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1.  Conditional and inducible transgene expression in endothelial and hematopoietic cells using Cre/loxP and tetracycline-off systems 
In the present study, the tetracycline-off and Cre/loxP systems were combined to gain temporal and spatial control of transgene expression. Mice were generated that carried three transgenes: Tie2-tTA, tet-O-Cre and either the ZEG or ZAP reporter. Tie2-tTA directs expression of tetracycline-controlled transactivator (tTA) in endothelial and hematopoietic cells under the control of the Tie2 promoter. Tet-O-Cre produces Cre recombinase from a minimal promoter containing the tet-operator (tetO). ZEG or ZAP contains a strong promoter and a loxP-flanked stop sequence, followed by an enhanced green fluorescence protein (EGFP) or human placental alkaline phosphatase (hPLAP) reporter. In the presence of tetracycline, the tTA transactivator produced by Tie-2-tTA is disabled and Cre is not expressed. In the absence of tetracycline, the tTA binds tet-O-Cre to drive the expression of Cre, which recombines the loxP sites of the ZEG or ZAP transgene and results in reporter gene expression. In the present study, the expression of the ZEG or ZAP reporter genes in embryos and adult animals with and without tetracycline treatment was examined. In the presence of tetracycline, no reporter gene expression was observed. When tetracycline was withdrawn, Cre excision was activated and the reporter genes were detected in endothelial and hematopoietic cells. These results demonstrate that this system may be used to bypass embryonic lethality and access adult phenotypes.
PMCID: PMC4186360  PMID: 25289022
Cre/loxP system; tetracycline; endothelial cells; hematopoietic cell; transgenic mice
2.  DARC shuttles inflammatory chemokines across the blood–brain barrier during autoimmune central nervous system inflammation 
Brain  2014;137(5):1454-1469.
Trafficking of T cells into the CNS is a pathophysiological hallmark of multiple sclerosis. Using an in vitro model of the blood-brain barrier, Minten et al. reveal that the atypical chemokine receptor DARC shuttles inflammatory chemokines across the barrier, where they contribute to immune cell trafficking into the brain.
The Duffy antigen/receptor for chemokines, DARC, belongs to the family of atypical heptahelical chemokine receptors that do not couple to G proteins and therefore fail to transmit conventional intracellular signals. Here we show that during experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, the expression of DARC is upregulated at the blood–brain barrier. These findings are corroborated by the presence of a significantly increased number of subcortical white matter microvessels staining positive for DARC in human multiple sclerosis brains as compared to control tissue. Using an in vitro blood–brain barrier model we demonstrated that endothelial DARC mediates the abluminal to luminal transport of inflammatory chemokines across the blood–brain barrier. An involvement of DARC in experimental autoimmune encephalomyelitis pathogenesis was confirmed by the observed ameliorated experimental autoimmune encephalomyelitis in Darc−/− C57BL/6 and SJL mice, as compared to wild-type control littermates. Experimental autoimmune encephalomyelitis studies in bone marrow chimeric Darc−/− and wild-type mice revealed that increased plasma levels of inflammatory chemokines in experimental autoimmune encephalomyelitis depended on the presence of erythrocyte DARC. However, fully developed experimental autoimmune encephalomyelitis required the expression of endothelial DARC. Taken together, our data show a role for erythrocyte DARC as a chemokine reservoir and that endothelial DARC contributes to the pathogenesis of experimental autoimmune encephalomyelitis by shuttling chemokines across the blood–brain barrier.
PMCID: PMC3999718  PMID: 24625696
DARC; chemokines; blood–brain barrier; multiple sclerosis; experimental autoimmune encephalomyelitis
3.  Angiopoietin 2 mediates microvascular and hemodynamic alterations in sepsis 
The Journal of Clinical Investigation  2013;123(8):3436-3445.
Septic shock is characterized by increased vascular permeability and hypotension despite increased cardiac output. Numerous vasoactive cytokines are upregulated during sepsis, including angiopoietin 2 (ANG2), which increases vascular permeability. Here we report that mice engineered to inducibly overexpress ANG2 in the endothelium developed sepsis-like hemodynamic alterations, including systemic hypotension, increased cardiac output, and dilatory cardiomyopathy. Conversely, mice with cardiomyocyte-restricted ANG2 overexpression failed to develop hemodynamic alterations. Interestingly, the hemodynamic alterations associated with endothelial-specific overexpression of ANG2 and the loss of capillary-associated pericytes were reversed by intravenous injections of adeno-associated viruses (AAVs) transducing cDNA for angiopoietin 1, a TIE2 ligand that antagonizes ANG2, or AAVs encoding PDGFB, a chemoattractant for pericytes. To confirm the role of ANG2 in sepsis, we i.p. injected LPS into C57BL/6J mice, which rapidly developed hypotension, acute pericyte loss, and increased vascular permeability. Importantly, ANG2 antibody treatment attenuated LPS-induced hemodynamic alterations and reduced the mortality rate at 36 hours from 95% to 61%. These data indicate that ANG2-mediated microvascular disintegration contributes to septic shock and that inhibition of the ANG2/TIE2 interaction during sepsis is a potential therapeutic target.
PMCID: PMC3726157  PMID: 23863629
4.  Junctional Adhesion Molecule (JAM)-C Deficient C57BL/6 Mice Develop a Severe Hydrocephalus 
PLoS ONE  2012;7(9):e45619.
The junctional adhesion molecule (JAM)-C is a widely expressed adhesion molecule regulating cell adhesion, cell polarity and inflammation. JAM-C expression and function in the central nervous system (CNS) has been poorly characterized to date. Here we show that JAM-C−/− mice backcrossed onto the C57BL/6 genetic background developed a severe hydrocephalus. An in depth immunohistochemical study revealed specific immunostaining for JAM-C in vascular endothelial cells in the CNS parenchyma, the meninges and in the choroid plexus of healthy C57BL/6 mice. Additional JAM-C immunostaining was detected on ependymal cells lining the ventricles and on choroid plexus epithelial cells. Despite the presence of hemorrhages in the brains of JAM-C−/− mice, our study demonstrates that development of the hydrocephalus was not due to a vascular function of JAM-C as endothelial re-expression of JAM-C failed to rescue the hydrocephalus phenotype of JAM-C−/− C57BL/6 mice. Evaluation of cerebrospinal fluid (CSF) circulation within the ventricular system of JAM-C−/− mice excluded occlusion of the cerebral aqueduct as the cause of hydrocephalus development but showed the acquisition of a block or reduction of CSF drainage from the lateral to the 3rd ventricle in JAM-C−/− C57BL/6 mice. Taken together, our study suggests that JAM-C−/− C57BL/6 mice model the important role for JAM-C in brain development and CSF homeostasis as recently observed in humans with a loss-of-function mutation in JAM-C.
PMCID: PMC3445510  PMID: 23029139
5.  Claudin-1 induced sealing of blood–brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis 
Acta Neuropathologica  2011;122(5):601-614.
In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood–brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have failed.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-011-0883-2) contains supplementary material, which is available to authorized users.
PMCID: PMC3207130  PMID: 21983942
Blood–brain barrier; Tight junction; Claudin-1; Experimental autoimmune encephalomyelitis; Vascular permeability
6.  VE-PTP controls blood vessel development by balancing Tie-2 activity 
The Journal of Cell Biology  2009;185(4):657-671.
Vascular endothelial protein tyrosine phosphatase (VE-PTP) is an endothelial-specific receptor-type tyrosine phosphatase that associates with Tie-2 and VE-cadherin. VE-PTP gene disruption leads to embryonic lethality, vascular remodeling defects, and enlargement of vascular structures in extraembryonic tissues. We show here that antibodies against the extracellular part of VE-PTP mimic the effects of VE-PTP gene disruption exemplified by vessel enlargement in allantois explants. These effects require the presence of the angiopoietin receptor Tie-2. Analyzing the mechanism we found that anti–VE-PTP antibodies trigger endocytosis and selectively affect Tie-2–associated, but not VE-cadherin–associated VE-PTP. Dissociation of VE-PTP triggers the activation of Tie-2, leading to enhanced endothelial cell proliferation and enlargement of vascular structures through activation of Erk1/2. Importantly, the antibody effect on vessel enlargement is also observed in newborn mice. We conclude that VE-PTP is required to balance Tie-2 activity and endothelial cell proliferation, thereby controlling blood vessel development and vessel size.
PMCID: PMC2711575  PMID: 19451274
7.  Pericyte Migration 
Diabetes  2008;57(9):2495-2502.
OBJECTIVE— The mechanism underlying pericyte loss during incipient diabetic retinopathy remains controversial. Hyperglycemia induces angiopoietin-2 (Ang-2) transcription, which modulates capillary pericyte coverage. In this study, we assessed loss of pericyte subgroups and the contribution of Ang-2 to pericyte migration.
RESEARCH DESIGN AND METHODS— Numbers of total pericytes and their subgroups were quantified in retinal digest preparations of spontaneous diabetic XLacZ mice. Pericytes were divided into subgroups according to their localization, their position relative to adjacent endothelial cells, and the expression of LacZ. The contribution of Ang-2 to pericyte migration was assessed in Ang-2 overexpressing (mOpsinhAng2) and deficient (Ang2LacZ) mice.
RESULTS— Pericyte numbers were reduced by 16% (P < 0.01) in XLacZ mice after 6 months of diabetes. Reduction of pericytes was restricted to pericytes on straight capillaries (relative reduction 27%, P < 0.05) and was predominantly observed in LacZ-positive pericytes (−20%, P < 0.01). Hyperglycemia increased the numbers of migrating pericytes (69%; P < 0.05), of which the relative increase due to diabetes was exclusively in LacZ-negative pericytes, indicating reduced adherence to the capillaries (176%; P < 0.01). Overexpression of Ang-2 in nondiabetic retinas mimicked diabetic pericyte migration of wild-type animals (78%; P < 0.01). Ang-2 deficient mice completely lacked hyperglycemia-induced increase in pericyte migration compared with wild-type littermates.
CONCLUSIONS— Diabetic pericyte loss is the result of pericyte migration, and this process is modulated by the Ang-Tie system.
PMCID: PMC2518502  PMID: 18559662
8.  VEGF-A stimulates ADAM17-dependent shedding of VEGFR2 and crosstalk between VEGFR2 and ERK signaling 
Circulation research  2008;103(9):916-918.
VEGF-A and the VEGF receptors are critical for regulating angiogenesis during development, homeostasis and in pathological conditions, such as cancer and proliferative retinopathies. Most effects of VEGF-A are mediated by the VEGFR2 and its co-receptor, Neuropilin-1 (NRP-1). Here, we show that VEGFR2 is shed from cells by the metalloprotease-disintegrin ADAM17, whereas NRP-1 is released by ADAM10. VEGF-A enhances VEGFR2 shedding by ADAM17, but not shedding of NRP-1 by ADAM10. VEGF-A activates ADAM17 via the ERK and MAP kinase pathways, thereby also triggering shedding of other ADAM17-substrates, including TNFα, TGFα, HB-EGF, and Tie-2. Interestingly, an ADAM17-selective inhibitor shortens the duration of VEGF-A-stimulated ERK phosphorylation in HUVECs, providing evidence for an ADAM17-dependent crosstalk between the VEGFR2 and ERK-signaling. Targeting the sheddases of VEGFR2 or NRP-1 might offer new opportunities to modulate VEGF-A signaling, an already established target for treatment of pathological neovascularization.
PMCID: PMC2574836  PMID: 18818406
ADAM17; Ectodomain shedding; VEGFR2; Tie-2; neuropilin

Results 1-8 (8)