The cellular architecture of the antimicrobial response network in human leprosy granulomas.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.

Stealing VEGF from thy neighbor.

The distribution and patterning of blood vessels is controlled by vascular endothelial growth factor (VEGF), which is precisely regulated throughout its life cycle. Okabe et al. show that VEGF is titrated away from the endothelium by adjacent neurons via endocytosis, regulating density and trajectory of blood vessels.

Progesterone receptor in the vascular endothelium triggers physiological uterine permeability preimplantation.

Vascular permeability is frequently associated with inflammation and is triggered by a cohort of secreted permeability factors such as vascular endothelial growth factor (VEGF). Here, we show that the physiological vascular permeability that precedes implantation is directly controlled by progesterone receptor (PR) and is independent of VEGF. Global or endothelial-specific deletion of PR blocks physiological vascular permeability in the uterus, whereas misexpression of PR in the endothelium of other organs results in ectopic vascular leakage. Integration of an endothelial genome-wide transcriptional profile with chromatin immunoprecipitation sequencing revealed that PR induces an NR4A1 (Nur77/TR3)-dependent transcriptional program that broadly regulates vascular permeability in response to progesterone. Silencing of NR4A1 blocks PR-mediated permeability responses, indicating a direct link between PR and NR4A1. This program triggers concurrent suppression of several junctional proteins and leads to an effective, timely, and venous-specific regulation of vascular barrier function that is critical for embryo implantation.

Mapping human haematopoietic stem cells from haemogenic endothelium to birth.

The ontogeny of human haematopoietic stem cells (HSCs) is poorly defined owing to the inability to identify HSCs as they emerge and mature at different haematopoietic sites1. Here we created a single-cell transcriptome map of human haematopoietic tissues from the first trimester to birth and found that the HSC signature RUNX1+HOXA9+MLLT3+MECOM+HLF+SPINK2+ distinguishes HSCs from progenitors throughout gestation. In addition to the aorta-gonad-mesonephros region, nascent HSCs populated the placenta and yolk sac before colonizing the liver at 6 weeks. A comparison of HSCs at different maturation stages revealed the establishment of HSC transcription factor machinery after the emergence of HSCs, whereas their surface phenotype evolved throughout development. The HSC transition to the liver marked a molecular shift evidenced by suppression of surface antigens reflecting nascent HSC identity, and acquisition of the HSC maturity markers CD133 (encoded by PROM1) and HLA-DR. HSC origin was tracked to ALDH1A1+KCNK17+ haemogenic endothelial cells, which arose from an IL33+ALDH1A1+ arterial endothelial subset termed pre-haemogenic endothelial cells. Using spatial transcriptomics and immunofluorescence, we visualized this process in ventrally located intra-aortic haematopoietic clusters. The in vivo map of human HSC ontogeny validated the generation of aorta-gonad-mesonephros-like definitive haematopoietic stem and progenitor cells from human pluripotent stem cells, and serves as a guide to improve their maturation to functional HSCs.

Scientific opportunities in resilience research for cardiovascular health and wellness. Report from a National Heart, Lung, and Blood Institute workshop.

Exposure of biological systems to acute or chronic insults triggers a host of molecular and physiological responses to either tolerate, adapt, or fully restore homeostasis; these responses constitute the hallmarks of resilience. Given the many facets, dimensions, and discipline-specific focus, gaining a shared understanding of "resilience" has been identified as a priority for supporting advances in cardiovascular health. This report is based on the working definition: "Resilience is the ability of living systems to successfully maintain or return to homeostasis in response to physical, molecular, individual, social, societal, or environmental stressors or challenges," developed after considering many factors contributing to cardiovascular resilience through deliberations of multidisciplinary experts convened by the National Heart, Lung, and Blood Institute during a workshop entitled: "Enhancing Resilience for Cardiovascular Health and Wellness." Some of the main emerging themes that support the possibility of enhancing resilience for cardiovascular health include optimal energy management and substrate diversity, a robust immune system that safeguards tissue homeostasis, and social and community support. The report also highlights existing research challenges, along with immediate and long-term opportunities for resilience research. Certain immediate opportunities identified are based on leveraging existing high-dimensional data from longitudinal clinical studies to identify vascular resilience measures, create a 'resilience index,' and adopt a life-course approach. Long-term opportunities include developing quantitative cell/organ/system/community models to identify resilience factors and mechanisms at these various levels, designing experimental and clinical interventions that specifically assess resilience, adopting global sharing of resilience-related data, and cross-domain training of next-generation researchers in this field.

Transcriptional Evaluation of the Ductus Arteriosus at the Single-Cell Level Uncovers a Requirement for Vim (Vimentin) for Complete Closure.

OBJECTIVE: Failure to close the ductus arteriosus, patent ductus arteriosus, accounts for 10% of all congenital heart defects. Despite significant advances in patent ductus arteriosus management, including pharmacological treatment targeting the prostaglandin pathway, a proportion of patients fail to respond and must undergo surgical intervention. Thus, further refinement of the cellular and molecular mechanisms that govern vascular remodeling of this vessel is required. METHODS: We performed single-cell RNA-sequencing of the ductus arteriosus in mouse embryos at E18.5 (embryonic day 18.5), and P0.5 (postnatal day 0.5), and P5 to identify transcriptional alterations that might be associated with remodeling. We further confirmed our findings using transgenic mouse models coupled with immunohistochemistry analysis. RESULTS: The intermediate filament vimentin emerged as a candidate that might contribute to closure of the ductus arteriosus. Indeed, mice with genetic deletion of vimentin fail to complete vascular remodeling of the ductus arteriosus. To seek mechanisms, we turned to the RNA-sequencing data that indicated changes in Jagged1 with similar profile to vimentin and pointed to potential links with Notch. In fact, Notch3 signaling was impaired in vimentin null mice and vimentin null mice phenocopies patent ductus arteriosus in Jagged1 endothelial and smooth muscle deleted mice. CONCLUSIONS: Through single-cell RNA-sequencing and by tracking closure of the ductus arteriosus in mice, we uncovered the unexpected contribution of vimentin in driving complete closure of the ductus arteriosus through a mechanism that includes deregulation of the Notch signaling pathway.

Perspectives on Cognitive Phenotypes and Models of Vascular Disease.

Clinical investigations have established that vascular-associated medical conditions are significant risk factors for various kinds of dementia. And yet, we are unable to associate certain types of vascular deficiencies with specific cognitive impairments. The reasons for this are many, not the least of which are that most vascular disorders are multi-factorial and the development of vascular dementia in humans is often a multi-year or multi-decade progression. To better study vascular disease and its underlying causes, the National Heart, Lung, and Blood Institute of the National Institutes of Health has invested considerable resources in the development of animal models that recapitulate various aspects of human vascular disease. Many of these models, mainly in the mouse, are based on genetic mutations, frequently using single-gene mutations to examine the role of specific proteins in vascular function. These models could serve as useful tools for understanding the association of specific vascular signaling pathways with specific neurological and cognitive impairments related to dementia. To advance the state of the vascular dementia field and improve the information sharing between the vascular biology and neurobehavioral research communities, National Heart, Lung, and Blood Institute convened a workshop to bring in scientists from these knowledge domains to discuss the potential utility of establishing a comprehensive phenotypic cognitive assessment of a selected set of existing mouse models, representative of the spectrum of vascular disorders, with particular attention focused on age, sex, and rigor and reproducibility. The workshop highlighted the potential of associating well-characterized vascular disease models, with validated cognitive outcomes, that can be used to link specific vascular signaling pathways with specific cognitive and neurobehavioral deficits.

Mechanisms of Endothelial Regeneration and Vascular Repair and Their Application to Regenerative Medicine.

Endothelial barrier integrity is required for maintaining vascular homeostasis and fluid balance between the circulation and surrounding tissues and for preventing the development of vascular disease. Despite comprehensive understanding of the molecular mechanisms and signaling pathways that mediate endothelial injury, the regulatory mechanisms responsible for endothelial regeneration and vascular repair are incompletely understood and constitute an emerging area of research. Endogenous and exogenous reparative mechanisms serve to reverse vascular damage and restore endothelial barrier function through regeneration of a functional endothelium and re-engagement of endothelial junctions. In this review, mechanisms that contribute to endothelial regeneration and vascular repair are described. Targeting these mechanisms has the potential to improve outcome in diseases that are characterized by vascular injury, such as atherosclerosis, restenosis, peripheral vascular disease, sepsis, and acute respiratory distress syndrome. Future studies to further improve current understanding of the mechanisms that control endothelial regeneration and vascular repair are also highlighted.

Single-cell RNA sequencing to study vascular diversity and function.

PURPOSE OF REVIEW: Single-cell RNA sequencing (scRNA-seq) can capture the transcriptional profile of thousands of individual cells concurrently from complex tissues and with remarkable resolution. Either with the goal of seeking information about distinct cell subtypes or responses to a stimulus, the approach has provided robust information and promoted impressive advances in cardiovascular research. The goal of this review is to highlight strategies and approaches to leverage this technology and bypass potential caveats related to evaluation of the vascular cells. RECENT FINDINGS: As the most recent technological development, details associated with experimental strategies, analysis, and interpretation of scRNA-seq data are still being discussed and scrutinized by investigators across the vascular field. Compilation of this information is valuable for those using the technology but particularly important to those about to start utilizing scRNA-seq to seek transcriptome information of vascular cells. SUMMARY: As our field progresses to catalog transcriptomes from distinct vascular beds, it is undeniable that scRNA-seq technology is here to stay. Sharing approaches to improve the quality of cell dissociation procedures, analysis, and a consensus of best practices is critical as information from this powerful experimental platform continues to emerge.

The many flavors of monocyte/macrophage--endothelial cell interactions.

PURPOSE OF REVIEW: The well recognized plasticity and diversity, typical of monocytes and macrophages have recently been expanded by the knowledge that additional macrophage lineages originated directly from embryonic progenitors, populate and establish residency in all tissues examined so far. This review aims to summarize our current understanding on the diversity of monocyte/macrophage subtypes associated with the vasculature, their specific origins, and nature of their cross-talk with the endothelium. RECENT FINDINGS: Taking stock of the many interactions between the endothelium and monocytes/macrophages reveals a far more intricate and ever-growing depth. In addition to circulating and surveilling the endothelium, monocytes can specifically be differentiated into patrolling cells that crawl on the surface of the endothelium and promote homeostasis. The conversion of classical to patrolling is endothelium-dependent uncovering an important functional link. In addition to patrolling cells, the endothelium also recruits and harbor an intimal-resident myeloid population that resides in the tunica intima in the absence of pathological insults. Moreover, the adventitia is populated with resident macrophages that support blood vessel integrity and prevent fibrosis. SUMMARY: The last few years have witnessed a significant expansion in our knowledge of the many subtypes of monocytes and macrophages and their corresponding functional interactions with the vascular wall. In addition to surveying the endothelium for opportunities of diapedeses, monocyte and macrophages take residence in both the intima (as patrolling or resident) and in the adventitia. Their contributions to vascular function are broad and critical to homeostasis, regeneration, and expansion.

Emerging molecular mechanisms of vascular dementia.

PURPOSE OF REVIEW: Microvascular ischemic disease of the brain is a common cause of cognitive impairment and dementia, particularly in the context of preexisting cardiovascular risk factors and aging. This review summarizes our current understanding of the emerging molecular themes that underlie progressive and irreparable vascular disease leading to neuronal tissue injury and dementia. RECENT FINDINGS: Cardiometabolic risk factors including diabetes and hypertension are known to contribute to vascular disease. Currently, the impact of these risk factors on the integrity and function of the brain vasculature has been target of intense investigation. Molecularly, the consequences associated with these risk factors indicate that reactive oxygen species are strong contributors to cerebrovascular dysfunction and injury. In addition, genetic linkage analyses have identified penetrant monogenic causes of vascular dementia. Finally, recent reports begun to uncover a large number of polymorphisms associated with a higher risk for cerebrovascular disease. SUMMARY: A comprehensive picture of key risk factors and genetic predispositions that contribute to brain microvascular disease and result in vascular dementia is starting to emerge. Understanding their relationships and cross-interactions will significantly aid in the development of preventive and intervention strategies for this devastating condition.

NOTCH regulation of the endothelial cell phenotype.

PURPOSE OF REVIEW: The formation of a hierarchical vascular network is a complex process that requires precise temporal and spatial integration of several signaling pathways. Amongst those, Notch has emerged as a key regulator of multiple steps that expand from endothelial sprouting to arterial specification and remains relevant in the adult. This review aims to summarize major concepts and rising hypotheses on the role of Notch signaling in the endothelium. RECENT FINDINGS: A wealth of new information has helped to clarify how Notch signaling cooperates with other pathways to orchestrate vascular morphogenesis, branching, and function. Endothelial vascular endothelial growth factor, C-X-C chemokine receptor type 4, and nicotinamide adenine dinucleotide phosphate oxidase 2 have been highlighted as key regulators of the pathway. Furthermore, blood flow forces during vascular development induce Notch1 signaling to suppress endothelial cell proliferation, enhance barrier function, and promote arterial specification. Importantly, Notch1 has been recently recognized as an endothelial mechanosensor that is highly responsive to the level of shear stress to enable differential Notch activation in distinct regions of the vessel wall and suppress inflammation. SUMMARY: Although it is well accepted that the Notch signaling pathway is essential for vascular morphogenesis, its contributions to the homeostasis of adult endothelium were uncovered only recently. Furthermore, its exquisite regulation by flow and impressive interface with multiple signaling pathways indicates that Notch is at the center of a highly interactive web that integrates both physical and chemical signals to ensure vascular stability.

Consensus guidelines for the use and interpretation of angiogenesis assays.

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

Transgenic tomatoes expressing the 6F peptide and ezetimibe prevent diet-induced increases of IFN-ß and cholesterol 25-hydroxylase in jejunum.

Feeding LDL receptor (LDLR)-null mice a Western diet (WD) increased the expression of IFN-ß in jejunum as determined by quantitative RT-PCR (RT-qPCR), immunohistochemistry (IHC), and ELISA (all P < 0.0001). WD also increased the expression of cholesterol 25-hydroxylase (CH25H) as measured by RT-qPCR (P < 0.0001), IHC (P = 0.0019), and ELISA (P < 0.0001), resulting in increased levels of 25-hydroxycholesterol (25-OHC) in jejunum as determined by LC-MS/MS (P < 0.0001). Adding ezetimibe at 10 mg/kg/day or adding a concentrate of transgenic tomatoes expressing the 6F peptide (Tg6F) at 0.06% by weight of diet substantially ameliorated these changes. Adding either ezetimibe or Tg6F to WD also ameliorated WD-induced changes in plasma lipids, serum amyloid A, and HDL cholesterol. Adding the same doses of ezetimibe and Tg6F together to WD (combined formulation) was generally more efficacious compared with adding either agent alone. Surprisingly, adding ezetimibe during the preparation of Tg6F, but before addition to WD, was more effective than the combined formulation for all parameters measured in jejunum (P = 0.0329 to P < 0.0001). We conclude the following: i) WD induces IFN-ß, CH25H, and 25-OHC in jejunum; and ii) Tg6F and ezetimibe partially ameliorate WD-induced inflammation by preventing WD-induced increases in IFN-ß, CH25H, and 25-OHC.

Autocrine VEGF maintains endothelial survival through regulation of metabolism and autophagy.

Autocrine VEGF is necessary for endothelial survival, although the cellular mechanisms supporting this function are unknown. Here, we show that--even after full differentiation and maturation--continuous expression of VEGF by endothelial cells is needed to sustain vascular integrity and cellular viability. Depletion of VEGF from the endothelium results in mitochondria fragmentation and suppression of glucose metabolism, leading to increased autophagy that contributes to cell death. Gene-expression profiling showed that endothelial VEGF contributes to the regulation of cell cycle and mitochondrial gene clusters, as well as several--but not all--targets of the transcription factor FOXO1. Indeed, VEGF-deficient endothelium in vitro and in vivo showed increased levels of FOXO1 protein in the nucleus and cytoplasm. Silencing of FOXO1 in VEGF-depleted cells reversed expression profiles of several of the gene clusters that were de-regulated in VEGF knockdown, and rescued both cell death and autophagy phenotypes. Our data suggest that endothelial VEGF maintains vascular homeostasis through regulation of FOXO1 levels, thereby ensuring physiological metabolism and endothelial cell survival.

Perivascular Macrophages Limit Permeability.

OBJECTIVE: Perivascular cells, including pericytes, macrophages, smooth muscle cells, and other specialized cell types, like podocytes, participate in various aspects of vascular function. However, aside from the well-established roles of smooth muscle cells and pericytes, the contributions of other vascular-associated cells are poorly understood. Our goal was to ascertain the function of perivascular macrophages in adult tissues under nonpathological conditions. APPROACH AND RESULTS: We combined confocal microscopy, in vivo cell depletion, and in vitro assays to investigate the contribution of perivascular macrophages to vascular function. We found that resident perivascular macrophages are associated with capillaries at a frequency similar to that of pericytes. Macrophage depletion using either clodronate liposomes or antibodies unexpectedly resulted in hyperpermeability. This effect could be rescued when M2-like macrophages, but not M1-like macrophages or dendritic cells, were reconstituted in vivo, suggesting subtype-specific roles for macrophages in the regulation of vascular permeability. Furthermore, we found that permeability-promoting agents elicit motility and eventual dissociation of macrophages from the vasculature. Finally, in vitro assays showed that M2-like macrophages attenuate the phosphorylation of VE-cadherin upon exposure to permeability-promoting agents. CONCLUSIONS: This study points to a direct contribution of macrophages to vessel barrier integrity and provides evidence that heterotypic cell interactions with the endothelium, in addition to those of pericytes, control vascular permeability.

Notch, lipids, and endothelial cells.

PURPOSE OF REVIEW: Notch signaling is an evolutionary conserved pathway critical for cardiovascular development and angiogenesis. More recently, the contribution of Notch signaling to the homeostasis of the adult vasculature has emerged as an important novel paradigm, but much remains to be understood. RECENT FINDINGS: Recent findings shed light on the impact of Notch in vascular and immune responses to microenvironmental signals as well as on the onset of atherosclerosis. In the past year, studies in human and mice explored the role of Notch in the maintenance of a nonactivated endothelium. Novel pieces of evidence suggest that this pathway is sensitive to environmental factors, including inflammatory mediators and diet-derived by-products. SUMMARY: An emerging theme is the ability of Notch to respond to changes in the microenvironment, including glucose and lipid metabolites. In turn, alterations in Notch enable an important link between metabolism and transcriptional changes, thus this receptor appears to function as a metabolic sensor with direct implications to gene expression.