Soluble Endoglin Stimulates Inflammatory and Angiogenic Responses in Microglia That Are Associated with Endothelial Dysfunction

Abstract

Increased soluble endoglin (sENG) has been observed in human brain arteriovenous malformations (bAVMs). In addition, the overexpression of sENG in concurrence with vascular endothelial growth factor (VEGF)-A has been shown to induce dysplastic vessel formation in mouse brains. However, the underlying mechanism of sENG-induced vascular malformations is not clear. The evidence suggests the role of sENG as a pro-inflammatory modulator, and increased microglial accumulation and inflammation have been observed in bAVMs. Therefore, we hypothesized that microglia mediate sENG-induced inflammation and endothelial cell (EC) dysfunction in bAVMs. In this study, we confirmed that the presence of sENG along with VEGF-A overexpression induced dysplastic vessel formation. Remarkably, we observed increased microglial activation around dysplastic vessels with the expression of NLRP3, an inflammasome marker. We found that sENG increased the gene expression of VEGF-A, pro-inflammatory cytokines/inflammasome mediators (TNF-α, IL-6, NLRP3, ASC, Caspase-1, and IL-1β), and proteolytic enzyme (MMP-9) in BV2 microglia. The conditioned media from sENG-treated BV2 (BV2-sENG-CM) significantly increased levels of angiogenic factors (Notch-1 and TGFβ) and pERK1/2 in ECs but it decreased the level of IL-17RD, an anti-angiogenic mediator. Finally, the BV2-sENG-CM significantly increased EC migration and tube formation. Together, our study demonstrates that sENG provokes microglia to express angiogenic/inflammatory molecules which may be involved in EC dysfunction. Our study corroborates the contribution of microglia to the pathology of sENG-associated vascular malformations.

Keywords: angiogenesis; brain arteriovenous malformation (bAVM); endothelial cells (ECs); inflammation; microglia; soluble endoglin (sENG).

Figure 1

Soluble ENG/VEGF-A induces the formation of dysplastic vessels in the mouse brain. (A) Experimental scheme for sENG and AAV1-VEGF-A injection. The mice were stereotaxically injected with AAV1-VEGF-A (or AAV1-LacZ) into the intra-striatum and administered recombinant sENG (or PBS) subcutaneously (s.c.) every day for two weeks beginning at six weeks after the AAV1-VEGF-A injection. At eight weeks after the AAV1-VEGF-A injection, the mice were sacrificed. (B) Representative images of latex cast-clarified brains in mice injected with AAV1-VEGF-A + PBS, AAV1-LacZ + sENG, and AAV1-VEGF-A + sENG. Coronal sections showed the enlarged abnormal vasculature (inset, arrows) in the AAV1-VEGF-A injected site (ipsilateral) compared to the non-injected site (contralateral) in mice injected with AAV1-VEGF-A and sENG. Scale bars: 2 mm (whole brain image) and 100 μm (inset). (C) The quantification of vessel volume in mouse brains. AAV-VEGF-A (or VEGF-A), mice injected with vehicle (PBS) and AAV1-VEGF-A, sENG, mice injected with sENG and AAV1-LacZ, AAV-VEGF-A (or VEGF-A) + sENG, mice injected with AAV1-VEGF-A and sENG. n = 3–4, * p < 0.05, ** p < 0.01, One-way ANOVA. (D) Image of CD31 immunostained brain from mice injected with sENG and AAV1-VEGF-A and the quantification of CD31 intensity. Scale bar: 50 µm, n = 3, data are presented as mean ± SEM, ** p < 0.01, Student’s t-test. Con, contralateral, Ipsi, ipsilateral.

Figure 2

Soluble ENG/VEGF-A induced microglial activation with expression of inflammasome marker around dysplastic capillaries in mouse brain. (A) Immunostaining of mouse brain with CD31, Iba-1, and NLRP3 antibodies. Iba-1+ activated microglia are distributed around sENG/VEGF-induced dysplastic vessels in the mouse brain. Iba1+/NLRP3+ cells (arrows) indicate the inflammasome activation within the activated microglia. Scale bar: 50 μm. (B,C) The quantification of intensity of Iba-1 (B) and NLRP3 (C) fluorescence normalized by CD31, respectively. n = 3; data are presented as mean ± SEM; * p < 0.05, ** p < 0.01 vs. contralateral; Student’s t-test.

Figure 3

Soluble ENG induced gene expression of angiogenic and inflammatory mediators in microglia. (A,B) Change of gene expression levels of angiogenic mediator and inflammatory cytokines (A) and inflammasome markers (B) in BV2 microglia by sENG treatment. BV2 cells were stimulated with 0.5 or 1 μg/mL of sENG for 24 h. Each mRNA level was normalized with GAPDH. n = 3; data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001; Student’s t-test.

Figure 4

Soluble ENG-stimulated microglia regulate the expression of angiogenic mediators in endothelial cells. (A) Gene expression of angiogenic mediators in ECs treated with sENG (0.5 or 1 μg/mL)-treated microglia conditioned medium (BV2-sENG-CM). Mouse brain vascular endothelial cells (ECs) were incubated in a medium containing BV2-CM, BV2-sENG-CM, PBS, or sENG for 24 h. Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001; one-way ANOVA. (B) BV2-sENG-CM or sENG induced p-ERK1/2 expression in ECs. n = 3; data are presented as mean ± SEM; * p < 0.05, ** p < 0.01; one-way ANOVA. (C) BV2-sENG-CM decreased IL-17RD expression in ECs. n = 3; data are presented as mean ± SEM; * p < 0.05; one-way ANOVA.

Figure 5

Soluble ENG-stimulated microglia induces a hyper-angiogenic phenotype of endothelial cells. (A) BV2-sENG-CM induced EC migration in a scratch-wound assay. The rate of EC migration in each treatment was quantified by measuring the distance between the edges of scratched area at 10 h compared to 0 h after the scratch. n = 3; data are presented as mean ± SEM; * p < 0.05, ** p < 0.01; scale bar: 400 μm. (B) BV2-sENG-CM increased endothelial tube formation. The tube length, branch count, and loop count were automatically and blindly measured using Ibidi software. Three independent experiments were performed, and representative images are shown. n = 3; data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, Student’s t-test; scale bar: 500 μm.

Figure 6

The role of soluble ENG in endothelial dysfunction and vascular malformation. (A) Endoglin (ENG) mediates the TGFβ signaling pathway in endothelial cells (ECs). The mutation of Eng (haploinsufficiency) reduces TGFβ signaling. MMP-14 cleaves to the extracellular domain of ENG in ECs, producing a soluble form of ENG (sENG). The sENG acts as a decoy receptor for the TGFβ, resulting in impaired TGFβ signaling. Therefore, both mutant ENG and sENG lead to the disruption of TGFβ signaling and subsequently mediate EC dysfunction and vascular malformation. TM, transmembrane. (B) Soluble ENG stimulates microglia (by an unknown mechanism) and leads to microglial activation with up-regulation of inflammatory/angiogenic mediators. The microglia-derived inflammatory/angiogenic mediators possibly induce hyper-angiogenic signaling on ECs and cause EC dysfunction. Taken together, the sENG-induced disruption of TGFβ signaling in ECs and the activation of microglia may work together to drive vascular malformations.