Osteoblasts Have a Neural Origin in Heterotopic Ossification.

BACKGROUND: Heterotopic ossification (HO) is the process of bone formation at a nonskeletal site. Recently, we showed that the earliest steps occur in sensory nerves. We now extend these studies by identifying unique osteogenic progenitors within the endoneurial compartment of sensory nerves. QUESTIONS/PURPOSES: We asked: (1) What is the nature of the osteoprogenitor in the endoneurium of peripheral nerves? (2) How do osteoprogenitors travel from the nerve to the site of new bone formation? METHODS: HO was induced by intramuscular injection of Ad5BMP-2-transduced cells in mice. Osteoprogenitors were identified through immunohistochemistry and then quantified and further characterized by fluorescence-activated cell sorting and immunocytochemistry. The kinetics of the appearance of markers of extravasation was determined by quantitative reverse transcription-polymerase chain reaction. In each experiment mice were injected with bone morphogenetic protein-2 (BMP-2)-producing cells (experimental) or with cells transduced with empty vector or, in some cases, a group receiving no injection (control). RESULTS: Induction of HO leads to the expression, within 24 hours, of osteoblast-specific transcription factors in cells in the endoneurium followed by their coordinate disappearance from the nerve at 48 hours. They reappear in blood also at 48 hours after induction. During vessel entrance they begin to express the tight junction molecule, claudin 5. The cells expressing both the osteoblast-specific transcription factor, osterix, as well as claudin 5, then disappear from circulation at approximately 3 to 4 days by extravasation into the site of new bone formation. These endoneurial osteoprogenitors express neural markers PDGFRα, musashi-1, and the low-affinity nerve growth factor receptor p75(NTR) as well as the endothelial marker Tie-2. In a key experiment, cells that were obtained from mice that were injected with cells transduced with an empty vector, at 2 days after injection, contained 0.83% (SD, 0.07; 95% confidence interval [CI], 0.59-1.05) cells expressing claudin 5. However, cells that were obtained from mice 2 days after injection of BMP-2-producing cells contained 4.5% cells expressing claudin 5 (SD, 0.72%; 95% CI, 2.01-6.94; p < 0.0015). Further analysis revealed that all of the cells expressing claudin 5 were found to be positive for osteoblast-specific markers, whereas cells not expressing claudin 5 were negative for these same markers. CONCLUSIONS: The findings suggest that the endoneurial progenitors are the major osteogenic precursors that are used for HO. They exit the nerve through the endoneurial vessels, flow through vessels to the site of new bone formation, and then extravasate out of the vessels into this site. CLINICAL RELEVANCE: The biogenesis of osteoblasts in HO is very different than expected and shows that HO is, at least in part, a neurological disorder. This could result in a major shift in orthopaedic methodologies to prevent or treat this disease. The fact that nerves are intimately involved in the process may also provide clues that will lead to an explanation of the clinical fact that HO often occurs as a result of traumatic brain injury.

Far-red fluorescence gene reporter tomography for determination of placement and viability of cell-based gene therapies.

Non-invasive injectable cellular therapeutic strategies based on sustained delivery of physiological levels of BMP-2 for spinal fusion are emerging as promising alternatives, which could provide sufficient fusion without the associated surgical risks. However, these injectable therapies are dependent on bone formation occurring only at the specific target region. In this study, we developed and deployed fluorescence gene reporter tomography (FGRT) to provide information on in vivo cell localization and viability. This information is sought to confirm the ideal placement of the materials with respect to the area where early bone reaction is required, ultimately providing three dimensional data about the future fusion. However, because almost all conventional fluorescence gene reporters require visible excitation wavelengths, current in vivo imaging of fluorescent proteins is limited by high tissue absorption and confounding autofluorescence. We previously administered fibroblasts engineered to produce BMP-2, but is difficult to determine 3-D information of placement prior to bone formation. Herein we used the far-red fluorescence gene reporter, IFP1.4 to report the position and viability of fibroblasts and developed 3-D tomography to provide placement information. A custom small animal, far-red fluorescence tomography system integrated into a commercial CT scanner was used to assess IFP1.4 fluorescence and to demark 3-D placement of encapsulated fibroblasts with respect to the vertebrae and early bone formation as assessed from CT. The results from three experiments showed that the placement of the materials within the spine could be detected. This work shows that in vivo fluorescence gene reporter tomography of cell-based gene therapy is feasible and could help guide cell-based therapies in preclinical models.

Estrogen and insulin-like growth factor-I (IGF-I) independently down-regulate critical repressors of breast cancer growth.

Although estrogen receptor alpha (ERα) and insulin-like growth factor (IGF) signaling are important for normal mammary development and breast cancer, cross-talk between these pathways, particularly at the level of transcription, remains poorly understood. We performed microarray analysis on MCF-7 breast cancer cells treated with estradiol (E2) or IGF-I for 3 or 24 h. IGF-I regulated mRNA of five to tenfold more genes than E2, and many genes were co-regulated by both ligands. Importantly, expression of these co-regulated genes correlated with poor prognosis of human breast cancer. Closer examination revealed enrichment of repressed transcripts. Interestingly, a number of potential tumor suppressors, for example, B-cell linker (BLNK), were down-regulated by IGF-I and E2. Analysis of three down-regulated genes showed that E2-mediated repression occurred independently of IGF-IR, and IGF-I-mediated repression occurred independently of ERα. However, repression by IGF-I or E2 required common kinases, such as PI3K and MEK, suggesting downstream convergence of the two pathways. In conclusion, E2 and IGF-I co-regulate a set of genes that affect breast cancer outcome. There is enrichment of repressed transcripts, and, for some genes, the down-regulation is independent at the receptor level. This may be important clinically, as tumors with active ERα and IGF-IR signaling may require co-targeting of both pathways.

Cell-based gene therapy for repair of critical size defects in the rat fibula.

More than a decade has passed since the first experiments using adenovirus-transduced cells expressing bone morphogenetic protein 2 were performed for the synthesis of bone. Since this time, the field of bone gene therapy has tackled many issues surrounding safety and efficacy of this type of strategy. We present studies examining the parameters of the timing of bone healing, and remodeling when heterotopic ossification (HO) is used for bone fracture repair using an adenovirus gene therapy approach. We use a rat fibula defect, which surprisingly does not heal even when a simple fracture is introduced. In this model, the bone quickly resorbs most likely due to the non-weight bearing nature of this bone in rodents. Using our gene therapy system robust HO can be introduced at the targeted location of the defect resulting in bone repair. The HO and resultant bone healing appeared to be dose dependent, based on the number of AdBMP2-transduced cells delivered. Interestingly, the HO undergoes substantial remodeling, and assumes the size and shape of the missing segment of bone. However, in some instances we observed some additional bone associated with the repair, signifying that perhaps the forces on the newly forming bone are inadequate to dictate shape. In all cases, the HO appeared to fuse into the adjacent long bone. The data collectively indicates that the use of BMP2 gene therapy strategies may vary depending on the location and nature of the defect. Therefore, additional parameters should be considered when implementing such strategies.

Characterization of chemical, radiochemical and optical properties of a dual-labeled MMP-9 targeting peptide.

Optical imaging possesses similar sensitivity to nuclear imaging and has led to the emergence of multimodal approaches with dual-labeled nuclear/near-infrared (NIR) agents. The growing impact of (68)Ga (t(1/2)=68 min) labeled peptides on preclinical and clinical research offers a promising opportunity to merge the high spatial resolution of NIR imaging with the clinically-accepted positron emission tomography (PET). Previously, dual-labeled agents have been prepared with longer-lived radiometals and showed no detrimental effects on optical properties as a result of radiolabeling. In this study, we selected a peptide (M(2)) that targets MMP-2/9 and is dual-labeled with IRDye 800 CW and (68)Ga. Since (68)Ga chelation typically requires low pH (3.5-4) and elevated heating temperatures (95 °C), we sought to evaluate the impact of (68)Ga labeling on the optical properties of M(2). An efficient method for preparation of (68)Ga-M(2) was developed and reaction conditions were optimized. Stability studies in PBS, DTPA, and serum were performed and high levels of intact agent were evident under each condition. The addition of multiple reporters to a targeting agent adds further complexity to the characterization and validation and thus requires not only testing to ensure the agent is stable chemically and radiochemically, but also optically. Therefore, fluorescence properties were evaluated using a spectrofluorometer as well as by fluorescence detection via HPLC. It was determined that (68)Ga-labeling conditions did not impair the fluorescent properties of the agent. The agent was then used for in vivo imaging in a mouse model of heterotopic ossification (HO) with activated MMP-9 expression as an early biomarker which precedes mineralization. Although (68)Ga-complexation greatly reduced binding affinity of the peptide and negated tracer uptake on PET, NIR imaging showed consistent fluorescent signal that correlated to MMP-9 expression. This attests to the feasibility of using (68)Ga/NIR for dual-labeling of other peptides or small molecules for multimodality molecular imaging.

Efficient and rapid osteoinduction in an immune-competent host.

Osteoinductive systems to induce targeted rapid bone formation hold clinical promise, but development of technologies for clinical use that must be tested in animal models is often a difficult challenge. We previously demonstrated that implantation of human cells transduced with Ad5F35BMP2 to express high levels of bone morphogenetic protein-2 (BMP2) resulted in rapid bone formation at targeted sites. Inclusion of human cells in this model precluded us from testing this system in an immune-competent animal model, thus limiting information about the efficacy of this approach. Here, for the first time we demonstrate the similarity between BMP2-induced endochondral bone formation in a system using human cells in an immune-incompetent mouse and a murine cell-based BMP2 gene therapy system in immune-competent animals. In both cases the delivery cells are rapidly cleared, within 5 days, and in neither case do they appear to contribute to any of the structures forming in the tissues. Endochondral bone formation progressed through a highly ordered series of stages that were both morphologically and temporally indistinguishable between the two models. Even longterm analysis of the heterotopic bone demonstrated similar bone volumes and the eventual remodeling to form similar structures. The results suggest that the ability of BMP2 to rapidly induce bone formation overrides contributions from either immune status or the nature of delivery cells.

Oxygen tension directs chondrogenic differentiation of myelo-monocytic progenitors during endochondral bone formation.

Synthesis of bone requires both essential progenitors to form the various structures and the correct microenvironment for their differentiation. To identify these factors, we have used a system that exploits bone morphogenetic protein's ability to induce endochondral bone formation rapidly. One of the earliest events observed was the influx and proliferation of fibroblastic cells that express both vascular smooth muscle cell markers, alpha smooth muscle actin (alpha SMA), smooth muscle myosin heavy chain, and the monocytic marker CD68. The expression of these factors was lost by days 4 to 5, coincident with the up-regulation of Sox9 and the appearance of chondrocytes. Studies with a cyclization recombination (Cre)/lox system, in which a myeloid-specific promoter driving Cre recombinase can irreversibly unblock expression of beta-galactosidase only in cells of myeloid origin, showed specific activity in the newly formed chondrocytes. These results suggest that early chondrocyte progenitors are of myeloid origin. Simultaneous with this recruitment, we determined that a numbers of these cells were in a hypoxic state, indicative of a low-oxygen environment. The cells in the hypoxic regions were undergoing chondrogenesis, whereas cells in adjacent normoxic regions appeared to be assembling into new vessels, suggesting that the oxygen microenvironment is critical for establishment of the cartilage.

Progenitors in Peripheral Nerves Launch Heterotopic Ossification.

Studies presented here, using a murine model of bone morphogenetic protein type 2 (BMP2)-induced heterotopic ossification (HO) show that the protein initiates HO by signaling through progenitors in the endoneurium of peripheral nerves. In the mouse, these cells were identified in the endoneurium one day after BMP2 induction using antibody against phosphoSMAD (PS) 1, 5, and 8. Studies conducted in a tracking mouse that contains a tamoxifen-regulated Wnt1-Cre recombinase crossed with a td Tomato red (TR) reporter (Wnt1CreErt :Ai9Tm) confirmed their neural origin. In this model both BMP2 induction and tamoxifen are absolutely required to induce TR. SP7+ (osterix+ )TR+ cells were found in the endoneurium on day 1 and associated with bone on day 7. Quantification of TR+ and TR- cells isolated by fluorescence-activated cell sorting showed that all SP7+ cells were found in the TR+ population, whereas only about 80% of the TR+ cells expressed SP7. Pre-chondrocytes (Sox 9+ ) and transient brown fat (tBAT, UCP1+ ) also coexpressed TR, suggesting that the progenitor in nerves is multi-potential. The endoneurium of human nerves near the site of HO contained many PS+ cells, and SP7+ cells were found in nerves and on bone in tissue from patients with HO. Control tissues and nerves did not contain these PS+ and SP7+ cells. Some osteoblasts on bone from patients with HO were positive for PS, suggesting the continued presence of BMP during bone formation. The data suggests that the progenitors for HO are derived from the endoneurium in both the mouse model of HO and in humans with HO. Stem Cells Translational Medicine 2017;6:1109-1119.

Location-dependent heterotopic ossification in the rat model: The role of activated matrix metalloproteinase 9.

Extremity amputation or traumatic injury can often lead to the formation of heterotopic ossification (HO). Studies to induce HO in rat muscle using cell-based gene therapy show that this process appears to be location dependent. In the present study, HO was induced in mice and rats through injection of immunologically matched cells transduced with either a replication-defective adenovirus possessing bone morphogenetic protein 2 (BMP2) or an empty adenovirus vector (control). Injection in rat near the skeletal bone resulted in HO, whereas cells injected into the same muscle group but distal from the bone did not result in bone formation. When cells were injected in the same limb at both locations at the same time, HO was formed at both sites. Characterization of the bone formation in rats versus mice demonstrated that different sources of osteogenic progenitors were involved, which may account for the location dependent bone formation observed in the rat. Further experimentation has shown that a potential reason for this difference may be the inability of rat to activate matrix metalloproteinase 9 (MMP9), an essential protease in mice necessary for recruitment of progenitors. Inhibition of active MMP9 in mice led to a significant decrease in HO. The studies reported here provide insight into the mechanisms and pathways leading to bone formation in different animals and species. It appears that not all animal models are appropriate for testing HO therapies, and our studies also challenge the conventional wisdom that larger animal models are better for testing treatments affecting bone. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1894-1904, 2016.

Progesterone crosstalks with insulin-like growth factor signaling in breast cancer cells via induction of insulin receptor substrate-2.

Both progesterone and the insulin-like growth factors (IGFs) are critically involved in mammary gland development and also in breast cancer progression. However, how the progesterone and IGF signaling pathways interact with each other to regulate breast cancer cell growth remains unresolved. In this study, we investigated progesterone regulation of IGF signaling components in breast cancer cells. We found that insulin receptor substrate-2 (IRS-2) levels were markedly induced by progesterone and the synthetic progestin R5020 in MCF-7 and other progesterone receptor (PR) positive breast cancer cell lines, whereas IRS-1 and the IGF-I receptor were not induced. The antiprogestin RU486 blocked the R5020 effect on IRS-2 expression. Ectopic expression of either PR-A or PR-B in C4-12 breast cancer cells (estrogen receptor and PR negative) showed that progestin upregulation of IRS-2 was mediated specifically by PR-B. The IRS-2 induction by R5020 occurred via an increase of IRS-2 mRNA levels. Furthermore, progestin treatment prior to IGF-I stimulation resulted in higher tyrosine-phosphorylated IRS-2 levels, increased binding of IRS-2 to Grb-2 and the PI3K regulatory subunit p85, and correspondingly enhanced ERK and Akt activation, as compared with IGF-I-only conditions. Taken together, our data suggest that IRS-2 may play an important role in crosstalk between progesterone and the IGFs in breast cancer cells.

Rapid healing of femoral defects in rats with low dose sustained BMP2 expression from PEGDA hydrogel microspheres.

Current strategies for bone regeneration after traumatic injury often fail to provide adequate healing and integration. Here, we combined the poly (ethylene glycol) diacrylate (PEGDA) hydrogel with allogeneic "carrier" cells transduced with an adenovirus expressing BMP2. The system is unique in that the biomaterial encapsulates the cells, shielding them and thus suppressing destructive inflammatory processes. Using this system, complete healing of a 5 mm-long femur defect in a rat model occurs in under 3 weeks, through secretion of 100-fold lower levels of protein as compared to doses of recombinant BMP2 protein used in studies which lead to healing in 2-3 months. New bone formation was evaluated radiographically, histologically, and biomechanically at 2, 3, 6, 9, and 12 weeks after surgery. Rapid bone formation bridged the defect area and reliably integrated into the adjacent skeletal bone as early as 2 weeks. At 3 weeks, biomechanical analysis showed the new bone to possess 79% of torsional strength of the intact contralateral femur. Histological evaluation showed normal bone healing, with no infiltration of inflammatory cells with the bone being stable approximately 1 year later. We propose that these osteoinductive microspheres offer a more efficacious and safer clinical option over the use of rhBMP2.

Transient brown adipocyte-like cells derive from peripheral nerve progenitors in response to bone morphogenetic protein 2.

Perineurial-associated brown adipocyte-like cells were rapidly generated during bone morphogenetic protein 2 (BMP2)-induced sciatic nerve remodeling in the mouse. Two days after intramuscular injection of transduced mouse fibroblast cells expressing BMP2 into wild-type mice, there was replication of beta-3 adrenergic receptor(+) (ADRB3(+)) cells within the sciatic nerve perineurium. Fluorescence-activated cell sorting and analysis of cells isolated from these nerves confirmed ADRB3(+) cell expansion and their expression of the neural migration marker HNK1. Similar analysis performed 4 days after BMP2 delivery revealed a significant decrease in ADRB3(+) cells from isolated sciatic nerves, with their concurrent appearance within the adjacent soft tissue, suggesting migration away from the nerve. These soft tissue-derived cells also expressed the brown adipose marker uncoupling protein 1 (UCP1). Quantification of ADRB3-specific RNA in total hind limb tissue revealed a 3-fold increase 2 days after delivery of BMP2, followed by a 70-fold increase in UCP1-specific RNA after 3 days. Expression levels then rapidly returned to baseline by 4 days. Interestingly, these ADRB3(+) UCP1(+) cells also expressed the neural guidance factor reelin. Reelin(+) cells demonstrated distinct patterns within the injected muscle, concentrated toward the area of BMP2 release. Blocking mast cell degranulation-induced nerve remodeling resulted in the complete abrogation of UCP1-specific RNA and protein expression within the hind limbs following BMP2 injection. The data collectively suggest that local BMP2 administration initiates a cascade of events leading to the expansion, migration, and differentiation of progenitors from the peripheral nerve perineurium to brown adipose-like cells in the mouse, a necessary prerequisite for associated nerve remodeling.

An injectable method for noninvasive spine fusion.

BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) induce bone formation but are difficult to localize, and subsequent diffusion from the site of interest and short half-life reduce the efficacy of the protein. Currently, spine fusion requires stripping, decortications of the transverse processes, and an autograft harvest procedure. Even in combination with BMPs, clinical spinal fusion has a high failure rate, presumably because of difficulties in localizing sufficient levels of BMP. PURPOSE: The goal was to achieve reliable spine fusion through a single injection of a cell-based gene therapy system without the need for any surgical intervention. STUDY DESIGN: Eighty-seven immunodeficient (n=44) and immune-competent (n=43) mice were injected along the paraspinous musculature to achieve rapid induction of heterotopic ossification (HO) and ultimately spinal arthrodesis. METHODS: Immunodeficient and immune-competent mice were injected with fibroblasts, transduced with an adenoviral vector to express BMP2, along the paraspinous musculature. Bone formation was evaluated via radiographs, microcomputed tomography, and biomechanical analysis. RESULTS: ew bridging bone between the vertebrae and the fusion to adjacent skeletal bone was obtained as early as 2 weeks. Reduction in spine flexion-extension also occurred as early as 2 weeks after injection of the gene therapy system, with greater than 90% fusion by 4 weeks in all animals regardless of their genetic background. CONCLUSIONS: Injection of our cell-based system into the paraspinous musculature induces spinal fusion that is dependent neither on the cell type nor on the immune status. These studies are the first to harness HO in an immune-competent model as a noninvasive injectable system for clinically relevant spinal fusion and may one day impact human spinal arthrodesis.

Matrix metalloproteinase-9 is a diagnostic marker of heterotopic ossification in a murine model.

Heterotopic ossification (HO) is a serious disorder that occurs when there is aberrant bone morphogenic protein (BMP) signaling in soft tissues. Currently, there are no methods to detect HO before mineralization occurs. Yet once mineralization occurs, there are no effective treatments, short of surgery, to reverse HO. Herein, we used in vivo molecular imaging and confirmatory ex vivo tissue analyses of an established murine animal model of BMP-induced HO to show that matrix metalloproteinase-9 (MMP-9) can be detected as an early-stage biomarker before mineralization. Ex vivo analyses show that active MMP-9 protein is significantly elevated within tissues undergoing HO as early as 48 h after BMP induction, with its expression co-localizing to nerves and vessels. In vivo molecular imaging with a dual-labeled near-infrared fluorescence and micro-positron emission tomography (µPET) agent specific to MMP-2/-9 expression paralleled the ex vivo observations and reflected the site of HO formation as detected from microcomputed tomography 7 days later. The results suggest that the MMP-9 is a biomarker of the early extracellular matrix (ECM) re-organization and could be used as an in vivo diagnostic with confirmatory ex vivo tissue analysis for detecting HO or conversely for monitoring the success of tissue-engineered bone implants that employ ECM biology for engraftment.

Assessing mechanical integrity of spinal fusion by in situ endochondral osteoinduction in the murine model.

BACKGROUND: Historically, radiographs, micro-computed tomography (micro-CT) exams, palpation and histology have been used to assess fusions in a mouse spine. The objective of this study was to develop a faster, cheaper, reproducible test to directly quantify the mechanical integrity of spinal fusions in mice. METHODS: Fusions were induced in ten mice spine using a previously described technique of in situ endochondral ossification, harvested with soft tissue, and cast in radiolucent alginate material for handling. Using a validated software package and a customized mechanical apparatus that flexed and extended the spinal column, the amount of intervertebral motion between adjacent vertebral discs was determined with static flexed and extended lateral spine radiographs. Micro-CT images of the same were also blindly reviewed for fusion. RESULTS: Mean intervertebral motion between control, non-fused, spinal vertebral discs was 6.1 +/- 0.2 degrees during spine flexion/extension. In fusion samples, adjacent vertebrae with less than 3.5 degrees intervertebral motion had fusions documented by micro-CT inspection. CONCLUSIONS: Measuring the amount of intervertebral rotation between vertebrae during spine flexion/extension is a relatively simple, cheap (<$100), clinically relevant, and fast test for assessing the mechanical success of spinal fusion in mice that compared favorably to the standard, micro-CT.

Hydrogel microsphere encapsulation of a cell-based gene therapy system increases cell survival of injected cells, transgene expression, and bone volume in a model of heterotopic ossification.

Bone morphogenetic proteins (BMPs) are well known for their osteoinductive activity, yet harnessing this capacity remains a high-priority research focus. We present a novel technology that delivers high BMP-2 levels at targeted locations for rapid endochondral bone formation, enhancing our preexisting cell-based gene therapy system by microencapsulating adenovirus-transduced cells in nondegradable poly(ethylene glycol) diacrylate (PEGDA) hydrogels before intramuscular delivery. This study evaluates the in vitro and in vivo viability, gene expression, and bone formation from transgenic fibroblasts encapsulated in PEGDA microspheres. Fluorescent viability and cytotoxicity assays demonstrated >95% viability in microencapsulated cells. ELISA and alkaline phosphatase assays established that BMP-2 secretion and specific activity from microencapsulated AdBMP2-transduced fibroblasts were not statistically different from monolayer. Longitudinal transgene expression studies of AdDsRed-transduced fibroblasts, followed through live animal optical fluorescent imaging, showed that microencapsulated cells expressed longer than unencapsulated cells. When comparable numbers of microencapsulated AdBMP2-transduced cells were intramuscularly injected into mice, microcomputed tomography evaluation demonstrated that the resultant heterotopic bone formation was approximately twice the volume of unencapsulated cells. The data suggest that microencapsulation protects cells and prolongs and spatially distributes transgene expression. Thus, incorporation of PEGDA hydrogels significantly advances current gene therapy bone repair approaches.

Vessel formation is induced prior to the appearance of cartilage in BMP-2-mediated heterotopic ossification.

Heterotopic ossification (HO), or endochondral bone formation at nonskeletal sites, often results from traumatic injury and can lead to devastating consequences. Alternatively, the ability to harness this phenomenon would greatly enhance current orthopedic tools for treating segmental bone defects. Thus, understanding the earliest events in this process potentially would allow us to design more targeted therapies to either block or enhance this process. Using a murine model of HO induced by delivery of adenovirus-transduced cells expressing bone morphogenetic protein 2 (BMP-2), we show here that one of the earliest stages in this process is the establishment of new vessels prior to the appearance of cartilage. As early as 48 hours after induction of HO, we observed the appearance of brown adipocytes expressing vascular endothelial growth factors (VEGFs) simultaneous with endothelial progenitor replication. This was determined by using a murine model that possesses the VEGF receptor 2 (Flk1) promoter containing an endothelial cell enhancer driving the expression of nuclear-localized yellow fluorescent protein (YFP). Expression of this marker has been shown previously to correlate with the establishment of new vasculature, and the nuclear localization of YFP expression allowed us to quantify changes in endothelial cell numbers. We found a significant increase in Flk1-H2B::YFP cells in BMP-2-treated animals compared with controls. The increase in endothelial progenitors occurred 3 days prior to the appearance of early cartilage. The data collectively suggest that vascular remodeling and growth may be essential to modify the microenvironment and enable engraftment of the necessary progenitors to form endochondral bone.

Insulin-like growth factor-I activates gene transcription programs strongly associated with poor breast cancer prognosis.

PURPOSE: Substantial evidence implicates insulin-like growth factor-I (IGF-I) signaling in the development and progression of breast cancer. To more clearly elucidate the role of IGF in human breast cancer, we identified and then examined gene expression patterns of IGF-I-treated breast cancer cells. METHODS: MCF-7 cells were stimulated with IGF-I for 3 or 24 hours and were profiled for greater than 22,000 RNA transcripts. We defined an IGF-I signature pattern of more than 800 genes that were up- or downregulated at both time points. The gene signature was examined in clinical breast tumors and in experimental models that represented other oncogenic pathways. The signature was correlated with clinical and pathologic variables and with patient outcome. RESULTS: IGF-I caused temporal changes in gene expression that were strongly associated with cell proliferation, metabolism, and DNA repair. Genes with early and sustained regulation by IGF-I were highly enriched for transcriptional targets of the estrogen receptor (ER), Ras/extracellular signal-related kinase 1/2, and phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathways. In three large, independent data sets of profiled human breast tumors, the IGF-I signature was manifested in the majority of ER-negative breast tumors and in a subset (approximately 25%) of ER-positive breast tumors. Patients who had tumors that manifested the IGF-I signature (including patients who did not receive adjuvant therapy) had a shorter time to a poor outcome event. The IGF gene signature was highly correlated with numerous poor prognostic factors and was one of the strongest indicators of disease outcome. CONCLUSION: Transcriptional targets of IGF-I represent pathways of increased aggressiveness and possibly hormone independence in clinical breast cancers.

The growth hormone receptor antagonist pegvisomant blocks both mammary gland development and MCF-7 breast cancer xenograft growth.

Mammary gland development is dependent upon the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis, this same axis has also been implicated in breast cancer progression. In this study we investigated the effect of a GH antagonist, pegvisomant (Somavert, Pfizer), on normal mammary gland development and breast cancer xenograft growth. Intraperitoneal administration of pegvisomant resulted in a 60% suppression of hepatic IGF-I mRNA levels and upto a 70-80% reduction of serum IGF-I levels. Pegvisomant administration to virgin female mice caused a significant delay of mammary ductal outgrowth that was associated with a decrease in the number of terminal end buds and reduced branching and complexity of the gland. This effect of pegvisomant was mediated by a complete inhibition of both GH and IGF-IR-mediated signaling within the gland. In breast cancer xenograft studies, pegvisomant caused shrinkage of MCF-7 xenografts, with an initial 30% reduction in tumor volume, which was associated with a 2-fold reduction in proliferation and a 2-fold induction of apoptosis. Long-term growth inhibition of MCF-7 xenografts was noted. In contrast, pegvisomant had no effect on MDA-231 or MDA-435 xenografts, consistent with primary growth of these xenografts being unresponsive to IGF-I both in vitro and in vivo. In MCF-7 xenografts that regressed, pegvisomant had only minor effects upon GHR and IGF-IR signaling. This data supports previous studies indicating a role for GH/IGF in mammary gland development, and suggests that pegvisomant maybe useful for the prevention and/or treatment of estrogen receptor positive breast cancer.