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Background: Whey protein isolate (WPI) is a by-product from the dairy industry, whose main component is β-lactoglobulin. Upon heating, WPI forms a hydrogel which can both support controlled drug delivery and enhance the proliferation and osteogenic differentiation of bone-forming cells. This study makes a novel contribution by evaluating the ability of WPI hydrogels to support the growth of endothelial cells, which are essential for vascularization, which in turn is a pre-requisite for bone regeneration., Methods: In this study, the proliferation and antioxidant levels in human umbilical vascular endothelial cells (HUVECs) cultured with WPI supplementation were evaluated using real-time cell analysis and flow cytometry. Further, the attachment and growth of HUVECs seeded on WPI-based hydrogels with different concentrations of WPI (15%, 20%, 30%, 40%) were investigated., Results: Supplementation with WPI did not affect the viability or proliferation of HUVECs monitored with real-time cell analysis. At the highest used concentration of WPI (500 µg/mL), a slight induction of ROS production in HUVECs was detected as compared with control samples, but it was not accompanied by alterations in cellular thiol levels. Regarding WPI-based hydrogels, HUVEC adhered and spread on all samples, showing good metabolic activity. Notably, cell number was highest on samples containing 20% and 30% WPI., Conclusions: The demonstration of the good compatibility of WPI hydrogels with endothelial cells in these experiments is an important step towards promoting the vascularization of hydrogels upon implantation in vivo, which is expected to improve implant outcomes in the future.

This study aimed to develop a suitable hydrogel-based 3D platform to support long-term culture of primary endothelial cells (ECs) and fibroblasts. Two hydrogel systems based on allyl-modified gelatin (gelAGE), G 1MM and G 2LH , were cross-linked via thiol-ene click reaction with a four-arm thiolated polyethylene glycol (PEG-4-SH). Compared to G 1MM , the G 2LH hydrogel was characterized by the lower polymer content and cross-linking density with a softer matrix and homogeneous and open porosity. Cell viability in both hydrogels was comparable, although the G 2LH -based platform supported better F-actin organization, cell-cell interactions, and collagen and fibronectin production. In co-cultures, early morphogenesis leading to tubular-like structures was observed within 2 weeks. Migration of fibroblasts out of spheroids embedded in the G 2LH hydrogels started after 5 days of incubation. Taken together, the results demonstrated that the G 2LH hydrogel fulfilled the demands of both ECs and fibroblasts to enable long-term culture and matrix remodeling.

A hydrogel system based on oxidized alginate covalently crosslinked with gelatin (ADA-GEL) has been utilized for different biofabrication approaches to design constructs, in which cell growth, proliferation and migration have been observed. However, cell-bioink interactions are not completely understood and the potential effects of free aldehyde groups on the living cells have not been investigated. In this study, alginate, ADA and ADA-GEL were characterized via FTIR and NMR, and their effect on cell viability was investigated. In the tested cell lines, there was a concentration-dependent effect of oxidation degree on cell viability, with the strongest cytotoxicity observed after 72 h of culture. Subsequently, primary human cells, namely fibroblasts and endothelial cells (ECs) were grown in ADA and ADA-GEL hydrogels to investigate the molecular effects of oxidized material. In ADA, an extremely strong ROS generation resulting in a rapid depletion of cellular thiols was observed in ECs, leading to rapid necrotic cell death. In contrast, less pronounced cytotoxic effects of ADA were noted on human fibroblasts. Human fibroblasts had higher cellular thiol content than primary ECs and entered apoptosis under strong oxidative stress. The presence of gelatin in the hydrogel improved the primary cell survival, likely by reducing the oxidative stress via binding to the CHO groups. Consequently, ADA-GEL was better tolerated than ADA alone. Fibroblasts were able to survive the oxidative stress in ADA-GEL and re-entered the proliferative phase. To the best of our knowledge, this is the first report that shows in detail the relationship between oxidative stress-induced intracellular processes and alginate di-aldehyde-based bioinks.

Tissue-engineered scaffolds require an effective colonization with cells. Superparamagnetic iron oxide nanoparticles (SPIONs) can enhance cell adhesion to matrices by magnetic cell seeding. We investigated the possibility of improving cell attachment and growth on different alginate-based hydrogels using fibroblasts and endothelial cells (ECs) loaded with SPIONs. Hydrogels containing pure alginate (Alg), alginate dialdehyde crosslinked with gelatin (ADA-G) and Alg blended with G or silk fibroin (SF) were prepared. Endothelial cells and fibroblasts loaded with SPIONs were seeded and grown on hydrogels for up to 7 days, in the presence of magnetic field during the first 24 h. Cell morphology (fluorescent staining) and metabolic activity (WST-8 assay) of magnetically-seeded versus conventionally seeded cells were compared. Magnetic seeding of ECs improved their initial attachment and further growth on Alg/G hydrogel surfaces. However, we did not achieve an efficient and stable colonization of ADA-G films with ECs even with magnetic cell seeding. Fibroblast showed good initial colonization and growth on ADA-G and on Alg/SF. This effect was further significantly enhanced by magnetic cell seeding. On pure Alg, initial attachment and spreading of magnetically-seeded cells was dramatically improved compared to conventionally-seeded cells, but the effect was transient and diminished gradually with the cessation of magnetic force. Our results demonstrate that magnetic seeding improves the strength and uniformity of initial cell attachment to hydrogel surface in cell-specific manner, which may play a decisive role for the outcome in tissue engineering applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2948-2956, 2017., (© 2017 Wiley Periodicals, Inc.)

Background: Hypoxia is a major driving force in vascularization and vascular remodeling. Pharmacological inhibition of prolyl hydroxylases (PHDs) leads to an oxygen-independent and long-lasting activation of hypoxia-inducible factors (HIFs). Whereas effects of HIF-stabilization on transcriptional responses have been thoroughly investigated in endothelial cells, the molecular details of cytoskeletal changes elicited by PHD-inhibition remain largely unknown. To investigate this important aspect of PHD-inhibition, we used a spheroid-on-matrix cell culture model., Results: Microvascular endothelial cells (glEND.2) were organized into spheroids. Migration of cells from the spheroids was quantified and analyzed by immunocytochemistry. The PHD inhibitor dimethyloxalyl glycine (DMOG) induced F-actin stress fiber formation in migrating cells, but only weakly affected microvascular endothelial cells firmly attached in a monolayer. Compared to control spheroids, the residual spheroids were larger upon PHD inhibition and contained more cells with tight VE-cadherin positive cell-cell contacts. Morphological alterations were dependent on stabilization of HIF-1α and not HIF-2α as shown in cells with stable knockdown of HIF-α isoforms. DMOG-treated endothelial cells exhibited a reduction of immunoreactive Rac-1 at the migrating front, concomitant with a diminished Rac-1 activity, whereas total Rac-1 protein remained unchanged. Two chemically distinct Rac-1 inhibitors mimicked the effects of DMOG in terms of F-actin fiber formation and orientation, as well as stabilization of residual spheroids. Furthermore, phosphorylation of p21-activated kinase PAK downstream of Rac-1 was reduced by DMOG in a HIF-1α-dependent manner. Stabilization of cell-cell contacts associated with decreased Rac-1 activity was also confirmed in human umbilical vein endothelial cells., Conclusions: Our data demonstrates that PHD inhibition induces HIF-1α-dependent cytoskeletal remodeling in endothelial cells, which is mediated essentially by a reduction in Rac-1 signaling.

Background: Atherosclerotic plaques are initiated by pro-inflammatory endothelial activation at arterial regions subjected to non-uniform shear stress. We applied the in vitro flow-through cell culture slides to investigate whether different patterns of shear stress affect the secreted cytokine and chemokine profile in endothelial cells., Methods: Human umbilical vein endothelial cells (ECs) were exposed to 24 h of flow in straight or bifurcating flow-through slides, in some experiments followed by 6 h stimulation with 2.5 ng/mL TNF-α. IL-1β, IL-6, IL-8, IL-12p70, MIP-1α, MCP-1 and sICAM-1 were measured in conditioned medium samples by flow cytometry using antibodies conjugated with fluorescent beads., Results: The release of IL-1β, IL-12p70 and MIP-1α from endothelial cells exposed to shear stress was below detectable levels. Strongly increased level of sICAM and significantly increased IL-8 concentration were detected in conditioned medium from endothelial cells exposed to flow in bifurcating slides as compared with cells grown under laminar flow in straight channels. The release of IL-6 and MCP-1 was not significantly induced in bifurcating slides. Treatment with TNF-α for 6 h induced 2-3 fold increase in secreted chemokines and cytokines. In particular, significantly increased MCP-1 and increased IL-8 levels were released from endothelial cells grown in bifurcating slides. This release was partly prevented in cells grown in straight channels, i.e. under exposure to laminar flow only., Conclusions: Although the endothelial monolayer areas exposed to non-uniform shear stress are relatively small, the activation of cells in these regions is strong enough to produce a detectable change in cytokine and chemokine profile, which represents the earliest step in atherogenic endothelial dysfunction.

Background: Atherosclerotic plaques develop at arterial regions subjected to non-uniform shear stress, and are initiated by increased leukocyte-endothelial interactions. Here we applied the in vitro model of arterial bifurcations to investigate whether telmisartan, an anti-inflammatory angiotensin II receptor blocker with PPAR-gamma activating ability, prevents monocyte recruitment by endothelium., Methods: Human umbilical vein endothelial cells (ECs) were exposed to 18 h non-uniform shear stress in bifurcating flow-through slides, followed by 2 h stimulation with 2.5 ng/mL TNF-alpha. During flow, cells were treated with telmisartan. To study cell adhesion, ECs were perfused with medium containing THP-1 monocytic cells. Adherent THP-1 monocytes were quantified by light microscopy. Endothelial protein expression was determined by immunofluorescence., Results: Non-uniform shear stress in combination with TNF-alpha dramatically induced monocytic cell recruitment by endothelial cells. In cells treated with telmisartan (0.5-2.5 μmol/L) during exposure to non-uniform shear stress, dose-dependent inhibition of monocytic cell adhesion was observed, with about 45% reduction at 1 μmol/L. This effect was mediated by a significant reduction of endothelial VCAM-1 expression. On the contrary, the induction of E-selectin by TNF-alpha in ECs exposed to non-uniform shear stress was not affected by telmisartan. The inhibitory effect of telmisartan on monocytic cell recruitment and VCAM-1 induction was prevented in the presence of the PPAR-gamma antagonist GW9662., Conclusions: Treatment with telmisartan decreases the TNF-α-induced recruitment of monocytic cells and endothelial expression of VCAM-1 in regions of non-uniform shear stress in vitro. This mechanism can contribute to the beneficial pleiotropic effects of telmisartan in atherosclerosis-prone arterial regions.

Hypoxia, a driving force in neovascularization, promotes alterations in gene expression mediated by hypoxia-inducible factor (HIF)-1alpha. Connective tissue growth factor (CTGF, CCN2) is a modulator of endothelial cell growth and migration, but its regulation by hypoxia is poorly understood. Therefore, we analyzed signaling pathways involved in the regulation of CTGF by hypoxia in endothelial cells. Exposure to low oxygen tension or treatment with the hypoxia-mimetic dimethyloxalyl glycine (DMOG) stabilized HIF-1alpha and up-regulated CTGF in human umbilical vein endothelial cells and in a murine microvascular endothelial cell line. Induction of CTGF correlated with a HIF-dependent increase in protein and mRNA levels, and nuclear accumulation of the transcription factor FoxO3a. By contrast, gene expression and cellular localization of FoxO1 were not significantly altered by hypoxia. Expression of CTGF was strongly reduced by siRNA silencing of FoxO1 or FoxO3a. Furthermore, nuclear exclusion of FoxO1/3a transcription factors by inhibition of serine/threonine protein phosphatases by okadaic acid inhibited CTGF expression, providing evidence for both FoxO proteins as regulators of CTGF expression. The DMOG-stimulated induction of CTGF was further increased when endothelial cells were co-incubated with transforming growth factor-beta, an activator of Smad signaling. Activation of RhoA-Rho kinase signaling by the microtubule-disrupting drug combretastatin A4 also enhanced the DMOG-induced CTGF expression, thus placing CTGF induction by hypoxia in a network of interacting signaling pathways. Our findings provide evidence that FoxO1, hypoxia-stimulated expression of FoxO3a and its nuclear accumulation are required for the induction of CTGF by hypoxia in endothelial cells.

Background: Atherosclerotic plaques develop at arterial regions subjected to non-uniform shear stress, and are initiated by increased leukocyte-endothelial interactions. In this study, we investigated the effects of distinct shear stress patterns on endothelial recruitment of monocytic cells., Methods: Human umbilical vein endothelial cells (ECs) were exposed to laminar or non-uniform shear stress in bifurcating flow-through slides, followed by 2 h stimulation with TNF-α. To study cell adhesion, ECs were perfused with medium containing THP-1 monocytic cells for 1 h. Endothelial protein expression was determined by immunofluorescence., Results: Exposure to non-uniform shear stress and TNF-α lead to progressive induction of adhesion molecules and increase in monocytic cell adhesion observed over 0.5-3 h. To investigate the relative role of the shear stress patterns in monocytic cell recruitment, ECs were exposed to reduced levels of shear stress, resulting in a reduced gradient steepness in the non-uniform shear stress regions. Lowering the shear stress from 10 to 5 and 2 dyne/cm2 resulted in increased monocytic cell adhesion under laminar shear stress. However, in these conditions, adherent monocytic cells under non-uniform shear stress were strongly reduced. Moreover, in the region exposed to shear stress gradient parallel to flow direction, monocytic cell adhesion was significantly lower than in the region of non-uniform shear stress, characterized by transversal gradient., Conclusion: Exposure to non-uniform shear stress results in progressive induction of adhesion molecules and monocytic cell recruitment in response to circulating TNF-α. Enhanced monocytic cell recruitment at bifurcations is affected not only by the magnitude and steepness of shear stress gradient, but also by its direction in relation to the flow.

Objective: Atherosclerotic plaque formation results from a combination of local shear stress patterns and inflammatory processes. This study investigated the endothelial response to shear stress in combination with the inflammatory cytokine TNF-alpha in a simplified model of arterial bifurcation., Methods: Human umbilical vein endothelial cells (ECs) were exposed to laminar or non-uniform shear stress in bifurcating flow-through slides, followed by stimulation with TNF-alpha. To study cell adhesion, ECs were perfused with medium containing THP-1 monocytic cells. Endothelial protein expression was determined by immunofluorescence., Results: Adhesion of monocytic cells to unstimulated ECs was nearly undetectable under laminar shear stress and was slightly increased under non-uniform shear stress. Exposure of ECs to non-uniform shear stress in combination with TNF-alpha induced a 12-fold increase in monocytic cell recruitment and a significant induction of endothelial E-selectin and VCAM-1 expression. Both these effects were prevented in ECs exposed to laminar shear stress. The significant differences in TNF-alpha-induced monocytic cell recruitment and adhesion molecule expression between laminar and non-uniform shear stress regions were abolished in the absence of shear stress preconditioning. Simvastatin (1 micromol/L) suppressed the non-uniform shear stress- and TNF-alpha-induced increase in monocytic cell adhesion by about 30% via inhibition of VCAM-1 expression. Resveratrol, the active component of red wine, inhibited the expression of both VCAM-1 and E-selectin, and reduced monocytic cell recruitment by 50% at 20 micromol/L., Conclusions: Non-uniform shear stress induces endothelial susceptibility to circulating TNF-alpha and adhesion of monocytic cells. Interference with this process may inhibit inflammatory response in atherosclerosis-prone regions.

Shear stress changes play an important role in atheroma formation. This study focussed on atherogenic protein expression under non-uniform shear stress and the pharmacological modulation of shear-related endothelial dysfunction. Bifurcating flow-through cell culture slides were used to expose HUVECs to steady laminar or non-uniform shear stress for 18 h at 10 dyn/cm(2). Protein expression was determined by immunofluorescence, and quantified using MetaVue software. Laminar shear stress resulted in cell alignment, reduced F-actin fibers, and significant induction of endothelial nitric oxide synthase expression. Under non-uniform shear stress at bifurcations, minor upregulation of adhesion molecules was observed. Connective tissue growth factor (CTGF) was significantly downregulated by laminar shear stress and induced in cells exposed to non-uniform shear stress. CTGF upregulation by non-uniform shear stress was RhoA-dependent, because it was almost completely inhibited in cells transfected with dominant negative RhoA-N19, and when cells were treated with 1 micromol/L simvastatin during flow. Pre-incubation of HUVECs with inhibitors of Rho-associated kinase before exposure to flow significantly suppressed the CTGF induction in regions of non-uniform shear stress. In conclusion, non-uniform shear stress-dependent CTGF expression requires active RhoA and can be prevented pharmacologically. Interference with shear stress-induced protein expression may inhibit endothelial dysfunction in atheroprone vessel regions.

Expression of connective tissue growth factor (CTGF) in endothelial cells is modulated by shear stress affecting the organization of the cytoskeleton. The molecular connection between alterations of actin and CTGF expression was investigated in human umbilical vein endothelial cells (HUVEC) and a microvascular endothelial cell line. Overexpression of nonpolymerizable monomeric actin R62D interfered with stress fiber formation in HUVEC and concomitantly reduced immunoreactive CTGF. In microvascular endothelial cells, flow-dependent upregulation of CTGF was prevented by this actin mutant. In contrast, overexpression of actin S14C strengthened filamentous actin and increased CTGF expression. These data indicated an inverse relationship between CTGF expression and monomeric actin. Coexpression of the mutant actins and different CTGF promoter constructs revealed an actin-sensitive site between 3 and 4.5 kb of the CTGF promoter. A CArG-like box at -3791 bp was responsible for actin-dependent CTGF induction as shown by mutagenesis. Overexpression of actin S14C activated the nonmutated promoter significantly more strongly than the mutated promoter. Actin polymerization is regulated by the small GTPase RhoA and activation of serum response factor (SRF). Overexpression of constitutively active RhoA or SRF significantly increased CTGF protein synthesis. The 4.5-kb promoter construct, but not the construct with a mutation in the CArG box, was activated by SRF or RhoA, providing evidence for a functional role of this site in CTGF induction. These findings provide novel evidence that monomeric actin is the connecting link between alterations in the cytoskeleton and CTGF gene expression and demonstrate the importance of SRF in regulating CTGF transcription.