Correction to: The role of fibrinolysis inhibition in engineered vascular networks derived from endothelial cells and adipose-derived stem cells.

The original article [1] contains numerous value errors in the graphs in Fig. 2b regarding the markers describing the values for total tubule length and mean tubule length without aprotinin at 2.5 mg/ml concentration of fibrinogen. The corrected version of this figure can be viewed ahead.

The role of fibrinolysis inhibition in engineered vascular networks derived from endothelial cells and adipose-derived stem cells.

BACKGROUND: Co-cultures of endothelial cells with mesenchymal stem cells currently represent one of the most promising approaches in providing oxygen and nutrient supply for microvascular tissue engineering. Still, to translate this model into clinics several in vitro parameters including growth medium and scaffold degradation need to be fine-tuned. METHODS: We recently described the co-culture of adipose-derived stem cells with endothelial cells in fibrin, resulting in capillary formation in vitro as well as their perfusion in vivo. Here, we aimed to further characterise microvascular tube formation in fibrin by determining the role of scaffold degradation, thrombin concentration and culture conditions on vascularisation. RESULTS: We observed that inhibition of cell-mediated fibrin degradation by the commonly used inhibitor aprotinin resulted in impaired vascular network formation. Aprotinin had no effect on laminin and collagen type IV deposition or formation of tube-like structures in scaffold-free co-culture, indicating that poor vascularisation of fibrin clots is primarily caused by inhibition of plasminogen-driven fibrinolysis. Co-culture in plasminogen- and factor XIII-depleted fibrin did not result in different vascular network density compared to controls. Furthermore, we demonstrate that thrombin negatively affects vascular network density at high concentrations. However, only transient activation of incorporated endothelial cells by thrombin could be observed, thus excluding a long-term inflammatory response in tissue-engineered micro-capillaries. Finally, we show that vascularisation of fibrin scaffolds in basal medium is undermined because of increased fibrinolytic activity leading to scaffold destabilisation without aprotinin. CONCLUSIONS: Taken together, our data reveal a critical role of fibrinolysis inhibition in in vitro cell-mediated vascularisation of fibrin scaffolds.

Selective IKK2 inhibitor IMD0354 disrupts NF-κB signaling to suppress corneal inflammation and angiogenesis.

Corneal neovascularization is a sight-threatening condition caused by angiogenesis in the normally avascular cornea. Neovascularization of the cornea is often associated with an inflammatory response, thus targeting VEGF-A alone yields only a limited efficacy. The NF-κB signaling pathway plays important roles in inflammation and angiogenesis. Here, we study consequences of the inhibition of NF-κB activation through selective blockade of the IKK complex IκB kinase ß (IKK2) using the compound IMD0354, focusing on the effects of inflammation and pathological angiogenesis in the cornea. In vitro, IMD0354 treatment diminished HUVEC migration and tube formation without an increase in cell death and arrested rat aortic ring sprouting. In HUVEC, the IMD0354 treatment caused a dose-dependent reduction in VEGF-A expression, suppressed TNFα-stimulated expression of chemokines CCL2 and CXCL5, and diminished actin filament fibers and cell filopodia formation. In developing zebrafish embryos, IMD0354 treatment reduced expression of Vegf-a and disrupted retinal angiogenesis. In inflammation-induced angiogenesis in the rat cornea, systemic selective IKK2 inhibition decreased inflammatory cell invasion, suppressed CCL2, CXCL5, Cxcr2, and TNF-α expression and exhibited anti-angiogenic effects such as reduced limbal vessel dilation, reduced VEGF-A expression and reduced angiogenic sprouting, without noticeable toxic effect. In summary, targeting NF-κB by selective IKK2 inhibition dampened the inflammatory and angiogenic responses in vivo by modulating the endothelial cell expression profile and motility, thus indicating an important role of NF-κB signaling in the development of pathologic corneal neovascularization.

Protective Effects of Oral Astaxanthin Nanopowder against Ultraviolet-Induced Photokeratitis in Mice.

PURPOSE: Astaxanthin (AST) has a strong antioxidant cellular membrane chaperone protective effect. Recently, a water-soluble nanosized AST (nano-AST) form was produced, which is expected to improve the efficacy of oral intake effects. The purpose of this study was to examine whether oral nano-AST has therapeutic effects on UV-induced photokeratitis in mice. METHODS: C57BL/6 mice were administered twice with either nano-AST, AST oil, lutein, or bilberry extracts 3 hours before and shortly before UV irradiation (dose: 400 mJ/cm2). The corneas were collected 24 hours after irradiation and stained with H&E and TUNEL. NF-κB, dihydroethidium (DHE), COX-2, p-IκB-α, TNFα, and CD45 expression were evaluated through immunohistochemistry, Western blot analysis, and qPCR. RESULTS: Corneal epithelium was significantly thicker in mice orally administered with nano-AST than in the others (p < 0.01), with significantly less NF-κB nucleus translocation (p < 0.001), and significantly fewer TUNEL cells (p < 0.01). Weaker DHE signals were detected in the nano-AST group (p < 0.05) relative to the others. Furthermore, reduced inflammation and decreased cell death in corneal tissue were observed in the nano-AST group, as indicated by a reduction in the expression of COX-2, p-IκB-α, TNFα, and CD45. CONCLUSIONS: Oral administration of nano-AST demonstrated a protective effect on UV-induced photokeratitis via antioxidative, anti-inflammatory, and antiapoptotic activity.

Genome-wide expression datasets of anti-VEGF and dexamethasone treatment of angiogenesis in the rat cornea.

Therapeutics against pathologic new blood vessel growth, particularly those targeting vascular endothelial growth factor (VEGF) are of enormous clinical interest. In the eye, where anti-VEGF agents are in widespread clinical use for treating retinal and corneal blindness, only partial or transient efficacy and resistance to anti-VEGF agents are among the major drawbacks. Conversely, corticosteroids have long been used in ophthalmology for their potency in suppressing inflammation and angiogenesis, but their broad biological activity can give rise to side effects such as glaucoma and cataract. To aid in the search for more targeted and effective anti-angiogenic therapies in the eye, we present here a dataset comparing gene expression changes in dexamethasone versus anti-Vegfa treatment of inflammation leading to angiogenesis in the rat cornea. Global gene expression analysis with GeneChip Rat 230 2.0 microarrays was conducted and the metadata submitted to Expression Omnibus repository. Here, we present a high-quality validated dataset enabling genome-wide comparison of genes differentially targeted by dexamethasone and anti-Vegf treatments, to identify potential alternative therapeutic targets for evaluation.

Genome-wide expression differences in anti-Vegf and dexamethasone treatment of inflammatory angiogenesis in the rat cornea.

Angiogenesis as a pathological process in the eye can lead to blindness. In the cornea, suppression of angiogenesis by anti-VEGF treatment is only partially effective while steroids, although effective in treating inflammation and angiogenesis, have broad activity leading to undesirable side effects. In this study, genome-wide expression was investigated in a suture-induced corneal neovascularization model in rats, to investigate factors differentially targeted by dexamethasone and anti-Vegf. Topical treatment with either rat-specific anti-Vegf, dexamethasone, or normal goat IgG (sham) was given to sutured corneas for 48 hours, after which in vivo imaging, tissue processing for RNA microarray, and immunofluorescence were performed. Dexamethasone suppressed limbal vasodilation (P < 0.01) and genes in PI3K-Akt, focal adhesion, and chemokine signaling pathways more effectively than anti-Vegf. The most differentially expressed genes were confirmed by immunofluorescence, qRTPCR and Western blot. Strong suppression of Reg3g and the inflammatory chemokines Ccl2 and Cxcl5 and activation of classical complement pathway factors C1r, C1s, C2, and C3 occurred with dexamethasone treatment, effects absent with anti-Vegf treatment. The genome-wide results obtained in this study provide numerous potential targets for specific blockade of inflammation and angiogenesis in the cornea not addressed by anti-Vegf treatment, as possible alternatives to broad-acting immunosuppressive therapy.

Engineering Blood and Lymphatic Microvascular Networks in Fibrin Matrices.

Vascular network engineering is essential for nutrient delivery to tissue-engineered constructs and, consequently, their survival. In addition, the functionality of tissues also depends on tissue drainage and immune cell accessibility, which are the main functions of the lymphatic system. Engineering both the blood and lymphatic microvasculature would advance the survival and functionality of tissue-engineered constructs. The aim of this study was to isolate pure populations of lymphatic endothelial cells (LEC) and blood vascular endothelial cells (BEC) from human dermal microvascular endothelial cells and to study their network formation in our previously described coculture model with adipose-derived stromal cells (ASC) in fibrin scaffolds. We could follow the network development over a period of 4 weeks by fluorescently labeling the cells. We show that LEC and BEC form separate networks, which are morphologically distinguishable and sustainable over several weeks. In addition, lymphatic network development was dependent on vascular endothelial growth factor (VEGF)-C, resulting in denser networks with increasing VEGF-C concentration. Finally, we confirm the necessity of cell-cell contact between endothelial cells and ASC for the formation of both blood and lymphatic microvascular networks. This model represents a valuable platform for in vitro drug testing and for the future in vivo studies on lymphatic and blood microvascularization.

Lymphatic Vessels in Regenerative Medicine and Tissue Engineering.

The lymphatic system is involved in maintaining interstitial fluid homeostasis, fat absorption, and immune surveillance. Dysfunction of lymphatic fluid uptake can lead to lymphedema. Worldwide up to 250 million people are estimated to suffer from this disfiguring and disabling disease, which places a strain on the healthcare system as well as on the affected patients. The severity of lymphatic diseases calls for the establishment of new treatment methods. One approach is to replace dysfunctional lymphatic vessels with bioengineered ones. In this study, we mainly focus on hydrogels, scaffolds with cellular constructs, interstitial flow, and extracorporeal shockwave therapy. This review provides an overview on the current status of lymphatic biology and approaches of reconstruction and regeneration of lymphatic vascular tissues.