Exosomal miRNA-155-5p from M1-polarized macrophages suppresses angiogenesis by targeting GDF6 to interrupt diabetic wound healing.

Unprogrammed macrophage polarization, especially prolonged activation of proinflammatory macrophages, is associated with delayed wound healing in diabetic objectives. Macrophage-derived exosomes cargo a variety of microRNAs (miRNAs), participating in different stages in wound healing. Here, exosomes were isolated from naive bone marrow-derived macrophages (BMDMs) (M0-Exos), interferon-γ plus lipopolysaccharide-polarized BMDMs (M1-Exos), and interleukin-4-polarized BMDMs (M2-Exos). M1-Exos impaired migration and tube formation in human umbilical vein endothelial cells (HUVECs) compared to M0-Exos, whereas M2-Exos exhibited the opposite effects. High-throughput sequencing was performed to decipher the miRNA expression profiles in M0-Exos, M1-Exos, and M2-Exos. A total of 63 miRNAs were identified to be differentially expressed in exosomes derived from polarized BMDMs. Among them, miRNA-155-5p is highly expressed in M1-Exos, which interrupted angiogenesis in HUVECs. Furthermore, miRNA-155-5p directly binds to the 3' UTR of growth differentiation factor 6 (GDF6) mRNA to suppress its protein expression. Lastly, local administration of a temperature-sensitive hydrogel Pluronic F-127 loading miRNA-155-5p antagomiR promoted angiogenesis and accelerated wound healing in diabetic db/db mice via enhancing GDF6. In summary, this study deciphered the miRNA expression profiles in exosomes from polarized macrophages. M2-like macrophage-derived exosomes and miRNA-155-5p inhibitors could be promising therapeutics against diabetic foot ulcers.

Defective Joint Development and Maintenance in GDF6-Related Multiple Synostoses Syndrome.

Multiple synostoses syndromes (SYNS) are a group of rare genetic bone disorders characterized by multiple joint fusions. We previously reported an SYNS4-causing GDF6 c.1330 T > A (p.Tyr444Asn) mutation, which reduced Noggin-induced GDF6 inhibition and enhanced SMAD1/5/8 signaling. However, the mechanisms by which GDF6 gain-of-function mutation alters joint formation and the comprehensive molecular portraits of SYNS4 remain unclear. Herein, we introduce the p.Tyr443Asn (orthologous to the human GDF6 p.Tyr444Asn) mutation into the mouse Gdf6 locus and report the results of extensive phenotype analysis, joint development investigation, and transcriptome profiling of Gdf6 p.Tyr443Asn limb buds. Gdf6 p.Tyr443Asn knock-in mice recapitulated the morphological features of human SYNS4, showing joint fusion in the wrists, ankles, phalanges, and auditory ossicles. Analysis of mouse embryonic forelimbs demonstrated joint interzone formation defects and excess chondrogenesis in Gdf6 p.Tyr443Asn knock-in mice. Further, RNA sequencing of forelimb buds revealed enhanced bone formation and upregulated bone morphogenetic protein (BMP) signaling in mice carrying the Gdf6 p.Tyr443Asn mutation. Because tightly regulated BMP signaling is critical for skeletal development and joint morphogenesis, our study shows that enhancing GDF6 activity has a significant impact on both prenatal joint development and postnatal joint maintenance. © 2023 American Society for Bone and Mineral Research (ASBMR).

Protective Effects of Growth Differentiation Factor-6 on the Intervertebral Disc: An In Vitro and In Vivo Study.

Growth differentiation factors (GDFs) regulate homeostasis by amplifying extracellular matrix anabolism and inhibiting pro-inflammatory cytokine production in the intervertebral disc (IVD). The aim of this study was to elucidate the effects of GDF-6 on human IVD nucleus pulposus (NP) cells using a three-dimensional culturing system in vitro and on rat tail IVD tissues using a puncture model in vivo. In vitro, Western blotting showed decreased GDF-6 expression with age and degeneration severity in surgically collected human IVD tissues (n = 12). Then, in moderately degenerated human IVD NP cells treated with GDF-6 (100 ng/mL), immunofluorescence demonstrated an increased expression of matrix components including aggrecan and type II collagen. Quantitative polymerase chain reaction analysis also presented GDF-6-induced downregulation of pro-inflammatory tumor necrosis factor (TNF)-α (p = 0.014) and interleukin (IL)-6 (p = 0.016) gene expression stimulated by IL-1ß (10 ng/mL). Furthermore, in the mitogen-activated protein kinase pathway, Western blotting displayed GDF-6-induced suppression of p38 phosphorylation (p = 0.041) under IL-1ß stimulation. In vivo, intradiscal co-administration of GDF-6 and atelocollagen was effective in alleviating rat tail IVD annular puncture-induced radiologic height loss (p = 0.005), histomorphological degeneration (p < 0.001), matrix metabolism (aggrecan, p < 0.001; type II collagen, p = 0.001), and pro-inflammatory cytokine production (TNF-α, p < 0.001; IL-6, p < 0.001). Consequently, GDF-6 could be a therapeutic growth factor for degenerative IVD disease.

A Novel Human Long Noncoding RNA SCDAL Promotes Angiogenesis through SNF5-Mediated GDF6 Expression.

Angiogenesis is essential for vascular development. The roles of regulatory long noncoding RNAs (lncRNAs) in mediating angiogenesis remain under-explored. Human embryonic stem cell-derived mesenchymal stem cells (hES-MSCs) are shown to exert more potent cardioprotective effects against cardiac ischemia than human bone marrow-derived MSCs (hBM-MSCs), associated with enhanced neovascularization. The purpose of this study is to search for angiogenic lncRNAs enriched in hES-MSCs, and investigate their roles and mechanisms. AC103746.1 is one of the most highly expressed intergenic lncRNAs detected in hES-MSCs versus hBM-MSCs, and named as SCDAL (stem cell-derived angiogenic lncRNA). SCDAL knockdown significantly reduce the angiogenic potential and reparative effects of hES-MSCs in the infarcted hearts, while overexpression of SCDAL in either hES-MSCs or hBM-MSCs exhibits augmented angiogenesis and cardiac function recovery. Mechanistically, SCDAL induces growth differentiation factor 6 (GDF6) expression via direct interaction with SNF5 at GDF6 promoter. Secreted GDF6 promotes endothelial angiogenesis via non-canonical vascular endothelial growth factor receptor 2 activation. Furthermore, SCDAL-GDF6 is expressed in human endothelial cells, and directly enhances endothelial angiogenesis in vitro and in vivo. Thus, these findings uncover a previously unknown lncRNA-dependent regulatory circuit for angiogenesis. Targeted intervention of the SCDAL-GDF6 pathway has potential as a therapy for ischemic heart diseases.

Growth differentiation factor-6 attenuates inflammatory and pain-related factors and degenerated disc-induced pain behaviors in rat model.

Previous studies have indicated that growth differentiation factor 6 (GDF6) is a potential candidate for intervertebral disc (IVD) degeneration (IDD) treatment. Here, we investigated the effect of GDF6 on IDD by examining changes in disc structure and the expression of inflammatory and pain-related factors. A rat posterior disc puncture model of single segments and three consecutive segments was constructed, and GDF6 or phosphate-buffered solution was administered via intradiscal injection 1 or 2 weeks after surgery. Magnetic resonance imaging showed a clear degeneration signal in the punctured disc, which was inhibited by GDF6. Histological staining revealed that GDF6 did not significantly improve the structure of IVDs in rats 8 weeks after puncture surgery, but it had an inhibitory effect on expression of the tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1ß in the IVD. Furthermore, GDF6 was found to protect the morphology and structure of the IVD 32 weeks after surgery. Mechanical and thermal hyperalgesia tests suggested that GDF6 injection can significantly improve mechanical and thermal-stimulated pain behavior in rats and inhibit the expression of inflammatory factors TNF-α and IL-1ß and the pain factor calcitonin gene-related peptide in the dorsal root ganglion. A rat protein array test indicated that GDF6 could reduce the expression of cytokines IL-6, intercellular cell adhesion molecule-1, matrix metalloproteinase-13, IL-1ß, and TNF-α and increase the expression of tissue inhibitor of metalloproteinases 1, Transforming growth factor-beta 2, IL-10, and resistin in a TNF-α-induced IDD cell model. Thus, our study demonstrates that GDF6 can improve the structure of the IVD, inhibit the expression of inflammatory and pain-related factors, and improve pain behavior in rats. Clinical Significance: To establish further preclinical research and clinical trials, comprehensive data are needed to validate the regenerative properties of GDF6. Ideally, a regenerative agent should also be able to relieve discogenic pain, achieving the best clinical outcomes.

Regenerative Response of Degenerate Human Nucleus Pulposus Cells to GDF6 Stimulation.

Growth differentiation factor (GDF) family members have been implicated in the development and maintenance of healthy nucleus pulposus (NP) tissue, making them promising therapeutic candidates for treatment of intervertebral disc (IVD) degeneration and associated back pain. GDF6 has been shown to promote discogenic differentiation of mesenchymal stem cells, but its effect on NP cells remains largely unknown. Our aim was to investigate GDF6 signalling in adult human NP cells derived from degenerate tissue and determine the signal transduction pathways critical for GDF6-mediated phenotypic changes and tissue homeostatic mechanisms. This study demonstrates maintained expression of GDF6 receptors in human NP and annulus fibrosus (AF) cells across a range of degeneration grades at gene and protein level. We observed an anabolic response in NP cells treated with recombinant GDF6 (increased expression of matrix and NP-phenotypic markers; increased glycosaminoglycan production; no change in catabolic enzyme expression), and identified the signalling pathways involved in these responses (SMAD1/5/8 and ERK1/2 phosphorylation, validated by blocking studies). These findings suggest that GDF6 promotes a healthy disc tissue phenotype in degenerate NP cells through SMAD-dependent and -independent (ERK1/2) mechanisms, which is important for development of GDF6 therapeutic strategies for treatment of degenerate discs.

High BMPR2 expression leads to enhanced SMAD1/5/8 signalling and GDF6 responsiveness in human adipose-derived stem cells: implications for stem cell therapies for intervertebral disc degeneration.

Stem cell-based regenerative strategies are promising for intervertebral disc degeneration. Stimulation of bone-marrow- and adipose-derived multipotent stem cells with recombinant human growth differentiation factor 6 (rhGDF6) promotes anabolic nucleus pulposus like phenotypes. In comparison to mesenchymal stem cells, adipose-derived multipotent stem cells exhibit greater NP-marker gene expression and proteoglycan-rich matrix production. To understand these response differences, we investigated bone morphogenetic protein receptor profiles in donor-matched human mesenchymal stem cells and adipose-derived multipotent stem cells, determined differences in rhGDF6 signalling and their importance in NP-like differentiation between cell populations. Bone morphogenetic protein receptor expression in mesenchymal stem cells and adipose-derived multipotent stem cells revealed elevated and less variable expression of BMPR2 in adipose-derived multipotent stem cells, which corresponded with increased downstream pathway activation (SMAD1/5/8, ERK1/2). Inhibitor studies demonstrated SMAD1/5/8 signalling was required for rhGDF6-induced nucleus-pulposus-like adipose-derived multipotent stem cell differentiation, while ERK1/2 contributed significantly to critical nucleus pulposus gene expression, aggrecan and type II collagen production. These data inform cell regenerative therapeutic choices for intervertebral disc degeneration regeneration and identify further potential optimisation targets.

Microparticles for controlled growth differentiation factor 6 delivery to direct adipose stem cell-based nucleus pulposus regeneration.

Currently, there is no effective long-term treatment for intervertebral disc (IVD) degeneration, making it an attractive candidate for regenerative therapies. Hydrogel delivery of adipose stem cells (ASCs) in combination with controlled release of bioactive molecules is a promising approach to halt IVD degeneration and promote regeneration. Growth differentiation factor 6 (GDF6) can induce ASC differentiation into anabolic nucleus pulposus (NP) cells and hence holds promise for IVD regeneration. Here, we optimised design of novel poly(DL-lactic acid-co-glycolic acid) (PLGA)-polyethylene glycol-PLGA microparticles to control GDF6 delivery and investigated effect of released GDF6 on human ASCs differentiation to NP cells. Recombinant human (rh)GDF6 was loaded into microparticles and total protein and rhGDF6 release assessed. The effect of microparticle loading density on distribution and gel formation was investigated through scanning electron microscopy. ASC differentiation to NP cells was examined after 14 days in hydrogel culture by quantitative polymerase chain reaction, histological, and immunohistochemical staining in normoxic and IVD-like hypoxic conditions. RhGDF6 microparticles were distributed throughout gels without disrupting gelation and controlled rhGDF6 release over 14 days. Released GDF6 significantly induced NP differentiation of ASCs, with expression comparable with or exceeding media supplemented rhGDF6. Microparticle-delivered rhGDF6 also up-regulated sulphated glycosaminoglycan and aggrecan secretion in comparison with controls. In hypoxia, microparticle-delivered rhGDF6 continued to effectively induce NP gene expression and aggrecan production. This study demonstrates the effective encapsulation and controlled delivery of rhGDF6, which maintained its activity and induced ASC differentiation to NP cells and synthesis of an NP-like matrix suggesting suitability of microparticles for controlled growth factor release in regenerative strategies for treatment of IVD degeneration.

ISSLS PRIZE IN BASIC SCIENCE 2018: Growth differentiation factor-6 attenuated pro-inflammatory molecular changes in the rabbit anular-puncture model and degenerated disc-induced pain generation in the rat xenograft radiculopathy model.

PURPOSE: To elucidate the effects of growth differentiation factor-6 (GDF6) on: (i) gene expression of inflammatory/pain-related molecules and structural integrity in the rabbit intervertebral disc (IVD) degeneration model, and (ii) sensory dysfunction and changes in pain-marker expression in dorsal nerve ganglia (DRGs) in the rat xenograft radiculopathy model. METHODS: Forty-six adolescent rabbits received anular-puncture in two non-consecutive lumbar IVDs. Four weeks later, phosphate-buffered saline (PBS) or GDF6 (1, 10 or 100 µg) was injected into the nucleus pulposus (NP) of punctured discs and followed for 4 weeks for gene expression analysis and 12 weeks for structural analyses. For pain assessment, eight rabbits were sacrificed at 4 weeks post-injection and NP tissues of injected discs were transplanted onto L5 DRGs of 16 nude rats to examine mechanical allodynia. The rat DRGs were analyzed immunohistochemically. RESULTS: In GDF6-treated rabbit NPs, gene expressions of interleukin-6, tumor necrosis factor-α, vascular endothelial growth factor, prostaglandin-endoperoxide synthase 2, and nerve growth factor were significantly lower than those in the PBS group. GDF6 injections resulted in partial restoration of disc height and improvement of MRI disc degeneration grades with statistical significance in rabbit structural analyses. Allodynia induced by xenograft transplantation of rabbit degenerated NPs onto rat DRGs was significantly reduced by GDF6 injection. Staining intensities for ionized calcium-binding adaptor molecule-1 and calcitonin gene-related peptide in rat DRGs of the GDF6 group were significantly lower than those of the PBS group. CONCLUSION: GDF6 injection may change the pathological status of degenerative discs and attenuate degenerated IVD-induced pain.

Growth Differentiation Factor 6 Promotes Vascular Stability by Restraining Vascular Endothelial Growth Factor Signaling.

OBJECTIVE: The assembly of a functional vascular system requires a coordinated and dynamic transition from activation to maturation. High vascular endothelial growth factor activity promotes activation, including junction destabilization and cell motility. Maturation involves junctional stabilization and formation of a functional endothelial barrier. The identity and mechanism of action of prostabilization signals are still mostly unknown. Bone morphogenetic protein receptors and their ligands have important functions during embryonic vessel assembly and maturation. Previous work has suggested a role for growth differentiation factor 6 (GDF6; bone morphogenetic protein 13) in vascular integrity although GDF6's mechanism of action was not clear. Therefore, we sought to further explore the requirement for GDF6 in vascular stabilization. APPROACH AND RESULTS: We investigated the role of GDF6 in promoting endothelial vascular integrity in vivo in zebrafish and in cultured human umbilical vein endothelial cells in vitro. We report that GDF6 promotes vascular integrity by counteracting vascular endothelial growth factor activity. GDF6-deficient endothelium has increased vascular endothelial growth factor signaling, increased vascular endothelial-cadherin Y658 phosphorylation, vascular endothelial-cadherin delocalization from cell-cell interfaces, and weakened endothelial cell adherence junctions that become prone to vascular leak. CONCLUSIONS: Our results suggest that GDF6 promotes vascular stabilization by restraining vascular endothelial growth factor signaling. Understanding how GDF6 affects vascular integrity may help to provide insights into hemorrhage and associated vascular pathologies in humans.