Endoglin expression is reduced in normal vessels but still detectable in arteriovenous malformations of patients with hereditary hemorrhagic telangiectasia type 1

Abstract

Endoglin is predominantly expressed on endothelium and is mutated in hereditary hemorrhagic telangiectasia (HHT) type 1 (HHT1). We report the analysis of endoglin in tissues of a newborn (family 2), who died of a cerebral arteriovenous malformation (CAVM), and in a lung specimen surgically resected from a 78-year-old patient (family 5), with a pulmonary AVM (PAVM). The clinically affected father of the newborn revealed a novel mutation that was absent in his parents and was identified as a duplication of exons 3 to 8, by quantitative multiplex polymerase chain reaction. The corresponding mutant protein (116-kd monomer) and the missense mutant protein (80-kd monomer) present in family 5 were detected only as transient intracellular species and were unreactive by Western blot analysis and immunostaining. Normal endoglin (90-kd monomer) was reduced by 50% on peripheral blood-activated monocytes of the HHT1 patients. When analyzed by immunostaining and densitometry, presumed normal blood vessels of the newborn lung and brain and vessels adjacent to the adult PAVM showed a 50% reduction in the endoglin/PECAM-1 ratio. A similar ratio was observed in the CAVM and PAVM, suggesting that all blood vessels of HHT1 patients express reduced endoglin in situ and that AVMs are not attributed to a focal loss of endoglin.

Figure 1.

A: Pedigrees of HHT Family 2. Patients H283 and H262 were both unaffected and are the biological parents of patient H3, who presented with telangiectases, nosebleeds, and PAVM. His wife, patient H4, was unaffected and had two miscarriages at 7 weeks of gestation. Their first child, patient H9, died at 7 days of a cerebral hemorrhage followed by heart failure caused by rupture of one of the two CAVMs present. Their second child, patient H11, was born normally and was confirmed unaffected. B: Pedigree of family 5 with a history of HHT1 for four generations. Patient H12 had nosebleeds, telangiectases, and a PAVM. Patient H150 presented with a PAVM and telangiectases, whereas patient H170 had nosebleeds and telangiectases. Black symbols, HHT1-affected individuals; gray symbols, HHT1-suspected individuals; white symbols, unaffected individuals.

Figure 2.

Analysis of peripheral blood-activated monocytes shows reduced levels of normal endoglin and presence of a mutant protein in patient H3 but not in his parents. A: Cells from the clinically affected patient H3 (family 2), and a normal control (C) were labeled with ³⁵S-methionine, solubilized in Triton X-100, and immunoprecipitated with mAb P3D1 or P4A4 to endoglin. Equivalent amounts of labeled proteins were fractionated on 4 to 12% gradient SDS-PAGE, under reducing (lanes 1–4) and nonreducing (lanes 9–12) conditions. Fully processed endoglin (E) and partially glycosylated precursor (P) were observed in patient H3 at reduced levels compared with control, and a mutant protein (M) is noted. Cells from individuals H262 and H283, mother and father of patient H3, were also analyzed in the same manner (lanes 5–8 and 13–16), normal E and P forms of endoglin were seen at normal levels, and no mutant protein was observed. B: Activated monocytes from patient H3 were lysed in Triton X-100, and various amounts of proteins were fractionated on 8% sodium dodecyl sulfate-polyacrylamide gels, under nonreducing conditions (without dithiothreitol). Gels were transferred and analyzed by Western blot analysis, using mAb SN6h to endoglin. The control (C) was an extract from HUVEC. Only the normal dimers of endoglin (E) are detected by Western blot analysis. Molecular mass markers in kilodaltons are indicated.

Figure 3.

QMPCR fragment analysis reveals a duplication of exons 3 to 8 in patient H3 of family 2. DNA samples were analyzed by QMPCR using five different reactions (R1–R5), optimized so that peak area is proportional to copy number for each exon. Illustrated here is DNA from patient H3 and control (c) for each of the five reactions. The exon number is indicated under the peak, and “st” refers to internal size standards. The ratio of each exon is expressed relative to c4 fragment, an internal copy number standard. In each experiment, the peak area was estimated for H3 relative to the mean ratio observed for 12 control samples. The ratios of H3 relative to these controls were 1.0 for exons 1 and 2 and ranged from 1.3 to 1.6 for exons 3 to 8 and from 0.84 to 1.0 for exons 9a to 14. Accordingly, peak surface area is greater for exons 3 to 8 in patient H3.

Figure 4.

Normal and mutant copies of endoglin DNA are present in the PAVM of patient H12. DNA extracted from the PAVM of patient H12, (family 5) was sequenced for exon 4. At position 447, G of the normal allele and C of the mutant allele are both present. This missense mutation was absent in the control DNA sample.

Figure 5.

Vessels of lung and central nervous system from the HHT1 newborn express less endoglin than control, compared with PECAM-1. Formalin-fixed, paraffin-embedded lung (A) and spinal cord (B) sections from control and patient H9 were stained with mAbs SN6h to endoglin and JC70A to PECAM-1, followed by an alkaline phosphatase-conjugated antibody. A: Endoglin and PECAM-1 staining on an artery (A, control; a, patient H9), vein (B; b) and capillaries (C; c) of lung tissues are illustrated for control and patient H9. B: Sections of dural vessels of spinal cord are shown. Two arteries (D; d and E; e), two veins (F; f and G; g) are represented for age-matched control and patient H9. PECAM-1 was also detected on platelets in vessels with blood infiltrates. Original magnification, ×194.

Figure 6.

Endoglin is expressed on the large abnormal feeding vessel of the CAVM in patient H9. Sections from the right middle cerebral artery feeding the CAVM that ruptured in patient H9 were stained with Van Giesen elastin stain. The adventitia (a), media (m), and intima (i) of the artery are marked. The lumen of this vessel is filled with blood (*). Sections stained for endoglin and PECAM-1 by alkaline phosphatase immunostaining are shown at higher magnification. The arrow outlines endoglin and PECAM-1 staining on the endothelium. Original magnifications: ×31 and ×78.

Figure 7.

Endoglin is detected on the endothelium and the adventitia of the aneurysmic dilatation of the CAVM. A: Sections of the aneurysm of patient H9, downstream from the right middle cerebral feeding artery, were stained for α-smooth muscle cell actin. ★, hemorrhage in the ruptured vessel. Original magnification, ×22. B: An area showing intense remodeling of the vessel wall in A (solid arrow) is shown at higher magnification (original magnification, ×194). The subsequent section stained for endoglin revealed expression on mesenchymal cells (solid arrowhead). C: The venous pouch shown in A (open arrow) was stained for PECAM-1, endoglin, and α-smooth muscle cell actin. The dispersed endothelium is indicated by the open arrowhead. Original magnification, ×194.

Figure 8.

Endoglin is still detectable on the endothelial cells of the PAVM. Sections from the resected lung right middle lobe of patient H12, containing a PAVM, were stained with Van Giesen elastin stain. The lung parenchyma (p) and the three layers of the abnormally dilated vessel—adventitia (a), media (m), and intima (i)—are shown. Subsequent sections of the PAVM were stained for endoglin and PECAM-1 by alkaline phosphatase immunostaining. The arrow shows the staining of the endothelial cells with both markers. Original magnifications: ×30 and ×165.