Arf GTPase-Activating proteins SMAP1 and AGFG2 regulate the size of Weibel-Palade bodies and exocytosis of von Willebrand factor

Arf GTPase-Activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors (Arfs), that are critical to form transport intermediates. ArfGAPs have been thought to be negative regulators of Arfs, however, accumulating evidence indicates that ArfGAPs are important for cargo sorting and promote membrane traffic. Weibel-Palade bodies (WPBs) are cigar-shaped secretory granules in endothelial cells that contain von Willebrand factor (vWF) as their main cargo. WPB biogenesis at the Golgi was reported to be regulated by Arf and their regulators, but the role of ArfGAPs has been unknown. In this study, we performed siRNA screening of ArfGAPs to investigate the role of ArfGAPs in the biogenesis of WPBs. We found two ArfGAPs, SMAP1 and AGFG2, to be involved in WPB size and vWF exocytosis, respectively. SMAP1 depletion resulted in small-sized WPBs, and the lysosomal inhibitor leupeptin recovered the size of WPBs. The results indicate that SMAP1 functions in preventing the degradation of cigar-shaped WPBs. On the other hand, AGFG2 downregulation resulted in the inhibition of vWF secretion upon Phorbol 12-myristate 13-acetate (PMA) or histamine stimulation, suggesting that AGFG2 plays a role in vWF exocytosis. Our study revealed unexpected roles of ArfGAPs in vWF transport.

Ca2+ release-activated Ca2+ channels are responsible for histamine-induced Ca2+ entry, permeability increase, and interleukin synthesis in lymphatic endothelial cells

The lymphatic functions in maintaining lymph transport, and immune surveillance can be impaired by infections and inflammation, thereby causing debilitating disorders, such as lymphedema and inflammatory bowel disease. Histamine is a key inflammatory mediator known to trigger vasodilation and vessel hyperpermeability upon binding to its receptors and evoking intracellular Ca2+ ([Ca2+]i) dynamics for downstream signal transductions. However, the exact molecular mechanisms beneath the [Ca2+]i dynamics and the downstream cellular effects have not been elucidated in the lymphatic system. Here, we show that Ca2+ release-activated Ca2+ (CRAC) channels, formed by Orai1 and stromal interaction molecule 1 (STIM1) proteins, are required for the histamine-elicited Ca2+ signaling in human dermal lymphatic endothelial cells (HDLECs). Blockers or antagonists against CRAC channels, phospholipase C, and H1R receptors can all significantly diminish the histamine-evoked [Ca2+]i dynamics in lymphatic endothelial cells (LECs), while short interfering RNA-mediated knockdown of endogenous Orai1 or STIM1 also abolished the Ca2+ entry upon histamine stimulation in LECs. Furthermore, we find that histamine compromises the lymphatic endothelial barrier function by increasing the intercellular permeability and disrupting vascular endothelial-cadherin integrity, which is remarkably attenuated by CRAC channel blockers. Additionally, the upregulated expression of inflammatory cytokines, IL-6 and IL-8, after histamine stimulation was abolished by silencing Orai1 or STIM1 with RNAi in LECs. Taken together, our data demonstrated the essential role of CRAC channels in mediating the [Ca2+]i signaling and downstream endothelial barrier and inflammatory functions induced by histamine in the LECs, suggesting a promising potential to relieve histamine-triggered vascular leakage and inflammatory disorders in the lymphatics by targeting CRAC channel functions.

Effect of the Fexofenadine on the expression of HRH-1 and HRH-4 receptor in Peripheral Blood Mononuclear Cell isolated from children with diagnosed allergy – in vitro study Short communication

Purpose: Fexofenadine (FXF) is the active metabolite of terfenadine with selective peripheral H1 receptor antagonist activity. FXF is a third-generation antihistamine, non-sedating, rapid and very long acting used in symptoms associated with allergic diseases such as allergic rhinitis, asthma and dermatitis. The pleiotropic effects of histamine are mediated by four types of receptors that belong to the G-protein-coupled receptor family: histamine H1 receptor (HRH-1), histamine H2 receptor, histamine H3 receptor, and histamine H4 receptor. Our hypothesis is that HRH-4 opens new possibility in treatment in allergy diseases and FXF could be the antagonist of both HRH-1 and HRH-4. Methods: We isolated a peripheral blood mononuclear cell (PBMC) from children with diagnosed allergies and healthy – control group and measured the HRH-1 and HRH-4 mRNA gene expression using Quantitive Real-Time PCR. We obtained the results from basal gene expression and after FXF and histamine stimulation. Results: HRH-1 mRNA basal gene expression shows significantly higher, and HRH-4 shows significantly lower expression in allergy group compared to control. In both groups HRH-1 mRNA gene expression was observed as statistically significant increased after histamine stimulation compared to cells not treated, while in HRH-4 only in allergy group we observed statistical increase. FXF successively blocked histamine affinity in HRH-1 mRNA gene expression but not in HRH-4, where we not observed any reaction. Conclusions: Results clearly overturned our hypothesis about the possibility of using FXF to block over-expression HRH-4 and open new way of treatment in allergy diseases.

Abstract WMP83: Extracellular Vimentin Contributes to Von Willebrand Factor (VWF) String Formation in the Cerebrovasculature Following Endothelial Activation

Introduction: VWF strings can form within the vascular lumen following endothelial activation and have been implicated in platelet adhesion and worsened outcome following stroke. However, the molecular interactions facilitating VWF string anchorage to the endothelial surface are currently unknown. Here we examined the novel role of endothelial vimentin in mediating the anchorage of VWF strings within the cerebrovasculature. We hypothesize that VWF released from activated endothelium remains anchored at the luminal surface (i.e. VWF strings) through direct interaction with extracellular vimentin. Methods: Cultured endothelial cell (EC) experiments were performed with human umbilical vein EC (HUVECs) and human brain microvascular EC (HBMVECs). EC were stimulated with histamine (10 uM) under flow conditions. Specific protein interactions were probed with recombinant vimentin and A2 domain of VWF. Mouse middle cerebral and superior cerebellar arteries from WT and vimentin KO mice were set up in a pressurized artery chamber, stimulated with histamine, and then processed for VWF immunofluorescence to quantify VWF strings. VWF string formation was further evaluated by cranial window preparation using labeled platelets to detect real-time in vivo string formation. Results: In cultured EC, histamine stimulation promoted the production of long VWF strings that were significantly attenuated in the presence of recombinant vimentin or A2 domain of VWF. In pressurized cerebral arteries, histamine stimulation promoted VWF strings that aligned along the luminal endothelial surface. VWF string formation was reduced 2.9 fold in arteries from vimentin knockout mice (P=0.02). Histamine stimulation in the cranial window produced platelet-adherent strings in the pial microvasculature. Conclusions: These studies provide in vitro and in vivo evidence for a novel interaction between vimentin and the A2 domain of VWF at the endothelial surface which contributes to the anchorage of VWF strings in the microvasculature. This vimentin/VWF interaction critically regulates VWF-mediated platelet adhesion at the surface of activated endothelium and could thus provide a novel therapeutic target in the treatment of acute ischemic stroke.

Regulation of mitogen-activated protein kinase by protein kinase C and mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle

Vascular smooth muscle contraction is primarily regulated by phosphorylation of myosin light chain. There are also modulatory pathways that control the final level of force development. We tested the hypothesis that protein kinase C (PKC) and mitogen-activated protein (MAP) kinase modulate vascular smooth muscle activity via effects on MAP kinase phosphatase-1 (MKP-1). Swine carotid arteries were mounted for isometric force recording and subjected to histamine stimulation in the presence and absence of inhibitors of PKC [bisindolylmaleimide-1 (Bis)], MAP kinase kinase (MEK) (U0126), and MKP-1 (sanguinarine) and flash frozen for measurement of MAP kinase, PKC-potentiated myosin phosphatase inhibitor 17 (CPI-17), and caldesmon phosphorylation levels. CPI-17 was phosphorylated in response to histamine and was inhibited in the presence of Bis. Caldesmon phosphorylation levels increased in response to histamine stimulation and were decreased in response to MEK inhibition but were not affected by the addition of Bis. Inhibition of PKC significantly increased p42 MAP kinase, but not p44 MAP kinase. Inhibition of MEK with U0126 inhibited both p42 and p44 MAP kinase activity. Inhibition of MKP-1 with sanguinarine blocked the Bis-dependent increase of MAP kinase activity. Sanguinarine alone increased MAP kinase activity due to its effects on MKP-1. Sanguinarine increased MKP-1 phosphorylation, which was inhibited by inhibition of MAP kinase. This suggests that MAP kinase has a negative feedback role in inhibiting MKP-1 activity. Therefore, PKC catalyzes MKP-1 phosphorylation, which is reversed by MAP kinase. Thus the fine tuning of vascular contraction is due to the concerted effort of PKC, MAP kinase, and MKP-1.