Collagenolytic Enzymes Research Articles

Collagen cross-linking of the Boston keratoprosthesis donor carrier to prevent corneal melting in high-risk patients.

To examine the clinical relevance and pathophysiology of Boston keratoprosthesis (B-KPro)-related corneal keratolysis (cornea melt) and to describe a novel method of preventing corneal melt using ex vivo crosslinked cornea tissue carrier. A review of B-KPro literature was performed to highlight cases of corneal melt. Studies examining the effect of corneal collagen cross-linking (CXL) on the biomechanical properties of corneal tissue are summarized. The use of crosslinked corneal tissue as a carrier to the B-KPro is illustrated with a case. Corneal melting after B-KPro is a relatively rare event, occurring in 3% of eyes during the first 3 years of postoperative follow-up. The risk of post-KPro corneal melting is heightened in eyes with chronic ocular surface inflammation such as eyes with Stevens-Johnson syndrome and mucous membrane pemphigoid. This chronic inflammation results in high tear levels of matrix metalloproteinases, the enzymes responsible for collagenolysis and corneal melt. Crosslinked corneal tissue has been shown to have stiffer biomechanical properties and to be more resistant to degradation by collagenolytic enzymes. We have previously optimized the technique for ex vivo corneal CXL and are currently studying its impact on the prevention of corneal melting after B-KPro surgery in high-risk eyes. Crosslinked carrier tissue was used in a 52-year-old man with familial aniridia and severe post-KPro corneal melt. The patient maintained his visual acuity and showed no evidence of corneal thinning or melt in the first postoperative year. Collagen crosslinking was previously shown to halt the enzymatic degradation of corneal buttons ex vivo. This study demonstrates the safety and potential benefit of using crosslinked corneal grafts as carriers for the B-KPro, especially in eyes at higher risk of postoperative melt.

37% Phosphoric Acid Induced Stronger Matrix Metalloproteinase-8 Expression of the Dental Pulp than 19% Ethylene Diamine Tetraacetic Acid

Etching agents such as ethylene diamine tetraacetic acid (EDTA) and phosphoric acid which are widely used in adhesive restoration system aimed to increase for retention of restorative materials, may act a chemical irritant that induce inflammation of dental pulp. Inflammation is a body response against irritant and infectious agents. Matrix metalloproteinase-8, the major collagenolytic enzyme, degrades collagen type 1. This enzyme is expressed in low level in normal condition, however, the expression will increase during inflammation. The purpose of the present research was to study the effect of 19% EDTA and 37% phosphoric acid application as an etching agents on the MMP-8 expression of dental pulp. Forty-five male Sprague Dawley rats were divided into 3 groups. Cavity preparation was made on the occlusal surface of maxillary first molar using a round diamond bur. 19% EDTA, 37% phosphoric acid, and distilled water were applied on the surface of the cavity of the teeth in group I, II, and III subsequently. The cavity then filed by glass ionomer cements. The rats were sacrified at 1, 3, 5, 7, and 14 days after the treatment (n=3 for each day). The specimens were then processed histologically. Immunohistochemical (IHC) analysis was performed using rabbit anti rat MMP-8 polyclonal antibody to examine MMP-8 expression and HE (Hematoxylen Eosin) staining to observe the number of macrophages. The results showed 37% phos­phoric acid application induced stronger expression of MMP-8 and higher number of macrophages than 19% EDTA. The strongest expression of MMP-8 seems on 5 days after the treatment where the highest number of macrophages were also found.

A reconstructed metastasis model to recapitulate the metastatic spread in vitro.

Metastasis remains a leading cause of morbidity and mortality from solid tumors. Lack of comprehensive systems to study the progression of metastasis contributes to the low success of treatment. We developed a novel three-dimensional in vitro reconstructed metastasis (rMet) model that incorporates extracellular matrix (ECM) elements characteristic of the primary (breast, prostate, or lung) and metastatic (bone marrow, BM) sites. A cytokine-rich liquid interphase separates the primary and distant sites, further recapitulating circulation. Similar to main events underlying the metastatic cascade, the rMet model fractionated human tumor cell lines into sub-populations with distinct invasive and migratory abilities: (i) a primary tumor-like fraction mainly consisting of non-migratory spheroids; (ii) an invasive fraction that invaded through the primary tumor ECM, but failed to acquire anchorage-independence and reach the BM; and (iii) a highly migratory BM-colonizing population that invaded the primary ECM, survived in the "circulation-like" media, and successfully invaded and proliferated within BM ECM. BM-colonizing fractions successfully established metastatic bone lesions in vivo, whereas the tumor-like spheroids failed to engraft the bones, showing the ability of the rMet model to faithfully select for highly aggressive sub-populations with a propensity to colonize a metastatic site. By applying the rMet model to study real-time ECM remodeling, we show that tumor cells secrete collagenolytic enzymes for invading the primary site ECM but not for entering the BM ECM, indicating possible differences in ECM remodeling mechanisms at primary tumor versus metastatic sites.

Glycation cross-linking induced mechanical–enzymatic cleavage of microscale tendon fibers

Recent molecular modeling data using collagen peptides predicted that mechanical force transmitted through intermolecular cross-links resulted in collagen triple helix unwinding. These simulations further predicted that this unwinding, referred to as triple helical microunfolding, occurred at forces well below canonical collagen damage mechanisms. Based in large part on these data, we hypothesized that mechanical loading of glycation cross-linked tendon microfibers would result in accelerated collagenolytic enzyme damage. This hypothesis is in stark contrast to reports in literature that indicated that individually mechanical loading or cross-linking each retards enzymatic degradation of collagen substrates. Using our Collagen Enzyme Mechano-Kinetic Automated Testing (CEMKAT) System we mechanically loaded collagen-rich tendon microfibers that had been chemically cross-linked with sugar and tested for degrading enzyme susceptibility. Our results indicated that cross-linked fibers were >5 times more resistant to enzymatic degradation while unloaded but became highly susceptible to enzyme cleavage when they were stretched by an applied mechanical deformation.

Histochemical examination of cathepsin K, MMP1 and MMP2 in compressed periodontal ligament during orthodontic tooth movement in periostin deficient mice

The purpose of this study was to investigate immunolocalization of collagenolytic enzymes including cathepsin K, matrix metalloproteinase (MMP) 1 and 2 in the compressed periodontal ligament (PDL) during orthodontic tooth movement using a periostin deficient (Pn-/-) mouse model. Twelve-week-old male mice homozygous for the disrupted periostin gene and their wild type (WT) littermates were used in these experiments. The tooth movement was performed according to Waldo's method, in which elastic bands of 0.5mm thickness were inserted between the first and second upper molars of mice under anesthesia. At 1 and 3days after orthodontic force application, mice were fixed with transcardial perfusion of 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4), and the first molars and peripheral alveolar bones were extracted for histochemical analyses. Compared with WT mice, immunolocalization of cathepsin K, MMP1 and MMP2 was significantly decreased at 1 and 3days after orthodontic tooth movement in the compressed PDL of Pn-/- mice, although MMP1-reactivity and MMP2-reactivity decreased at different amounts. Very little cathepsin K-immunoreactivity was observed in the assessed regions of Pn-/- mice, both before and after orthodontic force application. Furthermore, Pn-/- mice showed a much wider residual PDL than WT mice. Taken together, we concluded that periostin plays an essential role in the function of collagenolytic enzymes like cathepsin K, MMP1 and MMP2 in the compressed PDL after orthodontic force application.

Collagenases and cracks in the plaque

The core of an atheromatous plaque contains lipids, macrophages, and cellular debris, typically covered by a fibrous cap that separates the thrombogenic core from the blood. Rupture of the fibrous cap causes most fatal myocardial infarctions. Interstitial collagen confers tensile strength on the cap, as it does in skin and tendons. In 1994, Peter Libby and colleagues demonstrated overexpression of collagenolytic enzymes in atheromatous plaques and implicated MMPs in the destabilization of these lesions.

Metabolic regulation of osteoclast differentiation and function

The osteoclast is a giant cell that resorbs calcified matrix by secreting acids and collagenolytic enzymes. The molecular mechanisms underlying metabolic adaptation to the increased biomass and energetic demands of osteoclastic bone resorption remain elusive. Here we show that during osteoclastogenesis the expression of both glucose transporter 1 (Glut1) and glycolytic genes is increased, whereas the knockdown of hypoxia-inducible factor 1-alpha (Hif1α), as well as glucose deprivation, inhibits the bone-resorbing function of osteoclasts, along with a suppression of Glut1 and glycolytic gene expression. Furthermore, the expression of the glutamine transporter solute carrier family 1 (neutral amino acid transporter), member 5 (Slc1a5) and glutaminase 1 was increased early in differentiation, and a depletion of L-glutamine or pharmacological inhibition of the Slc1a5 transporter suppressed osteoclast differentiation and function. Inhibition of c-Myc function abrogated osteoclast differentiation and function, along with a suppression of Slc1a5 and glutaminase 1 gene expression. Genetic and pharmacological inhibition of mammalian target of rapamycin (mTOR), as well as the activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), inhibited osteoclastogenesis. Thus, the uptake of glucose and glutamine and utilization of the carbon sources derived from them, coordinated by HIF1α and c-Myc, are essential for osteoclast development and bone-resorbing activity through a balanced regulation of the nutrient and energy sensors, mTOR and AMPK.

The pleotropic role of statins: Could it be the imminent host modulation agent in periodontics?

Periodontal disease is a chronic inflammatory disease which represents a primarily anaerobic Gram-negative oral infection that results in gingival inflammation, loss of attachment, bone destruction. Bacterial endotoxins in the form of lipopolysaccharides (LPS) that are instrumental in generating a host-mediated tissue destructive immune response by mobilizing their defensive cells and releasing cytokines like Interleukin-1β (IL-1β), Tumor Necrosis Factor-α (TNF-α), and Interleukin-6 (IL-6), which lead to tissue destruction by stimulating the production of the collagenolytic enzymes: Matrix metalloproteinases (MMPs). Since the host-mediated tissue destruction is to be controlled, various means have been employed for modulating this response. Statins, 3-hydroxy-3-methylglutarylcoenzyme A (HMG CoA) reductase inhibitors, besides having lipid-lowering abilities also have antioxidant, antithrombotic, anti-inflammatory, immunomodulatory and osteomodulatory properties. All of these pleiotropic effects of statins point out to it perhaps becoming the novel host modulation agent in periodontics.

Differential distribution of cathepsin B in human umbilical cord tissues.

The extracellular matrix components are differentially distributed among various structures of the umbilical cord. Wharton's jelly is especially rich in collagens and growth factors. Cathepsin B is a major cysteine protease involved in collagen degradation, as well as in the activation of precursor forms of other collagenolytic enzymes and growth factors. We assessed the activity and expression of cathepsin B in the umbilical cord arteries, veins and Wharton's jelly. Extracts of separated umbilical cord components were subjected to an activity assay with the use of specific fluorogenic substrate. The expression of cathepsin B protein was qualitatively evaluated by Western immunoblotting and quantitatively determined with an immunoenzymatic method. The total cathepsin B activity and content calculated per gram of DNA were higher in Wharton's jelly than in the umbilical cord vessels, and the latter parameter was the lowest in the umbilical cord arteries. Moreover, the expression and the activity of latent cathepsin B (following activation by pepsin digestion) calculated per gram of DNA were the highest in Wharton's jelly and the lowest in the umbilical cord arteries. High expression and activity of latent, pepsin-activatable cathepsin B related to DNA content in Wharton's jelly seem to reflect the stimulation of its cells by high amounts of collagen I and growth factors.

Optimizing dentin bond durability: Control of collagen degradation by matrix metalloproteinases and cysteine cathepsins

ObjectivesContemporary adhesives lose their bond strength to dentin regardless of the bonding system used. This loss relates to the hydrolysis of collagen matrix of the hybrid layers. The preservation of the collagen matrix integrity is a key issue in the attempts to improve the dentin bonding durability. MethodsDentin contains collagenolytic enzymes, matrix metalloproteinases (MMPs) and cysteine cathepsins, which are responsible for the hydrolytic degradation of collagen matrix in the bonded interface. ResultsThe identities, roles and function of collagenolytic enzymes in mineralized dentin has been gathered only within last 15years, but they have already been demonstrated to have an important role in dental hard tissue pathologies, including the degradation of the hybrid layer. Identifying responsible enzymes facilitates the development of new, more efficient methods to improve the stability of dentin–adhesive bond and durability of bond strength. SignificanceUnderstanding the nature and role of proteolytic degradation of dentin–adhesive interfaces has improved immensely and has practically grown to a scientific field of its own within only 10years, holding excellent promise that stable resin–dentin bonds will be routinely available in a daily clinical setting already in a near future.

Bioactive effects of a calcium/sodium phosphosilicate on the resin–dentine interface: a microtensile bond strength, scanning electron microscopy, and confocal microscopy study

This study evaluated, through microtensile bond strength (μTBS) testing, the bioactive effects of a calcium/sodium phosphosilicate (BAG) at the resin-dentine interface after 6 months of storage in phosphate buffer solution (PBS). Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were also performed. Three bonding protocols were evaluated: (i) RES-Ctr (no use of BAG), (ii) BAG containing adhesive (BAG-AD), and (iii) BAG/H (3) PO (4) before adhesive (BAG-PR). The dentin-bonded specimens were prepared for μTBS testing, which was carried out after 24 h or 6 months of storage in PBS. Scanning electron microscopy ultramorphology analysis was performed after debonding. Confocal laser scanning microscopy was used to evaluate the morphological and nanoleakage changes induced by PBS storage. High μTBS values were achieved in all groups after 24 h of storage in PBS. Subsequent to 6 months of storage in PBS the specimens created using the BAG-AD bonding approach still showed no significant reduction in μTBS. Moreover, specimens created using the BAG-AD or the BAG-PR approach showed an evident reduction of nanoleakage after prolonged storage in PBS. The use of BAG-containing adhesive may enhance the durability of the resin-dentine bonds through therapeutic/protective effects associated with mineral deposition within the bonding interface and a possible interference with collagenolytic enzyme activity (matrix metalloproteinases) responsible for the degradation of the hybrid layer.

Tenderization effect of cold-adapted collagenolytic protease MCP-01 on beef meat at low temperature and its mechanism

The enzymes currently used to increase meat tenderness are all mesophilic or thermophilic proteases. This study provides insight into the tenderization effect and the mechanism of a cold-adapted collagenolytic enzyme MCP-01 on beef meat at low temperatures. MCP-01 (10 U of caseinolytic activity) reduced the meat shear force by 23% and increased the relative myofibrillar fragmentation index of the meat by 91.7% at 4 °C, and it also kept the fresh colour and moisture of the meat. Compared to the commercially used tenderizers papain and bromelain, MCP-01 showed a unique tenderization mechanism. MCP-01 had a strong selectivity for degrading collagen at 4 °C, showed a distinct digestion pattern on the myofibrillar proteins, and had a different disruption pattern on the muscle fibres under scanning electron micrograph. These results suggest that the cold-adapted collagenolytic protease MCP-01 may be promising for use as a meat tenderizer at low and moderate temperatures.

New and Paradoxical Roles of Matrix Metalloproteinases in the Tumor Microenvironment

Processes such as cell proliferation, angiogenesis, apoptosis, or invasion are strongly influenced by the surrounding microenvironment of the tumor. Therefore, the ability to change these surroundings represents an important property through which tumor cells are able to acquire specific functions necessary for tumor growth and dissemination. Matrix metalloproteinases (MMPs) constitute key players in this process, allowing tumor cells to modify the extracellular matrix (ECM) and release cytokines, growth factors, and other cell-surface molecules, ultimately facilitating protease-dependent tumor progression. Remodeling of the ECM by collagenolytic enzymes such as MMP1, MMP8, MMP13, or the membrane-bound MT1-MMP as well as by other membrane-anchored proteases is required for invasion and recruitment of novel blood vessels. However, the multiple roles of the MMPs do not all fit into a simple pattern. Despite the pro-tumorigenic function of certain metalloproteinases, recent studies have shown that other members of these families, such as MMP8 or MMP11, have a protective role against tumor growth and metastasis in animal models. These studies have been further expanded by large-scale genomic analysis, revealing that the genes encoding metalloproteinases, such as MMP8, MMP27, ADAM7, and ADAM29, are recurrently mutated in specific tumors, while several ADAMTSs are epigenetically silenced in different cancers. The importance of these proteases in modifying the tumor microenvironment highlights the need for a deeper understanding of how stroma cells and the ECM can modulate tumor progression.

Permeability Variation Study in Collagen-Based Polymeric Capsules

Nanomedicine, the application of nanotechnologies to healthcare, is an area of nanotechnology which is promising and has extraordinary and far-reaching implications on human health both from the points of view of disease diagnosis and treatment. With regard to disease treatment, the possibility of developing new nanosystems for efficient drug delivery has attracted great attention. A prerequisite for nanotechnology-based delivery systems, in order to be effective, is that the encapsulated drug molecules have to be released at the site of interest. One method to obtain specific release is based on the development of systems sensitive to disease-associated overexpressed enzymes. In this study, we report on the fabrication of collagen-based containers for drug encapsulation and release by cellular demand by the action of the collagenolytic enzymes matrix metalloproteinases (MMPs).Collagen is the major component of the connective tissue and is involved in many biological functions. Its degradation is at the basis of different pathological processes. The up-regulated expression of MMPs is the cause of such degradation. Collagen-based capsules were fabricated by the layer-by-layer technique and characterized by scanning electron microscopy and confocal microscopy. Real-time monitoring of the release kinetics of loaded gold nanoparticles, having different diameters, was done by total reflection X-ray fluorescence. Excitation of the fluorescence was performed by grazing incidence X-ray beam of synchrotron radiation. This study represents the first attempt in estimating the distribution and dimension of pore size in polyelectrolyte nanoshells.

Pulmonary fibroblasts mobilize the membrane-tethered matrix metalloprotease, MT1-MMP, to destructively remodel and invade interstitial type I collagen barriers

In acute and chronic lung disease, widespread disruption of tissue architecture underlies compromised pulmonary function. Pulmonary fibroblasts have been implicated as critical effectors of tissue-destructive extracellular matrix (ECM) remodeling by mobilizing a spectrum of proteolytic enzymes. Although efforts to date have focused on the catabolism of type I collagen, the predominant component of the lung interstitial matrix, the key collagenolytic enzymes employed by pulmonary fibroblasts remain unidentified. Herein, membrane type-1 matrix metalloprotease (MT1-MMP) is identified as the dominant and direct-acting protease responsible for the type I collagenolytic activity mediated by both mouse and human pulmonary fibroblasts. Furthermore, MT1-MMP is shown to be essential for pulmonary fibroblast migration within three-dimensional (3-D) hydrogels of cross-linked type I collagen that recapitulate ECM barriers encountered in the in vivo environment. Together, these findings demonstrate that MT1-MMP serves as a key effector of type I collagenolytic activity in pulmonary fibroblasts and earmark this pericellular collagenase as a potential target for therapeutic intervention.

Small-angle light scattering to detect strain-directed collagen degradation in native tissue

It has been demonstrated that there is a mechanochemical relationship between collagen and collagenolytic enzymes such that increased tensile mechanical strain reduces the enzymatic cutting rate. This mechanochemical relationship has the potential to permit directed remodelling of tissue-engineered constructs in vitro and to shed light on the generation of load-adapted collagen-based connective tissue. In this investigation, we demonstrate that small-angle light scattering (SALS) has the sensitivity to dynamically detect the preferential enzymatic degradation of a subset of unloaded collagen fibrils within differentially loaded native tissue. Detection of the difference in the relative degradation rate of unloaded fibrils versus loaded fibrils was manifested through changes in the spatial distribution of the SALS signal. Specifically, we found a linear increase in the eccentricity of the SALS data that was consistent with preferential retention of the collagen fibrils aligned with the applied tensile strain. We conclude that SALS is simple, inexpensive and may provide a useful optical screening method permitting real-time monitoring of strain-controlled tissue and construct remodelling.

A polyphasic approach to the exploration of collagenolytic activity in the bacterial community associated with the marine sponge Cymbastela concentrica

Collagen is an important, extracellular structural protein for metazoans and provides a rich nutrient source for bacteria that possess collagen-degrading enzymes. In a symbiotic host system, collagen degradation could benefit the bacteria, but would be harmful for the eukaryotic host. Using a polyphasic approach, we investigated the presence of collagenolytic activity in the bacterial community hosted by the marine sponge Cymbastela concentrica. Functional screening for collagenase activity using metagenomic library clones (227 Mbp) and cultured isolates of sponge's bacterial community, as well as bioinformatic analysis of metagenomic shotgun-sequencing data (106,679 predicted genes) were used. The results show that the abundant members of the bacterial community contain very few genes encoding for collagenolytic enzymes, while some low-abundance sponge isolates possess collagenolytic activities. These findings indicate that collagen is not a preferred nutrient source for the majority of the members of the bacterial community associated with healthy C. concentrica, and that some low-abundance bacteria have collagenase activities that have the potential to harm the sponge through tissue degradation.

Extracellular matrix remodeling of the umbilical cord in pre-eclampsia as a risk factor for fetal hypertension.

The human umbilical cord forms a connection between the placenta and the foetus. It is composed of two arteries and one vein surrounded by Wharton's jelly. Pre-eclampsia is accompanied by extensive remodeling of extracellular matrix of umbilical cord. Matrix metalloproteinases (MMPs) are engaged in degradation of extracellular matrix proteins and activation/inactivation of certain cytokines and enzymes. These enzymes will probably play a central role in the release of matrix-embedded cytokines and growth factors. MMP-2 (gelatinase A) is the main collagenolytic enzyme of both umbilical artery and vein. Other metalloproteinases are present in several times lower amounts. Reduced activity of collagen-degrading enzymes may be a factor, which enhances the accumulation of collagen and some other proteins in the pre-eclamptic umbilical cord tissues. It seems to be possible that similar alterations occur in other fetal blood vessels. It may result in an increase in peripheral resistance as well as an increase in the blood pressure in the fetal vascular system. Some observations suggest that the raised pressure may persist after birth. Pre-eclampsia may be a factor that evokes an initiation of hypertension in utero and its amplification through childhood and adulthood.

Host Defense Against Implant Infection: The Ambivalent Role of Phagocytosis

Bacteria embedded in biofilms resist both antibiotics and host defense mechanisms. However, biofilms are not inherently protected against the attack of phagocytic cells. The weapons that polymorphonuclear neutrophils employ against implant infection are phagocytosis, degranulation, with release of antimicrobial molecules, and formation of Neutrophil Extracellular Traps (NETs). NETs contain DNA, histones, and neutrophil elastase, which enable neutrophils to fulfill their role of limiting both microbial spread and the collateral damage from granular contents. It is not yet clear whether the DNA released by neutrophils would support biofilm formation by adding to bacterial extracellular DNA (eDNA), an integral part of the biofilm extracellular matrix. In spite of the evidence of somewhat effective phagocytosis around an implant infection, biofilm-embedded staphylococci persist, tissue destruction occurs and, in the case of orthopedic implant infection, osteolysis prevails. The mechanism for tissue destruction is based on the infiltration and persistence at the site of infection of neutrophils which are unable to effectively perform phagocytosis, but able to inflict tissue damage and cause osteolysis by the release of proteolytic and collagenolytic enzymes. Phagocytosis thus has an ambiguous and ambivalent role: it carries out an antibacterial strategy and at the same time is responsible for osteolysis.

Pathogenesis of parodontitis in rheumatic diseases

Inflammatory periodontal disease (PD) is a common disease worldwide that has a primarily bacterial aetiology and is characterized by dysregulation of the host inflammatory response. The degree of inflammation varies among individuals with PD independently of the degree of bacterial infection, suggesting that alteration of the immune function may substantially contribute to its extent. Factors such as smoking, education, and body mass index (BMI) are discussed as potential risk factors for PD. Most PD patients respond to bacterial invaders by mobilizing their defensive cells and releasing cytokines such as interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, and IL-6, which ultimately causes tissue destruction by stimulating the production of collagenolytic enzymes, such matrix metalloproteinases. Recently, there has been growing evidence suggesting an association between PD and the increased risk of systemic diseases, such ateriosclerosis, diabetes mellitus, stroke, and rheumatoid arthritis (RA). PD and rheumatologic diseases such as RA share many pathological aspects and immunological findings.