Soluble endoglin modulates aberrant cerebral vascular remodeling

Abstract

Objective: Brain arteriovenous malformations (AVMs) are an important cause of neurological morbidity in young adults. The pathophysiology of these lesions is poorly understood. A soluble form of endoglin (sEng) has been shown to cause endothelial dysfunction and induce preeclampsia. We tested if sEng would be elevated in brain AVM tissues relative to epilepsy brain tissues, and also investigated whether sEng overexpression via gene transfer in the mouse brain would induce vascular dysplasia and associated changes in downstream signaling pathways.

Methods: Expression levels of sEng in surgical specimens were determined by Western blot assay and enzyme-linked immunosorbent assay. Vascular dysplasia, levels of matrix metalloproteinase (MMP), and oxidative stress were determined by immunohistochemistry and gelatin zymography.

Results: Brain AVMs (n = 33) had higher mean sEng levels (245 +/- 175 vs 100 +/- 60, % of control, p = 0.04) compared with controls (n = 8), as determined by Western blot. In contrast, membrane-bound Eng was not significantly different (108 +/- 79 vs 100 +/- 63, % of control, p = 0.95). sEng gene transduction in the mouse brain induced abnormal vascular structures. It also increased MMP activity by 490 +/- 30% (MMP-9) and 220 +/- 30% (MMP-2), and oxidants by 260 +/- 20% (4-hydroxy-2-nonenal) at 2 weeks after injection, suggesting that MMPs and oxidative radicals may mediate sEng-induced pathological vascular remodeling.

Interpretation: The results suggest that elevated sEng may play a role in the generation of sporadic brain AVMs. Our findings may provide new targets for therapeutic intervention for patients with brain AVMs. Ann Neurol 2009;66:19-27.

Figure 1. Western blot analysis of endoglin in brain AVM and control tissues

A. Representative Western blot shows the membrane form of Endoglin (mEng) as a 90kDa monomer in all samples, while the 65kDa fragment corresponding to the soluble form (sEng) is seen only in AVMs. β-actin blot was used to control for protein loading and CD31 for measurement of endothelial cell mass. Lanes 1 and 2: AVMs; Lane 3: middle cerebral artery from autopsy; Lanes 4 and 5: epilepsy cortex samples. B. Quantification of sEng level. The sEng, actin and CD31 bands were quantified by densitometry and sEng levels normalized to those of β-actin and CD31. Brain AVMs (n=33) had higher sEng levels (0.98 ± 0.7, vs 0.4 ± 0.24, P< 0.05) compared with controls (n=8). We normalized sENG to CD31 expression to control for artifactual estimates of increased sENG caused by higher vascular density in AVM tissues. C. Quantification of mEng level. The bands corresponding to the plasma membrane form of Eng were quantified and expressed relative to β-actin and CD31. The values were not significantly different (2.6 ± 1.9, in brain AVMs vs 2.4 ± 1.5 in control samples, P=0.95).

Figure 2. Measurement of Eng protein levels

A. ELISA assay showing that brain AVM tissue (n=20) had higher average sEng protein levels (4.0 ± 3.6, vs 0.9 ± 0.5, ng/mg, P< 0.05) compared to control tissue (n=14). B. Western blotting confirmed absence of membrane Eng (90kDa) and presence of sEng (65kDa) in tissue lysates used for ELISA. C Immmunohistochemical staining indicates that Eng was present in endothelial cells (arrows) and adventitia (arrowheads) in both brain AVMs (a, b) and controls (c, d), and no obvious differences in staining intensity were noted. Images b and d are higher magnification of the boxed areas from images a and c, respectively. Size bar= 50µm.

Figure 3. Expression of human sEng in the adult mouse brain after AdsEng transduction

A Immunostaining of human sEng (brown) is shown for Ad-sEng-transduced mouse brain; Ad-lacZ-transduced mouse brain is used for control. Staining was performed 1 week after viral injection. Upper Bar = 1000 µm, lower bar=100µm. B Representative Western blots showing human sEng expression (65kDa) in the mouse brain after Ad-sEng transduction. Samples were collected at 1 and 2 weeks after adenoviral injection. Lanes 1 and 2: lacZ, 1 week; Lanes 3 and 4: lacZ, 2 weeks; Lanes 5 and 6: sEng, 1 week; Lanes 7 and 8: sEng, 2 weeks.

Figure 4. sEng induces dysplastic capillaries in the mouse brain

A Photomicrographs show lectin-stained blood vessels in brains injected with Ad-lacZ (a), or Ad-sEng (b, c, and d) following AAV-VEGF transduction. Many dysplastic capillaries were detected in Ad-sEng-injected brains (arrow pointing to enlarged giant capillaries and arrowhead pointing to morphologically changed capillaries). Bar = 100 µm. B. Bar graph shows the dysplasia index in the treated groups of mice. Data are mean ± SD. *P<0.01, Ad-sEng-transduced mice vs. Ad-lacZ-transduced mice.

Figure 5. Effects of sEng overexpression on MMP-9 and MMP-2 activity

A. Representative photograph of a zymogram gel. St: MMP standards. Lanes 1 and 2: lacZ -treated mice, 2 weeks; Lanes 3 and 4: sEng -treated mice, 2 weeks. B. Bar graph demonstrates densitometric analysis of the band mean intensity of MMP-9 activity. C. Quantitation of MMP-2 activity. Data are mean ± SD, n= 6, * P<0.05, sEng vs. lacZ

Figure 6. sEng overexpression induces production of HNE

A. Photograph represents HNE protein adduct expression (≈70 kDa) by Western blot analysis. B. Bar graph shows densitometric analysis of the major HNE band mean intensity. Lanes 1 and 2: lacZ treated, after 2 weeks; Lanes 3 and 4: sEng -treated, after 2weeks. * P<0.05, sEng vs. lacZ