Although left ventricular assist devices (LVADs) are an alternative to heart transplantation, their artificial surfaces often lead to serious thrombotic complications requiring high-risk device replacement. Coating blood-contacting surfaces with antithrombogenic endothelial cells is considered an effective strategy for preventing thrombus formation. However, this concept has not yet been successfully implemented in LVADs, as severe cell loss is to be expected, especially on the impeller surface with high prothrombogenic supraphysiological shear stress. This study presents a strategy that exploits the magnetic attraction of the impeller on ECs loaded with iron oxide nanoparticles (IONPs) to minimize shear stress-induced cell detachment from the rotating magnetic impeller while ensuring antithrombogenic EC adhesion, especially as a bridge until they formed their adhesion-promoting matrix. In contrast to polyvinylpyrrolidone (PVP)-coated IONPs, more efficient and safer cell loading is achieved with sodium citrate (Cit)-stabilized IONPs, where incubation with 6.6 µg iron mL-1 Cit-IONPs for 24 h resulting in an average internalization of 23 pg iron per cell. Internalization of Cit-IONP significantly improved cell attraction to the highly magnetic impeller surface without affecting cell viability or antithrombogenic function. This protocol is key for the development of a biohybrid LVAD impeller that can prevent life-threatening thrombosis and hemorrhage in a future clinical application.

Vascular scaffold implantation induces injury to the intimal layer and causes discontinuity of the regenerated endothelial monolayer, compromising barrier integrity, increasing permeability, and allowing the transmigration of leukocytes and lipoproteins into the subendothelial space. Mechanical vascular wall stretching triggers Ras homolog family member A (RhoA)/Rho kinase-mediated actomyosin contractility and destabilization of adherens junctions, leading to endothelial barrier dysfunction. Assembly of intercellular adhesion and actin cytoskeletal organization of interendothelial junctions are controlled by downregulation of RhoA guanosine triphosphatase (GTPase)-mediated barrier-disruptive activity and upregulation of repressor-activator protein 1 (Rap1) and Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase-mediated cytoskeletal reorganization, leading to endothelial barrier stabilization. This review highlights the involvement of Rho GTPases in the disruption of endothelial barrier integrity following vascular scaffold implantation and the targeting of downstream Rho-associated protein kinases, which signal the network to restore endothelial barrier integrity and stability.

The aim of this study was to investigate the ability of peptides and peptide combinations to support circulating endothelial colony forming cell (ECFC) rolling and adhesion under shear flow, informing biomaterial design in moving toward rapid cardiovascular device endothelialization. ECFCs have high proliferative capability and can differentiate into endothelial cells, making them a promising cell source for endothelialization. Both single peptides and peptide combinations designed to target integrins α₄β₁ and α₅β₁ were coupled to poly(ethylene glycol) hydrogels, and their performance was evaluated by monitoring velocity patterns during the ECFC rolling process, in addition to firm adhesion (capture). Tether percentage and velocity fluctuation, a parameter newly defined here, were found to be valuable in assessing cell rolling velocity patterns and when used in combination were able to predict cell capture. REDV-containing peptides binding integrin α₄β₁ have been previously shown to reduce ECFC rolling velocity but not to support firm adhesion. This study finds that the performance of REDV-containing peptides in facilitating ECFC dynamic adhesion and capture can be improved by combination with α₅β₁ integrin-binding peptides, which support ECFC static adhesion. Moreover, when similar in length, the peptide combinations may have synergistic effects in capturing ECFCs. With matching lengths, the peptide combinations including CRRETAWAC(cyclic)+REDV, P_RGDS+KSSP_REDV, and P_RGDS+P_REDV showed high values in both tether percentage and velocity fluctuation and improvement in ECFC capture compared to the single peptides at the shear rate of 20 s^-1. These newly identified peptide combinations have the potential to be used as vascular device coatings to recruit ECFCs. STATEMENT OF SIGNIFICANCE: Restoration of functional endothelium following placement of stents and vascular grafts is critical for maintaining long-term patency. Endothelial colony forming cells (ECFCs) circulating in blood flow are a valuable cell source for rapid endothelialization. Here we identify and test novel peptides and peptide combinations that can potentially be used as coatings for vascular devices to support rolling and capture of ECFCs from flow. In addition to the widely used assessment of final ECFC adhesion, we also recorded the rolling process to quantitatively evaluate the interaction between ECFCs and the peptides, obtaining detailed performance of the peptides and gaining insight into effective capture molecule design. Peptide combinations targeting both integrin α₄β₁ and integrin α₅β₁ showed the highest percentages of ECFC capture.

We have previously reported that outgrowth endothelial cells (OECs) restore cerebral endothelial cell integrity through effective homing to the injury site. This study further investigates whether treatment with OECs can restore blood-brain barrier (BBB) function in settings of ischemia-reperfusion injury both in vitro and in vivo.

An in vitro model of human BBB was established by co-culture of astrocytes, pericytes, and human brain microvascular endothelial cells (HBMECs) before exposure to oxygen-glucose deprivation alone or followed by reperfusion (OGD±R) in the absence or presence of exogenous OECs. Using a rodent model of middle cerebral artery occlusion (MCAO), we further assessed the therapeutic potential of OECs in vivo.

Owing to their prominent antioxidant, proliferative, and migratory properties, alongside their inherent capacity to incorporate into brain vasculature, treatments with OECs attenuated the extent of OGD±R injury on BBB integrity and function, as ascertained by increases in transendothelial electrical resistance and decreases in paracellular flux across the barrier. Similarly, intravenous delivery of OECs also led to better barrier protection in MCAO rats as evidenced by significant decreases in ipsilateral brain edema volumes on day 3 after treatment. Mechanistic studies subsequently showed that treatment with OECs substantially reduced oxidative stress and apoptosis in HBMECs subjected to ischemic damages.

This experimental study shows that OEC-based cell therapy restores BBB integrity in an effective manner by integrating into resident cerebral microvascular network, suppressing oxidative stress and cellular apoptosis.

Stem cell transplantation is an appealing potential therapy for vascular diseases and an indispensable key step in vascular tissue engineering. Substantial effort has been made to differentiate stem cells toward vascular cell phenotypes, including endothelial cells (ECs) and smooth muscle cells. The microenvironment of vascular cells not only contains biochemical factors that influence differentiation but also exerts hemodynamic forces, such as shear stress and cyclic strain. More recently, studies have shown that shear stress can influence the differentiation of stem cells toward ECs. A deep understanding of the responses and underlying mechanisms involved in this process is essential for clinical translation. This review highlights current data supporting the role of shear stress in stem cell differentiation into ECs. Potential mechanisms and signaling cascades for transducing shear stress into a biological signal are proposed. Further study of stem cell responses to shear stress will be necessary to apply stem cells for pharmacological applications and cardiovascular implants in the realm of regenerative medicine.

Lymphatic vessels (LVs) have been suggested as a preferential conduit for metastatic progression in breast cancer, where a correlation between the occurrence of lymph node metastasis and an increased extracellular matrix (ECM) density has been reported. However, the effect of ECM density on LV function is largely unknown. To better understand these effects, we used a microfluidic device to recreate tubular LVs in a collagen type I matrix. The density of the matrix was tailored to mimic normal breast tissue using a low-density collagen (LD-3 mg mL-1) and cancerous breast tissue using a high-density collagen (HD-6 mg mL-1). We investigated the effect of ECM density on LV morphology, growth, cytokine secretion, and barrier function. LVs cultured in HD matrices showed morphological changes as compared to LVs cultured in a LD matrix. Specifically, LVs cultured in HD matrices had a 3-fold higher secretion of the pro-inflammatory cytokine, IL-6, and a leakier phenotype, suggesting LVs acquired characteristics of activated vessels. Interestingly, LV leakiness was mitigated by blocking the IL-6 receptor on the lymphatic ECs, maintaining endothelium permeability at similar levels of LV cultured in a LD matrix. To recreate a more in vivo microenvironment, we incorporated metastatic breast cancer cells (MDA-MB-231) into the LD and HD matrices. For HD matrices, co-culture with MDA-MB-231 cells exacerbated vessel leakiness and secretion of IL-6. In summary, our data suggest that (1) ECM density is an important microenvironmental cue that affects LV function in the breast tumor microenvironment (TME), (2) dense matrices condition LVs towards an activated phenotype and (3) blockade of IL-6 signaling may be a potential therapeutic target to mitigate LV dysfunction. Overall, modeling LVs and their interactions with the TME can help identify novel therapeutic targets and, in turn, advance therapeutic discovery.

Appropriate mechanical properties and fast endothelialization of synthetic grafts are key to ensure long-term functionality of implants. We used a newly developed biostable polyurethane elastomer (TPCU) to engineer electrospun vascular scaffolds with promising mechanical properties (E-modulus: 4.8 ± 0.6 MPa, burst pressure: 3326 ± 78 mmHg), which were biofunctionalized with fibronectin (FN) and decorin (DCN). Neither uncoated nor biofunctionalized TPCU scaffolds induced major adverse immune responses except for minor signs of polymorph nuclear cell activation. The in vivo endothelial progenitor cell homing potential of the biofunctionalized scaffolds was simulated in vitro by attracting endothelial colony-forming cells (ECFCs). Although DCN coating did attract ECFCs in combination with FN (FN + DCN), DCN-coated TPCU scaffolds showed a cell-repellent effect in the absence of FN. In a tissue-engineering approach, the electrospun and biofunctionalized tubular grafts were cultured with primary-isolated vascular endothelial cells in a custom-made bioreactor under dynamic conditions with the aim to engineer an advanced therapy medicinal product. Both FN and FN + DCN functionalization supported the formation of a confluent and functional endothelial layer.

Human cytomegalovirus establishes a lifelong, latent infection in the human host and can cause significant morbidity and mortality, particularly, in immunocompromised individuals. One established site of HCMV latency and reactivation is in cells of the myeloid lineage. In undifferentiated myeloid cells, such as CD14+ monocytes, virus is maintained latently. We have recently reported an analysis of the total proteome of latently infected CD14+ monocytes, which identified an increase in hematopoietic lineage cell-specific protein (HCLS1). Here we show that this latency-associated upregulation of HCLS1 occurs in a US28-dependent manner and stabilizes actin structure in latently infected cells. This results in their increased motility and ability to transit endothelial cell layers. Thus, latency-associated increases in monocyte motility could aid dissemination of the latently infected reservoir, and targeting this increased motility could have an impact on the ability of latently infected monocytes to distribute to tissue sites of reactivation.

PolyJet three-dimensional (3D) printing allows for the rapid manufacturing of 3D moulds for the fabrication of cross-linked poly(dimethylsiloxane) microwell arrays (PMAs). As this 3D printing technique has a resolution on the micrometer scale, the moulds exhibit a distinct surface roughness. In this study, the authors demonstrate by optical profilometry that the topography of the 3D printed moulds can be transferred to the PMAs and that this roughness induced cell adhesive properties to the material. In particular, the topography facilitated immobilization of endothelial cells on the internal walls of the microwells. The authors also demonstrate that upon immobilization of endothelial cells to the microwells, a second population of cells, namely, pancreatic islets could be introduced, thus producing a 3D coculture platform.

The blood flow through vessels produces a tangential, or shear, stress sensed by their innermost layer (i.e., endothelium) and representing a major hemodynamic force. In humans, endothelial repair and blood vessel formation are mainly performed by circulating endothelial progenitor cells (EPCs) characterized by a considerable expression of vascular endothelial growth factor receptor 2 (VEGFR2), CD34, and CD133, pronounced tube formation activity in vitro, and strong reendothelialization or neovascularization capacity in vivo. EPCs have been proposed as a promising agent to induce reendothelialization of injured arteries, neovascularization of ischemic tissues, and endothelialization or vascularization of bioartificial constructs. A number of preconditioning approaches have been suggested to improve the regenerative potential of EPCs, including the use of biophysical stimuli such as shear stress. However, in spite of well-defined influence of shear stress on mature endothelial cells (ECs), articles summarizing how it affects EPCs are lacking. Here we discuss the impact of shear stress on homing, paracrine effects, and differentiation of EPCs. Unidirectional laminar shear stress significantly promotes homing of circulating EPCs to endothelial injury sites, induces anti-thrombotic and anti-atherosclerotic phenotype of EPCs, increases their capability to form capillary-like tubes in vitro, and enhances differentiation of EPCs into mature ECs in a dose-dependent manner. These effects are mediated by VEGFR2, Tie2, Notch, and β1/3 integrin signaling and can be abrogated by means of complementary siRNA/shRNA or selective pharmacological inhibitors of the respective proteins. Although the testing of sheared EPCs for vascular tissue engineering or regenerative medicine applications is still an unaccomplished task, favorable effects of unidirectional laminar shear stress on EPCs suggest its usefulness for their preconditioning.

Membrane regulatory proteins, such as CD46, CD55, and CD59, prevent excess complement activation and to protect cells from damage. Previous investigations confirmed that shear stress in the physiological range was more favorable for endothelial progenitor cells (EPCs) to repair injured vascular endothelial cells and operates mainly in atheroprotective actions. However, detailed events that contribute to shear stress-induced protection in EPCs, particularly the mechanisms of signal transduction, remain poorly understood. In this study, we observed shear stress-mediated changes in the expression of complement regulatory proteins CD46, CD55, and CD59 on human EPCs and focused on the mechanical transmission mechanism in transformed cells in response to the ECM-F-actin pathway in vitro. Shear stress was observed to promote the expression of complement regulatory protein CD59, but not CD46 or CD55, on EPCs. In addition, the shear stress-induced CD59 expression was confirmed to be associated with the ECM components and was alleviated in EPCs pretreated with GRGDSP, which inhibits ECM components-integrin interaction. Furthermore, shear stress also promotes the rearrangement and polymerization of F-actin. However, shear stress-induced CD59 expression was reduced when the F-actin stress fiber formation process was delayed by Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) or destroyed by cytochalasin D (Cyto D), while Jasplakinolide (JAS) reversed the expression of CD59 through promotion of F-actin polymerization and its stabilizing capacities. Our results indicates that shear stress is an important mediator in EPC expression of CD59 regulated by the ECM-F-actin pathway, which is a key factor in preventing membrane attack complex (MAC) -mediated cell autolysis.