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Organoids derived from human pluripotent stem cells are interesting models to study mechanisms of morphogenesis and promising platforms for disease modeling and drug screening. However, they mostly remain incomplete as they lack stroma, tissue resident immune cells and in particular vasculature, which create important niches during development and disease. We propose, that the directed incorporation of mesodermal progenitor cells (MPCs) into organoids will overcome the aforementioned limitations. In order to demonstrate the feasibility of the method, we generated complex human tumor as well as neural organoids. We show that the formed blood vessels display a hierarchic organization and mural cells are assembled into the vessel wall. Moreover, we demonstrate a typical blood vessel ultrastructure including endothelial cell-cell junctions, a basement membrane as well as luminal caveolae and microvesicles. We observe a high plasticity in the endothelial network, which expands, while the organoids grow and is responsive to anti-angiogenic compounds and pro-angiogenic conditions such as hypoxia. We show that vessels within tumor organoids connect to host vessels following transplantation. Remarkably, MPCs also deliver Iba1 + cells that infiltrate the neural tissue in a microglia-like manner.

Transition from the transient hyaloid vasculature to the mature retinal vascular network is a critical step in mammalian eye development. While WNT7B signaling in endothelial cells regulates programmed hyaloid vessel regression, the specific receptors and downstream mechanisms remain poorly defined. Here, we identify GPR124, a ligand-specific co-receptor for WNT7A/B, as a key regulator of this process. Using an endothelial-specific, tamoxifen-inducible Gpr124 knockout mouse model, we show that loss of GPR124 in hyaloid endothelium markedly impairs postnatal hyaloid vessel clearance. Notably, GPR124 function in the developing eye was highly compartment-specific: although crucial for hyaloid vessel regression, it was dispensable for retinal angiogenesis, despite being expressed in both vascular beds. Gpr124 deletion led to downregulation of several established WNT target genes in hyaloid endothelial cells, consistent with a role for GPR124 in mediating WNT/β-catenin signaling, as previously shown in brain endothelium. While GPR124 was not required for induction of isolated apoptotic events in hyaloid vessels, it promoted segmental vessel regression, characterized by linear clusters of apoptotic endothelial cells. Single-cell RNA sequencing of hyaloid endothelial cells, combined with immunostaining, revealed transcriptional changes consistent with partial endothelial-mesenchymal transition (EndMT). Our findings identify GPR124 as a key mediator of hyaloid vessel regression and suggest that GPR124-dependent WNT/β-catenin signaling and EndMT may contribute to vascular remodeling during ocular development.

3D cell culture models which closely resemble real human tissues are of high interest for disease modelling, drug screening as well as a deeper understanding of human developmental biology. Such structures are termed organoids. Within the last years, several human organoid models were described. These are usually stem cell derived, arise by self-organization, mimic mechanisms of normal tissue development, show typical organ morphogenesis and recapitulate at least some organ specific functions. Many tissues have been reproduced in vitro such as gut, liver, lung, kidney and brain. The resulting entities can be either derived from an adult stem cell population, or generated from pluripotent stem cells using a specific differentiation protocol. However, many organoid models only recapitulate the organs parenchyma but are devoid of stromal components such as blood vessels, connective tissue and inflammatory cells. Recent studies show that the incorporation of endothelial and mesenchymal cells into organoids improved their maturation and might be required to create fully functional micro-tissues, which will allow deeper insights into human embryogenesis as well as disease development and progression. In this review article, we will summarize and discuss recent works trying to incorporate stromal components into organoids, with a special focus on neural organoid models.

Background Vasculature is an essential part of all tissues and organs and is involved in a wide range of different diseases. However, available software for blood vessel image analysis is often limited: Some only process two-dimensional data, others lack batch processing, putting a time burden on the user, while still others require tightly defined culturing methods and experimental conditions. This highlights the need for software that has the ability to batch process three-dimensional image data and requires few and simple experimental preparation steps. Results We present VESNA, a Fiji (ImageJ) macro for automated segmentation and skeletonization of three-dimensional fluorescence images, enabling quantitative vascular network analysis. It requires only basic experimental preparation, making it highly adaptable to a wide range of possible applications across experimental goals and different tissue culturing methods. The macro’s potential is demonstrated on a range of different image data sets, from organoids with varying sizes, network complexities, and growth conditions, to expanding to other 3D tissue culturing methods, with an example of hydrogel-based cultures. Conclusions With its ability to process large amounts of 3D image data and its flexibility across experimental conditions, VESNA fulfills previously unmet needs in image processing of vascular structures and can be a valuable tool for a variety of experimental setups around three-dimensional vasculature, such as drug screening, research in tissue development and disease mechanisms.

Salivary gland tumors are a rare tumor entity within malignant tumors of all tissues. The most common are malignant mucoepidermoid carcinoma, adenoid cystic carcinoma, and acinic cell carcinoma. Pleomorphic adenoma is the most recurrent form of benign salivary gland tumor. Due to their low incidence rates and complex histological patterns, they are difficult to diagnose accurately. Malignant tumors of the salivary glands are challenging in terms of differentiation because of their variability in histochemistry and translocations. Therefore, the primary goal of the study was to review the current literature to identify the recent developments in histochemical diagnostics and translocations for differentiating salivary gland tumors.

Chronic inflammation with progressive age, called inflammaging, contributes to the pathogenesis of cardiovascular diseases. Previously, we have shown increased vascular expression of the Carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) in aged mice and humans, presumably via mutual upregulation with the pro‐inflammatory cytokine TNF‐α. CEACAM1 is critical for aging‐associated vascular alterations like endothelial dysfunction, fibrosis, oxidative stress, and sustained inflammation and can be regarded as a main contributor to vascular inflammaging. This study was conducted to elucidate the mechanisms underlying endothelial CEACAM1 upregulation by TNF‐α in detail. Using wildtype (WT) and TNF‐α knockout (Tnf−/−) mice, we confirmed that the aging‐related upregulation of endothelial CEACAM1 critically depends on TNF‐α. The underlying mechanisms were analyzed in an endothelial cell culture model. TNF‐α time‐dependently upregulated CEACAM1 in vitro. In pharmacological experiments, we identified an early NF‐κB‐ and a delayed β‐catenin‐mediated response. Involvement of β‐catenin was further substantiated by siRNA‐mediated knockdown of the β‐catenin‐targeted transcription factor TCF4. Both signaling pathways acted independent from each other. Elucidating the delayed response, co‐immunoprecipitation analysis revealed release of β‐catenin from adherens junctions by TNF‐α. Finally, TNF‐α activated Akt kinase by increasing its Ser473 phosphorylation. Consequently, Akt kinase facilitated β‐catenin signaling by inhibiting its degradation via phosphorylation of GSK3β at Ser9 and by increased phosphorylation of β‐catenin at Ser552 that augments its transcriptional activity. Taken together, our study provides novel mechanistic insights into the aging‐related, inflammation‐mediated endothelial upregulation of CEACAM1. Beyond the pathogenesis of cardiovascular diseases, these findings may be significant to all fields of inflammaging. Early response: TNF‐α activates NF‐κB signaling by IKK‐induced degradation of the inhibitor of nuclear factor kappa B (IκB). Released NF‐κB translocates into the nucleus and upregulates CEACAM1 expression detectable after 24 h of TNF‐α stimulation. Delayed response: TNF‐α‐induced adherens junction disassembly releases β‐catenin, promoting its cytosolic and nuclear accumulation. Nuclear β‐catenin binds to transcription factors of the TCF/LEF family to induce CEACAM1 expression. Akt kinase inhibits GSK3β by phosphorylation thereby stabilizing β‐catenin and directly phosphorylates β‐catenin which was reported to increase its transcriptional activity.

Salivary gland (SG) hypofunction is a common post-radiotherapy complication. Besides the parenchymal damage after irradiation (IR), there are also effects on mesenchymal stem cells (MSCs) which were shown to contribute to regeneration and repair of damaged tissues by differentiating into stromal cell types or releasing vesicles and soluble factors supporting the healing processes. However, there are no adequate reports about their roles during SG damage and regeneration so far. Using an irradiated SG mouse model, we performed certain immunostainings on tissue sections of submandibular glands at different time points after IR. Immunostaining for CD31 revealed that already one day after IR, vascular impairment was induced at the level of capillaries. In addition, the expression of CD44—a marker of acinar cells—diminished gradually after IR and, by 20 weeks, almost disappeared. In contrast, the number of CD34-positive cells significantly increased 4 weeks after IR and some of the CD34-positive cells were found to reside within the adventitia of arteries and veins. Laser confocal microscopic analyses revealed an accumulation of CD34-positive cells within the area of damaged capillaries where they were in close contact to the CD31-positive endothelial cells. At 4 weeks after IR, a fraction of the CD34-positive cells underwent differentiation into α-SMA-positive cells, which suggests that they may contribute to regeneration of smooth muscle cells and/or pericytes covering the small vessels from the outside. In conclusion, SG-resident CD34-positive cells represent a population of progenitors that could contribute to new vessel formation and/or remodeling of the pre-existing vessels after IR and thus, might be an important player during SG tissue healing.

: Pulmonary vascular endotheliitis, perivascular inflammation, and immune activation are observed in COVID-19 patients. While the initial SARS-CoV-2 infection mainly infects lung epithelial cells, whether it also infects endothelial cells (ECs) and to what extent SARS-CoV-2-mediated pulmonary vascular endotheliitis is associated with immune activation remain to be determined. : To address these questions, we studied SARS-CoV-2-infected ( ) mice, a severe COVID-19 mouse model, as well as lung samples from SARS-CoV-2-infected nonhuman primates (NHP) and patient deceased from COVID-19. We used immunostaining, RNAscope, and electron microscopy to analyze the organs collected from animals and patient. We conducted bulk and single cell (sc) RNA-seq analyses, and cytokine profiling of lungs or serum of the severe COVID-19 mice. : We show that SARS-CoV-2-infected mice develop severe COVID-19, including progressive body weight loss and fatality at 7 days, severe lung interstitial inflammation, edema, hemorrhage, perivascular inflammation, systemic lymphocytopenia, and eosinopenia. Body weight loss in mice correlated with the severity of pneumonia, but not with brain infection. We also observed endothelial activation and dysfunction in pulmonary vessels evidenced by the up-regulation of VCAM1 and ICAM1 and the downregulation of VE-cadherin. We detected SARS-CoV-2 in capillary ECs, activation and adhesion of platelets and immune cells to the vascular wall of the alveolar septa, and increased complement deposition in the lungs, in both COVID-19-murine and NHP models. We also revealed that pathways of coagulation, complement, K-ras signaling, and genes of ICAM1 and VCAM1 related to EC dysfunction and injury were upregulated, and were associated with massive immune activation in the lung and circulation. : Together, our results indicate that SARS-CoV-2 causes endotheliitis via both infection and infection-mediated immune activation, which may contribute to the pathogenesis of severe COVID-19 disease.

Pathological angiogenesis promotes tumor growth, metastasis, and atherosclerotic plaque rupture. Macrophages are key players in these processes. However, whether these macrophages differentiate from bone marrow-derived monocytes or from local vascular wall-resident stem and progenitor cells (VW-SCs) is an unresolved issue of angiogenesis. To answer this question, we analyzed vascular sprouting and alterations in aortic cell populations in mouse aortic ring assays (ARA). ARA culture leads to the generation of large numbers of macrophages, especially within the aortic adventitia. Using immunohistochemical fate-mapping and genetic in vivo-labeling approaches we show that 60% of these macrophages differentiate from bone marrow-independent Ly6c + /Sca-1 + adventitial progenitor cells. Analysis of the NCX −/− mouse model that genetically lacks embryonic circulation and yolk sac perfusion indicates that at least some of those progenitor cells arise yolk sac-independent. Macrophages represent the main source of VEGF in ARA that vice versa promotes the generation of additional macrophages thereby creating a pro-angiogenetic feedforward loop. Additionally, macrophage-derived VEGF activates CD34 + progenitor cells within the adventitial vasculogenic zone to differentiate into CD31 + endothelial cells. Consequently, depletion of macrophages and VEGFR2 antagonism drastically reduce vascular sprouting activity in ARA. In summary, we show that angiogenic activation induces differentiation of macrophages from bone marrow-derived as well as from bone marrow-independent VW-SCs. The latter ones are at least partially yolk sac-independent, too. Those VW-SC-derived macrophages critically contribute to angiogenesis, making them an attractive target to interfere with pathological angiogenesis in cancer and atherosclerosis as well as with regenerative angiogenesis in ischemic cardiovascular disorders.

Cardiac tissue engineering is a promising strategy to restore cardiac function in heart failure patients. Understanding the cardiac tissue architecture including the cardiac stroma is essential for developing not only advanced cardiac tissue engineering but also novel therapeutic strategies. One of the crucial components of the cardiac stroma is the myocardial vasculature. To enhance the spatial visualization of the cardiac stromal cytoarchitecture with a particular focus on myocardial vasculature, we performed 3D reconstructions of the murine cardiac micro vessels using Serial Block-Face Scanning Electron Microscopy (SBF-SEM). These analyses revealed that pericyte cell bodies were primarily oriented lengthwise and extended several cellular protrusions towards the endothelium. At capillary branching points, some pericytes made contact with both capillaries emerging from branching. In addition to pericytes that are completely encapsulated by the common basal lamina together with capillary endothelial cells, we identified other vascular-associated cells located outside this sheath. Based on marker expression, these cells were distinguished from fibroblasts and suggested to be telocytes. The vascular-associated cells formed electron-dense contact zones with endothelial cells, suggesting functional coupling between these both cell types. In conclusion, this study provides detailed three-dimensional visualizations of the cardiac stroma with a particular focus on cardiac microvasculature, offering enhanced insight into the cardiac stromal cytoarchitecture.