ATZ11 Recognizes Not Only Z-α₁-Antitrypsin-Polymers and Complexed Forms of Non-Z-α₁-Antitrypsin but Also the von Willebrand Factor

<div><p>Aims</p><p>The ATZ11 antibody has been well established for the identification of α₁-anti-trypsin (AAT) molecule type PiZ (Z-AAT) in blood samples and liver tissue. In this study, we systematically analyzed the antibody for additional binding sites in human tissue.</p><p>Methods and Results</p><p>Ultrastructural ATZ11 binding was investigated immunoelectron microscopically in human umbilical vein endothelial cells (HUVECs) and in platelets of a healthy individual. Human embryonic kidney (HEK293) cells were transiently transfected with Von Willebrand factor (VWF) and analyzed immunocytochemically using confocal microscopy and SDS-PAGE electrophoresis followed by western blotting (WB). Platelets and serum samples of VWF-competent and VWF-deficient patients were investigated using native PAGE and SDS-PAGE electrophoresis followed by WB. The specificity of the ATZ11 reaction was tested immunohistochemically by extensive antibody-mediated blocking of AAT- and VWF-antigens.</p><p>ATZ11-positive epitopes could be detected in Weibel-Palade bodies (WPBs) of HUVECs and α-granules of platelets. ATZ11 stains pseudo-WBP containing recombinant wild-type VWF (rVWF-WT) in HEK293 cells. In SDS-PAGE electrophoresis followed by WB, anti-VWF and ATZ11 both identified rVWF-WT. However, neither rVWF-WT-multimers, human VWF-multimers, nor serum proteins of VWF-deficient patients were detected using ATZ11 by WB, whereas anti-VWF antibody (anti-VWF) detected rVWF-WT-multimers as well as human VWF-multimers. In human tissue specimens, AAT-antigen blockade using anti-AAT antibody abolished ATZ11 staining of Z-AAT in a heterozygous AAT-deficient patient, whereas VWF-antigen blockade using anti-VWF abolished ATZ11 staining of endothelial cells and megakaryocytes.</p><p>Conclusions</p><p>ATZ11 reacts with cellular bound and denatured rVWF-WT and human VWF as shown using immunocytochemistry and subsequent confocal imaging, immunoelectron microscopy, SDS-PAGE and WB, and immunohistology. These immunoreactions are independent of the binding of Z-AAT-molecules and non-Z-AAT complexes.</p></div

Confocal imaging of VWF-transfected HEK293 cells: Pseudo-Weibel-Palade-Body (pseudo-WPB) granules formed after transfection of HEK293 cells using recombinant wild-type VWF (rVWF-WT) constructs: (A, B) Pseudo-WPB granules are shown in green (anti-VWF staining).

<p>(C) The same intracellular structures are stained with ATZ11 (red). (D) Small dot-like signals of less than .25 µm were found in very few HEK293 cells stained with anti-AAT (arrow). (E) Merged images of anti-VWF and ATZ11 stains highlight the co-localization of the antibody-binding sites. At a single cell level, small dot-like positive signals were found in the ATZ11 reaction, which were not co-localized with VWF staining (arrow). (F) Merged images of anti-VWF staining and anti-AAT signals demonstrated that the dot-like anti-AAT positive signals were not associated with pseudo-WPBs (arrow). Scale bar = 10 µm.</p

Protein-electrophoretic studies on VWF-transfected HEK293 cells and human serum samples:

<p>(A) <b>SDS-PAGE electrophoresis and subsequent western blotting (WB) and visualization using anti-VWF: (lane 1) cell lysates of recombinant wild-type VWF (rVWF-WT)-transfected HEK293 cells, (lane 2) mock-transfected HEK293 cells.</b> (B) SDS-PAGE electrophoresis and subsequent WB and visualization using ATZ11: (lane 1) of cell lysates of rVWF-WT-transfected HEK293 cells and (lane 2) mock-transfected HEK293 cells. A congruent single band of 225 kDa was detected in the VWF-transfected HEK293 cells using both anti-VWF (A) and ATZ11 (B). (C) SDS-PAGE electrophoresis and subsequent WB of human serum samples of a non-Z healthy individual (lanes 1–2) and of VWF-deficient patients (lanes 3–5) stained with anti-VWF. (D) SDS-PAGE electrophoresis and subsequent WB of serum samples of a non-Z healthy individual (lanes 1–2) and of VWF-deficient patients (lanes 3–5) stained with ATZ11. (E) Native PAGE electrophoresis and subsequent WB of a recombinant VWF (lanes 1–2) and serum samples of a non-Z healthy individual (lanes 3–5) stained with the anti-VWF antibody.</p

Human liver histology.

<p><b>a) Human hepatic Sirius Red staining.</b> Liver specimen of healthy control and NCIPH were stained with Sirius red to detect collagen fibers. The staining of Sirius red was increased in NCIPH patients compared to healthy controls. <b>b) Human hepatic αSMA staining.</b> Liver specimen of healthy control and NCIPH were stained with αSMA to detect activated hepatic stellate cells. The staining of αSMA was increased in NCIPH patients compared to healthy controls. <b>c) Human hepatic CD105 staining.</b> Liver specimen of healthy control and NCIPH were stained with CD105 to detect endoglin, which is involved in the cytoskeletal organization affecting cell morphology and migration of endothelial cells. The staining of CD105 was increased in NCIPH patients compared to healthy controls. <b>d) Quantification of human hepatic stainings.</b> Sirius red, αSMA and CD105 stainings were quantified in human NCIPH liver specimen and compared to healthy controls using computerized image capture (Histoquant; 3DHistech, Budapest, Hungary). All stainings were significantly increased in NCIPH liver specimens compared to healthy controls. */**/***p<0.05/0.001/0.0001.</p

Recombinant von Willebrand Factor Expression in four Transfection Experiments in HEK293 cell lines.

<p>rVFW – recombinant von Willebrand factor.</p

Portal and systemic hemodynamic assessment and the NCIPH model.

<p><b>a) Portal pressure.</b> Portal pressure was taken every week in weekly embolized and in control rats. In single embolized rats, the PP was taken at the beginning and the end of experiment. Portal pressures are shown in mmHg. Significant differences are evaluated using paired t-Test within the same group. <b>b) Portal pressure at the end of experiments.</b> At the end of experiments, the portal pressures were measured invasively in all rats. After weekly embolization, the PP was significantly higher compared to rats after a single embolization. The portal pressures are shown in mmHg. The significant difference to single embolized rats is evaluated using the nonparametric Mann-Whitney test. <b>c) Mesenteric blood flow.</b> The mesenteric blood flow was investigated at the end of experiment. The results are shown in ml/min/100g/kg body weight. The mesenteric blood flow was increased significantly in weekly embolized rats compared to single embolized and control rats. <b>d) Mesenteric shunt volume.</b> The mesenteric shunt volume was assessed at the end of experiment. The results of weekly, single embolized and control rats are shown in ml/min/g liver. The mesenteric shunt volume was increased most in weekly embolized rats and less in single embolized rats. In control rats the mesenteric shunt volume was lowest. <b>e) Splanchnic vascular resistance.</b> At the end of experiments the splanchnic vascular resistance was assessed using the coloured microsphere technique. The splanchnic vascular resistance was significantly decreased after weekly embolization in rats. The results of are shown in mmHg/min/100g/ml. *p<0.05 / **p<0.005 / ***p<0.0008 vs. Control; #p<0.05 vs. single embolization.</p

General characteristics of the patients with NCIPH.

<p>General characteristics of the patients have been investigated in all patients and are shown in all patients. Characteristics of the parenchyma and of the liver have been evaluated by senior pathologists in specimens of the liver biopsies.</p

Comparative immunostaining of temporary artery specimens.

<p>(A) Localization of AAT in the temporal artery. Specimen stained with polyclonal anti-AAT (1∶5000) showed immunoreactivity on the endothelial surface and a gradient of presumably soluble AAT within the vessel wall. (B) ATZ11 (1∶100) showed a distinct staining of the endothelial layer. (C) The endothelial layer is distinctly stained using anti-VFW (1∶500). (D) After saturation with anti-AAT antibody (1∶10), ATZ11 labeled a thin endothelial layer. (E) Blockade with anti-VWF antibody (1∶10) abolished ATZ11 staining (1∶100) of the endothelial layer. (F) Sequential blockade with anti-AAT (1∶10) and anti-VWF (1∶10) completely abolished ATZ11 staining (1∶100).</p

Phenotypic characterization of parental strains after six weeks of CCl₄ injections.

<p>Liver fibrosis was assessed by morphometric (A) and biochemical (B) measurement of hepatic collagen (Hyp) contents. Hepatic inflammation was measured by serum ALT activities (C). Sirius red staining of hepatic collagen showed circumferential fibrosis in C57BL/6J mice (D) and pronounced fibrosis in DBA/2J mice (E), corresponding to mean F-scores of 2.0±0.1 and 3.9±0.1, respectively.</p

Chromosomal regions of pQTLs with significant genome-wide LRS values determined by single QTL scans and CIM.

<p>Abbreviations and definitions: <b>pQTL (chr):</b> chromosomal position of quantitative trait locus; <b>LRS (max):</b> likelihood ratio statistic, maximum association between genotype and phenotype variation; <b>SNP (max):</b> single nucleotide polymorphism with maximum LRS in QTL region; <b>1.5 LOD support interval (Mb):</b> chromosomal region in Megabases spanning QTL position; <b>Additive allele effect</b>: estimate of a change in the average phenotype by substitution of one parental allele by another at a given marker position; <b>(−)</b> values indicate an increase of phenotype by C57BL/6J allele, <b>(+)</b> values an increase of phenotype by DBA/2J allele; <b>Dataset:</b> dataset in which the QTL was identified; <b>Hyp</b>: hydroxyproline; CIM: composite interval mapping.</p