Faculty Bibliography

Rationale: Vascular calcification is a significant contributor to cardiovascular morbidity and mortality. We recently reported that cartilage oligomeric matrix protein (COMP) is pivotal for maintaining the homeostasis of vascular smooth muscle cells (VSMCs). Whether COMP affects the process of vascular calcification is unknown. Objective: We aimed to test whether COMP modulates vascular calcification. Methods and Results: VSMC calcification in vitro was induced by calcifying media containing high inorganic phosphate or calcium. In vivo medial vessel calcification was induced in rats by 5/6 nephrectomy with a high-phosphate diet or by periadventitial application of CaCl(2) to the abdominal aorta. COMP protein level was markedly reduced in both calcified VSMCs and arteries. COMP deficiency remarkably exacerbated VSMC calcification, whereas ectopic expression of COMP greatly reduced calcification. Furthermore, COMP knockdown facilitated osteogenic markers expression by VSMCs even in the absence of calcifying media. By contrast, COMP overexpression significantly inhibited high phosphate- or high calcium-induced VSMC osteochondrogenic transition. Induction of osteogenic marker expression by COMP silencing was reversed by a soluble form of bone morphogenetic protein (BMP)-2 receptor IA, which suggests a BMP-2-dependent mechanism. Our data revealed that COMP bound directly to BMP-2 through the C terminus, inhibited BMP-2 receptor binding, and blocked BMP-2 osteogenic signaling, indicating COMP inhibits osteochondrogenic transition of VSMCs at least partially through inhibiting BMP-2. Conclusions: Our data strongly suggest that COMP is a novel inhibitor of vascular calcification. The imbalance between the effects of COMP and BMP-2 may provide new insights into the pathophysiology of vascular calcification

Granulin epithelin precursor (GEP) is a new growth factor that functions in brain development, chondrogenesis, tissue regeneration, tumorigenesis, and inflammation. The goal of this study was to study whether GEP was critical for odontogenesis and amelogenesis both in vivo and in vitro. The in situ hybridization and immunohistochemistry data showed that GEP was expressed in both odontoblast and ameloblast cells postnatally. Knockdown of GEP by crossing U6-ploxPneo-GEP and Sox2-Cre transgenic mice led to a reduction of dentin thickness, an increase in predentin thickness, and a reduction in mineral content in enamel. The in vitro application of recombinant GEP up-regulated molecular markers important for odontogenesis (DMP1, DSPP, and ALP) and amelogenesis (ameloblastin, amelogenin and enamelin). In conclusion, both the in vivo and the in vivo data support an important role of GEP in tooth formation during postnatal development

OBJECTIVES:: A delayed union or a nonunion of a fracture is a potentially adverse complication. Understanding the mechanisms of nonunion development may lead to improved treatment modalities. Proteases such as the matrix metalloproteinases play important roles in bone remodeling and repair, in which an imbalance or a nonfunctioning enzyme may lead to defects in bone healing (nonunion). The purpose of this pilot study was twofold: first to define an mRNA expression profile of all the matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases with thrombospondin motif (ADAMTS) enzymes, and their inhibitors (TIMPs) within fracture nonunion tissue, and second to compare this profile with mineralized fracture callus. METHODS:: Using a systematic real-time polymerase chain reaction, we screened the gene expression profiles of all members of the MMPs, ADAMTS, and their inhibitor TIMPs on human fracture nonunion tissue and matched mineralized callus tissue. Significant results were further analyzed using Western immunoblotting, immunohistochemistry, and in vitro protein interaction assays with bone morphogenetic protein-2. RESULTS:: This analysis confirmed MMP-7 and MMP-12 as two unidentified enzymes expressed in fracture nonunion tissue. Both MMP-7 and MMP-12 mRNAs were significantly elevated in nonunion tissue when compared with local mineralized callus from the same site (P < 0.001); the elevated protein levels of interest were visualized through immunoblotting and immunohistochemistry. In addition, these two MMPs were found to directly bind to and degrade bone morphogenetic protein-2 in vitro. CONCLUSION:: Collectively, our findings indicate that tissue present at the site of hypertrophic nonunions commonly expresses significantly higher levels of MMP-7 and MMP-12 in relation to mineralized fracture callus. Both were found to directly inactivate bone morphogenetic protein-2 in vitro, the best established growth factor in bone formation and repair

OBJECTIVE: To determine 1) whether a protein interaction network exists between granulin-epithelin precursor (GEP), ADAMTS-7/ADAMTS-12, and cartilage oligomeric matrix protein (COMP); 2) whether GEP interferes with the interactions between ADAMTS-7/ADAMTS-12 metalloproteinases and COMP substrate, including the cleavage of COMP; 3) whether GEP affects tumor necrosis factor alpha (TNFalpha)-mediated induction of ADAMTS-7/ADAMTS-12 expression and COMP degradation; and 4) whether GEP levels are altered during the progression of arthritis. METHODS: Yeast two-hybrid, in vitro glutathione S-transferase pull-down, and coimmunoprecipitation assays were used to 1) examine the interactions between GEP, ADAMTS-7/ADAMTS-12, and COMP, and 2) map the binding sites required for the interactions between GEP and ADAMTS-7/ADAMTS-12. Immunofluorescence cell staining was performed to visualize the subcellular localization of GEP and ADAMTS-7/ADAMTS-12. An in vitro digestion assay was employed to determine whether GEP inhibits ADAMTS-7/ADAMTS-12-mediated digestion of COMP. The role of GEP in inhibiting TNFalpha-induced ADAMTS-7/ADAMTS-12 expression and COMP degradation in cartilage explants was also analyzed. RESULTS: GEP bound directly to ADAMTS-7 and ADAMTS-12 in vitro and in chondrocytes, and the 4 C-terminal thrombospondin motifs of ADAMTS-7/ADAMTS-12 and each granulin unit of GEP mediated their interactions. Additionally, GEP colocalized with ADAMTS-7 and ADAMTS-12 on the cell surface of chondrocytes. More importantly, GEP inhibited COMP degradation by ADAMTS-7/ADAMTS-12 in a dose-dependent manner through 1) competitive inhibition through direct protein-protein interactions with ADAMTS-7/ADAMTS-12 and COMP, and 2) inhibition of TNFalpha-induced ADAMTS-7/ADAMTS-12 expression. Furthermore, GEP levels were significantly elevated in patients with either osteoarthritis or rheumatoid arthritis. CONCLUSION: Our observations demonstrate a novel protein-protein interaction network between GEP, ADAMTS-7/ADAMTS-12, and COMP. Furthermore, GEP is a novel specific inhibitor of ADAMTS-7/ADAMTS-12-mediated COMP degradation and may play a significant role in preventing the destruction of joint cartilage in arthritis

Granulin epithelin precursor (GEP) has been implicated in development, tissue regeneration, tumorigenesis, and inflammation. Herein we report that GEP stimulates chondrocyte differentiation from mesenchymal stem cells in vitro and endochondral ossification ex vivo, and GEP-knockdown mice display skeleton defects. Similar to bone morphogenic protein (BMP) 2, application of the recombinant GEP accelerates rabbit cartilage repair in vivo. GEP is a key downstream molecule of BMP2, and it is required for BMP2-mediated chondrocyte differentiation. We also show that GEP activates chondrocyte differentiation through Erk1/2 signaling and that JunB transcription factor is one of key downstream molecules of GEP in chondrocyte differentiation. Collectively, these findings reveal a novel critical role of GEP growth factor in chondrocyte differentiation and the molecular events both in vivo and in vitro.

In an effort to define the biological functions of COMP, a functional genetic screen was performed. This led to the identification of extracellular matrix protein 1 (ECM1) as a novel COMP-associated partner. COMP directly binds to ECM1 both in vitro and in vivo. The EGF domain of COMP and the C-terminus of ECM1 mediate the interaction between them. COMP and ECM1 colocalize in the growth plates invivo. ECM1 inhibits chondrocyte hypertrophy, matrix mineralization, and endochondral bone formation, and COMP overcomes the inhibition by ECM1. In addition, COMP-mediated neutralization of ECM1 inhibition depends on their interaction, since COMP largely fails to overcome the ECM1 inhibition in the presence of the EGF domain of COMP, which disturbs the association of COMP and ECM1. These findings provide the first evidence linking the association of COMP and ECM1 and the biological significance underlying the interaction between them in regulating endochondral bone growth

STUDY DESIGN: Retrospective chart review. OBJECTIVE: We reviewed the peri- and postoperative outcomes of our patients who had undergone lumbar and lumbosacral fusion both with and without recombinant human bone morphogenetic protein (rhBMP) over a period of 8 years to assess the frequency of complications and new diagnoses associated with the use of rhBMP2. SUMMARY OF BACKGROUND DATA: Administration of rhBMP2 for augmentation of lumbar and lumbosacral spinal fusion has not previously been associated with systemic complications. METHODS: A review of all patients undergoing lumbar and lumbosacral fusion over an 8-year period was performed to determine the frequency of postoperative complications and new diagnoses. Comparisons in complication frequency and new postoperative diagnoses between patients receiving rhBMP2 versus only allo- or autograft were made. Statistical methodology was applied to determine significance. RESULTS.: None of the 105 patients not receiving rhBMP2 and 3 of 24 patients receiving rhBMP2 had blood urea nitrogens and creatinines that more than doubled and reached values >30 and 1.5 mg/dL, respectively, after surgery (P = 0.006). Renal parameters returned to baseline within 45 days of surgery. Two of the 3 patients with postoperative renal insufficiency had been administered 16 mL (24 mg) of rhBMP2, whereas all other patients receiving rhBMP2 had received 8 mL (12 mg). Both of these patients also had supraventricular tachycardia, fever, and mental status changes after surgery. We recorded no significant increase in the incidence of new endocrinologic, autoimmune, neurologic, or neoplastic disorders associated with the use of rhBMP2 in our small patient population. CONCLUSION: A small subset of patients may develop transient renal insufficiency after rhBMP2 to augment spinal fusion. Higher doses of rhBMP2 may possibly increase the risk of developing renal insufficiency in particular patients; however, additional study is needed before all the risk factors are understood

Rational: Vascular smooth muscle cells (VSMCs) switching from a contractile/differentiated to a synthetic/dedifferentiated phenotype has an essential role in atherosclerosis, postangioplastic restenosis and hypertension. However, how normal VSMCs maintain the differentiated state is less understood. Objective: We aimed to indentify the effect of cartilage oligomeric matrix protein (COMP), a normal vascular extracellular matrix, on modulation of VSMCs phenotype. Methods and Results: We demonstrated that COMP was associated positively with the expression of VSMC differentiation marker genes during phenotype transition. Knockdown of COMP by small interfering (si)RNA favored dedifferentiation. Conversely, adenoviral overexpression of COMP markedly suppressed platelet-derived growth factor-BB-elicited VSMC dedifferentiation, characterized by altered VSMC morphology, actin fiber organization, focal adhesion assembly, and the expression of phenotype-dependent markers. Whereas alpha7 integrin coimmunoprecipitated with COMP in normal rat VSMCs and vessels, neutralizing antibody or siRNA against alpha7 integrin inhibited VSMC adhesion to COMP, which indicated that alpha7beta1 integrin is a potential receptor for COMP. As well, blocking or interference by siRNA of alpha7 integrin completely abolished the effect of COMP on conserving the contractile phenotype. In accordance, ectopic adenoviral overexpression of COMP greatly retarded VSMC phenotype switching, rescued contractility of carotid artery ring, and inhibited neointima formation in balloon-injured rats. Conclusions: Our data suggest that COMP is essential for maintaining a VSMC contractile phenotype and the protective effects of COMP are mainly mediated through interaction with alpha7beta1 integrin. Investigations to identify the factors affecting the expression and integrity of COMP may provide a novel therapeutic target for vascular disorders

The expression of the murine p200 family protein p204 in numerous tissues can be activated by a variety of distinct, tissue-specific transcription factors. p204 modulates cell proliferation, cell cycling, and the differentiation of various tissues, including skeletal muscle myotubes, beating cardiac myocytes, osteoblasts, chondrocytes, and macrophages. This protein modulates these processes in various ways, such as by (1) blocking ribosomal RNA synthesis in the nucleolus, (2) inhibiting Ras signaling in the cytoplasm, (3) promoting the activity of particular transcription factors in the nucleus by forming complexes with them, and (4) overcoming the block of the activity of other transcription factors by inhibitor of differentiation (Id) proteins. Much remains to be learned about p204, particularly with respect to its expected involvement in the differentiation of several as yet unexplored tissues

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family consists of 19 proteases. These enzymes are known to play important roles in development, angiogenesis and coagulation; dysregulation and mutation of these enzymes have been implicated in many disease processes, such as inflammation, cancer, arthritis and atherosclerosis. This review briefly summarizes the structural organization and functional roles of ADAMTSs in normal and pathological conditions, focusing on members that are known to be involved in the degradation of extracellular matrix and loss of cartilage in arthritis, including the aggrecanases (ADAMTS-4 and ADAMTS-5), ADAMTS-7 and ADAMTS-12, the latter two are associated with cartilage oligomeric matrix protein (COMP), a component of the cartilage extracellular matrix (ECM). We will discuss the expression pattern and the regulation of these metalloproteinases at multiple levels, including their interaction with substrates, induction by pro-inflammatory cytokines, protein processing, inhibition (e.g., TIMP-3, alpha-2-macroglobulin, GEP), and activation (e.g., syndecan-4, PACE-4)