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The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) will depend on the accurate recapitulation of embryonic haematopoiesis. In the early embryo, HSCs develop from the haemogenic endothelium (HE) and are specified in a Notch-dependent manner through a process named endothelial-to-haematopoietic transition (EHT). As HE is associated with arteries, it is assumed that it represents a subpopulation of arterial vascular endothelium (VE). Here we demonstrate at a clonal level that hPSC-derived HE and VE represent separate lineages. HE is restricted to the CD34(+)CD73(-)CD184(-) fraction of day 8 embryoid bodies and it undergoes a NOTCH-dependent EHT to generate RUNX1C(+) cells with multilineage potential. Arterial and venous VE progenitors, in contrast, segregate to the CD34(+)CD73(med)CD184(+) and CD34(+)CD73(hi)CD184(-) fractions, respectively. Together, these findings identify HE as distinct from VE and provide a platform for defining the signalling pathways that regulate their specification to functional HSCs.
The mapping and characterization of the vasculature using proteomics offers the opportunity to better understand the steps and molecular mechanisms involved in vascular development, and angiogenesis in particular. Proteomics has many key advantages over genomics, especially in directly determining protein expression. Such an approach offers researchers the opportunity to discover the proteins that make up the vasculature in an a priori manner facilitating the generation of hypothesis which can subsequently be validated by other methods. This chapter focuses on proteomic principles and methods, with a particular focus on their applications to characterizing the vascular endothelium (both tumor and normal), as it exists in vivo.
Vascular permeability factor/Vascular endothelial growth factor (VPF/VEGF), a multifunctional cytokine, is a potent inducer of vascular permeability, an important early step in angiogenesis. It is known that the neurotransmitter dopamine can inhibit VPF/VEGF mediated angiogenesis, in particular microvascular permeability, but the effectors of this action remain unclear.
Endothelial cells from rat brain microvessels, human aortic artery and human umbilical vein were examined, together with ex vivo rat brain capillaries and rat aortic ring sections, for the expression of opioid receptor-like OP-4 mRNA and protein. High levels of mRNA expression and an immunopositive reaction for the receptor protein were detected in the endothelial cells from primary and from established in vitro cultures, as well as in the intima of ex vivo rat aortic rings, where the signal was limited to the endothelial layer. Interaction of the OP4 receptor with its physiological ligand nociceptin caused, in cultured endothelial cells, the activation of a mitogen-activated protein (MAP) kinase cascade. Taken together, these results show that the OP4 receptor is synthesised and functionally expressed in endothelial cells, presumably as a starting point for some vasoactive mechanism(s).
Bioengineering of vascular grafts holds great potential to address the shortcomings associated with autologous and conventional synthetic vascular grafts used for small diameter grafting procedures. Lumen endothelialization of bioengineered vascular grafts is essential to provide an antithrombogenic graft surface to ensure long-term patency after implantation. Conventional methods used to assess endothelialization in vitro typically involve periodic harvesting of the graft for histological sectioning and staining of the lumen. Endpoint testing methods such as these are effective but do not provide real-time information of endothelial cells in their intact microenvironment, rather only a single time point measurement of endothelium development. Therefore, nondestructive methods are needed to provide dynamic information of graft endothelialization and endothelium maturation in vitro. To address this need, we have developed a nondestructive fiber optic based (FOB) imaging method that is capable of dynamic assessment of graft endothelialization without disturbing the graft housed in a bioreactor. In this study we demonstrate the capability of the FOB imaging method to quantify electrospun vascular graft endothelialization, EC detachment, and apoptosis in a nondestructive manner. The electrospun scaffold fiber diameter of the graft lumen was systematically varied and the FOB imaging system was used to noninvasively quantify the affect of topography on graft endothelialization over a 7-day period. Additionally, results demonstrated that the FOB imaging method had a greater imaging penetration depth than that of two-photon microscopy. This imaging method is a powerful tool to optimize vascular grafts and bioreactor conditions in vitro, and can be further adapted to monitor endothelium maturation and response to fluid flow bioreactor preconditioning.
Vascular endothelial cells respond to nitric oxide by activating MAPK pathways and upregulating stress-activated proteins such as gamma-glutamylcysteine synthetase (gamma-GCS) and heme oxygenase-1 (HO-1). Since consensus sequences for the antioxidant response element (ARE) are found in the promoters of the gamma-GCS and HO-1 genes, we examined nuclear translocation of Nrf2, a CNC-bZIP protein which binds to and activates the ARE. We found a dramatic increase in Nrf2 nuclear translocation 1-8h following the nitric oxide donor spermine NONOate. Translocation was inhibited by pretreatment of cells with N-acetylcysteine suggesting involvement of an oxidative mechanism in this response. Translocation was also blocked by PD 98059 and SB 203580, inhibitors of ERK and p38 pathways, respectively. In addition to effects on Nrf2 subcellular localization, spermine NONOate increased Nrf2 protein levels by a mechanism which was inhibited by PD 98059. Pretreatment with N-acetylcysteine, PD 98059, and SB 203580 decreased HO-1 upregulation in spermine NONOate-treated cells. These results suggest that ERK and p38 pathways may regulate nitric oxide-mediated adaptive responses in vascular endothelium via translocation of Nrf2 and activation of the ARE.
Vascular comorbidities are inherently linked to the pathogenesis of central retinal artery occlusion (CRAO) and central retinal vein occlusion (CRVO). However, the endothelial-mediated pathogenic mechanisms that precede, and therefore modulate, luminal occlusion have not been clarified. The aim of this study was to delineate the pattern of endothelial morphometric alteration in the central retinal artery and vein in patients with vascular comorbidities. Eyes with a previous history of vascular occlusion were not included in this study in order to avoid the confounding effects of post-occlusion endothelial changes. This study also sought to determine if vascular comorbidities had a disparate effect on arterial and venous endothelium in the optic nerve head. Comparisons were made between 13 human eyes from patients with vascular comorbidities and 22 control eyes from patients with no known systemic disease. Novel micro-cannulation techniques developed in our laboratory were used to label the cytoskeleton and nuclei of endothelial cells in the central retinal artery and vein following which images were captured using confocal microscopy. Endothelial and nuclear morphometric parameters were quantified in different laminar regions of the optic nerve head. F-actin stress fibre expression was also quantified. Analysis of covariance was used to determine statistical differences between the two groups. Interestingly, age did not influence endothelial morphometry, nuclear morphometry or f-actin expression in central retinal vessels. There were also no arterial endothelial differences between control and disease groups in any laminar region. Endothelial f-actin stress fibre expression increased significantly in the central retinal vein in patients with vascular comorbidities. The greatest change in these eyes was found to occur at the posterior lamina cribrosa. Increased venous endothelial f-actin stress fibre expression may reflect vascular comorbid disease-induced alterations to hemodynamic properties and coagulation cascades in the central retinal vein. The posterior lamina may be an important site for thrombus formation in CRVO as venous endothelia in this region are most influenced by the presence of vascular comorbidities. The findings of this study suggest that the role of endothelial dysfunction in CRVO and CRAO pathogenesis could be different.
Despite its known expression in both the vascular endothelium and the lung epithelium, until recently the physiological role of the adhesion receptor Gpr116/ADGRF5 has remained elusive. We generated a new mouse model of constitutive Gpr116 inactivation, with a large genetic deletion encompassing exon 4 to exon 21 of the Gpr116 gene. This model allowed us to confirm recent results defining Gpr116 as necessary regulator of surfactant homeostasis. The loss of Gpr116 provokes an early accumulation of surfactant in the lungs, followed by a massive infiltration of macrophages, and eventually progresses into an emphysema-like pathology. Further analysis of this knockout model revealed cerebral vascular leakage, beginning at around 1.5 months of age. Additionally, endothelial-specific deletion of Gpr116 resulted in a significant increase of the brain vascular leakage. Mice devoid of Gpr116 developed an anatomically normal and largely functional vascular network, surprisingly exhibited an attenuated pathological retinal vascular response in a model of oxygen-induced retinopathy. These data suggest that Gpr116 modulates endothelial properties, a previously unappreciated function despite the pan-vascular expression of this receptor. Our results support the key pulmonary function of Gpr116 and describe a new role in the central nervous system vasculature.
Collective migration of vascular endothelial cells is central for embryonic development, angiogenesis, and wound closure. Although physical confinement of cell assemblies has been shown to elicit specific patterns of collective movement in various cell types, endothelial migration in vivo often occurs without confinement. Here we show that unconfined endothelial cell monolayers on microgroove substrates that mimic the anisotropic organization of the extracellular matrix exhibit a specific type of collective movement that takes the form of a periodic pattern of antiparallel cell streams. We further establish that the development of these streams requires intact cell-cell junctions and that stream sizes are particularly sensitive to groove depth. Finally, we show that modeling the endothelial cell sheet as an active fluid with the microgrooves acting as constraints on cell orientation predicts the occurrence of the periodic antiparallel cell streams as well as their lengths and widths. We posit that in unconfined cell assemblies, physical factors that constrain or bias cellular orientation such as anisotropic extracellular matrix cues or directed flow-derived shear forces dictate the pattern of collective cell movement.
Toxoplasma gondii is a highly successful parasite that infects approximately one-third of the human population and can cause fatal disease in immunocompromised individuals. Systemic parasite dissemination to organs such as the brain and eye is critical to pathogenesis. T. gondii can disseminate via the circulation, and both intracellular and extracellular modes of transport have been proposed. However, the processes by which extracellular tachyzoites adhere to and migrate across vascular endothelium under the conditions of rapidly flowing blood remain unknown. We used microfluidics and time-lapse fluorescence microscopy to examine the interactions between extracellular T. gondii and primary human endothelial cells under conditions of physiologic shear stress. Remarkably, tachyzoites adhered to and glided on human vascular endothelium under shear stress conditions. Compared to static conditions, shear stress enhanced T. gondii helical gliding, resulting in a significantly greater displacement, and increased the percentage of tachyzoites that invaded or migrated across the endothelium. The intensity of the shear forces (from 0.5 to 10 dynes/cm(2)) influenced both initial and sustained adhesion to endothelium. By examining tachyzoites deficient in the T. gondii adhesion protein MIC2, we found that MIC2 contributed to initial adhesion but was not required for adhesion strengthening. These data suggest that under fluidic conditions, T. gondii adhesion to endothelium may be mediated by a multistep cascade of interactions that is governed by unique combinations of adhesion molecules. This work provides novel information about tachyzoite interactions with vascular endothelium and contributes to our understanding of T. gondii dissemination in the infected host. IMPORTANCE Toxoplasma gondii is a global parasite pathogen that can cause fatal disease in immunocompromised individuals. An unresolved question is how the parasites circulate in the body to tissues to cause disease. T. gondii parasites are found in the bloodstream of infected animals and patients, and they have been shown to adhere to and cross the endothelial cells that line blood vessel walls. To investigate these interactions, we devised a microfluidic system to visualize parasites interacting with vascular endothelium under conditions similar to those found in the bloodstream. Interestingly, parasite migration was significantly influenced by the mechanical force of shear flow. Furthermore, we identified a role for the parasite surface protein MIC2 in the initial phase of adhesion. Our study is the first to document T. gondii interactions with endothelium under shear stress conditions and provides a foundation for future studies on the molecules that mediate parasite interaction with the vasculature.
The present study was undertaken to investigate the influence of hypothermia on endothelium-independent vascular smooth muscle contractility and to determine the mechanism underlying the relaxation. Denuded aortic rings from male rats were used and isometric contractions were recorded and combined with molecular experiments. Hypothermia significantly inhibited fluoride-, thromboxane A2-, phenylephrine-, and phorbol ester-induced vascular contractions regardless of endothelial nitric oxide synthesis, suggesting that another pathway had a direct effect on vascular smooth muscle. Hypothermia significantly inhibited the fluoride-induced increase in pMYPT1 level and phorbol ester-induced increase in pERK1/2 level, suggesting inhibition of Rho-kinase and MEK activity and subsequent phosphorylation of MYPT1 and ERK1/2. These results suggest that the relaxing effect of moderate hypothermia on agonist-induced vascular contraction regardless of endothelial function involves inhibition of Rho-kinase and MEK activities.
The blood-brain barrier (BBB) limits the entry of leukocytes and potentially harmful substances from the circulation into the central nervous system (CNS). While BBB defects are a hallmark of many neurological disorders, the cellular heterogeneity at the neurovascular interface, and the mechanisms governing neuroinflammation are not fully understood. Through single-cell RNA sequencing of non-neuronal cell populations of the murine cerebral cortex during development, adulthood, ageing, and neuroinflammation, we identify reactive endothelial venules, a compartment of specialized postcapillary endothelial cells that are characterized by consistent expression of cell adhesion molecules, preferential leukocyte transmigration, association with perivascular macrophage populations, and endothelial activation initiating CNS immune responses. Our results provide novel insights into the heterogeneity of the cerebral vasculature and a useful resource for the molecular alterations associated with neuroinflammation and ageing.
Sodium overload stiffens vascular endothelial cells in vitro and promotes arterial hypertension in vivo. The hypothesis was tested that the endothelial glycocalyx (eGC), a mesh of anionic biopolymers covering the surface of the endothelium, participates in the stiffening process. By using a mechanical nanosensor, mounted on an atomic force microscope, height (∼400 nm) and stiffness (∼0.25 pN/nm) of the eGC on the luminal endothelial surface of split-open human umbilical arteries were quantified. In presence of aldosterone, the increase of extracellular sodium concentration from 135 to 150 mM over 5 days (sodium overload) led the eGC shrink by ∼50% and stiffening by ∼130%. Quantitative eGC analyses reveal that sodium overload caused a reduction of heparan sulphate residues by 68% which lead to destabilization and collapse of the eGC. Sodium overload transformed the endothelial cells from a sodium release into a sodium-absorbing state. Spironolactone, a specific aldosterone antagonist, prevented these changes. We conclude that the endothelial glycocalyx serves as an effective buffer barrier for sodium. Damaged eGC facilitates sodium entry into the endothelial cells. This could explain endothelial dysfunction and arterial hypertension observed in sodium abuse.
The decline of endothelial autophagy is closely related to vascular senescence and disease, although the molecular mechanisms connecting these outcomes in vascular endothelial cells (VECs) remain unclear. Here, we identify a crucial role for CD44, a multifunctional adhesion molecule, in controlling autophagy and ageing in VECs. The CD44 intercellular domain (CD44ICD) negatively regulates autophagy by reducing PIK3R4 and PIK3C3 levels and disrupting STAT3-dependent PtdIns3K complexes. CD44 and its homologue clec-31 are increased in ageing vascular endothelium and Caenorhabditis elegans, respectively, suggesting that an age-dependent increase in CD44 induces autophagy decline and ageing phenotypes. Accordingly, CD44 knockdown ameliorates age-associated phenotypes in VECs. The endothelium-specific CD44ICD knock-in mouse is shorter-lived, with VECs exhibiting obvious premature ageing characteristics associated with decreased basal autophagy. Autophagy activation suppresses the premature ageing of human and mouse VECs overexpressing CD44ICD, function conserved in the CD44 homologue clec-31 in C. elegans. Our work describes a mechanism coordinated by CD44 function bridging autophagy decline and ageing.
Increased pressure suppresses endothelial control of vascular tone but it remains uncertain (1) how pressure is sensed by the endothelium and (2) how the vascular response is inhibited. This study used a novel imaging method to study large numbers of endothelial cells in arteries that were in a physiological configuration and held at normal blood pressures. Increased pressure suppressed endothelial IP3 -mediated Ca(2+) signals. Pressure modulated endothelial cell shape. The changes in cell shape may alter endothelial Ca(2+) signals by modulating the diffusive environment for Ca(2+) near IP3 receptors. Endothelial pressure-dependent mechanosensing may occur without a requirement for a conventional molecular mechanoreceptor.
SIRT1 has many important molecular functions in aging, and the estrogen receptors (ERs) have a vasculoprotective effect, although the detailed mechanism for the roles of SIRT1 and ERs in vascular aging remains unclear. We found that ERβ expression in the endothelium was reduced in aging mice, and the expression of ERα and SIRT1 did not change, while SIRT1 activity declined. Further investigation showed that the ERβ expression was regulated by SIRT1 through complexes of SIRT1-PPARγ/RXR-p300 that bind to a PPRE (PPAR response element) site on the ERβ promoter, and the declined SIRT1 function in aging mice was due to compromised phosphorylation at S154. A single-mutant SIRT1-C152(D) restored the reduced ERβ expression in the endothelium with minimized reactive oxygen species generation and DNA damage and increased mitochondrial function and fatty acid metabolism. In high-fat diet aging mice, the endothelium-specific delivery of ERβ or SIRT1-C152(D) on the vascular wall reduced the circulating lipids with ameliorated vascular damage, including the restored vessel tension and blood pressure. We conclude that SIRT1-mediated ERβ suppression in the endothelium contributes to vascular aging, and the modulation of SIRT1 phosphorylation through a single-mutant SIRT1-C152(D) restores this effect.
The selective entry of nanoparticles into target tissues is the key factor which determines their tissue distribution. Entry is primarily controlled by microvascular endothelial cells, which have tissue-specific properties. This study investigated the cellular properties involved in selective transport of gold nanoparticles (<5 nm) coated with PEG-amine/galactose in two different human vascular endothelia. Kidney endothelium (ciGENC) showed higher uptake of these nanoparticles than brain endothelium (hCMEC/D3), reflecting their biodistribution in vivo. Nanoparticle uptake and subcellular localisation was quantified by transmission electron microscopy. The rate of internalisation was approximately 4x higher in kidney endothelium than brain endothelium. Vesicular endocytosis was approximately 4x greater than cytosolic uptake in both cell types, and endocytosis was blocked by metabolic inhibition, whereas cytosolic uptake was energy-independent. The cellular basis for the different rates of internalisation was investigated. Morphologically, both endothelia had similar profiles of vesicles and cell volumes. However, the rate of endocytosis was higher in kidney endothelium. Moreover, the glycocalyces of the endothelia differed, as determined by lectin-binding, and partial removal of the glycocalyx reduced nanoparticle uptake by kidney endothelium, but not brain endothelium. This study identifies tissue-specific properties of vascular endothelium that affects their interaction with nanoparticles and rate of transport.
A further understanding of tumor angiogenesis is urgently needed due to the limited therapeutic efficacy of anti-angiogenesis agents. However, the origin of endothelial cells (EC) in tumors remains widely elusive and controversial. Snail has been thoroughly elucidated as a master regulator of the epithelial-mesenchymal transition (EMT), but its role in endothelium generation is not yet established. In this study, we reported a new and unexpected function of Snail in endothelium generation by breast cancer cells. We showed that high Snail-expressing breast cancer cells isolated from patients showed more endothelium generated from these cells. Expression of Snail was positively correlated with endothelial markers in breast cancer patients. The ectopic expression of Snail induced endothelial marker expression, tube formation and DiI-AcLDL uptake of breast cancer cells in vitro, and enhanced tumor growth and microvessel density in vivo. Snail-mediated endothelium generation depended on VEGF and Sox2. Mechanistically, Snail promoted the expression of VEGF and Sox2 through recruiting the p300 activator complex to these promoters. We showed the dual function of Snail in tumor initiation and angiogenesis in vivo and in vitro through activation of Sox2 and VEGF, suggesting Snail may be an ideal target for cancer therapy.
Vascular permeability is frequently associated with inflammation and is triggered by a cohort of secreted permeability factors such as vascular endothelial growth factor (VEGF). Here, we show that the physiological vascular permeability that precedes implantation is directly controlled by progesterone receptor (PR) and is independent of VEGF. Global or endothelial-specific deletion of PR blocks physiological vascular permeability in the uterus, whereas misexpression of PR in the endothelium of other organs results in ectopic vascular leakage. Integration of an endothelial genome-wide transcriptional profile with chromatin immunoprecipitation sequencing revealed that PR induces an NR4A1 (Nur77/TR3)-dependent transcriptional program that broadly regulates vascular permeability in response to progesterone. Silencing of NR4A1 blocks PR-mediated permeability responses, indicating a direct link between PR and NR4A1. This program triggers concurrent suppression of several junctional proteins and leads to an effective, timely, and venous-specific regulation of vascular barrier function that is critical for embryo implantation.
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