Modified skulls but conservative brains? The palaeoneurology and endocranial anatomy of baryonychine dinosaurs (Theropoda: Spinosauridae).

The digital reconstruction of neurocranial endocasts has elucidated the gross brain structure and potential ecological attributes of many fossil taxa, including Irritator, a spinosaurine spinosaurid from the "mid" Cretaceous (Aptian) of Brazil. With unexceptional hearing capabilities, this taxon was inferred to integrate rapid and controlled pitch-down movements of the head that perhaps aided in the predation of small and agile prey such as fish. However, the neuroanatomy of baryonychine spinosaurids remains to be described, and potentially informs on the condition of early spinosaurids. Using micro-computed tomographic scanning (µCT), we reconstruct the braincase endocasts of Baryonyx walkeri and Ceratosuchops inferodios from the Wealden Supergroup (Lower Cretaceous) of England. We show that the gross endocranial morphology is similar to other non-maniraptoriform theropods, and corroborates previous observations of overall endocranial conservatism amongst more basal theropods. Several differences of unknown taxonomic utility are noted between the pair. Baryonychine neurosensory capabilities include low-frequency hearing and unexceptional olfaction, whilst the differing morphology of the floccular lobe tentatively suggests less developed gaze stabilisation mechanisms relative to spinosaurines. Given the morphological similarities observed with other basal tetanurans, baryonychines likely possessed comparable behavioural sophistication, suggesting that the transition from terrestrial hypercarnivorous ancestors to semi-aquatic "generalists" during the evolution of Spinosauridae did not require substantial modification of the brain and sensory systems.

Sexing Bones: Improving Transparency of Sex Reporting to Address Bias Within Preclinical Studies.

Despite knowledge that sexually dimorphic mechanisms regulate bone homeostasis, sex often remains unreported and unconsidered in preclinical experimental design. Failure to report sex could lead to inappropriate generalizations of research findings and less effective translation into clinical practice. Preclinical sex bias (preferential selection of one sex) is present across other fields, including neuroscience and immunology, but remains uninvestigated in skeletal research. For context, we first summarized key literature describing sexually dimorphic bone phenotypes in mice. We then investigated sex reporting practices in skeletal research, specifically how customary it is for murine sex to be included in journal article titles or abstracts and then determined whether any bias in sex reporting exists. Because sex hormones are important regulators of bone health (gonadectomy procedures, ie, ovariectomy [OVX] and orchidectomy [ORX], are common yet typically not reported with sex), we incorporated reporting of OVX and ORX terms, representing female and male mice, respectively, into our investigations around sex bias. Between 1999 and 2020, inclusion of sex in titles or abstracts was low in murine skeletal studies (2.6%-4.06%). Reporting of OVX and ORX terms was low (1.44%-2.64%) and reporting of OVX and ORX with sex uncommon (0.4%-0.3%). When studies were combined to include both sexes and OVX (representing female) and ORX terms (representing male), a bias toward reporting of female mice was evident. However, when the terms OVX and ORX were removed, a bias toward the use of male mice was identified. Thus, studies focusing on sex hormones are biased toward female reporting with all other studies biased in reporting of male mice. We now call upon journal editors to introduce consistent guidance for transparent and accessible reporting of murine sex in skeletal research to better monitor preclinical sex bias, to diversify development of treatments for bone health, and to enable global skeletal health equity. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

New spinosaurids from the Wessex Formation (Early Cretaceous, UK) and the European origins of Spinosauridae.

Spinosaurids are among the most distinctive and yet poorly-known of large-bodied theropod dinosaurs, a situation exacerbated by their mostly fragmentary fossil record and competing views regarding their palaeobiology. Here, we report two new Early Cretaceous spinosaurid specimens from the Wessex Formation (Barremian) of the Isle of Wight. Large-scale phylogenetic analyses using parsimony and Bayesian techniques recover the pair in a new clade within Baryonychinae that also includes the hypodigm of the African spinosaurid Suchomimus. Both specimens represent distinct and novel taxa, herein named Ceratosuchops inferodios gen. et sp. nov. and Riparovenator milnerae gen. et sp. nov. A palaeogeographic reconstruction suggests a European origin for Spinosauridae, with at least two dispersal events into Africa. These new finds provide welcome information on poorly sampled areas of spinosaurid anatomy, suggest that sympatry was present and potentially common in baryonychines and spinosaurids as a whole, and contribute to updated palaeobiogeographic reconstructions for the clade.

Synthetic Nanoclay Gels Do Not Cause Skin Irritation in Healthy Human Volunteers.

Synthetic clays are promising biomaterials for delivery of therapeutic molecules in regenerative medicine. However, before their use can be translated into clinical applications, their safety must be assessed in human volunteers. The aim of this study was to test the hypothesis that a synthetic nanoclay (LAPONITE) does not cause irritation to the human skin. To achieve this, a nanoclay gel at two different concentrations (1.5 and 3% w/v) was applied on the forearm of healthy volunteers for 24 h. 1% sodium lauryl sulfate (SLS) and 3% (w/v) polyacrylic acid were used as the positive and negative controls, respectively. The compromise in the skin barrier function was measured by trans-epidermal water loss (TEWL), erythema by spectroscopic measurements, and skin inflammatory biomarkers (IL-1α and IL-1RA) by the enzyme-linked immunosorbent assay. We found that the nanoclay caused no prolonged increase in TEWL, erythema, or induction of inflammatory cytokines. This was in contrast to 1% SLS, a known irritant, which induced significant increases in both skin erythema and TEWL. We conclude that the nanoclay is not an irritant and is thus suitable for therapeutic interventions at the skin surface.

Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition.

Collagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF; also known as VEGFA) in bone-forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we used murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Label-free and non-destructive polarisation-resolved second-harmonic generation (p-SHG) microscopy revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed that the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-strand stability, in addition to matrix carbonation and mineralisation. Vegf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure, including Adamts2, Spp1, Mmp9 and Lama1. Our results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders that clinically manifest in pathological nano- and macro-level disorganisation. This article has an associated First Person interview with the first author of the paper.

Raman spectroscopy links differentiating osteoblast matrix signatures to pro-angiogenic potential.

Mineralization of bone is achieved by the sequential maturation of the immature amorphous calcium phase to mature hydroxyapatite (HA) and is central in the process of bone development and repair. To study normal and dysregulated mineralization in vitro, substrates are often coated with poly-l-lysine (PLL) which facilitates cell attachment. This study has used Raman spectroscopy to investigate the effect of PLL coating on osteoblast (OB) matrix composition during differentiation, with a focus on collagen specific proline and hydroxyproline and precursors of HA. Deconvolution analysis of murine derived long bone OB Raman spectra revealed collagen species were 4.01-fold higher in OBs grown on PLL. Further, an increase of 1.91-fold in immature mineral species (amorphous calcium phosphate) was coupled with a 9.32-fold reduction in mature mineral species (carbonated apatite) on PLL versus controls. These unique low mineral signatures identified in OBs were linked with reduced alkaline phosphatase enzymatic activity, reduced Alizarin Red staining and altered osteogenic gene expression. The promotion of immature mineral species and restriction of mature mineral species of OB grown on PLL were linked to increased cell viability and pro-angiogenic vascular endothelial growth factor (VEGF) production. These results demonstrate the utility of Raman spectroscopy to link distinct matrix signatures with OB maturation and VEGF release. Importantly, Raman spectroscopy could provide a label-free approach to clinically assess the angiogenic potential of bone during fracture repair or degenerative bone loss.

Injectable nanoclay gels for angiogenesis.

The retention and sustained activity of therapeutic proteins at delivery sites are goals of regenerative medicine. Vascular endothelial growth factor (VEGF) has significant potential in promoting the growth and regeneration of blood vessels but is intrinsically labile. This is exacerbated by the inflammatory microenvironments at sites requiring regeneration. For VEGF to be efficacious, it may require a carrier that stabilises it, protects it from degradation and retains it at the site of interest. In this study, we tested the hypothesis that injectable nanoclay gels comprising Laponite™ XLG (a synthetic hectorite clay) can stabilise VEGF and retain it in the active form for therapeutic delivery. To achieve this, VEGF was incorporated in Laponite gels and its activity tested at a range of concentrations using in vitro cell culture tubulogenesis assays and in vivo angiogenesis assays. We found that VEGF-Laponite gels enhanced tubulogenesis in a dose-dependent manner in vitro. When administered subcutaneously in vivo, Laponite was retained at the injection site for up to a period of three weeks and promoted a 4-fold increase in blood vessel formation compared with that of alginate or vehicle controls as confirmed by CD31 staining. Notably, as compared to alginate, Laponite gels did not release VEGF, indicating a strong interaction between the growth factor and the nanoclay and suggesting that Laponite enhancement of VEGF efficacy is due to its retention at the implantation site for a prolonged period. Our approach provides a robust method for the delivery of bioactive recombinant VEGF without the necessity for complex hydrogel or protein engineering. STATEMENT OF SIGNIFICANCE: In medicine, it is important to deliver drugs to a particular location in the body. Often, however, the drugs are quickly broken down and carried away in the blood before they can exert their effect. In this study, we used a type of synthetic clay, called Laponite™, to preserve a molecule, named VEGF, that stimulates the growth of blood vessels. Previously, we have been able to bind VEGF to the surface of clays, but the clay is not effective when injected or applied as a gel. Herein, we show that we can mix VEGF with the clay and that it strongly stimulates blood vessel growth. We speculate that this would be a useful material for skin wound healing.

Regulation of the Bone Vascular Network is Sexually Dimorphic.

Osteoblast (OB) lineage cells are an important source of vascular endothelial growth factor (VEGF), which is critical for bone growth and repair. During bone development, pubertal differences in males and females exist, but little is known about whether VEGF signaling contributes to skeletal sexual dimorphism. We have found that in mice, conditional disruption of VEGF in osteocalcin-expressing cells (OcnVEGFKO) exerts a divergent influence on morphological, cellular, and whole bone properties between sexes. Furthermore, we describe an underlying sexual divergence in VEGF signaling in OB cultures in vitro independent of circulating sex hormones. High-resolution synchrotron computed tomography and backscattered scanning electron microscopy revealed, in males, extensive unmineralized osteoid encasing enlarged blood vessel canals and osteocyte lacunae in cortical bone after VEGF deletion, which contributed to increased porosity. VEGF was deleted in male and female long bone-derived OBs (OBVEGKO) in vitro and Raman spectroscopic analyses of mineral and matrix repertoires highlighted differences between male and female OBVEGFKO cells, with increased immature phosphate species prevalent in male OBVEGFKO cultures versus wild type (WT). Further sexual dimorphism was observed in bone marrow endothelial cell gene expression in vitro after VEGF deletion and in sclerostin protein expression, which was increased in male OcnVEGFKO bones versus WT. The impact of altered OB matrix composition after VEGF deletion on whole bone geometry was assessed between sexes, although significant differences between OcnVEGFKO and WT were identified only in females. Our results suggest that bone-derived VEGF regulates matrix mineralization and vascularization distinctly in males and females, which results in divergent physical bone traits.

Simultaneous visualisation of calcified bone microstructure and intracortical vasculature using synchrotron X-ray phase contrast-enhanced tomography.

3D imaging of the bone vasculature is of key importance in the understanding of skeletal disease. As blood vessels in bone are deeply encased in the calcified matrix, imaging techniques that are applicable to soft tissues are generally difficult or impossible to apply to the skeleton. While canals in cortical bone can readily be identified and characterised in X-ray computed tomographic data in 3D, the soft tissue comprising blood vessels that are putatively contained within the canal structures does not provide sufficient image contrast necessary for image segmentation. Here, we report an approach that allows for rapid, simultaneous visualisation of calcified bone tissue and the vasculature within the calcified bone matrix. Using synchrotron X-ray phase contrast-enhanced tomography we show exemplar data with intracortical capillaries uncovered at sub-micrometre level without the need for any staining or contrast agent. Using the tibiofibular junction of 15 week-old C57BL/6 mice post mortem, we show the bone cortical porosity simultaneously along with the soft tissue comprising the vasculature. Validation with histology confirms that we can resolve individual capillaries. This imaging approach could be easily applied to other skeletal sites and transgenic models, and could improve our understanding of the role the vasculature plays in bone disease.

Detection of early osteogenic commitment in primary cells using Raman spectroscopy.

Major challenges in the development of novel implant surfaces for artificial joints include osteoblast heterogeneity and the lack of a simple and sensitive in vitro assay to measure early osteogenic responses. Raman spectroscopy is a label-free, non-invasive and non-destructive vibrational fingerprinting optical technique that is increasingly being applied to detect biochemical changes in cells. In this study Raman spectroscopy has been used to obtain bone cell-specific spectral signatures and to identify any changes therein during osteoblast commitment and differentiation of primary cells in culture. Murine calvarial osteoblasts (COBs) were extracted and cultured and studied by Raman spectroscopy over a 14 day culture period. Distinct osteogenic Raman spectra were identified after 3 days of culture with strong bands detected for mineral: phosphate ν3 (1030 cm-1) and B-type carbonate (1072 cm-1), DNA (782 cm-1) and collagen matrix (CH2 deformation at 1450 cm-1) and weaker phosphate bands (948 and 970 cm-1). Early changes were detected by Raman spectroscopy compared to a standard enzymatic alkaline phosphatase (ALP) assay and gene expression analyses over this period. Proliferation of COBs was confirmed by fluorescence intensity measurements using the Picogreen dsDNA reagent. Changes in ALP levels were evident only after 14 days of culture and mRNA expression levels for ALP, Col1a1 and Sclerostin remained constant during the culture period. Sirius red staining for collagen deposition also revealed little change until day 14. In contrast Raman spectroscopy revealed the presence of amorphous calcium phosphate (945-952 cm-1) and carbonated apatite (957-962 cm-1) after only 3 days in culture and octacalcium phosphate (970 cm-1) considered a transient mineral phase, was detected after 5 days of COBs culture. PCA analysis confirmed clear separation between time-points. This study highlights the potential of Raman spectroscopy to be utilised for the early and specific detection of proliferation and differentiation changes in primary cultures of bone cells.

Are Mesenchymal Stem Cells So Bloody Great After All?

This Perspective discusses some activities of mesenchymal stem cells (MSCs) in the context of angiogenesis, focusing on contrasting effects that could call into question the extent to which MSCs can be used clinically in the future. We report on the antiangiogenic/antiproliferative effects of specific MSC populations (including bone marrow MSCs), their paracrine activity, tissue heterogeneity, and endothelial cell interactions. Also discussed are what could lead to contrasting effects of the influence of MSCs in regulating angiogenesis, pointing to some negative effects of these cells. In conclusion, this article highlights important aspects of MSC behavior within the perspective of translational medicine applications. Stem Cells Translational Medicine 2017;6:3-6.

Sost Deficiency does not Alter Bone's Lacunar or Vascular Porosity in Mice.

SCLEROSTIN (Sost) is expressed predominantly in osteocytes acting as a negative regulator of bone formation. In humans, mutations in the SOST gene lead to skeletal overgrowth and increased bone mineral density, suggesting that SCLEROSTIN is a key regulator of bone mass. The function of SCLEROSTIN as an inhibitor of bone formation is further supported by Sost knockout (KO) mice which display a high bone mass with elevated bone formation. Previous studies have indicated that Sost exerts its effect on bone formation through Wnt-mediated regulation of osteoblast differentiation, proliferation, and activity. Recent in vitro studies have also suggested that SCLEROSTIN regulates angiogenesis and osteoblast-to-osteocyte transition. Despite this wealth of knowledge of the mechanisms responsible for SCLEROSTIN action, no previous studies have examined whether SCLEROSTIN regulates osteocyte and vascular configuration in cortices of mouse tibia. Herein, we image tibiae from Sost KO mice and their wild-type (WT) counterparts with high-resolution CT to examine whether lack of SCLEROSTIN influences the morphometric properties of lacunae and vascular canal porosity relating to osteocytes and vessels within cortical bone. Male Sost KO and WT mice (n = 6/group) were sacrificed at 12 weeks of age. Fixed tibiae were analyzed using microCT to examine cortical bone mass and architecture. Then, samples were imaged by using benchtop and synchrotron nano-computed tomography at the tibiofibular junction. Our data, consistent with previous studies show that, Sost deficiency leads to significant enhancement of bone mass by cortical thickening and bigger cross-sectional area and we find that this occurs without modifications of tibial ellipticity, a measure of bone shape. In addition, our data show that there are no significant differences in any lacunar or vascular morphometric or geometric parameters between Sost KO mouse tibia and WT counterparts. We, therefore, conclude that the significant increases in bone mass induced by Sost deficiency are not accompanied by any significant modification in the density, organization, or shape of osteocyte lacunae or vascular content within the cortical bone. These data may imply that SCLEROSTIN does not modify the frequency of osteocytogenic recruitment of osteoblasts to initiate terminal osteocytic differentiation in mice.

Modulation of endoglin expression in islets of langerhans by VEGF reveals a novel regulator of islet endothelial cell function.

BACKGROUND: Endoglin/CD105 is an auxiliary receptor for transforming growth factor-ß with established roles in vascular remodelling. It has recently been shown that heterozygous endoglin deficiency in mice decreases insulin secretion in an animal model of obesity, highlighting a potential role for endoglin in the regulation of islet function. We have previously identified two different populations of endoglin expressing cells in human and mouse islets which are: (i) endothelial cells (ECs) and (ii) islet mesenchymal stromal cells. The contribution of islet EC endoglin expression to islet development and sensitivity to VEGF is unknown and is the focus of this study. RESULTS: In vitro culture of mouse islets with VEGF164 for 48 h increased endoglin mRNA levels above untreated controls but VEGF did not modulate VEGFR2, CD31 or CD34 mRNA expression or islet viability. Removal of EC-endoglin expression in vivo reduced islet EC area but had no apparent effect on islet size or architecture. CONCLUSION: EC-specific endoglin expression in islets is sensitive to VEGF and plays partial roles in driving islet vascular development, however such regulation appears to be distinct to mechanisms required to modulate islet viability and size.

VEGF and bone cell signalling: an essential vessel for communication?

Vascular endothelial growth factor (VEGF) is an endothelial cell survival factor and is required for effective coupling of angiogenesis and osteogenesis. Although central to bone homeostasis, repair and the pathobiology that affect these processes, the precise mechanisms coupling endothelial cell function within bone formation and remodelling remain unclarified. This review will (i) focus on the potential directionality of VEGF signalling in adult bone by identifying the predominant source of VEGF within the bone microenvironment, (ii) will summarize current VEGF receptor expression studies by bone cells and (iii) will provide evidence for a role for VEGF signalling during postnatal repair and osteoporosis. A means of understanding the directionality of VEGF signalling in adult bone would allow us to most effectively target angiogenic pathways in diseases characterized by changes in bone remodelling rates and enhance bone repair when compromised.

Activin receptor-like kinase 5 inhibition reverses impairment of endothelial cell viability by endogenous islet mesenchymal stromal cells.

Following islet transplantation, islet graft revascularization is compromised due to loss of endothelial cells (ECs) during islet culture. TGF-ß signaling pathways are essential for vascular homeostasis but their importance for islet EC function is unclear. We have identified a population of multipotent mesenchymal stromal cells (MSCs) within islets and investigated how modulation of TGF-ß signaling by these cells influences islet EC viability. Cultured islets exhibited reduced expression of EC markers (VEGFR2, VE-cadherin and CD31), which was associated with diminished but sustained expression of endoglin a marker of both ECs and MSCs. Double fluorescent labeling of islets in situ with the EC marker CD31 disclosed a population of CD31-negative cells which were positive for endoglin. In vitro coculture of microvascular ECs with endoglin-positive, CD31-negative islet MSCs reduced VEGFR2 protein expression, disrupted EC angiogenic behavior, and increased EC detachment. Medium conditioned by islet MSCs significantly decreased EC viability and increased EC caspase 3/7 activity. EC:MSC cocultures showed enhanced Smad2 phosphorylation consistent with altered ALK5 signaling. Pharmacological inhibition of ALK5 activity with SB431542 (SB) improved EC survival upon contact with MSCs, and SB-treated cultured islets retained EC marker expression and sensitivity to exogenous VEGF164 . Thus, endoglin-expressing islet MSCs influence EC ALK5 signaling in vitro, which decreases EC viability, and changes in ALK5 activity in whole cultured islets contribute to islet EC loss. Modifying TGF-ß signaling may enable maintenance of islet ECs during islet isolation and thus improve islet graft revascularization post-transplantation.

Regulation of UDP-glucose dehydrogenase is sufficient to modulate hyaluronan production and release, control sulfated GAG synthesis, and promote chondrogenesis.

Glycosaminoglycans (GAGs) are critical for extracellular matrix (ECM) integrity in cartilage but mechanisms regulating their synthesis are not defined. UDP-glucose dehydrogenase (UGDH) catalyses UDP-glucose oxidation to UDP-glucuronic acid, an essential monosaccharide in many GAGs. Our previous studies in articular surface (AS) cells from embryonic joints have established pivotal roles for mitogen-activated protein kinases (MAPK) in synthesis of the unsulfated GAG, hyaluronan (HA). We investigated the functional significance of UGDH in GAG production and chondrogenesis, and determined roles for MEK-ERK and p38MAPK pathways in regulating UGDH expression and function. Inhibitors of MEK and p38MAPK reduced UGDH protein in AS cells. Treatment with TGF-ß (archetypal growth factor) increased UGDH expression, sulfated (s)-GAG/HA release and pericellular matrix formation in a p38MAPK-dependent manner. Retroviral overexpression of UGDH augmented HA/sGAG release and pericellular matrix elaboration, which were blocked by inhibiting MEK but not p38MAPK. UGDH overexpression increased cartilage nodule size in bone marrow culture, promoted chondrogenesis in limb bud micromass culture and selectively suppressed medium HA levels and modified GAG sulfation, as assessed by FACE analysis. Our data provide evidence that: (i) TGF-ß regulates UGDH expression via p38MAPK to modulate sGAG/HA secretion, (ii) MEK-ERK, but not p38MAPK facilitates UGDH-induced HA and sGAG release, and (iii) increased UGDH expression promotes chondrogenesis directly and differential modifies GAG levels and sulfation. These results indicate a more diverse role for UGDH in the support of selective GAG production than previously described. Factors regulating UGDH may provide novel candidates for restoring ECM integrity in degenerative cartilage diseases, such as osteoarthritis.Arthritis Research Campaign.

Heterotypic contact reveals a COX-2-mediated suppression of osteoblast differentiation by endothelial cells: A negative modulatory role for prostanoids in VEGF-mediated cell: cell communication?

In bone, angiogenesis must be initiated appropriately, but limited once remodelling or repair is complete. Our recent findings have supported a role for prostaglandins (PG), known modulators of osteoblast (OB) and endothelial cell (EC) behaviour, in facilitating VEGF-mediated paracrine communication from OBs to 'remotely located' ECs, but the mechanism(s) regulating OB:EC crosstalk when these cells are closely opposed are undefined. In this study we have examined: (i) the effects of exogenous PGE(2) on VEGF-driven events in ECs, and (ii) the role of endogenous COX-2-derived prostanoids in mediating communication between intimately opposed OBs and ECs in direct contact. Exposure of ECs to PGE(2) increased ERK1/2 phosphorylation, COX-2 induction, 6-keto-PGF(1alpha) release and EC proliferation. In contrast, PGE(2) attenuated VEGF(165)-induced VEGFR2/Flk1 phosphorylation, ERK1/2 activation and proliferation of ECs, suggesting that exogenous PGE(2) restricts the actions of VEGF. However, the COX-2-selective inhibitor, NS398, also attenuated VEGF-induced proliferation, implying a distinct role for endogenous COX-2 activity in regulating EC behaviour. To examine the effect of OB:EC proximity and the role of COX-2 products further, we used a confrontational co-culture model. These studies showed that COX-2 blockade with NS398 enhanced EC-dependent increases in OB differentiation, that this effect was reversed by exogenous PGH(2) (immediate COX-2 product), and that exogenous VEGF did not influence EC-dependent OB differentiation under these conditions. Our findings indicate that locally produced prostanoids may serve distinct roles depending on OB:EC proximity and negatively modulate VEGF-mediated changes in EC behaviour when these cells are closely opposed to control angiogenesis during bone (re)modelling.

Evaluation of VEGF-mediated signaling in primary human cells reveals a paracrine action for VEGF in osteoblast-mediated crosstalk to endothelial cells.

Communication between endothelial and bone cells is crucial for controlling vascular supply during bone growth, remodeling, and repair but the molecular mechanisms coordinating this intercellular crosstalk remain ill-defined. We have used primary human and rat long bone-derived osteoblast-like cells (HOB and LOB) and human umbilical vein endothelial cells (HUVEC) to interrogate the potential autocrine/paracrine role of vascular endothelial cell growth factor (VEGF) in osteoblast:endothelial cell (OB:EC) communication and examined whether prostaglandins (PG), known modulators of both OB and EC behavior, modify VEGF production. We found that the stable metabolite of PGI2, 6-keto-PGF(1alpha) and PGE2, induced a concentration-dependent increase in VEGF release by HOBs but not ECs. In ECs, VEGF promoted early ERK1/2 activation, late cyclooxygenase-2 (COX-2) protein induction, and release of 6-keto-PGF1alpha. In marked contrast, no significant modulation of these events was observed in HOBs exposed to VEGF, but LOBs clearly exhibited COX-dependent prostanoid release (10-fold less than EC) following VEGF treatment. A low level of osteoblast-like cell responsiveness to exogenous VEGF was supported by VEGFR2/Flk-1 immunolabelling and by blockade of VEGF-mediated prostanoid generation by a VEGFR tyrosine kinase inhibitor (TKI). HOB alkaline phosphatase (ALP) activity was increased following long-term non-contact co-culture with ECs and exposure of ECs to VEGF in this system further increased OB-like cell differentiation and markedly enhanced prostanoid release. Our studies confirm a paracrine EC-mediated effect of VEGF on OB-like cell behavior and are the first supporting a model in which prostanoids may facilitate this unidirectional VEGF-driven OB:EC communication. These findings may offer novel regimes for modulating pathological bone remodeling anomalies through the control of the closely coupled vascular supply.