Inhibition of transglutaminase activity in periodontitis rescues periodontal ligament collagen content and architecture.

BACKGROUND AND OBJECTIVE: Periodontal disease (PD) afflicts approximately 50% of the population in the United States and is characterized by chronic inflammation of the periodontium that can lead to loss of the periodontal ligament through collagen degradation, loss of alveolar bone, and to eventual tooth loss. Previous studies have implicated transglutaminase (TG) activity in promoting thin collagen I fiber morphology and decreased mechanical strength in homeostatic PDL. The aim of this study was to determine whether TG activity influenced collagen assembly in PDL in the setting of periodontal disease. MATERIAL AND METHODS: A ligature model was used to induce clinically relevant PD in mice. Mice with ligature were assessed at 5 and 14 days to determine PDL collagen morphology, transglutaminase (TG) activity, and bone loss. The effects of inhibition of TG on PDL were assessed by immunohistochemistry and second-harmonic generation (SHG) to visualize collagen fibers in native tissue. RESULTS: Ligature placement around the 2nd molar resulted in significant bone loss and a decrease in total collagen content after 5 days of ligature placement. A significant increase in thin over thick fibers was also demonstrated in mice with ligature at 5 days associated with apparent increases in immunoreactivity for TG2 and for TG-mediated N-ε-γ-glutamyl cross-links in PDL. Inhibition of TG activity increased total collagen and thick collagen fiber content over vehicle control in mice with ligature for 5 days. SHG of PDL was used to visualize and quantify the effects of TG inhibition on enhanced collagen fiber organization in unfixed control and diseased PDL. CONCLUSION: These studies support a role of TG in regulating collagen fiber assembly and suggest that strategies to inhibit TG activity in disease might contribute to restoration of PDL tissue integrity.

SPARC and the N-propeptide of collagen I influence fibroblast proliferation and collagen assembly in the periodontal ligament.

The periodontal ligament (PDL) is a fibrous connective tissue that anchors tooth cementum into alveolar bone. Secreted protein acidic and rich in cysteine (SPARC) is a collagen-binding matricellular protein known to influence collagen fiber assembly in the PDL. In contrast, functional properties of the N-propeptide of collagen I, encoded in exon 2 of the COL1A1 gene, are poorly understood. In this study, the PDL of collagen I exon 2-deleted (wt/ko), SPARC-null (ko/wt), and double transgenic (ko/ko) mice were evaluated in terms of cellularity, collagen area, fiber morphology, and extraction force and compared to WT (wt/wt) mice. Picro sirius red staining indicated a decrease in total PDL collagen content in each of the transgenic mice compared to WT at 1 and 3 month age points. At 12 months, only SPARC-null (ko/wt) and double-null PDL demonstrated less total collagen versus WT. Likewise, an increase in thin PDL collagen fibers was observed at 1 and 3 months in each transgenic, with increases only in SPARC-null and double-null mice at 12 months. The force required for tooth extraction was significantly reduced in SPARC-null versus exon 2-deleted and WT mice, whereas double-null mice demonstrated further decreases in force required for tooth extraction. The number of proliferating fibroblasts and number and size of epithelial rests of Malassez were increased in each transgenic versus WT with double-null PDL exhibiting highest levels of proliferation and rests of Malassez at 1 month of age. Consistent with increases in PDL collagen in exon-2 deleted mice, with age, numbers of rests decreased at 12 months in this genotype. These results demonstrate for the first time a functional role of the N-propeptide in regulating collagen fiber assembly and cell behavior and suggest that SPARC and the N-propeptide of collagen I have distinct activities in regulating collagen fiber assembly and fibroblast function.

Decreased Mechanical Strength and Collagen Content in SPARC-Null Periodontal Ligament Is Reversed by Inhibition of Transglutaminase Activity.

The periodontal ligament (PDL) is a critical tissue that provides a physical link between the mineralized outer layer of the tooth and the alveolar bone. The PDL is composed primarily of nonmineralized fibrillar collagens. Expression of secreted protein acidic and rich in cysteine (SPARC/osteonectin), a collagen-binding matricellular protein, has been shown to be essential for collagen homeostasis in PDL. In the absence of SPARC, PDL collagen fibers are smaller and less dense than fibers that constitute WT PDL. The aim of this study was to identify cellular mechanisms by which SPARC affected collagen fiber assembly and morphology in PDL. Cross-linking of fibrillar collagens is one parameter that is known to affect insoluble collagen incorporation and fiber morphology. Herein, the reduction in collagen fiber size and quantity in the absence of SPARC expression was shown to result in a PDL with reduced molar extraction force in comparison to that of WT mice (C57Bl/6J). Furthermore, an increase in transglutaminase activity was found in SPARC-null PDL by biochemical analyses that was supported by immunohistochemical results. Specifically, collagen I was identified as a substrate for transglutaminase in PDL and transglutaminase activity on collagen I was found to be greater in SPARC-null tissues in comparison to WT. Strikingly, inhibition of transglutaminase activity in SPARC-null PDL resulted in increases in both collagen fiber thickness and in collagen content, whereas transglutaminase inhibitors injected into WT mice resulted in increases in collagen fiber thickness only. Furthermore, PDL treated with transglutaminase inhibitors exhibited increases in molar extraction force in WT and in SPARC-null mice. Thus, SPARC is proposed to act as a critical regulator of transglutaminase activity on collagen I with implications for mechanical strength of tissues.

The effects of age and the expression of SPARC on extracellular matrix production by cardiac fibroblasts in 3-D cultures.

Fibrillar collagen is the primary component of the cardiac interstitial extracellular matrix. This extracellular matrix undergoes dramatic changes from birth to adulthood and then into advanced age. As evidence, fibrillar collagen content was compared in sections from neonates, adult, and old hearts and was found to increase at each respective age. Cardiac fibroblasts are the principle cell type that produce and control fibrillar collagen content. To determine whether fibroblast production, processing, and deposition of collagen differed with age, primary cardiac fibroblasts from neonate, adult, and old mice were isolated and cultured in 3-dimensional (3D) fibrin gels. Fibroblasts from each age aligned in fibrin gels along points of tension and deposited extracellular matrix. By confocal microscopy, wild-type neonate fibroblasts appeared to deposit less collagen into fibrillar structures than fibroblasts from adults. However, by immunoblot analysis, differences in procollagen production and processing of collagen I were not detected in neonate versus adult fibroblasts. In contrast, fibroblasts from old mice demonstrated increased efficiency of procollagen processing coupled with decreased production of total collagen. SPARC is a collagen-binding protein previously shown to affect cardiac collagen deposition. Accordingly, in the absence of SPARC, less collagen appeared to be associated with fibroblasts of each age grown in fibrin gels. In addition, the increased efficiency of procollagen alpha 1(I) processing in old wild-type fibroblasts was not detected in old SPARC-null fibroblasts. Increased levels of fibronectin were detected in wild-type neonate fibroblasts over that of adult and old fibroblasts but not in SPARC-null neonate fibroblasts versus older ages. Immunostaining of SPARC overlapped with that of collagen I but not to that of fibronectin in 3D cultures. Hence, whereas increases in procollagen processing, influenced by SPARC expression, plausibly contribute to increased collagen deposition in old hearts, other cellular mechanisms likely affect differential collagen deposition by neonate fibroblasts.

The Function of SPARC as a Mediator of Fibrosis.

Fibrosis is a common end-point of a number of different diseases such as hypertension, diabetes, liver cirrhosis, and those associated with chronic inflammation. Fibrosis is characterized by excessive deposition of extracellular matrix that interferes with normal tissue architecture and function. Increased expression of secreted protein acidic and rich in cysteine (SPARC) in fibrotic tissues has been reported in numerous studies. SPARC is a 43 kDa collagen-binding protein secreted from several different cell types into the extracellular matrix and has been shown to be anti-proliferative and counter-adhesive in vitro. SPARC is a matricellular protein; meaning SPARC is secreted into the extracellular space but does not serve a structural function. Instead, SPARC modulates interactions between cells and the surrounding extracellular matrix. In animal models of fibrotic disease and in human fibrotic tissues, elevated expression of SPARC has been reported in many tissues including heart, lungs, kidneys, liver, dermis, intestine, and eyes. In this review, we will summarize current studies that have examined the expression and functional importance of SPARC in various animal models of fibrosis and in human tissues. Although cellular mechanisms of SPARC in fibrosis remain to be fully elucidated, the studies summarized here provide impetus to further explore the efficacy of SPARC as a potential target for reducing fibrosis.