Cleavage Of Extracellular Matrix Research Articles

Potential role of extracellular granzyme B in wet age-related macular degeneration and fuchs endothelial corneal dystrophy.

Age-related ocular diseases are the leading cause of blindness in developed countries and constitute a sizable socioeconomic burden worldwide. Age-related macular degeneration (AMD) and Fuchs endothelial corneal dystrophy (FECD) are some of the most common age-related diseases of the retina and cornea, respectively. AMD is characterized by a breakdown of the retinal pigment epithelial monolayer, which maintains retinal homeostasis, leading to retinal degeneration, while FECD is characterized by degeneration of the corneal endothelial monolayer, which maintains corneal hydration status, leading to corneal edema. Both AMD and FECD pathogenesis are characterized by disorganized local extracellular matrix (ECM) and toxic protein deposits, with both processes linked to aberrant protease activity. Granzyme B (GrB) is a serine protease traditionally known for immune-mediated initiation of apoptosis; however, it is now recognized that GrB is expressed by a variety of immune and non-immune cells and aberrant extracellular localization of GrB substantially contributes to various age-related pathologies through dysregulated cleavage of ECM, tight junction, and adherens junction proteins. Despite growing recognition of GrB involvement in multiple age-related pathologies, its role in AMD and FECD remains poorly understood. This review summarizes the pathophysiology of, and similarities between AMD and FECD, outlines the current knowledge of the role of GrB in AMD and FECD, as well as hypothesizes putative contributions of GrB to AMD and FECD pathogenesis and highlights the therapeutic potential of pharmacologically inhibiting GrB as an adjunctive treatment for AMD and FECD.

Effects of heavy ions (12C6+) on malignant melanoma B16F10 cells.

BackgroundThis study aimed to investigate the effects of heavy ion (12C6+) irradiation on the proliferation, apoptosis, cell cycle, migration, and epithelial-mesenchymal transition (EMT) of B16F10 cells.MethodsThe B16F10 cells, which is a malignant melanoma cell line widely used in research, irradiated by 12C6+ and X-ray were detected by Hoechst 33342/propidium iodide double staining, Western blot, flow cytometry, and cell scratch tests to evaluate cell proliferation, expression of apoptosis-related proteins, G2/M phase arrest, cell migration, cell invasion and EMT.ResultsCompared with the same physical X-ray dose, 12C6+ could effectively inhibit the proliferation of B16F10 cells, inhibit the expression of B-cell lymphoma-2 (Bcl-2) and cellular-myelocytomatosis viral oncogene (c-Myc), and induce the expression of Bax to promote the apoptosis of B16F10 cells. After 12C6+ irradiation, the B16F10 cells exhibited G2/M phase arrest. B16F10 cells were highly sensitive to 12C6+ irradiation. Moreover, compared with X-ray, the 12C6+ irradiation significantly inhibited the migration of B16F10 cells and inhibited extracellular matrix cleavage, induced E-cadherin expression, enhanced cell adhesion, and further inhibited cell invasion, migration, and EMT.ConclusionsThe B16F10 cells were highly radiosensitive to 12C6+. Compared with X-ray, B16F10 cells irradiated by 12C6+ significantly reduced the expressions of matrix metalloproteinases to inhibit extracellular matrix cleavage and, thus, effectively inhibit cell invasion and metastasis. However, although the issue of the different therapeutic effects of heavy ion and X-ray radiotherapy on malignant melanoma was investigated and preliminary research results were obtained, several problems must be further studied.

Acute Exhaustive Exercise under Normoxic and Normobaric Hypoxic Conditions Differentially Regulates Angiogenic Biomarkers in Humans.

Background and Objectives: Angiogenesis describes the outgrowth of new capillaries from already existing ones. Different biomarkers regulate this process. Physical exercise and hypoxia are key stimuli for the activation of different angiogenic molecules, such as the vascular endothelial growth factor (VEGF). matrix metalloproteases (MMPs)-2 and -9 or the extracellular matrix cleavage fragment endostatin. The present study aimed to investigate influences of short-term, intensive cycling exercise under both normoxic and normobaric hypoxic conditions on the mentioned parameters. Materials and Methods: Twelve male subjects (age: 23.3 ± 2.0 years) participated in the study. All subjects conducted four intensive cycling tests until individual exhaustion in a randomized order under the following conditions: normoxia, 2000 m, 3000 m and 4000 m above sea level. Blood samples were taken before (pre) and 10 min, 30 min, 60 min and 240 min post exercise and were analyzed by ELISA. Results: VEGF showed a significantly reduced concentration compared to the pre-value solely under 4000 m at 10 min post exercise. MMP-2 showed significantly reduced concentrations at 240 min post exercise under 4000 m. MMP-9 increased at 240 min post exercise under both 2000 m and 4000 m conditions. Endostatin was significantly increased at 10 min post exercise independently of the applied stimulus. Conclusions: The presented data show that intensive short-term exercise bouts facilitate the bioavailability of angiogenic, ECM (extracellular matrix)-related biomarkers. This finding is interesting for both health- and performance-related research as it demonstrates the positive effects of intensive short exercise interventions.

Bimodal Effects of P2Y12 Antagonism on Matrix Metalloproteinase–Associated Contractile Dysfunction in İnsulin-Resistant Mammalian Heart

The matrix metalloproteinases (MMPs) contribute to matrix remodeling in diabetes via tissue degradation; however, their contributions can be different depending on the pathology. For instance, MMPs are elevated in acute stress hyperglycemia, whereas they can be degraded in chronic hyperglycemia. Since studies emphasize the possible cardioprotective effect of ticagrelor (Tica) beyond its antiplatelet action, we aimed to examine whether Tica treatment can reverse the depressed heart function of metabolic syndrome (MetS) rats via affecting the expression levels of MMPs. Tica treatment of high-carbohydrate-induced MetS rats could not affect significantly the depressed contractile activity of Langendorff-perfused heart preparations. On the other hand, the Tica treatment provided a significant recovery in the reduced relaxation activity of the aortic preparations from the same animals. Histological examination of the hearts demonstrated marked damages in Mets rats, such as increases in the number of foamy cells and accumulation of collagen fiber and increases in the elastic lamellar irregularity of tunica media, while Tica treatment provided a slight improvement in the structure of left ventricle tissue. We also could not obtain a significant reverse in the high cytosolic labile Zn2+ ([Zn2+]i) with the treatment of cardiomyocytes with Tica. Furthermore, Tica treatment of MetS rats could not significantly reverse the degraded protein levels of MMP-2 and MMP-9 in the heart, as well. Overall, we demonstrated that Tica treatment of MetS rats has no significant benefits on the depressed heart function, although provide a significant beneficial impact on vascular relaxation. This action of Tica may be through its lack of action on both MMP degradation and high [Zn2+]i, which can further precipitate in cleavage of extracellular matrix in the heart.

The Modulatory effects of Hesperitin on the Expression of Oxidative Stress Associated Genes in LPS‐Activated BV‐2 Microglial Cells

Numerous natural compounds have been reported to protect against oxidative stress and decrease the mortality related to chronic diseases. Flavonoids are known to protect against reactive oxygen species (ROS) damage. Many studies suggested a possible protective role of flavonoids in neurodegenerative and inflammatory conditions associated with excessive oxidative stress in the brain. In this study, we evaluated the effect of hesperetin, a major flavonoid found in oranges and mandarins, on LPS-activated BV-2 microglial cells. The methodology included cell viability, ELISA, RT-PCR using individual primers, and PCR arrays to screen a range of genes associated with oxidative stress. Cell viability results showed that only the highest concentration of 200 µM of hesperitin had a toxic effect on the BV-2 microglial cells, causing 20% of cell death. The combination of LPS and hesperitin also exhibited no toxicity in concentrations ranging from 0.78 to 100 µM. Still, in the concentration of 200 µM, there was a reduction of 50% of cell viability after 24 h. The ELISA and RT-PCR data demonstrated that hesperitin increased the expression of PDL-1 in the protein and transcription level in the LPS-activated cells. Also, hesperitin induced the mRNA expression of Nrf2, which is involved in the transcription of several antioxidant genes, and up-regulated the expression of MMP-9, which participates in the cleavage of extracellular matrix and plays a critical role in the central nervous system function. Also, the data from the oxidative stress PCR arrays showed that after 24 h, of cell treatment with 100 µM of hesperitin modulated numerous genes that regulate the oxidative stress process. The results show that hesperitin down-regulated the mRNA expression of Nos2, Ptgs2, Ncf1, Actb, Txnip, Ucp2, and up-regulated expression Srxn1, Prdx3, and Txn1. These genes participate not only in excessive oxidative stress processes by also in exacerbated inflammatory states. These data show that hesperitin may have a potential in the prevention and treatment of oxidative stress-related neurodegenerative diseases.

Single nucleotide polymorphism (SNP) rs3751143 in P2RX7 is associated with therapy failure in chronic Q fever while rs7125062 in MMP1 is associated with fewer complications

ObjectivesChronic Q fever is a persistent infection with the intracellular bacterium Coxiella burnetii. Development of chronic Q fever is associated with single nucleotide polymorphisms (SNPs) in genes encoding for pattern recognition receptors, for phagolysosomal pathway components and for matrix metalloproteinases (MMPs). We evaluated the association of SNPs in these innate-immunity and MMP genes with clinical outcomes. MethodsSNPs were selected from previous association studies and analysed in a cohort of patients with chronic Q fever. The primary outcome was all-cause mortality; secondary outcomes were therapy failure and chronic Q fever–related complications. Subdistribution hazard ratios (SHR) were calculated. ResultsNineteen SNPs were analysed in 134 patients with proven and 29 with probable chronic Q fever. In multivariable analysis, none of the selected SNPs was associated with all-cause mortality. However, SNP rs3751143 located in P2RX7 appeared to be associated with therapy failure (SHR 2.42; 95% confidence interval, 1.16–5.05; p 0.02), which is in line with other reports, showing that a loss of function of the P2X7 receptor leads to inefficient killing of intracellular organisms. In addition, SNP rs7125062 located in MMP1, involved in the cleavage of extracellular matrix, was associated with fewer chronic Q fever–related complications such as acute aneurysms (SHR 0.49; 95% confidence interval, 0.29–0.83; p 0.008). ConclusionsA polymorphism in P2RX7, known to lead to loss of function of the receptor and inefficient killing of intracellular organisms, and a polymorphism in MMP1 were respectively associated with more therapy failures and fewer complications such as acute aneurysms in patients with chronic Q fever.

Granzyme B Contributes to Barrier Dysfunction in Oxazolone-Induced Skin Inflammation through E-Cadherin and FLG Cleavage

Atopic dermatitis (AD) is the most common inflammatory skin condition. Skin barrier dysfunction is of major importance in AD because it facilitates allergen sensitization and systemic allergic responses. Long regarded as a pro-apoptotic protease, emerging studies indicate granzyme B (GzmB) to have extracellular roles involving the proteolytic cleavage of extracellular matrix, cell adhesion proteins, and basement membrane proteins. Minimally expressed in normal skin, GzmB is elevated in AD and is positively correlated with disease severity and pruritus. We hypothesized that GzmB contributes to AD through extracellular protein cleavage. A causative role for GzmB was assessed in an oxazolone-induced murine model of dermatitis, comparing GzmB-/- mice with wild-type mice, showing significant reductions in inflammation, epidermal thickness, and lesion formation in GzmB-/- mice. Topical administration of a small-molecule GzmB inhibitor reduced disease severity compared with vehicle-treated controls. Mechanistically, GzmB impaired epithelial barrier function through E-cadherin and FLG cleavage. GzmB proteolytic activity contributes to impaired epidermal barrier function and represents a valid therapeutic target for AD.

Enhanced endothelial motility and multicellular sprouting is mediated by the scaffold protein TKS4

Endothelial cell motility has fundamental role in vasculogenesis and angiogenesis during developmental or pathological processes. Tks4 is a scaffold protein known to organize the cytoskeleton of lamellipodia and podosomes, and thus modulating cell motility and invasion. In particular, Tks4 is required for the localization and activity of membrane type 1-matrix metalloproteinase, a key factor for extracellular matrix (ECM) cleavage during cell migration. While its role in transformed cells is well established, little is known about the function of Tks4 under physiological conditions. In this study we examined the impact of Tks4 gene silencing on the functional activity of primary human umbilical vein endothelial cells (HUVEC) and used time-lapse videomicrosopy and quantitative image analysis to characterize cell motility phenotypes in culture. We demonstrate that the absence of Tks4 in endothelial cells leads to impaired ECM cleavage and decreased motility within a 3-dimensional ECM environment. Furthermore, absence of Tks4 also decreases the ability of HUVEC cells to form multicellular sprouts, a key requirement for angiogenesis. To establish the involvement of Tks4 in vascular development in vivo, we show that loss of Tks4 leads sparser vasculature in the fetal chorion in the Tks4-deficient ‘nee’ mouse strain.

Characterization and in vitro expression studies of a potential xenogeneic DNA vaccine against canine mammary tumours

Matrix metalloproteinases-7 (MMP-7) which is expressed in a wide variety of malignant cells has been seen tobe extensively up-regulated in mammary carcinomas. MMP-7 can promote cancer invasion and angiogenesis through proteolytic cleavage of extracellular matrix and basement membrane proteins. This property of MMP-7 makes it a promising target in the context of immunotherapy. Further, to enhance DNA-based immunization, a cytokine gene can be employed as an adjuvant. Interleukin-18 (IL-18) is a Th1-type cytokine that has been demonstrated as a potential biological adjuvant in murine tumour models. The present study was undertaken to clone murine MMP-7 (mMMP-7) and IL-18 genes in pVIVO2.mcs eukaryotic expression vector and to characterize their expression by immunofluorescence and Western blotting. This double gene construct now may be used as a potential xenogeneic DNA vaccine against canine tumour model.

Diverse impact of acute and long-term extracellular proteolytic activity on plasticity of neuronal excitability.

Learning and memory require alteration in number and strength of existing synaptic connections. Extracellular proteolysis within the synapses has been shown to play a pivotal role in synaptic plasticity by determining synapse structure, function, and number. Although synaptic plasticity of excitatory synapses is generally acknowledged to play a crucial role in formation of memory traces, some components of neural plasticity are reflected by nonsynaptic changes. Since information in neural networks is ultimately conveyed with action potentials, scaling of neuronal excitability could significantly enhance or dampen the outcome of dendritic integration, boost neuronal information storage capacity and ultimately learning. However, the underlying mechanism is poorly understood. With this regard, several lines of evidence and our most recent study support a view that activity of extracellular proteases might affect information processing in neuronal networks by affecting targets beyond synapses. Here, we review the most recent studies addressing the impact of extracellular proteolysis on plasticity of neuronal excitability and discuss how enzymatic activity may alter input-output/transfer function of neurons, supporting cognitive processes. Interestingly, extracellular proteolysis may alter intrinsic neuronal excitability and excitation/inhibition balance both rapidly (time of minutes to hours) and in long-term window. Moreover, it appears that by cleavage of extracellular matrix (ECM) constituents, proteases may modulate function of ion channels or alter inhibitory drive and hence facilitate active participation of dendrites and axon initial segments (AISs) in adjusting neuronal input/output function. Altogether, a picture emerges whereby both rapid and long-term extracellular proteolysis may influence some aspects of information processing in neurons, such as initiation of action potential, spike frequency adaptation, properties of action potential and dendritic backpropagation.

Granzyme B Mediates Both Direct and Indirect Cleavage of Extracellular Matrix in Skin After Chronic Low‐Dose Ultraviolet Light Irradiation

Extracellular matrix (ECM) degradation is a hallmark of tissue aging, as well as many other chronic inflammatory diseases that can lead to a loss of function. Granzyme B (GzmB), a serine protease that is expressed by a variety cells, has been shown to accumulate in the extracellular space during chronic inflammation and cleave a number of proteins. Using a chronic low‐grade UV‐irradiation murine model, we hypothesized that GzmB contributes to skin damage and ECM degradation through processes involving both direct ECM cleavage and indirect ECM cleavage through the induction of other proteinases. Wild‐type and GzmB‐knockout (KO) mice were repeatedly exposed to minimal erythemal doses of solar simulated UV‐irradiation for up to 20 weeks. GzmB expression was significantly increased in wild‐type UV‐treated skin compared to non‐irradiated controls. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density. GzmB‐KO mice were also protective against the loss of fibronectin (FN) in vivo, and FN fragments were released from the cell‐derived matrix of cultured fibroblasts after GzmB treatment. GzmB‐mediated FN fragments increased the release of metalloproteinase (MMP)‐1 and MMP‐3 from fibroblasts. Furthermore, GzmB cleavage of decorin predisposed collagen fibrils more susceptible to attack by MMP‐1. Finally, GzmB reduced the barrier function of a monolayer of keratinocytes in vitro as measured by electrical impedance. Collectively, these findings indicate a significant role for GzmB in ECM degradation, which may have implications in many aged‐related dermatological indications.

V‐ATPase‐mediated Activation of Cellular and Secreted Cathepsins: Exploring Their Role in Breast Cancer Metastasis

Vacuolar H+‐ATPases are essential H+ pumps that acidify cellular compartments. V‐ATPases at the cell surface of MDA‐MB231 breast cancer cells may contribute to a highly metastatic phenotype. We hypothesize that cell surface V‐ATPases create an acidic microenvironment that activates secreted cathepsins, which leads to cleavage of extracellular matrix, facilitating tumor cell invasion. Less is known about the activation of secreted cathepsins thus we looked into the role of V‐ATPases in secretion and activation of cathepsins B and L (CatB and CatL). Cathepsin activity was measured using a substrate which fluoresces when cleaved. The assays were performed + or ‐ cathepsin‐specific inhibitors. Activity levels were corroborated by immunoblotting for each cathepsin in cell lysates and conditioned media. CatB activity is much higher than CatL inside the cell and CatL activity was much higher extracellularly. Inhibition of V‐ATPases with concanamycin A (a general V‐ATPase inhibitor) leads to a large reduction in the intracellular activity of both CatB and CatL. In conditioned media, inhibition of V‐ATPases leads to a large reduction in CatB activity and a modest reduction in CatL activity. From our results it appears that V‐ATPases are playing a role in activation of both intracellular and secreted cathepsins. These results provide evidence that cell surface V‐ATPases play a role in activation of secreted cathepsins. However, there may be specificity in which secreted cathepsins they act on. Concanamycin can penetrate the cell membrane so we cannot rule out that intracellular V‐ATPases are activating CatB and CatL in secretory vesicles. Experiments with subunit “a” isoform‐specific siRNAs will help define the role that intra‐ and extracellular V‐ATPases play in activation of secreted CatB and CatL.

Fluid shear stress promotes proprotein convertase-dependent activation of MT1-MMP

During angiogenesis, endothelial cells (ECs1) initiate new blood vessel growth and invade into the extracellular matrix (ECM). Membrane type-1 matrix metalloproteinase (MT1-MMP) facilitates this process and translocates to the plasma membrane following activation to promote ECM cleavage. The N-terminal pro-domain within MT1-MMP must be processed for complete activity of the proteinase. This study investigated whether MT1-MMP activation was altered by sphingosine 1-phosphate (S1P) and wall shear stress (WSS), which combine to stimulate EC invasion in three dimensional (3D) collagen matrices. MT1-MMP was activated rapidly and completely by WSS but not S1P. Proprotein convertases (PCs) promoted MT1-MMP processing, prompting us to test whether WSS or S1P treatments increased PC activity. Like MT1-MMP, PC activity increased with WSS, while S1P had no effect. A pharmacological PC inhibitor completely blocked S1P- and WSS-induced EC invasion and MT1-MMP translocation to the plasma membrane. Further, a recombinant PC inhibitor reduced MT1-MMP activation and decreased lumen formation in invading ECs, a process known to be controlled by MT1-MMP. Thus, we conclude that PC and MT1-MMP activation are mechanosensitive events that are required for EC invasion into 3D collagen matrices.

Granzyme B mediates both direct and indirect cleavage of extracellular matrix in skin after chronic low-dose ultraviolet light irradiation.

Extracellular matrix (ECM) degradation is a hallmark of many chronic inflammatory diseases that can lead to a loss of function, aging, and disease progression. Ultraviolet light (UV) irradiation from the sun is widely considered as the major cause of visible human skin aging, causing increased inflammation and enhanced ECM degradation. Granzyme B (GzmB), a serine protease that is expressed by a variety of cells, accumulates in the extracellular milieu during chronic inflammation and cleaves a number of ECM proteins. We hypothesized that GzmB contributes to ECM degradation in the skin after UV irradiation through both direct cleavage of ECM proteins and indirectly through the induction of other proteinases. Wild-type and GzmB-knockout mice were repeatedly exposed to minimal erythemal doses of solar-simulated UV irradiation for 20 weeks. GzmB expression was significantly increased in wild-type treated skin compared to nonirradiated controls, colocalizing to keratinocytes and to an increased mast cell population. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density, which was related to the cleavage of decorin, an abundant proteoglycan involved in collagen fibrillogenesis and integrity. GzmB also cleaved fibronectin, and GzmB-mediated fibronectin fragments increased the expression of collagen-degrading matrix metalloproteinase-1 (MMP-1) in fibroblasts. Collectively, these findings indicate a significant role for GzmB in ECM degradation that may have implications in many age-related chronic inflammatory diseases.

Halogen Bonding Controls Selectivity of FRET Substrate Probes for MMP-9

Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endoproteases that catalyze cleavage of extracellular matrix and nonmatrix proteins. MMPs play a role in tissue remodeling, and their uncontrolled activity is associated with number of diseases, including tumor metastasis. Thus, there is a need to develop methods to monitor MMP activity, and number of probes has been previously described. The key problem many probes encounter is the issue of selectivity, since 23 human MMPs, despite playing different physiological roles, have structurally similar active sites. Here, we introduce the halogen bonding concept into the probe design and show that the probe containing iodine exhibits an unprecedented selectivity for MMP-9. We provide structure-based explanation for the selectivity, confirming that it is due to formation of the halogen bond that supports catalysis, and we highlight the value of exploring halogen bonding in the context of selective probe design.

Current microscopic methods for the neural ECM analysis.

The extracellular matrix (ECM) occupies the space between both neurons and glial cells and thus provides a microenvironment that regulates multiple aspects of neural activities. Because of the vital role of ECM as a natural environment of cells in vivo, there is a growing interest to develop methodology allowing for the detailed structural and functional analyses of ECM. In this chapter, we provide the detailed overview of current microscopic methods used for ECM analysis and also describe general labeling strategies for ECM visualization. Since ECM remodeling involves the proteolytic cleavage of ECM, we will also describe current experimental approaches to image the proteolytic reorganization and/or degradation of ECM. The special focus of this chapter is set to the application of Förster resonance energy transfer-based approaches to monitor intracellular and extracellular matrix functions with high spatiotemporal resolution.

Regression of Canine Cutaneous Histiocytoma is Associated with an Orchestrated Expression of Matrix Metalloproteinases

Canine cutaneous histiocytoma (CCH) is a common benign skin tumour originating from epidermal Langerhans cells. These tumours often display spontaneous regression and therefore represent a valuable animal model for investigation of tumour regression. Based on previous studies it was hypothesized that up-regulation of cytokines during CCH regression leads to up-regulation of matrix metalloproteinases (MMPs) favouring infiltration of lymphocytes and enhanced tumour regression. The expression of MMPs and their inhibitors (tissue inhibitors of metalloproteinases, TIMPs) was investigated immunohistochemically in 27 CCHs. The tumours were classified into four groups defined as having no regression (group 1), early regression (group 2), intermediate regression (group 3) or late regression (group 4). The distribution and expression intensity of MMP-1, -2, -3, -7, -9, -13 and -14 and TIMP-1 and -2 were determined in peripheral and central areas of each tumour. Group 3 and 4 CCHs showed up-regulation of expression of MMP-1, -9 and -14 at the periphery. Variable expression of MMP-2 and -3 was observed. Expression of the remaining MMPs and TIMPs showed no group-specific changes. Most MMPs and TIMPs displayed significantly higher expression at the tumour periphery compared with the centre, independently of the stage of regression and indicating more pronounced proteolysis in the peripheral areas. The results are consistent with cytokine-enhanced MMP expression, particularly of MMP-9, leading to enhanced lymphocyte recruitment in combination with elevated cleavage of extracellular matrix and basement membranes.

Substrate-Guided Proteomics Enhances Degradome Resolution

High-throughput technologies offer unrecognized insights into the genesis of human disease, propelling innovation across a spectrum of biomedical specialties.1 The evolution of molecular diagnostics continues to shape deployment of modern therapies, including identification of markers of disease prognosis, predictors of therapeutic response, and determinants of optimal clinical management, all recognized cornerstones of modern medical practice.2 In the postgenomic era, proteomic paradigms have demonstrated particular utility in advancing the practice of personalized disease prediction, diagnosis, and therapy,3 with systems-based approaches facilitating functional deconvolution of proteomes despite their inherent biological complexity.4 The ever-expanding proteomics toolkit encompasses an array of sophisticated technologies with the flexibility to interrogate a totality of putative protein targets, thereby enabling systematic resolution of proteome structures and functions. Indeed, mapping and decoding of proteome landscapes in health and disease provide the foundation to iteratively collate, integrate, and prioritize large-scale raw data.5 Thus, high-throughput proteomics offers a robust, adaptable technological foundation from which to systematically comprehend the underpinnings of (patho)physiological processes. Article see p 106 Innovative proteomic methods are designed to separate and identify proteins with high fidelity and to assess protein abundance, structure, posttranslational modifications, and interactions, enabling charting of the global proteome (ie, the protein complement of a genome; Figure). Progress in proteomic research has benefitted from the application of stringent criteria in assigning peptide and protein identities for comprehensive proteome cartography, such that perturbed or modified subproteomes within reconstituted protein networks can now be extracted at unprecedented rates for interpretation of their biological relevance. Expansion of proteomic studies has also been propelled by the concomitant development of high-resolution, high-mass-accuracy instrumentation, together with next-generation bioinformatics platforms for data analysis and systems interpretation. Existing approaches, however, may fall short of offering adequate experimental solutions arising from particular protein physicochemical constraints, limiting the otherwise far-reaching …

Cleavage of extracellular matrix in periodontitis: Gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C

Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease.

Matrix Metalloprotease 8-Dependent Extracellular Matrix Cleavage at the Blood-CSF Barrier Contributes to Lethality during Systemic Inflammatory Diseases

Systemic inflammatory response syndrome (SIRS) is a highly mortal inflammatory disease, associated with systemic inflammation and organ dysfunction. SIRS can have a sterile cause or can be initiated by an infection, called sepsis. The prevalence is high, and available treatments are ineffective and mainly supportive. Consequently, there is an urgent need for new treatments. The brain is one of the first organs affected during SIRS, and sepsis and the consequent neurological complications, such as encephalopathy, are correlated with decreased survival. The choroid plexus (CP) that forms the blood-CSF barrier (BCSFB) is thought to act as a brain "immune sensor" involved in the communication between the peripheral immune system and the CNS. Nevertheless, the involvement of BCSFB integrity in systemic inflammatory diseases is seldom investigated. We report that matrix metalloprotease-8 (MMP8) depletion or inhibition protects mice from death and hypothermia in sepsis and renal ischemia/reperfusion. This effect could be attributed to MMP8-dependent leakage of the BCSFB, caused by collagen cleavage in the extracellular matrix of CP cells, which leads to a dramatic change in cellular morphology. Disruption of the BCSFB results in increased CSF cytokine levels, brain inflammation, and downregulation of the brain glucocorticoid receptor. This receptor is necessary for dampening the inflammatory response. Consequently, MMP8(+/+) mice, in contrast to MMP8(-/-) mice, show no anti-inflammatory response and this results in high mortality. In conclusion, we identify MMP8 as an essential mediator in SIRS and, hence, a potential drug target. We also propose that the mechanism of action of MMP8 involves disruption of the BCSFB integrity.