Zonation, ligand and dose dependence of S1PR1 signalling in blood and lymphatic vasculature.

AIMS: Circulating levels of sphingosine 1-phosphate (S1P), an HDL-associated ligand for endothelial cell (EC) protective S1P receptor-1 (S1PR1), are reduced in disease states associated with endothelial dysfunction. Yet as S1PR1 has high affinity for S1P and can be activated by ligand-independent mechanisms and EC-autonomous S1P production, it is unclear if relative reductions in circulating S1P impact endothelial function. It is also unclear how EC S1PR1 insufficiency, whether induced by ligand deficiency or by S1PR1-directed immunosuppressive therapy, affects different vascular subsets. METHODS AND RESULTS: We here fine-map the zonation of S1PR1 signalling in the murine blood and lymphatic vasculature, superimpose cell type-specific and relative deficiencies in S1P production to define ligand source- and dose-dependence, and correlate receptor engagement to essential functions. In naïve blood vessels, despite broad expression, EC S1PR1 engagement was restricted to resistance-size arteries, lung capillaries and high-endothelial venules (HEV). Similar zonation was observed for albumin extravasation in EC S1PR1 deficient mice, and brain extravasation was reproduced with arterial EC-selective S1pr1 deletion. In lymphatic EC, S1PR1 engagement was high in collecting vessels and lymph nodes and low in terminal capillaries that drain tissue fluids. While EC S1P production sustained S1PR1 signaling in lymphatics and HEV, hematopoietic cells provided ∼90% of plasma S1P and sustained signaling in resistance arteries and lung capillaries. S1PR1 signaling and endothelial function were both surprisingly sensitive to reductions in plasma S1P with apparent saturation around 50% of normal levels. S1PR1 engagement did not depend on sex or age, but modestly increased in arteries in hypertension and diabetes. Sphingosine kinase (Sphk)-2 deficiency also increased S1PR1 engagement selectively in arteries, which could be attributed to Sphk1-dependent S1P release from perivascular macrophages. CONCLUSIONS: This study highlights vessel subtype-specific S1PR1 functions and mechanisms of engagement and supports the relevance of S1P as circulating biomarker for endothelial function.

ACVR1/ALK2-p21 signaling axis modulates proliferation of the venous endothelium in the retinal vasculature.

The proliferation of the endothelium is a highly coordinated process to ensure the emergence, expansion, and homeostasis of the vasculature. While Bone Morphogenetic Protein (BMP) signaling fine-tunes the behaviors of endothelium in health and disease, how BMP signaling influences the proliferation of endothelium and therefore, modulates angiogenesis remains largely unknown. Here, we evaluated the role of Activin A Type I Receptor (ACVR1/ALK2), a key BMP receptor in the endothelium, in modulating the proliferation of endothelial cells. We show that ACVR1/ALK2 is a key modulator for the proliferation of endothelium in the retinal vessels. Loss of endothelial ALK2 leads to a significant reduction in endothelial proliferation and results in fewer branches/endothelial cells in the retinal vessels. Interestingly, venous endothelium appears to be more susceptible to ALK2 deletion. Mechanistically, ACVR1/ALK2 inhibits the expression of CDKN1A/p21, a critical negative regulator of cell cycle progression, in a SMAD1/5-dependent manner, thereby enabling the venous endothelium to undergo active proliferation by suppressing CDKN1A/p21. Taken together, our findings show that BMP signaling mediated by ACVR1/ALK2 provides a critical yet previously underappreciated input to modulate the proliferation of venous endothelium, thereby fine-tuning the context of angiogenesis in health and disease.

The interactions between energy homeostasis and neurovascular plasticity.

Food intake and energy expenditure are sensed and processed by multiple brain centres to uphold energy homeostasis. Evidence from the past decade points to the brain vasculature as a new critical player in regulating energy balance that functions in close association with the local neuronal networks. Nutritional imbalances alter many properties of the neurovascular system (such as neurovascular coupling and blood-brain barrier permeability), thus suggesting a bidirectional link between the nutritional milieu and neurovascular health. Increasing numbers of people are consuming a Western diet (comprising ultra-processed food with high-fat and high-sugar content) and have a sedentary lifestyle, with these factors contributing to the current obesity epidemic. Emerging pharmacological interventions (for example, glucagon-like peptide 1 receptor agonists) successfully trigger weight loss. However, whether these approaches can reverse the detrimental effects of long-term exposure to the Western diet (such as neurovascular uncoupling, neuroinflammation and blood-brain barrier disruption) and maintain stable body weight in the long-term needs to be clarified in addition to possible adverse effects. Lifestyle interventions revert the nutritional trigger for obesity and positively affect our overall health, including the cardiovascular system. This Perspective examines how lifestyle interventions affect the neurovascular system and neuronal networks.

Notch signaling regulates UNC5B to suppress endothelial proliferation, migration, junction activity, and retinal plexus branching.

Notch signaling guides vascular development and function by regulating diverse endothelial cell behaviors, including migration, proliferation, vascular density, endothelial junctions, and polarization in response to flow. Notch proteins form transcriptional activation complexes that regulate endothelial gene expression, but few of the downstream effectors that enable these phenotypic changes have been characterized in endothelial cells, limiting our understanding of vascular Notch activities. Using an unbiased screen of translated mRNA rapidly regulated by Notch signaling, we identified novel in vivo targets of Notch signaling in neonatal mouse brain endothelium, including UNC5B, a member of the netrin family of angiogenic-regulatory receptors. Endothelial Notch signaling rapidly upregulates UNC5B in multiple endothelial cell types. Loss or gain of UNC5B recapitulated specific Notch-regulated phenotypes. UNC5B expression inhibited endothelial migration and proliferation and was required for stabilization of endothelial junctions in response to shear stress. Loss of UNC5B partially or wholly blocked the ability of Notch activation to regulate these endothelial cell behaviors. In the developing mouse retina, endothelial-specific loss of UNC5B led to excessive vascularization, including increased vascular outgrowth, density, and branchpoint count. These data indicate that Notch signaling upregulates UNC5B as an effector protein to control specific endothelial cell behaviors and inhibit angiogenic growth.

Macrophages preserve endothelial cell specialization in the adrenal gland to modulate aldosterone secretion and blood pressure.

Macrophages play crucial roles in organ-specific functions and homeostasis. In the adrenal gland, macrophages closely associate with sinusoidal capillaries in the aldosterone-producing zona glomerulosa. We demonstrate that macrophages preserve capillary specialization and modulate aldosterone secretion. Using macrophage-specific deletion of VEGF-A, single-cell transcriptomics, and functional phenotyping, we found that the loss of VEGF-A depletes PLVAP+ fenestrated endothelial cells in the zona glomerulosa, leading to increased basement membrane collagen IV deposition and subendothelial fibrosis. This results in increased aldosterone secretion, called "haptosecretagogue" signaling. Human aldosterone-producing adenomas also show capillary rarefaction and basement membrane thickening. Mice with myeloid cell-specific VEGF-A deletion exhibit elevated serum aldosterone, hypokalemia, and hypertension, mimicking primary aldosteronism. These findings underscore macrophage-to-endothelial cell signaling as essential for endothelial cell specialization, adrenal gland function, and blood pressure regulation, with broader implications for other endocrine organs.

Vascular development, remodeling and maturation.

The development of the vascular system is crucial in supporting the growth and health of all other organs in the body, and vascular system dysfunction is the major cause of human morbidity and mortality. This chapter discusses three successive processes that govern vascular system development, starting with the differentiation of the primitive vascular system in early embryonic development, followed by its remodeling into a functional circulatory system composed of arteries and veins, and its final maturation and acquisition of an organ specific semi-permeable barrier that controls nutrient uptake into tissues and hence controls organ physiology. Along these steps, endothelial cells forming the inner lining of all blood vessels acquire extensive heterogeneity in terms of gene expression patterns and function, that we are only beginning to understand. These advances contribute to overall knowledge of vascular biology and are predicted to unlock the unprecedented therapeutic potential of the endothelium as an avenue for treatment of diseases associated with dysfunctional vasculature.

VEGF-C prophylaxis favors lymphatic drainage and modulates neuroinflammation in a stroke model.

Meningeal lymphatic vessels (MLVs) promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelial growth factor-C (VEGF-C) regulates MLV development and maintenance and has therapeutic potential for treating neurological disorders. Herein, we investigated the effects of VEGF-C overexpression on brain fluid drainage and ischemic stroke outcomes in mice. Intracerebrospinal administration of an adeno-associated virus expressing mouse full-length VEGF-C (AAV-mVEGF-C) increased CSF drainage to the deep cervical lymph nodes (dCLNs) by enhancing lymphatic growth and upregulated neuroprotective signaling pathways identified by single nuclei RNA sequencing of brain cells. In a mouse model of ischemic stroke, AAV-mVEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage, associated with mitigated microglia-mediated inflammation and increased BDNF signaling in brain cells. Neuroprotective effects of VEGF-C were lost upon cauterization of the dCLN afferent lymphatics and not mimicked by acute post-stroke VEGF-C injection. We conclude that VEGF-C prophylaxis promotes multiple vascular, immune, and neural responses that culminate in a protection against neurological damage in acute ischemic stroke.

Complementary and Inducible creERT2 Mouse Models for Functional Evaluation of Endothelial Cell Subtypes in the Bone Marrow.

In the adult bone marrow (BM), endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche, which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM, distinct vascular arteriole, transitional, and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However, the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover, constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this, we describe two tamoxifen-inducible cre-expressing lines, Vegfr3-creERT2 and Cx40-creERT2, that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow, without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre-dependent recombination constitutively-activates MAPK signaling within adult endothelium, we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creERT2-expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM, providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis.

Compartmentalized ocular lymphatic system mediates eye-brain immunity.

The eye, an anatomical extension of the central nervous system (CNS), exhibits many molecular and cellular parallels to the brain. Emerging research demonstrates that changes in the brain are often reflected in the eye, particularly in the retina1. Still, the possibility of an immunological nexus between the posterior eye and the rest of the CNS tissues remains unexplored. Here, studying immune responses to herpes simplex virus in the brain, we observed that intravitreal immunization protects mice against intracranial viral challenge. This protection extended to bacteria and even tumours, allowing therapeutic immune responses against glioblastoma through intravitreal immunization. We further show that the anterior and posterior compartments of the eye have distinct lymphatic drainage systems, with the latter draining to the deep cervical lymph nodes through lymphatic vasculature in the optic nerve sheath. This posterior lymphatic drainage, like that of meningeal lymphatics, could be modulated by the lymphatic stimulator VEGFC. Conversely, we show that inhibition of lymphatic signalling on the optic nerve could overcome a major limitation in gene therapy by diminishing the immune response to adeno-associated virus and ensuring continued efficacy after multiple doses. These results reveal a shared lymphatic circuit able to mount a unified immune response between the posterior eye and the brain, highlighting an understudied immunological feature of the eye and opening up the potential for new therapeutic strategies in ocular and CNS diseases.

Stromal Cell-SLIT3/Cardiomyocyte-ROBO1 Axis Regulates Pressure Overload-Induced Cardiac Hypertrophy.

BACKGROUND: Recently shown to regulate cardiac development, the secreted axon guidance molecule SLIT3 maintains its expression in the postnatal heart. Despite its known expression in the cardiovascular system after birth, SLIT3's relevance to cardiovascular function in the postnatal state remains unknown. As such, the objectives of this study were to determine the postnatal myocardial sources of SLIT3 and to evaluate its functional role in regulating the cardiac response to pressure overload stress. METHODS: We performed in vitro studies on cardiomyocytes and myocardial tissue samples from patients and performed in vivo investigation with SLIT3 and ROBO1 (roundabout homolog 1) mutant mice undergoing transverse aortic constriction to establish the role of SLIT3-ROBO1 in adverse cardiac remodeling. RESULTS: We first found that SLIT3 transcription was increased in myocardial tissue obtained from patients with congenital heart defects that caused ventricular pressure overload. Immunostaining of hearts from WT (wild-type) and reporter mice revealed that SLIT3 is secreted by cardiac stromal cells, namely fibroblasts and vascular mural cells, within the heart. Conditioned media from cardiac fibroblasts and vascular mural cells both stimulated cardiomyocyte hypertrophy in vitro, an effect that was partially inhibited by an anti-SLIT3 antibody. Also, the N-terminal, but not the C-terminal, fragment of SLIT3 and the forced overexpression of SLIT3 stimulated cardiomyocyte hypertrophy and the transcription of hypertrophy-related genes. We next determined that ROBO1 was the most highly expressed roundabout receptor in cardiomyocytes and that ROBO1 mediated SLIT3's hypertrophic effects in vitro. In vivo, Tcf21+ fibroblast and Tbx18+ vascular mural cell-specific knockout of SLIT3 in mice resulted in decreased left ventricular hypertrophy and cardiac fibrosis after transverse aortic constriction. Furthermore, α-MHC+ cardiomyocyte-specific deletion of ROBO1 also preserved left ventricular function and abrogated hypertrophy, but not fibrosis, after transverse aortic constriction. CONCLUSIONS: Collectively, these results indicate a novel role for the SLIT3-ROBO1-signaling axis in regulating postnatal cardiomyocyte hypertrophy induced by pressure overload.

Alphastatin-C a new inhibitor of endothelial cell activation is a pro-arteriogenic agent in vivo and retards B16-F10 melanoma growth in a preclinical model

Most characterized angiogenic modulators are proteolytic fragments of structural plasma and/or matrix components. Herein, we have identified a novel anti-angiogenic peptide generated by the in vitro hydrolysis of the C-terminal moiety of the fibrinogen alpha chain, produced by the snake venom metalloprotease bothropasin (SVMP), a hemorrhagic proteinase in Bothrops jararaca venom. The 14-amino acids peptide (alphastatin-C) is a potent antagonist of basic fibroblast growth factor, induced endothelial cell (HUVEC-CS) proliferation, migration and capillary tube formation in matrigel. It also inhibits cell adhesion to fibronectin. The basis of the antagonism between bFGF and alphastatin-C is elucidated by the inhibition of various bFGF induced signaling pathways and their molecular components modification, whenever the combination of the stimuli is provided, in comparison to the treatment with bFGF only. To corroborate to the potential therapeutic use of alphastatin-C, we have chosen to perform in vivo assays in two distinct angiogenic settings. In chick model, alphastatin-C inhibits chorioallantoic membrane angiogenesis. In mouse, it efficiently reduces tumor number and volume in a melanoma model, due to the impairment of tumor neovascularization in treated mice. In contrast, we show that the alphastatin-C peptide induces arteriogenesis, increasing pial collateral density in neonate mice. alphastatin-C is an efficient new antiangiogenic FGF-associated agent in vitro, it is an inhibitor of embryonic and tumor vascularization in vivo while, it is an arteriogenic agent. The results also suggest that SVMPs can be used as in vitro biochemical tools to process plasma and/or matrix macromolecular components unraveling new angiostatic peptides.