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Thrombospondins (TSPs) belong to a functional class of ECM proteins called matricellular proteins that are not primarily structural, but instead influence cellular interactions within the local extracellular environment. The 3D arrangement of TSPs allow interactions with other ECM proteins, sequestered growth factors, and cell surface receptors. They are expressed in mesenchymal condensations and limb buds during skeletal development, but they are not required for patterning. Instead, when absent, there are alterations in musculoskeletal connective tissue ECM structure, organization, and function, as well as altered skeletal cell phenotypes. Both functional redundancies and unique contributions to musculoskeletal tissue structure and physiology are revealed in mouse models with compound TSP deletions. Crucial roles of individual TSPs are revealed during musculoskeletal injury and regeneration. The interaction of TSPs with mesenchymal stem cells (MSC), and their influence on cell fate, function, and ultimately, musculoskeletal phenotype, suggest that TSPs play integral, but as yet poorly understood roles in musculoskeletal health. Here, unique and overlapping contributions of trimeric TSP1/2 and pentameric TSP3/4/5 to musculoskeletal cell and matrix physiology are reviewed. Opportunities for new research are also noted., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Objectives: Synovium is acutely affected following joint trauma and contributes to post-traumatic osteoarthritis (PTOA) progression. Little is known about discrete cell types and molecular mechanisms in PTOA synovium. We aimed to describe synovial cell populations and their dynamics in PTOA, with a focus on fibroblasts. We also sought to define mechanisms of synovial Wnt/β-catenin signalling, given its emerging importance in arthritis., Methods: We subjected mice to non-invasive anterior cruciate ligament rupture as a model of human joint injury. We performed single-cell RNA-sequencing to assess synovial cell populations, subjected Wnt-GFP reporter mice to joint injury to study Wnt-active cells, and performed intra-articular injections of the Wnt agonist R-spondin 2 (Rspo2) to assess whether gain of function induced pathologies characteristic of PTOA. Lastly, we used cultured fibroblasts, macrophages and chondrocytes to study how Rspo2 orchestrates crosstalk between joint cell types., Results: We uncovered seven distinct functional subsets of synovial fibroblasts in healthy and injured synovium, and defined their temporal dynamics in early and established PTOA. Wnt/β-catenin signalling was overactive in PTOA synovium, and Rspo2 was strongly induced after injury and secreted exclusively by Prg4 hi lining fibroblasts. Trajectory analyses predicted that Prg4 hi lining fibroblasts arise from a pool of Dpp4+ mesenchymal progenitors in synovium, with SOX5 identified as a potential regulator of this emergence. We also showed that Rspo2 orchestrated pathological crosstalk between synovial fibroblasts, macrophages and chondrocytes., Conclusions: Synovial fibroblasts assume distinct functional identities during PTOA in mice, and Prg4 hi lining fibroblasts secrete Rspo2 that may drive pathological joint crosstalk after injury., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

The trimeric thrombospondin homologs, TSP1 and TSP2, are both components of bone tissue and contribute in redundant and distinct ways to skeletal physiology. TSP1-null mice display increased femoral cross-sectional area and thickness due to periosteal expansion, as well as diminished matrix quality and impaired osteoclast function. TSP2-null mice display increased femoral cross-sectional thickness and reduced marrow area due to increased endosteal osteoblast activity, with very little periosteal expansion. Osteoblast lineage cells are reduced in TSP2-null mice, but not in TSP1-null. The functional effects of combined TSP1 and TSP2 deficiency remain to be elucidated. Here, we examined the spectrum of detergent soluble proteins in diaphyseal cortical bone of growing (6-week old) male and female mice deficient in both thrombospondins (double knockout (DKO)). Of 3429 detected proteins, 195 were differentially abundant in both male and female DKO bones. Physiologically relevant annotation terms identified by Ingenuity Pathway Analysis included "ECM degradation" and "Quantity of Monocytes." Manual inspection revealed that a number of proteins with shared expression among osteoclasts and osteocytes were reduced in DKO bones. To associate changes in protein content with phenotype, we examined 12-week old male and female DKO and WT mice. DKO mice were smaller than WT and in male DKO, femoral cross section area was reduced. Some of the male DKO femora also had a flattened, less circular cross-section. Male DKO bones were less stiff in bending and they displayed reduced ultimate load. Displacements at yield load and at max load were both elevated in male DKO. However, the ratios of post-yield to pre-yield displacements significantly diminished in DKO suggesting proportionally reduced post-yield behavior. Male DKO mice also exhibited reductions in trabecular bone mass, which were surprisingly associated with equivalent osteoblast numbers and accordingly increased osteoblast surface. Marrow-derived colony forming unit-fibroblastic was reduced in male and female DKO mice. Together our data suggest that when both TSP1 and TSP2 are absent, a unique, sex-specific bone phenotype not predicted by the single knockouts, is manifested., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Fracture healing is a complex process that relies heavily on the carefully orchestrated expansion and differentiation of periosteal mesenchymal progenitor cells (MSC). Identification of new markers for periosteal MSCs is essential for the development of fracture therapeutics. Expression of the matricellular protein thrombospondin-2 (TSP2) increases during early fracture healing; however, it is currently unknown what cell population expresses TSP2. Using a TSP2 GFP reporter mouse and a stabilized murine fracture model, we characterized the expression of TSP2 during the inflammatory, soft callus formation, and hard callus formation phases of fracture healing. In addition, using TSP2 GFP positive cells harvested from reporter mouse cells, we characterized the cell population using flow cytometry and colony formation assays. In uninjured diaphyseal bone, we observed TSP2 expression in the cells located along the inner periosteum. We also observed a population of TSP2 expressing cells in undifferentiated regions of early fracture callus and along the periphery of the callus. Later in callus development, TSP2 cells were broadly distributed in the undifferentiated callus, but GFP was not expressed by chondrocytes. Flow cytometry confirmed that the majority of TSP2 expressing cells were positive for traditional murine MSC markers. Our in vitro assays further supported these findings by demonstrating all adherent and colony-forming cells expressed TSP2. Taken together, our results suggest that TSP2 is expressed by undifferentiated MSCs, but downregulated in chondrocytes. Clinical significance: expression of the matricellular protein TSP2 is a promising new marker to identify MSCs in early fracture healing., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Purpose: To determine thrombospondin-2 (TSP2) expression and its impact on postnatal retinal vascular development and retinal neovascularization., Methods: The TSP2-deficient (TSP2(-/-)) mice and a line of TSP2 reporter mice were used to assess the expression of TSP2 during postnatal retinal vascular development and neovascularization. The postnatal retinal vascularization was evaluated using immunostaining of wholemount retinas prepared at different postnatal days by collagen IV staining and/or TSP2 promoter driven green fluorescent protein (GFP) expression. The organization of astrocytes was evaluated by glial fibrillary acidic protein (GFAP) staining. Retinal vascular densities were determined using trypsin digestion preparation of wholemount retinas at 3- and 6-weeks of age. Retinal neovascularization was assessed during the oxygen-induced ischemic retinopathy (OIR). Choroidal neovascularization (CNV) was assessed using laser-induced CNV., Results: Using the TSP2-GFP reporter mice, we observed significant expression of TSP2 mRNA in retinas of postnatal day 5 (P5) mice, which increased by P7 and remained high up to P42. Similar results were observed in retinal wholemount preparations, and western blotting for GFP with the highest level of GFP was observed at P21. In contrast to high level of mRNA at P42, the GFP fluorescence or protein level was dramatically downregulated. The primary retinal vasculature developed at a faster rate in TSP2(-/-) mice compared with TSP2(+/+) mice up to P5. However, the developing retinal vasculature in TSP2(+/+) mice caught up with that of TSP2(-/-) mice after P7. No significant differences in retinal vascular density were observed at 3- or 6-weeks of age. TSP2(-/-) mice also exhibited a similar sensitivity to the hyperoxia-mediated vessel obliteration and similar level of neovascularization during OIR as TSP2(+/+) mice. Lack of TSP2 expression minimally affected laser-induced CNV compared with TSP2(+/+) mice., Conclusions: Lack of TSP2 expression was associated with enhanced retinal vascularization during early postnatal days but not at late postnatal times, and minimally affected retinal and CNV. However, the utility of TSP2 as a potential therapeutic target for inhibition of ocular neovascularization awaits further investigation.

R-spondins are a family of four matricellular proteins produced by a variety of cell-types. Structurally, R-spondins contain a TSR1 domain that retains the tryptophan structure and a modified cysteine-rich CSVCTG region. In addition, the R-spondins contain two furin repeats implicated in canonical Wnt signaling. R-spondins positively regulate canonical Wnt signaling by reducing Wnt receptor turnover and thereby increasing beta-catenin stabilization. R-spondins are prominently expressed in the developing skeleton and contribute to limb formation, particularly of the distal digit. Additionally, results suggest that R-spondins may contribute to the maintenance of adult bone mass by regulating osteoblastogenesis and bone formation., (Copyright © 2014. Published by Elsevier B.V.)

It is often difficult to decouple the relative importance of different factors in regulating MSC differentiation. Genetically modified mice provide model systems whereby some variables can be manipulated while others are kept constant. Fracture repair in thrombospondin-2 (TSP2)-null mice is characterized by reduced endochondral ossification and enhanced intramembranous bone formation. The proposed mechanism for this shift in MSC fate is that increased vascular density and hence oxygen availability in TSP2-null mice regulates differentiation. However, TSP2 is multifunctional and regulates other aspects of the regenerative cascade, such as MSC proliferation. The objective of this study is to use a previously developed computational model of tissue differentiation, in which substrate stiffness and oxygen tension regulate stem cell differentiation, to simulate potential mechanisms which may drive alterations in MSC fate in TSP2-null mice. Four models (increased cell proliferation, increased numbers of MSCs in the marrow decreased cellular oxygen consumption, and an initially stiffer callus) were not predictive of experimental observations in TSP2-null mice. In contrast, increasing the rate of angiogenic progression led to a prediction of greater intramembranous ossification, diminished endochondral ossification, and a reduced region of hypoxia in the fracture callus similar to that quantified experimentally by the immunohistochemical detection of pimonidazole adducts that develop with hypoxia. This study therefore provides further support for the hypothesis that oxygen availability during early fracture healing is a key regulator of MSC bipotential differentiation, and furthermore, it highlights the advantages of integrating computational models with genetically modified mouse studies for further elucidating mechanisms regulating stem cell fate., (Copyright © 2013 Orthopaedic Research Society.)

Thrombospondin-2 (TSP2) is a matricellular protein that is highly up-regulated during fracture healing. TSP2 negatively regulates vascularity, vascular reperfusion following ischemia, and cutaneous wound healing. As well, TSP2-null mice show increased endocortical bone formation due to an enhanced number of mesenchymal progenitor cells and show increased cortical thickness. Mice deficient in TSP2 (TSP2-null) show an alteration in fracture healing, that is unrelated to their cortical bone phenotype, which is characterized by enhanced vascularization with a shift towards an intramembranous healing phenotype; thus, we hypothesized that there would be enhanced ischemic fracture healing in the absence of TSP2. We investigated whether an absence of TSP2 would enhance ischemic fracture healing utilizing Laser doppler, µCT and histological analysis. Ischemic tibial fractures were created in wildtype (WT) and TSP2-null mice and harvested 10, 20, or 40 days post-fracture. TSP2-null mice show enhanced vascular perfusion following ischemic fracture. At day 10 post-fracture, TSP2-null mice have 115% greater bone volume than WT mice. This is associated with a 122% increase in vessel density, 20% increase in cell proliferation, and 15% decrease in apoptosis compared to WT. At day 20, TSP2-null mice have 34% more bone volume, 51% greater bone volume fraction, and 37% more bone tissue mineral density than WT. By 40 days after fracture the TSP2-null mice have a 24% increase in bone volume fraction, but other parameters show no significant differences. These findings indicate TSP2 is a negative regulator of ischemic fracture healing and that in the absence of TSP2 bone regeneration is enhanced., (Copyright © 2012 Orthopaedic Research Society.)

The matricellular protein thrombospondin-2 (TSP2) inhibits proliferation and enhances osteoblastogenesis of multipotent mesenchymal progenitor cells (MPC). Osteoblastogenesis and adipogenesis are reciprocally regulated, thus we hypothesized that TSP2 could be an inhibitor of adipogenesis. TSP2 gene expression is up-regulated during MPC osteoblast differentiation and down-regulated during adipogenic differentiation through a cAMP-PKA pathway, relative to control cells. Next, the importance of TSP2 in adipogenesis was studied by comparing gene-targeted knockout mice that lack TSP2 protein (TSP2-null) and control wild-type mice. TSP2-null marrow-derived MPC show 25% increased adipocytes. Similarly, TSP2-null adipose-derived MPC show increased adipocytes (25-50%) and proliferation (2-fold) relative to wild-type cells. However, the increase in TSP2-null adipocytes is not due to an increase in MPC number, because the percentage of adipocytes relative to total MPC is still significantly increased. Surprisingly, there are only minor alterations in the adipogenic transcriptional program (PPARγ and C/EBPα expression). Weight gain over time was evaluated in TSP2-null and control wild-type mice fed normal and high-fat diets. Female TSP2-null mice are 30% heavier than wild-type controls due to an increase in non-visceral adipose tissue, measured by dual-energy X-ray absorptiometry (DEXA) and direct weighing of fat pads. These results show that TSP2 is an endogenous matricellular protein produced by MPC that in addition to enhancing osteoblastogenesis, inhibits adipocytes and decreases subcutaneous adiposity., (Copyright © 2010 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Thrombospondins (TSPs) are a family of five secreted multimeric matricellular proteins that share homology in the type II and III repeats and carboxy-terminal region. Type I repeats, also known as properdin or thrombospondin repeats (TSRs), are found in TSP1/2, but not TSP3-5. A variety of other secreted proteins contain TSRs, including the novel extracellular molecules, R-spondins. TSP family and many TSR-containing proteins, including R-spondins, are highly expressed in skeletal tissues during development and postnatal. TSP2 regulates the osteoblast lineage, influencing bone mass and geometry, as well as response to fracture healing, ovariectomy, and mechanical loading. Compound knockout mice of TSPs have revealed important mechanistic insights. TSP1/2 knockout mice have craniofacial dysmorphism, and TSP1/3/5 compound knockout mice display growth plate abnormalities. R-spondins promote osteoblast differentiation and R-spondin-2 deficiency results in skeletal developmental defects. Overall, TSP and other TSR molecules influence multiple aspects of bone development and remodeling.

The matricellular protein thrombospondin-2 (TSP2) has context-dependent effects on osteoblast lineage proliferation and differentiation. Mice lacking TSP2 display increased endocortical bone thickness, which is associated with increased marrow stromal cell (MSC) number and in vitro proliferation. TSP2-null MSC also exhibit delayed osteoblastogenesis and enhanced adipogenesis compared to cells harvested from wild type mice. The goal of the present work was to more precisely characterize the contribution that TSP2 makes to the maturation of osteoblast-derived extracellular matrix (ECM) using a highly characterized pre-osteoblast cell line. Specifically, we asked whether TSP2 influences mineralization indirectly through its known effects on proliferation, or whether TSP2 directly promotes osteoblast differentiation. To pursue these questions, we used RNA-interference (RNAi) to inhibit TSP2 gene expression in MC3T3-E1 pre-osteoblasts. Introduction of siRNA oligonucleotides resulted in reduced TSP2 mRNA expression as early as 24 h post-transfection, and TSP2 mRNA levels remained low for 10 days. Similarly, TSP2 protein levels in both conditioned medium and the cell-matrix layer were reduced at 24 h post-transfection and remained reduced for 7 days. At day 21, mineralization was significantly reduced in cells transfected with TSP2 siRNA when compared to cells treated with scrambled siRNA. This decrease in mineralization occurred without a concomitant change in cell number. Twenty-four hours after transfection, runx2 gene expression was transiently enhanced in TSP2 siRNA-treated cultures. Between 6 and 14 days post-transfection, runx2, osterix, alkaline phosphatase, type I collagen, osteocalcin and bone sialoprotein all displayed moderate increases in gene expression with TSP2 RNAi. As well, soluble osteocalcin levels were markedly higher in the conditioned medium of cells treated with TSP2 siRNA than in control siRNA-treated cells. Increased soluble osteocalcin occurred without a concomitant change in the levels of osteocalcin in the cell-ECM layer. TSP2 reduction also elicited a transient change in the distribution of collagen between the acid soluble cell-ECM protein fraction and the insoluble matrix. Together, our data suggest that TSP2 may promote mineralization, by facilitating proper organization of the osteoblast-derived ECM., ((c) 2009 Elsevier Inc. All rights reserved.)

Thrombospondin-2 (TSP2) is a matricellular protein with increased expression during growth and regeneration. TSP2-null mice show accelerated dermal wound healing and enhanced bone formation. We hypothesized that bone regeneration would be enhanced in the absence of TSP2. Closed, semistabilized transverse fractures were created in the tibias of wildtype (WT) and TSP2-null mice. The fractures were examined 5, 10, and 20 days after fracture using microCT, histology, immunohistochemistry, quantitative RT-PCR, and torsional mechanical testing. Ten days after fracture, TSP2-null mice showed 30% more bone by microCT and 40% less cartilage by histology. Twenty days after fracture, TSP2-null mice showed reduced bone volume fraction and BMD. Mice were examined 5 days after fracture during the stage of neovascularization and mesenchymal cell influx to determine a cellular explanation for the phenotype. TSP2-null mice showed increased cell proliferation with no difference in apoptosis in the highly cellular fracture callus. Although mature bone and cartilage is minimal 5 days after fracture, TSP2-null mice had reduced expression of collagen IIa and Sox9 (chondrocyte differentiation markers) but increased expression of osteocalcin and osterix (osteoblast differentiation markers). Importantly, TSP2-null mice had a 2-fold increase in vessel density that corresponded with a reduction in vascular endothelial growth factor (VEGF) and Glut-1 (markers of hypoxia inducible factor [HIF]-regulated transcription). Finally, by expressing TSP2 using adenovirus starting 3 days after fracture, chondrogenesis was restored in TSP2-null mice. We hypothesize that TSP2 expressed by cells in the fracture mesenchyme regulates callus vascularization. The increase in vascularity increases tissue oxemia and decreases HIF; thus, undifferentiated cells in the callus develop into osteoblasts rather than chondrocytes. This leads to an alternative strategy for achieving fracture healing with reduced endochondral ossification and enhanced appositional bone formation. Controlling the ratio of cartilage to bone during fracture healing has important implications for expediting healing or promoting regeneration in nonunions.

Wnt11 signals through both canonical (beta-catenin) and non-canonical pathways and is up-regulated during osteoblast differentiation and fracture healing. In these studies, we evaluated the role of Wnt11 during osteoblastogenesis. Wnt11 overexpression in MC3T3E1 pre-osteoblasts increases beta-catenin accumulation and promotes bone morphogenetic protein (BMP)-induced expression of alkaline phosphatase and mineralization. Wnt11 dramatically increases expression of the osteoblast-associated genes Dmp1 (dentin matrix protein 1), Phex (phosphate-regulating endopeptidase homolog), and Bsp (bone sialoprotein). Wnt11 also increases expression of Rspo2 (R-spondin 2), a secreted factor known to enhance Wnt signaling. Overexpression of Rspo2 is sufficient for increasing Dmp1, Phex, and Bsp expression and promotes bone morphogenetic protein-induced mineralization. Knockdown of Rspo2 abrogates Wnt11-mediated osteoblast maturation. Antagonism of T-cell factor (Tcf)/beta-catenin signaling with dominant negative Tcf blocks Wnt11-mediated expression of Dmp1, Phex, and Rspo2 and decreases mineralization. However, dominant negative Tcf fails to block the osteogenic effects of Rspo2 overexpression. These studies show that Wnt11 signals through beta-catenin, activating Rspo2 expression, which is then required for Wnt11-mediated osteoblast maturation.

Extracellular thrombospondins (TSP or THBS) and the Notch family of transmembrane receptors share a role in multiple, overlapping cellular functions and participate in developmental signaling and pathological reactions to tissue injury. We demonstrate that TSP2, but not TSP1, enhances the potency of Notch3 signal transduction. In addition, TSP2 reduces cancer cell proliferation in a Notch-ligand dependent fashion. The loss of TSP2 in knock-out mice reduces Notch target gene expression. TSP2 binds directly to Notch3 and Jagged1. TSP1 also binds to Notch3 and Jagged1; however, only TSP2 augments the interaction between Notch3 and Jagged1. These studies demonstrate that the diverse functions of TSP2 may also include a role as an intermediary protein that facilitates transcellular receptor-ligand interactions.

Unlabelled: Fra1 transgenic (Tg) mice develop osteosclerosis and exhibit altered expression of bone matrix proteins. We found that expression of Thbs1 and Thbs2 was reduced in Fra1 Tg osteoblasts. Fra1 Tg and non-osteosclerotic Thbs1-/-Thbs2-/- mice share an edge-to-edge bite. Therefore, reduced expression of thrombospondins may contribute to craniofacial dysmorphism independently of osteosclerosis., Introduction: Tg mice overexpressing Fra1, a component of the transcription factor activator protein-1 (AP-1), show progressive osteosclerosis caused by cell autonomous abnormalities in osteoblasts. The expression of several bone matrix proteins, including matrix gla protein, is dysregulated in Fra1 Tg osteoblasts., Materials and Methods: In osteoblastogenic cultures, altered bone matrix production by Fra1 overexpression was monitored using Alizarin red staining, quantitative RT-PCR, and Western blotting. Responsiveness to ovariectomy was examined by bone histomorphometry. Craniofacial parameters were measured on radiographs and using CT., Results: Thrombospondin-1 (Thbs1) and thrombospondin-2 (Thbs2) were reduced in Fra1 Tg osteoblasts differentiated in vitro and in bones from Fra1 Tg mice. Despite alterations in bone matrix proteins, ovariectomy induces high turnover bone loss in Fra1 Tg mice as in wildtype mice. Fra1 Tg mice, as well as Thbs1-/- Thbs2-/- mice, which do not show osteosclerosis, exhibit an edge-to-edge bite phenotype associated with craniofacial dysmorphism., Conclusions: These data suggest that reduced expression of thrombospondins in Fra1 Tg mice underlies craniofacial dysmorphism, independent of osteosclerosis.

Thrombospondin 2 (TSP2) is an extracellular matrix (ECM) protein localized to bone. Since mice with a targeted disruption of the TSP2 gene (TSP2-null) have increased bone formation, we hypothesized that mice lacking TSP2 would show an enhanced osteogenic response to mechanical loading. We addressed our hypothesis by subjecting wild-type (WT) and TSP2-null mice to mechanical loading using the non-invasive murine tibia loading device, and statistical comparisons were made between loaded and unloaded bones within genotype, between genotypes, and between the periosteal and endocortical surfaces within genotype. Right tibiae of WT and TSP2-null mice received 5 days of a low-magnitude loading protocol. This low-magnitude loading (inducing approximately 900 and 500 muepsilon at periosteal and endocortical surfaces of WT bones, respectively) affected neither periosteal nor endocortical bone formation rate (BFR/BS) when comparing loaded to intact bones in either WT or TSP2-null mice, nor did it result in any significant differences between WT and TSP2-null. As well, there was no difference between loaded endocortical and periosteal surfaces in WT mice; however, endocortical BFR/BS in TSP2-null loaded tibia was significantly elevated relative to the periosteal BFR/BS-despite peak periosteal strains being significantly greater than endocortical strains in TSP2-null mice (690 versus 460 muepsilon). To confirm this counterintuitive surface-specific response in TSP2-null mice and to induce significant periosteal bone formation, osteogenic potency of the loading protocol was amplified by doubling the number of loading bouts (10 loading days) and loading magnitude (1 Hz, resulting in 1400 and 900 muepsilon peak strain at the periosteal and endocortical surfaces, respectively). Under load, both WT and TSP2-null mice showed significantly increased periosteal mineralizing surface (by nearly three-fold and five-fold, respectively), but mineral apposition rate (MAR) was not statistically changed. The increased MS/BS resulted in a five-fold increase in WT periosteal BFR/BS, but the TSP2-null periosteal BFR/BS was unchanged. Furthermore, this increase in WT loaded periosteal BFR/BS was statistically greater than the WT endocortical BFR/BS. At the endocortical surface of WT mice, loading did not significantly increase bone formation parameters (versus intact). In contrast, at the endocortical surface of TSP2-null mice, loading induced a significant two-fold increase in BFR/BS (versus intact), that was also significantly greater than the endocortical BFR/BS of loaded WT mice. Thus, exogenous loading of TSP2-null mice resulted in highly variable responses that did not reflect the induced strains at the periosteal and endocortical surfaces. While in WT mice, loading resulted in increased periosteal BFR/BS that was greater than the endocortical BFR/BS, in TSP2-null mice loading resulted in endocortical (not periosteal) BFR/BS that was elevated. This reversal in envelope-specific bone formation in TSP2-null mice occurred despite periosteal strains being significantly greater than endocortical (1290 versus 775 muepsilon) and strain distributions being similar to that of WT. These results show that the disruption of a single gene can lead to a reversal in normal pattern of load induced bone formation, and more specifically, that the functional interaction of TSP2 with mechanical loading is highly contextual and specific to the cortical bone envelope examined.

Thrombospondin 3 (TSP3) is structurally similar to cartilage oligomeric matrix protein (COMP/TSP5), but its function is unknown. To determine the functional significance of TSP3, we generated mice with a targeted disruption of Thbs3. TSP3-null mice are viable and fertile and show normal prenatal skeletal patterning, based on Alcian blue/Alizarin red S staining. However, subtle and transient abnormalities were detected in the developing postnatal skeleton. Young adult TSP3-null mice are heavier than controls, and analyses of the geometric and biomechanical properties of long bones show increases in the moments of inertia, endocortical and periostal radii, and failure load. The bones of 9-week-old TSP3-null male mice also have a significantly greater cortical area. Most of these differences were no longer detected in 15-week-old mice. Micro-computed tomography scans showed that the trabecular bone proximal to the femoral head growth plate developed at an earlier time in TSP3-null mice than in wild-type mice. Thus, vascular invasion and ossification start in the femoral heads of TSP3-null mice at 9 weeks, whereas the wild-type femoral head is still composed of hypertrophic chondroctyes in a calcified matrix at 15 weeks. These results provide evidence for a role for TSP3 in the regulation of skeletal maturation in mice.

Mice that lack the matricellular angiogenesis inhibitor, thrombospondin-2 (TSP2), display a bleeding diathesis, despite normal blood coagulation and the lack of thrombocytopenia. Although platelets do not contain detectable levels of TSP2, TSP2-null platelets are compromised in their ability to aggregate in vivo in response to denudation of the carotid artery endothelium, and in vitro following exposure to adenosine diphosphate (ADP). Megakaryocytes (MKs) show high levels of TSP2 by immunohistochemical analysis of bone marrow. However, when cultured in vitro, MKs contain little TSP2 protein or mRNA. These findings suggest that most TSP2 is acquired from the bone marrow microenvironment. Consistent with this hypothesis, MKs take up recombinant TSP2 in an integrin-dependent manner when it is supplied in the culture medium. Furthermore, uptake of TSP2 in vitro affects MK differentiation and proplatelet formation. The functional significance of this process is supported by the presence of ultrastructural abnormalities in TSP2-null bone marrow, including extensive fragmentation of the peripheral zone in MKs and failure of this zone to form close associations with vascular sinuses. We conclude that the uptake of TSP2 by MKs from the marrow milieu is required for proper MK function and the release of functionally competent platelets.

The matricellular protein thrombospondin 2 (TSP2) regulates a variety of cell-matrix interactions. A prominent feature of TSP2-null mice is increased microvascular density, particularly in connective tissues synthesized after injury. We investigated the cellular basis for the regulation of angiogenesis by TSP2 in cultures of murine and human fibroblasts and endothelial cells. Fibroblasts isolated from murine and human dermis synthesize TSP2 mRNA and secrete significant amounts of immunoreactive TSP2, whereas endothelial cells from mouse lung and human dermis did not synthesize TSP2 mRNA or protein. Recombinant mouse TSP2 inhibited growth of human microvascular endothelial cells (HMVECs) mediated by basic fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, and vascular endothelial growth factor (VEGF). HMVECs exposed to TSP2 in the presence of these growth factors had a decreased proportion of cells in S and G2/M phases. HMVECs cultured with a combination of basic fibroblast growth factor, insulin-like growth factor-1, and epidermal growth factor displayed an increased proportion of nonviable cells in the presence of TSP2, but the addition of VEGF blocked this TSP2-mediated impairment of cell viability. TSP2-mediated inhibition of DNA synthesis by HMVECs in the presence of VEGF was not affected by the broad-spectrum caspase inhibitor zVAD-fmk. Similar findings were obtained with TSP1. Taken together, these observations indicate that either TSP2 or TSP1 can inhibit HMVEC proliferation by inhibition of cell cycle progression and induction of cell death, but the mechanisms responsible for TSP2-mediated inhibition of cell cycle progression are independent from those leading to cell death.

Marrow stromal cells (MSCs) are obtained in increased number from mice in which the thrombospondin 2 (TSP2) gene is disrupted, and these cells show increased DNA synthesis in vitro. To examine more closely the role of TSP2 in the physiology and osteogenic differentiation of MSCs, an in-depth characterization of TSP2-null MSCs was conducted. Determination of TSP2 protein content by Western analysis and RNA levels by reverse-transcription polymerase chain reaction (RT-PCR) indicated that MSCs are the primary source of TSP2 in the marrow and secrete abundant TSP2 into culture medium. Morphologically, the TSP2-null and wild-type (WT) cell populations were similar and by flow cytometry contained equivalent numbers of CD44+, Mac1+, intercellular adhesion molecule-1 (ICAM-1+), and ScaI+ cells. TSP2-null cells showed delayed mineralization associated with an increased rate of proliferation. Consistent with this finding, there was a decrease in expression of collagen and osteocalcin RNA by TSP2-null MSCs on day 7 and increased osteopontin expression on day 7 and day 14. In add-back experiments, recombinant TSP2 produced a dose-dependent decrease in proliferation. This reduction was associated with an accumulation of TSP2-treated cells in the G1 phase of the cell cycle and did not result from an increase in apoptosis. When TSP2 treatment was terminated, the cell population reentered the S phase. We conclude that the increased endosteal bone formation observed in TSP2-null mice results primarily from the failure of TSP2 to regulate locally MSC cell cycle progression.

Nuclear factor-κB (NF-κB) is a master regulator of genes that control a large number of cellular processes, including angiogenesis and inflammation. We recently demonstrated that cytochrome P-450 1B1 (Cyp1B1) deficiency in endothelial cells (EC) and pericytes (PC) results in increased oxidative stress, alterations in migration, attenuation of capillary morphogenesis, sustained activation of NF-κB, and increased expression of thrombospondin-2 (TSP2), an endogenous inhibitor of angiogenesis. On the basis of a growing body of evidence that phenethyl isothiocyanate (PEITC) and pyrrolidine dithiocarbamate (PDTC) function as antioxidants and suppressors of NF-κB activation, we investigated their potential ability to restore a normal phenotype in Cyp1B1-deficient (cyp1b1-/-) vascular cells. PEITC and PDTC inhibited NF-κB activity and expression in cyp1b1-/- EC and PC. We also observed restoration of migration and capillary morphogenesis of cyp1b1-/- EC and decreased cellular oxidative stress in cyp1b1-/- EC and PC without restoration to normal TSP2 levels. In addition, expression of a dominant-negative inhibitor κB, a suppressor of NF-κB activation, decreased NF-κB activity without affecting TSP2 expression in these cells. In contrast, knockdown of TSP2 expression resulted in attenuation of NF-κB activity in cyp1b1-/- vascular cells. Furthermore, expression of TSP2 in wild-type (cyp1b1+/+) cells resulted in increased NF-κB activity. Together, our results demonstrate an important role for TSP2 in modulation of NF-κB activity and attenuation of angiogenesis. Thus Cyp1B1 expression in vascular cells plays an important role in the regulation of vascular homeostasis through modulation of the cellular reductive state, TSP2 expression, and NF-κB activation. [ABSTRACT FROM AUTHOR]

Abstract: Angiogenesis is a key component of bone repair. New blood vessels bring oxygen and nutrients to the highly metabolically active regenerating callus and serve as a route for inflammatory cells and cartilage and bone precursor cells to reach the injury site. Angiogenesis is regulated by a variety of growth factors, notably vascular endothelial growth factor (VEGF), which are produced by inflammatory cells and stromal cells to induce blood vessel in-growth. A variety of studies with transgenic and gene-targeted mice have demonstrated the importance of angiogenesis in fracture healing, and have provided insights into regulatory processes governing fracture angiogenesis. Indeed, in animal models enhancing angiogenesis promotes bone regeneration, suggesting that modifying fracture vascularization could be a viable therapeutic approach for accelerated/improved bone regeneration clinically. [Copyright &y& Elsevier]

Extracellular thrombospondins (TSP or THBS) and the Notch family of transmembrane receptors share a role in multiple, overlapping cellular functions and participate in developmental signaling and pathological reactions to tissue injury. We demonstrate that TSP2, but not TSP1, enhances the potency of Notch3 signal transduction, In addition, TSP2 reduces cancer cell proliferation in a Notch-ligand dependent fashion. The loss of TSP2 in knock-out mice reduces Notch target gene expression. TSP2 binds directly to Notch3 and Jaggedi. TSP1 also binds to Notch3 and Jagged 1; however, only TSP2 augments the interaction between Notch3 and Jagged 1. These studies demonstrate that the diverse functions of TSP2 may also include a role as an intermediary protein that facilitates transcellular receptor-ligand interactions. [ABSTRACT FROM AUTHOR]