Person:

Olsen, Bjorn

Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1). VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas.

Cranial base growth plates are important centers of longitudinal growth in the skull and are responsible for the proper anterior placement of the face and the stimulation of normal cranial vault development. We report that the presphenoidal synchondrosis (PSS), a midline growth plate of the cranial base, closes in the DBA/2J mouse strain but not in other common inbred strains. We investigated the genetics of PSS closure in DBA/2J mice by evaluating F1, F1 backcross, and/or F1 intercross offspring from matings with C57BL/6J and DBA/1J mice, whose PSS remain open. We observed that PSS closure is genetically determined, but not inherited as a simple Mendelian trait. Employing a genome-wide SNP array, we identified a region on chromosome 11 in the C57BL/6J strain that affected the frequency of PSS closure in F1 backcross and F1 intercross offspring. The equivalent region in the DBA/1J strain did not affect PSS closure in F1 intercross offspring. We conclude that PSS closure in the DBA/2J strain is complex and modified by different loci when outcrossed with C57BL/6J and DBA/1J mice. Electronic supplementary material The online version of this article (doi:10.1007/s00335-012-9437-8) contains supplementary material, which is available to authorized users.

To elucidate the transcriptional regulation of Bmp4 expression during organogenesis, we used phylogenetic footprinting and transgenic reporter analyses to identify Bmp4 cis-regulatory modules (CRMs). These analyses identified a regulatory region located (\sim)46 kb upstream of the mouse Bmp4 transcription start site that had previously been shown to direct expression in lateral plate mesoderm. We refined this regulatory region to a 396-bp minimal enhancer, and show that it recapitulates features of endogenous Bmp4 expression in developing mandibular arch ectoderm and incisor epithelium during the initiation-stage of tooth development. In addition, this enhancer directs expression in the apical ectodermal ridge (AER) of the developing limb and in anterior and posterior limb mesenchyme. Transcript profiling of E11.5 mouse incisor dental lamina, together with protein binding microarray (PBM) analyses, allowed identification of a conserved DNA binding motif in the Bmp4 enhancer for Pitx homeoproteins, which are also expressed in the developing mandibular and incisor epithelium. In vitro electrophoretic mobility shift assays (EMSA) and in vivo transgenic reporter mutational analyses revealed that this site supports Pitx binding and that the site is necessary to recapitulate aspects of endogenous Bmp4 expression in developing craniofacial and limb tissues. Finally, Pitx2 chromatin immunoprecipitation (ChIP) demonstrated direct binding of Pitx2 to this Bmp4 enhancer site in a dental epithelial cell line. These results establish a direct molecular regulatory link between Pitx family members and Bmp4 gene expression in developing incisor epithelium.

Cherubism is a skeletal dysplasia characterized by bilateral and symmetric fibro-osseous lesions limited to the mandible and maxilla. In most patients, cherubism is due to dominant mutations in the SH3BP2 gene on chromosome 4p16.3. Affected children appear normal at birth. Swelling of the jaws usually appears between 2 and 7 years of age, after which, lesions proliferate and increase in size until puberty. The lesions subsequently begin to regress, fill with bone and remodel until age 30, when they are frequently not detectable. Fibro-osseous lesions, including those in cherubism have been classified as quiescent, non-aggressive and aggressive on the basis of clinical behavior and radiographic findings. Quiescent cherubic lesions are usually seen in older patients and do not demonstrate progressive growth. Non-aggressive lesions are most frequently present in teenagers. Lesions in the aggressive form of cherubism occur in young children and are large, rapidly growing and may cause tooth displacement, root resorption, thinning and perforation of cortical bone. Because cherubism is usually self-limiting, operative treatment may not be necessary. Longitudinal observation and follow-up is the initial management in most cases. Surgical intervention with curettage, contouring or resection may be indicated for functional or aesthetic reasons. Surgical procedures are usually performed when the disease becomes quiescent. Aggressive lesions that cause severe functional problems such as airway obstruction justify early surgical intervention.

Cherubism is a rare bone dysplasia that is characterized by symmetrical bone resorption limited to the jaws. Bone lesions are filled with soft fibrous giant cell-rich tissue that can expand and cause severe facial deformity. The disorder typically begins in children at ages of 2-5 years and the bone resorption and facial swelling continues until puberty; in most cases the lesions regress spontaneously thereafter. Most patients with cherubism have germline mutations in the gene encoding SH3BP2, an adapter protein involved in adaptive and innate immune response signaling. A mouse model carrying a Pro416Arg mutation in SH3BP2 develops osteopenia and expansile lytic lesions in bone and some soft tissue organs. In this review we discuss the genetics of cherubism, the biological functions of SH3BP2 and the analysis of the mouse model. The data suggest that the underlying cause for cherubism is a systemic autoinflammatory response to physiologic challenges despite the localized appearance of bone resorption and fibrous expansion to the jaws in humans.

Progress in translational research has led to effective new treatments of a large number of diseases. Despite this progress, diseases including cancer and cardiovascular disorders still are at the top in death statistics and disorders such as osteoporosis and osteoarthritis represent an increasing disease burden in the aging population. Novel strategies in research are needed more than ever to overcome such diseases. The growing field of extracellular protein phosphorylation provides excellent opportunities to make major discoveries of disease mechanisms that can lead to novel therapies. Reversible phosphorylation/dephosphorylation of sites in the extracellular domains of matrix, cell-surface and trans-membrane proteins is emerging as a critical regulatory mechanism in health and disease. Moreover, a new concept is emerging from studies of extracellular protein phosphorylation: in cells where ATP is stored within secretory vesicles and released by exocytosis upon cell-stimulation, phosphorylation of extracellular proteins can operate as a messenger operating uniquely in signaling pathways responsible for long-term cellular adaptation. Here, we highlight new concepts that arise from this research, and discuss translation of the findings into clinical applications such as development of diagnostic disease markers and next-generation drugs.

It is well known that angiogenesis is linked to fibrotic processes in fibroproliferative diseases, but insights into pathophysiological processes are limited, due to lack of understanding of molecular mechanisms controlling endothelial and fibroblastic homeostasis. We demonstrate here that the matrix receptor anthrax toxin receptor 1 (ANTXR1), also known as tumor endothelial marker 8 (TEM8), is an essential component of these mechanisms. Loss of TEM8 function in mice causes reduced synthesis of endothelial basement membrane components and hyperproliferative and leaky blood vessels in skin. In addition, endothelial cell alterations in mutants are almost identical to those of endothelial cells in infantile hemangioma lesions, including activated VEGF receptor signaling in endothelial cells, increased expression of the downstream targets VEGF and CXCL12, and increased numbers of macrophages and mast cells. In contrast, loss of TEM8 in fibroblasts leads to increased rates of synthesis of fiber-forming collagens, resulting in progressive fibrosis in skin and other organs. Compromised interactions between TEM8-deficient endothelial and fibroblastic cells cause dramatic reduction in the activity of the matrix-degrading enzyme MMP2. In addition to insights into mechanisms of connective tissue homeostasis, our data provide molecular explanations for vascular and connective tissue abnormalities in GAPO syndrome, caused by loss-of-function mutations in ANTXR1. Furthermore, the loss of MMP2 activity suggests that fibrotic skin abnormalities in GAPO syndrome are, in part, the consequence of pathophysiological mechanisms underlying syndromes (NAO, Torg and Winchester) with multicentric skin nodulosis and osteolysis caused by homozygous loss-of-function mutations in MMP2.

Angiogenesis is an essential component of skeletal development and VEGF signaling plays an important if not pivotal role in this process. Previous attempts to examine the roles of VEGF in vivo have been largely unsuccessful because deletion of even one VEGF allele leads to embryonic lethality before skeletal development is initiated. The availability of mice expressing only the VEGF120 isoform (which do survive to term) has offered an opportunity to explore the function of VEGF during embryonic skeletal development. Our study of these mice provides new in vivo evidence for multiple important roles of VEGF in both endochondral and intramembranous bone formation, as well as some insights into isoform-specific functions. There are two key differences in vascularization of developing bones between wild-type and VEGF120/120 mice. VEGF120/120 mice have not only a delayed recruitment of blood vessels into the perichondrium but also show delayed invasion of vessels into the primary ossification center, demonstrating a significant role of VEGF at both an early and late stage of cartilage vascularization. These findings are the basis for a two-step model of VEGF-controlled vascularization of the developing skeleton, a hypothesis that is supported by the new finding that VEGF is expressed robustly in the perichondrium and surrounding tissue of cartilage templates of future bones well before blood vessels appear in these regions. We also describe new in vivo evidence for a possible role of VEGF in chondrocyte maturation, and document that VEGF has a direct role in regulating osteoblastic activity based on in vivo evidence and organ culture experiments.