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. 2012 Jan 19;119(3):884-93.
doi: 10.1182/blood-2011-03-344671. Epub 2011 Nov 21.

A novel pathway for human endothelial cell activation by antiphospholipid/anti-β2 glycoprotein I antibodies

Kristi L Allen  1 , Fabio V Fonseca, Venkaiah Betapudi, Belinda Willard, Jainwei Zhang, Keith R McCrae
Affiliations

A novel pathway for human endothelial cell activation by antiphospholipid/anti-β2 glycoprotein I antibodies

Kristi L Allen et al. Blood. .

Abstract

Antiphospholipid Abs (APLAs) are associated with thrombosis and recurrent fetal loss. These Abs are primarily directed against phospholipid-binding proteins, particularly β(2)GPI, and activate endothelial cells (ECs) in a β(2)GPI-dependent manner after binding of β(2)GPI to EC annexin A2. Because annexin A2 is not a transmembrane protein, the mechanisms of APLA/anti-β(2)GPI Ab-mediated EC activation are uncertain, although a role for a TLR4/myeloid differentiation factor 88-dependent pathway leading to activation of NF-κB has been proposed. In the present study, we confirm a critical role for TLR4 in anti-β(2)GPI Ab-mediated EC activation and demonstrate that signaling through TLR4 is mediated through the assembly of a multiprotein signaling complex on the EC surface that includes annexin A2, TLR4, calreticulin, and nucleolin. An essential role for each of these proteins in cell activation is suggested by the fact that inhibiting the expression of each using specific siRNAs blocked EC activation mediated by APLAs/anti-β(2)GPI Abs. These results provide new evidence for novel protein-protein interactions on ECs that may contribute to EC activation and the pathogenesis of APLA/anti-β(2)GPI-associated thrombosis and suggest potential new targets for therapeutic intervention in antiphospholipid syndrome.

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Figures

Figure 1
Figure 1
APLA/anti-β2GPI–mediated EC activation is mediated by TLR4. HUVECs were incubated in medium alone, treated with control RNA of random sequence but identical nucleotide composition as specific siRNA, or with specific siRNA against TLR2, TLR4, or apoER2. Twenty-four hours later, cells were replated in 6- or 96-well plates, incubated for an additional 24 hours, then harvested and analyzed for expression of the targeted protein or assessed for cell-surface E-selectin expression in response to β2GPI and anti-β2GPI Abs or TNFα. (A) Cells treated with siRNA to TLR2 or TLR4. (B) Cells treated with siRNA to apoER2. Error bars represent the mean ± SD of triplicate points. P = .0016 by ANOVA for cells treated with TLR4 siRNA versus control or TLR2 siRNA. P = .48 for cells treated with apoER2 siRNA versus control RNA. The results shown are from 1 representative experiment of 3.
Figure 2
Figure 2
Detection of annexin A2 and TLR4-associated proteins by immunoprecipitation. ECs were either untreated or incubated with β2GPI and anti-β2GPI Abs for 5 hours. Detergent extracts were prepared and analyzed by 4%-12% SDS-PAGE before (A) or after immunoprecipitation using Abs against nucleolin (B), annexin A2 (C), or TLR4 (D). Whole extracts and each of the immunoprecipitates were then immunoblotted using Abs against annexin A2, nucleolin, calreticulin, or TLR4. Extracts were also immunoprecipitated using control IgG, which did not precipitate any detectable protein. The results shown are from 1 representative experiment of 3.
Figure 3
Figure 3
Annexin A2, TLR4, calreticulin, and nucleolin are necessary for activation of ECs by β2GPI and anti-β2GPI Abs. ECs were either not pretreated or were pretreated with control RNA of random sequence but identical composition as specific siRNAs or with siRNAs specific for annexin A2, TLR4, calreticulin, or nucleolin. Twenty-four hours later, cells were replated in 6- or 96-well plates, incubated for an additional 24 hours, and then harvested and analyzed for protein expression (A) or assessed for cell-surface E-selectin expression in response to β2GPI and either control IgG or anti-β2GPI Abs (B). Error bars represent the means ± SD of quadruplicate points. Decreases in E-selectin expression in cells pretreated with control versus specific siRNA before incubation with β2GPI and anti-β2GPI Abs were highly significant (P < .0001 by ANOVA). The results shown are from 1 representative experiment of 3.
Figure 4
Figure 4
Annexin A2, TLR4, calreticulin, and nucleolin promote EC activation in response to β2GPI and anti-β2GPI Abs through regulation of mRNA levels. ECs were pretreated with either control RNA of random sequence but identical composition as specific siRNA or with specific siRNA against annexin A2, TLR4, calreticulin, or nucleolin. Twenty-four hours later, cells were replated in 6-well microplates, incubated for an additional 24 hours, and then either harvested and analyzed for expression of the targeted protein (not shown) or incubated with β2GPI and anti-β2GPI Abs or TNFα. The content of specific mRNAs was then analyzed by qPCR. Graphs depict the levels of E-selectin (A), ICAM-1 (B), VCAM-1 (C), or TF (D) mRNA. In all cases, specific siRNAs significantly reduced the levels of mRNA for each of the cell-adhesion molecules in response to β2GPI and anti-β2GPI Abs, but not in response to TNFα. Error bars represent the means ± SD of quadruplicate points. The results shown are from 1 representative experiment of 3.
Figure 5
Figure 5
Activation of the TLR4/MyD88/NF-κB signaling pathway is dependent on assembly of the multiprotein signaling complex. (A) ECs were plated in 96-well plates and either not pretreated or pretreated with a cell-permeable peptide that contains a sequence from the MyD88/TIR homodimerization domain that blocks MyD88-dependent TLR4 signaling. Cells were then exposed to LPS, β2GPI and anti-β2GPI Abs, or TNFα. The peptide inhibited EC activation in response to LPS and anti-β2GPI Abs, but not TNFα, demonstrating the dependence of activation on a TLR4/MyD88 pathway. Error bars represent the means ± SD of triplicate points. *P < .0001 for cells treated with β2GPI/anti-β2GPI Abs alone versus β2GPI/anti-β2GPI in the presence of the homodimerization domain peptide. (B) ECs were pretreated with control RNA of random sequence but identical composition as specific siRNA (lanes 1 and 2), TLR4 siRNA (lane 3), annexin A2 siRNA (lane 4), calreticulin siRNA (lane 5), nucleolin siRNA (lane 6), or all 4 siRNAs simultaneously (lane 7). Cells were then incubated with β2GPI and anti-β2GPI Abs, and cell lysates prepared and assessed for phosphorylated NF-κB p65 (S536) and total NF-κB content by immunoblotting. (C). ECs were transfected with a construct containing an NF-κB promoter sequence linked to a luciferase reporter gene. Cells were then treated with control RNA or siRNA directed against annexin A2, TLR4, nucleolin, or calreticulin. After incubation with β2GPI and anti-β2GPI Abs, cell lysates were prepared and luciferase activity determined. Pretreatment of cells with each of the siRNAs caused significant reductions in luciferase activity compared with cells pretreated with control RNA (P < .0001 by ANOVA). Error bars represent the means ± SD of triplicate points. The results shown are from 1 representative experiment of 3.
Figure 6
Figure 6
Activation of ECs by anti-β2GPI Abs induces increased expression of components of the multiprotein signaling complex and accessory proteins. (A) ECs were incubated with β2GPI and anti-β2GPI Abs or TNFα to induce activation. Total RNA was then isolated, reverse-transcribed, and analyzed for content of S100A10 and annexin A2 mRNA using qPCR. Significant increases in both mRNA species occurred in response to β2GPI and anti-β2GPI Abs, but not TNFα. (B) ECs were activated as described in panel A, and RNA isolated, reverse transcribed, and analyzed for content of MyD88, MD2, TLR4, calreticulin, and nucleolin mRNA using qPCR. Significant increases in mRNA species encoding MyD88 and MD2, with lesser increases in mRNA encoding TLR4, calreticulin, and nucleolin, were observed. These exceeded those observed in response to TNFα. Error bars represent the means ± SD of triplicate points.
Figure 7
Figure 7
Staining of human and murine tissues for nucleolin and calreticulin. Formalin-fixed, paraffin-embedded tissue was stained with Abs to nucleolin (A,C) calreticulin (E,G), or equal concentrations of control IgG (B,D,F,H). Strong endothelial staining for nucleolin can be observed in human tonsil (A) and mouse kidney (C). Endothelial staining for calreticulin can be observed in mouse kidney (E) and brain (G). Photomicrographs were taken at 63×.
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