BAMBI Is Expressed in Endothelial Cells and Is Regulated by Lysosomal/Autolysosomal Degradation

BACKGROUND: BAMBI (BMP and Activin Membrane Bound Inhibitor) is considered to influence TGFβ and Wnt signaling, and thereby fibrosis. Surprisingly data on cell type-specific expression of BAMBI are not available. We therefore examined the localization, gene regulation, and protein turnover of BAMBI in kidneys. METHODOLOGY/PRINCIPAL FINDINGS: By immunofluorescence microscopy and by mRNA expression, BAMBI is restricted to endothelial cells of the glomerular and some peritubular capillaries and of arteries and veins in both murine and human kidneys. TGFβ upregulated mRNA of BAMBI in murine glomerular endothelial cells (mGEC). LPS did not downregulate mRNA for BAMBI in mGEC or in HUVECs. BAMBI mRNA had a half-life of only 60 minutes and was stabilized by cycloheximide, indicating post-transcriptional regulation due to AU-rich elements, which we identified in the 3' untranslated sequence of both the human and murine BAMBI gene. BAMBI protein turnover was studied in HUVECs with BAMBI overexpression using a lentiviral system. Serum starvation as an inducer of autophagy caused marked BAMBI degradation, which could be totally prevented by inhibition of lysosomal and autolysosomal degradation with bafilomycin, and partially by inhibition of autophagy with 3-methyladenine, but not by proteasomal inhibitors. Rapamycin activates autophagy by inhibiting TOR, and resulted in BAMBI protein degradation. Both serum starvation and rapamycin increased the conversion of the autophagy marker LC3 from LC3-I to LC3-II and also enhanced co-staining for BAMBI and LC3 in autolysosomal vesicles. CONCLUSIONS/SIGNIFICANCE: 1. BAMBI localizes to endothelial cells in the kidney and to HUVECs. 2. BAMBI mRNA is regulated by post-transcriptional mechanisms. 3. BAMBI protein is regulated by lysosomal and autolysosomal degradation. The endothelial localization and the quick turnover of BAMBI may indicate novel, yet to be defined functions of this modulator for TGFβ and Wnt protein actions in the renal vascular endothelium in health and disease

BAMBI Regulates Angiogenesis and Endothelial Homeostasis through Modulation of Alternative TGFβ Signaling

BACKGROUND: BAMBI is a type I TGFβ receptor antagonist, whose in vivo function remains unclear, as BAMBI(-/-) mice lack an obvious phenotype. METHODOLOGY/PRINCIPAL FINDINGS: Identifying BAMBI's functions requires identification of cell-specific expression of BAMBI. By immunohistology we found BAMBI expression restricted to endothelial cells and by electron microscopy BAMBI(-/-) mice showed prominent and swollen endothelial cells in myocardial and glomerular capillaries. In endothelial cells over-expression of BAMBI reduced, whereas knock-down enhanced capillary growth and migration in response to TGFβ. In vivo angiogenesis was enhanced in matrigel implants and in glomerular hypertrophy after unilateral nephrectomy in BAMBI(-/-) compared to BAMBI(+/+) mice consistent with an endothelial phenotype for BAMBI(-/-) mice. BAMBI's mechanism of action in endothelial cells was examined by canonical and alternative TGFβ signaling in HUVEC with over-expression or knock-down of BAMBI. BAMBI knockdown enhanced basal and TGFβ stimulated SMAD1/5 and ERK1/2 phosphorylation, while over-expression prevented both. CONCLUSIONS/SIGNIFICANCE: Thus we provide a first description of a vascular phenotype for BAMBI(-/-) mice, and provide in vitro and in vivo evidence that BAMBI contributes to endothelial and vascular homeostasis. Further, we demonstrate that in endothelial cells BAMBI interferes with alternative TGFβ signaling, most likely through the ALK 1 receptor, which may explain the phenotype observed in BAMBI(-/-) mice. This newly described role for BAMBI in regulating endothelial function has potential implications for understanding and treating vascular disease and tumor neo-angiogenesis

Accelerated Reendothelialization, Increased Neovascularization and Erythrocyte Extravasation after Arterial Injury in BAMBI^−/− Mice

<div><p>Background</p><p>Intimal injury rapidly activates TGFβ and enhances vascular repair by the growth of endothelial (EC) and vascular smooth muscle cells (VSMC). The response to the TGFβ family of growth factors can be modified by BAMBI (BMP, Activin, Membrane Bound Inhibitor) acting as a non-signaling, competitive antagonist of TGFβ type I receptors such as ALK 1 and 5. In vivo the effect of BAMBI will depend on its cell-specific expression and of that of the ALK type receptors. We recently reported EC restricted BAMBI expression and genetic elimination of BAMBI resulting in an in vitro and in vivo phenotype characterized by endothelial activation and proliferation involving alternative pathway activation by TGFβ through ALK 1.</p> <p>Methodology/Principal Findings</p><p>To test the hypothesis that BAMBI modulates arterial response to injury via its effects on endothelial repair and arterial wall neovascularization we used a model of femoral arterial denudation injury in wild type (WT) and BAMBI^−/− mice. Arterial response was evaluated at 2 and 4 weeks after luminal endothelial denudation of femoral arteries. The BAMBI^−/− genotype mice showed accelerated luminal endothelial repair at 2 weeks and a highly unusual increase in arterial wall neovascularization compared to WT mice. The exuberant intimal and medial neovessel formation with BAMBI^−/− genotype was also associated with significant red blood cell extravasation. The bleeding into the neointima at 2 weeks transiently increased it’s area in the BAMBI^−/−genotype despite the faster luminal endothelial repair in this group. Vascular smooth muscle cells were decreased at 2 weeks in BAMBI^−/− mice, but comparable to wild type at 4 weeks.</p> <p>Conclusions/Significance</p><p>The absence of BAMBI results in a highly unusual surge in arterial wall neovascularization that surprisingly mimiks features of intra-plaque hemorrhage of advanced atheroma in a mechanical injury model. This suggests important effects of BAMBI on arterial EC homeostasis that need to be further studied in a model of inflammatory atherosclerosis.</p> </div

BAMBI deficiency results in neo-intimal neovasculariztion with proliferating endothelial cells.

<p>Proliferation of endothelial cells was evaluated by double staining for the proliferation marker Ki67 (green) and the endothelial cell marker vWF (red); co-localization of both signals showing as yellow (overlay) The lamina elastica interna shows typical green autofluorescence. At two weeks double positive cells (white arrows) were restricted to the luminal surface in the BAMBI^−/− mice, whereas at 4 weeks double positive cells also appeared deep in the neointima in a pattern consistent with ongoing neo-vessel formation (original magnification ×1000).</p

Smooth muscle cells in neointima and media from BAMBI^+/+ and BAMBI ^−/− mice.

<p>Staining for alphaSMA in the femoral arteries from BAMBI^+/+ (A) and BAMBI^−/− (B) mice 2 and 4 weeks after injury. (Original magnification ×400 and ×1000; bar = 50 µm; Ni: neointima, Me: media and Ad: adventitia). (C) and (D) alphaSMA positive areas in media and neointima of the femoral arteries. Data are mean ± SEM, n = 5–9, *P<0.05 compared to respective wild type.</p

BAMBI deficiency accelerates reendothelialization and increased neo-intimal neovascularization and accumulation of erythrocytes.

<p>(<b>A</b>) Endothelial cells of femoral arteries were stained by isolectin B4 in BAMBI^+/+ and BAMBI^−/− mice 2 and 4 weeks after intimal denudation. (Ni: neointima, Me: media and Ad: adventitia. Black arrows indicate lectin positive luminal endothelial cells and intimal microvessels, original magnification ×400 upper panel and ×1000 lower panel; bar = 50 µm). (<b>B</b>) Reendothelialization was also determined by staining with von Willebrand factor (black arrows) magnification ×1000.(<b>C</b>) Fibrin and erythrocytes were visualized by Carstairs’ staining of femoral arteries at the same time point (Original magnification ×400 upper panel and ×1000 lower panel; bar = 50 µm; black arrows indicate RBC infiltration). (<b>D</b>) Quantification of the reendothelialization of the femoral arteries. (<b>E</b>) Microvascular density in the different layers of the femoral arteries 4 weeks after injury. (<b>F</b>) Erythrocytes accumulation in neointima 2 and 4 weeks after injury. Data are mean ± SEM, n = 5–9, *<i>P</i><0.05 compared to respective wild type.</p

Deficiency of BAMBI increases early neo-intimal hyperplasia after intima denudation.

<p>Representative pictures of femoral arteries from BAMBI^+/+ (<b>A</b>) and BAMBI^−/− (<b>B</b>) mice at 2 and 4 weeks after injury. (Hematoxylin and eosin stains. Original magnification ×400 and ×1000; bar = 50 µm; Ni: neointima, Me: media and Ad: adventitia). (<b>C</b>). Morphometric analysis of the femoral arteries. Data are mean ± SEM; n = 5–9; *<i>P</i><0.05 compared to respective wild type.</p

Targeting the Alternative Complement Pathway With Iptacopan to Treat IgA Nephropathy: Design and Rationale of the APPLAUSE-IgAN Study

Introduction: Targeting the alternative complement pathway (AP) is an attractive therapeutic strategy because of its role in immunoglobulin A nephropathy (IgAN) pathophysiology. Iptacopan (LNP023), a proximal complement inhibitor that specifically binds to factor B and inhibits the AP, reduced proteinuria and attenuated AP activation in a Phase 2 study of patients with IgAN, thereby supporting the rationale for its evaluation in a Phase 3 study. Methods: APPLAUSE-IgAN (NCT04578834) is a multicenter, randomized, double-blind, placebo-controlled, parallel-group, Phase 3 study enrolling approximately 450 adult patients (aged ≥18 years) with biopsy-confirmed primary IgAN at high risk of progression to kidney failure despite optimal supportive treatment. Eligible patients receiving stable and maximally tolerated doses of angiotensin-converting enzyme inhibitors (ACEis) or angiotensin receptor blockers (ARBs) will be randomized 1:1 to either iptacopan 200 mg or placebo twice daily for a 24-month treatment period. A prespecified interim analysis (IA) will be performed when approximately 250 patients from the main study population complete the 9-month visit. The primary objective is to demonstrate superiority of iptacopan over placebo in reducing 24-hour urine protein-to-creatinine ratio (UPCR) at the IA and demonstrate the superiority of iptacopan over placebo in slowing the rate of estimated glomerular filtration rate (eGFR) decline (total eGFR slope) estimated over 24 months at study completion. The effect of iptacopan on patient-reported outcomes, safety, and tolerability will be evaluated as secondary outcomes. Conclusions: APPLAUSE-IgAN will evaluate the benefits and safety of iptacopan, a novel targeted therapy for IgAN, in reducing complement-mediated kidney damage and thus slowing or preventing disease progression

Electron microscopy pictures show an activated endothelial phenotype in tissues from BAMBI^−/− as compared to BAMBI^+/+ mice.

<p><b>A</b>. Myocardium of the BAMBI<b>⁻</b>^/<b>−</b> mice is notable for the number of endothelial cells per capillary cross-section and the prominent nuclei. In BAMBI^+/+ mice myocardial capillaries show one endothelial cell per cross-section, while those from BAMBI<b>⁻</b>^/<b>−</b> mice show two nuclei per cross-section in 70% of the capillaries (scale bar 2 µm). <b>B</b> and <b>C</b>. glomeruli from BAMBI<b>⁻</b>^/<b>−</b> mice show a prominent number of endothelial cells, which also appear swollen as compared to those from BAMBI^+/+ mice. The glomerular capillary lumen is almost obliterated by the endothelial cells in the BAMBI<b>⁻</b>^/<b>−</b> mice <i>vs.</i> BAMBI^+/+ (low magnification).The activated endothelial phenotype in the BAMBI<b>⁻</b>^/<b>−</b> glomeruli is also apparent at higher magnification (bottom row). <b>D.</b> Peritubular capillaries of BAMBI<b>⁻</b>^/<b>−</b> mice also show a prominent and swollen endothelial cells impinging on the capillary lumen as compared to BAMBI ^+/+ mice.</p