FGF receptor inhibitor BGJ398 partially rescues osteoarthritis-like phenotype in older high molecular weight FGF2 transgenic mice via multiple mechanisms.

We have used Basic Fibroblast Growth Factor (FGF2) transgenic mice as experimental models for human X-linked hypophosphatemia (XLH)-related degenerative osteoarthritis (OA) to investigate the pathogenesis of the disease and to test potential pharmacotherapies for treatment. This study tested the efficacy of BJG398, a small molecule fibroblast growth factor receptor tyrosine kinase (FGFRTK) inhibitor, to rescue the knee joint osteoarthritis phenotype in High Molecular Weight fibroblast growth factor 2 transgenic (HMWTgFGF2) mice. BJG398 was administered in vivo to 8-month-old female HMWTgFGF2 mice for six weeks. Histomorphometry, immunohistochemistry and micro-CT were used to examine the knee joints in BGJ398-treated and control mice. We assessed: Fibroblast Growth Factor 23 (FGF23) expression and FGFR1 activity; Matrix metalloproteinase 13 (MMP13) and Aggrecanase2 (ADAMTS5) expression; then signaling by SMAD1/5/8-pSMAD6, pERK1/2 and Runt-related transcription factor 2 (RUNX2). Using PrimePCR arrays, we identified a contributing role for major target genes in the TGFB/BMP2 signaling pathway that were regulated by BGJ398. BGJ398 inhibited HMWFGF2/FGF23-induced increase in bone morphogenic protein receptor-1, bone morphogenic protein-2 and 4 and Serine peptidase inhibitor, clade E, member 1. The results from Micro-CT and histology show BGJ398 treatment rescued the OA changes in subchondral bone and knee articular cartilage of HMWTgFGF2 mice. The gene expression and signal transduction results provide convincing evidence that HMWFGF2 generates OA through FGFRTK with characteristic downstream signaling that defines OA, namely: increased FGF23-FGFR1 activity with BMP-BMPR, activation of pSMAD1/5/8-RUNX2 and pERK signaling pathways, then upregulation of MMP13 and ADAMTS5 to degrade matrix. BGJ398 treatment effectively reversed these OA molecular phenotypes, providing further evidence that the OA generated by HMWFGF2 in the transgenic mice is FGFR-mediated and phenocopies the OA found in the Hyp mouse homolog of XLH with a spontaneous mutation in the Phex (phosphate regulating endopeptidase on the X chromosome) gene and human XLH-OA. Overall, the results obtained here explain how the pleotropic effects of FGF2 emanate from the different functions of HMW protein isoforms for cartilage and bone homeostasis, and the pathogenesis of XLH-degenerative osteoarthropathy. BGJ398 inhibits HMWFGF2-induced osteoarthritis via multiple mechanisms. These results provided important scientific evidence for the potential application of BGJ398 as a therapeutic agent for osteoarthritis in XLH.

Fibroblast Growth Factor 2 and Its Receptors in Bone Biology and Disease.

The fibroblast growth factor (FGF) regulatory axis is phylogenetically ancient, evolving into a large mammalian/human gene family of 22 ligands that bind to four receptor tyrosine kinases for a complex physiologic system controlling cell growth, differentiation, and metabolism. The tissue targets for the primary FGF function are mainly in cartilage and in bone for morphogenesis, mineralization, and metabolism. A multitude of complexities in the FGF ligand-receptor signaling pathways have made translation into therapies for FGF-related bone disorders such as osteomalacia, osteoarthritis, and osteoporosis difficult but not impossible.

BMP-2 differentially modulates FGF-2 isoform effects in osteoblasts from newborn transgenic mice.

We previously generated separate lines of transgenic mice that specifically overexpress either the Fibroblast growth factor (FGF)-2 low-molecular-mass isoform (Tg(LMW)) or the high-mass isoforms (Tg(HMW)) in the osteoblast lineage. Vector/control (Tg(Vector)) mice were also made. Here we report the use of isolated calvarial osteoblasts (COBs) from those mice to investigate whether the FGF-2 protein isoforms differentially modulate bone formation in vitro. Our hypothesis states that FGF-2 isoforms specifically modulate bone morphogenetic protein 2 (BMP-2) function and subsequently bone differentiation genes and their related signaling pathways. We found a significant increase in alkaline phosphatase-positive colonies in Tg(LMW) COBs compared with Tg(Vector) controls. BMP-2 treatment significantly increased mineralized colonies in Tg(Vector) and Tg(LMW) COBs. BMP-2 caused a further significant increase in mineralized colonies in Tg(LMW) COBs compared with Tg(Vector) COBs but did not increase alkaline phosphatase-positive colonies in Tg(HMW) COBs. Time-course studies showed that BMP-2 caused a sustained increase in phosphorylated mothers against decapentaplegic-1/5/8 (Smad/1/5/8), runt-related transcription factor-2 (Runx-2), and osterix protein in Tg(LMW) COBs. BMP-2 caused a sustained increase in phospho-p38 MAPK in Tg(Vector) but only a transient increase in Tg(LMW) and Tg(HMW) COBs. BMP-2 caused a transient increase in phospho-p44/42 MAPK in Tg(Vector) COBs and no increase in Tg(LMW) COBs, but a sustained increase was found in Tg(HMW) COBs. Basal expression of FGF receptor 1 protein was significantly increased in Tg(LMW) COBs relative to Tg(Vector) COBs, and although BMP-2 caused a transient increase in FGF receptor 1 expression in Tg(Vector) COBs and Tg(HMW) COBs, there was no further increase Tg(LMW) COBs. Interestingly, although basal expression of FGF receptor 2 was similar in COBs from all genotypes, BMP-2 treatment caused a sustained increase in Tg(LMW) COBs but decreased FGF receptor 2 in Tg(Vector) COBs and Tg(HMW) COBs.

Disruption of the Fgf2 gene activates the adipogenic and suppresses the osteogenic program in mesenchymal marrow stromal stem cells.

Here we determine the Fibroblast Growth Factor-2 (FGF2) dependency of the time course of changes in bone mass in female mice. This study extends our earlier reports that knockout of the FGF2 gene (Fgf2) caused low turnover bone loss in Fgf2(-/-) male mice by examining bone loss with age in Fgf2(-/-) female mice, and by assessing whether reduced bone formation is associated with differentiation of bone marrow stromal cells (BMSCs) towards the adipocyte lineage. Bone mineral density (BMD) was similar in 3-month-old female Fgf2(+/+) and Fgf2(-/-) mice but was significantly reduced as early as 5 months of age in Fgf2(-/-) mice. In vivo studies showed that there was a greater accumulation of marrow fat in long bones of 14 and 20 month old Fgf2(-/-) mice compared with Fgf2(+/+) littermates. To study the effect of disruption of FGF2 on osteoblastogenesis and adipogenesis, BMSCs from both genotypes were cultured in osteogenic or adipogenic media. Reduced alkaline phosphatase positive (ALP), mineralized colonies and a marked increase in adipocytes were observed in Fgf2(-/-) BMSC cultures. These cultures also showed an increase in the mRNA of the adipogenic transcription factor PPARgamma2 as well as the downstream target genes aP2 and adiponectin. Treatment with exogenous FGF2 blocked adipocyte formation and increased ALP colony formation and ALP activity in BMSC cultures of both genotypes. These results support an important role for endogenous FGF2 in osteoblast (OB) lineage determination. Alteration in FGF2 signaling may contribute to impaired OB bone formation capacity and to increased bone marrow fat accumulation both of which are characteristics of aged bone.

The influence of FGF2 high molecular weight (HMW) isoforms in the development of cardiac ischemia-reperfusion injury.

Fibroblast growth factor 2 (FGF2) consists of multiple protein isoforms (low [LMW] and high molecular weight [HMW]), which are localized to different cellular compartments, indicating unique biological activity. We previously showed that the LMW isoform is important in protecting the heart from myocardial dysfunction associated with ischemia-reperfusion (I/R) injury, but the roles of the HMW isoforms remain unknown. To elucidate the role of HMW isoforms in I/R and cardioprotection, hearts from novel mouse models, in which the murine FGF2 HMWs are knocked out (HMWKO) or the human FGF2 24 kDa HMW isoform is overexpressed (HMW Tg) and their wildtype (Wt) or non-transgenic (NTg) cohorts were subjected to an ex vivo work-performing heart model of I/R. There was a significant improvement in post-ischemic recovery of cardiac function in HMWKO hearts (76+/-5%, p<0.05) compared to Wt hearts (55+/-5%), with a corresponding decrease in HMW Tg function (line 20: 38+/-6% and line 28: 33+/-4%, p<0.05) compared to non-transgenic hearts (68+/-9%). FGF2 LMW isoform was secreted from Wt and HMWKO hearts during I/R, and a FGF receptor (FGFR) inhibitor, PD173074 caused a decrease in cardiac function when administered in I/R in Wt and FGF2 HMWKO hearts (p<0.05), indicating that FGFR is involved in FGF2 LMW isoform's biological effect in ischemia-reperfusion injury. Moreover, overexpression of HMW isoform reduced FGFR1 phosphorylation/activation with no further decrease in the phosphorylation state in the presence of the FGFR inhibitor. Overall, our data indicate that HMW isoforms have a detrimental role in the development of post-ischemic myocardial dysfunction.

Nuclear isoforms of fibroblast growth factor 2 are novel inducers of hypophosphatemia via modulation of FGF23 and KLOTHO.

FGF2 transgenic mice were developed in which type I collagen regulatory sequences drive the nuclear high molecular weight FGF2 isoforms in osteoblasts (TgHMW). The phenotype of TgHMW mice included dwarfism, decreased bone mineral density (BMD), osteomalacia, and decreased serum phosphate (P(i)). When TgHMW mice were fed a high P(i) diet, BMD was increased, and dwarfism was partially reversed. The TgHMW phenotype was similar to mice overexpressing FGF23. Serum FGF23 was increased in TgHMW mice. Fgf23 mRNA in bones and fibroblast growth factor receptors 1c and 3c and Klotho mRNAs in kidneys were increased in TgHMW mice, whereas the renal Na(+)/P(i) co-transporter Npt2a mRNA was decreased. Immunohistochemistry and Western blot analyses of TgHMW kidneys showed increased KLOTHO and decreased NPT2a protein. The results suggest that overexpression of HMW FGF2 increases FGF23/FGFR/KLOTHO signaling to down-regulate NPT2a, causing P(i) wasting, osteomalacia, and decreased BMD. We assessed whether HMW FGF2 expression was altered in the Hyp mouse, a mouse homolog of the human disease X-linked hypophosphatemic rickets/osteomalacia. Fgf2 mRNA was increased in bones, and Western blots showed increased FGF2 protein in nuclear fractions from osteoblasts of Hyp mice. In addition, immunohistochemistry demonstrated co-localization of FGF23 and HMW FGF2 protein in osteoblasts and osteocytes from Hyp mice. This study reveals a novel mechanism of regulation of the FGF23-P(i) homeostatic axis.

Endogenous FGF-2 is critically important in PTH anabolic effects on bone.

Parathyroid hormone (PTH) increases fibroblast growth factor receptor-1 (FGFR1) and fibroblast growth factor-2 (FGF-2) expression in osteoblasts and the anabolic response to PTH is reduced in Fgf2-/- mice. This study examined whether candidate factors implicated in the anabolic response to PTH were modulated in Fgf2-/- osteoblasts. PTH increased Runx-2 protein expression in Fgf2+/+ but not Fgf2-/- osteoblasts. By immunocytochemistry, PTH treatment induced nuclear accumulation of Runx-2 only in Fgf2+/+ osteoblasts. PTH and FGF-2 regulate Runx-2 via activation of the cAMP response element binding proteins (CREBs). Western blot time course studies showed that PTH increased phospho-CREB within 15 min that was sustained for 24 h in Fgf2+/+ but had no effect in Fgf2-/- osteoblasts. Silencing of FGF-2 in Fgf2+/+ osteoblasts blocked the stimulatory effect of PTH on Runx-2 and CREBs phosphorylation. Studies of the effects of PTH on proteins involved in osteoblast precursor proliferation and apoptosis showed that PTH increased cyclinD1-cdk4/6 protein in Fgf2+/+ but not Fgf2-/- osteoblasts. Interestingly, PTH increased the cell cycle inhibitor p21/waf1 in Fgf2-/- osteoblasts. PTH increased Bcl-2/Bax protein ratio in Fgf2+/+ but not Fgf2-/- osteoblasts. In addition PTH increased cell viability in Fgf2+/+ but not Fgf2-/- osteoblasts. These data suggest that endogenous FGF-2 is important in PTH effects on osteoblast proliferation, differentiation, and apoptosis. Reduced expression of these factors may contribute to the reduced anabolic response to PTH in the Fgf2-/- mice. Our results strongly indicate that the anabolic PTH effect is dependent in part on FGF-2 expression.

Exported 18-kDa isoform of fibroblast growth factor-2 is a critical determinant of bone mass in mice.

The role of the 18-kDa isoform of fibroblast growth factor-2 (FGF2) in the maintenance of bone mass was examined in Col3.6-18-kDa FGF2-IRES-GFPsaph transgenic (18-kDa TgFGF2) mice in which a 3.6-kb fragment of the type I collagen 5'-regulatory region (Col3.6) drives the expression of only the 18-kDa isoform of FGF2 with green fluorescent protein-sapphire (GFPsaph). Vector only transgenic mice (Col3.6-IRES-GFPsaph, VTg) were also developed as a control, and mice specifically deficient in 18-kDa FGF2 (FGF2(lmw)(-/-)) were also examined. Bone mineral density, femoral bone volume, trabecular thickness, and cortical bone area and thickness were significantly increased in 18-kDa TgFGF2 mice compared with VTg. Bone marrow cultures (BMSC) from 18-kDa TgFGF2 mice produced more mineralized nodules than VTg. Increased bone formation was associated with reduced expression of the Wnt antagonist secreted frizzled receptor 1 (sFRP-1). In contrast to 18-kDa TgFGF2 mice, FGF2(lmw)(-/-) mice have significantly reduced bone mineral density and fewer mineralized nodules, coincident with increased expression of sFRP-1 in bones and BMSC. Moreover, silencing of sFRP-1 in BMSC from FGF2(lmw)(-/-) mice reversed the decrease in beta-catenin and Runx2 mRNA. Assay of Wnt/beta-catenin-mediated transcription showed increased and decreased TCF-luciferase activity in BMSC from 18-kDa TgFGF2 and FGF2(lmw)(-/-) mice, respectively. Collectively, these results demonstrate that the 18-kDa FGF2 isoform is a critical determinant of bone mass in mice by modulation of the Wnt signaling pathway.

Fgf-2 overexpression increases excitability and seizure susceptibility but decreases seizure-induced cell loss.

Fibroblast growth factor 2 (FGF-2) has multiple, pleiotropic effects on the nervous system that include neurogenesis, neuroprotection and neuroplasticity. Thus, alteration in FGF-2 expression patterns may have a profound impact in brain function, both in normal physiology and in pathology. Here, we used FGF-2 transgenic mice (TgFGF2) to study the effects of endogenous FGF-2 overexpression on susceptibility to seizures and to the pathological consequences of seizures. TgFGF2 mice display increased FGF-2 expression in hippocampal pyramidal neurons and dentate granule cells. Increased density of glutamatergic synaptic vesicles was observed in the hippocampus of TgFGF2 mice, and electrophysiological data (input/output curves and patch-clamp recordings in CA1) confirmed an increase in excitatory inputs in CA1, suggesting the presence of a latent hyperexcitability. Indeed, TgFGF2 mice displayed increased susceptibility to kainate-induced seizures compared with wild-type (WT) littermates, in that latency to generalized seizure onset was reduced, whereas behavioral seizure scores and lethality were increased. Finally, WT and TgFGF2 mice with similar seizure scores were used for examining seizure-induced cellular consequences. Neurogenesis and mossy fiber sprouting were not significantly different between the two groups. In contrast, cell damage (assessed with Fluoro-Jade B, silver impregnation and anti-caspase 3 immunohistochemistry) was significantly lower in TgFGF2 mice, especially in the areas of overexpression (CA1 and CA3), indicating reduction of seizure-induced necrosis and apoptosis. These data suggest that FGF-2 may be implicated in seizure susceptibility and in seizure-induced plasticity, exerting different, and apparently contrasting effects: favoring ictogenesis but reducing seizure-induced cell death.

Arsenic exposure in pregnant mice disrupts placental vasculogenesis and causes spontaneous abortion.

Arsenic is an abundant toxicant in ground water and soil around areas with extractive industries. Human epidemiological studies have shown that arsenic exposure is linked to developmental defects and miscarriage. The placenta is known to utilize vasculogenesis to develop its circulation. The hypothesis tested here states the following: arsenic exposure causes placental dysmorphogenesis and defective placental vasculogenesis resulting in placental insufficiency and subsequent spontaneous abortion. To test this hypothesis, pregnant mice were exposed to sodium arsenite (AsIII) through drinking water from conception through weanling stages. Neonatal assessment of birth rates, pup weights, and litter sizes in arsenic exposed and control mothers revealed that AsIII-exposed mothers had only 40% the fecundity of controls. Preterm analysis at E12.5 revealed a loss of fecundity at E12.5 from either 20 ppm or greater exposures to AsIII. There was no loss of fecundity at E7.5 suggesting that spontaneous abortion occurs during placentation. Histomorphometry on E12.5 placentae from arsenic-exposed mice revealed placental dysplasia especially in the vasculature. These results suggest that arsenic toxicity is causative for mammalian spontaneous abortion by virtue of aberrant placental vasculogenesis and placental insufficiency.

Activation of protein kinase C and disruption of endothelial monolayer integrity by sodium arsenite--Potential mechanism in the development of atherosclerosis.

Arsenic exposure has been shown to exacerbate atherosclerosis, beginning with activation of the endothelium that lines the vessel wall. Endothelial barrier integrity is maintained by proteins of the adherens junction (AJ) such as vascular endothelial cadherin (VE-cadherin) and beta-catenin and their association with the actin cytoskeleton. In the present study, human aortic endothelial cells (HAECs) were exposed to 1, 5 and 10 microM sodium arsenite [As(III)] for 1, 6, 12 and 24 h, and the effects on endothelial barrier integrity were determined. Immunofluorescence studies revealed formation of actin stress fibers and non-uniform VE-cadherin and beta-catenin staining at cell-cell junctions that were concentration- and time-dependent. Intercellular gaps were observed with a measured increase in endothelial permeability. In addition, concentration-dependent increases in tyrosine phosphorylation (PY) of beta-catenin and activation of protein kinase Calpha (PKCalpha) were observed. Inhibition of PKCalpha restored VE-cadherin and beta-catenin staining at cell-cell junctions and abolished the As(III)-induced formation of actin stress fibers and intercellular gaps. Endothelial permeability and PY of beta-catenin were also reduced to basal levels. These results demonstrate that As(III) induces activation of PKCalpha, which leads to increased PY of beta-catenin downstream of PKCalpha activation. Phosphorylation of beta-catenin plausibly severs the association of VE-cadherin and beta-catenin, which along with formation of actin stress fibers, results in intercellular gap formation and increased endothelial permeability. To the best of our knowledge, this is the first report demonstrating that As(III) causes a loss of endothelial monolayer integrity, which potentially could contribute to the development of atherosclerosis.

Transitional angiogenesis and vascular remodeling during coronary angiogenesis in response to myocardial infarction.

Coronary artery disease (CAD) is a major source of morbidity and mortality in the industrialized world. CAD causes ischemia as a prelude to angina, myocardial infarction and heart failure as specific forms of heart disease causing a decline in the quality of life. CAD or atherosclerosis and the resulting myocardial ischemia trigger a natural angiogenic response that generates collateral circulations. The long-term goal for these studies is to develop therapeutic angiogenesis that augments the natural coronary angiogenesis. This project makes use of an infarcted transgenic mouse model to characterize formation of those collateral circulations in the post-infraction heart. The experiments utilized thoracotomy and a microcauterizer to produce an infarct in transgenic mice and this stimulated neovascularization and allowed labeling of the coronary vessels, thereby defining the morphogenic processes involved in formation of collateral circulations. The results show that the heart consistently responds to infarcts with angiogenesis at 1d post-treatment (PT) that undergoes transition into vascular remodeling at 7d PT with complete remodeling at 14d PT. The vascular remodeling appears to mitigate any net increase in perfusion that may be achieved early in coronary angiogenesis. The results suggest that therapeutic approaches need to shift from an exclusive focus on stimulating angiogenesis to include modulation of vascular remodeling for increased long-term myocardial perfusion.

Manganese enhances peroxynitrite and leukotriene E4 formation in bovine aortic endothelial cells exposed to arsenic.

Long-term exposure to arsenic in drinking water has been linked to cancer and other health effects, including cardiovascular disease. Arsenic in the environment is found in combination with a range of metals that could influence its toxicity. Manganese, in particular, is a metal that is typically found in conjunction with arsenic in contaminated groundwater. Peroxynitrite is a powerful oxidant formed from the reaction between nitric oxide and superoxide anion. Arsenic has been shown to increase the formation of peroxynitrite in bovine aortic endothelial cells (BAECs) and promote the formation of 3-nitrotyrosine (3-NY) in the atherosclerotic plaque of ApoE-/-/LDLr-/- mice. Arsenic exposure also increases leukotriene E4 (LTE4) formation in both the mice and BAECs, an effect that is partially reversed by the addition of Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor. In the present study, we investigated the effect of adding nontoxic concentrations of manganese along with arsenic to BAEC cultures. Manganese increased arsenic toxicity and enhanced peroxynitrite, 3-NY, and LTE4 formation in BAECs. Addition of LNAME reduced 3-NY formation induced by arsenic/manganese mixtures, but in contrast to its effect on arsenic alone, L-NAME actually increased LTE4 synthesis in BAECs treated with the arsenic/manganese combination. Overall, these data suggest that manganese may exacerbate the toxic effects of arsenic on the vascular system.

Arsenic exposure exacerbates atherosclerotic plaque formation and increases nitrotyrosine and leukotriene biosynthesis.

A correlation between arsenic and cardiovascular disease (CVD) has been established through epidemiological studies, although the mechanisms are unknown. Using a mouse model that develops atherosclerotic lesions on a normal chow diet, we have confirmed a connection between long-term arsenic intake and CVD. Our results reveal a significant increase in the degree of atherosclerotic plaque stenosis within the innominate artery of ApoE-/-/LDLr-/- mice treated with 10 ppm sodium arsenite (133 microM) in drinking water for 18 weeks compared to controls. Immunohistochemistry shows nitrotyrosine formation, a marker of reactive nitrogen species generation, is significantly higher within the atherosclerotic plaque of arsenic-treated mice. In addition, there is a significant increase in the 5-lipoxygenase (5-LO) product, leukotriene E4 (LTE4), in the serum of arsenic-treated mice. This is supported by induction of the 5-LO protein and subsequent increases in LTE4 synthesis in bovine aortic endothelial cells. This increase in LTE4 is partially inhibited by inhibitors of nitric oxide synthase, suggesting a link between reactive nitrogen species and arsenic-induced inflammation. Furthermore, there is a significant increase in prostacyclin (PGI2) in the serum of arsenic-treated mice. We conclude that changes in specific inflammatory mediators such as LTE4 and PGI2 are related to arsenic-induced atherosclerosis. In addition, amplified synthesis of reactive species such as peroxynitrite results in increased protein nitration in response to arsenic exposure. This finding is consistent with the pathology seen in human atherosclerotic plaques.

Stat1 controls postnatal bone formation by regulating fibroblast growth factor signaling in osteoblasts.

Activation of the signal transducers and activators of transcription (STAT) pathway is important in fibroblast growth factor (FGF) modulation of chondrocyte proliferation and endochondral bone formation during embryogenesis. However, it is not known if the FGF/STAT signaling pathway is important for postnatal bone formation. To examine this, we have characterized a novel skeletal phenotype in Stat1-/- mice in which we find a significant increase in bone mineral density, bone mineral content, and other parameters of bone growth. The data show that osteoblasts derived from Stat1-/- mice have decreased expression of cell cycle inhibitor p21WAF/CIP and FGF receptor 3, a known negative regulator of chondrocyte proliferation. Interestingly, Stat1-/- osteoblasts showed increased expression of FGF18 in vivo and increased responsiveness to FGF18 in vitro. These results suggest a mechanism for the regulation of the osteoblast in which Stat1 functions not only to directly regulate the cell cycle but also to modify the repertoire of FGF receptor expression from a potentially inhibitory receptor, FGFR3 to a stimulatory receptor such as FGFR1 or FGFR2.

Impaired osteoclast formation in bone marrow cultures of Fgf2 null mice in response to parathyroid hormone.

Fibroblast growth factor (FGF)-2 and parathyroid hormone (PTH) are potent inducers of osteoclast (OCL) formation, and PTH increases FGF-2 mRNA and protein expression in osteoblasts. To elucidate the role of endogenous FGF-2 in PTH responses, we examined PTH-induced OCL formation in bone marrow cultures from wild type and mice with a disruption of the Fgf2 gene. FGF-2-induced OCL formation was similar in marrow culture from both genotypes. In contrast, PTH-stimulated OCL formation in bone marrow cultures or co-cultures of osteoblast-spleen cells from Fgf2-/mice was significantly impaired. PTH increased RANKL mRNA expression in osteoblasts cultures from both genotypes. After 6 days of treatment, osteoprotegerin protein in cell supernatants was 40-fold higher in vehicle-treated and 30-fold higher in PTH-treated co-cultures of osteoblast and spleen cells from Fgf2-/mice compared with Fgf2+/+ mice. However, a neutralizing antibody to osteoprotegerin did not rescue reduced OCL formation in response to PTH. Injection of PTH caused hypercalcemia in Fgf2+/+ but not Fgf2-/mice. We conclude that PTH stimulates OCL formation and bone resorption in mice in part by endogenous FGF-2 synthesis by osteoblasts. Because RANKL- and interleukin-11-induced OCL formation was also reduced in bone marrow cultures from Fgf2-/mice, we further conclude that endogenous FGF-2 is necessary for maximal OCL formation by multiple bone resorbing factors.

Immunohistologic labeling of murine endothelium.

BACKGROUND: Reliable identification of endothelial cells is a prerequisite for understanding vascularity changes in many cardiovascular diseases and therapeutic interventions. With the rising use of mouse models of disease and genetic manipulation, a consistent system to label murine endothelial cells in normal and diseased tissues would be an invaluable tool. METHODS: Immunohistologic and histochemical methods were used to label endothelial cells in normal and infarcted heart as well as small intestine. Isolectin B(4) or antibodies to thrombomodulin, vWF, Tie-2, VE-cadherin, CD34, and CD31 were used to immunostain tissues fixed in either 4% formaldehyde (prepared fresh from powdered paraformaldehyde, PF), methyl Carnoy's (MC), zinc (Zn) (all paraffin-embedded), or frozen sections. In addition, we used beta-galactosidase histochemistry in frozen sections from the Tie-2/beta-galactosidase transgenic mouse, in which the lacZ reporter gene is driven by the endothelial-specific Tie-2 promoter. RESULTS AND CONCLUSIONS: Immunohistologic localization of endothelial cells was best accomplished using anti-CD31 in Zn-fixed, paraffin-embedded tissues. Antithrombomodulin staining also worked in Zn-fixed tissues as well as frozen tissues, but additional steps were required to reduce background. The beta-galactosidase reporter system also marked endothelial cells in frozen Tie-2 transgenic mouse tissues; however, this system required careful control of fixation and optimization of histochemistry and was not amenable to double labeling. Lectin staining gave complete labeling of endothelial cells but cross-reacted intensely with macrophages in the infarcted heart. We conclude that anti-CD31 immunostaining in Zn-fixed, paraffin-embedded murine tissue offered superior morphology and permitted optimal identification of proliferating endothelial cells during infarct repair.

Arsenic induces peroxynitrite generation and cyclooxygenase-2 protein expression in aortic endothelial cells: possible role in atherosclerosis.

Epidemiological evidence suggests that exposure to the metalloid arsenic constitutes a risk factor for cardiovascular disease. The purpose of this study was to determine whether arsenic could stimulate generation of factors involved in oxidative stress and inflammation, conditions associated with atherosclerosis, or coronary artery disease. We found that production of peroxynitrite, a strong oxidant formed from the coupling of nitric oxide and superoxide anion, was significantly increased in bovine aortic endothelial (BAE) cells exposed to sodium arsenite at concentrations as low as 0.5 microM. Expression of the inflammatory mediator cyclooxygenase-2 (COX-2) was also upregulated in response to arsenite exposure as demonstrated by Western blot analysis. The increase in COX-2 protein was time dependent with highest levels at 30 min and 48 h. This result was supported by an increase in the generation of prostaglandin E(2) following exposure to arsenic. Nitrotyrosine residues in proteins are indicative of peroxynitrite generation, and extensive nitrotyrosine formation has been detected in atherosclerotic plaques. Therefore, COX-2 protein was immunoprecipitated from BAE cells and submitted to Western blot analysis using an antibody to nitrotyrosine. Nitration of COX-2 was detected in arsenic-treated cells, but not in untreated control cells. The findings in this report suggest an increase in reactive species, notably peroxynitrite, in BAE cells exposed to arsenic. Furthermore, induction of important inflammatory mediators such as COX-2 may exacerbate the inflammatory state typical of atherosclerosis.