Resilient anatomy and local plasticity of naive and stress haematopoiesis.

The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis, erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis1 to define the anatomy of normal and stress haematopoiesis. In the steady state, across the skeleton, single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels, where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells, which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults, as it was maintained after haemorrhage, systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment, and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF, and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis, define the anatomy of normal and stress responses, identify discrete microanatomical production sites that confer plasticity to haematopoiesis, and uncover unprecedented heterogeneity of stress responses across the skeleton.

Identification of distinct vascular mural cell populations during zebrafish embryonic development.

BACKGROUND: Mural cells are an essential perivascular cell population that associate with blood vessels and contribute to vascular stabilization and tone. In the embryonic zebrafish vasculature, pdgfrb and tagln are commonly used as markers for identifying pericytes and vascular smooth muscle cells. However, the overlapping and distinct expression patterns of these markers in tandem have not been fully described. RESULTS: Here, we used the Tg(pdgfrb:Gal4FF; UAS:RFP) and Tg(tagln:NLS-EGFP) transgenic lines to identify single- and double-positive perivascular cell populations on the cranial, axial, and intersegmental vessels between 1 and 5 days postfertilization. From this comparative analysis, we discovered two novel regions of tagln-positive cell populations that have the potential to function as mural cell precursors. Specifically, we found that the hypochord-a reportedly transient structure-contributes to tagln-positive cells along the dorsal aorta. We also identified a unique mural cell progenitor population that resides along the midline between the neural tube and notochord and contributes to intersegmental vessel mural cell coverage. CONCLUSION: Together, our findings highlight the variability and versatility of tracking both pdgfrb and tagln expression in mural cells of the developing zebrafish embryo and reveal unexpected embryonic cell populations that express pdgfrb and tagln.

Molecular mechanism of the interaction between Megalocytivirus-induced virus-mock basement membrane (VMBM) and lymphatic endothelial cells.

IMPORTANCE: Viruses are able to mimic the physiological or pathological mechanism of the host to favor their infection and replication. Virus-mock basement membrane (VMBM) is a Megalocytivirus-induced extracellular structure formed on the surface of infected cells and structurally and functionally mimics the basement membrane of the host. VMBM provides specific support for lymphatic endothelial cells (LECs) rather than blood endothelial cells to adhere to the surface of infected cells, which constitutes a unique phenomenon of Megalocytivirus infection. Here, the structure of VMBM and the interactions between VMBM components and LECs have been analyzed at the molecular level. The regulatory effect of VMBM components on the proliferation and migration of LECs has also been explored. This study helps to understand the mechanism of LEC-specific attachment to VMBM and to address the issue of where the LECs come from in the context of Megalocytivirus infection.

Endothelial mechanical stretch regulates the immunological synapse interface of renal endothelial cells in a sex-dependent manner.

Increased mechanical endothelial cell stretch contributes to the development of numerous cardiovascular and renal pathologies. Recent studies have shone a light on the importance of sex-dependent inflammation in the pathogenesis of renal disease states. The endothelium plays an intimate and critical role in the orchestration of immune cell activation through upregulation of adhesion molecules and secretion of cytokines and chemokines. While endothelial cells are not recognized as professional antigen-presenting cells, in response to cytokine stimulation, endothelial cells can express both major histocompatibility complex (MHC) I and MHC II. MHCs are essential to forming a part of the immunological synapse interface during antigen presentation to adaptive immune cells. Whether MHC I and II are increased under increased mechanical stretch is unknown. Due to hypertension being multifactorial, we hypothesized that increased mechanical endothelial stretch promotes the regulation of MHCs and key costimulatory proteins on mouse renal endothelial cells (MRECs) in a stretch-dependent manner. MRECs derived from both sexes underwent 5%, 10%, or 15% uniaxial cyclical stretch, and immunological synapse interface proteins were determined by immunofluorescence microscopy, immunoblot analysis, and RNA sequencing. We found that increased endothelial mechanical stretch conditions promoted downregulation of MHC I in male MRECs but upregulation in female MRECs. Moreover, MHC II was upregulated by mechanical stretch in both male and female MRECs, whereas CD86 and CD70 were regulated in a sex-dependent manner. By bulk RNA sequencing, we found that increased mechanical endothelial cell stretch promoted differential gene expression of key antigen processing and presentation genes in female MRECs, demonstrating that females have upregulation of key antigen presentation pathways. Taken together, our data demonstrate that mechanical endothelial stretch regulates endothelial activation and immunological synapse interface formation in renal endothelial cells in a sex-dependent manner.NEW & NOTEWORTHY Endothelial cells contribute to the development of renal inflammation and have the unique ability to express antigen presentation proteins. Whether increased endothelial mechanical stretch regulates immunological synapse interface proteins remains unknown. We found that antigen presentation proteins and costimulatory proteins on renal endothelial cells are modulated by mechanical stretch in a sex-dependent manner. Our data provide novel insights into the sex-dependent ability of renal endothelial cells to present antigens in response to endothelial mechanical stimuli.

Generation of specialized blood vessels via lymphatic transdifferentiation.

The lineage and developmental trajectory of a cell are key determinants of cellular identity. In the vascular system, endothelial cells (ECs) of blood and lymphatic vessels differentiate and specialize to cater to the unique physiological demands of each organ1,2. Although lymphatic vessels were shown to derive from multiple cellular origins, lymphatic ECs (LECs) are not known to generate other cell types3,4. Here we use recurrent imaging and lineage-tracing of ECs in zebrafish anal fins, from early development to adulthood, to uncover a mechanism of specialized blood vessel formation through the transdifferentiation of LECs. Moreover, we demonstrate that deriving anal-fin vessels from lymphatic versus blood ECs results in functional differences in the adult organism, uncovering a link between cell ontogeny and functionality. We further use single-cell RNA-sequencing analysis to characterize the different cellular populations and transition states involved in the transdifferentiation process. Finally, we show that, similar to normal development, the vasculature is rederived from lymphatics during anal-fin regeneration, demonstrating that LECs in adult fish retain both potency and plasticity for generating blood ECs. Overall, our research highlights an innate mechanism of blood vessel formation through LEC transdifferentiation, and provides in vivo evidence for a link between cell ontogeny and functionality in ECs.

Oxidative pentose phosphate pathway controls vascular mural cell coverage by regulating extracellular matrix composition.

Vascular mural cells (vMCs) play an essential role in the development and maturation of the vasculature by promoting vessel stabilization through their interactions with endothelial cells. Whether endothelial metabolism influences mural cell recruitment and differentiation is unknown. Here, we show that the oxidative pentose phosphate pathway (oxPPP) in endothelial cells is required for establishing vMC coverage of the dorsal aorta during early vertebrate development in zebrafish and mice. We demonstrate that laminar shear stress and blood flow maintain oxPPP activity, which in turn, promotes elastin expression in blood vessels through production of ribose-5-phosphate. Elastin is both necessary and sufficient to drive vMC recruitment and maintenance when the oxPPP is active. In summary, our work demonstrates that endothelial cell metabolism regulates blood vessel maturation by controlling vascular matrix composition and vMC recruitment.

A single-cell atlas of the normal and malformed human brain vasculature.

Cerebrovascular diseases are a leading cause of death and neurologic disability. Further understanding of disease mechanisms and therapeutic strategies requires a deeper knowledge of cerebrovascular cells in humans. We profiled transcriptomes of 181,388 cells to define a cell atlas of the adult human cerebrovasculature, including endothelial cell molecular signatures with arteriovenous segmentation and expanded perivascular cell diversity. By leveraging this reference, we investigated cellular and molecular perturbations in brain arteriovenous malformations, which are a leading cause of stroke in young people, and identified pathologic endothelial transformations with abnormal vascular patterning and the ontology of vascularly derived inflammation. We illustrate the interplay between vascular and immune cells that contributes to brain hemorrhage and catalog opportunities for targeting angiogenic and inflammatory programs in vascular malformations.

3D printing of self-standing and vascular supportive multimaterial hydrogel structures for organ engineering.

Three dimensional printable formulation of self-standing and vascular-supportive structures using multi-materials suitable for organ engineering is of great importance and highly challengeable, but, it could advance the 3D printing scenario from printable shape to functional unit of human body. In this study, the authors report a 3D printable formulation of such self-standing and vascular-supportive structures using an in-house formulated multi-material combination of albumen/alginate/gelatin-based hydrogel. The rheological properties and relaxation behavior of hydrogels were analyzed before the printing process. The suitability of the hydrogel in 3D printing of various customizable and self-standing structures, including a human ear model, was examined by extrusion-based 3D printing. The structural, mechanical, and physicochemical properties of the printed scaffolds were studied systematically. Results supported the 3D printability of the formulated hydrogel with self-standing structures, which are customizable to a specific need. In vitro cell experiment showed that the formulated hydrogel has excellent biocompatibility and vascular supportive behavior with the extent of endothelial sprout formation when tested with human umbilical vein endothelial cells. In conclusion, the present study demonstrated the suitability of the extrusion-based 3D printing technique for manufacturing complex shapes and structures using multi-materials with high fidelity, which have great potential in organ engineering.

Distinct Characteristics Between Perivascular and Subcutaneous Adipose-Derived Stem Cells.

Adipose-derived stem cells (ADSCs) can differentiate into vascular lineages and participate in vascular remodeling. Perivascular ADSCs (PV-ADSCs) draw attention because of their unique location. The heterogeneity of subcutaneous (SUB) and abdominal ADSCs were well addressed, but PV-ADSCs' heterogeneity has not been investigated. In this study, we applied single-cell analysis to compare SUB-ADSCs and PV-ADSCs regarding their subpopulations, functions, and cell fates. We uncovered four subpopulations of PV-ADSCs (Dpp4+, Col4a2+/Icam1+, Clec11a+/Cpe+, and Sult1e1+ cells), among which the Clec11a+ subpopulation potentially participated in and regulated PV-ADSC differentiation toward a smooth muscle cell (SMC) phenotype. Distinct characteristics between PV-ADSCs and SUB-ADSCs were revealed.

3D biomimetic platform reveals the first interactions of the embryo and the maternal blood vessels.

The process of implantation and the cellular interactions at the embryo-maternal interface are intrinsically difficult to analyze, as the implanting embryo is concealed by the uterine tissues. Therefore, the mechanisms mediating the interconnection of the embryo and the mother are poorly understood. Here, we established a 3D biomimetic culture environment that harbors the key features of the murine implantation niche. This culture system enabled direct analysis of trophoblast invasion and revealed the first embryonic interactions with the maternal vasculature. We found that implantation is mediated by the collective migration of penetrating strands of trophoblast giant cells, which acquire the expression of vascular receptors, ligands, and adhesion molecules, assembling a network for communication with the maternal blood vessels. In particular, Pdgf signaling cues promote the establishment of the heterologous contacts. Together, the biomimetic platform and our findings thereof elucidate the hidden dynamics of the early interactions at the implantation site.

Pathophysiology and inflammatory biomarkers of sulfur mustard-induced corneal injury in rabbits.

Sulfur mustard (SM) is a cytotoxic, vesicating, chemical warfare agent, first used in 1917; corneas are particularly vulnerable to SM exposure. They may develop inflammation, ulceration, neovascularization (NV), impaired vision, and partial/complete blindness depending upon the concentration of SM, exposure duration, and bio-physiological conditions of the eyes. Comprehensive in vivo studies have established ocular structural alterations, opacity, NV, and inflammation upon short durations (<4 min) of SM exposure. In this study, detailed analyses of histopathological alterations in corneal structure, keratocytes, inflammatory cells, blood vessels, and expressions of cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), and cytokines were performed in New Zealand white rabbits, in a time-dependent manner till 28 days, post longer durations (5 and 7 min) of ocular SM exposure to establish quantifiable endpoints of injury and healing. Results indicated that SM exposure led to duration-dependent increases in corneal thickness, opacity, ulceration, epithelial-stromal separation, and epithelial degradation. Significant increases in NV, keratocyte death, blood vessels, and inflammatory markers (COX-2, MMP-9, VEGF, and interleukin-8) were also observed for both exposure durations compared to the controls. Collectively, these findings would benefit in temporal delineation of mechanisms underlying SM-induced corneal toxicity and provide models for testing therapeutic interventions.

Programmatic introduction of parenchymal cell types into blood vessel organoids.

Pluripotent stem cell-derived organoids have transformed our ability to recreate complex three-dimensional models of human tissue. However, the directed differentiation methods used to create them do not afford the ability to introduce cross-germ-layer cell types. Here, we present a bottom-up engineering approach to building vascularized human tissue by combining genetic reprogramming with chemically directed organoid differentiation. As a proof of concept, we created neuro-vascular and myo-vascular organoids via transcription factor overexpression in vascular organoids. We comprehensively characterized neuro-vascular organoids in terms of marker gene expression and composition, and demonstrated that the organoids maintain neural and vascular function for at least 45 days in culture. Finally, we demonstrated chronic electrical stimulation of myo-vascular organoid aggregates as a potential path toward engineering mature and large-scale vascularized skeletal muscle tissue from organoids. Our approach offers a roadmap to build diverse vascularized tissues of any type derived entirely from pluripotent stem cells.

NLRP3 Inflammasome Mediates Immune-Stromal Interactions in Vasculitis.

[Figure: see text].

Engineering the Dynamics of Cell Adhesion Cues in Supramolecular Hydrogels for Facile Control over Cell Encapsulation and Behavior.

The extracellular matrix (ECM) forms through hierarchical assembly of small and larger polymeric molecules into a transient, hydrogel-like fibrous network that provides mechanical support and biochemical cues to cells. Synthetic, fibrous supramolecular networks formed via non-covalent assembly of various molecules are therefore potential candidates as synthetic mimics of the natural ECM, provided that functionalization with biochemical cues is effective. Here, combinations of slow and fast exchanging molecules that self-assemble into supramolecular fibers are employed to form transient hydrogel networks with tunable dynamic behavior. Obtained results prove that modulating the ratio between these molecules dictates the extent of dynamic behavior of the hydrogels at both the molecular and the network level, which is proposed to enable effective incorporation of cell-adhesive functionalities in these materials. Excitingly, the dynamic nature of the supramolecular components in this system can be conveniently employed to formulate multicomponent supramolecular hydrogels for easy culturing and encapsulation of single cells, spheroids, and organoids. Importantly, these findings highlight the significance of molecular design and exchange dynamics for the application of supramolecular hydrogels as synthetic ECM mimics.

Human Blood Vessel Organoids Penetrate Human Cerebral Organoids and Form a Vessel-Like System.

Vascularization of tissues, organoids and organ-on-chip models has been attempted using endothelial cells. However, the cultured endothelial cells lack the capacity to interact with other somatic cell types, which is distinct from developing vascular cells in vivo. Recently, it was demonstrated that blood vessel organoids (BVOs) recreate the structure and functions of developing human blood vessels. However, the tissue-specific adaptability of BVOs had not been assessed in somatic tissues. Herein, we investigated whether BVOs infiltrate human cerebral organoids and form a blood-brain barrier. As a result, vascular cells arising from BVOs penetrated the cerebral organoids and developed a vessel-like architecture composed of CD31+ endothelial tubes coated with SMA+ or PDGFR+ mural cells. Molecular markers of the blood-brain barrier were detected in the vascularized cerebral organoids. We revealed that BVOs can form neural-specific blood-vessel networks that can be maintained for over 50 days.

Characterization of the growth plate-bone interphase region using cryo-FIB SEM 3D volume imaging.

The interphase region at the base of the growth plate includes blood vessels, cells and mineralized tissues. In this region, cartilage is mineralized and replaced with bone. Blood vessel extremities permeate this space providing nutrients, oxygen and signaling factors. All these different components form a complex intertwined 3D structure. Here we use cryo-FIB SEM to elaborate this 3D structure without removing the water. As it is challenging to image mineralized and unmineralized tissues in a hydrated state, we provide technical details of the parameters used. We obtained two FIB SEM image stacks that show that the blood vessels are in intimate contact not only with cells, but in some locations also with mineralized tissues. There are abundant red blood cells at the extremities of the vessels. We also documented large multinucleated cells in contact with mineralized cartilage and possibly also with bone. We observed membrane bound mineralized particles in these cells, as well as in blood serum, but not in the hypertrophic chondrocytes. We confirm that there is an open pathway from the blood vessel extremities to the mineralizing cartilage. Based on the sparsity of the mineralized particles, we conclude that mainly ions in solution are used for mineralizing cartilage and bone, but these are augmented by the supply of mineralized particles.

Niches that regulate stem cells and hematopoiesis in adult bone marrow.

In mammals, hematopoietic stem cells (HSCs) engage in hematopoiesis throughout adult life within the bone marrow, where they produce the mature cells necessary to maintain blood cell counts and immune function. In the bone marrow and spleen, HSCs are sustained in perivascular niches (microenvironments) associated with sinusoidal blood vessels-specialized veins found only in hematopoietic tissues. Endothelial cells and perivascular leptin receptor+ stromal cells produce the known factors required to maintain HSCs and many restricted progenitors in the bone marrow. Various other cells synthesize factors that maintain other restricted progenitors or modulate HSC or niche function. Recent studies identified new markers that resolve some of the heterogeneity among stromal cells and refine the localization of restricted progenitor niches. Other recent studies identified ways in which niches regulate HSC function and hematopoiesis beyond growth factors. We summarize the current understanding of hematopoietic niches, review recent progress, and identify important unresolved questions.

General sites of nanoparticle biodistribution as a novel opportunity for nanomedicine.

The development of nanomedical devices has led to a considerable number of clinically applied nanotherapeutics. Yet, the overall poor translation of nanoparticular concepts into marketable systems has not met the initial expectations and led to increasing criticism in recent years. Most novel nano approaches thereby use highly refined formulations including a plethora of active targeting sequences, but ultimately fail to reach their target due to a generally high off-target deposition in organs such as the liver or kidney. In this context, we argue that initial nanoparticle (NP) development should not entirely become set on conventional formulation aspects. In contrast, we propose a change of focus towards a prior analysis of general sites of NP in vivo deposition and an assessment of how accumulation in these organs or tissues can be harnessed to develop therapies for site-related pathologies. We therefore give a comprehensive overview of existing nanotherapeutic targeting strategies for specific cell types within three of the usual suspects, i.e. the liver, kidney and the vascular system. We discuss the physiological surroundings and relevant pathologies of described tissues as well as the implications for NP-mediated drug delivery. Additionally, successful cell-selective NP concepts using active targeting strategies are assessed. By bringing together both (patho)physiological aspects and concepts for cell-selective NP formulations, we hope to show a novel opportunity for the development of more promising nanotherapeutic devices.

Advances in tissue engineering of vasculature through three-dimensional bioprinting.

BACKGROUND: A significant challenge facing tissue engineering is the fabrication of vasculature constructs which contains vascularized tissue constructs to recapitulate viable, complex and functional organs or tissues, and free-standing vascular structures potentially providing clinical applications in the future. Three-dimensional (3D) bioprinting has emerged as a promising technology, possessing a number of merits that other conventional biofabrication methods do not have. Over the last decade, 3D bioprinting has contributed a variety of techniques and strategies to generate both vascularized tissue constructs and free-standing vascular structures. RESULTS: This review focuses on different strategies to print two kinds of vasculature constructs, namely vascularized tissue constructs and vessel-like tubular structures, highlighting the feasibility and shortcoming of the current methods for vasculature constructs fabrication. Generally, both direct printing and indirect printing can be employed in vascularized tissue engineering. Direct printing allows for structural fabrication with synchronous cell seeding, while indirect printing is more effective in generating complex architecture. During the fabrication process, 3D bioprinting techniques including extrusion bioprinting, inkjet bioprinting and light-assisted bioprinting should be selectively implemented to exert advantages and obtain the desirable tissue structure. Also, appropriate cells and biomaterials matter a lot to match various bioprinting techniques and thus achieve successful fabrication of specific vasculature constructs. CONCLUSION: The 3D bioprinting has been developed to help provide various fabrication techniques, devoting to producing structurally stable, physiologically relevant, and biologically appealing constructs. However, although the optimization of biomaterials and innovation of printing strategies may improve the fabricated vessel-like structures, 3D bioprinting is still in the infant period and has a great gap between in vitro trials and in vivo applications. The article reviews the present achievement of 3D bioprinting in generating vasculature constructs and also provides perspectives on future directions of advanced vasculature constructs fabrication.