Incongruence between transcriptional and vascular pathophysiological cell states.

The Notch pathway is a major regulator of endothelial transcriptional specification. Targeting the Notch receptors or Delta-like ligand 4 (Dll4) dysregulates angiogenesis. Here, by analyzing single and compound genetic mutants for all Notch signaling members, we find significant differences in the way ligands and receptors regulate liver vascular homeostasis. Loss of Notch receptors caused endothelial hypermitogenic cell-cycle arrest and senescence. Conversely, Dll4 loss triggered a strong Myc-driven transcriptional switch inducing endothelial proliferation and the tip-cell state. Myc loss suppressed the induction of angiogenesis in the absence of Dll4, without preventing the vascular enlargement and organ pathology. Similarly, inhibition of other pro-angiogenic pathways, including MAPK/ERK and mTOR, had no effect on the vascular expansion induced by Dll4 loss; however, anti-VEGFA treatment prevented it without fully suppressing the transcriptional and metabolic programs. This study shows incongruence between single-cell transcriptional states, vascular phenotypes and related pathophysiology. Our findings also suggest that the vascular structure abnormalization, rather than neoplasms, causes the reported anti-Dll4 antibody toxicity.

Role of Notch in endothelial biology.

The Notch signalling pathway is one of the main regulators of endothelial biology. In the last 20 years the critical function of Notch has been uncovered in the context of angiogenesis, participating in tip-stalk specification, arterial-venous differentiation, vessel stabilization, and maturation processes. Importantly, pharmacological compounds targeting distinct members of the Notch signalling pathway have been used in the clinics for cancer therapy. However, the underlying mechanisms that support the variety of outcomes triggered by Notch in apparently opposite contexts such as angiogenesis and vascular homeostasis remain unknown. In recent years, advances in -omics technologies together with mosaic analysis and high molecular, cellular and temporal resolution studies have allowed a better understanding of the mechanisms driven by the Notch signalling pathway in different endothelial contexts. In this review we will focus on the main findings that revisit the role of Notch signalling in vascular biology. We will also discuss potential future directions and technologies that will shed light on the puzzling role of Notch during endothelial growth and homeostasis. Addressing these open questions may allow the improvement and development of therapeutic strategies based on modulation of the Notch signalling pathway.

Cre mRNA Is Not Transferred by EVs from Endothelial and Adipose-Derived Stromal/Stem Cells during Vascular Network Formation.

Coculture systems employing adipose tissue-derived mesenchymal stromal/stem cells (ASC) and endothelial cells (EC) represent a widely used technique to model vascularization. Within this system, cell-cell communication is crucial for the achievement of functional vascular network formation. Extracellular vesicles (EVs) have recently emerged as key players in cell communication by transferring bioactive molecules between cells. In this study we aimed to address the role of EVs in ASC/EC cocultures by discriminating between cells, which have received functional EV cargo from cells that have not. Therefore, we employed the Cre-loxP system, which is based on donor cells expressing the Cre recombinase, whose mRNA was previously shown to be packaged into EVs and reporter cells containing a construct of floxed dsRed upstream of the eGFP coding sequence. The evaluation of Cre induced color switch in the reporter system via EVs indicated that there is no EV-mediated RNA transmission either between EC themselves or EC and ASC. However, since Cre mRNA was not found present in EVs, it remains unclear if Cre mRNA is generally not packaged into EVs or if EVs are not taken up by the utilized cell types. Our data indicate that this technique may not be applicable to evaluate EV-mediated cell-to-cell communication in an in vitro setting using EC and ASC. Further investigations will require a functional system showing efficient and specific loading of Cre mRNA or protein into EVs.

Arterialization requires the timely suppression of cell growth.

The formation of arteries is thought to occur by the induction of a highly conserved arterial genetic programme in a subset of vessels that will later experience an increase in oxygenated blood flow1,2. The initial steps of arterial specification require both the VEGF and Notch signalling pathways3-5. Here, we combine inducible genetic mosaics and transcriptomics to modulate and define the function of these signalling pathways in cell proliferation, arteriovenous differentiation and mobilization. We show that endothelial cells with high levels of VEGF or Notch signalling are intrinsically biased to mobilize and form arteries; however, they are not genetically pre-determined, and can also form veins. Mechanistically, we found that increased levels of VEGF and Notch signalling in pre-arterial capillaries suppresses MYC-dependent metabolic and cell-cycle activities, and promotes the incorporation of endothelial cells into arteries. Mosaic lineage-tracing studies showed that endothelial cells that lack the Notch-RBPJ transcriptional activator complex rarely form arteries; however, these cells regained the ability to form arteries when the function of MYC was suppressed. Thus, the development of arteries does not require the direct induction of a Notch-dependent arterial differentiation programme, but instead depends on the timely suppression of endothelial cell-cycle progression and metabolism, a process that precedes arterial mobilization and complete differentiation.

Endothelial sprouting, proliferation, or senescence: tipping the balance from physiology to pathology.

Therapeutic modulation of vascular cell proliferation and migration is essential for the effective inhibition of angiogenesis in cancer or its induction in cardiovascular disease. The general view is that an increase in vascular growth factor levels or mitogenic stimulation is beneficial for angiogenesis, since it leads to an increase in both endothelial proliferation and sprouting. However, several recent studies showed that an increase in mitogenic stimuli can also lead to the arrest of angiogenesis. This is due to the existence of intrinsic signaling feedback loops and cell cycle checkpoints that work in synchrony to maintain a balance between endothelial proliferation and sprouting. This balance is tightly and effectively regulated during tissue growth and is often deregulated or impaired in disease. Most therapeutic strategies used so far to promote vascular growth simply increase mitogenic stimuli, without taking into account its deleterious effects on this balance and on vascular cells. Here, we review the main findings on the mechanisms controlling physiological vascular sprouting, proliferation, and senescence and how those mechanisms are often deregulated in acquired or congenital cardiovascular disease leading to a diverse range of pathologies. We also discuss alternative approaches to increase the effectiveness of pro-angiogenic therapies in cardiovascular regenerative medicine.

Purinergic P2Y2 receptors modulate endothelial sprouting.

Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth.

Fabrication of biomimetic placental barrier structures within a microfluidic device utilizing two-photon polymerization.

The placenta is a transient organ, essential for development and survival of the unborn fetus. It interfaces the body of the pregnant woman with the unborn child and secures transport of endogenous and exogenous substances. Maternal and fetal blood are thereby separated at any time, by the so-called placental barrier. Current in vitro approaches fail to model this multifaceted structure, therefore research in the field of placental biology is particularly challenging. The present study aimed at establishing a novel model, simulating placental transport and its implications on development, in a versatile but reproducible way. The basal membrane was replicated using a gelatin-based material, closely mimicking the composition and properties of the natural extracellular matrix. The microstructure was produced by using a high-resolution 3D printing method - the two-photon polymerization (2PP). In order to structure gelatin by 2PP, its primary amines and carboxylic acids are modified with methacrylamides and methacrylates (GelMOD-AEMA), respectively. High-resolution structures in the range of a few micrometers were produced within the intersection of a customized microfluidic device, separating the x-shaped chamber into two isolated cell culture compartments. Human umbilical-vein endothelial cells (HUVEC) seeded on one side of this membrane simulate the fetal compartment while human choriocarcinoma cells, isolated from placental tissue (BeWo B30) mimic the maternal syncytium. This barrier model in combination with native flow profiles can be used to mimic the microenvironment of the placenta, investigating different pharmaceutical, clinical and biological scenarios. As proof-of-principle, this bioengineered placental barrier was used for the investigation of transcellular transport processes. While high molecular weight substances did not permeate, smaller molecules in the size of glucose were able to diffuse through the barrier in a time-depended manner. We envision to apply this bioengineered placental barrier for pathophysiological research, where altered nutrient transport is associated with health risks for the fetus.

The impact of wavelengths of LED light-therapy on endothelial cells.

Low level light therapy receives increasing interest in the fields of tissue regeneration and wound healing. Several in vivo studies demonstrated the positive effects of LLLT on angiogenesis. This study aimed to investigate the underlying properties in vitro by comparing the effects of light therapy by light emitting diodes of different wavelengths on endothelial cells in vitro. Human umbilical vein endothelial cells were treated with either 475 nm, 516 nm or 635 nm light. Control cells were not illuminated. 2D proliferation was quantified by manual counting. HUVEC migration was analyzed by performing a 2D wound scratch assay and a 3D bead assay. The influence of LLLT on early vasculogenic events was determined in a 3D fibrin co-culture model with adipose-derived stem cells. Stimulation with both red and green pulsed LED light significantly increased HUVEC proliferation and 3D migration. Moreover, HUVEC showed increased 2D migration potential with green light stimulation. The treatment with blue light was ineffective. Several parameters showed that green light was even more potent to stimulate proliferation and migration of endothelial cells than clinically well-established red light therapy. Further studies have to focus on intracellular mechanisms induced by different wavelengths in order to optimize this promising therapy in tissue regeneration.

Laser photofabrication of cell-containing hydrogel constructs.

The two-photon polymerization (2PP) of photosensitive gelatin in the presence of living cells is reported. The 2PP technique is based on the localized cross-linking of photopolymers induced by femtosecond laser pulses. The availability of water-soluble photoinitiators (PI) suitable for 2PP is crucial for applying this method to cell-containing materials. Novel PIs developed by our group allow 2PP of formulations with up to 80% cell culture medium. The cytocompatibility of these PIs was evaluated by an MTT assay. The results of cell encapsulation by 2PP show the occurrence of cell damage within the laser-exposed regions. However, some cells located in the immediate vicinity and even within the 2PP-produced structures remain viable and can further proliferate. The control experiments demonstrate that the laser radiation itself does not damage the cells at the parameters used for 2PP. On the basis of these findings and the reports by other groups, we conclude that such localized cell damage is of a chemical origin and can be attributed to reactive species generated during 2PP. The viable cells trapped within the 2PP structures but not exposed to laser radiation continued to proliferate. The live/dead staining after 3 weeks revealed viable cells occupying most of the space available within the 3D hydrogel constructs. While some of the questions raised by this study remain open, the presented results indicate the general practicability of 2PP for 3D processing of cell-containing materials. The potential applications of this highly versatile approach span from precise engineering of 3D tissue models to the fabrication of cellular microarrays.