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Alzheimer’s disease (AD) is a common disorder of progressive cognitive decline among elderly subjects. Angiogenesis-related factors including vascular endothelial growth factor (VEGF) might be involved in the pathogenesis of AD. Soluble form of the VEGF receptor is likely to be an intrinsic negative counterpart of VEGF. We measured the plasma levels of VEGF and its two soluble receptors (sVEGFR1 and sVEGFR2) in 120 control subjects, 75 patients with mild cognitive impairment, and 76 patients with AD using ELISA. Plasma levels of VEGF in patients with AD were higher than those in healthy control subjects. However, plasma levels of sVEGFR1 and sVEGFR2 were lower in patients with AD than in healthy control subjects. Levels of VEGFR2 mRNA were significantly decreased in human umbilical vein endothelial cells after amyloid-beta treatment. Further, protein levels of VEGFR2 were also decreased in the brains of AD model mice. In addition, we show that the expression of sVEGFR2 and VEGFR2 was also decreased by the transfection with the Notch intracellular domain. These results indicate that the alterations of VEGF and its two receptors levels might be associated with those at risk for Alzheimer’s disease.

Arterial networks enlarge in response to increase in tissue metabolism to facilitate flow and nutrient delivery. Typically, the transition of a growing artery with a small diameter into a large caliber artery with a sizeable diameter occurs upon the blood flow driven change in number and shape of endothelial cells lining the arterial lumen. Here, using zebrafish embryos and endothelial cell models, we describe an alternative, flow independent model, involving enlargement of arterial endothelial cells, which results in the formation of large diameter arteries. Endothelial enlargement requires the GEF1 domain of the guanine nucleotide exchange factor Trio and activation of Rho-GTPases Rac1 and RhoG in the cell periphery, inducing F-actin cytoskeleton remodeling, myosin based tension at junction regions and focal adhesions. Activation of Trio in developing arteries in vivo involves precise titration of the Vegf signaling strength in the arterial wall, which is controlled by the soluble Vegf receptor Flt1. Arterial flow regulates artery diameter but other mechanisms may also affect this. Here, the authors show that the guanine nucleotide exchange factor Trio and GTPases Rac1 and RhoG, triggers F-actin remodeling in arterial endothelial cells, independent of flow, to enhance lumen diameter in zebrafish and cell models.

A branched vascular network is crucial to placental development and is dependent on factors such as vascular endothelial growth factor (VEGF), placental growth factor (PlGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) to regulate blood vessel growth. Imbalances in these factors can lead to aberrant placental vascular development. Throughout pregnancy, these factors are also released into the maternal circulation to aid in adapting the maternal cardiovascular system to pregnancy. Increased secretion of anti-angiogenic factors can lead to the development of an anti-angiogenic state in the mother and contribute to the development of pregnancy pathologies such as pre-eclampsia and foetal growth restriction (FGR). Thus, what are commonly referred to as ‘angiogenic factors’ have distinct functions in the maternal and placental circulations making this a misnomer. Indeed, technical issues in this field such as assay methodology and lack of data considering different placental cell types mean that the physiological roles of these factors in the maternal and placental circulations are frequently muddled in the literature. This review aims to (1) unpick the distinct roles of factors that influence placental vascular development and separate these from the roles of the same factors within the maternal circulation in normal pregnancy and (2) critically assess how imbalances may contribute to the distinct pathophysiological mechanisms underlying pregnancy disorders. Together, this critical assessment of the field endeavours to improve our ability to accurately use these factors as predictive/diagnostic biomarkers in the future.

Key Points Angiogenesis inhibitors that target vascular endothelial growth factor (VEGF) signalling pathways have proved successful for the clinical treatment of various types of cancer. However, a substantial fraction of tumours is resistant or escapes current anti-angiogenic therapies. Moreover, VEGFA is a trophic factor for healthy vessels, and therefore anti-angiogenic therapies cause side effects that although well managed, can also lead to life-threatening conditions in a subset of patients with cancer. VEGFR2 (also known as FLK1) is the primary receptor that binds VEGFA, and therefore controls angiogenesis in both healthy and diseased tissues. By contrast, VEGFR1 (also known as FLT1) binds VEGFA, VEGFB and placental growth factor (PlGF). The expression of FLT1 and its two ligands, PlGF and VEGFB, is increased in various tumours, which correlates with disease progression and can predict poor prognosis, metastasis and recurrent disease in humans. PlGF signals directly through FLT1 in various cell types, including endothelial cells, smooth-muscle cells, fibroblasts, angiogenesis-competent myeloid progenitors, macrophages and tumour cells, and thereby promotes tumour angiogenesis, lymphangiogenesis, tumour growth and the formation of the premetastatic niche. Therefore deletion of tyrosine-kinase activity in Flt1 TK−/− mice or treatment with FLT1- and PlGF-specific inhibitors, such as a monoclonal antibody against PlGF (αPlGF), impairs inflammation and pathological angiogenesis, and suppresses tumour growth and metastasis. Myeloid cells confer resistance to therapies that target VEGF by secreting additional pro-angiogenic factors. αPlGF enhances the responsiveness to VEGF-targeted therapies by inhibiting macrophage recruitment. Therefore, owing to its complementary activities, αPlGF eliminates the source of angiogenic factors that contributes to anti-angiogenic escape of tumours. Gene-deletion studies have revealed that PlGF is redundant for vascular development and physiological vessel maintenance in healthy adults, but contributes to the angiogenic and inflammatory switch in cancer. In support of the concept that PlGF is a disease-specific factor, αPlGF selectively inhibits pathological angiogenesis without affecting healthy vessels and thus does not cause the side effects that are typically observed during current anti-angiogenic therapies. Genetic and pharmacological studies have thus identified FLT1 and PlGF as attractive therapeutic targets for anticancer therapy, which might provide an answer to some of the challenges and unmet needs that are faced by current anti-angiogenic therapies: how to increase efficacy, avoid resistance and minimize toxicity. Anti-angiogenic drugs have become part of the standard therapeutics used to treat cancer. Despite this milestone, anti-angiogenic therapy still faces a number of clinical hurdles. Will other agents with complementary mechanisms offer novel opportunities for improved treatment? Less than 5 years ago, it was still not clear whether anti-angiogenic drugs would prove successful in the clinic. After numerous patients with cancer or age-related macular degeneration have been treated with these drugs, they have now become part of the standard range of therapeutic tools. Despite this milestone, anti-angiogenic therapy still faces a number of clinical hurdles, such as improving efficacy, avoiding escape and resistance, and minimizing toxicity. Hopefully, other agents with complementary mechanisms, such as those that target placental growth factor, will offer novel opportunities for improved treatment.

Tumor progression and metastatic dissemination are driven by cell-intrinsic and biomechanical cues that favor the growth of life-threatening secondary tumors. We recently identified pro-metastatic vascular regions with blood flow profiles that are permissive for the arrest of circulating tumor cells. We have further established that such flow profiles also control endothelial remodeling, which favors extravasation of arrested CTCs. Yet, how shear forces control endothelial remodeling is unknown. In the present work, we aimed at dissecting the cellular and molecular mechanisms driving blood flow-dependent endothelial remodeling. Transcriptomic analysis of endothelial cells revealed that blood flow enhanced VEGFR signaling, among others. Using a combination of in vitro microfluidics and intravital imaging in zebrafish embryos, we now demonstrate that the early flow-driven endothelial response can be prevented upon specific inhibition of VEGFR tyrosine kinase and subsequent signaling. Inhibitory targeting of VEGFRs reduced endothelial remodeling and subsequent metastatic extravasation. These results confirm the importance of VEGFR-dependent endothelial remodeling as a driving force of CTC extravasation and metastatic dissemination. Furthermore, the present work suggests that therapies targeting endothelial remodeling might be a relevant clinical strategy in order to impede metastatic progression.

•  Introduction ‐  Angiogenesis in physiology and pathology ‐  Angiogenesis in current medicine ‐  VEGF ligand and receptor system: where does sVEGFR1 fit? ‐  Ligands: the human VEGF family ‐  Membrane‐bound signalling receptors: VEGFRs ‐  Non‐signalling co‐receptors and matrix proteins: HSPGs and NRPs ‐  Soluble receptors: sVEGFR1, sVEGFR2, sNRP1 •  Molecular biology of sVEGFR1 •  Physiological and pathophysiological roles of sVEGFR1 •  Molecular mechanism of sVEGFR1’s anti‐angiogenic potential •  sVEGFR1 as a clinical marker for disease •  Plasma VEGF and sVEGFR1: non‐uniform predictors of angiogenic status across all diseases •  Systems biology perspective: unifying interpretation of plasma angiogenic markers ‐  Baseline heterogeneity in clinical measurements of healthy VEGF and sVEGFR1 levels in plasma ‐  Effect of sVEGFR1 on VEGF bioavailability: VEGF‐sVEGFR1 complexes ‐  Compartmental analysis: biotransport and biodistribution ‐  Pathogenic phenomenon versus compensatory response •  Concluding remarks Angiogenesis is the growth of new capillaries from pre‐existent microvasculature. A wide range of pathological conditions, from atherosclerosis to cancer, can be attributed to either excessive or deficient angiogenesis. Central to the physiological regulation of angiogenesis is the vascular endothelial growth factor (VEGF) system – its ligands and receptors (VEGFRs) are thus prime molecular targets of pro‐angiogenic and anti‐angiogenic therapies. Of growing interest as a prognostic marker and therapeutic target in angiogenesis‐dependent diseases is soluble VEGF receptor‐1 (sVEGFR1, also known as sFlt‐1) – a truncated version of the cell membrane‐spanning VEGFR1. For instance, it is known that sVEGFR1 is involved in the endothelial dysfunction characterizing the pregnancy disorder of pre‐eclampsia, and sVEGFR1’s therapeutic potential as an anti‐angiogenic agent is being evaluated in pre‐clinical models of cancer. This mini review begins with an examination of the protein domain structure and biomolecular interactions of sVEGFR1 in relation to the full‐length VEGFR1. A synopsis of known and inferred physiological and pathological roles of sVEGFR1 is then given, with emphasis on the utility of computational systems biology models in deciphering the molecular mechanisms by which sVEGFR1’s purported biological functions occur. Finally, we present the need for a systems biology perspective in interpreting circulating VEGF and sVEGFR1 concentrations as surrogate markers of angiogenic status in angiogenesis‐dependent diseases.

While vascular endothelial growth factor (VEGF) expression in breast tumors has been correlated with a poor outcome in the pathogenesis of breast cancer, the expression, localization, and function of VEGF receptors VEGFR1 (also known as FLT1) and VEGFR2 (also known as KDR or FLK1), as well as neuropilin 1 (NRP1), in breast cancer are controversial. We investigated the expression and function of VEGF and VEGF receptors in breast cancer cells. We observed that VEGFR1 expression was abundant, VEGFR2 expression was low, and NRP1 expression was variable. MDA-MB-231 and MCF-7 breast cancer cells, transfected with antisense VEGF cDNA or with siVEGF (VEGF-targeted small interfering RNA), showed a significant reduction in VEGF expression and increased apoptosis as compared to the control cells. Additionally, specifically targeted knockdown of VEGFR1 expression by siRNA (siVEGFR1) significantly decreased the survival of breast cancer cells through down-regulation of protein kinase B (AKT) phosphorylation, while targeted knockdown of VEGFR2 or NRP1 expression had no effect on the survival of these cancer cells. Since a VEGFR1-specific ligand, placenta growth factor (PGF), did not, as expected, inhibit the breast cancer cell apoptosis induced by siVEGF, and since VEGFR1 antibody also had no effects on the survival of these cells, we examined VEGFR1 localization. VEGFR1 was predominantly expressed internally in MDA-MB-231 and MCF-7 breast cancer cells. Specifically, VEGFR1 was found to be colocalized with lamin A/C and was expressed mainly in the nuclear envelope in breast cancer cell lines and primary breast cancer tumors. Breast cancer cells treated with siVEGFR1 showed significantly decreased VEGFR1 expression levels and a lack of VEGFR1 expression in the nuclear envelope. This study provides, to our knowledge for the first time, evidence of a unique survival system in breast cancer cells by which VEGF can act as an internal autocrine (intracrine) survival factor through its binding to VEGFR1. These results may lead to an improved strategy for tumor therapy based on the inhibition of angiogenesis.

Background: In healthy pregnancies, components of the Renin-Angiotensin system (RAS) are present in the placental villi and contribute to invasion, migration, and angiogenesis. At the same time, soluble fms-like tyrosine kinase 1 (sFlt-1) production is induced after binding of ANG-II to its receptor (AT-1R) in response to hypoxia. As RAS plays an essential role in the pathogenesis of COVID-19, we hypothesized that angiogenic marker (sFlt-1) and RAS components (ANG-II and ACE-2) may be related to adverse outcomes in pregnant women with COVID-19; Methods: Prospective cohort study. Primary outcome was severe pneumonia. Secondary outcomes were ICU admission, intubation, sepsis, and death. Spearman’s Rho test was used to analyze the correlation between sFlt-1 and ANG-II levels. The sFlt-1/ANG-II ratio was determined and the association with each adverse outcome was explored by logistic regression analysis and the prediction was assessed using receiver-operating-curve (ROC); Results: Among 80 pregnant women with COVID-19, the sFlt-1/ANG-II ratio was associated with an increased probability of severe pneumonia (odds ratio [OR]: 1.31; p = 0.003), ICU admission (OR: 1.05; p = 0.007); intubation (OR: 1.09; p = 0.008); sepsis (OR: 1.04; p = 0.008); and death (OR: 1.04; p = 0.018); Conclusion: sFlt-1/ANG-II ratio is a good predictor of adverse events such as pneumonia, ICU admission, intubation, sepsis, and death in pregnant women with COVID-19.

Studies on mechanisms underlying the differentiation of dental pulp stem cells are critical for the understanding of the biology of odontogenesis and for dental tissue engineering. Here, we tested the hypothesis that stem cells from exfoliated deciduous teeth (SHED) differentiate into functional odontoblasts and endothelial cells. SHED were seeded in tooth slice/scaffolds and implanted subcutaneously into immunodeficient mice. SHED differentiated into functional odontoblasts that generated tubular dentin, as determined by tetracycline staining and confocal microscopy. These cells also differentiated into vascular endothelial cells, as determined by beta-galactosidase staining of LacZ-tagged SHED. In vitro, vascular endothelial growth factor (VEGF) induced SHED to express VEGFR2, CD31, and VE-Cadherin (markers of endothelium) and to organize into capillary-like sprouts. VEGF induced ERK and AKT phosphorylation (indicative of differentiation), while inhibiting phosphorylation of STAT3 (indicative of ‘stemness’). Collectively, this work demonstrates that SHED can differentiate into angiogenic endothelial cells and odontoblasts capable of generating tubular dentin.

Accumulating evidence suggests that extracellular galectin-1 and galectin-3 promote angiogenesis. Increased expression of galectin-1 and/or galectin-3 has been reported to be associated with tumour progression. Thus, it is critical to identify their influence on angiogenesis. We examined the individual and combined effects of galectin-1 and galectin-3 on endothelial cell (EC) growth and tube formation using two EC lines, EA.hy926 and HUVEC. The activation of vascular endothelial growth factor receptors (VEGFR1 and VEGFR2) was determined by ELISA and Western blots. We evaluated the VEGFR1 and VEGFR2 levels in endosomes by proximity ligation assay. We observed different responses to exogenous galectins depending on the EC line. An enhanced effect on EA.hy926 cell growth and tube formation was observed when both galectins were added together. Focusing on this enhanced effect, we observed that together galectins induced the phosphorylation of both VEGFR1 and VEGFR2, whereas galectin-1 and -3 alone induced VEGFR2 phosphorylation only. In the same way, the addition of a blocking VEGFR1 antibody completely abolished the increase in tube formation induced by the combined addition of both galectins. In contrast, the addition of a blocking VEGFR2 antibody only partially inhibited this effect. Finally, the addition of both galectins induced a decrease in the VEGFR1 and VEGFR2 endocytic pools, with a significantly enhanced effect on the VEGFR1 endocytic pool. These results suggest that the combined action of galectin-1 and galectin-3 has an enhanced effect on angiogenesis via VEGFR1 activation, which could be related to a decrease in receptor endocytosis.