Enhanced Biological Activity of BMP‐2 Bound to Surface‐Grafted Heparan Sulfate

Over the last decade, there has been a growing interest in the development of new materials to improve bone morphogenetic protein‐2 (BMP‐2) delivery for tissue regeneration. This study reports the development and application of model surfaces that present BMP‐2 via heparan sulfate (HS), a ubiquitous component of the extracellular matrix (ECM). On these surfaces, HS is grafted by its reducing end, to mimic the natural arrangement of HS proteoglycans in the ECM. The binding of each component on these biomimetic surfaces is highly controlled, in terms of stoichiometry of molecules and BMP‐2/grafted‐HS affinity, as determined by surface‐sensitive techniques. For comparison, this study also uses surfaces presenting immobilized BMP‐2 alone. Functional validations of the surfaces are performed using a murine myoblast cell line (C2C12) and primary human mesenchymal stromal cells. In both cell types, HS‐bound BMP‐2 and surface‐immobilized BMP‐2 significantly prolong SMAD 1/5 phosphorylation, compared to BMP‐2 added to the culture media. Moreover, HS‐bound BMP‐2 enhances p‐SMAD 1/5 levels in C2C12 cells and reduces noggin antagonistic activity. Thus, grafted HS positively affects BMP‐2 cellular activity. This innovative surface design, which mimics natural interactions of growth factors with ECM components, constitutes a promising candidate for future regenerative medicine applications

Bone morphogenetic protein signaling in bone homeostasis

Cancer Signaling networks and Molecular Therapeutic

Emerging regulators of BMP bioavailability

Cancer Signaling networks and Molecular Therapeutic

Role of bone morphogenetic proteins in sprouting angiogenesis: differential BMP receptor-dependent signaling pathways balance stalk vs. tip cell competence

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 (BMP) 2 and BMP6 are proangiogenic in vitro and ex vivo and that the BMP type I receptors, activin receptor-like kinase (ALK)3 and ALK2, play crucial and distinct roles in this process. BMP2 activates the expression of tip cell–associated genes, such as DLL4 (delta-like ligand 4) and KDR (kinase insert domain receptor), 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 HES1 (hairy and enhancer of split 1) and FLT1 (fms-like tyrosine kinase 1). 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

Nanoscale Control of Surface Immobilized BMP-2: Toward a Quantitative Assessment of BMP-Mediated Signaling Events

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

New insights into the molecular mechanism of multiple synostoses syndrome (SYNS): Mutation within the GDF5 knuckle epitope causes noggin-resistance

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 non-Smad 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

The role of titanium surface nanotopography on preosteoblast morphology, adhesion and migration

Surface structuring of titanium-based implants with appropriate nanotopographies can significantly modulate their impact on the biological behavior of cells populating these implants. Implant assisted bone tissue repair and regeneration require functional adhesion and expansion of bone progenitors. The surface nanotopography of implant materials used to support bone healing and its effect on cell behavior, in particular cell adhesion, spreading, expansion, and motility, is still not clearly understood. The aim of this study is to investigate preosteoblast proliferation, adhesion, morphology, and migration on different titanium materials with similar surface chemistry, but distinct nanotopographical features. Sonochemical treatment and anodic oxidation were employed to fabricate disordered – mesoporous titania (TMS), and ordered – titania nanotubular (TNT) topographies respectively. The morphological evaluation revealed a surface dependent shape, thickness, and spreading of cells owing to different adherence behavior. Cells were polygonal-shaped and well-spread on glass and TMS, but displayed an elongated fibroblast-like morphology on TNT surfaces. The cells on glass however, were much flatter than on nanostructured surfaces. Both nanostructured surfaces impaired cell adhesion, but TMS was more favorable for cell growth due to its support of cell attachment and spreading in contrast to TNT. Quantitative wound healing assay in combination with live-cell imaging revealed that cells seeded on TMS surfaces migrated in close proximity to neighboring cells and less directed when compared to the migratory behavior on other surfaces. The results indicate distinctly different cell adhesion and migration on ordered and disordered titania nanotopographies, providing important information that could be used in optimizing titanium-based scaffold design to foster bone tissue growth and repair

The role of titanium surface nanostructuring on preosteoblast morphology, adhesion, and migration

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

BMPR2 acts as a gatekeeper to protect endothelial cells from increased TGF beta responses and altered cell mechanics

Balanced transforming growth factor-beta (TGF beta)/bone morphogenetic protein (BMP)-signaling is essential for tissue formation and homeostasis. While gain in TGF beta 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/TGF beta R1/TGF beta R2 that enable enhanced cellular responses toward TGF beta. These include canonical TGF beta-SMAD2/3 and lateral TGF beta-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 beta 1-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 TGF beta 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 TGF beta signaling. In summary, our findings highlight a crucial role for BMPR2 as a gatekeeper of endothelial homeostasis protecting cells from increased TGF beta responses and integrin-mediated mechano-transduction