Rab35 Governs Apicobasal Polarity Through Regulation of Actin Dynamics During Sprouting Angiogenesis

In early blood vessel development, trafficking programs, such as those using Rab GTPases, are tasked with delivering vesicular cargo with high spatiotemporal accuracy. However, the function of many Rab trafficking proteins remain ill-defined in endothelial tissue; therefore, their relevance to blood vessel development is unknown. Rab35 has been shown to play an enigmatic role in cellular behaviors which differs greatly between tissue-type and organism. Importantly, Rab35 has never been characterized for its potential contribution in sprouting angiogenesis; thus, our goal was to map Rab35s primary function in angiogenesis. Our results demonstrate that Rab35 is critical for sprout formation; in its absence apicobasal polarity is entirely lost in vitro and in vivo. To determine mechanism, we systematically explored established Rab35 effectors and show that none are operative in endothelial cells. However, we find that Rab35 partners with DENNd1c, an evolutionarily divergent guanine exchange factor, to localize to actin. Here, Rab35 regulates actin polymerization, which is required to setup proper apicobasal polarity during sprout formation. Our findings establish that Rab35 is a potent regulator of actin architecture during blood vessel development.

eNOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity

The roles of nitric oxide (NO) and endothelial NO synthase (eNOS) in the regulation of angiogenesis are well documented. However, the involvement of eNOS in the sprouting of endothelial tip-cells at the vascular front during sprouting angiogenesis remains poorly defined. In this study, we show that downregulation of eNOS markedly inhibits VEGF-stimulated migration of endothelial cells but increases their polarization, as evidenced by the reorientation of the Golgi in migrating monolayers and by the fewer filopodia on tip cells at ends of sprouts in endothelial cell spheroids. The effect of eNOS inhibition on EC polarization was prevented in Par3-depleted cells. Importantly, downregulation of eNOS increased the expression of polarity genes, such as PARD3B, PARD6A, PARD6B, PKCΖ, TJP3, and CRB1 in endothelial cells. In retinas of eNOS knockout mice, vascular development is retarded with decreased vessel density and vascular branching. Furthermore, tip cells at the extremities of the vascular front have a marked reduction in the number of filopodia per cell and are more oriented. In a model of oxygen-induced retinopathy (OIR), eNOS deficient mice are protected during the initial vaso-obliterative phase, have reduced pathological neovascularization, and retinal endothelial tip cells have fewer filopodia. Single-cell RNA sequencing of endothelial cells from OIR retinas revealed enrichment of genes related to cell polarity in the endothelial tip-cell subtype of eNOS deficient mice. These results indicate that inhibition of eNOS alters the polarity program of endothelial cells, which increases cell polarization, regulates sprouting angiogenesis and normalizes pathological neovascularization during retinopathy.

Heterotypic Multicellular Spheroids as Experimental and Preclinical Models of Sprouting Angiogenesis

Sprouting angiogenesis is the common response of live tissues to physiological and pathological angiogenic stimuli. Its accurate evaluation is of utmost importance for basic research and practical medicine and pharmacology and requires adequate experimental models. A variety of assays for angiogenesis were developed, none of them perfect. In vitro approaches are generally less physiologically relevant due to the omission of essential components regulating the process. However, only in vitro models can be entirely non-xenogeneic. The limitations of the in vitro angiogenesis assays can be partially overcome using 3D models mimicking tissue O2 and nutrient gradients, the influence of the extracellular matrix (ECM), and enabling cell-cell interactions. Here we present a review of the existing models of sprouting angiogenesis that are based on the use of endothelial cells (ECs) co-cultured with perivascular or other stromal cells. This approach provides an excellent in vitro platform for further decoding of the cellular and molecular mechanisms of sprouting angiogenesis under conditions close to the in vivo conditions, as well as for preclinical drug testing and preclinical research in tissue engineering and regenerative medicine.

Ultrastructural Study of Platelet Behavior and Interrelationship in Sprouting and Intussusceptive Angiogenesis during Arterial Intimal Thickening Formation

Platelets in atherosclerosis, bypass stenosis, and restenosis have been extensively assessed. However, a sequential ultrastructural study of platelets in angiogenesis during the early phases of these lesions has received less attention. Our objective was the study of platelets in angiogenesis and vessel regression during intimal thickening (IT) formation, a precursor process of these occlusive vascular diseases. For this purpose, we used an experimental model of rat occluded arteries and procedures for ultrastructural observation. The results show (a) the absence of platelet adhesion in the de-endothelialized occluded arterial segment isolated from the circulation, (b) that intraarterial myriad platelets contributed from neovessels originated by sprouting angiogenesis from the periarterial microvasculature, (c) the association of platelets with blood components (fibrin, neutrophils, macrophages, and eosinophils) and non-polarized endothelial cells (ECs) forming aggregates (spheroids) in the arterial lumen, (d) the establishment of peg-and-socket junctions between platelets and polarized Ecs during intussusceptive angiogenesis originated from the EC aggregates, with the initial formation of IT, and (e) the aggregation of platelets in regressing neovessels (‘transitory paracrine organoid’) and IT increases. In conclusion, in sprouting and intussusceptive angiogenesis and vessel regression during IT formation, we contribute sequential ultrastructural findings on platelet behavior and relationships, which can be the basis for further studies using other procedures.

Competition for endothelial cell polarity drives vascular morphogenesis

Blood vessel formation generates unique vascular patterns in each individual. The principles governing the apparent stochasticity of this process remain to be elucidated. Using mathematical methods, we find that the transition between two fundamental vascular morphogenetic programs, sprouting angiogenesis and vascular remodeling, is established by a shift on collective front-rear polarity of endothelial cells. We demonstrate that the competition between biochemical (VEGFA) and mechanical (blood flow-induced shear stress) cues controls this collective polarity shift. Shear stress increases tension at focal adhesions overriding VEGFA-driven collective polarization, which relies on tension at adherens junctions. We propose that vascular morphogenetic cues compete to regulate individual cell polarity and migration through tension shifts that translates into tissue-level emergent behaviors, ultimately leading to uniquely organized vascular patterns.

The people behind the papers – Zoe Grant, Tim Thomas, Anne Voss and Leigh Coultas

The histone acetyltransferase HBO1 (KAT1) is required for histone H3 lysine 14 acetylation, which is crucial for embryonic development. A new paper in Development reveals that, in the vascular system, HBO1 is required in endothelial cells for sprouting angiogenesis regulation. To hear more about the story, we caught up with first author Zoe Grant and senior authors Professor Anne Voss, Associate Professor Tim Thomas and Leigh Coultas, Business Development Manager, from the Walter and Eliza Hall Institute of Medical Research (WEHI), Australia.

Disproportion in Pericyte/Endothelial Cell Proliferation and Mechanisms of Intussusceptive Angiogenesis Participate in Bizarre Vessel Formation in Glioblastoma

Glioblastoma (GBM) is the most malignant tumor in the brain. In addition to the vascular pattern with thin-walled vessels and findings of sprouting angiogenesis, GBM presents a bizarre microvasculature (BM) formed by vascular clusters, vascular garlands, and glomeruloid bodies. The mechanisms in BM morphogenesis are not well known. Our objective was to assess the role of pericyte/endothelial proliferation and intussusceptive angiogenic mechanisms in the formation of the BM. For this purpose, we studied specimens of 66 GBM cases using immunochemistry and confocal microscopy. In the BM, the results showed (a) transitional forms between the BM patterns, mostly with prominent pericytes covering all the abluminal endothelial cell (EC) surface of the vessels, (b) a proliferation index high in the prominent pericytes and low in ECs (47.85 times higher in pericytes than in ECs), (c) intravascular pillars (hallmark of intussusceptive angiogenesis) formed by transcapillary interendothelial bridges, endothelial contacts of opposite vessel walls, and vessel loops, and (d) the persistence of these findings in complex glomeruloid bodies. In conclusion, disproportion in pericyte/EC proliferation and mechanisms of intussusceptive angiogenesis participate in BM formation. The contributions have morphogenic and clinical interest since pericytes and intussusceptive angiogenesis can condition antiangiogenic therapy in GBM.

The histone acetyltransferase HBO1 (KAT7) promotes efficient tip cell sprouting during angiogenesis

Blood vessel growth and remodelling are essential during embryonic development and disease pathogenesis. The diversity of endothelial cells (ECs) is transcriptionally evident and ECs undergo dynamic changes in gene expression during vessel growth and remodelling. Here, we investigated the role of the histone acetyltransferase HBO1 (KAT7), which is important for activating genes during development and histone H3 lysine 14 acetylation (H3K14ac). Loss of HBO1 and H3K14ac impaired developmental sprouting angiogenesis and reduced pathological EC overgrowth in the retinal endothelium. Single-cell RNA-sequencing of retinal ECs revealed an increased abundance of tip cells in Hbo1 deleted retinas, which lead to EC overcrowding in the retinal sprouting front and prevented efficient tip cell migration. We found that H3K14ac was highly abundant in the endothelial genome in both intra- and intergenic regions suggesting that the role of HBO1 is as a genome organiser that promotes efficient tip cell behaviour necessary for sprouting angiogenesis.