SummaryWeibel-Palade bodies (WPBs) are endothelial-cell-specific organelles that, upon fusion with the plasma membrane, release cargo molecules that are essential in blood vessel abnormalities, such as thrombosis and inflammation, as well as in angiogenesis. Despite the importance of WPBs, the basic mechanisms that mediate their secretion are only poorly understood. Rab GTPases play fundamental role in the trafficking of intracellular organelles. Yet, the only known WPB-associated Rabs are Rab27a and Rab3d. To determine the full spectrum of WPB-associated Rabs we performed a complete Rab screening by analysing the localisation of all Rabs in WPBs and their involvement in the secretory process in endothelial cells. Apart from Rab3 and Rab27, we identified three additional Rabs, Rab15 (a previously reported endocytic Rab), Rab33 and Rab37, on the WPB limiting membrane. A knockdown approach using siRNAs showed that among these five WPB Rabs only Rab3, Rab27 and Rab15 are required for exocytosis. Intriguingly, we found that Rab15 cooperates with Rab27a in WPB secretion. Furthermore, a specific effector of Rab27, Munc13-4, appears to be also an effector of Rab15 and is required for WPB exocytosis. These data indicate that WPB secretion requires the coordinated function of a specific group of Rabs and that, among them, Rab27a and Rab15, as well as their effector Munc13-4, cooperate to drive exocytosis.

Endocytosis plays a crucial role in receptor signalling. VEGFR2 (also known as KDR) and its ligand VEGFA are fundamental in neovascularisation. However, our understanding of the role of endocytosis in VEGFR2 signalling remains limited. Despite the existence of diverse internalisation routes, the only known endocytic pathway for VEGFR2 is the clathrin-mediated pathway. Here, we show that this pathway is the predominant internalisation route for VEGFR2 only in the absence of ligand. Intriguingly, VEGFA induces a new internalisation itinerary for VEGFR2, the pathway of macropinocytosis, which becomes the prevalent endocytic route for the receptor in the presence of ligand, whereas the contribution of the clathrin-mediated route becomes minor. Macropinocytic internalisation of VEGFR2, which mechanistically is mediated through the small GTPase CDC42, takes place through macropinosomes generated at ruffling areas of the membrane. Interestingly, macropinocytosis plays a crucial role in VEGFAinduced signalling, endothelial cell functions in vitro and angiogenesis in vivo, whereas clathrin-mediated endocytosis is not essential for VEGFA signalling. These findings expand our knowledge on the endocytic pathways of VEGFR2 and suggest that VEGFA-driven internalisation of VEGFR2 through macropinocytosis is essential for endothelial cell signalling and angiogenesis.

VEGFR2 is a critical angiogenic receptor playing a key role in vascular homeostasis. Upon activation by VEGF, VEGFR2 becomes endocytosed. Internalisation of VEGFR2 is facilitated, in part, through clathrin mediated endocytosis (CME), the role of which in VEGFR2 function is debated. Here, we confirm the contribution of CME in VEGFR2 uptake. However, curiously, we find that different approaches of inhibition of CME exert contradictory effects on VEGF signalling; knockdown of clathrin, or of dynamin, or overexpression of dynamin K44A, do not affect VEGF-induced phosphorylation of ERK1/2, while dynasore causes strong inhibition. We resolve this discrepancy by showing that although dynasore inhibits CME of VEGFR2, its inhibitory action in ERK1/2 phosphorylation is not related to attenuation of VEGFR2 endocytosis; it is rather due to an off-target effect of the drug. Dynasore inhibits VEGF-induced calcium release, a signalling event that lies upstream of ERK1/2, which implies that this effect could be responsible, at least in part, for the inhibitory action of the drug on VEGF-to-ERK1/2 signalling. These results raise caution that although dynasore is specific in inhibiting clathrin- and dynamin-mediated endocytosis, it may also exert off-target effects on signalling molecules, hence influencing the interpretation of the role of endocytosis in signalling.

Endocytosis plays a crucial role in receptor signalling. VEGFR2 (also known as KDR) and its ligand VEGFA are fundamental in neovascularisation. However, our understanding of the role of endocytosis in VEGFR2 signalling remains limited. Despite the existence of diverse internalisation routes, the only known endocytic pathway for VEGFR2 is the clathrin-mediated pathway. Here, we show that this pathway is the predominant internalisation route for VEGFR2 only in the absence of ligand. Intriguingly, VEGFA induces a new internalisation itinerary for VEGFR2, the pathway of macropinocytosis, which becomes the prevalent endocytic route for the receptor in the presence of ligand, whereas the contribution of the clathrin-mediated route becomes minor. Macropinocytic internalisation of VEGFR2, which mechanistically is mediated through the small GTPase CDC42, takes place through macropinosomes generated at ruffling areas of the membrane. Interestingly, macropinocytosis plays a crucial role in VEGFAinduced signalling, endothelial cell functions in vitro and angiogenesis in vivo, whereas clathrin-mediated endocytosis is not essential for VEGFA signalling. These findings expand our knowledge on the endocytic pathways of VEGFR2 and suggest that VEGFA-driven internalisation of VEGFR2 through macropinocytosis is essential for endothelial cell signalling and angiogenesis.

Background: A study was conducted at the University Hospital of Patras between January 2002 and December 2003 to investigate antibiotic resistance patterns and clonality of Salmonella enterica in southwestern Greece. Methods: Ninety-five isolates recovered from different outpatients were characterized by specific antisera and were tested for their susceptibility to various antimicrobial agents. Clones were identified by pulsed-field gel electrophoresis (PFGE) of XbaI chromosomal DNA digests. Results: Five serotypes were characterized among 95 isolates, recovered mainly from children, with the predominance of Salmonellaenteritidis followed by Salmonellatyphimurium. The strains were further distinguished by PFGE to 16 clones. The majority of S.enteritidis (61 strains) belonged to clone A, already characterized in Greece, while S.typhimurium (18 isolates) belonged to 3 clones, exhibiting multiresistant phenotypes. Conclusions: One clone S.enteritidis, type A, circulates in southwestern Greece, mainly during summer, while clonality was also observed among S.typhimurium.

Human embryonic stem cells (hESCs) are promising in regenerative medicine (RM) due to their differentiation plasticity and proliferation potential. However, a major challenge in RM is the generation of a vascular system to support nutrient flow to newly synthesized tissues. Here we refined an existing method to generate tight vessels by differentiating hESCs in CD34(+) vascular progenitor cells using chemically defined media and growth conditions. We selectively purified these cells from CD34(-) outgrowth populations also formed. To analyze these differentiation processes, we compared the proteomes of the hESCs with those of the CD34(+) and CD34(-) populations using high resolution mass spectrometry, label-free quantification, and multivariate analysis. Eighteen protein markers validate the differentiated phenotypes in immunological assays; nine of these were also detected by proteomics and show statistically significant differential abundance. Another 225 proteins show differential abundance between the three cell types. Sixty-three of these have known functions in CD34(+) and CD34(-) cells. CD34(+) cells synthesize proteins implicated in endothelial cell differentiation and smooth muscle formation, which support the bipotent phenotype of these progenitor cells. CD34(-) cells are more heterogeneous synthesizing muscular/osteogenic/chondrogenic/adipogenic lineage markers. The remaining >150 differentially abundant proteins in CD34(+) or CD34(-) cells raise testable hypotheses for future studies to probe vascular morphogenesis.

VEGFR2 is the main receptor and mediator of the vasculogenic and angiogenic activity of VEGF. Activated VEGFR2 internalizes through clathrin-mediated endocytosis and macropinocytosis. As dynamin is a key regulator of the clathrin pathway, chemical inhibitors of dynamin are commonly used to assess the role of the clathrin route in receptor signaling. However, drugs may also exert off-target effects. Here, we compare the effects of three dynamin inhibitors, dynasore, dyngo 4a and dynole, on VEGFR2 internalization and signaling. Although these drugs consistently inhibit clathrin-mediated endocytosis of both transferrin (a typical cargo of this route) and VEGFR2, surprisingly, they exert contradictory effects in receptor signaling. Thus, while dynasore has no effect on phosphorylation of VEGFR2, the other two drugs are strong inhibitors. Furthermore, although dyngo does not interfere with phosphorylation of Akt, dynasore and dynole have a strong inhibitory effect. These inconsistent effects suggest that the above dynamin blockers, besides inhibiting dynamin-dependent endocytosis of VEGFR2, exert additional inhibitory effects on signaling that are independent of endocytosis; i.e., they are due to off-target effects. Using a recently developed protocol, we comparatively validate the specificity of two endocytic inhibitors, dynasore and EIPA. Our findings highlight the importance of assessing whether the effect of an endocytic drug on signaling is specifically due to its interference with endocytosis or due to off-targets.

Peptide analogs/conjugates Abbreviation Ac-(Lys-Aib-Cys) 4 -NH 2 CPSOC CF-Ahx-[Lys-Aib-Cys(S-CH 2 CONH 2 )] 4 -NH 2 CF-Ahx-CPSOC(S-CH 2 CONH 2 ) CF-G 48 RKKRRQRRRPPQ 60 -Cys (CH 2 CONH 2 )-NH 2 CF-Tat 48-60 -Cys (CH 2 CONH 2 ) CF-Ahx-S 158 ALTQKGLKNVFDEA 172 -NH 2 CF-Ahx-Cdc42 158-172 CF-Ahx-[Lys-Aib-Cys(S-CH 2 CO-V 181 IKKSKK 187 -NH 2 )] 4 -NH 2 CF-Ahx-CPSOC(S-CH 2 CO-Cdc42 181-187 ) CF-Ahx-[Lys-Aib-Cys(S-CH 2 CO-I 173 VAALEPPVIKKSKK 187 -NH 2 )] 4 -NH 2 CF-Ahx-CPSOC(S-CH 2 CO-Cdc42 173-187