Biomolecular Research Center Publications and Presentations

Osteochondritis Dissecans Knee Histology Studies Have Variable Findings and Theories of Etiology

Kevin G. Shea et al. — Fri, 03 May 2013 11:13:51 PDT

Background

Although many etiological theories have been proposed for osteochondritis dissecans (OCD), its etiology remains unclear. Histological analysis of the articular cartilage and subchondral bone tissues of OCD lesions can provide useful information about the cellular changes and progression of OCD. Previous research is predominantly comprised of retrospective clinical studies from which limited conclusions can be drawn.

Questions/purposes

The purposes of this study were threefold: (1) Is osteonecrosis a consistent finding in OCD biopsy specimens? (2) Is normal articular cartilage a consistent finding in OCD biopsy specimens? (3) Do histological studies propose an etiology for OCD based on the tissue findings?

Methods

We searched the PubMed, Embase, and CINAHL databases for studies that conducted histological analyses of OCD lesions of the knee and identified 1560 articles. Of these, 11 met our inclusion criteria: a study of OCD lesions about the knee, published in the English language, and performed a histological analysis of subchondral bone and articular cartilage. These 11 studies were assessed for an etiology proposed in the study based on the study findings.

Results

Seven of 11 studies reported subchondral bone necrosis. Four studies reported normal articular cartilage, two studies reported degenerated or irregular articular cartilage, and five studies found a combination of normal and degenerated or irregular articular cartilage. Five studies proposed trauma or repetitive stress and two studies proposed poor blood supply as possible etiologies.

Conclusions

We found limited research on histological analysis of OCD lesions of the knee. Future studies with consistent methodology are necessary to draw major conclusions about the histology and progression of OCD lesions. Inconsistent histologic findings have resulted in a lack of consensus regarding the presence of osteonecrosis, whether the necrosis is primary or secondary, the association of cartilage degeneration, and the etiology of OCD. Such studies could use a standardized grading system to allow better comparison of findings.

Minor Fibrillar Collagens, Variable Regions Alternative Splicing, Intrinsic Disorder, and Tyrosine Sulfation

Ming Fang et al. — Wed, 24 Oct 2012 13:50:33 PDT

Minor fibrillar collagen types V and XI, are those less abundant than the fibrillar collagens types I, II and III. The alpha chains share a high degree of similarity with respect to protein sequence in all domains except the variable region. Genomic variation and, in some cases, extensive alternative splicing contribute to the unique sequence characteristics of the variable region. While unique expression patterns in tissues exist, the functions and biological relevance of the variable regions have not been elucidated. In this review, we summarize the existing knowledge about expression patterns and biological functions of the collagen types V and XI alpha chains. Analysis of biochemical similarities among the peptides encoded by each exon of the variable region suggest the potential for shared function. The alternative splicing, conservation of biochemical characteristics in light of low sequence conservation, and evidence for intrinsic disorder, suggests modulation of binding events between the surface of collagen fibrils and surrounding extracellular molecules as a shared function.

Collagen Type XI α1 Chain Amino Propeptide Structural Model and Glycosaminoglycan Interactions in Silico

Christopher Mallory et al. — Fri, 07 Sep 2012 11:45:34 PDT

Modeling of the collagen α1(XI) amino propeptide (NPP) domain was performed to better understand how dimerization and glycosaminoglycan binding are coordinated. The program MODELLER was used to generate a homology model of collagen α1(XI) NPP domain based on the crystal structure of the closely related NC4 domain of collagen α1 (IX) (PDB:2UUR) to a root mean square deviation (rmsd) of 0.785 Å resolution. A model of collagen α1(XI) NPP domain dimer was constructed in two alternative templates; 1) the thrombospondin dimer template (PDB:1Z78), and 2) by submission of two monomer subunits based on PDB:2UUR to ClusPro. Calculation of relative binding energy for the interaction between each collagen α1(XI) NPP model and glycosaminoglycans as ligands was performed using AutoDock4. Results support a higher affinity between heparan sulfate and the dimer compared to the monomer. Sequential point mutation studies in the putative binding site (147-KKKITK-152) indicated the importance of each basic lysine residue in the binding of heparan sulfate. Two orders of magnitude change in binding affinity was predicted when comparing wild type to the mutation K152A.

Proteomic Analysis of Col11a1-associated Protein Complexes

Raquel Brown et al. — Wed, 16 Nov 2011 13:18:35 PST

Cartilage plays an essential role during skeletal development within the growth plate and in articular joint function. Interactions between the collagen fibrils and other extracellular matrix molecules maintain structural integrity of cartilage, orchestrate complex dynamic events during embryonic development, and help to regulate fibrillogenesis. To increase our understanding of these events, affinity chromatography and liquid chromatography/tandem mass spectrometry were used to identify proteins that interact with the collagen fibril surface via the amino terminal domain of collagen alpha 1(XI) a protein domain that is displayed at the surface of heterotypic collagen fibrils of cartilage. Proteins extracted from fetal bovine cartilage using homogenization in high ionic strength buffer were selected based on affinity for the amino terminal noncollagenous domain of collagen alpha 1(XI). Mass spectrometry was used to determine the amino acid sequence of tryptic fragments for protein identification. Extracellular matrix molecules and cellular proteins that were identified as interacting with the amino terminal domain of collagen alpha 1(XI) directly or indirectly, included proteoglycans, collagens, and matricellular molecules, some of which also play a role in fibrillogenesis, while others are known to function in the maintenance of tissue integrity. Characterization of these molecular interactions will provide a more thorough understanding of how the extracellular matrix molecules of cartilage interact and what role collagen XI plays in the process of fibrillogenesis and maintenance of tissue integrity. Such information will aid tissue engineering and cartilage regeneration efforts to treat cartilage tissue damage and degeneration

Deamidation Destabilizes and Triggers Aggregation of a Lens Protein, βA3-crystallin

Takumi Takata et al. — Wed, 16 Nov 2011 08:44:32 PST

Protein aggregation is a hallmark of several neurodegenerative diseases and also of cataracts. The major proteins in the lens of the eye are crystallins, which accumulate throughout life and are extensively modified. Deamidation is the major modification in the lens during aging and cataracts. Among the crystallins, the βA3-subunit has been found to have multiple sites of deamidation associated with the insoluble proteins in vivo. Several sites were predicted to be exposed on the surface of βA3 and were investigated in this study. Deamidation was mimicked by site-directed mutagenesis at Q42 and N54 on the N-terminal domain, N133 and N155 on the C-terminal domain, and N120 in the peptide connecting the domains. Deamidation altered the tertiary structure without disrupting the secondary structure or the dimer formation of βA3. Deamidations in the C-terminal domain and in the connecting peptide decreased stability to a greater extent than deamidations in the N-terminal domain. Deamidation at N54 and N155 also disrupted the association with the βB1-subunit. Sedimentation velocity experiments integrated with high-resolution analysis detected soluble aggregates at 15%–20% in all deamidated proteins, but not in wild-type βA3. These aggregates had elevated frictional ratios, suggesting that they were elongated. The detection of aggregates in vitro strongly suggests that deamidation may contribute to protein aggregation in the lens. A potential mechanism may include decreased stability and/or altered interactions with other β-subunits. Understanding the role of deamidation in the long-lived crystallins has important implications in other aggregation diseases.

Immunohistochemical Localization of Collagen Type XI α1 and α2 Chains in Human Colon Tissue

Karen B. Bowen et al. — Wed, 16 Nov 2011 07:03:20 PST

In previous studies, collagen XI mRNA has been detected in colon cancer, but its location in human colon tissue has not been determined. The heterotrimeric collagen XI consists of three a chains. While it is known that collagen XI plays a regulatory role in collagen fibril formation, its function in the colon is unknown. The characterization of normal human colon tissue will allow a better understanding of the variance of collagen XI in abnormal tissues. Grossly normal and malignant human colon tissue was obtained from pathology archives. Immunohistochemical staining with a 58K Golgi marker and α1(XI) and α2(XI) antisera was used to specifically locate their presence in normal colon tissue. A comparative bright field microscopic analysis showed the presence of collagen XI in human colon. The juxtanuclear, dot-like collagen XI staining in the Golgi apparatus of goblet cells in normal tissue paralleled the staining of the 58K Golgi marker. Ultra light microscopy verified these results. Staining was also confirmed in malignant colon tissue. This study is the first to show that collagen XI is present in the Golgi apparatus of normal human colon goblet cells and localizes collagen XI in both normal and malignant tissue. Although the function of collagen XI in the colon is unknown, our immunohistochemical characterization provides the foundation for future immunohistopathology studies of the colon.

Characterization of Collagenous Matrix Assembly in a Chondrocyte Model System

Sorcha Yingst et al. — Thu, 07 Jul 2011 13:36:49 PDT

Collagen is a major component of the newly synthesized pericellular microenvironment of chondrocytes. Collagen types II, IX, and XI are synthesized and assembled into higher ordered complexes by a mechanism in which type XI collagen plays a role in nucleation of new fibrils, and in limiting fibril diameter. This study utilizes a cell line derived from the Swarm rat chondrosarcoma that allows the accumulation and assembly of pericellular matrix. Immunofluorescence and atomic force microscopy were used to assess early intermediates of fibril formation. Results indicate that this cell line synthesizes and secretes chondrocyte-specific pericellular matrix molecules including types II, IX, and XI collagen and is suitable for the study of newly synthesized collagen matrix under the experimental conditions used. AFM data indicate that small fibrils or assemblies of microfibrils are detectable and may represent precursors of the ∼20 nm thin fibrils reported in cartilage. Treatment with hyaluronidase indicates that the dimensions of the small fibrils may be dependent upon the presence of hyaluronan within the matrix. This study provides information on the composition and organization of the newly synthesized extracellular matrix that plays a role in establishing the material properties and performance of biological materials such as cartilage.

Immuno-SEM Characterization of Developing Bovine Cartilage

Michelle Gerritsen et al. — Mon, 06 Jun 2011 16:29:12 PDT

Collagen is a vital material in the tissues of living organisms. Found almost everywhere in the human body, collagen is important in connective tissues, bone growth, and cartilage. Collagen XI makes up a very small portion of the cartilaginous tissue; however, it plays a key role in cartilaginous tissue. Collagen XI and two collagen XI isoforms, V1b and V2, are critical in the ossification process. The location of collagen XI, V1b, V2, and their specific functions in the ossification process within developing bovine cartilage are not well characterized. In this work, the location of collagens I, II, XI and two collagen XI isoforms, V1b and V2, present in developing bovine cartilage are investigated using the immuno-SEM technique. The results for the locations of collagen I and II indicate a high level of consistency with previous work, thus showing that the technique of immuno-SEM can be used with confidence to determine the location of various collagen types within cartilaginous and mineralized tissue. This work has shown that collagen XI is present in the lower hypertrophic region and also in a pericellular arrangement, within about two microns of cell walls, throughout the cartilaginous tissue. V1b is expressed in the articular surface, mineralized region, resting zone, and the distal edge of the diaphysis. The V2 isoform is most strongly expressed in areas of newly forming cartilage, and disappears with chondrocyte maturation. V2 is present in the distal edge of the epiphysis, as well as in mineralized tissue. Collagen XI and two of its isoforms, V1b and V2, are thought to play a critical role in the ossification process. However, this role is not well understood, and is still being characterized. The detection of collagen XI and two of its isoforms in the osteo-chondral junction as well as at a joint surface further point to collagen XI, V1b, and V2 playing a vital role in the ossification process, and warrants further research as to their specific function within the ossification process.

The Expression Patterns of Minor Fibrillar Collagens During Development in Zebrafish

Ming Fang et al. — Thu, 17 Mar 2011 13:45:45 PDT

Minor fibrillar collagens are recognized as the organizers and nucleators during collagen fibrillogenesis but likely serve additional functions. The minor fibrillar collagens include collagens type V and type XI. Mutations of collagen type V and XI can cause Ehlers Danlos, Stickler's, and Marshall's syndromes in human. We have characterized the spatiotemporal expression patterns of Col11a1, Col11a2, Col5a1 as well as Col5a3 in zebrafish embryos by in situ hybridization. Col5a1 is expressed in developing somites, neural crest, the head mesenchyme, developing cranial cartilage, pharyngeal arches and vertebrae. Col5a3 is detected in the notochord, mesenchyme cells in the eyes and lens. Both Col11a1 and Col11a2 have similar expression patterns, including notochord, otic vesicle, and developing cranial cartilages. Zebrafish may therefore serve as a valuable vertebrate model system for the study of diseases associated with collagens type V and XI mutations.

Inhibition of Proprotein Convertase Ski-1 Blocks Transcription of Key Extracellular Matrix Genes Regulating Osteoblastic Mineralization

Jeff P. Gorski et al. — Wed, 16 Mar 2011 14:51:04 PDT

Mineralization, a characteristic phenotypic property of osteoblastic lineage cells, was blocked by AEBSF and dec-RRLL-cmk, inhibitors of SKI-1 (site 1; subtilisin kexin like-1) protease. Since SKI-1 is required for activation of SREBP and CREB/ATF family transcription factors, we tested the effect of these inhibitors on gene expression. AEBSF decreased expression of 140 genes by 1.5- to 3.0-fold including Phex, Dmp1, COL1A1, COL11A1 and fibronectin. Direct comparison of AEBSF and dec-RRLL-cmk, a more specific SKI-1 inhibitor, demonstrated that expression of Phex, Dmp1, COL11A1 and fibronectin was reduced by both while COL1A2 and HMGCS1 were reduced only by AEBSF. AEBSF and dec-RRLL-cmk decreased the nuclear content of SKI-1 activated forms of transcription factors SREBP-1, SREBP-2, and OASIS. In contrast to AEBSF, the actions of dec-RRLL-cmk represent the sum of its direct actions on SKI-1 and indirect actions on caspase-3. Specifically, dec-RRLL-cmk reduced intracellular caspase-3 activity by blocking the formation of activated 19 kDa caspase-3. Conversely, over-expression of SKI-1 activated SREBP-1a and CREB-H in UMR106-01 osteoblastic cells increased the number of mineralized foci and altered their morphology to yield mineralization nodules, respectively. In summary, SKI-1 regulates the activation of transmembrane transcription factor precursors required for expression of key genes required for mineralization of osteoblastic cultures in vitro and bone formation in vivo. Our results indicate that the differentiated phenotype of osteoblastic cells, and possibly osteocytes, depends upon the non-apoptotic actions of SKI-1.

Differential Expression of Collagen Type V and XI α-1 in Human Ascending Thoracic Aortic Aneurysms

Julia Oxford — Thu, 24 Feb 2011 11:50:23 PST

Background—The molecular mechanisms leading to ascending thoracic aortic aneurysms (ATAAs) remain unknown. We hypothesized that alterations in expression levels of specific fibrillar collagens occur during the aneurysmal process.

Methods—Surgical samples from ascending aortas from patients with degenerative ATAAs were subdivided by aneurysm diameter: small, 5 to 6 cm; medium, 6 to 7 cm; and large, greater than 7 cm; and compared with nonaneurysmal aortas (mean diameter, 2.3 cm).

Results—Histology, immunofluorescence, and electron microscopy demonstrated greater disorganization of extracellular matrix constituents in ATAAs as compared with control with an increase in collagen α1(XI) within regions of cystic medial degenerative lesions. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed collagens type V and α1(XI) were significantly and linearly increased in ATAAs as compared with control (p < 0.001). There was no change in the messenger ribonucleic acid (mRNA) expression levels of collagens type I and III. Western blot analysis showed collagens type I and III were significantly decreased and collagens α1(XI) and V were significantly increased and were linearly correlated with the size of the aneurysm (p < 0.001 for both).

Conclusions—These results demonstrate that increased collagen α1(XI) and collagen V mRNA and protein levels are linearly correlated with the size of the aneurysm and provide a potential mechanism for the generation and progression of aneurysmal enlargement.

Collagen 11a1 is Indirectly Activated by Lymphocyte Enhancer-Binding Factor 1 (Lef1) and Negatively Regulates Osteoblast Maturation

Rachel A. Kahler et al. — Mon, 16 Nov 2009 15:33:31 PST

Alpha 1 (XI) collagen (Col11a1) is essential for normal skeletal development. Mutations in Col11a1 cause Marshall and Stickler syndromes, characterized by craniofacial abnormalities, nearsightedness and hearing abnormalities. Despite its link to human diseases, few studies have characterized the factors that control Col11a1 transcription. We previously identified Col11a1 as a differentially expressed gene in Lef1-suppressed MC3T3 preosteoblasts. Here we report that Lef1 activates the Col11a1 promoter. This activation is dependent upon the DNA binding domain of Lef1, but does not require the ß-catenin interaction domain, suggesting that it is not responsive to Wnt signals. Targeted deletion of Col11a1 with an antisense morpholino accelerated osteoblastic differentiation and mineralization in C2C12 cells, similar to what was observed in Lef1-suppressed MC3T3 cells. Moreover incubation with a purified Col11a1 N-terminal fragment, V1B, prevented alkaline phosphatase expression in MC3T3 and C2C12 cells. These results suggest that Lef1 is an activator of the Col11a1 promoter and that Col11a1 suppresses terminal osteoblast differentiation.

Breast Cancer Cells Stimulate Neutrophils to Produce Oncostatin M: Potential Implications for Tumor Progression

Marisa M. Queen et al. — Thu, 22 Jan 2009 18:05:49 PST

Tumor-associated and tumor-infiltrating neutrophils (TAN) and macrophages (TAM) can account for as much as 50% of the total tumor mass in invasive breast carcinomas. It is thought that tumors secrete factors that elicit a woundrepair response from TAMs and TANs and that this response inadvertently stimulates tumor progression. Oncostatin M is a pleiotropic cytokine belonging to the interleukin-6 family that is expressed by several cell types including activated human T lymphocytes, macrophages, and neutrophils. Whereas oncostatin M can inhibit the proliferation of breast cancer cells in vitro, recent studies suggest that oncostatin M may promote tumor progression by enhancing angiogenesis and metastasis. In addition, neutrophils can be stimulated to synthesize and rapidly release large quantities of oncostatin M. In this article, we show that human neutrophils secrete oncostatin M when cocultured with MDA-MB-231 and T47D human breast cancer cells. Neutrophils isolated from whole blood or breast cancer cells alone express little oncostatin M by immunocytochemistry and ELISA, but neutrophils express and release high levels of oncostatin M when they are cocultured with breast cancer cells. In addition, we show that granulocyte-macrophage colony-stimulating factor produced by breast cancer cells and cell-cell contact are both necessary for the release of oncostatin M from neutrophils. Importantly, neutrophilderived oncostatin M induces vascular endothelial growth factor from breast cancer cells in coculture and increases breast cancer cell detachment and invasive capacity, suggesting that neutrophils and oncostatin M may promote tumor progression in vivo.

Interaction Between Amino Propeptides of Type XI Procollagen 1 Chains

Julia T. Oxford — Thu, 15 Jan 2009 16:52:59 PST

Type XI collagen is a quantitatively minor yet essential constituent of the cartilage extracellular matrix. The amino propeptide of the 1 chain remains attached to the rest of the molecule for a longer period of time after synthesis than the other amino propeptides of type XI collagen and has been localized to the surface of thin collagen fibrils. Yeast two-hybrid system was used to demonstrate that a homodimer of 1(XI) amino propeptide (1(XI)Npp) could form in vivo. Interaction was also confirmed using multi-angle laser light scattering, detecting an absolute weight average molar mass ranging from the size of a monomer to the size of a dimer (25,000–50,000 g/mol), respectively. Binding was shown to be saturable by ELISA. An interaction between recombinant 1(XI)Npp and the endogenous 1(XI)Npp was observed, and specificity for 1(XI)Npp but not 2(XI)Npp was demonstrated by co-precipitation. The interaction between the recombinant form of 1(XI)Npp and the endogenous 1(XI)Npp resulted in a stable association during the regeneration of cartilage extracellular matrix by fetal bovine chondrocytes maintained in pellet culture, generating a protein that migrated with an apparent molecular mass of 50–60 kDa on an SDS-polyacrylamide gel.