Tumor endothelial cell-derived Sfrp1 supports the maintenance of cancer stem cells via Wnt signaling.

Cancer stem cells (CSCs), which are critical targets for cancer therapy as they are involved in drug resistance to anticancer drugs, and metastasis, are maintained by angiocrine factors produced by particular niches that form within tumor tissue. Secreted frizzled-related protein 1 (Sfrp1) is an extracellular protein that modulates Wnt signaling. However, the cells that produce Sfrp1 in the tumor environment and its function remain unclear. We aimed to elucidate angiocrine factors related to CSC maintenance, focusing on Sfrp1. Although Sfrp1 is a Wnt pathway-related factor, its impact on tumor tissues remains unknown. We investigated the localization of Sfrp1 in tumors and found that it is expressed in some tumor vessels. Analysis of mice lacking Sfrp1 showed that tumor growth was suppressed in Sfrp1-deficient tumor tissues. Flow cytometry analysis indicated that CSCs were maintained in the early tumor growth phase in the Sfrp1 knockout (KO) mouse model of tumor-bearing cancer. However, tumor growth was inhibited in the late tumor growth phase because of the inability to maintain CSCs. Real-time PCR results from tumors of Sfrp1 KO mice showed that the expression of Wnt signaling target genes significantly decreased in the late stage of tumor growth. This suggests that Sfrp1, an angiocrine factor produced by the tumor vascular niche, is involved in Wnt signaling-mediated mechanisms in tumor tissues.

Brain-derived endothelial cells are neuroprotective in a chronic cerebral hypoperfusion mouse model.

Whether organ-specific regeneration is induced by organ-specific endothelial cells (ECs) remains unelucidated. The formation of white matter lesions due to chronic cerebral hypoperfusion causes cognitive decline, depression, motor dysfunction, and even acute ischemic stroke. Vascular ECs are an important target for treating chronic cerebral hypoperfusion. Brain-derived ECs transplanted into a mouse chronic cerebral hypoperfusion model showed excellent angiogenic potential. They were also associated with reducing both white matter lesions and brain dysfunction possibly due to the high expression of neuroprotective humoral factors. The in vitro coculture of brain cells with ECs from several diverse organs suggested the function of brain-derived endothelium is affected within a brain environment due to netrin-1 and Unc 5B systems. We found brain CD157-positive ECs were more proliferative and beneficial in a mouse model of chronic cerebral hypoperfusion than CD157-negative ECs upon inoculation. We propose novel methods to improve the symptoms of chronic cerebral hypoperfusion using CD157-positive ECs.

Discovery of Transcription Factors Involved in the Maintenance of Resident Vascular Endothelial Stem Cell Properties.

A resident vascular endothelial stem cell (VESC) population expressing CD157 has been identified recently in mice. Herein, we identified transcription factors (TFs) regulating CD157 expression in endothelial cells (ECs) that were associated with drug resistance, angiogenesis, and EC proliferation. In the first screening, we detected 20 candidate TFs through the CD157 promoter and gene expression analyses. We found that 10 of the 20 TFs induced CD157 expression in ECs. We previously reported that 70% of CD157 VESCs were side population (SP) ECs that abundantly expressed ATP-binding cassette (ABC) transporters. Here, we found that the 10 TFs increased the expression of several ABC transporters in ECs and increased the proportion of SP ECs. Of these 10 TFs, we found that six (Atf3, Bhlhe40, Egr1, Egr2, Elf3, and Klf4) were involved in the manifestation of the SP phenotype. Furthermore, the six TFs enhanced tube formation and proliferation in ECs. Single-cell RNA sequence data in liver ECs suggested that Atf3 and Klf4 contributed to the production of CD157+ VESCs in the postnatal period. We concluded that Klf4 might be important for the development and maintenance of liver VESCs. Our work suggests that a TF network is involved in the differentiation hierarchy of VESCs.

[Vascular biology and hemophilia].

By carrying a systemic circulation, hematopoietic and vascular systems coordinately govern the functional organ connections in the body. Blood vessels play an important role in the development, regeneration, and maintenance of organs by acting as conduits for environmental factors in the blood to tissues and secreting organ-specific cytokines as angiocrine signals. Recently, it has become clear that vascular endothelial cells, which are the main constituent cells of the blood vessels and play a role in homeostasis, are diverse. It has also been established that the cells of stem cell fraction exist in endothelial cells. The vascular endothelial cells in various organs are functionally different. For example, it has been discovered that sinusoidal blood vessels in the liver produce coagulation factor VIII as an organ-specific vascular function. Determining how such tissue-/organ-specific function of the endothelial cells is induced is a topic of interest in the vascular field of study.

Single-cell sequencing reveals the existence of fetal vascular endothelial stem cell-like cells in mouse liver.

BACKGROUND: A resident vascular endothelial stem cell (VESC) population expressing CD157 and CD200 has been identified recently in the adult mouse. However, the origin of this population and how it develops has not been characterized, nor has it been determined whether VESC-like cells are present during the perinatal period. Here, we investigated the presence of perinatal VESC-like cells and their relationship with the adult VESC-like cell population. METHODS: We applied single-cell RNA sequencing of endothelial cells (ECs) from embryonic day (E) 14, E18, postnatal day (P) 7, P14, and week (W) 8 liver and investigated transcriptomic changes during liver EC development. We performed flow cytometry, immunofluorescence, colony formation assays, and transplantation assays to validate the presence of and to assess the function of CD157+ and CD200+ ECs in the perinatal period. RESULTS: We identified CD200- expressing VESC-like cells in the perinatal period. These cells formed colonies in vitro and had high proliferative ability. The RNA velocity tool and transplantation assay results indicated that the projected fate of this population was toward adult VESC-like cells expressing CD157 and CD200 1 week after birth. CONCLUSION: Our study provides a comprehensive atlas of liver EC development and documents VESC-like cell lineage commitment at single-cell resolution.

Aging impairs the ability of vascular endothelial stem cells to generate endothelial cells in mice.

Tissue-resident vascular endothelial stem cells (VESCs), marked by expression of CD157, possess long-term repopulating potential and contribute to vascular regeneration and homeostasis in mice. Stem cell exhaustion is regarded as one of the hallmarks of aging and is being extensively studied in several types of tissue-resident stem cells; however, how aging affects VESCs has not been clarified yet. In the present study, we isolated VESCs from young and aged mice to compare their potential to differentiate into endothelial cells in vitro and in vivo. Here, we report that the number of liver endothelial cells (ECs) including VESCs was lower in aged (27-28 month-old) than young (2-3 month-old) mice. In vitro culture of primary VESCs revealed that the potential to generate ECs is impaired in aged VESCs isolated from liver and lung relative to young VESCs. Orthotopic transplantation of VESCs showed that aged VESCs and their progeny expand less efficiently than their young counterparts when transplanted into aged mice, but they are equally functional in young recipients. Gene expression analysis indicated that inflammatory signaling was more activated in aged ECs including VESCs. Using single-cell RNA sequencing data from the Tabula Muris Consortium, we show that T cells and monocyte/macrophage lineage cells including Kupffer cells are enriched in the aged liver. These immune cells produce IL-1ß and several chemokines, suggesting the possible involvement of age-associated inflammation in the functional decline of VESCs with age.

Cancer apelin receptor suppresses vascular mimicry in malignant melanoma.

Several reports indicate that apelin is often over-expressed in tumors, and therefore it has been suggested that the apelin-apelin receptor (APJ) system may induce tumor progression. In contrast, our previous research revealed high expression of the apelin-APJ system in tumor blood vessels, suggesting its involvement in the regulation of tumor vessel formation and normalization, resulting in the suppression of tumor growth by promoting the infiltration of T cells. Thus, the effect of the apelin-APJ system on tumors remains controversial. In this report, to clarify the effect of apelin in tumor cells, we analyzed the function of APJ in tumor cells using APJ knock out (KO) mice. In APJ-KO mice, Apelin overexpression in B16/BL6 (B16) melanoma cells induced greater tumor growth than controls. In an APJ-KO melanoma inoculation model, although angiogenesis is suppressed compared to wild type, no difference is evident in tumor growth. We found that APJ deficiency promoted vascular mimicry in tumors. In vitro, cultured APJ-KO B16 cells demonstrated a spindle-like shape. This phenotypic change was thought to be induced by epithelial-mesenchymal transition (EMT) based on evidence that APJ-KO B16 cells show persistently high levels of the mesenchymal maker, Zeb1; however, we found that EMT did not correlate with the transforming growth factor-ß/smad signaling pathway in our model. We propose that apelin-APJ system in cancer cells induces tumor growth but negatively regulates EMT and tumor malignancy.

Upregulation of Robo4 expression by SMAD signaling suppresses vascular permeability and mortality in endotoxemia and COVID-19 models.

There is an urgent need to develop novel drugs to reduce the mortality from severe infectious diseases with the emergence of new pathogens, including Coronavirus disease 2019 (COVID-19). Although current drugs effectively suppress the proliferation of pathogens, immune cell activation, and inflammatory cytokine functions, they cannot completely reduce mortality from severe infections and sepsis. In this study, we focused on the endothelial cell-specific protein, Roundabout 4 (Robo4), which suppresses vascular permeability by stabilizing endothelial cells, and investigated whether enhanced Robo4 expression could be a novel therapeutic strategy against severe infectious diseases. Endothelial-specific overexpression of Robo4 suppresses vascular permeability and reduces mortality in lipopolysaccharide (LPS)-treated mice. Screening of small molecules that regulate Robo4 expression and subsequent analysis revealed that two competitive small mothers against decapentaplegic (SMAD) signaling pathways, activin receptor-like kinase 5 (ALK5)-SMAD2/3 and ALK1-SMAD1/5, positively and negatively regulate Robo4 expression, respectively. An ALK1 inhibitor was found to increase Robo4 expression in mouse lungs, suppress vascular permeability, prevent extravasation of melanoma cells, and decrease mortality in LPS-treated mice. The inhibitor suppressed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced endothelial barrier disruption and decreased mortality in mice infected with SARS-CoV-2. These results indicate that enhancing Robo4 expression is an efficient strategy to suppress vascular permeability and mortality in severe infectious diseases, including COVID-19, and that small molecules that upregulate Robo4 can be potential therapeutic agents against these diseases.

Identification of CD157-Positive Vascular Endothelial Stem Cells in Mouse Retinal and Choroidal Vessels: Fluorescence-Activated Cell Sorting Analysis.

Purpose: CD157 (also known as Bst1) positive vascular endothelial stem cells (VESCs), which contribute to vascular regeneration, have been recently identified in mouse organs, including the retinas, brain, liver, lungs, heart, and skin. However, VESCs have not been identified in the choroid. The purpose of this study was to identify VESCs in choroidal vessels and to establish the protocol to isolate retinal and choroidal VESCs. Methods: We established an efficient protocol to create single-cell suspensions from freshly isolated mouse retina and choroid by enzymatic digestion using dispase, collagenase, and type II collagenase. CD157-positive VESCs, defined as CD31+CD45-CD157+ cells, were sorted using fluorescence-activated cell sorting (FACS). Results: In mouse retina, among CD31+CD45- endothelial cells (ECs), 1.6 ± 0.2% were CD157-positive VESCs, based on FACS analysis. In mouse choroid, among CD31+CD45- ECs, 4.5 ± 0.4% were VESCs. The CD157-positive VESCs generated a higher number of EC networks compared with CD157-negative non-VESCs under vascular endothelial growth factor (VEGF) in vitro cultures. The EC network area, defined as the ratio of the CD31-positive area to the total area in each field, was 4.21 ± 0.39% (retinal VESCs) and 0.27 ± 0.12% (retinal non-VESCs), respectively (P < 0.01). The EC network area was 8.59 ± 0.78% (choroidal VESCs) and 0.14 ± 0.04% (choroidal non-VESCs), respectively (P < 0.01). The VESCs were located in large blood vessels but not in the capillaries. Conclusions: We confirmed distinct populations of CD157-positive VESCs in both mouse retina and choroid. VESCs are located in large vessels and have the proliferative potential. The current results may open new avenues for the research and treatment of ocular vascular diseases.

Apelin Resistance Contributes to Muscle Loss during Cancer Cachexia in Mice.

Cancer cachexia consists of dramatic body weight loss with rapid muscle depletion due to imbalanced protein homeostasis. We found that the mRNA levels of apelin decrease in muscles from cachectic hepatoma-bearing rats and three mouse models of cachexia. Furthermore, apelin expression inversely correlates with MuRF1 in muscle biopsies from cancer patients. To shed light on the possible role of apelin in cachexia in vivo, we generated apelin 13 carrying all the last 13 amino acids of apelin in D isomers, ultimately extending plasma stability. Notably, apelin D-peptides alter cAMP-based signaling in vitro as the L-peptides, supporting receptor binding. In vitro apelin 13 protects myotube diameter from dexamethasone-induced atrophy, restrains rates of degradation of long-lived proteins and MuRF1 expression, but fails to protect mice from atrophy. D-apelin 13 given intraperitoneally for 13 days in colon adenocarcinoma C26-bearing mice does not reduce catabolic pathways in muscles, as it does in vitro. Puzzlingly, the levels of circulating apelin seemingly deriving from cachexia-inducing tumors, increase in murine plasma during cachexia. Muscle electroporation of a plasmid expressing its receptor APJ, unlike apelin, preserves myofiber area from C26-induced atrophy, supporting apelin resistance in vivo. Altogether, we believe that during cachexia apelin resistance occurs, contributing to muscle wasting and nullifying any possible peptide-based treatment.

Blood vessels sense dermal stiffness via a novel mechanotransducer, APJ.

Microvascular dysfunction accompanied by a dramatic alteration of stable capillary structure is a major hallmark of numerous age-related diseases. In skin, although the role of angiogenesis during dermal reconstitution is well documented, the functional relevance of the extracellular matrix (ECM) stiffness to vascular remodeling and its molecular mechanisms was poorly understood. Here, we developed an ex vivo 3-dimensional angiogenic model using human fat, revealing that "appropriate" stiffness induces vascular maturation associated with upregulated APJ expression, whereas the overexpression of APJ promotes the formation of large vessels even in the absence of the "appropriate" stiffness. Taken together, APJ could be a novel mechanotransducer that accelerates the maturation of cutaneous blood vessels, leading to the prevention of human skin aging.

Lead Optimization of Influenza Virus RNA Polymerase Inhibitors Targeting PA-PB1 Interaction.

Influenza viruses are responsible for contagious respiratory illnesses in humans and cause seasonal epidemics and occasional pandemics worldwide. Previously, we identified a quinolinone derivative PA-49, which inhibited the influenza virus RNA-dependent RNA polymerase (RdRp) by targeting PA-PB1 interaction. This paper reports the structure optimization of PA-49, which resulted in the identification of 3-((dibenzylamino)methyl)quinolinone derivatives with more potent anti-influenza virus activity. During the optimization, the hit compound 89, which was more active than PA-49, was identified. Further optimization and scaffold hopping of 89 led to the most potent compounds 100 and a 1,8-naphthyridinone derivative 118, respectively. We conclusively determined that compounds 100 and 118 suppressed the replication of influenza virus and exhibited anti-influenza virus activity against both influenza virus types A and B in the range of 50% effective concentration (EC50) = 0.061-0.226 µM with low toxicity (50% cytotoxic concentration (CC50) >10 µM).

Identification of the first highly selective inhibitor of human lactate dehydrogenase B.

Lactate dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate and NADH to NAD+; it has two isoforms, LDHA and LDHB. LDHA is a promising target for cancer therapy, whereas LDHB is necessary for basal autophagy and cancer cell proliferation in oxidative and glycolytic cancer cells. To the best of our knowledge, selective inhibitors for LDHB have not yet been reported. Here, we developed a high-throughput mass spectrometry screening system using an LDHB enzyme assay by detecting NADH and NAD+. As a result, we identified a small-molecule LDHB selective inhibitor AXKO-0046, an indole derivative. This compound exhibited uncompetitive LDHB inhibition (EC50 = 42 nM). X-ray crystallography revealed that AXKO-0046 bound to the potential allosteric site away from the LDHB catalytic active site, suggesting that targeting the tetramerisation interface of the two dimers is critical for the enzymatic activity. AXKO-0046 and its derivatives can be used to validate LDHB-associated pathways in cancer metabolism.

Endothelial cell-derived Apelin inhibits tumor growth by altering immune cell localization.

The Apelin/APJ signalling pathway, involved in multiple physiological and pathological processes, has been attracting increasing interest recently. In our previous study, Apelin overexpression in colon26 tumor cells suppressed tumor growth by inducing vascular maturation. Here, we found that MC38 and LLC tumor growth were greater in the absence of Apelin than in wild-type (WT) mice, suggesting that Apelin acts as a tumor suppressor. Consistent with this, treating WT mice with [Pyr1]Apelin-13 inhibited tumor growth. In MC38 tumors, only endothelial cells (ECs) strongly express APJ, a cognate receptor for Apelin, indicating that EC-derived Apelin might regulate tumor formation in an autocrine manner. Comparing with WT mice, larger numbers of vessels with narrower diameters were observed in tumors of Apelin knockout mice and lack of Apelin enhanced tumor hypoxia. Investigating immune cells in the tumor revealed that [Pyr1]Apelin-13 infusion induced the accumulation of CD8+ and CD4+ T cells in central areas. Moreover, RNA-sequencing analysis showed that Apelin induces chemokine CCL8 expression in ECs. Thus, enhancing anti-tumor immunity might be one of the mechanisms by which Apelin is involved in tumor growth. Our result indicated that increased CCL8 expression might induce CD8 + T cells infiltration into tumor and tumor inhibition.

Indispensable role of Galectin-3 in promoting quiescence of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) in adult bone marrow (BM) are usually maintained in a state of quiescence. The cellular mechanism coordinating the balance between HSC quiescence and differentiation is not fully understood. Here, we report that galactose-binding lectin-3 (galectin-3; Gal-3) is upregulated by Tie2 or Mpl activation to maintain quiescence. Conditional overexpression of Gal-3 in mouse HSCs under the transcriptional control of Tie2 or Vav1 promoters (Gal-3 Tg) causes cell cycle retardation via induction of p21. Conversely, the cell cycle of long-term repopulating HSCs (LT-HSCs) in Gal-3-deficient (Gal-3-/-) mice is accelerated, resulting in their exhaustion. Mechanistically, Gal-3 regulates p21 transcription by forming a complex with Sp1, thus blocking cell cycle entry. These results demonstrate that Gal-3 is a negative regulator of cell-cycling in HSCs and plays a crucial role in adult hematopoiesis to prevent HSC exhaustion.

Reduction in miR-219 expression underlies cellular pathogenesis of oligodendrocytes in a mouse model of Krabbe disease.

Krabbe disease (KD), also known as globoid cell leukodystrophy, is an inherited demyelinating disease caused by the deficiency of lysosomal galactosylceramidase (GALC) activity. Most of the patients are characterized by early-onset cerebral demyelination with apoptotic oligodendrocyte (OL) death and die before 2 years of age. However, the mechanisms of molecular pathogenesis in the developing OLs before death and the exact causes of white matter degeneration remain largely unknown. We have recently reported that OLs of twitcher mouse, an authentic mouse model of KD, exhibit developmental defects and endogenous accumulation of psychosine (galactosylsphingosine), a cytotoxic lyso-derivative of galactosylceramide. Here, we show that attenuated expression of microRNA (miR)-219, a critical regulator of OL differentiation and myelination, mediates cellular pathogenesis of KD OLs. Expression and functional activity of miR-219 were repressed in developing twitcher mouse OLs. By using OL precursor cells (OPCs) isolated from the twitcher mouse brain, we show that exogenously supplemented miR-219 effectively rescued their cell-autonomous developmental defects and apoptotic death. miR-219 also reduced endogenous accumulation of psychosine in twitcher OLs. Collectively, these results highlight the role of the reduced miR-219 expression in KD pathogenesis and suggest that miR-219 has therapeutic potential for treating KD OL pathologies.

Vascular reconstitution in the tumor for more effective tumor immunotherapy.

It has been widely accepted that the regulation of the tumor microenvironment is an important strategy in cancer treatment. Particularly, control of the tumor vasculature has been suggested to be critical for antitumor immunotherapy. Effectiveness of cancer immunotherapy depends on the quality and quantity of immune cells infiltrating into tumor tissues, which may be affected by the status of the tumor vasculature. Under physiological conditions, immune cells migrate from the intravascular lumen into the parenchyma especially by passing through the vascular wall of venulae. Extravasation of immune cells is induced from venulae where endothelial cells (ECs) are fully covered with pericytes from the basal side. Interaction of pericytes with ECs contributes to immune cell extravasation by several steps, ie, adhesion of immune cells to intraluminal ECs, transmigration, and chemotaxis of immune cells. Blood vessels are structurally immature and non-functional in tumors, and therefore, induction of maturation in the tumor vasculature is a promising strategy for effective cancer therapies and is relevant not only for immune cell migration but also drug delivery.

An in vivo model allowing continuous observation of human vascular formation in the same animal over time.

Angiogenesis contributes to numerous pathological conditions. Understanding the molecular mechanisms of angiogenesis will offer new therapeutic opportunities. Several experimental in vivo models that better represent the pathological conditions have been generated for this purpose in mice, but it is difficult to translate results from mouse to human blood vessels. To understand human vascular biology and translate findings into human research, we need human blood vessel models to replicate human vascular physiology. Here, we show that human tumor tissue transplantation into a cranial window enables engraftment of human blood vessels in mice. An in vivo imaging technique using two-photon microscopy allows continuous observation of human blood vessels until at least 49 days after tumor transplantation. These human blood vessels make connections with mouse blood vessels as shown by the finding that lectin injected into the mouse tail vein reaches the human blood vessels. Finally, this model revealed that formation and/or maintenance of human blood vessels depends on VEGFR2 signaling. This approach represents a useful tool to study molecular mechanisms of human blood vessel formation and to test effects of drugs that target human blood vessels in vivo to show proof of concept in a preclinical model.

Age-dependent decline in remyelination capacity is mediated by apelin-APJ signaling.

Age-related regeneration failure in the central nervous system can occur as a result of a decline in remyelination efficacy. The responsiveness of myelin-forming cells to signals for remyelination is affected by aging-related epigenetic modification; however, the molecular mechanism is not fully clarified. In the present study, we report that the apelin receptor (APJ) mediates remyelination efficiency with age. APJ expression in myelin-forming cells is correlated with age-associated changes in remyelination efficiency, and the activation of APJ promotes remyelination through the translocation of myelin regulatory factor. APJ signaling activation promoted remyelination in both aged mice with toxin-induced demyelination and mice with experimental autoimmune encephalomyelitis. In human cells, APJ activation enhanced the expression of remyelination markers. Impaired oligodendrocyte function in aged animals can be reversibly reactivated; thus, the results demonstrate that dysfunction of the apelin-APJ system mediates remyelination failure in aged animals, and that their myelinating function can be reactivated by APJ activation.