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Background: Elevated endothelial microparticles (EMPs) levels are surrogate markers of vascular dysfunction. We analyzed EMPs with apoptotic characteristics and assessed the angiogenic contents of microparticles in the blood of patients with type 2 diabetes (T2D) according to the presence of coronary artery disease (CAD)., Methods: A total of 80 participants were recruited and equally classified as (1) healthy without T2D, (2) T2D without cardiovascular complications, (3) T2D and chronic coronary artery disease (CAD), and (4) T2D and acute coronary syndrome (ACS). MPs were isolated from the peripheral circulation, and EMPs were characterized using flow cytometry of CD42 and CD31. CD62E was used to determine EMPs' apoptotic/activation state. MPs content was extracted and profiled using an angiogenesis array., Results: Levels of CD42- CD31 + EMPs were significantly increased in T2D with ACS (257.5 ± 35.58) when compared to healthy subjects (105.7 ± 12.96, p < 0.01). There was no significant difference when comparing T2D with and without chronic CAD. The ratio of CD42-CD62 +/CD42-CD31 + EMPs was reduced in all T2D patients, with further reduction in ACS when compared to chronic CAD, reflecting a release by apoptotic endothelial cells. The angiogenic content of the full population of MPs was analyzed. It revealed a significant differential expression of 5 factors in patients with ACS and diabetes, including TGF-β1, PD-ECGF, platelet factor 4, serpin E1, and thrombospondin 1. Ingenuity Pathway Analysis revealed that those five differentially expressed molecules, mainly TGF-β1, inhibit key pathways involved in normal endothelial function. Further comparison of the three diabetes groups to healthy controls and diabetes without cardiovascular disease to diabetes with CAD identified networks that inhibit normal endothelial cell function. Interestingly, DDP-IV was the only differentially expressed protein between chronic CAD and ACS in patients with diabetes., Conclusion: Our data showed that the release of apoptosis-induced EMPs is increased in diabetes, irrespective of CAD, ACS patients having the highest levels. The protein contents of MPs interact in networks that indicate vascular dysfunction., (© 2022. The Author(s).)

Increased soluble endoglin (sENG) has been observed in human brain arteriovenous malformations (bAVMs). In addition, the overexpression of sENG in concurrence with vascular endothelial growth factor (VEGF)-A has been shown to induce dysplastic vessel formation in mouse brains. However, the underlying mechanism of sENG-induced vascular malformations is not clear. The evidence suggests the role of sENG as a pro-inflammatory modulator, and increased microglial accumulation and inflammation have been observed in bAVMs. Therefore, we hypothesized that microglia mediate sENG-induced inflammation and endothelial cell (EC) dysfunction in bAVMs. In this study, we confirmed that the presence of sENG along with VEGF-A overexpression induced dysplastic vessel formation. Remarkably, we observed increased microglial activation around dysplastic vessels with the expression of NLRP3, an inflammasome marker. We found that sENG increased the gene expression of VEGF-A, pro-inflammatory cytokines/inflammasome mediators (TNF-α, IL-6, NLRP3, ASC, Caspase-1, and IL-1β), and proteolytic enzyme (MMP-9) in BV2 microglia. The conditioned media from sENG-treated BV2 (BV2-sENG-CM) significantly increased levels of angiogenic factors (Notch-1 and TGFβ) and pERK1/2 in ECs but it decreased the level of IL-17RD, an anti-angiogenic mediator. Finally, the BV2-sENG-CM significantly increased EC migration and tube formation. Together, our study demonstrates that sENG provokes microglia to express angiogenic/inflammatory molecules which may be involved in EC dysfunction. Our study corroborates the contribution of microglia to the pathology of sENG-associated vascular malformations.

Capillary endothelial cells possess a specialized metabolism necessary to adapt to the unique alveolar-capillary environment. Here, we highlight how endothelial metabolism preserves the integrity of the pulmonary circulation by controlling vascular permeability, defending against oxidative stress, facilitating rapid migration and angiogenesis in response to injury, and regulating the epigenetic landscape of endothelial cells. Recent reports on single-cell RNA-sequencing reveal subpopulations of pulmonary capillary endothelial cells with distinctive reparative capacities, which potentially offer new insight into their metabolic signature. Lastly, we discuss broad implications of pulmonary vascular metabolism on acute respiratory distress syndrome, touching on emerging findings of endotheliitis in coronavirus disease 2019 (COVID-19) lungs.

Vascular ischemia/reperfusion injury (IRI) contributes to graft failure and adverse clinical outcomes following coronary artery bypass grafting. Sodium-glucose-cotransporter (SGLT)-2-inhibitors have been shown to protect against myocardial IRI, irrespective of diabetes. We hypothesized that adding canagliflozin (CANA) (an SGLT-2-inhibitor) to saline protects vascular grafts from IRI. Aortic rings from non-diabetic rats were isolated and immediately mounted in organ bath chambers (control, n = 9-10 rats) or underwent cold ischemic preservation in saline, supplemented either with a DMSO vehicle (IR, n = 8-10 rats) or 50µM CANA (IR + CANA, n = 9-11 rats). Vascular function was measured, the expression of 88 genes using PCR-array was analyzed, and feature selection using machine learning was applied. Impaired maximal vasorelaxation to acetylcholine in the IR-group compared to controls was significantly ameliorated by CANA (IR 31.7 ± 3.2% vs. IR + CANA 51.9 ± 2.5%, p < 0.05). IR altered the expression of 17 genes. Ccl2 , Ccl3 , Ccl4 , CxCr4 , Fos , Icam1 , Il10 , Il1a and Il1b have been found to have the highest interaction. Compared to controls, IR significantly upregulated the mRNA expressions of Il1a and Il6 , which were reduced by 1.5- and 1.75-fold with CANA, respectively. CANA significantly prevented the upregulation of Cd40, downregulated NoxO1 gene expression, decreased ICAM-1 and nitrotyrosine, and increased PECAM-1 immunoreactivity. CANA alleviates endothelial dysfunction following IRI.

The vascular endothelium is characterized by a remarkable level of plasticity, which is the driving force not only of physiological repair/remodeling of adult tissues but also of pathological angiogenesis. The resulting heterogeneity of endothelial cells (ECs) makes targeting the endothelium challenging, no less because many EC phenotypes are yet to be identified and functionally inventorized. Efforts to map the vasculature at the single-cell level have been instrumental to capture the diversity of EC types and states at a remarkable depth in both normal and pathological states. Here, we discuss new EC subtypes and functions emerging from recent single-cell studies in health and disease. Interestingly, such studies revealed distinct metabolic gene signatures in different EC phenotypes, which deserve further consideration for therapy. We highlight how this metabolic targeting strategy could potentially be used to promote (for tissue repair) or block (in tumor) angiogenesis in a tissue or even vascular bed-specific manner.

Chitinase 3-like 1 (CHI3L1) is an enzymatically inactive mammalian chitinase that is associated with tumor inflammation. Previous research indicated that CHI3L1 is able to interact with different extracellular matrix components, such as heparan sulfate. In the present work, we investigated whether the interaction of CHI3L1 with the extracellular matrix of melanoma cells can trigger an inflammatory activation of endothelial cells. The analysis of the melanoma cell secretome indicated that CHI3L1 increases the abundance of various cytokines, such as CC-chemokine ligand 2 (CCL2), and growth factors, such as vascular endothelial growth factor A (VEGF-A). Using a solid-phase binding assay, we found that heparan sulfate-bound VEGF-A and CCL2 were displaced by recombinant CHI3L1 in a dose-dependent manner. Microfluidic experiments indicated that the CHI3L1 altered melanoma cell secretome promoted immune cell recruitment to the vascular endothelium. In line with the elevated VEGF-A levels, CHI3L1 was also able to promote angiogenesis through the release of extracellular matrix-bound pro-angiogenic factors. In conclusion, we showed that CHI3L1 is able to affect the tumor cell secretome, which in turn can regulate immune cell recruitment and blood vessel formation. Accordingly, our data suggest that the molecular targeting of CHI3L1 in the course of cancer immunotherapies can tune patients' response and antitumoral inflammation.

The role of angiogenesis in the growth of organs and tumors is widely recognized. Vascular-organ interaction is a key mechanism and a concept that enables an understanding of all biological phenomena and normal physiology that is essential for human survival under pathological conditions. Recently, vascular endothelial cells have been classified as a type of innate immune cells that are dependent on the pathological situations. Moreover, inflammatory cytokines and signaling regulators activated upon exposure to infection or various stresses play crucial roles in the pathological function of parenchymal cells, peripheral immune cells, stromal cells, and cancer cells in tissues. Therefore, vascular-organ interactions as a vascular microenvironment or tissue microenvironment under physiological and pathological conditions are gaining popularity as an interesting research topic. Here, we review vascular contribution as a major factor in microenvironment homeostasis in the pathogenesis of normal as well as cancerous tissues. Furthermore, we suggest that the normalization strategy of pathological angiogenesis could be a promising therapeutic target for various diseases, including cancer.

Endothelial cells (ECs) are subjected to physical forces such as shear stress (SS) induced by blood flow that leads to significant changes in morphology, physiology and gene expression. The abnormal mechanical forces applied in the cardiovascular system can influence the development of conditions and diseases such as thrombosis, hypertension and atherosclerosis. This study investigated the expression of glycosaminoglycans (GAGs), proteoglycans and extracellular matrix molecules in ECs exposed to normal and altered SS. ECs were exposed to SS of 12 dyn/cm2 (artery physiological condition) and 4 dyn/cm2 (artery pathological condition). Subsequently, ECs were subjected to immunofluorescence, qPCR, GAG biosynthesis analyses and cell-based assays. SS induced changes in ECs morphology. There were other pathological consequences of altered SS, including inhibited adhesion, stimulation of migration and capillary-like tube formation, as well as increases of GAG synthesis. We observed higher expression of syndecan-4, perlecan, decorin, fibronectin and collagen III α1 and growth factors, including VEGF-A and TGFβ-1. ECs exposed to SS displayed extracellular matrix remodeling as well as expression of cell-matrix and cell-cell interaction molecules. This study contributes to the understanding of how vascular biology is affected by mechanical forces and how these molecules can be affected in cardiovascular diseases., Competing Interests: The authors have declared that no competing interests exist.

Endothelial cells (ECs) play an important role in the pathogenesis of cardiovascular disease, especially atherosclerosis (AS). The abnormal wall shear stress (WSS) which directly contacts with ECs is the key stimulating factor leading to AS. However, the underlying mechanism of ECs responding to WSS is still incompletely understood. This study aims to explore the novel mechano-sensitive genes and its potential mechanism in response to WSS in ECs by employing bioinformatics methods based on previously available high-throughput data from zebrafish embryos, both before and after blood flow formation. Six common differentially expressed genes (DEGs) (SRGN, SLC12A3, SLC25A4, PVALB1, ITGAE.2, zgc:198419) were selected out from two high-throughput datasets (GSE126617 and GSE20707) in the GEO database. Among them, SRGN was chosen for further verification through the in vitro shear stress loading experiments with human umbilical vein endothelial cells (HUVECs) and the in vivo partial ligation of carotid artery in mice. Our data indicated that low shear stress (LSS) could enhance the expression of SRGN via the PKA/CREB-dependent signaling pathway. The proportion of Ki67 + cells and the concentration of nitric oxide (NO) were high in SRGN high expression cells, suggesting that SRGN may be involved in the proliferation of HUVECs. Furthermore, in the partial ligation of the carotid artery mice model, we observed that the expression of SRGN was significantly increased in atherosclerotic plaques induced by abnormal shear stress. Taken together, this study demonstrated that SRGN is a key gene in the response of ECs to WSS and could be involved in AS.

Background and Aims: External counter-pulsation (ECP) generates sheer stress thereby improving endothelial function and anginal symptoms in coronary artery disease. Endothelial dysfunction is also involved in the pathogenesis of T2DM. The aim of this pilot study was to investigate the use of ECP at different doses in improving endothelial function and glycaemic markers in T2DM., Methods: This prospective study involved 46 subjects with T2DM randomly assigned to receive 35 sessions of ECP at different regimens (0.5 h versus 1 h) and duration (7 versus 12 weeks). Endothelial function was evaluated by reactive hyperaemia index (RHI) via peripheral arterial tonometry at the start, midpoint and end of study. Other secondary outcomes included fasting glucose, HOMA-IR, HbA1c, blood pressure, lipid profile, weight and vibration sense., Results: There was no change in RHI across all 3 regimens of ECP individually or collectively at the end of the study (ΔRHI +0.01%, p = 0.458). Glycaemic markers also remained unchanged at endpoint. Subgroup analysis showed an improvement in RHI (ΔRHI +20.6%, p = 0.0178) in subjects with more severe endothelial dysfunction at baseline., Conclusion: ECP did not show a beneficial effect on endothelial function or glycemic control in this South-East Asian population with T2DM at any of the three regimens. This may partly be explained by less severe endothelial dysfunction and less insulin resistance in our population at baseline., Competing Interests: Declaration of competing interest This is a Principal Investigator-initiated study, with research support from Renew Group Private Limited. The terms of this arrangement have been reviewed and approved by the National University Hospital Singapore in accordance with its policy on objectivity in research and with the funding source. The author has no conflict of interest., (Copyright © 2020 Diabetes India. Published by Elsevier Ltd. All rights reserved.)

Angiogenesis in arterial intimal thickening (AIT) has been considered mainly in late AIT stages and only refers to sprouting angiogenesis. We assess angiogenesis during early AIT development and the occurrence of the intussusceptive type. For this purpose, we studied AIT development in (a) human arteries with vasculitis in gallbladders with acute cholecystitis and urgent ( n = 25) or delayed ( n = 20) cholecystectomy, using immunohistochemical techniques and (b) experimentally occluded arterial segments ( n = 56), using semithin and ultrathin sections and electron microscopy. The results showed transitory angiogenic phenomena, with formation of an important microvasculature, followed by vessel regression. In addition to the sequential description of angiogenic and regressive findings, we mainly contribute (a) formation of intravascular pillars (hallmarks of intussusception) during angiogenesis and vessel regression and (b) morphological interrelation between endothelial cells (ECs) in the arterial wall and vascular smooth muscle cells (VSMCs), which adopt a pericytic arrangement and establish peg-and-socket junctions with ECs. In conclusion, angiogenesis and vessel regression play an important role in AIT development in the conditions studied, with participation of intussusceptive angiogenesis during the formation and regression of a provisional microvasculature and with morphologic interrelation between ECs and VSMCs.

Angiogenesis and vascular maturation play important roles in tumorigenesis and tumor development. The expression of neuropilin 1 (NRP1) is closely associated with angiogenesis in tumors; however, the molecular mechanisms of action in angiogenesis and tumor maturation, as well as the potential clinical value of NRP1 remain unclear. The importance of NRP1 expression in tumor progression was determined using The Cancer Genome Atlas (TCGA) database analysis. Gain‑ and loss‑of‑function experiments of NRP1 were performed in vascular endothelial cells (ECs) to investigate the functions in angiogenesis. CCK‑8, flow cytometry, Transwell experiments and a series of in vitro experiments were used to detect cell functions. A combination of angiogenesis antibody arrays and RNA‑Seq analyses were performed to reveal the proangiogenic mechanisms of action. The function of semaphorin 4D (SEMA4D) was also investigated separately. NRP1 mRNA levels were significantly increased in primary tumors compared with normal tissues based on TCGA data (P<0.01) and were associated with tumor development in patients. Gain‑ and loss‑of‑function experiments highlighted the function of NRP1 in promoting EC proliferation, motility and capillary‑like tube formation and in reducing apoptosis. NRP1 overexpression led to significantly decreased EC markers (PECAM‑1, angiogenin, PIGF and MMP‑9) expression levels and reduced the vascular maturity. MAPK7, TPM1, RRBP1, PTPRK, HSP90A, PRKD2, PFKFB3, RGS4 and SPARC were revealed to play important roles in this process. SEMA4D was revealed to be a key protein associated with NRP1 in ECs. These data indicated that NRP1‑promoted angiogenesis may be induced at the cost of reducing maturity of the ECs. NRP1 may also be a therapeutic target for antiangiogenic strategies and a candidate prognostic marker for tumors.

Angiogenesis inhibitors, such as the receptor tyrosine kinase (RTK) inhibitor sunitinib, target vascular endothelial growth factor (VEGF) signaling in cancers. However, only a fraction of patients respond, and most ultimately develop resistance to current angiogenesis inhibitor therapies. Activity of alternative pro-angiogenic growth factors, acting via RTK or G-protein coupled receptors (GPCR), may mediate VEGF inhibitor resistance. The phosphoinositide 3-kinase (PI3K)β isoform is uniquely coupled to both RTK and GPCRs. We investigated the role of endothelial cell (EC) PI3Kβ in tumor angiogenesis. Pro-angiogenic GPCR ligands were expressed by patient-derived renal cell carcinomas (PD-RCC), and selective inactivation of PI3Kβ reduced PD-RCC-stimulated EC spheroid sprouting. EC-specific PI3Kβ knockout (ΕC-βKO) in mice potentiated the sunitinib-induced reduction in subcutaneous growth of LLC1 and B16F10, and lung metastasis of B16F10 tumors. Compared to single-agent sunitinib treatment, tumors in sunitinib-treated ΕC-βKO mice showed a marked decrease in microvessel density, and reduced new vessel formation. The fraction of perfused mature tumor microvessels was increased in ΕC-βKO mice suggesting immature microvessels were most sensitive to combined sunitinib and PI3Kβ inactivation. Taken together, EC PI3Kβ inactivation with sunitinib inhibition reduces microvessel turnover and decreases heterogeneity of the tumor microenvironment, hence PI3Kβ inhibition may be a useful adjuvant antiangiogenesis therapy with sunitinib.

Angiogenesis is essential for cardiac functional recovery after myocardial infarction (MI). HSPA12B is predominately expressed in endothelial cells and required for angiogenesis. Yes-associated protein (YAP) plays an important role in tumor angiogenesis. This study investigated the cooperative role of HSPA12B and YAP in angiogenesis after MI. Silencing of either HSPA12B or YAP impaired hypoxia-promoted endothelial cell proliferation and angiogenesis. Deficiency of HSPA12B suppressed YAP expression and nuclear translocation after hypoxia. Knockdown of YAP attenuated hypoxia-stimulated HSPA12B nuclear translocation and abrogated HSPA12B-promoted endothelial cell angiogenesis. Mechanistically, hypoxia induced an interaction between endothelial HSPA12B and YAP. ChIP assay showed that HSPA12B is a target gene of YAP/transcriptional enhanced associated domain 4 (TEAD4) and a coactivator in YAP-associated angiogenesis. In vivo studies using the MI model showed that endothelial cell-specific deficiency of HSPA12B (eHspa12b-/-) or YAP (eYap-/-) impaired angiogenesis and exacerbated cardiac dysfunction compared with WT mice. MI increased YAP expression and nuclear translocation in WT hearts but not eHspa12b-/- hearts. HSPA12B expression and nuclear translocation were upregulated in WT MI hearts but not eYap-/- MI myocardium. Our data demonstrate that endothelial HSPA12B is a target and coactivator for YAP/TEAD4 and cooperates with YAP to regulate endothelial angiogenesis after MI.

Adaptive angiogenesis is necessary for tissue repair, however, it may also be associated with the exacerbation of injury and development of chronic disease. In these studies, we demonstrate that lung mesenchymal vascular progenitor cells (MVPC) modulate adaptive angiogenesis via lineage trace, depletion of MVPC, and modulation of β-catenin expression. Single cell sequencing confirmed MVPC as multipotential vascular progenitors, thus, genetic depletion resulted in alveolar simplification with reduced adaptive angiogenesis. Following vascular endothelial injury, Wnt activation in MVPC was sufficient to elicit an emphysema-like phenotype characterized by increased MLI, fibrosis, and MVPC driven adaptive angiogenesis. Lastly, activation of Wnt/β-catenin signaling skewed the profile of human and murine MVPC toward an adaptive phenotype. These data suggest that lung MVPC drive angiogenesis in response to injury and regulate the microvascular niche as well as subsequent distal lung tissue architecture via Wnt signaling., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Bone microenvironment provides growth and survival signals essential for osteosarcoma (OS) initiation and progression. OS cells regulate communications inside tumor microenvironment through different ways and, among all, tumor-derived exosomes support cancer progression and metastasis. To define the contribution of OS-derived exosomes inside the microenvironment, we investigated the effects induced in bone remodeling mechanism and tumor angiogenesis. We demonstrated that exosomes promoted osteoclasts differentiation and bone resorption activity. Furthermore, exosomes potentiated tube formation of endothelial cells and increased angiogenic markers expression. We therefore investigated the micro RNA (miRNA) cargo from exosomes and their parental cells by performing small RNA sequencing through NGS Illumina platform. Hierarchical clustering highlighted a unique molecular profile of exosomal miRNA; bioinformatic analysis by DIANA-mirPath revealed that miRNAs identified take part in various biological processes and carcinogenesis. Among these miRNAs, some were already known for their involvement in the tumor microenvironment establishment, as miR-148a and miR-21-5p. Enforced expression of miR-148a and miR-21-5p in Raw264.7 and hTert immortalized umbilical vein endothelial cells recapitulated the effects induced by exosomes. Overall, our study highlighted the importance of OS exosomes in tumor microenvironment also by a specific packaging of miRNAs., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Progressive respiratory failure is the primary cause of death in the coronavirus disease 2019 (Covid-19) pandemic. Despite widespread interest in the pathophysiology of the disease, relatively little is known about the associated morphologic and molecular changes in the peripheral lung of patients who die from Covid-19., Methods: We examined 7 lungs obtained during autopsy from patients who died from Covid-19 and compared them with 7 lungs obtained during autopsy from patients who died from acute respiratory distress syndrome (ARDS) secondary to influenza A(H1N1) infection and 10 age-matched, uninfected control lungs. The lungs were studied with the use of seven-color immunohistochemical analysis, micro-computed tomographic imaging, scanning electron microscopy, corrosion casting, and direct multiplexed measurement of gene expression., Results: In patients who died from Covid-19-associated or influenza-associated respiratory failure, the histologic pattern in the peripheral lung was diffuse alveolar damage with perivascular T-cell infiltration. The lungs from patients with Covid-19 also showed distinctive vascular features, consisting of severe endothelial injury associated with the presence of intracellular virus and disrupted cell membranes. Histologic analysis of pulmonary vessels in patients with Covid-19 showed widespread thrombosis with microangiopathy. Alveolar capillary microthrombi were 9 times as prevalent in patients with Covid-19 as in patients with influenza (P<0.001). In lungs from patients with Covid-19, the amount of new vessel growth - predominantly through a mechanism of intussusceptive angiogenesis - was 2.7 times as high as that in the lungs from patients with influenza (P<0.001)., Conclusions: In our small series, vascular angiogenesis distinguished the pulmonary pathobiology of Covid-19 from that of equally severe influenza virus infection. The universality and clinical implications of our observations require further research to define. (Funded by the National Institutes of Health and others.)., (Copyright © 2020 Massachusetts Medical Society.)

Background: Malformation of blood vessels represents a hallmark of cancers, but the role and regulation of vascular mural cells (vMCs), including vascular smooth muscle cells (vSMCs) and pericytes, in tumors has not been fully understood. SM22α has been identified as a marker of vSMCs. This study aims at elucidating the function and regulation of SM22α + mural cells (SM22-MCs) in tumor stroma., Methods: Gene-modified mice with a SM22α-CreER T2 transgene were employed to deplete SM22-MCs or activate/block Notch signaling in these cells. vSMCs from mouse dorsal aorta (vSMCs-DA) were cultured in vitro. RNA-seq was used to compare gene expression profiles. qRT-PCR and western blotting were used to determine gene expression level. Immunofluorescence was used to observe morphological alterations in tumors., Results: SM22-MCs are essential for stabilizing tumor vasculature. Notch signaling was downregulated in tumor-derived SM22-MCs and vSMCs-DA treated with cancer cell-derived conditioned medium. Notch activation in SM22-MCs normalized tumor vasculature and repressed tumor growth. On the other hand, Notch disruption aggravated abnormal tumor vasculature and promoted growth and metastasis. Gene expression profiling of vSMCs-DA showed that Notch activation enhances their contractile phenotype and suppresses their secretory phenotype, further attenuating the invasion and proliferation of tumor cells. In contrast, Notch blockade in vSMCs-DA mitigated their contractile phenotype while strengthened the secretory phenotype., Conclusion: SM22-MCs facilitate vessel stability in tumors, and they gain a secretory phenotype and promote tumor malignancy in the absence of Notch signaling.

Combretastatin A-4 phosphate (CA4P) is a microtubule-disrupting tumour-selective vascular disrupting agent (VDA). CA4P activates the actin-regulating RhoA-GTPase/ ROCK pathway, which is required for full vascular disruption. While hypoxia renders tumours resistant to many conventional therapies, little is known about its influence on VDA activity. Here, we found that active RhoA and ROCK effector phospho-myosin light chain (pMLC) were downregulated in endothelial cells by severe hypoxia. CA4P failed to activate RhoA/ROCK/pMLC but its activity was restored upon reoxygenation. Hypoxia also inhibited CA4P-mediated actinomyosin contractility, VE-cadherin junction disruption and permeability rise. Glucose withdrawal downregulated pMLC, and coupled with hypoxia, reduced pMLC faster and more profoundly than hypoxia alone. Concurrent inhibition of glycolysis (2-deoxy-D-glucose, 2DG) and mitochondrial respiration (rotenone) caused profound actin filament loss, blocked RhoA/ROCK signalling and rendered microtubules  CA4P-resistant. Withdrawal of the metabolism inhibitors restored the cytoskeleton and CA4P activity. The AMP-activated kinase AMPK was investigated as a potential mediator of pMLC downregulation. Pharmacological AMPK activators that generate AMP, unlike allosteric activators, downregulated pMLC but only when combined with 2DG and/or rotenone. Altogether, our results suggest that Rho/ROCK and actinomyosin contractility are regulated by AMP/ATP levels independently of AMPK, and point to hypoxia/energy depletion as potential modifiers of CA4P response.

Gastrointestinal tumors are responsible for more cancer-related fatalities than any other type of tumors, and colorectal and gastric malignancies account for a large part of these diseases. Thus, there is an urgent need to develop new therapeutic approaches to improve the patients' outcome and the tumor microenvironment is a promising arena for the development of such treatments. In fact, the nature of the microenvironment in the different gastrointestinal tracts may significantly influence not only tumor development but also the therapy response. In particular, an important microenvironmental component and a potential therapeutic target is the vasculature. In this context, the extracellular matrix is a key component exerting an active effect in all the hallmarks of cancer, including angiogenesis. Here, we summarized the current knowledge on the role of extracellular matrix in affecting endothelial cell function and intratumoral vascularization in the context of colorectal and gastric cancer. The extracellular matrix acts both directly on endothelial cells and indirectly through its remodeling and the consequent release of growth factors. We envision that a deeper understanding of the role of extracellular matrix and of its remodeling during cancer progression is of chief importance for the development of new, more efficacious, targeted therapies.

Angiomatoid Spitz nevus (ASN) is a rare histological variant of Spitz nevus (SN) that is characterized by prominent blood vessel proliferation around the intradermal melanocytes of SN. In contrast, SN may have pagetoid components, which are characterized by epidermal proliferation of single melanocytes. However, cases of ASN with predominant pagetoid melanocytic proliferation in the epidermis have not been reported. Here, we report a case of ASN with surrounding pagetoid melanocytic proliferation without formation of tumor nests in the epidermis in the plantar region. A 12-year-old girl presented with a bright red nodule surrounded by a brown macule on the sole of her right foot. Histologically, the nodule showed tumor nests in the dermis, composed of spindle or epithelioid melanocytes containing abundant cytoplasm and large nuclei. Around the nests, numerous blood vessels were seen. In the overlying epidermis of the nodule, numerous eosinophilic Kamino bodies were found along the dermal-epidermal interface. In the macule, proliferation of oval melanocytes was present as single-cell units in the epidermis. Theses melanocytes had abundant cytoplasm with large nuclei, which were larger than those of the surrounding keratinocytes. From these findings, a diagnosis of ASN with surrounding pagetoid melanocytic proliferation was made. Vascular endothelial growth factor and fibroblast growth factor 2 were strongly expressed in the melanocytes as well as in the endothelial cells in our case. Therefore, angiogenic factors produced by the melanocytes of SN might have played important roles in the surrounding angiogenesis of this case., (© 2020 Japanese Dermatological Association.)

Fibrillins constitute a family of large extracellular glycoproteins which multimerize to form microfibrils, an important structure in the extracellular matrix. It has long been assumed that fibrillin-2 was barely present during postnatal life, but it is now clear that fibrillin-2 molecules form the structural core of microfibrils, and are masked by an outer layer of fibrillin-1. Mutations in fibrillins give rise to heritable connective tissue disorders, including Marfan syndrome and congenital contractural arachnodactyly. Fibrillins also play an important role in matrix sequestering of members of the transforming growth factor-β family, and in context of Marfan syndrome excessive TGF-β activation has been observed. TGF-β activation is highly dependent on integrin binding, including integrin αvβ8 and αvβ6, which are upregulated upon TGF-β exposure. TGF-β is also involved in tumor progression, metastasis, epithelial-to-mesenchymal transition and tumor angiogenesis. In several highly vascularized types of cancer such as hepatocellular carcinoma, a positive correlation was found between increased TGF-β plasma concentrations and tumor vascularity. Interestingly, fibrillin-1 has a higher affinity to TGF-β and, therefore, has a higher capacity to sequester TGF-β compared to fibrillin-2. The previously reported downregulation of fibrillin-1 in tumor endothelium affects the fibrillin-1/fibrillin-2 ratio in the microfibrils, exposing the normally hidden fibrillin-2. We postulate that fibrillin-2 exposure in the tumor endothelium directly stimulates tumor angiogenesis by influencing TGF-β sequestering by microfibrils, leading to a locally higher active TGF-β concentration in the tumor microenvironment. From a therapeutic perspective, fibrillin-2 might serve as a potential target for future anti-cancer therapies., Competing Interests: Declaration of Competing Interest The authors declare to have no conflict of interest.This work was supported by the KWF Cancer Society [2018–11651]., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In lobular capillary hemangioma (LCH), misnamed pyogenic granuloma, only sprouting angiogenesis (SA) has been considered. We assess the occurrence of intussusceptive angiogenesis (IA) in LCH and whether IA determines the specific and other focal patterns in the lesion. For this purpose, we study specimens of 120 cases of LCH, using semithin sections (in 10), immunohistochemistry, and confocal microscopy (in 20). In addition to SA, the results in LCH showed (1) intussusceptive phenomena, including pillars/folds and associated vessel loops, which encircled interstitial tissue structures (ITSs). (2) Two types of evolved loops depending on interendothelial contacts from opposite walls: (a) numerous interendothelial contacts, alternating with capillary-sized lumens (main capillary pattern of the lesion) and (b) few interendothelial contacts, wide open lumens, and intravascular transport of pillars/folds, which were arranged linearly, forming septa (focal sinusoidal-like pattern) or were irregularly grouped (focal intravascular papillary endothelial hyperplasia, IPEH-like pattern). In conclusion, we demonstrate that IA participates in synergistic interaction with SA in LCH development and that the prevalence of specific intussusceptive phenomena determines the predominant capillary pattern and associated sinusoidal hemangioma-like and IPEH-like patterns in the lesion, which suggest a role of IA as conditioner of vessel tumour/pseudo-tumour morphology.

(1) Background: Pericytes are involved in intraplaque neovascularization of advanced and complicated atherosclerotic lesions. However, the role of pericytes in human carotid plaques is unclear. An unstable carotid plaque that shows high-intensity signals on time-of-flight (TOF) magnetic resonance angiography (MRA) is often a cause of ischemic stroke. The aim of the present study is to examine the relationship between the pericytes in intraplaque neovessels and MRA findings. (2) Methods: A total of 46 patients with 49 carotid artery stenoses who underwent carotid endarterectomy at our hospitals were enrolled. The patients with carotid plaques that were histopathologically evaluated were retrospectively analyzed. Intraplaque hemorrhage was evaluated using glycophorin A staining, and intraplaque neovessels were evaluated using CD34 (Cluster of differentiation) stain as an endothelial cell marker or NG2 (Neuron-glial antigen 2) and CD146 stains as pericyte markers. Additionally, the relationships between the TOF-MRA findings and the carotid plaque pathologies were evaluated. (3) Results: Of the 49 stenoses, 28 had high-intensity signals (TOF-HIS group) and 21 had iso-intensity signals (TOF-IIS group) on TOF-MRA. The density of the CD34-positive neovessels was equivalent in both groups. However, the NG2- and CD146-positive neovessels had significantly higher densities in the TOF-HIS group than in the TOF-IIS group. (4) Conclusion: The presence of a high-intensity signal on TOF-MRA in carotid plaques was associated with intraplaque hemorrhage and few pericytes in intraplaque neovessels. These findings may contribute to the development of new therapeutic strategies focusing on pericytes., Competing Interests: The authors declare no conflict of interest.

Angiogenesis is defined as the growth of new capillaries sprouting from pre-existing vasculature. Pathological angiogenesis signals can lead to dysregulated development of new vessels. Inflammation is accompanied by pathological angiogenesis. During an inflammatory process, newly formed blood vessels provide oxygen and nutrients to the inflamed tissue, facilitating the transport of inflammatory cells. Therefore, angiogenesis is closely related to pathogenesis of inflammatory autoimmune diseases. As a member of the angiopoietin family, Angiopoietin-2 (Ang-2) plays an irreplaceable role in angiogenesis. This review will narrate the expression of Ang-2 and its role in inflammatory autoimmune diseases. Collecting this information may improve the acquaintance of Ang-2 and provide a theoretical foundation for clinical trials and drug development in the future., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Angiogenesis represents a hallmark of cancer. Several proteoglycans associate with cell surface receptors and regulate angiogenesis within the tumor microenvironment (TME). We highlight the recent discovery that the proteoglycan Agrin cross talks between the tumor and the endothelium to promote an angiogenesis privileged niche during cancer progression., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The vascular endothelial growth factor-A (VEGF-A)-VEGFR2 pathway drives tumor vascularization by activating proangiogenic signaling in endothelial cells (ECs). Here, we show that EC-sphingosine-1-phosphate receptor 1 (S1PR1) amplifies VEGFR2-mediated angiogenic signaling to enhance tumor growth. We show that cancer cells induce S1PR1 activity in ECs, and thereby, conditional deletion of S1PR1 in ECs (EC-S1pr1 -/- mice) impairs tumor vascularization and growth. Mechanistically, we show that S1PR1 engages the heterotrimeric G-protein Gi, which amplifies VEGF-VEGFR2 signaling due to an increase in the activity of the tyrosine kinase c-Abl1. c-Abl1, by phosphorylating VEGFR2 at tyrosine-951, prolongs VEGFR2 retention on the plasmalemma to sustain Rac1 activity and EC migration. Thus, S1PR1 or VEGFR2 antagonists, alone or in combination, reverse the tumor growth in control mice to the level seen in EC-S1pr1 -/- mice. Our findings suggest that blocking S1PR1 activity in ECs has the potential to suppress tumor growth by preventing amplification of VEGF-VEGFR2 signaling., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hepatopulmonary syndrome (HPS) is a serious vascular complication in the setting of liver disease. Factors produced by the liver are essential to regulate pulmonary angiogenesis in the pathogenesis of HPS; however, the pathogenic mechanisms of pulmonary angiogenesis are not fully understood. We investigated the role of HPS rat serum exosomes (HEs) and sham-operated rat serum exosomes (SEs) in the regulation of angiogenesis. We found that HEs significantly enhance PMVEC proliferation, migration, and tube formation. We further identified miR-194 was the most notably increased miRNA in HEs compared to SEs. Once released, hepatocyte-derived exosomal miR-194 was internalized by PMVECs, leading to the promotion of PMVEC proliferation, migration, and tube formation through direct targeting of THBS1, STAT1, and LIF. Importantly, the pathogenic role of exosomal miR-194 in initiating angiogenesis was reversed by P53 inhibition, exosome secretion inhibition or miR-194 inhibition. Additionally, high levels of miR-194 were found in serum exosomes and were positively correlated with P(A-a)O 2 in HPS patients and rats. Thus, our results highlight that the exosome/miR-194 axis plays a critical pathologic role in pulmonary angiogenesis, representing a new therapeutic target for HPS.

The assessment of vascular permeability of malignant tumor plays an important role in the diagnosis and treatment of cancer. Dynamic contrast-enhanced magnetic resonance image (DCE-MRI) using Gd-encapsulated carbonaceous dots and Gd-DTPA-BMA as contrast agents was performed in 4T1 mouse breast cancer and HCC827 human non-small-cell lung cancer (NSNLC) xenograft models. Histopathological parameters of tumor vascularity microvessel density (MVD), microvessel area (MVA), endothelial area (EA) and α-SMA CD31 Co-expression (α-SMA/CD31%) were compared with the DCE-MRI parameters. Results demonstrated that DCE-MRI with the new nanoparticle Gd@C-dots can noninvasively evaluate vascular permeability. K trans measured by DCE-MRI with Gd@C-dots is an accurate parameter for the characterization of tumor permeability. EA is a reliable microvessel parameter to evaluate vessel permeability., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Angiogenesis is a hallmark of cancer. However, current antivascular therapies that primarily target angiogenic factors have faced difficulties and failures in treating most cancers. It is proposed that endothelial transformation acts as an alternative driver of excessive and abnormal vascularity that fuels cancer progression and immunosuppression, and also induces resistance to therapy. Thus, vascular detransformation may serve as a promising therapeutic strategy against cancer., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Chemerin is a multifunctional adipokine with established roles in inflammation, adipogenesis and glucose homeostasis. Increasing evidence suggest an important function of chemerin in cancer. Chemerin's main cellular receptors, chemokine-like receptor 1 (CMKLR1), G-protein coupled receptor 1 (GPR1) and C-C chemokine receptor-like 2 (CCRL2) are expressed in most normal and tumor tissues. Chemerin's role in cancer is considered controversial, since it is able to exert both anti-tumoral and tumor-promoting effects, which are mediated by different mechanisms like recruiting innate immune defenses or activation of endothelial angiogenesis. For this review article, original research articles on the role of chemerin and its receptors in cancer were considered, which are listed in the PubMed database. Additionally, we included meta-analyses of publicly accessible DNA microarray data to elucidate the association of expression of chemerin and its receptors in tumor tissues with patients' survival.

The formation of new blood vessels is essential for normal development, tissue repair and tumor growth. Here we show that inhibition of the kinase p38α enhances angiogenesis in human and mouse colon tumors. Mesenchymal cells can contribute to tumor angiogenesis by regulating proliferation and migration of endothelial cells. We show that p38α negatively regulates an angiogenic program in mesenchymal stem/stromal cells (MSCs), multipotent progenitors found in perivascular locations. This program includes the acquisition of an endothelial phenotype by MSCs mediated by both TGF-β and JNK, and negatively regulated by p38α. Abrogation of p38α in mesenchymal cells increases tumorigenesis, which correlates with enhanced angiogenesis. Using genetic models, we show that p38α regulates the acquisition of an endothelial-like phenotype by mesenchymal cells in colon tumors and damage tissue. Taken together, our results indicate that p38α in mesenchymal cells restrains a TGF-β-induced angiogenesis program including their ability to transdifferentiate into endothelial cells.

B-cell lymphoma 2 (Bcl-2) protein is the founding member of a group of proteins known to modulate apoptosis. Its discovery set the stage for identification of family members with either pro- or anti-apoptotic properties. Expression of Bcl-2 plays an important role during angiogenesis by influencing not only vascular cell survival, but also migration and adhesion. Although apoptosis and migration are postulated to have roles during vascular remodeling and regression, the contribution of Bcl-2 continues to emerge. We previously noted that the impaired retinal vascularization and an inability to undergo pathologic neovascularization observed in mice globally lacking Bcl-2 did not occur when mice lacked the expression of Bcl-2 only in endothelial cells. To further examine the effect of Bcl-2 expression during vascularization of the retina, we assessed its contribution in pericytes or astrocytes by generating mice with a conditional Bcl-2 allele (Bcl-2 Flox/Flox ) and Pdgfrb-cre (Bcl-2 PC mice) or Gfap-cre (Bcl-2 AC mice). Bcl-2 PC and Bcl-2 AC mice demonstrated increased retinal vascular cell apoptosis, reduced numbers of pericytes and endothelial cells and fewer arteries and veins in the retina. Bcl-2 PC mice also demonstrated delayed advancement of the superficial retinal vascular layer and aberrant vascularization of the deep vascular plexus and central retina. Although pathologic neovascularization in oxygen-induced ischemic retinopathy (OIR) was not affected by lack of expression of Bcl-2 in either pericytes or astrocytes, laser-induced choroidal neovascularization (CNV) was significantly reduced in Bcl-2 PC mice compared to littermate controls. Together these studies begin to reveal how cell autonomous modulation of apoptosis in vascular cells impacts development and homeostasis.

Perigestational alcohol consumption up to early organogenesis can produce abnormal maternal vascularization via altered decidual VEGF/receptor expression. CF-1 female mice were administered with 10% ethanol in drinking water for 17 days prior to and up to day 10 of gestation. Control females received water without ethanol. Treated females had reduced frequency of implantation sites with expanded vascular lumen (P < 0.05), α-SMA-immunoreactive spiral arteries in proximal mesometrial decidua, reduced PCNA-positive endothelial cells (P < 0.01) and diminished uterine NK cell numbers (P < 0.05) in proximal decidua compared to controls. The VEGF expression (laser capture microscopy, RT-PCR, western blot and immunohistochemistry) was reduced in decidual tissue after perigestational alcohol consumption (P < 0.05). The uNK-DBA+ cells of treated females had reduced VEGF immunoexpression compared to controls (P < 0.01). Very low decidual and endothelial cell KDR immunoreactivity and reduced decidual gene and protein KDR expression was found in treated females compared to controls (P < 0.001). Instead, strong FLT-1 immunoexpression was detected in decidual and uNK cells (P < 0.05) in the proximal decidua from treated females compared to controls. In conclusion, perigestational alcohol ingestion induces the reduction of lumen expansion of spiral arteries, concomitant with reduced endothelial cell proliferation and uNK cell population, and uncompleted remodeling of the artery smooth muscle. These effects were supported by low decidual VEGF and KDR gene and protein expression and increased FLT-1 expression, suggesting that VEGF and KDR reduction may contribute, in part, to mechanisms involved in deficient decidual angiogenesis after perigestational alcohol consumption in mouse.

Appropriate therapeutic modulation of endothelial proliferation and sprouting is essential for the effective inhibition of angiogenesis in cancer or its induction in cardiovascular disease. The current view is that an increase in growth factor concentration, and the resulting mitogenic activity, increases both endothelial proliferation and sprouting. Here, we modulate mitogenic stimuli in different vascular contexts by interfering with the function of the VEGF and Notch signalling pathways at high spatiotemporal resolution in vivo. Contrary to the prevailing view, our results indicate that high mitogenic stimulation induced by VEGF, or Notch inhibition, arrests the proliferation of angiogenic vessels. This is due to the existence of a bell-shaped dose-response to VEGF and MAPK activity that is counteracted by Notch and p21, determining whether endothelial cells sprout, proliferate, or become quiescent. The identified mechanism should be considered to achieve optimal therapeutic modulation of angiogenesis.

Long non-coding RNAs (lncRNAs) have been identified as playing critical roles in multiple diseases. However, little is known regarding their roles and mechanisms in post-stroke angiogenesis. Our studies focused on deciphering the functional roles and the underlying mechanisms of the lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in the process of angiogenesis following oxygen-glucose deprivation/reoxygenation (OGD/R). We characterized the up-regulation of MALAT1 expression in the process of angiogenesis after hypoxic injury in vivo and in vitro. We further showed that compared with the empty vector, MALAT1 knockdown had significantly reduced the capacity for angiogenesis, which was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), scratching, cell cycle and immunofluorescent staining. Thus, our findings suggest that MALAT1 may mediate proangiogenic function in OGD/R. To further explore the potential mechanisms, we used lentiviruses expressing shMALAT1 and empty vector; the results revealed that shMALAT1 reduced the expression of 15-lipoxygenase 1 (15-LOX1), vascular endothelial growth factor (VEGF) and the phosphorylation of signal transducers and activators of transcription 3 (pSTAT3). Taken together, our results are the first to propose that MALAT1 may regulate angiogenesis through the 15-LOX1/STAT3 signalling pathway, and they may provide a critical target for the treatment of hypoxic injury and an avenue for therapeutic angiogenesis., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

CCM3, originally described as PDCD10, regulates blood-brain barrier integrity and vascular maturation in vivo. CCM3 loss-of-function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral- and plasmid-free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss-of-function CCM3 variants into human ECs and studied the molecular and functional effects of long-term CCM3 inactivation. Induction of apoptosis, sprouting, migration, network and spheroid formation were significantly impaired upon prolonged CCM3 deficiency. Real-time deformability cytometry demonstrated that loss of CCM3 induces profound changes in cell morphology and mechanics: CCM3-deficient ECs have an increased cell area and elastic modulus. Small RNA profiling disclosed that CCM3 modulates the expression of miRNAs that are associated with endothelial ageing. In conclusion, the use of CRISPR/Cas9 genome editing provides new insight into the consequences of long-term CCM3 inactivation in human ECs and supports the hypothesis that clonal expansion of CCM3-deficient dysfunctional ECs contributes to CCM formation., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: Glioma, characterized by its undesirable prognosis and poor survival rate, is a serious threat to human health and lives. MicroRNA-9 (miR-9) is implicated in the regulation of multiple tumors, while the mechanisms underlying its aberrant expression and functional alterations in human glioma are still controversial., Methods: Expressions of miR-9 were measured in GEO database, patient specimens and glioma cell lines. Gain- and loss-of-function assays were applied to identify the effects of miR-9 on glioma cells and HUVECs in vitro and in vivo. Potential targets of miR-9 were predicted by bioinformatics and further verified via in vitro experiments. Transcriptional regulation of miR-9 by MYC and OCT4 was determined in glioma cells., Results: MiR-9 was frequently up-regulated in glioma specimens and cells, and could significantly enhance proliferation, migration and invasion of glioma cells. In addition, miR-9 could be secreted from glioma cells via exosomes and was then absorbed by vascular endothelial cells, leading to an increase in angiogenesis. COL18A1, THBS2, PTCH1 and PHD3 were verified as the direct targets of miR-9, which could elucidate the miR-9-induced malignant phenotypes in glioma cells. MYC and OCT4 were able to bind to the promoter region of miR-9 to trigger its transcription., Conclusions: Our results highlight that miR-9 is pivotal for glioma pathogenesis and can be treated as a potential therapeutic target for glioma.

Background : Yanghe Huayan Decoction (YHD), a traditional Chinese medicine, is one of the most common complementary medicine currently used in the treatment of breast cancer (BC). It has been recently linked to suppress precancerous lesion and tumor development. The current study sought to explore the role of YHD on trans-endothelium and angiogenesis of BC. Methods: HER2+ BC cells were treated with YHD, Trastuzumab, or the combination in vitro and in vivo to compare the effects of them on trans-endothelium and angiogenesis features. The present study also investigated the potential molecular mechanism of YHD in inhibiting angiogenesis of BC. Results : YHD significantly suppressed the invasion and angiogenesis of BC cells via elevated pAkt signaling. Administration of YHD in vivo also strikingly repressed angiogenesis in tumor grafts. Conclusion: YHD could partially inhibit and reverse tumorigenesis of BC. It also could inhibit Akt activation and angiogenesis in vitro and in vivo Its effect was superior to trastuzumab. Thus it was suitable for prevention and treatment of BC., (© 2019 The Author(s).)

A hallmark of proliferative retinopathies, such as retinopathy of prematurity (ROP), is a pathological neovascularization orchestrated by hypoxia and the resulting hypoxia-inducible factor (HIF)-dependent response. We studied the role of Hif2α in hematopoietic cells for pathological retina neovascularization in the murine model of ROP, the oxygen-induced retinopathy (OIR) model. Hematopoietic-specific deficiency of Hif2α ameliorated pathological neovascularization in the OIR model, which was accompanied by enhanced endothelial cell apoptosis. That latter finding was associated with up-regulation of the apoptosis-inducer FasL in Hif2α-deficient microglia. Consistently, pharmacological inhibition of the FasL reversed the reduced pathological neovascularization from hematopoietic-specific Hif2α deficiency. Our study found that the hematopoietic cell Hif2α contributes to pathological retina angiogenesis. Our findings not only provide novel insights regarding the complex interplay between immune cells and endothelial cells in hypoxia-driven retina neovascularization but also may have therapeutic implications for proliferative retinopathies.-Korovina, I., Neuwirth, A., Sprott, D., Weber, S., Sardar Pasha, S. P. B., Gercken, B., Breier, G., El-Armouche, A., Deussen, A., Karl, M. O., Wielockx, B., Chavakis, T., Klotzsche-von Ameln, A. Hematopoietic hypoxia-inducible factor 2α deficiency ameliorates pathological retinal neovascularization via modulation of endothelial cell apoptosis.

Tumor vascularization is a hallmark of cancer central to disease progression and metastasis. Current anti-angiogenic therapies have limited success prompting the need to better understand the cellular origin of tumor vessels. Using fate-mapping analysis of endothelial cell populations in melanoma, we report the very early infiltration of endovascular progenitors (EVP) in growing tumors. These cells harbored self-renewal and reactivated the expression of SOX18 transcription factor, initiating a vasculogenic process as single cells, progressing towards a transit amplifying stage and ultimately differentiating into more mature endothelial phenotypes that comprised arterial, venous and lymphatic subtypes within the core of the tumor. Molecular profiling by RNA sequencing of purified endothelial fractions characterized EVPs as quiescent progenitors remodeling the extracellular matrix with significant paracrine activity promoting growth. Functionally, EVPs did not rely on VEGF-A signaling whereas endothelial-specific loss of Rbpj depleted the population and strongly inhibited metastasis. The understanding of endothelial heterogeneity opens new avenues for more effective anti-vascular therapies in cancer.

Roundabout guidance receptor 4 (Robo4) is an endothelial cell-specific receptor that stabilizes the vasculature in pathological angiogenesis. Although Robo4 has been shown to suppress vascular hyperpermeability induced by vascular endothelial growth factor (VEGF) in angiogenesis, the role of Robo4 in inflammation is poorly understood. In this study, we investigated the role of Robo4 in vascular hyperpermeability during inflammation. Endotoxemia models using Robo4 -/- mice showed increased mortality and vascular leakage. In endothelial cells, Robo4 suppressed tumor necrosis factor α (TNFα)-induced hyperpermeability by stabilizing VE-cadherin at cell junctions, and deletion assays revealed that the C-terminus of Robo4 was involved in this suppression. Through binding and localization assays, we demonstrated that in endothelial cells, Robo4 binds to TNF receptor-associated factor 7 (TRAF7) through interaction with the C-terminus of Robo4. Gain- and loss-of-function studies of TRAF7 with or without Robo4 expression showed that TRAF7 is required for Robo4-mediated suppression of hyperpermeability. Taken together, our results demonstrate that the Robo4-TRAF7 complex is a novel negative regulator of inflammatory hyperpermeability. We propose this complex as a potential future target for protection against inflammatory diseases., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Renal cell carcinoma (RCC) is highly dependent on angiogenesis, due to the overactivation of the VHL/HIF/VEGF/VEGFRs axis; this justifies the marked sensitivity of this neoplasm to antiangiogenic agents which, however, ultimately fail to control tumor growth. RCC also frequently shows alterations in the mTOR signaling pathway, and mTOR inhibitors have shown a similar pattern of initial activity/late failure as pure antiangiogenic agents. Understanding mechanisms of resistance to these agents would be key to improve the outcome of our patients. Circulating endothelial cells are a family of mainly bone marrow-derived progenitors, which have been postulated to be responsible of the reactivation of angiogenesis in different tumors. In this review, we shall discuss the complex nature and function of these cells, the evidence pro and contra their contribution to tumor vascularization, especially as far as RCC is concerned, and their possible role in determining resistance to presently available treatments., (Copyright © 2018. Published by Elsevier B.V.)

Objective: In systemic sclerosis (SSc), early microvascular injury is followed by impaired angiogenesis and peripheral capillary loss. Here, we investigated the possible contribution of the neurovascular guidance molecule Slit2 and its Roundabout (Robo) receptors to SSc-related endothelial cell dysfunction., Methods: Circulating Slit2 levels were measured in patients with SSc and healthy controls. Slit2, Robo1 and Robo4 expression was investigated in SSc and healthy skin biopsies and explanted dermal microvascular endothelial cells (MVECs). Slit2/Robo4 function in MVEC angiogenesis was studied by cell viability, wound healing and capillary-like tube formation assays., Results: Circulating Slit2 was significantly increased in either SSc or patients with a very early diagnosis of SSc (VEDOSS) compared with controls. Interestingly, serum Slit2 levels were raised in patients with VEDOSS with nailfold videocapillaroscopy (NVC) abnormalities, while they were similar in VEDOSS with normal NVC and controls. In SSc, Slit2 and Robo4 expression was upregulated in clinically affected skin and explanted MVECs in respect to controls. The angiogenic performance of healthy MVECs was significantly reduced after challenge with recombinant human Slit2 or SSc sera. These inhibitory effects were significantly attenuated when SSc sera were preincubated with an anti-Slit2 blocking antibody. In vitro angiogenesis was severely compromised in SSc-MVECs and could be significantly ameliorated by Slit2 neutralisation or ROBO4 gene silencing. Slit2/Robo4 axis interfered with angiogenesis through the inhibition of Src kinase phosphorylation., Conclusions: In SSc, increased circulating levels of Slit2 and activation of the Slit2/Robo4 antiangiogenic axis may contribute to peripheral microangiopathy since the very early phase of the disease., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2018. No commercial re-use. See rights and permissions. Published by BMJ.)

Estradiol (E2) is a prime culprit for enhancing the progression of female hormone‑related cancers. Bone marrow‑derived cells (BMDCs) have been found to play a pivotal role in tumor growth. Estrogen receptors (ERs) are also found on certain subtypes of BMDCs, in addition to endothelial cells (ECs) and certain tumor cells. However, the role of BMDCs in E2‑induced tumor biology is still unclear. Thus, the effects of E2 on ER‑negative 4T1 breast cancer growth, the mobilization and recruitment of BMDCs, and interactions among BMDCs, ECs, and 4T1 cells were investigated. The results showed that E2 potentiated 4T1 tumor growth and angiogenesis in mice subjected to sham operation, ovariectomy (OVX), or OVX and E2 replacement treatment. E2 supplementation in mice with OVX upregulated the transcription of stromal cell‑derived factor‑1 (SDF‑1) mRNA in tumor tissues and enhanced the recruitment of BMDCs into tumor tissues in vivo. E2 deficiency significantly decreased proangiogenic CXCR4+, β3+, Sca‑1+ and CXCR4+β3+ BMDCs circulating in the peripheral blood. Cell‑based system analyses showed that E2 augmented the transcription of β3 mRNA in ECs, increased the adhesion of BMDCs to ECs. In addition, E2 enhanced the BMDC‑induced EC proliferation and migration, the BMDC‑induced 4T1 proliferation and the 4T1‑stimulated EC proliferation in addition to enhancing the proliferation of tumor cells and the migration of ECs in vitro. Therefore, E2 enhanced the growth of breast tumors by stimulating tumor cells and ECs directly, as well as by increasing proangiogenic BMDC mobilization and recruitment leading to augmentation of the tumor and EC functions indirectly by cell proliferation assay. These findings reveal a separate mechanism via which E2 promotes the growth of female hormone‑dependent tumors, which may be useful in explorations of new therapies for related cancers.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at a post-transcriptional level via either the degradation or translational repression of a target mRNA. They play an irreplaceable role in angiogenesis by regulating the proliferation, differentiation, apoptosis, migration and tube formation of angiogenesis-related cells, which are indispensable for multitudinous physiological and pathological processes, especially for the occurrence and development of vascular diseases. Imbalance between the regulation of miRNAs and angiogenesis may cause many diseases such as cancer, cardiovascular disease, aneurysm, Kawasaki disease, aortic dissection, phlebothrombosis and diabetic microvascular complication. Therefore, it is important to explore the essential role of miRNAs in angiogenesis, which might help to uncover new and effective therapeutic strategies for vascular diseases. This review focuses on the interactions between miRNAs and angiogenesis, and miRNA-based biomarkers in the diagnosis, treatment and prognosis of angiogenesis-related diseases, providing an update on the understanding of the clinical value of miRNAs in targeting angiogenesis., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Angiogenesis, the growth of capillaries from pre-existing ones, plays a key role in cancer progression. Tumours release tumour angiogenic factors (TAFs) into the extracellular matrix (ECM) that trigger angiogenesis once they reach the vasculature. The neovasculature provides nutrients and oxygen to the tumour. In the ECM, the interstitial fluid moves driven by pressure differences and may affect the distribution of tumour TAFs, and, in turn, tumour vascularization. In this work, we propose a hybrid mathematical model to investigate the influence of fluid flow in tumour angiogenesis. Our model shows the impact of interstitial flow in a time-evolving capillary network using a continuous approach. The flow model is coupled to a model of angiogenesis that includes tip endothelial cells, filopodia, capillaries and TAFs. The TAF transport equation considers not only diffusive mechanisms but also the convective transport produced by interstitial flow. Our simulations predict a significant alteration of the new vascular networks, which tend to grow more prominently against the flow. The model suggests that interstitial flow may produce increased tumour malignancies and hindered treatments., (© 2018 The Author(s).)