Presence Of Matrix Metalloproteinases Research Articles

Correlation between Matrix Metalloproteinase presence and caries surface appearance.

Cariogenic bacterial acids dissolve the inorganic elements in dentine, leaving the dentine matrix exposed. Host-derived matrix metalloproteinases (MMPs) play an essential role in caries progression as they are significant regulators of extracellular matrix turnover and can degrade exposed collagen. This paper investigates the expression of MMP2 and MMP9 across various stages of caries in primary human teeth and relate this with a diagnosis recorded by The International Caries Detection and Assessment System (ICDAS). Twenty-four sections (150um in thickness) from extracted teeth, clinically diagnosed using ICDAS, were immunohistochemically treated with monoclonal anti-MMP2 and anti-MMP9 antibodies. Positive staining was visualised by immunofluorescence using a VectorFluor Duet Double Labeling Kit. Images from triplicate samples for each ICDAS score were analysed using ImageJ software. Collagen degradation in caries lesions was detected using a hydroxyproline assay. MMPs were weakly detected in caries with ICDAS 1-2 scores, and an insignificant increase was detected in ICDAS 3. However, a significant increase in MMP expression was seen in caries with an ICDAS score of 4-6. There was a strong positive correlation between the ICDAS score and MMP2, [r(6) = .86, p = .002] and between ICDAS and MMP9, [r(6) = .82, p = .004]. Data were analysed using two-way ANOVA followed by Tukey multiple comparison test (*p < 0.05). The use of ICDAS to assess the severity of caries lesions and how this correlates with the presence of MMP in these lesions validates the modern approach to caries management with a minimally invasive concept.

Willis Lecture: Biomarkers for Inflammatory White Matter Injury in Binswanger Disease Provide Pathways to Precision Medicine.

Binswanger disease is the small vessel form of vascular cognitive impairment and dementia. Deposition of Alzheimer disease proteins can begin in midlife and progress slowly, whereas aging of the vasculature also can begin in midlife, continuing to progress into old age, making mixed dementia the most common type of dementia. Biomarkers facilitate the early diagnosis of dementias. It is possible to diagnose mixed dementia before autopsy with biomarkers for vascular disease derived from diffusor tensor images on magnetic resonance imaging and Alzheimer disease proteins, Aβ (amyloid β), and phosphorylated tau, in cerebrospinal fluid or in brain with positron emission tomography. The presence of vascular disease accelerates cognitive decline. Both misfolded proteins and vascular disease promote inflammation, which can be detected in cerebrospinal fluid by the presence of MMPs (matrix metalloproteinases), angiogenic growth factors, and cytokines. MMPs disrupt the blood-brain barrier and break down myelin, producing Binswanger disease's 2 main pathological features. Advances in detecting biomarkers in plasma will provide early detection of dementia and aided by machine learning and artificial intelligence, will enhance diagnosis and form the basis for early treatments.

The IRE1α-XBP1s Arm of the Unfolded Protein Response Activates N-Glycosylation to Remodel the Subepithelial Basement Membrane in Paramyxovirus Infection.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections (LRTI) associated with decreased pulmonary function, asthma, and allergy. Recently, we demonstrated that RSV induces the hexosamine biosynthetic pathway via the unfolded protein response (UPR), which is a pathway controlling protein glycosylation and secretion of the extracellular matrix (ECM). Because the presence of matrix metalloproteinases and matricellular growth factors (TGF) is associated with severe LRTI, we studied the effect of RSV on ECM remodeling and found that RSV enhances the deposition of fibronectin-rich ECM by small airway epithelial cells in a manner highly dependent on the inositol requiring kinase (IRE1α)–XBP1 arm of the UPR. To understand this effect comprehensively, we applied pharmacoproteomics to understand the effect of the UPR on N-glycosylation and ECM secretion in RSV infection. We observe that RSV induces N-glycosylation and the secretion of proteins related to ECM organization, secretion, or proteins integral to plasma membranes, such as integrins, laminins, collagens, and ECM-modifying enzymes, in an IRE1α–XBP1 dependent manner. Using a murine paramyxovirus model that activates the UPR in vivo, we validate the IRE1α–XBP1-dependent secretion of ECM to alveolar space. This study extends understanding of the IRE1α–XBP1 pathway in regulating N-glycosylation coupled to structural remodeling of the epithelial basement membrane in RSV infection.

Tumor-Associated Enzyme-Activatable Spherical Nucleic Acids.

Maximizing the tissue-targeting efficiency of nanomaterials while also protecting them from rapid clearance from the bloodstream and limiting their immunogenicity remains a central problem in the field of systemic-administered nanomedicine. Herein, we introduce a generalizable strategy to simultaneously increase tumor accumulation, prolong blood circulation, and limit nonspecific immune activation of nanomaterials via peptide-based, tumor-responsive, "sheddable" coatings. Spherical nucleic acids (SNAs) were designed and synthesized to contain an exterior coating composed of zwitterionic polypeptides with recognition sequences for tumor-associated proteases. In the presence of matrix metalloproteinases (MMPs), the polypetide coating is rapidly cleaved, leading to increased cellular uptake of these SNAs, relative to SNAs containing nonsheddable shells. Moreover, the zwitterionic nature of the polypeptide shell shields the SNAs from immune system recognition, which extends their blood circulation time and improves tumor accumulation and in vivo cellular uptake relative to control SNAs with no protective coating. Taken together, these results indicate that this strategy is a viable method for increasing nanoparticle tumor accumulation and can have utility for the systemic delivery of oligonucleotides and nanomaterials to target cells in vivo with low immunogenicity.

A Nano Drug Delivery System Based on Angelica sinensis Polysaccharide for Combination of Chemotherapy and Immunotherapy.

Combination of chemotherapy and immunotherapy has been a promising strategy in cancer treatment. Polysaccharides from Angelica sinensis (AP), a well-known Chinese herbal medicine, have been proved to have good immunomodulatory activity. In the present study, an enzyme-sensitive tumor-targeting nano drug delivery system (AP-PP-DOX (doxorubicin), PP stood for peptide) was constructed. In this system, Angelica polysaccharides act as not only carriers to targeted delivery of drugs to tumor tissue but also effectors to improve tumor microenvironment and enhance immune function, resulting in synergistic antitumor effect with chemotherapy drugs. The structure of this conjugate was confirmed by FI-IR and 1H-NMR. The particle size and zeta potential of the nanoparticles were 129.00 ± 3.32 nm and −28.45 ± 0.22 mV, respectively. Doxorubicin (DOX) and AP could be quickly released from the AP-PP-DOX under the presence of matrix metalloproteinase 2 (MMP2). The released DOX showed good antitumor efficacy in vitro. The treatment of released AP moiety increased the expression of IL-2, while that of IL-10 was decreased, showing potential in restoring Th1/Th2 immune balance in tumor microenvironment. In a word, this drug delivery system, with specific tissue targeting and tumor microenvironment improvement, will open a new avenue for combination treatment of cancer.

Expression of Matrix Metalloproteinase-9 and Tissue Inhibitor of Metalloproteinase-1 in Endometriosis Menstrual Blood.

Endometriosis is a gynecological disease characterized by the presence of endometrial-like tissue outside the uterine cavity. Remodeling of the extracellular matrix (ECM) is a prerequisite for tissue implantation. The presence of matrix metalloproteinase (MMP) and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), in shed endometrium cells has a significant role in ECM degradation. A case–control study was performed to find other diagnostic markers using menstrual blood. We examined a sample of 68 women who visited the gynecology clinic in Dr Hasan Sadikin General Hospital, 40% of whom were confirmed to have endometriosis, and the rest tested negative by histopathological examination. All endometriotic cases presented MMP-9 and TIMP-1 expression with different cell distribution. MMP-9 expression in endometriosis patients was increased compared to the controls (p = 0.002). Expression of MMP-9 in >80% of endometrial cells was associated with a higher risk for endometriosis (OR 4.44 95% CI 1.31 to 15.56) compared to MMP-9 expression in 50%–80% of cells. TIMP-1 cell expression in women with endometriosis was lower than in the control group (p = 0.030). Subjects with TIMP-1 expression in 20%–50% of endometrial cells had a higher risk for endometriosis (OR 4.5, 95%CI 1.21–17.42) compared those with TIMP-1 expression in 50%–80% of cells. These expressions levels can be useful to predict endometriosis.

MiR-21 nanocapsules promoteearly bone repair of osteoporotic fractures by stimulating the osteogenic differentiation of bone marrow mesenchymal stem cells.

ObjectiveThe healing of osteoporotic fractures in the elderly patients is a difficult clinical problem. Currently, based on the internal fixation of fractures, the available drug treatments mainly focus on either inhibiting osteoclast function, such as bisphosphonate, calcitonin, oestrogen or promoting osteogenesis, such as parathyroid hormones. However, the availability of current antiosteoporotic drugs in promoting osteoporotic fracture healing is limited. The objective of the present study was to investigate the ability of the MiR-21/nanocapsule to enhance the early bone repair of osteoporotic fractures.MethodsBased on the presence of matrix metalloproteinases that are overexpressed at the fracture site, we designed the matrix metalloproteinase–sensitive nanocapsules which were formed by in situ free radical polymerisation on the surface of MiR-21 with 2-(methacryloyloxy) ethyl phosphorylcholine and the bisacryloylated VPLGVRTK peptide. The MiR-21/nanocapsule [n (miR-21)] and O-carboxymethyl chitosan (CMCS) were mixed until they formed a gel-like material [CMCS/n (miR-21)] with good fluidity and injectability. Thirty elderly Sprague Dawley (SD) rats (female, 14-month-old, 380 ± 10 g) were subjected to bilateral removal of the ovaries (ovariectomised). All rats were subjected to bilateral bone defects (2 mm diameter) of the proximal tibia and randomly divided into three groups (groups A, B, and C): separately injected with CMCS/n (miR-21), CMCS/n (NC-miR), and saline. Micro-computed tomography (CT) imaging was performed to evaluate newly formed bone volume and connectivity. Nondecalcified histology and toluidine blue staining were performed to measure the effects of CMCS/n (miR-21) on bone repair. In vitro, the effect of n (miR-21) on osteogenic differentiation to bone marrow mesenchymal stem cells (BMSCs) which derived from the ovariectomised rat model was observed.ResultsThe morphology of n (miR-21) was a regular spherical nanocapsule with a uniform small size (25–35 nm). The results confirmed that n (miR-21) could be efficiently phagocytosed by BMSCs and released in the cytoplasm to promote osteogenesis. The expression level of alkaline phosphatase and Runt-related transcription factor 2 mRNA in the n (miR-21) group was higher than that in the n (NC-miR) group. Animal experiments proved that CMCS/n (miR-21) produced better bone repair compared with the CMCS/n (NC-miR) group in the early stages of fracture healing at 4 weeks. In the late stage of fracture healing (8 weeks), micro-CT quantitative analysis showed that the new bone trabeculae in the CMCS/n (miR-21) group has decreased compared with the CMCS/n (NC-miR) group. In the CMCS/n (miR-21) group, the new cancellous bone had been absorbed, and the process of bone healing was almost completed. In contrast, the new bone in the CMCS/n (NC-miR) and the control groups was still in the healing process.ConclusionThe cytological tests confirmed that n (miR-21) can promote osteogenic differentiation of BMSCs derived from the osteoporosis rat model. Furthermore, the results of animal tests demonstrated that local injection of CMCS/n (miR-21) promoted the early healing of osteoporotic bone defects. Consequently CMCS/n (miR-21) promoted the bone repair process to enter the moulding phase earlier.The translational potential of this articleCMCS/n (miR-21) can be widely applied to elderly patients with osteoporotic fractures. This method can help patients with osteoporotic fractures recover earlier and avoid serious complications. It provides a potential approach for the clinical treatment of osteoporotic fractures in the elderly.

Injectable MMP-Responsive Nanotube-Modified Gelatin Hydrogel for Dental Infection Ablation.

A photocrosslinkable gelatin methacryloyl (GelMA) hydrogel has been widely examined in regenerative engineering because of its good cell-tissue affinity and degradability in the presence of matrix metalloproteinases. A halloysite aluminosilicate nanotube (HNT) is a known reservoir for the loading and sustained delivery of therapeutics. Here, we formulate injectable chlorhexidine (CHX)-loaded nanotube-modified GelMA hydrogel that is cytocompatible and biodegradable and provides sustained release of CHX for infection ablation while displaying good biocompatibility. The effects of HNTs and CHX on hydrogel degradability and mechanical properties, as well as on the kinetics of CHX release, and on the antimicrobial efficacy against oral pathogens were systematically assessed. Cytocompatibility in stem cells from human exfoliated deciduous teeth and inflammatory response in vivo using a subcutaneous rat model were determined. Our hydrogel system, that is, (CHX)-loaded nanotube-modified GelMA showed minimum localized inflammatory responses, supporting its ability for drug delivery applications. Moreover, we showed that the incorporation of CHX-loaded nanotubes reduces the mechanical properties, increases the swelling ratio, and diminishes the degradation rate of the hydrogels. Importantly, the presence of CHX-loaded nanotubes inhibits bacterial growth with minimal cell toxicity. Our findings provide a new strategy to modify GelMA hydrogel with chlorhexidine-loaded nanotubes for clinical use as an injectable drug delivery strategy for dental infection ablation.

Acute Exercise in Hypobaric Hypoxia Attenuates Endothelial Shedding in Subjects Unacclimatized to High Altitudes.

Travel of unacclimatized subjects to a high altitude has been growing in popularity. Changes in endothelial shedding [circulating endothelial cells (ECs)] and hematopoietic stem and progenitor cells (CPCs) during physical exercise in hypobaric hypoxia, however, are not well understood. We investigated the change in ECs and CPCs when exposed to high altitude, after acute exercise therein, and after an overnight stay in hypobaric hypoxia in 11 healthy unacclimatized subjects. Blood withdrawal was done at baseline (520 m a.s.l.; baseline), after passive ascent to 3,883 m a.s.l. (arrival), after acute physical exercise (±400 m, postexercise) and after an overnight stay at 3,883 m a.s.l. (24 h). Mature blood cells, ECs, and CPCs were assessed by a hematology analyzer and flow cytometry, respectively. The presence of matrix metalloproteinases (MMPs), their activity, and hematopoietic cytokines were assessed in serum and plasma. EC and CPC concentrations significantly decreased after exercise (p = 0.019, p = 0.007, respectively). CPCs remained low until the next morning (24 h, p = 0.002), while EC concentrations returned back to baseline. MMP-9 decreased at arrival (p = 0.021), stayed low postexercise (p = 0.033), and returned to baseline at 24 h (p = 0.035 to postexercise). MMP-activity did not change throughout the study. Circulating MMP-9 concentrations, but not MMP-activity, were associated with EC concentrations (rrm = 0.48, p = 0.010). CPC concentrations were not linked to hematopoietic cytokines. Acute exercise at high altitude attenuated endothelial shedding, but did not enhance regenerative CPCs. Results were not linked to endothelial matrix remodeling or CPC mobilization. These results provide information to better understand the endothelium and immature immune system during an active, short-term sojourn at high altitude.

Nanocatalyst sensor produces simple cancer test

Detecting some cancers early can improve a patient’s chance of survival, but conventional detection methods can involve costly tests and equipment, making them inaccessible for people in low-resource areas. Researchers have now developed a diagnostic test that can detect a molecular signature of cancer and other diseases with a simple readout: it turns urine blue (Nat. Nanotechol. 2019, DOI: 10.1038/s41565-019-0527-6). Molly Stevens of Imperial College London teamed up with Sangeeta Bhatia of the Massachusetts Institute of Technology to create nanoparticle-protein complexes that, once injected, can reveal the presence of disease-related enzymes through a simple urine test. The team used peptides to link gold nanoclusters to neutravidin, a protein carrier. They designed the linkers so that enzymes called matrix metalloproteinases (MMPs) can cleave them. The presence of MMPs has been proposed as an early warning sign of several cancers. To test their sensors, the team injected the complexes into healthy mice

HPMA Polymeric Nanocarriers for Anticancer Drugs with Tumor Microenvironment-Responsive Extracellular Biodegradation and Intracellular Drug Release.

The balance between systemic toxicity and circulation time for a polymeric nanocarrier to deliver antitumor drugs has been trialed. A new approach to break the balance was proposed in this study by significantly improving its biosafety and prolonging the circulation time, hence, to enhance its anti-tumor efficacy. A matrix metalloproteinases (MMPs)-sensitive peptide (PVGLIGK) was introduced to cross-link the N-(2-hydroxypropyl) methylacrylamide polymer-doxorubicin conjugates (HPMA-Dox) conjugate to construct a nano-size polymeric nanocarrier-Dox assembly (PMD) with a molecular weight (MW) of 73 kDa and this modification has resulted in a prolonged circulation time (a half-time of 20.1 h) and enhanced accumulation of PMD at the tumor site, while negligible systemic toxicity and excellent biocompatibility were displayed after injection of PMD into the mice. The cross-linked nanoassembly was unpacking in the presence of MMPs in the extracellular microenvironment, and the conjugated Dox was released from the nanoassembly in the lysosome/endosome due to an intracellular low pH microenvironment. The released Dox from PMD inhibited tumor cells very efficiently with a tumor growth inhibition of around 70%. The outstanding performance of the dual stimuli-responsive biodegradable polymeric nanocarriers may open a door for other hydrophobic anti-tumor drugs.

Matrix metalloproteinase inhibitors prevent the release and proteolytic activity of monocyte/macrophage-derived microparticles.

The role of monocyte/macrophage-derived microparticles (MPs) in the pathophysiology of cancer and chronic inflammatory diseases has been reported; nevertheless, the mechanism underlying microparticles release is currently unclear. The aim of the current study was to investigate whether matrix metalloproteinase (MMP) inhibitors could prevent MP shedding from stimulated human monocyte/macrophage. Microparticles were obtained by isolated peripheral blood mononuclear cells after stimulation with the calcium ionophore, A23187. MP shedding, intracellular calcium concentration, analysis of RhoA expression, and proteolytic activities of isolated MPs were assessed in the absence or presence of MMP inhibitors. We demonstrated that MMP inhibitors remarkably prevented MP shedding in a concentration-dependent manner with IC50 values in the nano- to micromolar range. Such an effect was related to their ability to reduce the intracellular Ca2+ levels induced by the calcium ionophore and the consequent translocation of RhoA from cytosol to membrane. Furthermore, MMP inhibitors could inhibit the proteolytic activity of cell-derived MPs. The current study provide evidence that MMP inhibitors can prevent MPs shedding from stimulated human monocyte/macrophage and the proteolytic activity of released MPs. Finally, the most active compound tested might represent the lead compound of a new class of molecules with therapeutic potential in cancer and chronic inflammatory diseases.

High modulus, enzyme-degradable poly(trimethylene carbonate)-peptide biohybrid networks formed from triblock prepolymers.

Biohybrid networks have the potential to have stiffnesses equivalent to that of native soft connective tissues as well as cell-mediated degradation behavior. Most strategies to generate such materials to date have utilized crosslinking of two separate and orthogonally functionalized polymers. Herein we describe a triblock prepolymer consisting of a central enzyme degradable peptide block flanked by two synthetic, hydrolysis resistant poly(trimethylene carbonate) blocks (PTMC) or poly(ethylene glycol)-PTMC blocks terminated in methacrylate groups. To form these prepolymers heterobifunctional PTMC and PEG-PTMC were prepared, possessing a vinyl sulfone terminus and a methacrylate terminus. These polymers were conjugated to a di-cysteine containing peptide through a Michael-type addition to form cross-linkable prepolymers. These prepolymers were then photo-cured to form enzyme degradable networks. The compressive moduli of the resulting water swollen networks was within the range of many soft connective tissues and was inversely proportional to the water solubility of the prepolymers. The prepolymer water solubility in turn could be tuned by adjusting PTMC molecular weight or by the addition of a PEG block. In vitro degradation only occurred in the presence of matrix metalloproteinases, and was fastest for networks prepared with prepolymers of higher water solubility.

Sustained and Bioresponsive Two-Stage Delivery of Therapeutic miRNA via Polyplex Micelle-Loaded Injectable Hydrogels for Inhibition of Intervertebral Disc Fibrosis.

Intervertebral disc degeneration (IDD) is frequently caused by gradual pathological changes inside intervertebral discs (IVDs) and progressive fibrosis. MicroRNA-29 (miR-29) family possesses potent fibrosis suppression capability, but their application for treatment of chronic IDD is limited due to lack of suitable local delivery systems. In this report, given various overexpressed matrix metalloproteinases (MMPs) during IDD, injectable MMP-degradable hydrogels encapsulating MMP-responsive polyplex micelles are developed for sustained and bioresponsive delivery of miR-29a into nucleus pulposus cells via a two-stage process. Cationic block copolymers are designed to complex miR-29a, and subsequently mixed with the poly(ethylene glycol) (PEG) gelation precursors and MMP-cleavable peptide cross-linkers for in situ formation of polyplex micelle-encapsulated hydrogels in the diseased IVDs. In the presence of MMPs, the polyplex micelles are first released by MMP cleavage of the hydrogels, and subsequently, MMPs-responsive detachment of PEG shells from polyplex micelles contributes to efficient cellular uptake and endosomal escape. MiR-29a is demonstrated to effectively silence the expression of MMP-2, inhibit the fibrosis process, and reverse IDD in animal models through blocking the β-catenin translocation pathway from the cytoplasm to the nucleus. This two-stage bioresponsive local miRNA delivery system represents a novel and promising strategy for the treatment of chronic IDD.

Role of matrix metalloproteinases in tumour invasion: immunohistochemistry of peritoneum from peritoneal carcinomatosis

Colorectal cancer is one of the most common forms of cancer. Spread of tumour to the peritoneal cavity may lead to seeding of cancer cells that adhere to and invade the peritoneal membrane causing peritoneal carcinomatosis. Matrix metalloproteinases (MMPs) play an essential role in cancer cell invasion and dissemination. The aim of this study was to evaluate the morphology and presence of matrix metalloproteinases in peritoneal carcinomatosis. Biopsy samples of the parietal peritoneum were taken from patients undergoing cytoreductive surgery for peritoneal carcinomatosis. The samples were fixed in formalin, dehydrated and embedded in paraffin prior to cutting into 4-µm slices. Staining with haematoxylin/eosin was used for morphology studies, and MMP-1, MMP-2 and TIMP-1 levels were evaluated using immunohistochemistry and light microscopy. The microscopically tumour-free areas of the peritoneal membrane were thin compared to the peripheral invasion zone and the areas invaded by tumour. Peritoneum invaded by tumour was richly vascularised and contained inflammatory cells. MMP-1 was expressed in tumour-free peritoneum and in the invasion zone between tumour and peritoneal tissue, but not in tumour-invaded areas. MMP-2 and TIMP-1 were mostly expressed in the proximity of blood vessels and inflammatory cells in tumour-invaded areas, but was not seen in tumour-free areas. MMPs play an important role in the process of cancer cell invasion of the peritoneum in peritoneal carcinomatosis. The peripheral zone of the tumour appears to be of importance for tumour invasion.

Fucus vesiculosus extract inhibits the proteolytic activity and gene expression of matrix metalloproteinases in Atlantic salmon (Salmo salar L.)

Fucoidans are sulfated fucose-rich polysaccharides found in macroalgae. Multiple biological functions have been attributed to fucoidans including inhibitory activity against matrix metalloproteinases (MMPs). A recent study showed that kidney and blood remains left in the abdominal cavity after gutting had a significant negative effect on gaping and fillet firmness in Atlantic salmon (Salmo salar L.). The authors hypothesized that one reason could be that MMPs from kidney and blood remains break down the connective tissue in Atlantic salmon fillets. The objectives of this study were to investigate the presence of MMPs in Atlantic salmon kidney and the inhibitory effect of a Fucus vesiculosus extract (FVE) on the proteolytic activity and gene expression of MMPs in Atlantic salmon. The inhibitory effect of FVE was compared to the inhibitory effect of the synthetic broad-spectrum MMP inhibitor marimastat. In this study, we found that FVE inhibits the proteolytic activity of MMP-1, MMP-2, MMP-9, and MMP-13 in Atlantic salmon kidney as well as MMP-2 gene expression in Atlantic salmon liver. Although the inhibitory effect of FVE on the proteolytic activity of MMPs is small compared to the inhibitory effect of marimastat, both FVE and marimastat inhibit 80% of the DQ-gelatin breakdown caused by Atlantic salmon kidney at a concentration of 0.1 mg mL−1. This indicates that FVE may also be capable of inhibiting other proteinases present in Atlantic salmon kidney. Overall, our findings suggest that FVE could be used in the fish processing industry to limit collagenase and gelatinase activity, thus reducing the connective tissue degradation in Atlantic salmon fillets.

89. Expanding the Toolkit of Protease-Activatable Viruses to Optimize Their Versatility and Efficiency

Adeno-associated virus (AAV) has emerged as a promising gene delivery vector because of its non-pathogenicity, simple structure and genome, and low immunogenicity compared to other viral vectors. However, its wide adoption as a safe and effective gene therapy treatment for disease may rely on targeting the vector to deliver transgenes to desired cell populations. To this end, our group has developed a protease-activatable virus (PAV) based on AAV that responds to elevated protease activity commonly found in many diseased tissue microenvironments. Small peptide “locks” were inserted into the viral capsid near its primary receptor binding domain, interfering with the virus-cell receptor interaction and preventing binding and transduction. The peptide locks are flanked by two proteolytically cleavable peptides, which are cleaved off in the presence of matrix metalloproteinases (MMPs), restoring virus-receptor interaction and allowing transduction to resume. Cleavage sequences were chosen from literature based on well-characterized linear peptide substrates. Initial testing demonstrated the desired MMP-activated cellular binding and transduction behavior, but it was discovered that cleavage efficiency and transduction activity of the PAV variants were uncorrelated with the reported specificity constants of the cleavage sequences. We therefore devised several ways to tune the sensitivity and specificity of our PAV. For example, by controlling the ratio of wild type to MMP-activatable protein subunits in the self-assembling 60-mer capsid, we can modulate the level of transduction achieved by the PAVs to attain targeted gene delivery to a range of human pathologies. A new lock format was also designed to allow for more predictable kinetics. This new “linearizable” lock format is modified by replacing one of the two flanking MMP cleavage motifs with the enterokinase cleavage motif, DDDDK, on the C-terminal side of the lock. When the enterokinase-linearized PAVs (ePAVs) are pretreated with enterokinase, the peptide locks are cleaved on one side, allowing the peptides to assume a more flexible conformation protruding from the capsid. The newly linearized peptide locks are cleaved with greater efficiency and kinetics that more closely correlate with expected activity. The PAV platform was also expanded into several AAV serotypes to allow for more diverse clinical applications. Our PAVs have demonstrated targeted delivery to several cancer models, including ovarian and pancreatic cancers, as well as ischemic tissue resulting from myocardial infarction.

In vivo monitoring of angiogenesis in a mouse hindlimb ischemia model using fluorescent peptide-based probes.

Vascular endothelial growth factor receptor (VEGFR) and matrix metalloproteinase (MMP) are up-regulated in ischemic tissue and play pivotal roles in promoting angiogenesis. The purpose of the present study was to evaluate two fluorophore-conjugated peptide probes specific to VEGFR and MMP for dual-targeted in vivo monitoring of angiogenesis in a murine model of hindlimb ischemia. To this end, VEGFR-Probe and MMP-Probe were developed by conjugating distinct near-infrared fluorophores to VEGFR-binding and MMP substrate peptides, respectively. VEGFR-Probe exhibited specific binding to VEGFR on HUVECs, and self-quenched MMP-Probe produced strong fluorescence intensity in the presence of MMPs in vitro. Subsequently, VEGFR-Probe and MMP-Probe were successfully utilized for time course in vivo visualization of VEGFR or MMP, respectively. Simultaneous visualization provided information regarding the spatial distribution of these proteins, including areas of co-localization. This dual-targeted in vivo imaging approach will be useful for understanding the detailed mechanism of angiogenesis and for evaluating therapeutic angiogenesis.

Do matrix metalloproteinases play a role in degenerative disease of temporomandibular joint? A systematic review

Objective: To evaluate whether the high expression of metalloproteinases observed in the temporomandibular joint can be considered as an indicator of degenerative disease. Methods: The PubMed, BVS, Embase, and ScienceDirect databases were systematically searched for articles written in English, regarding human studies that were published from 2003 to 2013. After applying the exclusion and inclusion criteria, four articles were selected. Results: Two studies analyzed the presence of matrix metalloproteinases (MMPs) through histology and immunohistochemical evaluation of articular discs; both observed a higher concentration of MMPs in the discs of ill patients. Another study, using genomic DNA from oral cells of ill and healthy patients, found a higher presence of MMP-1G/2G in affected individuals. A fourth study examined the synovial fluid of patients and control groups and concluded that MMPs were present in most patients with articular changes. Discussion: Despite the lack of methodology standardization,...

In vivo evaluation of matrix metalloproteinase responsive silk–elastinlike protein polymers for cancer gene therapy

Silk–elastinlike protein polymers (SELPs) have been effectively used as controlled release matrices for the delivery of viruses for cancer gene therapy in preclinical models. However, the degradability of these polymers needs to be tuned for improved localized intratumoral gene delivery. Using recombinant techniques, systematic modifications in distinct regions of the polymer backbone, namely, within the elastin blocks, silk blocks, and adjacent to silk and elastin blocks, have been made to impart sensitivity to specific matrix metalloproteinases (MMPs) known to be overexpressed in the tumor environment. In this report we investigated the structure–function relationship of MMP-responsive SELPs for viral mediated gene therapy of head and neck cancer. These polymers showed significant degradation in vitro in the presence of MMPs. Their degradation rate was a function of the location of the MMP-responsive sequence in the polymer backbone when in hydrogel form. Treatment efficacy of the adenoviral vectors released from the MMP responsive SELP analogs in a xenograft mouse model of head and neck squamous cell carcinoma (HNSCC) was shown to be polymer structure dependent. These results demonstrate the tunable nature of MMP-responsive SELPs for localized matrix-mediated gene delivery.