The effect of rewarming ischemia on tissue transcriptome and metabolome signatures: a clinical observational study in lung transplantation.

BACKGROUND: In lung transplantation (LuTx), various ischemic phases exist, yet the rewarming ischemia time (RIT) during implantation has often been overlooked. During RIT, lungs are deflated and exposed to the body temperature in the recipient's chest cavity. Our prior clinical findings demonstrated that prolonged RIT increases the risk of primary graft dysfunction. However, the molecular mechanisms of rewarming ischemic injury in this context remain unexplored. We aimed to characterize the rewarming ischemia phase during LuTx by measuring organ temperature and comparing transcriptome and metabolome profiles in tissue obtained at the end versus the start of implantation. METHODS: In a clinical observational study, 34 double-LuTx with ice preservation were analyzed. Lung core and surface temperature (n=65 and 55 lungs) was measured during implantation. Biopsies (n=59 lungs) were wedged from right middle lobe and left lingula at start and end of implantation. Tissue transcriptomic and metabolomic profiling were performed. RESULTS: Temperature increased rapidly during implantation, reaching core/surface temperatures of 21.5°C/25.4°C within 30min. Transcriptomics showed increased pro-inflammatory signaling and oxidative stress at the end of implantation. Upregulation of NLRP3 and NFKB1 correlated with RIT. Metabolomics indicated elevated levels of amino acids, hypoxanthine, uric acid, cysteineglutathione disulfide alongside decreased levels of glucose and carnitines. Arginine, tyrosine, and 1-carboxyethylleucine showed correlation with incremental RIT. CONCLUSIONS: The final rewarming ischemia phase in LuTx involves rapid organ rewarming, accompanied by transcriptomic and metabolomic changes indicating pro-inflammatory signaling and disturbed cell metabolism. Limiting implantation time and lung cooling represent potential interventions to alleviate rewarming ischemic injury.

The serine synthesis pathway drives osteoclast differentiation through epigenetic regulation of NFATc1 expression.

Bone-resorbing osteoclasts are vital for postnatal bone health, as increased differentiation or activity results in skeletal pathologies such as osteoporosis. The metabolism of mature osteoclasts differs from their progenitor cells, but whether the observed metabolic changes are secondary to the altered cell state or actively drive the process of cell differentiation is unknown. Here, we show that transient activation of the serine synthesis pathway (SSP) is essential for osteoclastogenesis, as deletion of the rate-limiting enzyme phosphoglycerate dehydrogenase in osteoclast progenitors impairs their differentiation and results in increased bone mass. In addition, pharmacological phosphoglycerate dehydrogenase inhibition abrogated bone loss in a mouse model of postmenopausal osteoporosis by blocking bone resorption. Mechanistically, SSP-derived α-ketoglutarate is necessary for histone demethylases that remove repressive histone methylation marks at the nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1) gene locus, thereby inducing NFATc1 expression and consequent osteoclast maturation. Taken together, this study reveals a metabolic-epigenetic coupling mechanism that directs osteoclast differentiation and suggests that the SSP can be therapeutically targeted to prevent osteoporotic bone loss.

Skeletal progenitors preserve proliferation and self-renewal upon inhibition of mitochondrial respiration by rerouting the TCA cycle.

A functional electron transport chain (ETC) is crucial for supporting bioenergetics and biosynthesis. Accordingly, ETC inhibition decreases proliferation in cancer cells but does not seem to impair stem cell proliferation. However, it remains unclear how stem cells metabolically adapt. In this study, we show that pharmacological inhibition of complex III of the ETC in skeletal stem and progenitor cells induces glycolysis side pathways and reroutes the tricarboxylic acid (TCA) cycle to regenerate NAD+ and preserve cell proliferation. These metabolic changes also culminate in increased succinate and 2-hydroxyglutarate levels that inhibit Ten-eleven translocation (TET) DNA demethylase activity, thereby preserving self-renewal and multilineage potential. Mechanistically, mitochondrial malate dehydrogenase and reverse succinate dehydrogenase activity proved to be essential for the metabolic rewiring in response to ETC inhibition. Together, these data show that the metabolic plasticity of skeletal stem and progenitor cells allows them to bypass ETC blockade and preserve their self-renewal.

De novo serine synthesis regulates chondrocyte proliferation during bone development and repair.

The majority of the mammalian skeleton is formed through endochondral ossification starting from a cartilaginous template. Cartilage cells, or chondrocytes, survive, proliferate and synthesize extracellular matrix in an avascular environment, but the metabolic requirements for these anabolic processes are not fully understood. Here, using metabolomics analysis and genetic in vivo models, we show that maintaining intracellular serine homeostasis is essential for chondrocyte function. De novo serine synthesis through phosphoglycerate dehydrogenase (PHGDH)-mediated glucose metabolism generates nucleotides that are necessary for chondrocyte proliferation and long bone growth. On the other hand, dietary serine is less crucial during endochondral bone formation, as serine-starved chondrocytes compensate by inducing PHGDH-mediated serine synthesis. Mechanistically, this metabolic flexibility requires ATF4, a transcriptional regulator of amino acid metabolism and stress responses. We demonstrate that both serine deprivation and PHGDH inactivation enhance ATF4 signaling to stimulate de novo serine synthesis and serine uptake, respectively, and thereby prevent intracellular serine depletion and chondrocyte dysfunction. A similar metabolic adaptability between serine uptake and de novo synthesis is observed in the cartilage callus during fracture repair. Together, the results of this study reveal a critical role for PHGDH-dependent serine synthesis in maintaining intracellular serine levels under physiological and serine-limited conditions, as adequate serine levels are necessary to support chondrocyte proliferation during endochondral ossification.

Glutamine Metabolism Controls Chondrocyte Identity and Function.

Correct functioning of chondrocytes is crucial for long bone growth and fracture repair. These cells are highly anabolic but survive and function in an avascular environment, implying specific metabolic requirements that are, however, poorly characterized. Here, we show that chondrocyte identity and function are closely linked with glutamine metabolism in a feedforward process. The master chondrogenic transcription factor SOX9 stimulates glutamine metabolism by increasing glutamine consumption and levels of glutaminase 1 (GLS1), a rate-controlling enzyme in this pathway. Consecutively, GLS1 action is critical for chondrocyte properties and function via a tripartite mechanism. First, glutamine controls chondrogenic gene expression epigenetically through glutamate dehydrogenase-dependent acetyl-CoA synthesis, necessary for histone acetylation. Second, transaminase-mediated aspartate synthesis supports chondrocyte proliferation and matrix synthesis. Third, glutamine-derived glutathione synthesis avoids harmful reactive oxygen species accumulation and allows chondrocyte survival in the avascular growth plate. Collectively, our study identifies glutamine as a metabolic regulator of cartilage fitness during bone development.

Nestin-GFP transgene labels skeletal progenitors in the periosteum.

The periosteum is critical for bone repair and contains skeletal stem cells (SSCs), but these cells are still poorly characterized. In the bone marrow, cells expressing the Nes-GFP transgene have been described to be SSCs. Here, we investigated whether Nes-GFP expression also typifies SSCs in the periosteum. We show that in adult mice, Nes-GFP cells are present in the periosteum and localize closely to blood vessels, but periosteal Nes-GFP cells express SSC and progenitor markers differently compared to Nes-GFP cells in the bone marrow. Periosteal Nes-GFP cells show in vitro clonogenicity and tri-lineage differentiation potential and they can form bone in vivo. Shortly after fracture, they start to proliferate and they contribute to the osteoblast pool during the repair process. However, periosteal Nes-GFP cells are not slow dividing nor self-renewing in vivo. These results indicate that in adult mice, periosteal Nes-GFP expressing cells are skeletal progenitors rather than true SSCs, and they participate in the fracture healing process.

An Ectopic Imaging Window for Intravital Imaging of Engineered Bone Tissue.

Tissue engineering is a promising branch of regenerative medicine, but its clinical application remains limited because thorough knowledge of the in vivo repair processes in these engineered implants is limited. Common techniques to study the different phases of bone repair in mice are destructive and thus not optimal to gain insight into the dynamics of this process. Instead, multiphoton-intravital microscopy (MP-IVM) allows visualization of (sub)cellular processes at high resolution and frequency over extended periods of time when combined with an imaging window that permits optical access to implants in vivo. In this study, we have developed and validated an ectopic imaging window that can be placed over a tissue-engineered construct implanted in mice. This approach did not interfere with the biological processes of bone regeneration taking place in these implants, as evidenced by histological and micro-computed tomography (µCT)-based comparison to control ectopic implants. The ectopic imaging window permitted tracking of individual cells over several days in vivo. Furthermore, the use of fluorescent reporters allowed visualization of the onset of angiogenesis and osteogenesis in these constructs. Taken together, this novel imaging window will facilitate further analysis of the spatiotemporal regulation of cellular processes in bone tissue-engineered implants and provides a powerful tool to enhance the therapeutic potential of bone tissue engineering.

Osteocytic oxygen sensing controls bone mass through epigenetic regulation of sclerostin.

Preservation of bone mass is crucial for healthy ageing and largely depends on adequate responses of matrix-embedded osteocytes. These cells control bone formation and resorption concurrently by secreting the WNT/ß-catenin antagonist sclerostin (SOST). Osteocytes reside within a low oxygen microenvironment, but whether and how oxygen sensing regulates their function remains elusive. Here, we show that conditional deletion of the oxygen sensor prolyl hydroxylase (PHD) 2 in osteocytes results in a high bone mass phenotype, which is caused by increased bone formation and decreased resorption. Mechanistically, enhanced HIF-1α signalling increases Sirtuin 1-dependent deacetylation of the Sost promoter, resulting in decreased sclerostin expression and enhanced WNT/ß-catenin signalling. Additionally, genetic ablation of PHD2 in osteocytes blunts osteoporotic bone loss induced by oestrogen deficiency or mechanical unloading. Thus, oxygen sensing by PHD2 in osteocytes negatively regulates bone mass through epigenetic regulation of sclerostin and targeting PHD2 elicits an osteo-anabolic response in osteoporotic models.

Regulatory elements driving the expression of skeletal lineage reporters differ during bone development and adulthood.

To improve bone healing or regeneration more insight in the fate and role of the different skeletal cell types is required. Mouse models for fate mapping and lineage tracing of skeletal cells, using stage-specific promoters, have advanced our understanding of bone development, a process that is largely recapitulated during bone repair. However, validation of these models is often only performed during development, whereas proof of the activity and specificity of the used promoters during the bone regenerative process is limited. Here, we show that the regulatory elements of the 6kb collagen type II promoter are not adequate to drive gene expression during bone repair. Similarly, the 2.3kb promoter of collagen type I lacks activity in adult mice, but the 3.2kb promoter is suitable. Furthermore, Cre-mediated fate mapping allows the visualization of progeny, but this label retention may hinder to distinguish these cells from ones with active expression of the marker at later time points. Together, our results show that the lineage-specific regulatory elements driving gene expression during bone development differ from those required later in life and during bone repair, and justify validation of lineage-specific cell tracing and gene silencing strategies during fracture healing and bone regenerative applications.

SRC kinase inhibition with saracatinib limits the development of osteolytic bone disease in multiple myeloma.

Multiple myeloma (MM)-associated osteolytic bone disease is a major cause of morbidity and mortality in MM patients and the development of new therapeutic strategies is of great interest. The proto-oncogene SRC is an attractive target for such a strategy. In the current study, we investigated the effect of treatment with the SRC inhibitor saracatinib (AZD0530) on osteoclast and osteoblast differentiation and function, and on the development of MM and its associated bone disease in the 5TGM.1 and 5T2MM murine MM models. In vitro data showed an inhibitory effect of saracatinib on osteoclast differentiation, polarization and resorptive function. In osteoblasts, collagen deposition and matrix mineralization were affected by saracatinib. MM cell proliferation and tumor burden remained unaltered following saracatinib treatment and we could not detect any synergistic effects with drugs that are part of standard care in MM. We observed a marked reduction of bone loss after treatment of MM-bearing mice with saracatinib as reflected by a restoration of trabecular bone parameters to levels observed in naive control mice. Histomorphometric analyses support that this occurs through an inhibition of bone resorption. In conclusion, these data further establish SRC inhibition as a promising therapeutic approach for the treatment of MM-associated osteolytic bone disease.

Extra-intestinal calcium handling contributes to normal serum calcium levels when intestinal calcium absorption is suboptimal.

The active form of vitamin D, 1,25(OH)2D, is a crucial regulator of calcium homeostasis, especially through stimulation of intestinal calcium transport. Lack of intestinal vitamin D receptor (VDR) signaling does however not result in hypocalcemia, because the increased 1,25(OH)2D levels stimulate calcium handling in extra-intestinal tissues. Systemic VDR deficiency, on the other hand, results in hypocalcemia because calcium handling is impaired not only in the intestine, but also in kidney and bone. It remains however unclear whether low intestinal VDR activity, as observed during aging, is sufficient for intestinal calcium transport and for mineral and bone homeostasis. To this end, we generated mice that expressed the Vdr exclusively in the gut, but at reduced levels. We found that ~15% of intestinal VDR expression greatly prevented the Vdr null phenotype in young-adult mice, including the severe hypocalcemia. Serum calcium levels were, however, in the low-normal range, which may be due to the suboptimal intestinal calcium absorption, renal calcium loss, insufficient increase in bone resorption and normal calcium incorporation in the bone matrix. In conclusion, our results indicate that low intestinal VDR levels improve intestinal calcium absorption compared to Vdr null mice, but also show that 1,25(OH)2D-mediated fine-tuning of renal calcium reabsorption and bone mineralization and resorption is required to maintain fully normal serum calcium levels.

Expansion of murine periosteal progenitor cells with fibroblast growth factor 2 reveals an intrinsic endochondral ossification program mediated by bone morphogenetic protein 2.

The preservation of the bone-forming potential of skeletal progenitor cells during their ex vivo expansion remains one of the major challenges for cell-based bone regeneration strategies. We report that expansion of murine periosteal cells in the presence of FGF2, a signal present during the early stages of fracture healing, is necessary and sufficient to maintain their ability to organize in vivo into a cartilage template which gives rise to mature bone. Implantation of FGF2-primed cells in a large bone defect in mice resulted in complete healing, demonstrating the feasibility of using this approach for bone tissue engineering purposes. Mechanistically, the enhanced endochondral ossification potential of FGF2-expanded periosteal cells is predominantly driven by an increased production of BMP2 and is additionally linked to an improved preservation of skeletal progenitor cells in the cultures. This characteristic is unique for periosteal cells, as FGF2-primed bone marrow stromal cells formed significantly less bone and progressed exclusively through the intramembranous pathway, revealing essential differences between both cell pools. Taken together, our findings provide insight in the molecular regulation of fracture repair by identifying a unique interaction between periosteal cells and FGF2. These insights may promote the development of cell-based therapeutic strategies for bone regeneration which are independent of the in vivo use of growth factors, thus limiting undesired side effects.

Engineering vascularized bone: osteogenic and proangiogenic potential of murine periosteal cells.

One of the key challenges in bone tissue engineering is the timely formation of blood vessels that promote the survival of the implanted cells in the construct. Fracture healing largely depends on the presence of an intact periosteum but it is still unknown whether periosteum-derived cells (PDC) are critical for bone repair only by promoting bone formation or also by inducing neovascularization. We first established a protocol to specifically isolate murine PDC (mPDC) from long bones of adult mice. Mesenchymal stem cells were abundantly present in this cell population as more than 50% of the mPDC expressed mesenchymal markers (CD73, CD90, CD105, and stem cell antigen-1) and the cells exhibited trilineage differentiation potential (chondrogenic, osteogenic, and adipogenic). When transplanted on a collagen-calcium phosphate scaffold in vivo, mPDC attracted numerous blood vessels and formed mature bone which comprises a hematopoiesis-supportive stroma. We explored the proangiogenic properties of mPDC using in vitro culture systems and showed that mPDC promote the survival and proliferation of endothelial cells through the production of vascular endothelial growth factor. Coimplantation with endothelial cells demonstrated that mPDC can enhance vasculogenesis by adapting a pericyte-like phenotype, in addition to their ability to stimulate blood vessel ingrowth from the host. In conclusion, these findings demonstrate that periosteal cells contribute to fracture repair, not only through their strong osteogenic potential but also through their proangiogenic features and thus provide an ideal cell source for bone regeneration therapies.

NuSAP is essential for chromatin-induced spindle formation during early embryogenesis.

Mitotic spindle assembly is mediated by two processes: a centrosomal and a chromosomal pathway. RanGTP regulates the latter process by releasing microtubule-associated proteins from inhibitory complexes. NuSAP, a microtubule- and DNA-binding protein, is a target of RanGTP and promotes the formation of microtubules near chromosomes. However, the contribution of NuSAP to cell proliferation in vivo is unknown. Here, we demonstrate that the expression of NuSAP highly correlates with cell proliferation during embryogenesis and adult life, making it a reliable marker of proliferating cells. Additionally, we show that NuSAP deficiency in mice leads to early embryonic lethality. Spindle assembly in NuSAP-deficient cells is highly inefficient and chromosomes remain dispersed in the mitotic cytoplasm. As a result of sustained spindle checkpoint activity, the cells are unable to progress through mitosis, eventually leading to caspase activation and apoptotic cell death. Together, our findings demonstrate that NuSAP is essential for proliferation of embryonic cells and, simultaneously, they underscore the importance of chromatin-induced spindle assembly.

Increased skeletal VEGF enhances beta-catenin activity and results in excessively ossified bones.

Vascular endothelial growth factor (VEGF) and beta-catenin both act broadly in embryogenesis and adulthood, including in the skeletal and vascular systems. Increased or deregulated activity of these molecules has been linked to cancer and bone-related pathologies. By using novel mouse models to locally increase VEGF levels in the skeleton, we found that embryonic VEGF over-expression in osteo-chondroprogenitors and their progeny largely pheno-copied constitutive beta-catenin activation. Adult induction of VEGF in these cell populations dramatically increased bone mass, associated with aberrant vascularization, bone marrow fibrosis and haematological anomalies. Genetic and pharmacological interventions showed that VEGF increased bone mass through a VEGF receptor 2- and phosphatidyl inositol 3-kinase-mediated pathway inducing beta-catenin transcriptional activity in endothelial and osteoblastic cells, likely through modulation of glycogen synthase kinase 3-beta phosphorylation. These insights into the actions of VEGF in the bone and marrow environment underscore its power as pleiotropic bone anabolic agent but also warn for caution in its therapeutic use. Moreover, the finding that VEGF can modulate beta-catenin activity may have widespread physiological and clinical ramifications.

TRPV4-mediated calcium influx regulates terminal differentiation of osteoclasts.

Calcium signaling controls multiple cellular functions and is regulated by the release from internal stores and entry from extracellular fluid. In bone, osteoclast differentiation is induced by RANKL (receptor activator of NF-kappaB ligand)-evoked intracellular Ca(2+) oscillations, which trigger nuclear factor-activated T cells (NFAT) c1-responsive gene transcription. However, the Ca(2+) channels involved remain largely unidentified. Here we show that genetic ablation in mice of Trpv4, a Ca(2+)-permeable channel of the transient receptor potential (TRP) family, increases bone mass by impairing bone resorption. TRPV4 mediates basolateral Ca(2+) influx specifically in large osteoclasts when Ca(2+) oscillations decline. TRPV4-mediated Ca(2+) influx hereby secures intracellular Ca(2+) concentrations, ensures NFATc1-regulated gene transcription, and regulates the terminal differentiation and activity of osteoclasts. In conclusion, our data indicate that Ca(2+) oscillations and TRPV4-mediated Ca(2+) influx are sequentially required to sustain NFATc1-dependent gene expression throughout osteoclast differentiation, and we propose TRPV4 as a therapeutic target for bone diseases.

Placental growth factor mediates mesenchymal cell development, cartilage turnover, and bone remodeling during fracture repair.

Current therapies for delayed- or nonunion bone fractures are still largely ineffective. Previous studies indicated that the VEGF homolog placental growth factor (PlGF) has a more significant role in disease than in health. Therefore we investigated the role of PlGF in a model of semi-stabilized bone fracture healing. Fracture repair in mice lacking PlGF was impaired and characterized by a massive accumulation of cartilage in the callus, reminiscent of delayed- or nonunion fractures. PlGF was required for the early recruitment of inflammatory cells and the vascularization of the fracture wound. Interestingly, however, PlGF also played a role in the subsequent stages of the repair process. Indeed in vivo and in vitro findings indicated that PlGF induced the proliferation and osteogenic differentiation of mesenchymal progenitors and stimulated cartilage turnover by particular MMPs. Later in the process, PlGF was required for the remodeling of the newly formed bone by stimulating osteoclast differentiation. As PlGF expression was increased throughout the process of bone repair and all the important cell types involved expressed its receptor VEGFR-1, the present data suggest that PlGF is required for mediating and coordinating the key aspects of fracture repair. Therefore PlGF may potentially offer therapeutic advantages for fracture repair.

Soluble VEGF isoforms are essential for establishing epiphyseal vascularization and regulating chondrocyte development and survival.

VEGF is crucial for metaphyseal bone vascularization. In contrast, the angiogenic factors required for vascularization of epiphyseal cartilage are unknown, although this represents a developmentally and clinically important aspect of bone growth. The VEGF gene is alternatively transcribed into VEGF(120), VEGF(164), and VEGF(188) isoforms that differ in matrix association and receptor binding. Their role in bone development was studied in mice expressing single isoforms. Here we report that expression of only VEGF(164) or only VEGF(188) (in VEGF(188/188) mice) was sufficient for metaphyseal development. VEGF(188/188) mice, however, showed dwarfism, disrupted development of growth plates and secondary ossification centers, and knee joint dysplasia. This phenotype was at least partly due to impaired vascularization surrounding the epiphysis, resulting in ectopically increased hypoxia and massive chondrocyte apoptosis in the interior of the epiphyseal cartilage. In addition to the vascular defect, we provide in vitro evidence that the VEGF(188) isoform alone is also insufficient to regulate chondrocyte proliferation and survival responses to hypoxia. Consistent herewith, chondrocytes in or close to the hypoxic zone in VEGF(188/188) mice showed increased proliferation and decreased differentiation. These findings indicate that the insoluble VEGF(188) isoform is insufficient for establishing epiphyseal vascularization and regulating cartilage development during endochondral bone formation.

Impaired angiogenesis and endochondral bone formation in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188.

Vascular endothelial growth factor (VEGF)-mediated angiogenesis is an important part of bone formation. To clarify the role of VEGF isoforms in endochondral bone formation, we examined long bone development in mice expressing exclusively the VEGF120 isoform (VEGF120/120 mice). Neonatal VEGF120/120 long bones showed a completely disturbed vascular pattern, concomitant with a 35% decrease in trabecular bone volume, reduced bone growth and a 34% enlargement of the hypertrophic chondrocyte zone of the growth plate. Surprisingly, embryonic hindlimbs at a stage preceding capillary invasion exhibited a delay in bone collar formation and hypertrophic cartilage calcification. Expression levels of marker genes of osteoblast and hypertrophic chondrocyte differentiation were significantly decreased in VEGF120/120 bones. Furthermore, inhibition of all VEGF isoforms in cultures of embryonic cartilaginous metatarsals, through the administration of a soluble receptor chimeric protein (mFlt-1/Fc), retarded the onset and progression of ossification, suggesting that osteoblast and/or hypertrophic chondrocyte development were impaired. The initial invasion by osteoclasts and endothelial cells into VEGF120/120 bones was retarded, associated with decreased expression of matrix metalloproteinase-9. Our findings indicate that expression of VEGF164 and/or VEGF188 is important for normal endochondral bone development, not only to mediate bone vascularization but also to allow normal differentiation of hypertrophic chondrocytes, osteoblasts, endothelial cells and osteoclasts.