Objective: Vascular lumen formation requires the redistribution of intracellular proteins to instruct apicobasal polarity, thereby enforcing maturation of both luminal and basal domains. In the absence of proper apical signaling, lumen formation can be distorted leading to lumen collapse and cessation of blood flow. Slp2a (synaptotagmin-like protein-2a) has been implicated in apical membrane signaling; however, the role of Slp2a in vascular lumen formation has never been assessed. Approach and Results: Our results demonstrate that Slp2a is required for vascular lumen formation. Using a 3-dimensional sprouting assay, sub-cellular imaging, and zebrafish blood vessel development, we establish that Slp2a resides at the apical membrane acting as a tether for Rab27a that decorates Weibel-Palade bodies (WPBs). We show that Slp2a regulates exocytic activity of WPBs, thus regulating release of WPB contents into the luminal space during angiogenesis. Angiopoietin-2 is a Tie-2 receptor ligand that is selectively released from WPB secretory granules. We identify a critical role for angiopoietin-2 in regulating endothelial lumenization and show that in the absence of Slp2a, WPB contents cannot fuse with the apical membrane. This disrupts the release of angiopoietin-2 and blocks Tie-2 signaling necessary for proper lumen formation. Conclusions: Our results demonstrate a novel requirement of Slp2a for vascular lumen formation. Moreover, we show that Slp2a is required for the exocytic release of WPB secretory granule cargo during vascular lumen development, and thus is a core upstream component of the WPB secretory pathway. Furthermore, we provide evidence that WPB-housed angiopoietin-2 is required for vascular lumen formation.

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 Rab35’s 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 through limiting Rac1 and RhoA activity, which is required to set up proper apicobasal polarity during sprout formation. Our findings establish that Rab35 is a potent brake of actin remodeling during blood vessel development.

Objectives Vesicular trafficking dictates protein localization, functional activity, and half‐life, providing a critically important regulatory step in tissue development; however, there is little information detailing endothelial‐specific trafficking signatures. This is due, in part, to limitations in visualizing trafficking events in endothelial tissues. Our aim in this investigation was to explore the use of a 3‐dimensional (3D) in vitro sprouting model to image endothelial membrane trafficking events. Methods Endothelial cells were challenged to grow sprouts in a fibrin bead assay. Thereafter, spouts were transfected with fluorescent proteins and stained for various cell markers. Sprouts were then imaged for trafficking events using live and fixed‐cell microscopy. Results Our results demonstrate that fibrin bead sprouts have a strong apicobasal polarity marked by apical localization of proteins moesin and podocalyxin. Comparison of trafficking mediators Rab27a and Rab35 between 3D sprouts and 2D culture showed that vesicular carriers can be imaged at high resolution, exhibiting proper membrane polarity solely in 3D sprouts. Lastly, we imaged exocytic events of von Willebrand Factor and demonstrated a distinct imaging advantage for monitoring secretion events in 3D sprouts as compared with 2D culture. Conclusions Our results establish that the fibrin bead sprouting assay is well‐suited for imaging of trafficking events during angiogenic growth.

Blood vessels demonstrate a multitude of complex signaling programs that work in concert to produce functional vasculature networks during development. A known, but less widely studied, area of endothelial cell regulation is vesicular trafficking, also termed sorting. After moving through the Golgi apparatus, proteins are shuttled to organelles, plugged into membranes, recycled, or degraded depending on the internal and extrinsic cues. A snapshot of these protein-sorting systems can be viewed as a trafficking signature that is not only unique to endothelial tissue, but critically important for blood vessel form and function. In this review, we will cover how vesicular trafficking impacts various aspects of angiogenesis, such as sprouting, lumen formation, vessel stabilization, and secretion, emphasizing the role of Rab GTPase family members and their various effectors.

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.