Marina D'Angelo

Joint and Bone Disease in Mucopolysaccharidoses VI and VII: Identification of New Therapeutic Targets and BioMarkers Using Animal Models

The mucopolysaccharidoses (MPS) are inherited metabolic disorders resulting from the defective catabolism of glycosaminoglycans. In this report, we find that the stimulation of MPS connective tissue cells by the inflammatory cytokines causes enhanced secretion of several matrix-degrading metalloproteinases (MMPs). In addition, expression of tissue inhibitor of metalloproteinase-1 was elevated, consistent with the enhanced MMP activity. These findings were not restricted to one particular MPS disorder or species, and are consistent with previous observations in animal models with chemically induced arthritis. Bromodeoxyuridine incorporation studies also revealed that MPS chondrocytes proliferated up to 5-fold faster than normal chondrocytes, and released elevated levels of transforming growth factor-beta, presumably to counteract the marked chondrocyte apoptosis and matrix degradation associated with MMP expression. Despite this compensatory mechanism, studies of endochondral ossification revealed a reduction in chondro-differentiation in the growth plates. Thus, although MPS chondrocytes grew faster, most of the newly formed cells were immature and could not mineralize into bone. Our studies suggest that altered MMP expression, most likely stimulated by inflammatory cytokines and nitric oxide, is an important feature of the MPS disorders. These data also identify several proinflammatory cytokines, nitric oxide, and MMPs as novel therapeutic targets and/or biomarkers of MPS joint and bone disease. This information should aid in the evaluation of existing therapies for these disorders, such as enzyme replacement therapy and bone marrow transplantation, and may lead to the development of new therapeutic approaches.

MMP-13 is induced during chondrocyte hypertrophy

During development, mRNA for matrix metalloproteinase‐13 (MMP‐13) is found associated with cartilage undergoing hypertrophy, suggesting that this collagenase plays a role in cell enlargement and/or cartilage calcification. Using chondrocytes from prehypertrophic cartilage of chick embryo sternae, we have examined the relationship between MMP‐13 expression and the transition to hypertrophy. When hypertrophy was induced by serum‐free culture with ascorbate and bone morphogenetic protein‐2 (BMP‐2), MMP‐13 mRNA levels paralleled those for type X collagen. Chondrocytes from the caudal, nonhypertrophying portion of chick sternae expressed neither type X collagen nor MMP‐13, confirming that MMP‐13 mRNA is a marker for hypertrophy. Zymography with conditioned medium yielded a proteinase band at 59 kDa, which was absent in nonhypertrophic chondrocytes. A polyclonal antibody raised against chick MMP‐13 reacted with the 59‐kDa protein, confirming that it is MMP‐13. Although mRNA for MMP‐13 peaked at days 4–5 of culture, only low levels of MMP‐13 activity were present, and the activity increased gradually in parallel with later increases in MMP‐2. These results suggest that MMP‐13 is activated by MMP‐2 during chondrocyte maturation, and that the combination of both proteinases is required to prepare cartilage matrix for subsequent calcification, before endochondral ossification. J. Cell. Biochem. 77:678–693, 2000.

Smad-Runx interactions during chondrocyte maturation.

Background: Intracellular signaling triggered by bone morphogenetic proteins (BMPs) results in activated Smad complexes that regulate transcription of BMP-responsive genes. However, the low specificity of Smad binding to regulatory sequences implies that additional tissue-specific transcription factors are also needed. Runx2 (Cbfa1) is a transcription factor required for bone formation. We have examined the role of Smads and Runx2 in BMP induction of type X collagen, which is a marker of chondrocyte hypertrophy leading to endochondral bone formation. Methods: Pre-hypertrophic chondrocytes from the cephalic portion of the chick embryo sternum were placed in culture in the presence or absence of rhBMP-2. Cultures were transiently transfected with DNA containing the BMP-responsive type X collagen promoter upstream of the luciferase gene. The cultures were also transfected with plasmids, causing over-expression of Smads or Runx2, or both. After 24-48 hours, cell extracts were examined for levels of luciferase expression. Results: In the presence of BMP-2, chondrocytes over-expressing BMP-activated Smad1 or Smad5 showed significant enhancement of luciferase production compared with that seen with BMP alone. This enhancement was not observed with over-expression of Smad2, a transforming growth factor beta (TGF-&bgr;)-activated Smad. Over-expression of Runx2 in BMP-treated cultures increased transcriptional activity to levels similar to those seen with Smads 1 or 5. When chondrocytes were simultaneously transfected with both Runx2 and Smad 1 or 5, promoter activity was further increased, indicating that BMP-stimulated Smad activity can be augmented by increasing the levels of Runx2. Conclusions: These results implicate the skeletal tissue transcription factor Runx2 in regulation of chondrocyte hypertrophy and suggest that maximal transcription of the type X collagen gene in pre-hypertrophic chondrocytes involves interaction of BMP-stimulated Smads with Runx2. Clinical Relevance: Many skeletal abnormalities are associated with impaired regulation of chondrocyte hypertrophy in growth plates. These studies demonstrate that both BMP-activated Smads and Runx2 levels can modulate chondrocyte transition to hypertrophy.

Activation of transforming growth factor β in chondrocytes undergoing endochondral ossification

Transforming growth factor β (TGF‐β) has well‐documented roles in chondrocyte maturation and endochondral ossification, but the mechanisms of TGF‐β activation during these processes remain unclear. In this study, we analyzed TGF‐β activation in chick embryo resting, proliferating, and hypertrophic chondrocytes in culture. We found that both levels and activation of TGF‐β increased substantially with maturation. The majority of TGF‐β produced by resting cells over culture time remained latent, but a larger portion produced by proliferating and hypertrophic cells was activated with increasing maturation. Zymography of gelatin gels revealed that matrix metalloprotease 2 (MMP‐2) and MMP‐9 were expressed by each population and that MMP‐13 characterized hypertrophic chondrocytes and to a lesser extent proliferating chondrocytes in late cultures. Treatment with pharmacologic agents revealed that both MMPs and serine proteases are involved in activation. However, because inhibition of MMPs almost completely prevented TGF‐β activation, MMPs appear crucial for activation. During culture, inclusion of the tetracycline‐derived, collagenase/gelatinase inhibitor chemically modified nonantimicrobial tetracycline (CMT‐8) at concentrations specific for MMP‐13 inhibition resulted in complete inhibition of TGF‐β activation by proliferating and hypertrophic chondrocytes. These results show that TGF‐β production, release, and activation are regulated developmentally in chondrocytes. Our findings point to a strict mode of regulation of this potent factor to elicit diverse and highly specific effects during chondrocyte maturation and ossification.

Transforming growth factor-β receptor profiles of human and murine embryonic palate mesenchymal cells☆

Cell signalling in the developing mammalian palate appears to involve various growth factors and hormones. An important developmental role for the transforming growth factor-beta (TGF-beta) class of growth factors is suggested by the immunolocalization of TGF-beta 1 in the palate during its ontogeny. This study examined the effects of TGF-beta stimulation of, as well as TGF-beta receptor profiles in, murine embryonic palate mesenchymal (MEPM) and human embryonic palate mesenchymal (HEPM) cells. Results showed that TGF-beta 1 (1 ng/ml) stimulated proliferation of HEPM cells and inhibited proliferation of MEPM cells in a dose-dependent manner. The time course of 125I-TGF-beta 1 binding to specific receptors was determined by incubating cells in the presence of 170 pM 125I-TGF-beta 1 for up to 4 h. In both cell types, at 37 degrees C, the binding of 125I-TGF-beta decreased linearly over 4 h, while at 4 degrees C, binding increased with time of incubation. Incubation of both cell types at 4 degrees C for 4 h, with increasing concentrations of 125I-TGF-beta 1, resulted in binding which demonstrated saturation kinetics. Scatchard analyses revealed one class of receptors for HEPM (K 32.3 pM) and MEPM (K 26.3 pM). However, SDS-PAGE analyses of 125I-TGF-beta chemically crosslinked to specific receptor sites revealed that both cell types contained the types I (65,000 Mr) and III (230,000 Mr) TGF-beta receptors while MEPM also contained the type II (86,000 Mr) receptor. Binding studies further demonstrated the ability of platelet-derived growth factor to transmodulate TGF-beta binding. These results indicate that the HEPM cell line and primary cultures of MEPM cells, although obtained from palates at similar developmental stages, are dramatically different in their responsiveness to TGF-beta and have disparate TGF-beta receptor profiles.

Articular Chondrocytes Produce Factors That Inhibit Maturation of Sternal Chondrocytes in Serum‐Free Agarose Cultures: A TGF‐β Independent Process

Under normal conditions, articular chondrocytes persist throughout postnatal life, whereas “transient” chondrocytes, which constitute the bulk of prenatal and early postnatal cartilaginous skeleton, undergo maturation, hypertrophy, and replacement by bone cells. The mechanisms regulating the markedly different behavior and fate of articular and transient chondrocytes are largely unclear. In the present study, we asked whether articular chondrocytes possess dominant antimaturation properties which may subtend their ability to persist throughout life. Adult chicken articular chondrocytes and transient maturing chondrocytes from the core region of day 17 chick embryo cephalic sternum were cultured or cocultured in serum‐free agarose conditions. When the sternal cells were grown by themselves, they quickly developed into hypertrophic type X collagen‐synthesizing cells; however, when they were cocultured with as few as 10% articular chondrocytes or fed with articular chondrocyte‐conditioned medium, their maturation was markedly impaired, as revealed by a sharp drop in type X collagen synthesis. A similar, albeit less potent, antimaturation activity characterized resting and proliferating immature chondrocytes isolated from other regions of embryonic sternum. Transforming growth factor‐β2 (TGF‐β2) was previously suggested to be an inhibitor of chondrocyte maturation. We found, however, that treatment with a neutralizing antiserum to TGF‐β did not counteract the inhibition of maturation in cocultures of articular and maturing core sternal chondrocytes. Indeed, articular chondrocytes produced and accumulated relatively low levels of TGF‐β in their culture medium, about 15 ng/ml/48 h, of which over 90% was latent; surprisingly, maturing sternal core chondrocytes accumulated over 10‐fold more TGF‐β in the medium, about 150 ng/ml/48 h, of which over 20% was endogenously active. These results indicate that articular chondrocytes do possess dominant antimaturation properties which appear to be TGF‐β independent. The TGF‐βs may thus have a more prominent role in the terminal phases of chondrocyte maturation, as indicated by their abundance and greater activity in hypertrophic chondrocytes.

Transforming growth factor-β modulation of glycosaminoglycan production by mesenchymal cells of the developing murine secondary palate☆

Development of the mammalian secondary palate requires proper production of the extracellular matrix, particularly glycosaminoglycans (GAGs) and collagen. Endogenous factors that regulate the metabolism of these molecules are largely undefined. A candidate for a locally derived molecule would be transforming growth factor beta 1 (TGF beta 1) by virtue of its potency as a modulator of extracellular matrix metabolism by several cell lines. We have thus attempted to assign a regulatory role for TGF beta 1 in modulation of GAG production and degradation by mesenchymal cells of the murine embryonic palate (MEPM). Treatment with TGF beta 1 or TGF beta 2, but not IGF-II, resulted in a stimulation of total GAG synthesis. Furthermore, cells treated with both TGF beta 1 and TGF alpha showed a synergistic increase in GAG synthesis if pretreated with TGF beta 1 but not TGF alpha. Simultaneous stimulation with TGF beta 1 and TGF beta 2 did not elicit a synergistic response. These studies demonstrate the ability of TGF beta, synthesized by embryonic palatal cells, to specifically stimulate GAG synthesis by MEPM cells. Other growth factors present in the developing craniofacial region may also modulate TGF beta-induced GAG synthesis, a biosynthetic process critical to normal development of the embryonic palate.

Radiographic Evaluation of Bones and Joints in Mucopolysaccharidosis I and VII Dogs after Neonatal Gene Therapy

Mucopolysaccharidosis I (MPS I) and MPS VII are due to deficient activity of the glycosaminoglycan-degrading lysosomal enzymes alpha-L-iduronidase and beta-glucuronidase, respectively, and result in abnormal bones and joints. Here, the severity of skeletal disease in MPS I and MPS VII dogs and the effects of neonatal gene therapy were evaluated. For untreated MPS VII dogs, the lengths of the second cervical vertebrae (C2) and the femur were only 56% and 84% of normal, respectively, and bone dysplasia and articular erosions, and joint subluxation were severe. Previously, we reported that neonatal intravenous injection of a retroviral vector (RV) with the appropriate gene resulted in expression in liver and blood cells, and high serum enzyme activity. In this study, we demonstrate that C2 and femurs of RV-treated MPS VII dogs were longer at 82% and 101% of normal, respectively, and there were partial improvements of qualitative abnormalities. For untreated MPS I dogs, the lengths of C2 and femurs (91% and 96% of normal, respectively) were not significantly different from normal dogs. Qualitative changes in MPS I bones and joints were generally modest and were partially improved with RV treatment, although cervical spine disease was severe and was difficult to correct with gene therapy in both models. The greater severity of skeletal disease in MPS VII than in MPS I dogs may reflect accumulation of chondroitin sulfate in cartilage in MPS VII, or could relate to the specific mutations. Neonatal RV-mediated gene therapy ameliorates, but does not prevent, skeletal disease in MPS I and MPS VII dogs.

Anatomy in a New Curriculum: Facilitating the Learning of Gross Anatomy Using Web Access Streaming Dissection Videos

As anatomy course hours have decreased, it has become increasingly important to provide tools that facilitate laboratory task efficiency. Digital video clips were created to present dissection guidance to medical students. The video clips communicate challenging aspects of the dissection process with succinct visual demonstrations easily accessed via an online course site. Students were asked to complete a survey designed to assess the quality and utility of the videos. Survey respondents indicated that the videos enhanced the quality of the anatomy course as well as their individual performances. This teaching tool enhances student competencies in human gross anatomy.

Utilization of Bone Morphogenetic Protein Receptors during Chondrocyte Maturation.

Cartilage from the upper, cephalic portion of embryonic chick sternums undergoes hypertrophy, while the lower, caudal portion of the sternum remains as cartilage. Bone morphogenetic proteins (BMPs) induce type X collagen (colX) in cultured upper but not lower sternal chondrocytes (LSCs). We have examined the utilization of BMP receptors (BMPRs) by upper sternal chondrocytes (USCs) and LSCs both by analyzing receptor expression and by overexpressing mutant BMPRs. Reverse‐transcription polymerase chain reaction (RT‐PCR) analyses indicate that both upper and lower chondrocytes produce messenger RNA (mRNA) for all three receptors: BMPR type IA (BMPR‐IA), BMPR type IB (BMPR‐IB), and BMPR type II (BMPR‐II). Infection of USC with retroviral vectors expressing constitutively active (CA) BMPRs showed that CA‐BMPR‐IB, like exogenous BMP‐4, induced both colX mRNA and elevated alkaline phosphatase (AP), while CA‐BMPR‐IA was markedly less potent. However, expression of activated receptors in LSC cultures resulted in only minimal induction of hypertrophic markers. Consistent with the results seen for CA receptors, dominant negative (DN) BMPR‐IB blocked BMP‐induced hypertrophy in USCs more effectively than DN‐BMPR‐IA. These results imply that the major BMPR required for BMP induction of chondrocyte hypertrophy is BMPR‐IB, and that difference between permanent and prehypertrophic chondrocytes is not caused by absence of receptors required for BMP signaling.