Decreased blood vessel density and endothelial cell subset dynamics during ageing of the endocrine system.

Age-associated alterations of the hormone-secreting endocrine system cause organ dysfunction and disease states. However, the cell biology of endocrine tissue ageing remains poorly understood. Here, we perform comparative 3D imaging to understand age-related perturbations of the endothelial cell (EC) compartment in endocrine glands. Datasets of a wide range of markers highlight a decline in capillary and artery numbers, but not of perivascular cells in pancreas, testis and thyroid gland, with age in mice and humans. Further, angiogenesis and ß-cell expansion in the pancreas are coupled by a distinct age-dependent subset of ECs. While this EC subpopulation supports pancreatic ß cells, it declines during ageing concomitant with increased expression of the gap junction protein Gja1. EC-specific ablation of Gja1 restores ß-cell expansion in the aged pancreas. These results provide a proof of concept for understanding age-related vascular changes and imply that therapeutic targeting of blood vessels may restore aged endocrine tissue function. This comprehensive data atlas offers over > 1,000 multicolour volumes for exploration and research in endocrinology, ageing, matrix and vascular biology.

Haplodeficiency of the 9p21 tumor suppressor locus causes myeloid disorders driven by the bone marrow microenvironment.

The chromosome 9p21 locus comprises several tumor suppressor genes including MTAP, CDKN2A, and CDKN2B, and its homo- or heterozygous deletion is associated with reduced survival in multiple cancer types. We report that mice with germ line monoallelic deletion or induced biallelic deletion of the 9p21-syntenic locus (9p21s) developed a fatal myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN)-like disease associated with aberrant trabecular bone formation and/or fibrosis in the bone marrow (BM). Reciprocal BM transfers and conditional targeting of 9p21s suggested that the disease originates in the BM stroma. Single-cell analysis of 9p21s-deficient BM stroma revealed the expansion of chondrocyte and osteogenic precursors, reflected in increased osteogenic differentiation in vitro. It also showed reduced expression of factors maintaining hematopoietic stem/progenitor cells, including Cxcl12. Accordingly, 9p21s-deficient mice showed reduced levels of circulating Cxcl12 and concomitant upregulation of the profibrotic chemokine Cxcl13 and the osteogenesis- and fibrosis-related multifunctional glycoprotein osteopontin/Spp1. Our study highlights the potential of mutations in the BM microenvironment to drive MDS/MPN-like disease.

Molecular Deconvolution of Bone Marrow Adipose Tissue Interactions with Malignant Hematopoiesis: Potential for New Therapy Development.

PURPOSE OF REVIEW: Along with a strong impact on skeletal integrity, bone marrow adipose tissue (BMAT) is an important modulator of the adult hematopoietic system. This review will summarize the current knowledge on the causal relationship between bone marrow (BM) adipogenesis and the development and progression of hematologic malignancies. RECENT FINDINGS: BM adipocytes (BMAds) support a number of processes promoting oncogenesis, including the evolution of clonal hematopoiesis, malignant cell survival, proliferation, angiogenesis, and chemoresistance. In addition, leukemic cells manipulate surrounding BMAds by promoting lipolysis and release of free fatty acids, which are then utilized by leukemic cells via ß-oxidation. Therefore, limiting BM adipogenesis, blocking BMAd-derived adipokines, or lipid metabolism obstruction have been considered as potential treatment options for hematological malignancies. Leukemic stem cells rely heavily on BMAds within the structural BM microenvironment for necessary signals which foster disease progression. Further development of 3D constructs resembling BMAT at different skeletal regions are critical to better understand these relationships in geometric space and may provide essential insight into the development of hematologic malignancies within the BM niche. In turn, these mechanisms provide promising potential as novel approaches to targeting the microenvironment with new therapeutic strategies.

Zoledronic Acid in a Mouse Model of Human Fibrous Dysplasia: Ineffectiveness on Tissue Pathology, Formation of "Giant Osteoclasts" and Pathogenetic Implications.

We compared the effects of a nitrogen-containing bisphosphonate (N-BP), zoledronic acid (ZA), and an anti-mouse RANKL antibody (anti-mRANKL Ab) on the bone tissue pathology of a transgenic mouse model of human fibrous dysplasia (FD). For comparison, we also reviewed the histological samples of a child with McCune-Albright syndrome (MAS) treated with Pamidronate for 3 years. EF1α-GsαR201C mice with FD-like lesions in the tail vertebrae were treated with either 0.2 mg/kg of ZA at day 0, 7, and 14 or with 300 µg/mouse of anti-mRANKL Ab at day 0 and 21. All mice were monitored by Faxitron and histological analysis was performed at day 42. ZA did not affect the progression of the radiographic phenotype in EF1α-GsαR201C mice. FD-like lesions in the ZA group showed the persistence of osteoclasts, easily detectable osteoclast apoptotic activity and numerous "giant osteoclasts". In contrast, in the anti-mRANKL Ab-treated mice, osteoclasts were markedly reduced/absent, the radiographic phenotype reverted and the FD-like lesions were extensively replaced by newly formed bone. Numerous "giant osteoclasts" were also detected in the samples of the child with MAS. This study supports the hypothesis that osteoclasts per se, independently of their resorptive activity, are essential for development and expansion of FD lesions.

Bone Marrow Adipose Tissue: Regulation of Osteoblastic Niche, Hematopoiesis and Hematological Malignancies.

Bone marrow adipose tissue (BMAT) creates a specific microniche within multifunctional bone marrow (BM) ecosystem which imposes changes in surrounding cells and at systemic level. Moreover, BMAT contributes to spatial and temporal separation and metabolic compartmentalization of BM, thus regulating BM homeostasis and diseases. Recent findings have identified novel progenitor subsets of bone marrow adipocytes (BMAd)s recruited during the BM adipogenesis within different skeletal and hematopoietic stem cell niches. Potential of certain mesenchymal BM cells to differentiate into both osteogenic and adipogenic lineages, contributes to the complex interplay of BMAT with endosteal (osteoblastic) niche compartments as an important cellular player in bone tissue homeostasis. Targeting and ablation of BMAT cells at certain states might be an optional and promising strategy for improvement of bone health. Additionally, recent findings demonstrated spatial distribution of BMAds related to hematopoietic cells and pointed out important functional roles in the vital processes such as long-term hematopoiesis. BM adipogenesis appears to be an emergency phenomenon that follows the production of hematopoietic stem and progenitor cell niche factors, thus regulating physiological, stressed, and malignant hematopoiesis. Lipolytic and secretory activity of BMAds can influence survival and proliferation of hematopoietic cells at different maturation stages. Due to their different lipid status, constitutive and regulated BMAds are important determinants of normal and malignant hematopoietic cells. Further elucidation of cellular and molecular players involved in BMAT expansion and crosstalk with malignant cells is of paramount importance for conceiving the new therapies for improvement of BM health.

Next Generation Bone Marrow Adiposity Researchers: Report From the 1st BMAS Summer School 2021.

The first International Summer School on Bone Marrow Adiposity was organized by members of Bone Marrow Adiposity Society and held virtually on September 6-8 2021. The goal of this meeting was to bring together young scientists interested in learning about bone marrow adipose tissue biology and pathology. Fifty-two researchers from different backgrounds and fields, ranging from bone physiopathology to adipose tissue biology and hematology, participated in the summer school. The meeting featured three keynote lectures on the fundamentals of bone marrow adiposity, three scientific workshops on technical considerations in studying bone marrow adiposity, and six motivational and career development lectures, spanning from scientific writing to academic career progression. Moreover, twenty-one participants presented their work in the form of posters. In this report we highlight key moments and lessons learned from the event.

GsαR201C and estrogen reveal different subsets of bone marrow adiponectin expressing osteogenic cells.

The Gsα/cAMP signaling pathway mediates the effect of a variety of hormones and factors that regulate the homeostasis of the post-natal skeleton. Hence, the dysregulated activity of Gsα due to gain-of-function mutations (R201C/R201H) results in severe architectural and functional derangements of the entire bone/bone marrow organ. While the consequences of gain-of-function mutations of Gsα have been extensively investigated in osteoblasts and in bone marrow osteoprogenitor cells at various differentiation stages, their effect in adipogenically-committed bone marrow stromal cells has remained unaddressed. We generated a mouse model with expression of GsαR201C driven by the Adiponectin (Adq) promoter. Adq-GsαR201C mice developed a complex combination of metaphyseal, diaphyseal and cortical bone changes. In the metaphysis, GsαR201C caused an early phase of bone resorption followed by bone deposition. Metaphyseal bone formation was sustained by cells that were traced by Adq-Cre and eventually resulted in a high trabecular bone mass phenotype. In the diaphysis, GsαR201C, in combination with estrogen, triggered the osteogenic activity of Adq-Cre-targeted perivascular bone marrow stromal cells leading to intramedullary bone formation. Finally, consistent with the previously unnoticed presence of Adq-Cre-marked pericytes in intraosseous blood vessels, GsαR201C caused the development of a lytic phenotype that affected both cortical (increased porosity) and trabecular (tunneling resorption) bone. These results provide the first evidence that the Adq-cell network in the skeleton not only regulates bone resorption but also contributes to bone formation, and that the Gsα/cAMP pathway is a major modulator of both functions.

Subversion of Serotonin Receptor Signaling in Osteoblasts by Kynurenine Drives Acute Myeloid Leukemia.

Remodeling of the microenvironment by tumor cells can activate pathways that favor cancer growth. Molecular delineation and targeting of such malignant-cell nonautonomous pathways may help overcome resistance to targeted therapies. Herein we leverage genetic mouse models, patient-derived xenografts, and patient samples to show that acute myeloid leukemia (AML) exploits peripheral serotonin signaling to remodel the endosteal niche to its advantage. AML progression requires the presence of serotonin receptor 1B (HTR1B) in osteoblasts and is driven by AML-secreted kynurenine, which acts as an oncometabolite and HTR1B ligand. AML cells utilize kynurenine to induce a proinflammatory state in osteoblasts that, through the acute-phase protein serum amyloid A (SAA), acts in a positive feedback loop on leukemia cells by increasing expression of IDO1-the rate-limiting enzyme for kynurenine synthesis-thereby enabling AML progression. This leukemia-osteoblast cross-talk, conferred by the kynurenine-HTR1B-SAA-IDO1 axis, could be exploited as a niche-focused therapeutic approach against AML, opening new avenues for cancer treatment. SIGNIFICANCE: AML remains recalcitrant to treatments due to the emergence of resistant clones. We show a leukemia-cell nonautonomous progression mechanism that involves activation of a kynurenine-HTR1B-SAA-IDO1 axis between AML cells and osteoblasts. Targeting the niche by interrupting this axis can be pharmacologically harnessed to hamper AML progression and overcome therapy resistance. This article is highlighted in the In This Issue feature, p. 873.

High-resolution 3D imaging uncovers organ-specific vascular control of tissue aging.

Blood vessels provide supportive microenvironments for maintaining tissue functions. Age-associated vascular changes and their relation to tissue aging and pathology are poorly understood. Here, we perform 3D imaging of young and aging vascular beds. Multiple organs in mice and humans demonstrate an age-dependent decline in vessel density and pericyte numbers, while highly remodeling tissues such as skin preserve the vasculature. Vascular attrition precedes the appearance of cellular hallmarks of aging such as senescence. Endothelial VEGFR2 loss-of-function mice demonstrate that vascular perturbations are sufficient to stimulate cellular changes coupled with aging. Age-associated tissue-specific molecular changes in the endothelium drive vascular loss and dictate pericyte to fibroblast differentiation. Lineage tracing of perivascular cells with inducible PDGFRß and NG2 Cre mouse lines demonstrated that increased pericyte to fibroblast differentiation distinguishes injury-induced organ fibrosis and zymosan-induced arthritis. To spur further discoveries, we provide a freely available resource with 3D vascular and tissue maps.

Reporting Guidelines, Review of Methodological Standards, and Challenges Toward Harmonization in Bone Marrow Adiposity Research. Report of the Methodologies Working Group of the International Bone Marrow Adiposity Society.

The interest in bone marrow adiposity (BMA) has increased over the last decade due to its association with, and potential role, in a range of diseases (osteoporosis, diabetes, anorexia, cancer) as well as treatments (corticosteroid, radiation, chemotherapy, thiazolidinediones). However, to advance the field of BMA research, standardization of methods is desirable to increase comparability of study outcomes and foster collaboration. Therefore, at the 2017 annual BMA meeting, the International Bone Marrow Adiposity Society (BMAS) founded a working group to evaluate methodologies in BMA research. All BMAS members could volunteer to participate. The working group members, who are all active preclinical or clinical BMA researchers, searched the literature for articles investigating BMA and discussed the results during personal and telephone conferences. According to the consensus opinion, both based on the review of the literature and on expert opinion, we describe existing methodologies and discuss the challenges and future directions for (1) histomorphometry of bone marrow adipocytes, (2) ex vivo BMA imaging, (3) in vivo BMA imaging, (4) cell isolation, culture, differentiation and in vitro modulation of primary bone marrow adipocytes and bone marrow stromal cell precursors, (5) lineage tracing and in vivo BMA modulation, and (6) BMA biobanking. We identify as accepted standards in BMA research: manual histomorphometry and osmium tetroxide 3D contrast-enhanced µCT for ex vivo quantification, specific MRI sequences (WFI and H-MRS) for in vivo studies, and RT-qPCR with a minimal four gene panel or lipid-based assays for in vitro quantification of bone marrow adipogenesis. Emerging techniques are described which may soon come to complement or substitute these gold standards. Known confounding factors and minimal reporting standards are presented, and their use is encouraged to facilitate comparison across studies. In conclusion, specific BMA methodologies have been developed. However, important challenges remain. In particular, we advocate for the harmonization of methodologies, the precise reporting of known confounding factors, and the identification of methods to modulate BMA independently from other tissues. Wider use of existing animal models with impaired BMA production (e.g., Pfrt-/-, KitW/W-v) and development of specific BMA deletion models would be highly desirable for this purpose.

Angiocrine signals regulate quiescence and therapy resistance in bone metastasis.

Bone provides supportive microenvironments for hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) and is a frequent site of metastasis. While incidences of bone metastases increase with age, the properties of the bone marrow microenvironment that regulate dormancy and reactivation of disseminated tumor cells (DTCs) remain poorly understood. Here, we elucidate the age-associated changes in the bone secretome that trigger proliferation of HSCs, MSCs, and DTCs in the aging bone marrow microenvironment. Remarkably, a bone-specific mechanism involving expansion of pericytes and induction of quiescence-promoting secretome rendered this proliferative microenvironment resistant to radiation and chemotherapy. This bone-specific expansion of pericytes was triggered by an increase in PDGF signaling via remodeling of specialized type H blood vessels in response to therapy. The decline in bone marrow pericytes upon aging provides an explanation for loss of quiescence and expansion of cancer cells in the aged bone marrow microenvironment. Manipulation of blood flow - specifically, reduced blood flow - inhibited pericyte expansion, regulated endothelial PDGF-B expression, and rendered bone metastatic cancer cells susceptible to radiation and chemotherapy. Thus, our study provides a framework to recognize bone marrow vascular niches in age-associated increases in metastasis and to target angiocrine signals in therapeutic strategies to manage bone metastasis.

RANKL Inhibition in Fibrous Dysplasia of Bone: A Preclinical Study in a Mouse Model of the Human Disease.

Fibrous dysplasia of bone/McCune-Albright syndrome (Polyostotic FD/MAS; OMIM#174800) is a crippling skeletal disease caused by gain-of-function mutations of Gs α. Enhanced bone resorption is a recurrent histological feature of FD and a major cause of fragility of affected bones. Previous work suggests that increased bone resorption in FD is driven by RANKL and some studies have shown that the anti-RANKL monoclonal antibody, denosumab, reduces bone turnover and bone pain in FD patients. However, the effect of RANKL inhibition on the histopathology of FD and its impact on the natural history of the disease remain to be assessed. In this study, we treated the EF1α-Gs αR201C mice, which develop an FD-like phenotype, with an anti-mouse RANKL monoclonal antibody. We found that the treatment induced marked radiographic and microscopic changes at affected skeletal sites in 2-month-old mice. The involved skeletal segments became sclerotic due to the deposition of new, highly mineralized bone within developing FD lesions and showed a higher mechanical resistance compared to affected segments from untreated transgenic mice. Similar changes were also detected in older mice with a full-blown skeletal phenotype. The administration of anti-mouse RANKL antibody arrested the growth of established lesions and, in young mice, prevented the appearance of new ones. However, after drug withdrawal, the newly formed bone was remodelled into FD tissue and the disease progression resumed in young mice. Taken together, our results show that the anti-RANKL antibody significantly affected the bone pathology and natural history of FD in the mouse. Pending further work on the prevention and management of relapse after treatment discontinuation, our preclinical study suggests that RANKL inhibition may be an effective therapeutic option for FD patients. © 2019 American Society for Bone and Mineral Research.