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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Cell-matrix signals specify bone endothelial cells during developmental osteogenesis.

Blood vessels in the mammalian skeletal system control bone formation and support haematopoiesis by generating local niche environments. While a specialized capillary subtype, termed type H, has been recently shown to couple angiogenesis and osteogenesis in adolescent, adult and ageing mice, little is known about the formation of specific endothelial cell populations during early developmental endochondral bone formation. Here, we report that embryonic and early postnatal long bone contains a specialized endothelial cell subtype, termed type E, which strongly supports osteoblast lineage cells and later gives rise to other endothelial cell subpopulations. The differentiation and functional properties of bone endothelial cells require cell-matrix signalling interactions. Loss of endothelial integrin ß1 leads to endothelial cell differentiation defects and impaired postnatal bone growth, which is, in part, phenocopied by endothelial cell-specific laminin α5 mutants. Our work outlines fundamental principles of vessel formation and endothelial cell differentiation in the developing skeletal system.

Flow Dynamics and HSPC Homing in Bone Marrow Microvessels.

Measurements of flow velocities at the level of individual arterial vessels and sinusoidal capillaries are crucial for understanding the dynamics of hematopoietic stem and progenitor cell homing in the bone marrow vasculature. We have developed two complementary intravital two-photon imaging approaches to determine blood flow dynamics and velocities in multiple vessel segments by capturing the motion of red blood cells. High-resolution spatiotemporal measurements through a cranial window to determine short-time dynamics of flowing blood cells and repetitive centerline scans were used to obtain a detailed flow-profile map with hemodynamic parameters. In addition, we observed the homing of individual hematopoietic stem and progenitor cells and obtained detailed information on their homing behavior. With our imaging setup, we determined flow patterns at cellular resolution, blood flow velocities and wall shear stress in small arterial vessels and highly branched sinusoidal capillaries, and the cellular dynamics of hematopoietic stem and progenitor cell homing.

Confocal/two-photon microscopy in studying colonisation of cancer cells in bone using xenograft mouse models.

Confocal and two-photon microscopy has been widely used in bone research to not only produce high quality, three-dimensional images but also to provide valuable structural and quantitative information. In this article, we describe step-by-step protocols for confocal and two-photon microscopy to investigate earlier cellular events during colonisation of cancer cells in bone using xenograft mouse models. This includes confocal/two-photon microscopy imaging of paraformaldehyde fixed thick bone sections and frozen bone samples.

Blood flow controls bone vascular function and osteogenesis.

While blood vessels play important roles in bone homeostasis and repair, fundamental aspects of vascular function in the skeletal system remain poorly understood. Here we show that the long bone vasculature generates a peculiar flow pattern, which is important for proper angiogenesis. Intravital imaging reveals that vessel growth in murine long bone involves the extension and anastomotic fusion of endothelial buds. Impaired blood flow leads to defective angiogenesis and osteogenesis, and downregulation of Notch signalling in endothelial cells. In aged mice, skeletal blood flow and endothelial Notch activity are also reduced leading to decreased angiogenesis and osteogenesis, which is reverted by genetic reactivation of Notch. Blood flow and angiogenesis in aged mice are also enhanced on administration of bisphosphonate, a class of drugs frequently used for the treatment of osteoporosis. We propose that blood flow and endothelial Notch signalling are key factors controlling ageing processes in the skeletal system.

Vascular niches for disseminated tumour cells in bone.

The vasculature of the skeletal system regulates osteogenesis and hematopoiesis, in addition to its primary function as a transportation network. Recent studies suggest that the vasculature in bone regulates multiple steps involved in the metastatic cascade. Matrix and growth factor abundant vascular microenvironments in bone not only provide a fertile soil for the metastatic growth but also support the dormancy of Disseminated Tumour Cells (DTCs). Interestingly, vasculature also seems to direct the reactivation of dormant DTCs. Targeting such early steps of bone metastasis by directing therapies against vascular niches can lead to the development of effective therapeutic strategies that delay or even prevent the metastatic relapse. However, this would require a detailed understanding of the regulatory mechanisms that govern the interaction between endothelial cells and DTCs in the early stages of bone metastasis. This review aims to highlight the importance of vascular niches and outline their newly identified roles during bone metastasis.

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.