S. Christoforidis Laboratory

Research
Interplay between endocytosis, signaling and exocytosis in endothelial cells: Molecular mechanisms and role in blood vessel physiology.

Endothelial cells, which cover the inner wall of blood vessels, play key role in blood vessel homeostasis. Activated endothelium and/or endothelial dysfunction are implicated in the most life-threatening diseases, such as atherosclerosis, cardiovascular dysfunctions, stroke, bleeding and cancer angiogenesis, which are responsible for the vast majority of deaths in modern societies.  However, the exact mechanisms that govern, 1st, the properties of healthy endothelium, and 2nd, endothelial dysfunction in vascular diseases, are only poorly understood.

Intriguingly, many endothelial molecules, that play important role in the above pathophysiological processes, are stored in specialized organelles of endothelial cells, called Weibel Palade bodies, which are formed at the trans-Golgi network. Upon activation of endothelial cells, Weibel Palade bodies travel to the cell surface and fuse with plasma membrane, thereby releasing they cargo molecules in the blood stream. Among the secreted proteins are several key players of inflammation, thrombosis, angiogenesis and vessel tone, such as P-selectin, von willebrand factor (vWF), Angiopoeitin-2, endothelin, and others.

Activated endothelium plays critical role in inflammation and thrombosis. Under resting conditions, endothelial cells form a tight barrier which exerts anti-thrombotic and anti-inflammatory properties. However, upon activation (1). endothelium secretes molecules that are localized in the storage organelles Weibel Palade bodies (2). These molecules participate in processes that lead to loosening of intercellular contacts and migration of lymphocytes across the endothelial wall, causing inflammation (3). as well as in activation of platelets and thrombosis (4).

One of the most important activators of Weibel Palade body secretion is VEGF (Vascular Endothelial Growth Factor), a growth factor that plays important role in tumour angiogenesis. In general, upon their activation at the plasma membrane, growth factor receptors become internalized into endosomes. It has been traditionally thought that this is a process that ceases signaling, since, endocytosis was believed to serve solely as a process that delivers molecules into lysosomes for degradation. However, studies in the last two decades have clearly shown that ligand/receptor complexes are endocytosed towards diverse endosomal organelles, from where they control the duration, nature, and intensity of signaling pathways. Our group is interested in understanding the interconnections between endocytosis, signaling and exocytosis in endothelial cells and their importance in vascular physiology.
    
    To study endothelial cell activation, we are working on two types of receptors: Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), which belongs in the family of growth factor receptors, and purinergic receptors, which are G-protein coupled receptors that are activated by nucleotides. Both VEGFR2 and purinergic receptors regulate endothelial cell proliferation, migration, apoptosis and vascular permeability/inflammation. At least part of these functions is achieved via the ability of the above receptors to activate secretion of Weibel Palade bodies (described above). We are interested in understanding the mechanisms that control signaling of VEGFR2 and purinergic receptors, the role of endocytosis of these receptors in signaling, and the mechanisms responsible for Weibel Palade body exocytosis.
 
To achieve these goals, we are working on the following three projects:

Project No 1. Molecular mechanisms of Weibel Palade body exocytosis and role in vascular remodeling.

To get insights into the mechanisms of Weibel Palade body exocytosis, we focused on Rab GTPases, since these molecules possess central role in almost every aspect of vesicle trafficking, that is in vesicle formation, transport, docking and fusion with the target membrane. Specific localization of different members of the Rab family in individual vesicles provides specificity to the trafficking process, thereby ensuring correct delivery of cargo to the appropriate destination. The human genome contains over 60 members of Rabs. Given the importance of Rabs in membrane trafficking, we aimed to identify the full spectrum of Rabs that specifically regulate Weibel Palade body trafficking. To this end, our group has undertaken an unbiased approach by screening the localization of all Rab members of the human genome, tagged with GFP, in endothelial cells, using confocal fluorescence microscopy (Zografou et al, 2012, J Cell Sci, 125, 4780-4790). This analysis revealed that five Rabs are localized at Weibel Palade bodies, Rab 3, 27, 15, 33 and 37, and that three of them, Rab3, Rab27 and Rab15, are necessary for exocytosis of the bodies. Therefore, it appears that Weibel Palade body exocytosis is not a one's Rab case, but rather requires the coordinated function of a specific group of Rabs.

Rab3 and Rab15 (green) are co-localized with Rab27 (red) on vWF-positive (blue) Weibel Palade bodies of endothelial cells.

Furthermore, we found that a specific effector of Rab27, Munc13-4, is also an effector of Rab15 and is required for WPB exocytosis.

Munc13-4 (red) co-localises with Rab15 (green) on vWF-positive WPBs (blue) in endothelial cells. The lower panels are enlarged images of the area enclosed by the white box in the GFP-Rab15 image.

These data indicated that Rab27a and Rab15, as well as their effector Munc13-4, cooperate to drive exocytosis of Weibel Palade bodies.

Current studies in the lab aim to address the specific role of each of the five Rabs (Rab3, 15, 33, 27, 37) on formation, maturation, transport and exocytosis of Weibel Palade bodies.

Project No 2. Role of endocytosis in receptor signalling in endothelial cells.

(in collaboration with two other groups of IMBB/BR, Carol Murphy's and Theodore Fotsis's groups).

Activation of growth factor receptors at the plasma membrane results in their internalization and compartmentalization into endosomes. It has been traditionally thought that this is a process that ceases signaling, since, endocytosis was believed to serve solely as a process that delivers molecules into lysosomes for degradation. However, studies in the last two decades have shown that several receptors continue their signalling activity after they become delivered into the endosomal compartments, and that receptor endocytosis is a process that regulates the nature, duration and intensity of receptor signaling. To achieve this outcome, receptors explore a complex network of internalization routes that lead to diverse endosomal organelles.
    VEGFR2 is the main receptor and mediator of the vasculogenic and angiogenic properties of endothelial cells. We found recently that constitutive endocytosis of VEGFR2 protects the receptor against shedding (Basagiannis D and Christoforidis S, J Biol Chem, 2016, 291, 16892-16903), while addition of VEGF reroutes VEGFR2 towards macropinocytosis, a critical event for VEGF-induced signalling in vitro and for angiogenesis in vivo (Basagiannis D et al., J Cell Sci, 2016, 129, 4091-4104;  Basagiannis D. et al., Sci. Rep., 2017, Mar 22;7:45035). For a summary of these findings, see scheme below.

Left scheme, in blue background: Constitutive endocytosis via the clathrin route protects VEGFR2 against shedding (CME: clathrin-mediated endocytosis). Left scheme, in pink background: VEGF switches internalization of VEGFR2 towards macropinosomes (MP), which are imaged on the right via confocal microscopy.

Following these findings, we are currently aiming in elucidating the mechanism by which macropinocytosis controls VEGFR2 signalling and how the signals are conveyed to Weibel Palade bodies, for triggering exocytosis.

Project No 3. Purinergic receptor signaling and regulation by ecto-nucleotidases.

This project started already back in 1995, when we purified and characterized the prototype member of ecto-nucleotidases, ATP-diphosphohydrolase/NTPdase1 (Christoforidis et al. EJB, 1995, 234, 66-74). This is an endothelial ecto-enzyme that, by hydrolysing ATP and ADP in the blood stream, prevents platelet aggregation and ensures normal blood flow. Based on sequencing data of the purified enzyme and the available databases of that time, we proposed (for the first time for this enzyme) that this enzyme was a novel protein. Subsequent studies from other groups showed that this enzyme is identical to CD39, a marker of activated B and T lymphocytes. In a later work, we found that the localization of CD39 at the plasma membrane is polarized, with a high preference for the apical side of the membrane (Papanikolaou et al. 2005, JBC, 280, 26406-26414).

CD39 is preferentially targeted to the apical side of the plasma membrane in polarized MDCK cells. Optical xz sectioning of polarized MDCK cells, grown on filters, showing the plasma membrane localization of stably expressed CD39 (red), caveolin-1 (green), and the two together (merge).

For apical targeting, CD39/NTPdase1 utilizes two independent signals, one that is present at its Nt-transmembrane domain and one at its ecto-domain (Papanikolaou et al. 2011, Traffic, 12, 1148-1165).

The ecto-domain of CD39 possesses an apical targeting signal. Removal of either N- or C- terminal transmembrane domains (TM) does not interfere with apical targeting of CD39. The two bottom images show optical xz sections of polarized MDCK cells, grown on filters, stably expressing deletion mutants of CD39 (shown in red), ΔNt (at the left) and ΔCt (at the right). The basolateral marker is shown in blue.

We also found that CD39 (NTPdase1) is localized at lipid raft microdomains, which are membrane platforms enriched in cholesterol and sphingolipids, and that the enzymatic activity of CD39 is absolutely dependent on the levels of cholesterol in the membrane (Papanikolaou et al. 2005, JBC, 280, 26406-26414). Optimal enzymatic activity and cholesterol-dependence of CD39 rely on its two transmembrane domains (Papanikolaou et al. 2011, Traffic, 12, 1148-1165). Based on these findings, we propose that CD39 is an excellent model protein to study the mechanisms by which specialized membrane domains and their specific lipids regulate the function of transmembrane proteins. Current studies in the lab aim to shed light in this mechanism.

Links between the above projects
Although the above three projects are studied in parallel, special emphasis is given on the identification of molecular links between the three processes: receptor activation and signaling, receptor endocytosis, and stimulated exocytosis of storage organelles. The scheme below provides an overview of these links.

Receptor signaling can take place either at the plasma membrane or upon receptor internalization into early endosomes. Growth factor receptors and G-protein coupled receptors can initiate signaling at the plasma membrane (red arrows) or/and after their endocytosis at the early endosomes (purple arrows). Endocytosis is indicated by blue arrows. In our lab, we are studying the role of endocytosis in VEGFR2 and purinergic receptor signaling in endothelial cells. More specifically we are studying the role of endocytosis in signaling that triggers transcriptional initiation in the nucleus, as well as signaling cascades that result in Weibel Palade body exocytosis. Purinergic signaling is initiated by ATP (bottom left of the scheme), which is secreted by damaged or activated cells. Sequential hydrolysis of ATP to adenosine, by the ecto-enzymes CD39 (ecto-ATPase) and CD73 (ecto-AMPase), counteracts activation of purinergic receptors and promotes adenosine signaling. Reduction of purinergic signaling together with activation of adenosine receptors provide a strong beneficial effect in blood flow, since they result in vasodilation and prevention of platelet aggregation and thrombosis.

These studies will provide insights into the molecular and functional links between endocytosis, signalling and exocytosis, as well as on their spatial and temporal coordination.

Finally, in a new project in the lab, we aim to extend the above studies in stem cell research, focusing on the role of endocytosis and secretion in self-renewal of stem cells as well as on their differentiation towards the vascular lineages. (in collaboration with three other groups of IMBB/BR, Carol Murphy's, Theodore Fotsis's, and Panos Kouklis's groups).

Ultimately, the findings of our studies will contribute to the design of more effective therapeutic approaches in vascular diseases.

Methodology
To achieve the objectives described above, besides using standard techniques of the field of Molecular and Cell Biology, we employ leading edge technology such as super resolution STED confocal microscopy, Total Internal Reflection Fluorescence Microscopy (TIRF-M), quantitative image analysis and proteomics by high resolution mass spectrometry.