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.

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.

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.

Vasculature-driven stem cell population coordinates tissue scaling in dynamic organs.

Stem cell (SC) proliferation and differentiation organize tissue homeostasis. However, how SCs regulate coordinate tissue scaling in dynamic organs remain unknown. Here, we delineate SC regulations in dynamic skin. We found that interfollicular epidermal SCs (IFESCs) shape basal epidermal proliferating clusters (EPCs) in expanding abdominal epidermis of pregnant mice and proliferating plantar epidermis. EPCs consist of IFESC-derived Tbx3+-basal cells (Tbx3+-BCs) and their neighboring cells where Adam8-extracellular signal-regulated kinase signaling is activated. Clonal lineage tracing revealed that Tbx3+-BC clones emerge in the abdominal epidermis during pregnancy, followed by differentiation after parturition. In the plantar epidermis, Tbx3+-BCs are sustained as long-lived SCs to maintain EPCs invariably. We showed that Tbx3+-BCs are vasculature-dependent IFESCs and identified mechanical stretch as an external cue for the vasculature-driven EPC formation. Our results uncover vasculature-mediated IFESC regulations, which explain how the epidermis adjusts its size in orchestration with dermal constituents in dynamic skin.

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.

Regnase-1-mediated post-transcriptional regulation is essential for hematopoietic stem and progenitor cell homeostasis.

The balance between self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) maintains hematopoietic homeostasis, failure of which can lead to hematopoietic disorder. HSPC fate is controlled by signals from the bone marrow niche resulting in alteration of the stem cell transcription network. Regnase-1, a member of the CCCH zinc finger protein family possessing RNAse activity, mediates post-transcriptional regulatory activity through degradation of target mRNAs. The precise function of Regnase-1 has been explored in inflammation-related cytokine expression but its function in hematopoiesis has not been elucidated. Here, we show that Regnase-1 regulates self-renewal of HSPCs through modulating the stability of Gata2 and Tal1 mRNA. In addition, we found that dysfunction of Regnase-1 leads to the rapid onset of abnormal hematopoiesis. Thus, our data reveal that Regnase-1-mediated post-transcriptional regulation is required for HSPC maintenance and suggest that it represents a leukemia tumor suppressor.

Galectin-3 Inhibits Cancer Metastasis by Negatively Regulating Integrin ß3 Expression.

Galectin-3 (Gal-3; gene LGALS3) is a member of the ß-galactose-binding lectin family. Previous studies showed that Gal-3 is expressed in several tissues across species and functions as a regulator of cell proliferation, apoptosis, adhesion, and migration, thus affecting many aspects of events, such as angiogenesis and tumorigenesis. Although several reports have suggested that the level of Gal-3 expression correlates positively with tumor progression, herein we show that highly metastatic mouse melanoma B16/BL6 cells express less Gal-3 than B16 cells with a lower metastatic potential. It was found that overexpression of Gal-3 in melanoma cells in fact suppresses metastasis. In contrast, knocking out Gal-3 expression in cancer cells promoted cell aggregation mediated through interactions with platelets and fibrinogen in vitro and increased the number of metastatic foci in vivo. Thus, reduced Gal-3 expression results in the up-regulation of ß3 integrin expression, and this contributes to metastatic potential. These findings indicate that changes of Gal-3 expression in cancer cells during tumor progression influence the characteristics of metastatic cells.

TAK1 Prevents Endothelial Apoptosis and Maintains Vascular Integrity.

TNF-α is a pleiotropic cytokine that has the potential to induce apoptosis under inflammation. How endothelial cells (ECs) are spared from this fate in inflammatory environments where TNF-α is present is not known. Here, we show that TGF-ß-activated kinase 1 (TAK1) ensures EC survival and maintains vascular integrity upon TNF-α stimulation. Endothelial-specific TAK1 knockout mice exhibit intestinal and liver hemorrhage due to EC apoptosis, leading to vascular destruction and rapid death. This EC apoptosis was induced by TNF-α from myeloid cells responding to intestinal microbiota. TNF-α secretion associated with inflammation also induced vascular defects in inflamed organs. Additionally, we determined that TAK1 deletion in tumor ECs resulted in blood vessel and hence tumor regression. Our results illuminate mechanisms ensuring survival of intestinal and liver ECs under physiological conditions and ECs of other organs under inflammatory conditions that could be exploited for anti-angiogenic therapy to treat cancer.

LPA4-Mediated Vascular Network Formation Increases the Efficacy of Anti-PD-1 Therapy against Brain Tumors.

: The structure and function of tumor blood vessels profoundly affects the tumor microenvironment. Signals mediated through the lysophosphatidic acid receptor 4 (LPA4) promote vascular network formation to restore normal vascular barrier function in subcutaneous tumors and thus improve drug delivery. However, the characteristics of the vasculature vary by organ and tumor types, and how drug delivery and leukocyte trafficking are affected by modification of vascular function by LPA in different cancers is unclear. Here, we show that LPA4 activation promotes the formation of fine vascular structures in brain tumors. RhoA/ROCK signaling contributed to LPA-induced endothelial cell-cell adhesion, and RhoA/ROCK activity following LPA4 stimulation regulated expression of VCAM-1. This resulted in increased lymphocyte infiltration into the tumor. LPA improved delivery of exogenous IgG into brain tumors and enhanced the anticancer effect of anti-programmed cell death-1 antibody therapy. These results indicate the effects of LPA on vascular structure and function apply not only to chemotherapy but also to immunotherapy. SIGNIFICANCE: These findings demonstrate that lysophosphatidic acid, a lipid mediator, promotes development of a fine capillary network in brain tumors by inducing tightening of endothelial cell-to-cell adhesion, facilitating improved drug delivery, and lymphocyte penetration.

CD157 Marks Tissue-Resident Endothelial Stem Cells with Homeostatic and Regenerative Properties.

The generation of new blood vessels via angiogenesis is critical for meeting tissue oxygen demands. A role for adult stem cells in this process remains unclear. Here, we identified CD157 (bst1, bone marrow stromal antigen 1) as a marker of tissue-resident vascular endothelial stem cells (VESCs) in large arteries and veins of numerous mouse organs. Single CD157+ VESCs form colonies in vitro and generate donor-derived portal vein, sinusoids, and central vein endothelial cells upon transplantation in the liver. In response to injury, VESCs expand and regenerate entire vasculature structures, supporting the existence of an endothelial hierarchy within blood vessels. Genetic lineage tracing revealed that VESCs maintain large vessels and sinusoids in the normal liver for more than a year, and transplantation of VESCs rescued bleeding phenotypes in a mouse model of hemophilia. Our findings show that tissue-resident VESCs display self-renewal capacity and that vascular regeneration potential exists in peripheral blood vessels.

Lysophosphatidic Acid Receptor 4 Activation Augments Drug Delivery in Tumors by Tightening Endothelial Cell-Cell Contact.

Vascular normalization in tumors may improve drug delivery and anti-tumor immunity. Angiogenesis inhibitors induce hypoxia, which may facilitate malignant progression; therefore, we investigated other methods to promote vascular maturation. Here, we show that lysophosphatidic acid (LPA) enhances blood flow by promoting fine vascular networks, thereby improving vascular permeability and suppressing tumor growth when combined with anti-cancer drug treatment. Six different G protein-coupled receptors have been identified as LPA receptors (LPA1-6). In studies using mutant mice, we found that LPA4 is involved in vascular network formation. LPA4 activation induces circumferential actin bundling beneath the cell membrane and enhances linear adherens junction formation by VE-cadherin in endothelial cells. Therefore, we conclude that activation of LPA4 is a promising approach for vascular regulation.

Plakoglobin maintains the integrity of vascular endothelial cell junctions and regulates VEGF-induced phosphorylation of VE-cadherin.

Plakoglobin, also known as γ-catenin, is a close homolog of ß-catenin and interacts with shared protein partners. Functions of ß-catenin in cell adhesion are well-documented in terms of maintaining endothelial barrier function by interacting with vascular endothelial (VE)-cadherin. Plakoglobin also interacts with VE-cadherin, but its function in cell adhesion is not well understood. Here, we investigated plakoglobin function in vascular endothelial cell (ECs)-cell junction integrity. Knock-down of plakoglobin expression in ECs did not prevent cell proliferation or cell migration, but induced destabilization of the membrane distribution of VE-cadherin and resulted in increased permeability. Plakoglobin contributes to VE-cadherin-dependent adhesion in the steady state, but on stimulation with vascular endothelial growth factor (VEGF), it is essential for inducing sufficient VE-cadherin phosphorylation on VEGF signaling, thereby destabilizing cell-cell junctions. Furthermore, knock-down of plakoglobin expression increased vascular endothelial protein tyrosine phosphatase activity, an endothelial-specific membrane protein associating with VE-cadherin. These results indicate that plakoglobin plays multiple roles in regulation of cell-cell adhesion in a context dependent manner.

Regulation of SLD5 gene expression by miR-370 during acute growth of cancer cells.

SLD5 is a member of the GINS complex, essential for DNA replication in eukaryotes. It has been reported that SLD5 is involved in early embryogenesis in the mouse, and cell cycle progression and genome integrity in Drosophila. SLD5 may be involved in malignant tumor progression, but its relevance in human cancer has not been determined. Here, we found strong SLD5 expression in both human bladder cancer tissues from patients and cell lines. Knockdown of SLD5 using small interfering RNA resulted in reduction of cell growth both in vitro and an in vivo xenograft model. Moreover, we found that high levels of SLD5 in bladder cancer cells result from downregulation of microRNA (miR)-370 that otherwise suppresses its expression. High level expression of DNA-methyltransferase (DNMT) 1 and IL-6 were also observed in bladder cancer cells. Knockdown of IL-6 led to downregulation of DNMT1 and SLD5 expression, suggesting that IL-6-induced overexpression of DNMT1 suppresses miR-370, resulting in high SLD5 expression. Our findings could contribute to understanding tumorigenic processes and progression of human bladder cancer, whereby inhibition of SLD5 could represent a novel strategy to prevent tumor growth.

Endothelial Side Population Cells Contribute to Tumor Angiogenesis and Antiangiogenic Drug Resistance.

Angiogenesis plays a crucial role in tumor growth, with an undisputed contribution of resident endothelial cells (EC) to new blood vessels in the tumor. Here, we report the definition of a small population of vascular-resident stem/progenitor-like EC that contributes predominantly to new blood vessel formation in the tumor. Although the surface markers of this population are similar to other ECs, those from the lung vasculature possess colony-forming ability in vitro and contribute to angiogenesis in vivo These specific ECs actively proliferate in lung tumors, and the percentage of this population significantly increases in the tumor vasculature relative to normal lung tissue. Using genetic recombination and bone marrow transplant models, we show that these cells are phenotypically true ECs and do not originate from hematopoietic cells. After treatment of tumors with antiangiogenic drugs, these specific ECs selectively survived and remained in the tumor. Together, our results established that ECs in the peripheral vasculature are heterogeneous and that stem/progenitor-like ECs play an indispensable role in tumor angiogenesis as EC-supplying cells. The lack of susceptibility of these ECs to antiangiogenic drugs may account for resistance of the tumor to this drug type. Thus, inhibiting these ECs might provide a promising strategy to overcome antiangiogenic drug resistance. Cancer Res; 76(11); 3200-10. ©2016 AACR.

APJ Regulates Parallel Alignment of Arteries and Veins in the Skin.

Molecular pathways regulating the development of arterial and venous endothelial cells (ECs) are now well established, but control of parallel arterial-venous alignment is unclear. Here we report that arterial-venous alignment in the skin is determined by apelin receptor (APJ) expression in venous ECs. One of the activators of APJ is apelin. We found that apelin is produced by arterial ECs during embryogenesis, induces chemotaxis of venous ECs, and promotes the production of secreted Frizzled-related protein 1 (sFRP1) by APJ(+) ECs. sFRP1 stimulates matrix metalloproteinase production by Ly6B.2(+) neutrophil-like cells located between the arteries and veins, resulting in remodeling of extracellular matrices to support venous displacement. Moreover, using apelin- or APJ-deficient mice, which exhibit arterial-venous disorganization, we found that arterial-venous alignment is involved in thermoregulation, possibly by regulating countercurrent heat exchange. We hypothesize that the evolution of parallel juxtapositional arterial-venous alignment was an adaptation to reduce body heat loss.

Apelin inhibits diet-induced obesity by enhancing lymphatic and blood vessel integrity.

Angiogenesis is tightly associated with the outgrowth of adipose tissue, leading to obesity, which is a risk factor for type 2 diabetes and hypertension, mainly because expanding adipose tissue requires an increased nutrient supply from blood vessels. Therefore, induction of vessel abnormality by adipokines has been well-studied, whereas how altered vascular function promotes obesity is relatively unexplored. Also, surviving Prox1 heterozygous mice have shown abnormal lymphatic patterning and adult-onset obesity, indicating that accumulation of adipocytes could be closely linked with lymphatic function. Here, we propose a new antiobesity strategy based on enhancement of lymphatic and blood vessel integrity with apelin. Apelin knockout (KO) mice fed a high-fat diet (HFD) showed an obese phenotype associated with abnormal lymphatic and blood vessel enlargement. Fatty acids present in the HFD induced hyperpermeability of endothelial cells, causing adipocyte differentiation, whereas apelin promoted vascular stabilization. Moreover, treatment of apelin KO mice with a selective cyclooxygenase-2 inhibitor, celecoxib, that were fed an HFD improved vascular function and also attenuated obesity. Finally, apelin transgenic mice showed decreased subcutaneous adipose tissue attributable to inhibition of HFD-induced hyperpermeability of vessels. These results indicate that apelin inhibits HFD-induced obesity by enhancing vessel integrity. Apelin could serve as a therapeutic target for treating obesity and related diseases.

Apelin attenuates UVB-induced edema and inflammation by promoting vessel function.

Apelin, the ligand of the G protein-coupled receptor APJ, is involved in the regulation of cardiovascular functions, fluid homeostasis, and vessel formation. Recent reports indicate that apelin secreted from endothelial cells mediates APJ regulation of blood vessel caliber size; however, the function of apelin in lymphatic vessels is unclear. Here we report that APJ was expressed by human lymphatic endothelial cells and that apelin induced migration and cord formation of lymphatic endothelial cells dose-dependently in vitro. Furthermore, permeability assays demonstrated that apelin stabilizes lymphatic endothelial cells. In vivo, transgenic mice harboring apelin under the control of keratin 14 (K14-apelin) exhibited attenuated UVB-induced edema and a decreased number of CD11b-positive macrophages. Moreover, activation of apelin/APJ signaling inhibited UVB-induced enlargement of lymphatic and blood vessels. Finally, K14-apelin mice blocked the hyperpermeability of lymphatic vessels in inflamed skin. These results indicate that apelin plays a functional role in the stabilization of lymphatic vessels in inflamed tissues and that apelin might be a suitable target for prevention of UVB-induced inflammation.