Endoplasmic Reticulum Stress Induces Vasodilation in Liver Vessels That Is Not Mediated by Unfolded Protein Response.

There is a growing body of evidence that ER stress and the unfolded protein response (UPR) play a key role in numerous diseases. Impaired liver perfusion and ER stress often accompany each other in liver diseases. However, the exact impact of ER stress and UPR on the hepatic perfusion is not fully understood. The aim of this study was to disclose the effect of ER stress and UPR on the size of liver vessels and on the levels of Ca2+ and nitric oxide (NO), critical regulators of vascular tonus. This study was carried out in precisely cut liver tissue slices. Confocal microscopy was used to create 3D images of vessels. NO levels were determined either using either laser scan microscopy (LSM) in cells or by NO-analyser in medium. Ca2+ levels were analysed by LSM. We show that tunicamycin, an inducer of ER stress, acts similarly with vasodilator acetylcholine. Both exert a similar effect on the NO and Ca2+ levels; both induce significant vasodilation. Notably, this vasodilative effect persisted despite individual inhibition of UPR pathways-ATF-6, PERK, and IRE1-despite confirming the activation of UPR. Experiments with HUVEC cells showed that elevated NO levels did not result from endothelial NO synthase (eNOS) activation. Our study suggests that tunicamycin-mediated ER stress induces liver vessel vasodilation in an NO-dependent manner, which is mediated by intracellular nitrodilator-activatable NO store (NANOS) in smooth muscle cells rather than by eNOS.

Engineering blood and lymphatic microvascular networks.

The human vasculature plays a crucial role in the blood supply of nearly all organs as well as the drainage of the interstitial fluid. Consequently, if these physiological systems go awry, pathological changes might occur. Hence, the regeneration of existing vessels, as well as approaches to engineer artificial blood and lymphatic structures represent current challenges within the field of vascular research. In this review, we provide an overview of both the vascular blood circulation and the long-time neglected but equally important lymphatic system, with regard to their organotypic vasculature. We summarize the current knowledge within the field of vascular tissue engineering focusing on the design of co-culture systems, thereby mainly discussing suitable cell types, scaffold design and disease models. This review will mainly focus on addressing those subjects concerning atherosclerosis. Moreover, current technological approaches such as vascular organ-on-a-chip models and microfluidic devices will be discussed.

Circulating endothelial extracellular vesicle signatures correspond with ICU requirement: an exploratory study in COVID-19 patients.

Extracellular vesicles (EVs) represent nanometer-sized, subcellular spheres, that are released from almost any cell type and carry a wide variety of biologically relevant cargo. In severe cases of coronavirus disease 2019 (COVID-19) and other states of systemic pro-inflammatory activation, EVs, and their cargo can serve as conveyors and indicators for disease severity and progression. This information may help distinguish individuals with a less severe manifestation of the disease from patients who exhibit severe acute respiratory distress syndrome (ARDS) and require intensive care measures. Here, we investigated the potential of EVs and associated miRNAs to distinguish normal ward patients from intensive care unit (ICU) patients (N = 10/group), with 10 healthy donors serving as the control group. Blood samples from which plasma and subsequently EVs were harvested by differential ultracentrifugation (UC) were obtained at several points in time throughout treatment. EV-enriched fractions were characterized by flow cytometry (FC), nanoparticle tracking analysis (NTA), and qPCR to determine the presence of selected miRNAs. Circulating EVs showed specific protein signatures associated with endothelial and platelet origin over the course of the treatment. Additionally, significantly higher overall EV quantities corresponded with increased COVID-19 severity. MiR-223-3p, miR-191-5p, and miR-126-3p exhibited higher relative expression in the ICU group. Furthermore, EVs presenting endothelial-like protein signatures and the associated miR-126-3p showed the highest area under the curve in terms of receiver operating characteristics regarding the requirement for ICU treatment. In this exploratory investigation, we report that specific circulating EVs and miRNAs appear at higher levels in COVID-19 patients, especially when critical care measures are indicated. Our data suggest that endothelial-like EVs and associated miRNAs likely represent targets for future laboratory assays and may aid in clinical decision-making in COVID-19.

Occurrence of Lymphangiogenesis in Peripheral Nerve Autografts Contrasts Schwann Cell-Induced Apoptosis of Lymphatic Endothelial Cells In Vitro.

Peripheral nerve injuries pose a major clinical concern world-wide, and functional recovery after segmental peripheral nerve injury is often unsatisfactory, even in cases of autografting. Although it is well established that angiogenesis plays a pivotal role during nerve regeneration, the influence of lymphangiogenesis is strongly under-investigated. In this study, we analyzed the presence of lymphatic vasculature in healthy and regenerated murine peripheral nerves, revealing that nerve autografts contained increased numbers of lymphatic vessels after segmental damage. This led us to elucidate the interaction between lymphatic endothelial cells (LECs) and Schwann cells (SCs) in vitro. We show that SC and LEC secretomes did not influence the respective other cell types' migration and proliferation in 2D scratch assay experiments. Furthermore, we successfully created lymphatic microvascular structures in SC-embedded 3D fibrin hydrogels, in the presence of supporting cells; whereas SCs seemed to exert anti-lymphangiogenic effects when cultured with LECs alone. Here, we describe, for the first time, increased lymphangiogenesis after peripheral nerve injury and repair. Furthermore, our findings indicate a potential lymph-repellent property of SCs, thereby providing a possible explanation for the lack of lymphatic vessels in the healthy endoneurium. Our results highlight the importance of elucidating the molecular mechanisms of SC-LEC interaction.

Multi-Level Analysis of Adipose Tissue Reveals the Relevance of Perivascular Subpopulations and an Increased Endothelial Permeability in Early-Stage Lipedema.

Lipedema is a chronic, progressive disease of adipose tissue with unknown etiology. Based on the relevance of the stromal vascular fraction (SVF) cell population in lipedema, we performed a thorough characterization of subcutaneous adipose tissue, SVF isolated thereof and the sorted populations of endothelial cells (EC), pericytes and cultured adipose-derived stromal/stem cells (ASC) of early-stage lipedema patients. We employed histological and gene expression analysis and investigated the endothelial barrier by immunofluorescence and analysis of endothelial permeability in vitro. Although there were no significant differences in histological stainings, we found altered gene expression of factors relevant for local estrogen metabolism (aromatase), preadipocyte commitment (ZNF423) and immune cell infiltration (CD11c) in lipedema on the tissue level, as well as in distinct cellular subpopulations. Machine learning analysis of immunofluorescence images of CD31 and ZO-1 revealed a morphological difference in the cellular junctions of EC cultures derived from healthy and lipedema individuals. Furthermore, the secretome of lipedema-derived SVF cells was sufficient to significantly increase leakiness of healthy human primary EC, which was also reflected by decreased mRNA expression of VE-cadherin. Here, we showed for the first time that the secretome of SVF cells creates an environment that triggers endothelial barrier dysfunction in early-stage lipedema. Moreover, since alterations in gene expression were detected on the cellular and/or tissue level, the choice of sample material is of high importance in elucidating this complex disease.

Lymphatic and Blood Endothelial Extracellular Vesicles: A Story Yet to Be Written.

Extracellular vesicles (EVs), such as exosomes, microvesicles, and apoptotic bodies, are cell-derived, lipid bilayer-enclosed particles mediating intercellular communication and are therefore vital for transmitting a plethora of biological signals. The vascular endothelium substantially contributes to the circulating particulate secretome, targeting important signaling pathways that affect blood cells and regulate adaptation and plasticity of endothelial cells in a paracrine manner. Different molecular signatures and functional properties of endothelial cells reflect their heterogeneity among different vascular beds and drive current research to understand varying physiological and pathological effects of blood and lymphatic endothelial EVs. Endothelial EVs have been linked to the development and progression of various vascular diseases, thus having the potential to serve as biomarkers and clinical treatment targets. This review aims to provide a brief overview of the human vasculature, the biology of extracellular vesicles, and the current knowledge of endothelium-derived EVs, including their potential role as biomarkers in disease development.

Guiding cell migration in 3D with high-resolution photografting.

Multi-photon lithography (MPL) has proven to be a suitable tool to precisely control the microenvironment of cells in terms of the biochemical and biophysical properties of the hydrogel matrix. In this work, we present a novel method, based on multi-photon photografting of 4,4'-diazido-2,2'-stilbenedisulfonic acid (DSSA), and its capabilities to induce cell alignment, directional cell migration and endothelial sprouting in a gelatin-based hydrogel matrix. DSSA-photografting allows for the fabrication of complex patterns at a high-resolution and is a biocompatible, universally applicable and straightforward process that is comparably fast. We have demonstrated the preferential orientation of human adipose-derived stem cells (hASCs) in response to a photografted pattern. Co-culture spheroids of hASCs and human umbilical vein endothelial cells (HUVECs) have been utilized to study the directional migration of hASCs into the modified regions. Subsequently, we have highlighted the dependence of endothelial sprouting on the presence of hASCs and demonstrated the potential of photografting to control the direction of the sprouts. MPL-induced DSSA-photografting has been established as a promising method to selectively alter the microenvironment of cells.

Establishment of a human three-dimensional chip-based chondro-synovial coculture joint model for reciprocal cross talk studies in arthritis research.

Rheumatoid arthritis is characterised by a progressive, intermittent inflammation at the synovial membrane, which ultimately leads to the destruction of the synovial joint. The synovial membrane as the joint capsule's inner layer is lined with fibroblast-like synoviocytes that are the key player supporting persistent arthritis leading to bone erosion and cartilage destruction. While microfluidic models that model molecular aspects of bone erosion between bone-derived cells and synoviocytes have been established, RA's synovial-chondral axis has not yet been realised using a microfluidic 3D model based on human patient in vitro cultures. Consequently, we established a chip-based three-dimensional tissue coculture model that simulates the reciprocal cross talk between individual synovial and chondral organoids. When co-cultivated with synovial organoids, we could demonstrate that chondral organoids induce a higher degree of cartilage physiology and architecture and show differential cytokine response compared to their respective monocultures highlighting the importance of reciprocal tissue-level cross talk in the modelling of arthritic diseases.

Cre mRNA Is Not Transferred by EVs from Endothelial and Adipose-Derived Stromal/Stem Cells during Vascular Network Formation.

Coculture systems employing adipose tissue-derived mesenchymal stromal/stem cells (ASC) and endothelial cells (EC) represent a widely used technique to model vascularization. Within this system, cell-cell communication is crucial for the achievement of functional vascular network formation. Extracellular vesicles (EVs) have recently emerged as key players in cell communication by transferring bioactive molecules between cells. In this study we aimed to address the role of EVs in ASC/EC cocultures by discriminating between cells, which have received functional EV cargo from cells that have not. Therefore, we employed the Cre-loxP system, which is based on donor cells expressing the Cre recombinase, whose mRNA was previously shown to be packaged into EVs and reporter cells containing a construct of floxed dsRed upstream of the eGFP coding sequence. The evaluation of Cre induced color switch in the reporter system via EVs indicated that there is no EV-mediated RNA transmission either between EC themselves or EC and ASC. However, since Cre mRNA was not found present in EVs, it remains unclear if Cre mRNA is generally not packaged into EVs or if EVs are not taken up by the utilized cell types. Our data indicate that this technique may not be applicable to evaluate EV-mediated cell-to-cell communication in an in vitro setting using EC and ASC. Further investigations will require a functional system showing efficient and specific loading of Cre mRNA or protein into EVs.

Fluorescence-Based Nanoparticle Tracking Analysis and Flow Cytometry for Characterization of Endothelial Extracellular Vesicle Release.

As extracellular vesicles (EVs) have become a prominent topic in life sciences, a growing number of studies are published on a regular basis addressing their biological relevance and possible applications. Nevertheless, the fundamental question of the true vesicular nature as well as possible influences on the EV secretion behavior have often been not adequately addressed. Furthermore, research regarding endothelial cell-derived EVs (EndoEVs) often focused on the large vesicular fractions comprising of microvesicles (MV) and apoptotic bodies. In this study we aimed to further extend the current knowledge of the influence of pre-isolation conditions, such as cell density and conditioning time, on EndoEV release from human umbilical vein endothelial cells (HUVECs). We combined fluorescence nanoparticle tracking analysis (NTA) and the established fluorescence-triggered flow cytometry (FT-FC) protocol to allow vesicle-specific detection and characterization of size and surface markers. We found significant effects of cell density and conditioning time on both abundance and size distribution of EndoEVs. Additionally, we present detailed information regarding the surface marker display on EVs from different fractions and size ranges. Our data provide crucial relevance for future projects aiming to elucidate EV secretion behavior of endothelial cells. Moreover, we show that the influence of different conditioning parameters on the nature of EndoEVs has to be considered.

Purinergic P2Y2 receptors modulate endothelial sprouting.

Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth.

Ex vivo engineering of blood and lymphatic microvascular networks.

Upon implantation, engineered tissues rely on the supply with oxygen and nutrients as well as the drainage of interstitial fluid. This prerequisite still represents one of the current challenges in the engineering and regeneration of tissues. Recently, different vascularization strategies have been developed. Besides technical approaches like 3D printing or laser processing and de-/recelluarization of natural scaffolds, mainly co-cultures of endothelial cells (ECs) with supporting cell types are being used. This mini-review provides a brief overview of different co-culture systems for the engineering of blood and lymphatic microvascular networks.

Microvascular Networks From Endothelial Cells and Mesenchymal Stromal Cells From Adipose Tissue and Bone Marrow: A Comparison.

A promising approach to overcome hypoxic conditions in tissue engineered constructs is to use the potential of endothelial cells (EC) to form networks in vitro when co-cultured with a supporting cell type in a 3D environment. Adipose tissue-derived stromal cells (ASC) as well as bone marrow-derived stromal cells (BMSC) have been shown to support vessel formation of EC in vitro, but only very few studies compared the angiogenic potential of both cell types using the same model. Here, we aimed at investigating the ability of ASC and BMSC to induce network formation of EC in a co-culture model in fibrin. While vascular structures of BMSC and EC remained stable over the course of 3 weeks, ASC-EC co-cultures developed more junctions and higher network density within the same time frame. Both co-cultures showed positive staining for neural glial antigen 2 (NG2) and basal lamina proteins. This indicates that vessels matured and were surrounded by perivascular cells as well as matrix molecules involved in stabilization. Gene expression analysis revealed a significant increase of vascular endothelial growth factor (VEGF) expression in ASC-EC co-culture compared to BMSC-EC co-culture. These observations were donor-independent and highlight the importance of organotypic cell sources for vascular tissue engineering.

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.

Every Breath You Take: Non-invasive Real-Time Oxygen Biosensing in Two- and Three-Dimensional Microfluidic Cell Models.

Knowledge on the availability of dissolved oxygen inside microfluidic cell culture systems is vital for recreating physiological-relevant microenvironments and for providing reliable and reproducible measurement conditions. It is important to highlight that in vivo cells experience a diverse range of oxygen tensions depending on the resident tissue type, which can also be recreated in vitro using specialized cell culture instruments that regulate external oxygen concentrations. While cell-culture conditions can be readily adjusted using state-of-the-art incubators, the control of physiological-relevant microenvironments within the microfluidic chip, however, requires the integration of oxygen sensors. Although several sensing approaches have been reported to monitor oxygen levels in the presence of cell monolayers, oxygen demands of microfluidic three-dimensional (3D)-cell cultures and spatio-temporal variations of oxygen concentrations inside two-dimensional (2D) and 3D cell culture systems are still largely unknown. To gain a better understanding on available oxygen levels inside organ-on-a-chip systems, we have therefore developed two different microfluidic devices containing embedded sensor arrays to monitor local oxygen levels to investigate (i) oxygen consumption rates of 2D and 3D hydrogel-based cell cultures, (ii) the establishment of oxygen gradients within cell culture chambers, and (iii) influence of microfluidic material (e.g., gas tight vs. gas permeable), surface coatings, cell densities, and medium flow rate on the respiratory activities of four different cell types. We demonstrate how dynamic control of cyclic normoxic-hypoxic cell microenvironments can be readily accomplished using programmable flow profiles employing both gas-impermeable and gas-permeable microfluidic biochips.

Engineering of three-dimensional pre-vascular networks within fibrin hydrogel constructs by microfluidic control over reciprocal cell signaling.

Reengineering functional vascular networks in vitro remains an integral part in tissue engineering, since the incorporation of non-perfused tissues results in restricted nutrient supply and limited waste removal. Microfluidic devices are routinely used to mimic both physiological and pathological vascular microenvironments. Current procedures either involve the investigation of growth factor gradients and interstitial flow on endothelial cell sprouting alone or on the heterotypic cell-cell interactions between endothelial and mural cells. However, limited research has been conducted on the influence of flow on co-cultures of these cells. Here, we exploited the ability of microfluidics to create and monitor spatiotemporal gradients to investigate the influence of growth factor supply and elution on vascularization using static as well as indirect and direct flow setups. Co-cultures of human adipose-derived stem/stromal cells and human umbilical vein endothelial cells embedded in fibrin hydrogels were found to be severely affected by diffusion limited growth factor gradients as well as by elution of reciprocal signaling molecules during both static and flow conditions. Static cultures formed pre-vascular networks up to a depth of 4 mm into the construct with subsequent decline due to diffusion limitation. In contrast, indirect flow conditions enhanced endothelial cell sprouting but failed to form vascular networks. Additionally, complete inhibition of pre-vascular network formation was observable for direct application of flow through the hydrogel with decline of endothelial cell viability after seven days. Using finite volume CFD simulations of different sized molecules vital for pre-vascular network formation into and out of the hydrogel constructs, we found that interstitial flow enhances growth factor supply to the cells in the bulk of the chamber but elutes cellular secretome, resulting in truncated, premature vascularization.

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.