Lymphatic vessels in bone support regeneration after injury.

Blood and lymphatic vessels form a versatile transport network and provide inductive signals to regulate tissue-specific functions. Blood vessels in bone regulate osteogenesis and hematopoiesis, but current dogma suggests that bone lacks lymphatic vessels. Here, by combining high-resolution light-sheet imaging and cell-specific mouse genetics, we demonstrate presence of lymphatic vessels in mouse and human bones. We find that lymphatic vessels in bone expand during genotoxic stress. VEGF-C/VEGFR-3 signaling and genotoxic stress-induced IL6 drive lymphangiogenesis in bones. During lymphangiogenesis, secretion of CXCL12 from proliferating lymphatic endothelial cells is critical for hematopoietic and bone regeneration. Moreover, lymphangiocrine CXCL12 triggers expansion of mature Myh11+ CXCR4+ pericytes, which differentiate into bone cells and contribute to bone and hematopoietic regeneration. In aged animals, such expansion of lymphatic vessels and Myh11-positive cells in response to genotoxic stress is impaired. These data suggest lymphangiogenesis as a therapeutic avenue to stimulate hematopoietic and bone regeneration.

Regulation of Hematopoiesis and Osteogenesis by Blood Vessel-Derived Signals.

In addition to their conventional role as a versatile transport system, blood vessels provide signals controlling organ development, regeneration, and stem cell behavior. In the skeletal system, certain capillaries support perivascular osteoprogenitor cells and thereby control bone formation. Blood vessels are also a critical component of niche microenvironments for hematopoietic stem cells. Here we discuss key pathways and factors controlling endothelial cell behavior in bone, the role of vessels in osteogenesis, and the nature of vascular stem cell niches in bone marrow.

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.

Age-dependent modulation of vascular niches for haematopoietic stem cells.

Blood vessels define local microenvironments in the skeletal system, play crucial roles in osteogenesis and provide niches for haematopoietic stem cells. The properties of niche-forming vessels and their changes in the ageing organism remain incompletely understood. Here we show that Notch signalling in endothelial cells leads to the expansion of haematopoietic stem cell niches in bone, which involves increases in CD31-positive capillaries and platelet-derived growth factor receptor-ß (PDGFRß)-positive perivascular cells, arteriole formation and elevated levels of cellular stem cell factor. Although endothelial hypoxia-inducible factor signalling promotes some of these changes, it fails to enhance vascular niche function because of a lack of arterialization and expansion of PDGFRß-positive cells. In ageing mice, niche-forming vessels in the skeletal system are strongly reduced but can be restored by activation of endothelial Notch signalling. These findings indicate that vascular niches for haematopoietic stem cells are part of complex, age-dependent microenvironments involving multiple cell populations and vessel subtypes.

Distinct bone marrow blood vessels differentially regulate haematopoiesis.

Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols.

Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone.

The mammalian skeletal system harbours a hierarchical system of mesenchymal stem cells, osteoprogenitors and osteoblasts sustaining lifelong bone formation. Osteogenesis is indispensable for the homeostatic renewal of bone as well as regenerative fracture healing, but these processes frequently decline in ageing organisms, leading to loss of bone mass and increased fracture incidence. Evidence indicates that the growth of blood vessels in bone and osteogenesis are coupled, but relatively little is known about the underlying cellular and molecular mechanisms. Here we identify a new capillary subtype in the murine skeletal system with distinct morphological, molecular and functional properties. These vessels are found in specific locations, mediate growth of the bone vasculature, generate distinct metabolic and molecular microenvironments, maintain perivascular osteoprogenitors and couple angiogenesis to osteogenesis. The abundance of these vessels and associated osteoprogenitors was strongly reduced in bone from aged animals, and pharmacological reversal of this decline allowed the restoration of bone mass.

Endothelial Notch activity promotes angiogenesis and osteogenesis in bone.

Blood vessel growth in the skeletal system and osteogenesis seem to be coupled, suggesting the existence of molecular crosstalk between endothelial and osteoblastic cells. Understanding the nature of the mechanisms linking angiogenesis and bone formation should be of great relevance for improved fracture healing or prevention of bone mass loss. Here we show that vascular growth in bone involves a specialized, tissue-specific form of angiogenesis. Notch signalling promotes endothelial cell proliferation and vessel growth in postnatal long bone, which is the opposite of the well-established function of Notch and its ligand Dll4 in the endothelium of other organs and tumours. Endothelial-cell-specific and inducible genetic disruption of Notch signalling in mice not only impaired bone vessel morphology and growth, but also led to reduced osteogenesis, shortening of long bones, chondrocyte defects, loss of trabeculae and decreased bone mass. On the basis of a series of genetic experiments, we conclude that skeletal defects in these mutants involved defective angiocrine release of Noggin from endothelial cells, which is positively regulated by Notch. Administration of recombinant Noggin, a secreted antagonist of bone morphogenetic proteins, restored bone growth and mineralization, chondrocyte maturation, the formation of trabeculae and osteoprogenitor numbers in endothelial-cell-specific Notch pathway mutants. These findings establish a molecular framework coupling angiogenesis, angiocrine signals and osteogenesis, which may prove significant for the development of future therapeutic applications.

Type H blood vessels in coupling angiogenesis-osteogenesis and its application in bone tissue engineering.

One specific capillary subtype, termed type H vessel, has been found with unique functional characteristics in coupling angiogenesis with osteogenesis. Researchers have fabricated a variety of tissue engineering scaffolds to enhance bone healing and regeneration through the accumulation of type H vessels. However, only a limited number of reviews discussed the tissue engineering strategies for type H vessel regulation. The object of this review is to summary the current utilizes of bone tissue engineering to regulate type H vessels through various signal pathways including Notch, PDGF-BB, Slit3, HIF-1α, and VEGF signaling. Moreover, we give an insightful overview of recent research progress about the morphological, spatial and age-dependent characteristics of type H blood vessels. Their unique role in tying angiogenesis and osteogenesis together via blood flow, cellular microenvironment, immune system and nervous system are also summarized. This review article would provide an insight into the combination of tissue engineering scaffolds with type H vessels and identify future perspectives for vasculized tissue engineering research.

Heterogeneity and Dynamics of Vasculature in the Endocrine System During Aging and Disease.

The endocrine system consists of several highly vascularized glands that produce and secrete hormones to maintain body homeostasis and regulate a range of bodily functions and processes, including growth, metabolism and development. The dense and highly vascularized capillary network functions as the main transport system for hormones and regulatory factors to enable efficient endocrine function. The specialized capillary types provide the microenvironments to support stem and progenitor cells, by regulating their survival, maintenance and differentiation. Moreover, the vasculature interacts with endocrine cells supporting their endocrine function. However, the structure and niche function of vasculature in endocrine tissues remain poorly understood. Aging and endocrine disorders are associated with vascular perturbations. Understanding the cellular and molecular cues driving the disease, and age-related vascular perturbations hold potential to manage or even treat endocrine disorders and comorbidities associated with aging. This review aims to describe the structure and niche functions of the vasculature in various endocrine glands and define the vascular changes in aging and endocrine disorders.

Diversity of Vascular Niches in Bones and Joints During Homeostasis, Ageing, and Diseases.

The bones and joints in the skeletal system are composed of diverse cell types, including vascular niches, bone cells, connective tissue cells and mineral deposits and regulate whole-body homeostasis. The capacity of maintaining strength and generation of blood lineages lies within the skeletal system. Bone harbours blood and immune cells and their progenitors, and vascular cells provide several immune cell type niches. Blood vessels in bone are phenotypically and functionally diverse, with distinct capillary subtypes exhibiting striking changes with age. The bone vasculature has a special impact on osteogenesis and haematopoiesis, and dysregulation of the vasculature is associated with diverse blood and bone diseases. Ageing is associated with perturbed haematopoiesis, loss of osteogenesis, increased adipogenesis and diminished immune response and immune cell production. Endothelial and perivascular cells impact immune cell production and play a crucial role during inflammation. Here, we discuss normal and maladapted vascular niches in bone during development, homeostasis, ageing and bone diseases such as rheumatoid arthritis and osteoarthritis. Further, we discuss the role of vascular niches during bone malignancy.

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.

CD133-expressing stem cells associated with ovarian metastases establish an endothelial hierarchy and contribute to tumor vasculature.

Recruitment and localization of endothelial precursors within tumors is a potential area for the development of therapeutics, because their functional contribution to tumor vasculature is realized to be important for cancer cell survival. However, the exact nature of the recruited cell type and cellular events orchestrating the entire phenomenon remains obscure. We report that human ovarian cancer is frequently associated with cells expressing the stem cell surface marker CD133. We further show that these CD133-expressing cells are nontumorigenic in nature, and they augment tumor development through their vasculogenic potential. This cell population is attracted by cancer stem cells (CSCs) and retains a direct physical association within the CSC-derived spheroids. Our study further delineates the contribution of these vasculogenic CD133(+) stem cells, termed by us as endothelial stem cells (EnSCs) to the developing tumor vasculature during disease progression. In support of their being stem cells, the EnSCs have a capability of establishing an entire endothelial cell hierarchy. We conclude that such EnSCs play a crucial role in ensuring the development of long-term tumor vasculature to complement CSC-driven tumor development and disease progression.

Bone Angiogenesis and Vascular Niche Remodeling in Stress, Aging, and Diseases.

The bone marrow (BM) vascular niche microenvironments harbor stem and progenitor cells of various lineages. Bone angiogenesis is distinct and involves tissue-specific signals. The nurturing vascular niches in the BM are complex and heterogenous consisting of distinct vascular and perivascular cell types that provide crucial signals for the maintenance of stem and progenitor cells. Growing evidence suggests that the BM niche is highly sensitive to stress. Aging, inflammation and other stress factors induce changes in BM niche cells and their crosstalk with tissue cells leading to perturbed hematopoiesis, bone angiogenesis and bone formation. Defining vascular niche remodeling under stress conditions will improve our understanding of the BM vascular niche and its role in homeostasis and disease. Therefore, this review provides an overview of the current understanding of the BM vascular niches for hematopoietic stem cells and their malfunction during aging, bone loss diseases, arthritis and metastasis.

Bone Vasculature and Bone Marrow Vascular Niches in Health and Disease.

Bone vasculature and bone marrow vascular niches supply oxygen, nutrients, and secrete angiocrine factors required for the survival, maintenance, and self-renewal of stem and progenitor cells. In the skeletal system, vasculature creates nurturing niches for bone and blood-forming stem cells. Blood vessels regulate hematopoiesis and drive bone formation during development, repair, and regeneration. Dysfunctional vascular niches induce skeletal aging, bone diseases, and hematological disorders. Recent cellular and molecular characterization of the bone marrow microenvironment has provided unprecedented insights into the complexity, heterogeneity, and functions of the bone vasculature and vascular niches. The bone vasculature is composed of distinct vessel subtypes that differentially regulate osteogenesis, hematopoiesis, and disease conditions in bones. Further, bone marrow vascular niches supporting stem cells are often complex microenvironments involving multiple different cell populations and vessel subtypes. This review provides an overview of the emerging vascular cell heterogeneity in bone and the new roles of the bone vasculature and associated vascular niches in health and disease. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Role of angiocrine signals in bone development, homeostasis and disease.

Skeletal vasculature plays a central role in the maintenance of microenvironments for osteogenesis and haematopoiesis. In addition to supplying oxygen and nutrients, vasculature provides a number of inductive factors termed as angiocrine signals. Blood vessels drive recruitment of osteoblast precursors and bone formation during development. Angiogenesis is indispensable for bone repair and regeneration. Dysregulation of the angiocrine crosstalk is a hallmark of ageing and pathobiological conditions in the skeletal system. The skeletal vascular bed is complex, heterogeneous and characterized by distinct capillary subtypes (type H and type L), which exhibit differential expression of angiocrine factors. Furthermore, distinct blood vessel subtypes with differential angiocrine profiles differentially regulate osteogenesis and haematopoiesis, and drive disease states in the skeletal system. This review provides an overview of the role of angiocrine signals in bone during homeostasis and disease.

Endothelial proteolytic activity and interaction with non-resorbing osteoclasts mediate bone elongation.

Growth plate cartilage contributes to the generation of a large variety of shapes and sizes of skeletal elements in the mammalian system. The removal of cartilage and how this process regulates bone shape are not well understood. Here we identify a non-bone-resorbing osteoclast subtype termed vessel-associated osteoclast (VAO). Endothelial cells at the bone/cartilage interface support VAOs through a RANKL-RANK signalling mechanism. In contrast to classical bone-associated osteoclasts, VAOs are dispensable for cartilage resorption and regulate anastomoses of type H vessels. Remarkably, proteinases including matrix metalloproteinase-9 (Mmp9) released from endothelial cells, not osteoclasts, are essential for resorbing cartilage to lead directional bone growth. Importantly, disrupting the orientation of angiogenic blood vessels by misdirecting them results in contorted bone shape. This study identifies proteolytic functions of endothelial cells in cartilage and provides a framework to explore tissue-lytic features of blood vessels in fracture healing, arthritis and cancer.

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.

Inhibition of Endosteal Vascular Niche Remodeling Rescues Hematopoietic Stem Cell Loss in AML.

Bone marrow vascular niches sustain hematopoietic stem cells (HSCs) and are drastically remodeled in leukemia to support pathological functions. Acute myeloid leukemia (AML) cells produce angiogenic factors, which likely contribute to this remodeling, but anti-angiogenic therapies do not improve AML patient outcomes. Using intravital microscopy, we found that AML progression leads to differential remodeling of vasculature in central and endosteal bone marrow regions. Endosteal AML cells produce pro-inflammatory and anti-angiogenic cytokines and gradually degrade endosteal endothelium, stromal cells, and osteoblastic cells, whereas central marrow remains vascularized and splenic vascular niches expand. Remodeled endosteal regions have reduced capacity to support non-leukemic HSCs, correlating with loss of normal hematopoiesis. Preserving endosteal endothelium with the small molecule deferoxamine or a genetic approach rescues HSCs loss, promotes chemotherapeutic efficacy, and enhances survival. These findings suggest that preventing degradation of the endosteal vasculature may improve current paradigms for treating AML.