Purpose of review

The formation of a hierarchical vascular network is a complex process that requires precise temporal and spatial integration of several signaling pathways. Amongst those, Notch has emerged as a key regulator of multiple steps that expand from endothelial sprouting to arterial specification and remains relevant in the adult. This review aims to summarize major concepts and rising hypotheses on the role of Notch signaling in the endothelium.

Recent findings

A wealth of new information has helped to clarify how Notch signaling cooperates with other pathways to orchestrate vascular morphogenesis, branching, and function. Endothelial vascular endothelial growth factor, C-X-C chemokine receptor type 4, and nicotinamide adenine dinucleotide phosphate oxidase 2 have been highlighted as key regulators of the pathway. Furthermore, blood flow forces during vascular development induce Notch1 signaling to suppress endothelial cell proliferation, enhance barrier function, and promote arterial specification. Importantly, Notch1 has been recently recognized as an endothelial mechanosensor that is highly responsive to the level of shear stress to enable differential Notch activation in distinct regions of the vessel wall and suppress inflammation.

Summary

Although it is well accepted that the Notch signaling pathway is essential for vascular morphogenesis, its contributions to the homeostasis of adult endothelium were uncovered only recently. Furthermore, its exquisite regulation by flow and impressive interface with multiple signaling pathways indicates that Notch is at the center of a highly interactive web that integrates both physical and chemical signals to ensure vascular stability.

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and β-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Mechanical forces such as fluid shear have been shown to enhance cell growth and differentiation, but knowledge of their mechanistic effect on cells is limited because the local flow patterns and associated metrics are not precisely known. Here we present real-time, non-invasive measures of local hydrodynamics in 3D biomaterials based on nuclear magnetic resonance. Microflow maps were further used to derive pressure, shear and fluid permeability fields. Finally, remodeling of collagen gels in response to precise fluid flow parameters was correlated with structural changes. It is anticipated that accurate flow maps within 3D matrices will be a critical step towards understanding cell behavior in response to controlled flow dynamics.

The cellular and mechanistic bases underlying endothelial regeneration of adult large vessels have proven challenging to study. Using a reproducible in vivo aortic endothelial injury model, we characterized cellular dynamics underlying the regenerative process through a combination of multi-color lineage tracing, parabiosis, and single-cell transcriptomics. We found that regeneration is a biphasic process driven by distinct populations arising from differentiated endothelial cells. The majority of cells immediately adjacent to the injury site re-enter the cell cycle during the initial damage response, with a second phase driven by a highly proliferative subpopulation. Endothelial regeneration requires activation of stress response genes including Atf3, and aged aortas compromised in their reparative capacity express less Atf3. Deletion of Atf3 reduced endothelial proliferation and compromised the regeneration. These findings provide important insights into cellular dynamics and mechanisms that drive responses to large vessel injury.

Objective

Perivascular cells, including pericytes, macrophages, smooth muscle cells, and other specialized cell types, like podocytes, participate in various aspects of vascular function. However, aside from the well-established roles of smooth muscle cells and pericytes, the contributions of other vascular-associated cells are poorly understood. Our goal was to ascertain the function of perivascular macrophages in adult tissues under nonpathological conditions.

Approach and results

We combined confocal microscopy, in vivo cell depletion, and in vitro assays to investigate the contribution of perivascular macrophages to vascular function. We found that resident perivascular macrophages are associated with capillaries at a frequency similar to that of pericytes. Macrophage depletion using either clodronate liposomes or antibodies unexpectedly resulted in hyperpermeability. This effect could be rescued when M2-like macrophages, but not M1-like macrophages or dendritic cells, were reconstituted in vivo, suggesting subtype-specific roles for macrophages in the regulation of vascular permeability. Furthermore, we found that permeability-promoting agents elicit motility and eventual dissociation of macrophages from the vasculature. Finally, in vitro assays showed that M2-like macrophages attenuate the phosphorylation of VE-cadherin upon exposure to permeability-promoting agents.

Conclusions

This study points to a direct contribution of macrophages to vessel barrier integrity and provides evidence that heterotypic cell interactions with the endothelium, in addition to those of pericytes, control vascular permeability.

Objective

For tissue engineering, there is a need for quantitative methods to map cell density inside three-dimensional (3-D) bioreactors to assess tissue growth over time. The current cell mapping methods in 2-D cultures are based on optical microscopy. However, optical methods fail in 3-D due to increased opacity of the tissue. We present an approach for measuring the density of cells embedded in a hydrogel to generate quantitative maps of cell density in a living, 3-D tissue culture sample.

Methods

Quantification of cell density was obtained by calibrating the ¹H T₂, magnetization transfer (MT) and diffusion-weighted nuclear magnetic resonance (NMR) signals to samples of known cell density. Maps of cell density were generated by weighting NMR images by these parameters post-calibration.

Results

The highest sensitivity weighting arose from MT experiments, which yielded a limit of detection (LOD) of [Formula: see text] cells/mL/ √{Hz} in a 400 MHz (9.4 T) magnet.

Conclusion

This mapping technique provides a noninvasive means of visualizing cell growth within optically opaque bioreactors.

Significance

We anticipate that such readouts of tissue culture growth will provide valuable feedback for controlled cell growth in bioreactors.

Having demonstrated that apolipoprotein A-I (apoA-I) mimetic peptides ameliorate cancer in mouse models, we sought to determine the mechanism for the anti-tumorigenic function of these peptides. CT-26 cells (colon cancer cells that implant and grow into tumors in the lungs) were injected into wild-type BALB/c mice. The day after injection, mice were either continued on chow or switched to chow containing 0.06% of a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F). After four weeks, the number of lung tumors was significantly lower in Tg6F-fed mice. Gene expression array analyses of jejunum and lung identified Notch pathway genes significantly upregulated, whereas osteopontin (Spp1) was significantly downregulated by Tg6F in both jejunum and lung. In jejunum, Tg6F increased protein levels for Notch1, Notch2, Dll1, and Dll4. In lung, Tg6F increased protein levels for Notch1 and Dll4 and decreased Spp1. Tg6F reduced oxidized phospholipid levels (E06 immunoreactivity) and reduced 25-hydroxycholesterol (25-OHC) levels, which are known to inhibit Notch1 and induce Spp1, respectively. Notch pathway promotes anti-tumorigenic patrolling monocytes, while Spp1 facilitates pro-tumorigenic myeloid derived suppressor cells (MDSCs) formation. Tg6F-fed mice had higher numbers of patrolling monocytes in jejunum and in lung (p < 0.02), and lower plasma levels of Spp1 with reduced numbers of MDSCs in jejunum and in lung (p < 0.03). We conclude that Tg6F alters levels of specific oxidized lipids and 25-OHC to modulate Notch pathways and Spp1, which alter small intestine immune cells, leading to similar changes in lung that reduce tumor burden.

We previously reported that adding a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to a Western diet (WD) ameliorated systemic inflammation. To determine the mechanism(s) responsible for these observations, Ldlr^-/- mice were fed chow, a WD, or WD plus Tg6F. We found that a WD altered the taxonomic composition of bacteria in jejunum mucus. For example, Akkermansia muciniphila virtually disappeared, while overall bacteria numbers and lipopolysaccharide (LPS) levels increased. In addition, gut permeability increased, as did the content of reactive oxygen species and oxidized phospholipids in jejunum mucus in WD-fed mice. Moreover, gene expression in the jejunum decreased for multiple peptides and proteins that are secreted into the mucous layer of the jejunum that act to limit bacteria numbers and their interaction with enterocytes including regenerating islet-derived proteins, defensins, mucin 2, surfactant A, and apoA-I. Following WD, gene expression also decreased for Il36γ, Il23, and Il22, cytokines critical for antimicrobial activity. WD decreased expression of both Atoh1 and Gfi1, genes required for the formation of goblet and Paneth cells, and immunohistochemistry revealed decreased numbers of goblet and Paneth cells. Adding Tg6F ameliorated these WD-mediated changes. Adding oxidized phospholipids ex vivo to the jejunum from mice fed a chow diet reproduced the changes in gene expression in vivo that occurred when the mice were fed WD and were prevented with addition of 6F peptide. We conclude that Tg6F ameliorates the WD-mediated increase in oxidized phospholipids that cause changes in jejunum mucus, which induce dysbiosis and systemic inflammation.

Endothelial cells transduce mechanical forces from blood flow into intracellular signals required for vascular homeostasis. Here we show that endothelial NOTCH1 is responsive to shear stress, and is necessary for the maintenance of junctional integrity, cell elongation, and suppression of proliferation, phenotypes induced by laminar shear stress. NOTCH1 receptor localizes downstream of flow and canonical NOTCH signaling scales with the magnitude of fluid shear stress. Reduction of NOTCH1 destabilizes cellular junctions and triggers endothelial proliferation. NOTCH1 suppression results in changes in expression of genes involved in the regulation of intracellular calcium and proliferation, and preventing the increase of calcium signaling rescues the cell-cell junctional defects. Furthermore, loss of Notch1 in adult endothelium increases hypercholesterolemia-induced atherosclerosis in the descending aorta. We propose that NOTCH1 is atheroprotective and acts as a mechanosensor in adult arteries, where it integrates responses to laminar shear stress and regulates junctional integrity through modulation of calcium signaling.

Background and aims

Phagocytosis (efferocytosis) of apoptotic neutrophils by macrophages anchors the resolution of intestinal inflammation. Efferocytosis prevents secondary necrosis and inhibits further inflammation, and also reprograms macrophages to facilitate tissue repair and promote resolution function. Macrophage efferocytosis and efferocytosis-dependent reprogramming are implicated in the pathogenesis of inflammatory bowel disease. We previously reported that absence of macrophage cyclooxygenase 2 (COX2) exacerbates inflammatory bowel disease-like intestinal inflammation. To elucidate the underlying pathogenic mechanism, we investigated here whether COX2 mediates macrophage efferocytosis and efferocytosis-dependent reprogramming, including intestinal epithelial repair capacity.

Methods

Using apoptotic neutrophils and synthetic apoptotic targets, we determined the effects of macrophage specific Cox2 knockout and pharmacological COX2 inhibition on the efferocytosis capacity of mouse primary macrophages. COX2-mediated efferocytosis-dependent eicosanoid lipidomics was determined by liquid chromatography tandem mass spectrometry. Small intestinal epithelial organoids were employed to assay the effects of COX2 on efferocytosis-dependent intestinal epithelial repair.

Results

Loss of COX2 impaired efferocytosis in mouse primary macrophages, in part, by affecting the binding capacity of macrophages for apoptotic cells. This effect was comparable to that of high-dose lipopolysaccharide and was accompanied by both dysregulation of macrophage polarization and the inhibited expression of genes involved in apoptotic cell binding. COX2 modulated the production of efferocytosis-dependent lipid inflammatory mediators that include the eicosanoids prostaglandin I2, prostaglandin E2, lipoxin A4, and 15d-PGJ2; and further affected secondary efferocytosis. Finally, macrophage efferocytosis induced, in a macrophage COX2-dependent manner, a tissue restitution and repair phenotype in intestinal epithelial organoids.

Conclusions

Macrophage COX2 potentiates efferocytosis capacity and efferocytosis-dependent reprogramming, facilitating macrophage intestinal epithelial repair capacity.