Confluent Endothelial Monolayers Research Articles

Nanotexture and crystal phase regulation for synergistic enhancement in re-endothelialization on medical pure titanium surface

Re-endothelialization has been recognized as a promising strategy to address the tissue hyperplasia and subsequent restenosis which are major complications associated with vascular implant/interventional titanium devices. However, the uncontrollable over-proliferation of smooth muscle cells (SMCs) limits the clinical application of numerous modified strategies. Herein, a novel modified strategy involving with a two-step anodic oxidation and annealing treatment was proposed to achieve rapid re-endothelialization function regulated by regular honeycomb nanotexture and specific anatase phase on the titanium surface. Theoretical calculation revealed that the presence of nanotexture reduced the polar component of surface energy, while the generation of anatase significantly enhanced the polar component and total surface energy. Meanwhile, the modified surface with regular nanotexture and anatase phase produced positive effect on the expression of CD31, VE-Cadherin and down-regulated α-SMA proteins expression, indicating excellent capacity of pro-endothelial regeneration and inhibition of SMCs proliferation and migration. One-month in vivo implantation in rabbit carotid arteries further confirmed that modified tube implant surface effectively accelerated confluent endothelial monolayer formation and promoted native-like endothelium tissue regeneration. By contrast, original titanium tube implant induced a disorganized tissue proliferation in the lumen with a high risk of restenosis. Collectively, this study opens us an alternative route to achieve the function that selectively promotes endothelial cells (ECs) growth and suppresses SMCs on the medical titanium surface, which has a great potential in facilitating re-endothelialization on the surface of blood-contacting titanium implant.

Facile Photopatterning of Perfusable Microchannels in Synthetic Hydrogels to Recreate Microphysiological Environments.

The fabrication of perfusable hydrogels is crucial for recreating in vitro microphysiological environments. Existing strategies to fabricate complex microchannels in hydrogels involve sophisticated equipment/techniques. A cost-effective, facile, versatile, and ultra-fast methodology is reported to fabricate perfusable microchannels of complex shapes in photopolymerizable hydrogels without the need of specialized equipment or sophisticated protocols. The methodology utilizes one-step ultraviolet (UV) light-triggered cross-linking and a photomask printed on inexpensive transparent films to photopattern PEG-norbornene hydrogels. Complex and intricate patterns with high resolution, including perfusable microchannels, can be fabricated in <1s. The perfusable hydrogel is integrated into a custom-made microfluidic device that permits connection to external pump systems, allowing continuous fluid perfusion into the microchannels. Under dynamic culture, human endothelial cells form a functional and confluent endothelial monolayer that remains viable for at least 7 days and respond to inflammatory stimuli. Finally, approach to photopattern norbornene hyaluronic acid hydrogels is adapted, highlighting the versatility of the technique. This study presents an innovative strategy to simplify and reduce the cost of biofabrication techniques for developing functional in vitro models using perfusable three-dimensional (3D) hydrogels. The approach offers a novel solution to overcome the complexities associated with existing methods, allowing engineering advanced in vitro microphysiological environments.

Remote Outcomes with Poly-ε-Caprolactone Aortic Grafts in Rats.

Poly-ε-caprolactone ((1,7)-polyoxepan-2-one; PCL) is a biodegradable polymer widely used in various fields of bioengineering, but its behavior in long-term studies appears to depend on many conditions, such as application specificity, chemical structure, in vivo test systems, and even environmental conditions in which the construction is exploited in. In this study, we offer an observation of the remote outcomes of PCL tubular grafts for abdominal aorta replacement in an in vivo experiment on a rat model. Adult Wistar rats were implanted with PCL vascular matrices and observed for 180 days. The results of ultrasound diagnostics and X-ray tomography (CBCT) show that the grafts maintained patency for the entire follow-up period without thrombosis, leakage, or interruptions, but different types of tissue reactions were found at this time point. By the day of examination, all the implants revealed a confluent endothelial monolayer covering layers of hyperplastic neointima formed on the luminal surface of the grafts. Foreign body reactions were found in several explants including those without signs of stenosis. Most of the scaffolds showed a pronounced infiltration with fibroblastic cells. All the samples revealed subintimal calcium phosphate deposits. A correlation between chondroid metaplasia in profound cells of neointima and the process of mineralization was supported by immunohistochemical (IHC) staining for S100 proteins and EDS mapping. Microscopy showed that the scaffolds with an intensive inflammatory response or formed fibrotic capsules retain their fibrillar structure even on day 180 after implantation, but matrices infiltrated with viable cells partially save the original fibrillary network. This research highlights the advantages of PCL vascular scaffolds, such as graft permeability, revitalization, and good surgical outcomes. The disadvantages are low biodegradation rates and exceptionally high risks of mineralization and intimal hyperplasia.

Rho-associated coiled-coil kinase contributes an important role in Xingnaojing injection-induced adverse drug reactions

Xingnaojing injection (XNJI) is widely used for acute cerebral hemorrhage. However, the adverse drug reactions (ADRs) caused by XNJI have attracted broad attention. In this study, mice were intravenously administrated with a single dose of XNJI (10 or 20 mL/kg) to evaluate the vascular permeability. Then, pathological examinations on ears and lungs were performed to further determine the characteristics of ADRs induced by XNJI. In addition, human umbilical vein endothelial cells (HUVECs) were stimulated with XNJI (1/128, 1/64, and 1/32 XNJI pure product dilution) and stained with rhodamine-phalloidin to analyze the alterations on cytoskeleton. In addition, endothelial monolayer was incubated with XNJI and the effect on permeability was tested. Western blot testing was conducted to measure the involvement of Rho-associated coiled-coil kinase (ROCK) in XNJI-induced ADRs and fasudil hydrochloride was applied to additionally confirm the contribution of ROCK. The results indicated that XNJI may lead to dose-associated vascular leakage. None visibly abnormal symptom was noticed in the mice treated with XNJI at 10 mL/kg, while inflammation in ears and lungs was observed in the mice administrated with 20 mL/kg XNJI. In in vitro experiment, stimulation with high concentration of XNJI (1/32 XNJI pure product dilution) could enhance the permeability of confluent endothelial monolayer and induce rearrangement of actin cytoskeleton. Activation of ROCK, which was shown as augmented expressions of p-MLC2 and p-MYPT1, was observed in cells and mice treated with XNJI. ROCK inhibitor could attenuate XNJI-induced dysfunction of endothelial barrier in in vitro experiment and alleviate the vascular leakage and inflammation noticed in mice treated with XNJI. This study indicates that overdose of XNJI can destroy vascular endothelial function and induce increased vascular permeability, which may cause ADRs in clinical practice. Meanwhile, ROCK plays an important role in XNJI-induced ADRs.

Endothelial progenitor cells overexpressing Grb2-associated binder 1 for in vitro-constructed tissue-engineered heart valves.

Aim Endothelial progenitor cells (EPCs) play important roles in heart valve replacement surgery. Up-regulation of Grb2‑associated binder 1 (Gab1) promotes hepatocyte growth factor (HGF) - induced endothelial progenitor cell proliferation and migration. This study aimed to investigate the effects of up-regulation of Gab1 in hepatocyte growth factor-induced EPCs in tissue-engineered heart valves (TEHV).Material and methods Fresh porcine aortic valves were placed in 1 % Triton X-100 and trypsin buffer for decellularization. EPCs in the control group were cultured normally, whereas those in the experimental group were both HGF stimulated and transfected with adenovirus containing the Gab1 gene. Cells in the two groups were seeded onto the decellularized valve scaffolds and cultured for 3 or 7 days. TEHV were analyzed by HE and AB-PAS staining.Results By day 3, the experimental group had formed confluent endothelial monolayers on top of the decellularized valves, on the basis of by HE staining and AB-PAS staining. One week later, the control group showed a imperfect endothelial layer.Conclusion HGF-induced EPCs overexpressing Gab1 can endothelialize the decellularized matrix and create functional TEHV, which may then be preconditioned in a bioreactor before clinical implantation.

Electrospun Scaffolds Functionalized with a Hydrogen Sulfide Donor Stimulate Angiogenesis.

Tissue-engineered constructs are currently limited by the lack of vascularization necessary for the survival and integration of implanted tissues. Hydrogen sulfide (H2S), an endogenous signaling gas (gasotransmitter), has been recently reported as a promising alternative to growth factors to mediate and promote angiogenesis in low concentrations. Yet, sustained delivery of H2S remains a challenge. Herein, we have developed angiogenic scaffolds by covalent attachment of an H2S donor to a polycaprolactone (PCL) electrospun scaffold. These scaffolds were engineered to include azide functional groups (on 1, 5, or 10% of the PCL end groups) and were modified using a straightforward click reaction with an alkyne-functionalized N-thiocarboxyanhydride (alkynyl-NTA). This created H2S-releasing scaffolds that rely on NTA ring-opening in water followed by conversion of released carbonyl sulfide into H2S. These functionalized scaffolds showed dose-dependent release of H2S based on the amount of NTA functionality within the scaffold. The NTA-functionalized fibrous scaffolds supported human umbilical vein endothelial cell (HUVEC) proliferation, formed more confluent endothelial monolayers, and facilitated the formation of tight cell–cell junctions to a greater extent than unfunctionalized scaffolds. Covalent conjugation of H2S donors to scaffolds not only promotes HUVEC proliferation in vitro, but also increases neovascularization in ovo, as observed in the chick chorioallantoic membrane assay. NTA-functionalized scaffolds provide localized control over vascularization through the sustained delivery of a powerful endogenous angiogenic agent, which should be further explored to promote angiogenesis in tissue engineering.

Quantifying Endothelial Transcytosis with Total Internal Reflection Fluorescence Microscopy (TIRF).

Apical-to-basal transcytosis by endothelial cells can be visualized and quantified using total internal reflection fluorescence (TIRF) microscopy of the basal membrane. Past techniques to study transcytosis including electron microscopy and transwells have several limitations such as confounding from paracellular leakage, low transfection efficiency, and the largely descriptive nature of electron microscopy. After the addition of a fluorescent ligand to the apical endothelial surface, using TIRF to measure exocytosis at the basal membrane bypasses these issues by studying transcytosis across a single cell of a confluent endothelial monolayer in real time. A major benefit of TIRF is that only a small volume of the cell is illuminated, thus greatly reducing background noise from the overlying cytosol in the images. This protocol outlines the steps to image and quantify exocytosis of apically applied fluorophore-tagged low-density lipoprotein (LDL) using TIRF microscopy and MATLAB. A similar approach can be used to study endothelial transcytosis of other ligands such as albumin or high-density lipoprotein.

Rapid assembly of PMMA microfluidic devices with PETE membranes for studying the endothelium

Biomicrofluidic devices and organ-on-a-chip (OOC) systems with integrated membranes are often fabricated from two different thermoplastic materials but bonding of such dissimilar thermoplastics remains challenging to manufacture at scale. Here, we present a method to bond poly(methylmethacrylate) layers to a polyethylene terephthalate porous membrane to create membrane-based microfluidic devices for biological barrier modeling. By combining milling, laser cutting and chlorocarbon-based solvent bonding supported by retention grooves, we achieved a fabrication rate of 36 devices in 5 h. Chlorocarbon-based solvent bonding resulted in bond strength of ~10 J/m2 and did not adversely affect the membrane pore structure or the channel cross-sectional shape. The bonded devices were found to support long term culture of human endothelial cells that developed expected morphology and cell-cell adhesion contacts as evidenced by immunofluorescent labeling of VE-cadherin. Barrier permeability was measured to be 3.38 × 106 cm/s for 10 kDa dextran using a sampling-based method compatible with mass spectrometry and scintillation techniques and was in agreement with literature. To validate the devices for cell migration experiments, THP-1 monocytes were introduced into devices with confluent endothelial monolayers. Monocytes adhered to and migrated through the endothelium. Activation of the endothelium with TNF-α prior to introducing monocytes significantly increased monocyte adhesion. Overall, the rapid device fabrication method achieved medium-volume production rates and was found to support both cell culture and experiments associated with measuring barrier and endothelial function. This fabrication method has potential to both accelerate biomicrofluidics and OOC research in the lab and accelerate development of commercialized microfluidic membrane devices.

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.

Lysine-doped polydopamine coating enhances antithrombogenicity and endothelialization of an electrospun aligned fibrous vascular graft

Accelerating in situ endothelialization while inhibiting thrombosis is critical for the success of small-caliber tissue-engineered vascular grafts (TEVGs). In this study, a lysine (Lys) doped polydopamine (PDA) coating strategy was exploited to functionalize the TEVGs for vascular tissue regeneration. Using electrospun aligned poly(L-lactide-co-caprolactone) (PLCL) fiber arrays as the demonstrating biomaterial, introduction of Lys to the dopamine polymerization process was found to promote the formation of PDA-Lys composite coating layer atop the fiber surface via Schiff base and Michael addition reactions. Such a surface functionalization remarkably improved the coating layer uniformity, wettability, and protein adsorption capacity. In vitro assessment showed that the PDA-Lys coating layer was anti-thrombogenic and anti-inflammatory, and accelerated the formation of confluent endothelial monolayer by promoting cell-fiber and cell-cell interactions. Upon implantation in a rabbit carotid artery replacement model for 3 months, the constructed TEVGs (2 mm inner diameter), with oriented fiber architecture parallel to the longitudinal axis of grafts, were confirmed to significantly suppress thrombosis and accelerate regeneration of endothelium and tunica media layer, thereby exhibiting a positive vascular remodeling and integration capability in revascularization. This study demonstrated that the lysine-doped PDA coating provided a facile and effective platform for the improvement of antithrombogenicity and endothelialization of TEVGs.

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.

Biomechanical Characterization of Endothelial Cells Exposed to Shear Stress Using Acoustic Force Spectroscopy.

Characterizing mechanical properties of cells is important for understanding many cellular processes, such as cell movement, shape, and growth, as well as adaptation to changing environments. In this study, we explore the mechanical properties of endothelial cells that form the biological barrier lining blood vessels, whose dysfunction leads to development of many cardiovascular disorders. Stiffness of living endothelial cells was determined by Acoustic Force Spectroscopy (AFS), by pull parallel multiple functionalized microspheres located at the cell-cell periphery. The unique configuration of the acoustic microfluidic channel allowed us to develop a long-term dynamic culture protocol exposing cells to laminar flow for up to 48 h, with shear stresses in the physiological range (i.e., 6 dyn/cm2). Two different Endothelial cells lines, Human Aortic Endothelial Cells (HAECs) and Human Umbilical Vein Endothelial Cells (HUVECs), were investigated to show the potential of this tool to capture the change in cellular mechanical properties during maturation of a confluent endothelial monolayer. Immunofluorescence microscopy was exploited to follow actin filament rearrangement and junction formation over time. For both cell types we found that the application of shear-stress promotes the typical phenotype of a mature endothelium expressing a linear pattern of VE-cadherin at the cell-cell border and actin filament rearrangement along the perimeter of Endothelial cells. A staircase-like sequence of increasing force steps, ranging from 186 pN to 3.5 nN, was then applied in a single measurement revealing the force-dependent apparent stiffness of the membrane cortex in the kPa range. We also found that beads attached to cells cultured under dynamic conditions were harder to displace than cells cultured under static conditions, showing a stiffer membrane cortex at cell periphery. All together these results demonstrate that the AFS can identify changes in cell mechanics based on force measurements of adherent cells under conditions mimicking their native microenvironment, thus revealing the shear stress dependence of the mechanical properties of neighboring endothelial cells.

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.

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.

One-Pot Covalent Grafting of Gelatin on Poly(Vinyl Alcohol) Hydrogel to Enhance Endothelialization and Hemocompatibility for Synthetic Vascular Graft Applications.

Cardiovascular diseases remain the leading cause of death worldwide. Patency rates of clinically-utilized small diameter synthetic vascular grafts such as Dacron® and expanded polytetrafluoroethylene (ePTFE) to treat cardiovascular disease are inadequate due to lack of endothelialization. Sodium trimetaphosphate (STMP) crosslinked PVA could be potentially employed as blood-compatible small diameter vascular graft for the treatment of cardiovascular disease. However, PVA severely lacks cell adhesion properties, and the efforts to endothelialize STMP-PVA have been insufficient to produce a functioning endothelium. To this end, we developed a one-pot method to conjugate cell-adhesive protein via hydroxyl-to-amine coupling using carbonyldiimidazole by targeting residual hydroxyl groups on crosslinked STMP-PVA hydrogel. Primary human umbilical vascular endothelial cells (HUVECs) demonstrated significantly improved cells adhesion, viability and spreading on modified PVA. Cells formed a confluent endothelial monolayer, and expressed vinculin focal adhesions, cell-cell junction protein zonula occludens 1 (ZO1), and vascular endothelial cadherin (VE-Cadherin). Extensive characterization of the blood-compatibility was performed on modified PVA hydrogel by examining platelet activation, platelet microparticle formation, platelet CD61 and CD62P expression, and thrombin generation, which showed that the modified PVA was blood-compatible. Additionally, grafts were tested under whole, flowing blood without any anticoagulants in a non-human primate, arteriovenous shunt model. No differences were seen in platelet or fibrin accumulation between the modified-PVA, unmodified PVA or clinical, ePTFE controls. This study presents a significant step in the modification of PVA for the development of next generation in situ endothelialized synthetic vascular grafts.

Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption.

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular stress and traction generation is currently unknown. Therefore, we present here a mechanics-based protocol to probe the influence of gap junction connexin 43 (Cx43) has on endothelial biomechanics by exposing confluent endothelial monolayers to a known Cx43 inhibitor 2,5-dihydroxychalcone (chalcone) and measuring the impact this inhibitor has on tractions and intercellular stresses. We present representative results, which show a decrease in both tractions and intercellular stresses under a high chalcone dosage (2 µg/mL) when compared to control. This protocol can be applied to not just Cx43, but also other gap junctions as well, assuming the appropriate inhibitor is used. We believe this protocol will be useful in the fields of cardiovascular and mechanobiology research.

Leaf-Inspired Authentically Complex Microvascular Networks for Deciphering Biological Transport Process.

The vascular transport of molecules, cells, and nanoconstructs is a fundamental biophysical process impacting tissue regeneration, delivery of nutrients and therapeutic agents, and the response of the immune system to external pathogens. This process is often studied in single-channel microfluidic devices lacking the complex tridimensional organization of vascular networks. Here, soft lithography is employed to replicate the vein system of a Hedera elix leaf on a polydimethilsiloxane (PDMS) template. The replica is then sealed and connected to an external pumping system to realize an authentically complex microvascular network. This satisfies energy minimization criteria by Murray's law and comprises a network of channels ranging in size from capillaries (∼50 μm) to large arterioles and venules (∼400 μm). Micro-PIV (micro-particle image velocimetry) analysis is employed to characterize flow conditions in terms of streamlines, fluid velocity, and flow rates. To demonstrate the ability to reproduce physiologically relevant transport processes, two different applications are demonstrated: vascular deposition of tumor cells and lysis of blood clots. To this end, conditions are identified to culture cells within the microvasculature and realize a confluent endothelial monolayer. Then, the vascular deposition of circulating breast (MDA-MB 231) cancer cells is documented throughout the network under physiologically relevant flow conditions. Firm cell adhesion mostly occurs in channels with low mean blood velocity. As a second application, blood clots are formed within the chip by mixing whole blood with a thrombin solution. After demonstrating the blood clot stability, tissue plasminogen activator (tPA) and tPA-carrying nanoconstructs (tPA-DPNs) are employed as thrombolytics. In agreement with previous data, clot dissolution is equally induced by tPA and tPA-DPNs. The proposed leaf-inspired chip can be efficiently used to study a variety of vascular transport processes in complex microvascular networks, where geometry and flow conditions can be modulated and monitored throughout the experimental campaign.

Differential outcomes of venous and arterial tissue engineered vascular grafts highlight the importance of coupling long-term implantation studies with computational modeling

Electrospinning is commonly used to generate polymeric scaffolds for tissue engineering. Using this approach, we developed a small-diameter tissue engineered vascular graft (TEVG) composed of poly-ε-caprolactone-co-l-lactic acid (PCLA) fibers and longitudinally assessed its performance within both the venous and arterial circulations of immunodeficient (SCID/bg) mice. Based on in vitro analysis demonstrating complete loss of graft strength by 12 weeks, we evaluated neovessel formation in vivo over 6-, 12- and 24-week periods. Mid-term observations indicated physiologic graft function, characterized by 100% patency and luminal matching with adjoining native vessel in both the venous and arterial circulations. An active and robust remodeling process was characterized by a confluent endothelial cell monolayer, macrophage infiltrate, and extracellular matrix deposition and remodeling. Long-term follow-up of venous TEVGs at 24 weeks revealed viable neovessel formation beyond graft degradation when implanted in this high flow, low-pressure environment. Arterial TEVGs experienced catastrophic graft failure due to aneurysmal dilatation and rupture after 14 weeks. Scaffold parameters such as porosity, fiber diameter, and degradation rate informed a previously described computational model of vascular growth and remodeling, and simulations predicted the gross differential performance of the venous and arterial TEVGs over the 24-week time course. Taken together, these results highlight the requirement for in vivo implantation studies to extend past the critical time period of polymer degradation, the importance of differential neotissue deposition relative to the mechanical (pressure) environment, and further support the utility of predictive modeling in the design, use, and evaluation of TEVGs in vivo. Statement of SignificanceHerein, we apply a biodegradable electrospun vascular graft to the arterial and venous circulations of the mouse and follow recipients beyond the point of polymer degradation. While venous implants formed viable neovessels, arterial grafts experienced catastrophic rupture due to aneurysmal dilation. We then inform a previously developed computational model of tissue engineered vascular graft growth and remodeling with parameters specific to the electrospun scaffolds utilized in this study. Remarkably, model simulations predict the differential performance of the venous and arterial constructs over 24 weeks. We conclude that computational simulations should inform the rational selection of scaffold parameters to fabricate tissue engineered vascular grafts that must be followed in vivo over time courses extending beyond polymer degradation.

VE-PTP stabilizes VE-cadherin junctions and the endothelial barrier via a phosphatase-independent mechanism.

Vascular endothelial (VE) protein tyrosine phosphatase (PTP) is an endothelial-specific phosphatase that stabilizes VE-cadherin junctions. Although studies have focused on the role of VE-PTP in dephosphorylating VE-cadherin in the activated endothelium, little is known of VE-PTP's role in the quiescent endothelial monolayer. Here, we used the photoconvertible fluorescent protein VE-cadherin-Dendra2 to monitor VE-cadherin dynamics at adherens junctions (AJs) in confluent endothelial monolayers. We discovered that VE-PTP stabilizes VE-cadherin junctions by reducing the rate of VE-cadherin internalization independently of its phosphatase activity. VE-PTP serves as an adaptor protein that through binding and inhibiting the RhoGEF GEF-H1 modulates RhoA activity and tension across VE-cadherin junctions. Overexpression of the VE-PTP cytosolic domain mutant interacting with GEF-H1 in VE-PTP-depleted endothelial cells reduced GEF-H1 activity and restored VE-cadherin dynamics at AJs. Thus, VE-PTP stabilizes VE-cadherin junctions and restricts endothelial permeability by inhibiting GEF-H1, thereby limiting RhoA signaling at AJs and reducing the VE-cadherin internalization rate.