of HIT-thrombosis. Figure 1, upper panel: Maximal thrombus surface area coverage of TNF-α primed endothelial cells, perfused with unstimulated, TRAP-6 activated or HIT-like IgG/heparin challenged whole blood. HIT-like IgG did not increase thrombus formation in absence of heparin. Addition of low-dose heparin exerted a strong pro-thrombotic effect, that was fully reversible with a super-therapeutic concentration of heparin. Lower panel: representative images of thrombi formed by HIT-like IgG in absence and under low or high-dose heparin. Platelets: green, Calcein-AM. Scale bar: 200 µm.

Biohybrid lung Development: Towards Complete Endothelialization of an Assembled Extracorporeal Membrane Oxygenator.

Towards the establishment of a long-term lung-assist device to be used both as a bridge and as an alternative to lung transplantation according to final destination therapy, we develop the biohybrid lung (BHL) on the technical basis of contemporary extracorporeal membrane oxygenation (ECMO). Here, to overcome the significant drawbacks of ECMO, in particular the missing hemocompatibility of the artificial surfaces, all blood-contacting areas need to be endothelialized sufficiently. In continuation of our recent accomplishments, demonstrating the feasibility of establishing a physiological acting endothelial cell (EC) monolayer on the hollow fiber membranes (HFMs) of the ECMO in vitro, the next step towards BHL translation is the endothelialization of the complete oxygenator, consisting of HFMs and the surrounding housing. Therefore, we assessed EC seeding inside our model oxygenator (MOx), which simulated the conditions in the assembled HFM oxygenators in order to identify the most important factors influencing efficient endothelialization, such as cell seeding density, cell distribution, incubation time and culture medium consumption. Overall, upon adjusting the concentration of infused ECs to 15.2 × 104/cm2 and ensuring optimal dispersion of cells in the MOx, viable and confluent EC monolayers formed on all relevant surfaces within 24 h, even though they comprised different polymers, i.e., the fibronectin-coated HFMs and the polysulfone MOx housing. Periodic medium change ensured monolayer survival and negligible apoptosis rates comparable to the reference within the assembled system. By means of these results, revealing essential implications for BHL development, their clinical translation is coming one step closer to reality.

Poly(2-Methoxyethyl Acrylate) (PMEA)-Coated Anti-Platelet Adhesive Surfaces to Mimic Native Blood Vessels through HUVECs Attachment, Migration, and Monolayer Formation

Confluent monolayers of human umbilical vein endothelial cells (HUVECs) on a poly(2-methoxyethyl acrylate) (PMEA) antithrombogenic surface play a major role in mimicking the inner surface of native blood vessels. In this study, we extensively investigated the behavior of cell–polymer and cell–cell interactions by measuring adhesion strength using single-cell force spectroscopy. In addition, the attachment and migration of HUVECs on PMEA-analogous substrates were detected, and the migration rate was estimated. Moreover, the bilateral migration of HUVECs between two adjacent surfaces was observed. Furthermore, the outer surface of HUVEC was examined using frequency-modulation atomic force microscopy (FM-AFM). Hydration was found to be an indication of a healthy glycocalyx layer. The results were compared with the hydration states of individual PMEA-analogous polymers to understand the adhesion mechanism between the cells and substrates in the interface region. HUVECs could attach and spread on the PMEA surface with stronger adhesion strength than self-adhesion strength, and migration occurred over the surface of analogue polymers. We confirmed that platelets could not adhere to HUVEC monolayers cultured on the PMEA surface. FM-AFM images revealed a hydration layer on the HUVEC surfaces, indicating the presence of components of the glycocalyx layer in the presence of intermediate water. Our findings show that PMEA can mimic original blood vessels through an antithrombogenic HUVEC monolayer and is thus suitable for the construction of artificial small-diameter blood vessels.

Proteomic Analysis of Control and Disrupted Endothelial Monolayers

The microvascular wall regulates normal blood‐tissue exchange, but under inflammatory conditions, elevated endothelial permeability leads to edema, poor tissue oxygenation, and disrupted tissue homeostasis. Much remains unknown about the mechanisms involved. Using an unbiased proteomic analysis of confluent human umbilical vein endothelial cell (HUVEC) monolayers that were stimulated with 0.1 U/mL thrombin, which produces a time‐dependent increase in permeability that peaks 30 min and recovers to baseline levels at 120 min, novel candidate proteins that may participate in this mechanism were identified. Cell lysates were collected, proteins digested, and the peptides were characterized using a Thermo Q‐exactive‐HF mass spectrometer coupled to a Thermo Easy nLC 1200 to get label‐free quantitation (LFQ) of relative protein expression. High‐resolution mass spectrometric data were analyzed on the MaxQuant processing suite, and the protein groups file subsequently analyzed with the Perseus processing suite. The analysis revealed identified 4627 unique proteins present in HUVEC. Ratios of the LFQ intensities of 30 min. thrombin vs. control and 120 min. thrombin vs. control were generated. Statistical significance (p &lt; 0.05) for 30 min. thrombin vs. control or 120 min. thrombin vs. control was determined using Welch’s t‐test of log(2)‐transformed LFQ intensities. In addition, a z‐score cutoff calculated as [(t test difference of individual protein) − (median t test difference of dataset)] / (standard deviation of t test difference of dataset) was used. The criteria of |z‐score| &gt; 1 and p &lt; 0.05 with Welch’s t test were utilized to filter the dataset, identifying 214 proteins to have altered expression after 30 min thrombin treatment, and 172 proteins having altered expression after 120 min thrombin. When entering the filtered datasets into String‐db, tight junction signaling was identified as enriched with thrombin treatment. Employing a more stringent filtering approach to identify candidate proteins for further study (|z‐score| &gt; 2), we found that the tight junction protein claudin‐11 (CLDN11) had significantly decreased expression after 30 min thrombin (73% decrease from control) but was not significant changed at 120 min. Expression of CLDN11 was confirmed to be decreased by thrombin at 30 min using SimpleWestern (WES) analysis. To determine whether experimental depletion of CLDN11 expression decreases endothelial barrier function, selective CLDN11 siRNA was used. However, evaluation of barrier function using electric cell‐substrate impedance sensing showed no significant changes for up to 48 h after transfection with the siRNA, when CLDN11 protein levels were significantly decreased. Immunofluorescence labeling of CLDN11 in confluent HUVEC revealed that while some CLDN11 could be found in junctions, most labeling was punctate in the cytoplasm, possibly residing in vesicles. Collectively, the results suggest that thrombin significantly alters endothelial tight junctions and reduces CLDN11 expression. However, selective reduction of CLDN11 levels alone does not appear to be sufficient to alter HUVEC monolayer barrier function, and this may be because most CLDN11 fails to localize in junctions in this cultured endothelial cell model. Future studies will be directed at evaluating CLDN11 localization in postcapillary venules and determining its potential role in microvascular barrier function using intact microvessels and in vivo.

ArhGEF12 activates Rap1A and not RhoA in human dermal microvascular endothelial cells to reduce tumor necrosis factor-induced leak.

Overwhelming inflammation in the setting of acute critical illness induces capillary leak resulting in hypovolemia, edema, tissue dysoxia, organ failure and even death. The tight junction (TJ)-dependent capillary barrier is regulated by small GTPases, but the specific regulatory molecules most active in this vascular segment under such circumstances are not well described. We set out to identify GTPase regulatory molecules specific to endothelial cells (EC) that form TJs. Transcriptional profiling of confluent monolayers of TJ-forming human dermal microvascular ECs (HDMECs) and adherens junction only forming-human umbilical vein EC (HUVECs) demonstrate ARHGEF12 is basally expressed at higher levels and is only downregulated in HDMECs by junction-disrupting tumor necrosis factor (TNF). HDMECs depleted of ArhGEF12 by siRNA demonstrate a significantly exacerbated TNF-induced decrease in trans-endothelial electrical resistance and disruption of TJ continuous staining. ArhGEF12 is established as a RhoA-GEF in HUVECs and its knock down would be expected to reduce RhoA activity and barrier disruption. Pulldown of active GEFs from HDMECs depleted of ArhGEF12 and treated with TNF show decreased GTP-bound Rap1A after four hours but increased GTP-bound RhoA after 12h. In cell-free assays, ArhGEF12 immunoprecipitated from HDMECs is able to activate both Rap1A and RhoA, but not act on Rap2A-C, RhoB-C, or even Rap1B which shares 95% sequence identity with Rap1A. We conclude that in TJ-forming HDMECs, ArhGEF12selectively activates Rap1A to limit capillary barrier disruption in a mechanism independent of cAMP-mediated Epac1 activation.

The crescendo pulse frequency of shear stress stimulates the endothelialization of bone marrow mesenchymal stem cells on the luminal surface of decellularized scaffold in the bioreactor

ABSTRACT A completely confluent endothelial cell (EC) monolayer is required to maintain proper vascular function in small diameter tissue-engineered vascular graft (TEVG). However, the most effective method for EC attachment to the luminal surface and formation of an entire endothelium layer that works in vitro remains a complicated challenge that requires urgent resolution. Although pulsatile flow has been shown to be better suited for the generation of functional endothelium, the optimal frequency setting is unknown. Several pulsatile flow frequencies were used to implant rat bone mesenchymal stem cells (MSC) into the lumen of decellularized porcine carotid arteries. The endothelium's integrity and cell activity were investigated in order to determine the best pulse frequency settings. The results showed that MSC were maximally preserved and exhibited maximal morphological changes with improved endothelialization performance in response to increased pulse stimulation frequency. Increased pulse frequency stimulation stimulates the expression of mechanoreceptor markers, cytoskeleton reorganization in the direction of blood flow, denser skeletal proteins fibronectin, and stronger intercellular connections when compared to constant pulse frequency stimulation. MSC eventually develops an intact endothelial layer with anti-thrombotic properties on the inner wall of the decellularized tubular lumen. Conclusion: The decellularized vessels retain the three-dimensional structure of the vasculature, have a surface topography suitable for MSC growth, and have good mechanical properties. By increasing the frequency of pulsed stimulation, MSC endothelialize the lumen of the decellularized vasculature. It is expected to have anti-thrombotic and anti-neointimal hyperplasia properties after implantation, ultimately improving the patency of TEVG.

Atomic Level Dissection of the Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) Homophilic Binding Interface: Implications for Endothelial Cell Barrier Function.

PECAM-1 (platelet endothelial cell adhesion molecule 1) is a 130 kDa member of the immunoglobulin (Ig) gene superfamily that is expressed on the surfaces of platelets and leukocytes and concentrated at the intercellular junctions of confluent endothelial cell monolayers. PECAM-1 Ig domains 1 and 2 (IgD1 and IgD2) engage in homophilic interactions that support a host of vascular functions, including support of leukocyte transendothelial migration and the maintenance of endothelial junctional integrity. The recently solved crystal structure of PECAM-1 IgD1 and IgD2 revealed a number of intermolecular interfaces predicted to play important roles in stabilizing PECAM-1/PECAM-1 homophilic interactions and in formation and maintenance of endothelial cell-cell contacts. We sought to determine whether the protein interfaces implicated in the crystal structure reflect physiologically important interactions. Approach and Results: We assessed the impact of single amino acid substitutions at the interfaces between opposing PECAM-1 molecules on homophilic binding and endothelial cell function. Substitution of key residues within the IgD1-IgD1 and IgD1-IgD2 interfaces but not those within the smaller IgD2-IgD2 interface, markedly disrupted PECAM-1 homophilic binding and its downstream effector functions, including the ability of PECAM-1 to localize at endothelial cell-cell borders, mediate the formation of endothelial tubes, and restore endothelial barrier integrity. Taken together, these results validate the recently described PECAM-1 IgD1/IgD2 crystal structure by demonstrating that specific residues visualized within the IgD1-IgD1 and IgD1-IgD2 interfaces of opposing molecules in the crystal are required for functionally important homophilic interactions. This information can now be exploited to modulate functions of PECAM-1 in vivo.

A biomimetic shock model on the effect of endothelial aging on vascular barrier properties.

Aging is characterized by a decline in cellular function, which has an adverse effect on the biologic response to injury. Both aging and trauma/hemorrhagic shock (T/HS) increase oxidative stress which impairs the vascular endothelium (EC) and glycocalyx (EG). The additive effect of aging on EC and EG damage following T/HS are unknown. This was studied in an in vitro model. Confluent endothelial cell monolayers from primary aortic endothelial cells from 10-week-old mice ("young" cells) or primary aortic cells from 65-week-old mice ("aged" cells) were established in microfluidic devices (MFDs) and perfused at constant shear conditions overnight. Mouse endothelial cell monolayers were then exposed to hypoxia/reoxygenation alone and/or epinephrine or norepinephrine. Endothelial glycocalyx degradation was indexed as well as subsequent endothelial injury/activation. Aged endothelial cells showed increase glycocalyx shedding and subsequent loss of glycocalyx thickness. This lead to a more pronounced level of EC injury/activation compared with young endothelial cells. Although exposure to biomimetic shock conditions exacerbated both endothelial glycocalyx shedding and endothelial injury in both aged and young endothelial cells, the effect was significantly more pronounced in aged cells. Advanced age is associated with worse outcomes in severely injured trauma patients. Our study demonstrates that there is increased EG shedding and a diminished EG layer in aged compared to "young" endothelial cell layers. Biomimetic shock conditions lead to an even greater impairment of the endothelial glycocalyx in aged versus young endothelial cell monolayers. It appears that these effects are a consequence of aging related oxidative stress at both baseline and shock conditions. This exacerbates shock-induced endotheliopathy and may contribute to untoward effects on patient outcomes in this population.

Response of Endothelial Cells to Gelatin-Based Hydrogels

The establishment of confluent endothelial cell (EC) monolayers on implanted materials has been identified as a concept to avoid thrombus formation but is a continuous challenge in cardiovascular device engineering. Here, material properties of gelatin-based hydrogels obtained by reacting gelatin with varying amounts of lysine diisocyanate ethyl ester were correlated with the functional state of hydrogel contacting venous EC (HUVEC) and HUVEC's ability to form a monolayer on these hydrogels. The density of adherent HUVEC on the softest hydrogel at 37 °C (G' = 1.02 kPa, E = 1.1 ± 0.3 kPa) was significantly lower (125 mm-1) than on the stiffer hydrogels (920 mm-1; G' = 2.515 and 5.02 kPa, E = 4.8 ± 0.8 and 10.3 ± 1.2 kPa). This was accompanied by increased matrix metalloprotease activity (9 pmol·min-2 compared to 0.6 pmol·min-2) and stress fiber formation, while cell-to-cell contacts were comparable. Likewise, release of eicosanoids (e.g., prostacyclin release of 1.7 vs 0.2 pg·mL-1·cell-1) and the pro-inflammatory cytokine MCP-1 (8 vs <1.5 pg·mL-1·cell-1) was higher on the softer than on the stiffer hydrogels. The expressions of pro-inflammatory markers COX-2, COX-1, and RAGE were slightly increased on all hydrogels on day 2 (up to 200% of the control), indicating a weak inflammation; however, the levels dropped to below the control from day 6. The study revealed that hydrogels with higher moduli approached the status of a functionally confluent HUVEC monolayer. The results indicate the promising potential especially of the discussed gelatin-based hydrogels with higher G' as biomaterials for implants foreseen for the venous system.

Catecholaminergic Vasopressors Reduce Toll-Like Receptor Agonist-Induced Microvascular Endothelial Cell Permeability But Not Cytokine Production.

Catecholaminergic vasopressors are the cornerstone of circulatory shock management. Nevertheless, catecholamines have problematic side effects, arousing a growing interest in noncatecholaminergic agents such as vasopressin or angiotensin-II. However, their respective effects on sepsis-associated microvascular endothelial dysfunction such as permeability or inflammation remain elusive. We investigated the role of catecholamines and other vasopressors on Toll-like receptor agonists-induced microvascular endothelial permeability and inflammation. University research laboratory/cell research. Human pulmonary microvascular endothelial cells from multiple donors. Confluent monolayers of human pulmonary microvascular endothelial cells were treated with Toll-like receptor agonists (lipopolysaccharide, Poly[I:C], or tripalmitoyl-S-glyceryl cysteine) in the presence or absence of epinephrine, norepinephrine, vasopressin, and angiotensin-II. Permeability was inferred from transendothelial resistance, measured using electrical cell impedance sensing, where decreased transendothelial resistance is consistent with increased permeability. Cell-cell junction molecule expression was assessed via immunofluorescence microscopy and flow cytometry. We quantified cytokines in supernatants of Toll-like receptor agonist-treated human pulmonary microvascular endothelial cells. Epinephrine and norepinephrine both ameliorate lipopolysaccharide, polyinosinic:polycytidylic acid, or tripalmitoyl-S-glyceryl cysteine-induced reductions in transendothelial resistance, a surrogate for endothelial permeability. In contrast, the noncatecholaminergic agents, vasopressin, and angiotensin-II did not affect Toll-like receptor agonists-induced reductions in transendothelial resistance. β1- and β2-adrenergic receptor antagonists reduced the effects of the catecholamines on transendothelial resistance, whereas α-adrenergic receptor antagonists did not. We observed that epinephrine and norepinephrine induced actin cytoskeletal rearrangement and normalized the membrane expression of proteins involved with adherens-junctions (vascular endothelial-cadherin) and tight-junctions (zona occludens-1). Despite having a substantial effect on endothelial permeability, epinephrine and norepinephrine did not affect human pulmonary microvascular endothelial cell survival or production of interleukin-8, interleukin-6, or monocyte chemoattractant protein-1 (CCL-2) induced by Toll-like receptor agonists, suggesting that these functions are regulated separately from permeability. Our findings demonstrate that treatment with epinephrine or norepinephrine strongly reduces endothelial permeability induced by agonists of multiple Toll-like receptors (Toll-like receptor-2, Toll-like receptor-3, Toll-like receptor-4) in vitro. Our studies suggest that both β1- and β2-adrenergic receptors mediate the stabilizing effects of epinephrine and norepinephrine on the endothelial barrier.

Analysis of Thickness and Roughness Effects of Artificial Basement Membranes on Endothelial Cell Functions.

Various cells and tissues are highly organized in vivo by basement membranes (BMs) and thus promising artificial BMs (A-BMs) constructed by electrospinning and layer-by-layer (LbL) assembly have recently attracted much attention in the tissue engineering field. However, control of cell adhesion, morphology, and migration of the attached cells on the A-BMs has not been reported yet. In this study, we investigated both thickness and roughness-dependent effects of A-BMs on the functions of endothelial cells (ECs), which resulted from different assembly concentrations. The results indicated that the roughness of A-BMs increased gradually with the increase of nanofilm thickness. EC adhesion, spreading and proliferation were inhibited on thicker A-BM surfaces with larger roughness, while interendothelial junctions and the barrier effect of confluent EC monolayers on thicker A-BM surfaces were compensated by increasing seeding cell number and expanding culture time. Our study highlights the influence of LbL assembly conditions on endothelial functions, which offers a new criterion for the design of A-BMs in well-organized 3D tissues.

Porphyromonas gingivalis Outer Membrane Vesicles Increase Vascular Permeability

Periodontitis is increasingly associated with increased risk of cardiovascular and other systemic diseases. The Gram-negative anaerobe, Porphyromonas gingivalis, is a key periodontal pathogen, and several lines of evidence link the presence of this bacterium in the circulation with vascular disease. The outer membrane vesicles (OMVs) produced by P. gingivalis have been shown to play a role in periodontitis, although, to date, little is known about their interaction with the vasculature; therefore, this study assessed the effects of P. gingivalis OMVs on the endothelium. OMVs were isolated from wild-type strain W83 and the gingipain-deficient strain ΔK/R-ab. Immunoblotting along with cryo-EM showed gingipain expression in W83 but not ΔK/R-ab-derived OMVs, where gingipains were localized to the cell wall surface. Confluent endothelial cell monolayers infected with either W83 or W83-derived OMV displayed significantly increased dextran permeability over those infected with ΔK/R-ab or its OMV. Moreover, W83-derived OMVs induced significantly more vascular disease in a zebrafish larvae systemic infection model over 72 h compared to those injected with gingipain-deficient OMVs or controls. In line with these data, human microvascular endothelial cells (HMEC-1) displayed an OMV-associated, gingipain-dependent decrease in cell surface levels of the intercellular adhesion molecule PECAM-1 (CD31) when examined by flow cytometry. These data show, for the first time, that OMVs from P. gingivalis mediate increased vascular permeability, leading to a diseased phenotype both in vitro and in vivo. Moreover, these data strongly implicate gingipains present on the OMV surface in mediating these vascular events, most likely via a mechanism that involves proteolytic cleavage of endothelial cell-cell adhesins such as PECAM-1. These data provide important evidence for the role of bacterial-derived OMVs in mediating systemic disease.

Transport and Preservation Comparison of Preloaded and Prestripped-Only DMEK Grafts

This study compares the effect of the transport of conventionally prestripped Descemet membrane endothelial keratoplasty (DMEK) tissue with the DMEK revolutionary advanced Preloadable Injection Device (RAPID) preloaded transport system from Geuder AG (Heidelberg, Germany). Endothelial cell loss, tissue integrity, endothelial cell phenotype, and viability were assessed and compared. Twelve DMEK grafts were prestripped by the cornea bank and transported using the following 2 conditions: conventional flask (n = 6) or a preloaded transport cartridge (DMEK RAPID, n = 6). After transport, tissues were analyzed for cell density; denuded areas; immunolocalization of corneal endothelial markers, such as ZO-1, CD166, and Na/K ATPase; histology analysis; and cell viability staining with Hoechst, calcein AM, and ethidium homodimer. Endothelial cell loss (10.35% vs. 9.15%) did not differ between transport conditions. Histological analysis confirmed the integrity of the Descemet membrane and endothelial cell layer with both transport conditions. Similarly, the corneal endothelial cell mosaic was conserved in both conditions. The ZO-1 tight junctions confirmed the integrity of the confluent corneal endothelial cell monolayer. CD166 and Na/K ATPase detection with immunofluorescence was also comparable. A similar percentage of dead cells was reported in both conditions (18.1% vs. 16.73%). Moreover, the surface covered with calcein-positive cells (59.02% vs. 61.95%) did not differ between transport conditions. Our results suggest that DMEK grafts can be prestripped or preloaded into a novel transport cartridge and shipped to the clinic with comparable endothelial cell loss, phenotypical marker expression, and viability to the conventional prestripped donor tissue.

Apolipoprotein‐M bound sphingosine‐1 phosphate enhances heparan sulfate expression and endothelial nitric oxide synthase phosphorylation in cultured endothelial cells

Sphingosine‐1 phosphate (S1P) is blood borne bioactive sphingolipid metabolite that has protective properties against microvascular leakage under inflammatory conditions and also can promote endothelial NO production. Previous investigations suggest that functions are mediated by S1P receptor‐1 (S1pr1) but are also limited by action of S1P on other S1P receptors. Apolipoprotein‐M (ApoM), which has been shown to act as a carrier of S1P, may help target S1P to S1pr1, producing biased S1pr1 signaling. In the current study, we hypothesized that combination of ApoM will improve the ability of S1P to enhance 1) endothelial barrier function, and 2) phosphorylation of eNOS on its activation site. We used confluent monolayers of human umbilical vein endothelial cells (HUVEC) to model the endothelial barrier. Transendothelial electrical resistance (TER), determined by electric cell‐substrate impedance sensing, served as an indicator of barrier function. Because endothelial glycocalyx health is related to barrier function, we also determined the degree of heparan sulfate expression on the cell surface, visualized by immunofluorescence microscopy of fixed, non‐permeabilized HUVEC. Western blotting was used to measure eNOS phosphorylation on its activation site (S1177). These endpoints were evaluated in control cells and cells that were treated with ApoM (0.5 μM), S1P (1 μM), or the combination of both. The results show that the addition of ApoM‐bound S1P to HUVEC significantly increased and sustained the increase in TER of the HUVEC monolayers compared to the vehicle and S1P alone. ApoM‐bound S1P also increased heparan sulfate expression on the plasma membrane and elicited enhanced phosphorylation of eNOS on S1177 compared to either ApoM or S1P alone. Collectively, the findings support that ApoM enhances the ability of S1P to promote endothelial barrier function, surface heparan sulfate expression, and activation of eNOS. These findings suggest high potential for using ApoM‐bound S1P as a therapeutic target for ameliorating endothelial dysfunction.Support or Funding InformationThis work was supported by NIH R01GM120774 and American Heart Association 18UFEL33960365. Tabitha E. Norton and Taylor E. Collignon contributed equally to this work.

Towards the Development of a Biohybrid Lung as Alternative to Lung Transplantation

Lung transplantation is the only curative therapy option for patients suffering from end stage lung diseases. However, it is associated with high risks and only available for selected patients. In order to provide an alternative treatment option, this project aims for the development of an implantable biohybrid lung (BL), based on hollow fibre membrane (HFM) technology used in extracorporeal membrane oxygenators. A crucial requirement to achieve long-lasting durability of the BL is the optimized bio- and haemocompatibility of all blood contacting surfaces, which can be achieved by endothelialisation. Albumin/Heparin (A/H) and Fibronectin (FN) coated HFM were compared for their eligibility to mediate the establishment of a viable, confluent and non-thrombogenic endothelial cell (EC) monolayer. Activation status of the seeded ECs was analyzed and verified by leukocyte- and thrombocyte adhesion assays. The behavior of HFM-seeded ECs under workload conditions, i.e. fluid flow and oxidative stress exposure, was assessed in a customized flow chamber setup. To identify a clinically relevant EC source, comparative studies including human cord blood derived endothelial cells (hCBECs) induced pluripotent stem cell derived endothelial cells and immunotolerable MHC-silenced hCBECs were conducted. A physiologic, viable and confluent EC monolayer could be sustained under both, static and flow dynamic culture conditions. All tested EC types preserved their non-thrombogenic and non-inflammatory status on both tested HMF. Exposure to relevant levels of hyperoxia or hypoxia did not affect the physiological function of the ECs. However, FN coated HFMs demonstrated an improved EC-monolayer resistance towards flow conditions. Endothelialisation of HFM improves their hemocompatibility. Moreover, the endothelial monolayer on HFM can resist clinically relevant settings, like flow exposure and hyperoxia/hypoxia. MHC-silenced ECs could be considered as suitable cell source, escaping the recipient's immune response. These results may bring BL as alternative of lung transplantation for patients with end-stage lung diseases closer to reality.

Edema and Weight Increase during Organ Preservation - Identification of Solution-Specific Endothelial Leakage

Lung transplantation remains the only treatment option for patients with end-stage lung disease. Although surgical techniques and organ preservation significantly improved over the last decades, ischemia and reperfusion injuries, which inter alia manifest in edema formation, still represent major limitations affecting clinical outcome. Here we investigated the hypothesis that different lung preservation solutions contribute to solution-specific degrees of transient leaks within the endothelial cell (EC) monolayer, therefore allowing liquid influx into the interstitial space leading to edema formation. Intercellular connectivity within confluent EC monolayers was assessed after 1h, respectively 5.5h storage in the respective preservation solutions OCS™, Steen™, Perfadex™, PerfadexPlus™ or Celsior™ ± 10µM or 100µM 17β-Estradiol (17ß), which was used to deliberate disintegration of the monolayer. Furthermore, 5.5h and 24h of regeneration in the preservation solution, respectively EC medium without 17ß was analyzed. Monolayer permeability was evaluated using FITC-Dextran transwell-assays, cell viability (Calcein AM) and monolayer integrity (VE-Cadherin) were examined by fluorescence microscopy. Realtime qRT-PCR analyses were carried out to investigate responses on gene expression levels. FITC-Dextran assay indicated solution-specific monolayer permeability, after 1h incubation highest was identified for Celsior™, followed by Perfadex™ and OCS™, Steen™ and PerfadexPlus™ showed up with the lowest permeability. While addition of 10µM 17ß did not affect solution-specific permeability, all groups treated with 100µM 17ß become durable leaky. Comparable results were seen after 5.5h and 24h of regeneration, while previous incubation with 100µM 17ß induced irreversible effects. These results could be confirmed by immunofluorescence staining. By contrast, independent of the treatment qRT-PCR analyses indicated negligible effects on gene expressions of activation and thrombogenic state markers. The application of different preservation solutions results in solution-specific, partly irreversible endothelial leakage, which may partly explain edema and weight increase during organ preservation. Subsequent analysis will correlate these results with clinical parameters.

Rapid Fabrication of Ready-to-Use Gelatin Scaffolds with Prevascular Networks Using Alginate Hollow Fibers as Sacrificial Templates.

In this study, we developed a facile manufacturing method for interconnected prevascular networks using calcium chloride (CaCl2) cross-linked alginate hollow fibers as sacrificial templates. The resulting network can be used to deliver oxygen and nutrients and remove waste for embedded cells in large-volume gelatin scaffolds during in vitro culturing. The sacrificial templates were printed by customized coaxial nozzles and embedded in scaffolds made of a mixture of gelatin, microbial transglutaminase (mTG), and sodium citrate. During the cross-linking of gelatin and mTG, the sacrificial templates started to dissolve from the scaffold-template interface due to the presence of the sodium citrate in the gelatin. The embedded sacrificial templates were completely dissolved without any postprocessing, and the designed prevascular networks successfully retained their geometries and dimensions. No residue of the template was observed at the scaffold-template interface after dissolution, which promoted cell adhesion. This manufacturing method has a high degree of freedom in templates' geometry, which was demonstrated by fabricating prevascular networks with various designs, including grid, branched, and dendritic networks. The effects of hollow fiber size and sodium citrate concentration on the dissolution time were analyzed. Human umbilical vein endothelial cells were injected into the aforementioned networks and formed a confluent endothelial cell monolayer with high viability during the culture process. The results suggest great promise to rapidly build large-scale ready-to-use gelatin scaffolds with prevascular networks for the applications in tissue engineering.

Two-Channel Compartmentalized Microfluidic Chip for Real-Time Monitoring of the Metastatic Cascade.

Metastases are the primary cause of death in cancer patients. Small animal models are helping in dissecting some key features in the metastatic cascade. Yet, tools for systematically analyzing the contribution of blood flow, vascular permeability, inflammation, tissue architecture, and biochemical stimuli are missing. In this work, a microfluidic chip is designed and tested to replicate in vitro key steps in the metastatic cascade. It comprises two channels, resting on the same plane, connected via an array of rounded pillars to form a permeable micromembrane. One channel acts as a vascular compartment and is coated by a fully confluent monolayer of endothelial cells, whereas the other channel is filled with a mixture of matrigel and breast cancer cells (MDA-MB-231) and reproduces the malignant tissue. The vascular permeability can be finely modulated by inducing pro-inflammatory conditions in the tissue compartment, which transiently opens up the tight junctions of endothelial cells. Permeability ranges from 1 μm/s (tight endothelium) to 5 μm/s (TNF-α at 50 ng/mL overnight) and up to ∼10 μm/s (no endothelium). Fresh medium flowing continuously in the vascular compartment is sufficient to induce cancer cell intravasation at rates of 8 cells/day with an average velocity of ∼0.5 μm/min. On the other hand, the vascular adhesion and extravasation of circulating cancer cells require TNF-α stimulation. Extravasation occurs at lower rates with 4 cells/day and an average velocity of ∼0.1 μm/min. Finally, the same chip is completely filled with matrigel and the migration of cancer cells from one channel to the other is monitored over a region of about 400 μm. Invasion rates of 12 cells/day are documented upon TNF-α stimulation. This work demonstrates that the proposed compartmentalized microfluidic chip can efficiently replicate in vitro, under controlled biophysical and biochemical conditions, the multiple key steps in the cancer metastatic cascade.

The role of mutations associated with familial neurodegenerative disorders on blood\u2013brain barrier function in an iPSC model

BackgroundBlood–brain barrier dysfunction is associated with many late-stage neurodegenerative diseases. An emerging question is whether the mutations associated with neurodegenerative diseases can independently lead to blood–brain barrier (BBB) dysfunction. Studies from patient-derived induced pluripotent stem cells suggest that mutations associated with neurodegenerative disease are non-cell autonomous, resulting in gain of toxic function in derived neurons and astrocytes. Here we assess whether selected mutations associated with neurodegenerative diseases can contribute to impairment of the blood–brain barrier.MethodsWe assessed barrier function of confluent monolayers of human brain microvascular endothelial cells (hBMECs) derived from induced pluripotent stem cells (iPSC) from three healthy individuals and eight individuals with neurodegenerative disease. We systematically assessed protein and gene expression of BBB biomarkers, transendothelial resistance (TEER), permeability of Lucifer yellow, permeability of d-glucose, permeability of rhodamine 123, the efflux ratio of rhodamine 123, and P-gp inhibition using Tariquidar for confluent monolayers of human brain microvascular endothelial cell (hBMECs).ResultsWe provide evidence supporting the hypothesis that mutations associated with neurodegenerative disease can independently cause BBB dysfunction. These functional changes are not catastrophic since barrier breakdown would result in BBB impairment during development. Synergistic interactions between non-cell autonomous cerebrovascular dysfunction and the effects of gain-of-toxic function in neurons (e.g. toxic oligomers) are likely to increase disease burden through a positive feedback mechanism.ConclusionsThese results suggest that the accumulation of defects in brain microvascular endothelial cells may ultimately lead to impairment of the BBB. Small changes in barrier function over time could lead to accumulated defects that result in positive feedback to unrelated central nervous system diseases.