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Balanced transforming growth factor-beta (TGFβ)/bone morphogenetic protein (BMP)-signaling is essential for tissue formation and homeostasis. While gain in TGFβ signaling is often found in diseases, the underlying cellular mechanisms remain poorly defined. Here we show that the receptor BMP type 2 (BMPR2) serves as a central gatekeeper of this balance, highlighted by its deregulation in diseases such as pulmonary arterial hypertension (PAH). We show that BMPR2 deficiency in endothelial cells (ECs) does not abolish pan-BMP-SMAD1/5 responses but instead favors the formation of mixed-heteromeric receptor complexes comprising BMPR1/TGFβR1/TGFβR2 that enable enhanced cellular responses toward TGFβ. These include canonical TGFβ-SMAD2/3 and lateral TGFβ-SMAD1/5 signaling as well as formation of mixed SMAD complexes. Moreover, BMPR2-deficient cells express genes indicative of altered biophysical properties, including up-regulation of extracellular matrix (ECM) proteins such as fibrillin-1 (FBN1) and of integrins. As such, we identified accumulation of ectopic FBN1 fibers remodeled with fibronectin (FN) in junctions of BMPR2-deficient ECs. Ectopic FBN1 deposits were also found in proximity to contractile intimal cells in pulmonary artery lesions of BMPR2-deficient heritable PAH (HPAH) patients. In BMPR2-deficient cells, we show that ectopic FBN1 is accompanied by active β1-integrin highly abundant in integrin-linked kinase (ILK) mechano-complexes at cell junctions. Increased integrin-dependent adhesion, spreading, and actomyosin-dependent contractility facilitates the retrieval of active TGFβ from its latent fibrillin-bound depots. We propose that loss of BMPR2 favors endothelial-to-mesenchymal transition (EndMT) allowing cells of myo-fibroblastic character to create a vicious feed-forward process leading to hyperactivated TGFβ signaling. In summary, our findings highlight a crucial role for BMPR2 as a gatekeeper of endothelial homeostasis protecting cells from increased TGFβ responses and integrin-mediated mechano-transduction.

In this work we determine the impact of surface density of immobilized BMP-2 on intracellular signal transduction. We use block copolymer micellar nanolithography to fabricate substrates with precisely spaced and tunable gold nanoparticle arrays carrying single BMP-2 molecules. We found that the immobilized growth factor triggers prolonged and elevated Smad signaling pathway activation compared to the same amount of soluble protein. This approach is suitable for achieving controlled and sustained local delivery of BMP-2 and other growth factors.

Before the onset of sprouting angiogenesis, the endothelium is prepatterned for the positioning of tip and stalk cells. Both cell identities are not static, as endothelial cells (ECs) constantly compete for the tip cell position in a dynamic fashion. Here, we show that both bone morphogenetic protein 2 (BMP2) and BMP6 are proangiogenic in vitro and ex vivo and that the BMP type I receptors, activin receptor-like kinase 3 (ALK3) and ALK2, play crucial and distinct roles in this process. BMP2 activates the expression of tip cell–associated genes, such as delta-like ligand 4 (DLL4) and kinase insert domain receptor (KDR), and p38-heat shock protein 27 (HSP27)–dependent cell migration, thereby generating tip cell competence. Whereas BMP6 also triggers collective cell migration via the p38-HSP27 signaling axis, BMP6 induces in addition SMAD1/5 signaling, thereby promoting the expression of stalk cell–associated genes, such as hairy and enhancer of split 1 (HES1) and fms-like tyrosine kinase 1 (FLT1). Specifically, ALK3 is required for sprouting from HUVEC spheroids, whereas ALK2 represses sprout formation. We demonstrate that expression levels and respective complex formation of BMP type I receptors in ECs determine stalk vs. tip cell identity, thus contributing to endothelial plasticity during sprouting angiogenesis. As antiangiogenic monotherapies that target the VEGF or ALK1 pathways have not fulfilled efficacy objectives in clinical trials, the selective targeting of the ALK2/3 pathways may be an attractive new approach.—Benn, A., Hiepen, C., Osterland, M., Schütte, C., Zwijsen, A., Knaus, P. Role of bone morphogenetic proteins in sprouting angiogenesis: differential BMP receptor-dependent signaling pathways balance stalk vs. tip cell competence.

Surface structuring of titanium-based implants is known to modulate the behavior of adherent cells, but the influence of different nanotopographies is poorly understood. The aim is to investigate preosteoblast proliferation, adhesion, morphology, and migration on surfaces with similar surface chemistry but distinct nanotopographical features. Sonochemical treatment and anodic oxidation are employed to fabricate disordered, mesoporous titania (TMS) and ordered titania nanotubular (TNT) topographies on titanium, respectively. Morphological evaluation reveals that cells are polygonal and well-spread on TMS, but display an elongated, fibroblast-like morphology on TNT surfaces, while they are much flatter on glass. Both nanostructured surfaces impair cell adhesion, but TMS is more favorable for cell growth due to its support of cell attachment and spreading in contrast to TNT. A quantitative wound healing assay in combination with live-cell imaging reveals that cell migration on TMS surfaces has a more collective character than on other surfaces, probably due to a closer proximity between neighboring migrating cells on TMS. The results indicate distinctly different cell adhesion and migration on ordered and disordered titania nanotopographies, providing important information that can be used in optimizing titanium-based scaffold design to foster bone tissue growth and repair while allowing for the encapsulation of drugs into porous titania layer

Growth and differentiation factor 5 (GDF5), a member of the bone morphogenetic protein (BMP) family, is essential for cartilage, bone, and joint formation. Antagonists such as noggin counteract BMP signaling by covering the ligand's BMP type I (BMPRI) and type II (BMPRII, ActRII, ActRIIB) interaction sites. The mutation GDF5-S94N is located within the BMPRII interaction site, the so-called knuckle epitope, and was identified in patients suffering from multiple synostoses syndrome (SYNS). SYNS is characterized by progressive symphalangism, carpal/tarsal fusions, deafness and mild facial dysmorphism. Here we present a novel molecular mechanism of a GDF5 mutation affecting chondrogenesis and osteogenesis. GDF5-S94N exhibits impaired binding to BMPRII causing alleviated Smad and nonSmad signaling and reduced chondrogenic differentiation of ATDC5 cells. Surprisingly, chondrogenesis in mouse micromass cultures was strongly enhanced by GDF5-S94N. By using quantitative techniques (SPR, reporter gene assay, ALP assay, qPCR), we uncovered that this gain of function is caused by strongly reduced affinity of GDF5-S94N to the BMP/GDF antagonist noggin and the consequential lack of noggin inhibition. Thus, since noggin is upregulated during chondrogenic differentiation, GDF5-S94N exceeds the GDF5 action, which results in the phenotypic outcome of SYNS. The detailed molecular characterization of GDF5-S94N as a noggin-resistant growth factor illustrates the potential of GDF5 mutants in applications with defined therapeutical needs. ß

Bone Morphogenetic Proteins (BMPs) are members of the transforming growth factor-β superfamily of secreted polypeptide growth factors and are important regulators in a multitude of cellular processes. To ensure the precise and balanced propagation of their pleiotropic signaling responses, BMPs and their corresponding signaling pathways are subject to tight control. A large variety of regulatory mechanisms throughout different biological levels combines into a complex network and provides the basis for physiological BMP function. This regulatory network not only includes biochemical factors but also mechanical cues. Both BMP signaling and mechanotransduction pathways are tightly interconnected and represent an elaborate signaling network active during development but also during organ homeostasis. Moreover, its dysregulation is associated with a number of human pathologies. A more detailed understanding of this crosstalk in respect to molecular interactions will be indispensable in the future, in particular to understand BMP-related diseases as well as with regard to an efficient clinical application of BMP ligands.