Mechanotransductive feedback control of endothelial cell motility and vascular morphogenesis

Vascular morphogenesis requires persistent endothelial cell motility that is responsive to diverse and dynamic mechanical stimuli. Here, we interrogated the mechanotransductive feedback dynamics that govern endothelial cell motility and vascular morphogenesis. We show that the transcriptional regulators, YAP and TAZ, are activated by mechanical cues to transcriptionally limit cytoskeletal and focal adhesion maturation, forming a conserved mechanotransductive feedback loop that mediates human endothelial cell motility in vitro and zebrafish intersegmental vessel (ISV) morphogenesis in vivo. This feedback loop closes in 4 hours, achieving cytoskeletal equilibrium in 8 hours. Feedback loop inhibition arrested endothelial cell migration in vitro and ISV morphogenesis in vivo. Inhibitor washout at 3 hrs, prior to feedback loop closure, restored vessel growth, but washout at 8 hours, longer than the feedback timescale, did not, establishing lower and upper bounds for feedback kinetics in vivo. Mechanistically, YAP and TAZ induced transcriptional suppression of myosin II activity to maintain dynamic cytoskeletal equilibria. Together, these data establish the mechanoresponsive dynamics of a transcriptional feedback loop necessary for persistent endothelial cell migration and vascular morphogenesis

YAP and TAZ Mediate Osteocyte Perilacunar/Canalicular Remodeling

Bone fragility fractures are caused by low bone mass or impaired bone quality. Osteoblast/osteoclast coordination determines bone mass, but the factors that control bone quality are poorly understood. Osteocytes regulate osteoblast and osteoclast activity on bone surfaces but can also directly reorganize the bone matrix to improve bone quality through perilacunar/canalicular remodeling; however, the molecular mechanisms remain unclear. We previously found that deleting the transcriptional regulators Yes-associated protein (YAP) and Transcriptional co-activator with PDZ-motif (TAZ) from osteoblast-lineage cells caused lethality in mice due to skeletal fragility. Here, we tested the hypothesis that YAP and TAZ regulate osteocyte-mediated bone remodeling by conditional ablation of both YAP and TAZ from mouse osteocytes using 8kb-DMP1-Cre. Osteocyte-conditional YAP/TAZ deletion reduced bone mass and dysregulated matrix collagen content and organization, which together decreased bone mechanical properties. Further, YAP/TAZ deletion impaired osteocyte perilacunar/canalicular remodeling by reducing canalicular network density, length, and branching, as well as perilacunar flourochrome-labeled mineral deposition. Consistent with recent studies identifying TGF-β as a key inducer of osteocyte expression of matrix-remodeling enzymes, YAP/TAZ deletion in vivo decreased osteocyte expression of matrix proteases MMP13, MMP14, and CTSK. In vitro, pharmacologic inhibition of YAP/TAZ transcriptional activity in osteocyte-like cells abrogated TGF-β-induced matrix protease gene expression. Together, these data show that YAP and TAZ control bone matrix accrual, organization, and mechanical properties by regulating osteocyte-mediated bone remodeling. Elucidating the signaling pathways that control perilacunar/canalicular remodeling may enable future therapeutic targeting of bone quality to reverse skeletal fragility

Heterogeneity of Cellular Hypoxia in Murine Bone Marrow

<p>This abstract (Paper No. 482) was presented at the Annual Meeting of the Orthopedic Research Society (ORS) - February 10 – 14, 2023, Dallas, Texas, USA.</p><p>The abstract has been published previously online here https://www.ors.org/transactions/2023/482.pdf</p&gt

BMP-SMAD1/5 Signaling Is Required For Adequate Coupling Of Angiogenesis And Osteogenesis In Long Bones

The bone vasculature is essential for skeletal development, homeostasis, and regeneration. In long bones, specific capillary subtypes couple angiogenesis to osteogenesis, especially at the growth plate (metaphysis) and endosteum. These metaphyseal and endosteal vessels, termed “type H,” express high levels of CD31 and endomucin (CD31hiEmcnhi), show a columnar structure and are closely related to Osterix (Osx)- and Runt-related transcription factor 2 (Runx2)-expressing osteoblasts and their progenitors [1]. Sinusoidal vessels with low expression of CD31 and Emcn (CD31lowEmcnlow, type L vessels) are mainly found in the diaphysis/bone marrow cavity [1]. The crosstalk between endothelial cells (ECs) of type H vessels and bone-related cells is characterized by osteogenesis-promoting factors provided by ECs and angiogenic mediators secreted by osteoblast-lineage cells. Bone morphogenetic proteins (BMP) are major regulators of vessel formation, however their role within angiogenic and osteogenic coupling and in particular, the involvement of BMP-related intracellular effectors SMAD1 and SMAD5 (SMAD1/5) is unclear. Previously, we demonstrated that during mouse embryonic development, the synergy of Notch and SMAD1/5 is responsible for the balanced selection of tip and stalk cells in vascular sprouting [2]. Furthermore, we reported that endothelium-specific deletion of SMAD1/5 during early postnatal retinal angiogenesis resulted in arteriovenous malformations, a reduced number of tip cells and a hyperdensity in the vascular plexus [3]. Therefore, we hypothesized that functional EC-specific SMAD1/5 signaling is required for (i) formation and maturation of long bone vasculature and (ii) coupling of osteogenesis and angiogenesis, specifically of type H vessel formation in the metaphysis

Mechanoregulation of MSC spheroid immunomodulation

Mesenchymal stromal cells (MSCs) are widely used in cell-based therapies and tissue regeneration for their potent secretome, which promotes host cell recruitment and modulates inflammation. Compared to monodisperse cells, MSC spheroids exhibit improved viability and increased secretion of immunomodulatory cytokines. While mechanical stimulation of monodisperse cells can increase cytokine production, the influence of mechanical loading on MSC spheroids is unknown. Here, we evaluated the effect of controlled, uniaxial cyclic compression on the secretion of immunomodulatory cytokines by human MSC spheroids and tested the influence of load-induced gene expression on MSC mechanoresponsiveness. We exposed MSC spheroids, entrapped in alginate hydrogels, to three cyclic compressive regimes with varying stress (L) magnitudes (i.e., 5 and 10 kPa) and hold (H) durations (i.e., 30 and 250 s) L5H30, L10H30, and L10H250. We observed changes in cytokine and chemokine expression dependent on the loading regime, where higher stress regimes tended to result in more exaggerated changes. However, only MSC spheroids exposed to L10H30 induced human THP-1 macrophage polarization toward an M2 phenotype compared to static conditions. Static and L10H30 loading facilitated a strong, interlinked F-actin arrangement, while L5H30 and L10H250 disrupted the structure of actin filaments. This was further examined when the actin cytoskeleton was disrupted via Y-27632. We observed downregulation of YAP-related genes, and the levels of secreted inflammatory cytokines were globally decreased. These findings emphasize the essential role of mechanosignaling in mediating the immunomodulatory potential of MSC spheroids

Effects of Bone Morphogenetic Protein-2 on Neovascularization During Large Bone Defect Regeneration

Insufficient blood vessel supply is a primary limiting factor for regenerative approaches to large bone defect repair. Recombinant bone morphogenetic protein-2 (BMP-2) delivery induces robust bone formation and has been observed to enhance neovascularization, but whether the angiogenic effects of BMP-2 are due to direct endothelial cell stimulation or due to indirect paracrine signaling remain unclear. In this study, we evaluated the effects of BMP-2 delivery on vascularized bone regeneration and tested whether BMP-2 induces neovascularization directly or indirectly. We found that delivery of BMP-2 (5 μg) enhanced both bone formation and neovascularization in critically sized (8 mm) rat femoral bone defects; however, BMP-2 did not directly stimulate angiogenesis in vitro. In contrast, conditioned medium from both mesenchymal progenitor cells and osteoblasts induced endothelial cell migration in vitro, suggesting a paracrine mechanism of BMP-2 action. Consistent with this inference, codelivery of BMP-2 with endothelial colony forming cells to a heterotopic site, distant from the skeletal stem cell-rich bone marrow niche, induced ossification but had no effect on neovascularization. Taken together, these data suggest that paracrine activation of osteoprogenitor cells is an important contributor to neovascularization during BMP-2-mediated bone regeneration. Impact Statement In this study, we show that bone morphogenetic protein-2 (BMP-2) robustly induces neovascularization during tissue-engineered large bone defect regeneration, and we found that BMP-2 induced angiogenesis, in part, through paracrine signaling from osteoprogenitor cells

Skeletal cell YAP and TAZ combinatorially promote bone development

The functions of the paralogous transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in bone are controversial. Each has been observed to promote or inhibit osteogenesis in vitro, with reports of both equivalent and divergent functions. Their combinatorial roles in bone physiology are unknown. We report that combinatorial YAP/TAZ deletion from skeletal lineage cells, using Osterix-Cre, caused an osteogenesis imperfecta-like phenotype with severity dependent on allele dose and greater phenotypic expressivity with homozygous TAZ vs. YAP ablation. YAP/TAZ deletion decreased bone accrual and reduced intrinsic bone material properties through impaired collagen content and organization. These structural and material defects produced spontaneous fractures, particularly in mice with homozygous TAZ deletion and caused neonatal lethality in dual homozygous knockouts. At the cellular level in vivo, YAP/TAZ ablation reduced osteoblast activity and increased osteoclast activity, in an allele dose-dependent manner, impairing bone accrual and remodeling. Transcriptionally, YAP/TAZ deletion and small-molecule inhibition of YAP/TAZ interaction with the transcriptional coeffector TEAD reduced osteogenic and collagen-related gene expression, both in vivo and in vitro. These data demonstrate that YAP and TAZ combinatorially promote bone development through regulation of osteoblast activity, matrix quality, and osteoclastic remodeling.-Kegelman, C. D., Mason, D. E., Dawahare, J. H., Horan, D. J., Vigil, G. D., Howard, S. S., Robling, A. G., Bellido, T. M., Boerckel, J. D. Skeletal cell YAP and TAZ combinatorially promote bone development

Human stem cell delivery for treatment of large segmental bone defects

Local or systemic stem cell delivery has the potential to promote repair of a variety of damaged or degenerated tissues. Although various stem cell sources have been investigated for bone repair, few comparative reports exist, and cellular distribution and viability postimplantation remain key issues. In this study, we quantified the ability of tissue-engineered constructs containing either human fetal or adult stem cells to enhance functional repair of nude rat critically sized femoral defects. After 12 weeks, defects treated with cell-seeded polymer scaffolds had significantly higher bone ingrowth and torsional strength compared to those receiving acellular scaffolds, although there were no significant differences between the cell sources. Next, stem cells were labeled with fluorescent quantum dots (QDs) in an attempt to noninvasively track their distribution after delivery on scaffolds. Clear fluorescence was observed at implantation sites throughout the study; however, beginning 7–10 days after surgery, signals were also observed at contralateral sites treated with acellular QD-free scaffolds. Although immunostaining for human nuclei revealed retention of some cells at the implantation site, no human cells were detected in the control limb defects. Additional histological analysis of implantation and control defect tissues revealed macrophages containing endocytosed QDs. Furthermore, QD-labeling appeared to diminish transplanted cell function resulting in reduced healing responses. In summary, augmentation of polymeric scaffolds with stem cells derived from fetal and adult tissues significantly enhanced healing of large segmental bone defects; however, QD labeling of stem cells eliminated the observed therapeutic effect and failed to conclusively track stem cell location long-term in vivo