scholarly journals Detection and monitoring of localized matrix metalloproteinase upregulation in a murine model of asthma

2014 ◽  
Vol 306 (8) ◽  
pp. L764-L774 ◽  
Author(s):  
Csilla N. Felsen ◽  
Elamprakash N. Savariar ◽  
Michael Whitney ◽  
Roger Y. Tsien
Keyword(s):  
Disease Progression ◽  
Protease Activity ◽  
Lung Parenchyma ◽  
Cell Penetrating Peptides ◽  
Normal Lung ◽  
Therapeutic Drug ◽  
Extracellular Proteases ◽  
Gelatinase Activity ◽  
Chronic Lung Diseases

Extracellular proteases including matrix metalloproteinases (MMPs) are speculated to play a significant role in chronic lung diseases, such as asthma. Although increased protease expression has been correlated with lung pathogenesis, the relationship between localized enzyme activity and disease progression remains poorly understood. We report the application of MMP-2/9 activatable cell-penetrating peptides (ACPPs) and their ratiometric analogs (RACPPs) for in vivo measurement of protease activity and distribution in the lungs of mice that were challenged with the allergen ovalbumin. MMP-2/9 activity was increased greater than twofold in whole, dissected lungs from acutely challenged mice compared with control mice ( P = 1.8 × 10−4). This upregulation of MMP-2/9 activity was localized around inflamed airways with 1.6-fold higher protease-dependent ACPP uptake surrounding diseased airways compared with adjacent, pathologically normal lung parenchyma ( P = 0.03). MMP-2/9 activity detected by ACPP cleavage colocalized with gelatinase activity measured with in situ dye-quenched gelatin. For comparison, neutrophil elastase activity and thrombin activity, detected with elastase- and thrombin-cleavable RACPPs, respectively, were not significantly elevated in acutely allergen-challenged mouse lungs. The results demonstrate that ACPPs, like the MMP-2/9-activated and related ACPPs, allow for real-time detection of protease activity in a murine asthma model, which should improve our understanding of protease activation in asthma disease progression and help elucidate new therapy targets or act as a mechanism for therapeutic drug delivery.

scholarly journals The perplexing role of RAGE in pulmonary fibrosis: causality or casualty?

2021 ◽  
Vol 15 ◽  
pp. 175346662110160
Author(s):  
Timothy N. Perkins ◽  
Tim D. Oury
Keyword(s):  
Respiratory Failure ◽  
Pulmonary Fibrosis ◽  
Disease Progression ◽  
Advanced Glycation Endproducts ◽  
Experimental Studies ◽  
Normal Lung ◽  
Unknown Etiology ◽  
Chronic Lung Diseases ◽  
Incidence And Mortality

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease in which most patients die within 3 years of diagnosis. With an unknown etiology, IPF results in progressive fibrosis of the lung parenchyma, diminishing normal lung function, which results in respiratory failure, and eventually, death. While few therapies are available to reduce disease progression, patients continue to advance toward respiratory failure, leaving lung transplantation the only viable option for survival. As incidence and mortality rates steadily increase, the need for novel therapeutics is imperative. The receptor for advanced glycation endproducts (RAGE) is most highly expressed in the lungs and plays a significant role in a number of chronic lung diseases. RAGE has long been linked to IPF; however, confounding data from both human and experimental studies have left an incomplete and perplexing story. This review examines the present understanding of the role of RAGE in human and experimental models of IPF, drawing parallels to recent advances in RAGE biology. Moreover, this review discusses the role of RAGE in lung injury response, type 2 immunity, and cellular senescence, and how such mechanisms may relate to RAGE as both a biomarker of disease progression and potential therapeutic target in IPF. The reviews of this paper are available via the supplemental material section.

Matrix metalloproteinase-9 : surrogate marker and therapeutic target against neurovascular impairment after cerebral ischemia

2015 ◽  
Author(s):  
◽  
Shanyan Chen
Keyword(s):  
Endothelial Cells ◽  
Ischemic Stroke ◽  
Cerebral Ischemia ◽  
Molecular Mechanisms ◽  
Combined Treatment ◽  
Cell Penetrating Peptides ◽  
Therapeutic Window ◽  
Gelatinase Activity

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Stroke ranks fourth among all causes of death, and acute ischemic stroke is the most common form. The neurovascular unit (NVU) describes a basic functional structure in the brain and is primarily composed of endothelial cells, pericytes, astrocytes, microglia and neurons. The dynamic structure of the NVU is highly regulated due to interactions between different cells and extracellular matrix (ECM) components. Proteolysis of the ECM by matrix metalloproteinases (MMPs), especially MMP-9, plays an important role in the pathophysiology of cerebral ischemia and administration of tissue plasminogen activator (tPA). The activation of gelatinases (MMP-2/9) is considered a key mechanism involved in the impairment of NVU. The overall goal of this research project is to examine the role of MMP-9 in the neurovascular impairment after ischemic stroke in mice. In this project, we implemented a new strategy using gelatinase-activatable cell-penetrating peptides (ACPPs) tagged with fluorescence and/or gadolinium-based contrast agents to investigate proteolysis of gelatinases as surrogate markers of neurovascular integrity. We presented evidence that the combination of a sensitive fluorescent chromatophore and MRI contrast enhancement agent can be used to monitor gelatinase activity and its distribution in cultured neurons as well as in mice after focal cerebral ischemia. Detection of the activity of gelatinases in vivo using ACPPs could provide insights into the underlying mechanism for gelatinase proteolysis that mediate ischemia-related neurovascular impairment. We also applied a two-dimensional (2D) gelatin zymography technique that combines isoelectric focusing (IEF) with zymographic electrophoresis. We demonstrated that the 2D zymography approach can improve separation of different isoforms of gelatinases in both in vitro and in vivo conditions. 2D zymography is an effective method to separate posttranslational modification isoforms of gelatinases and to identify modifications that regulate their enzymatic activity in acute brain injuries. In work that follows, we used a fibrin-rich blood clot to occlude the middle cerebral artery (MCA) in mice as a model to represent the critical thromboembolic features of ischemic stroke in humans. In this study, we evaluated effects of SB-3CT, a mechanism-based inhibitor selective for gelatinases. We demonstrated MMP-9 activation and neurovasculature impairment in this stroke model, and showed the ability of SB-3CT to inhibit MMP-9 activity in vivo, which in turn resulted in maintenance of laminin, antagonism of pericyte contraction and loss, preservation of laminin-positive pericytes and endothelial cells, and thus rescuing neurons from apoptosis and preventing intracerebral hemorrhage. We further demonstrated that SB-3CT/tPA combined treatment could attenuate MMP-9 -- mediated degradation of endothelial laminin, impairment of endothelial cells, and decrease of caveolae -- mediated transcytosis. Early inhibition of MMP-9 proteolysis by SB-3CT decreased brain damage, reduced BBB disruption, and prevented hemorrhagic transformation after delayed tPA treatment. Therefore usage of SB-3CT will be helpful in accessing combination therapy with tPA in ischemic stroke. Results from these studies indicate the important role of MMP-9 in cerebral ischemia and thus the need for further studies to explore the molecular mechanisms underlying its activation and regulation. Results further demonstrated that the combined use of MMP-9 inhibitor with tPA may extend tPA therapeutic window for mitigating stroke damage.

scholarly journals Gelatinase activity imaged by activatable cell-penetrating peptides in cell-based and in vivo models of stroke

2016 ◽  
Vol 37 (1) ◽  
pp. 188-200 ◽  
Author(s):  
Shanyan Chen ◽  
Jiankun Cui ◽  
Tao Jiang ◽  
Emilia S Olson ◽  
Quan-Yu Cai ◽  
...  
Keyword(s):  
Inductively Coupled Plasma ◽  
Cell Injury ◽  
Fluorescence Analysis ◽  
Cell Penetrating Peptides ◽  
In Vivo Models ◽  
Gelatinase Activity ◽  
Blood Brain Barrier Disruption ◽  
Ischemic Region ◽  
Cell Penetrating

Matrix metalloproteinases (MMPs), particularly gelatinases (MMP-2/-9), are involved in neurovascular impairment after stroke. Detection of gelatinase activity in vivo can provide insight into blood–brain barrier disruption, hemorrhage, and nerve cell injury or death. We applied gelatinase-activatable cell-penetrating peptides (ACPP) with a cleavable l-amino acid linker to examine gelatinase activity in primary neurons in culture and ischemic mouse brain in vivo. We found uptake of Cy5-conjugated ACPP (ACPP-Cy5) due to gelatinase activation both in cultured neurons exposed to n-methyl-d-aspartate and in mice after cerebral ischemia. Fluorescence intensity was significantly reduced when cells or mice were treated with MMP inhibitors or when a cleavage-resistant ACPP-Cy5 was substituted. We also applied an ACPP dendrimer (ACPPD) conjugated with multiple Cy5 and/or gadolinium moieties for fluorescence and magnetic resonance imaging (MRI) in intact animals. Fluorescence analysis showed that ACPPD was detected in sub-femtomole range in ischemic tissues. Moreover, MRI and inductively coupled plasma mass spectrometry revealed that ACPPD produced quantitative measures of gelatinase activity in the ischemic region. The resulting spatial pattern of gelatinase activity and neurodegeneration were very similar. We conclude that ACPPs are capable of tracing spatiotemporal gelatinase activity in vivo, and will therefore be useful in elucidating mechanisms of gelatinase-mediated neurodegeneration after stroke.

Cellular Localization of Enzymatically Active Thrombin in Intact Human Tissues by Hirudin Binding

1995 ◽  
Vol 73 (05) ◽  
pp. 793-797 ◽  
Author(s):  
Leo R Zacharski ◽  
Vincent A Memoli ◽  
William D Morain ◽  
Jean-Marc Schlaeppi ◽  
Sandra M Rousseau
Keyword(s):  
Tumor Cell ◽  
Cell Carcinoma ◽  
Cellular Localization ◽  
Thrombin Generation ◽  
Normal Lung ◽  
Cancer Tissue ◽  
Immunohistochemical Techniques ◽  
Tumor Types ◽  
Negative Controls

SummaryCellular sites of coagulation activation within complex, intact tissues have been studied by immunohistochemical techniques. Hirudin, a specific and high affinity inihibitor of the active site of thrombin, together with antibody to hirudin were applied to sections of AMeX-fixed specimens of normal lung, kidney, placenta, freshly incised skin and unperturbed skin obtained at fresh autopsy; to rheumatoid synovial tissue; and to malignant tissue from a variety of tumor types. Staining for thrombin was observed selectively on pulmonary alveolar, rheumatoid synovial, and placental macrophages that express an intact extrinsic coagulation pathway. Staining was also observed restricted to the endothelium of capillaries in freshly incised skin but not in either unperturbed skin or in aged incisions. Staining of tumor cell bodies was observed in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma tissues that we found previously to show tumor cell-associated procoagulant activity. This staining occurred commonly on cells within the tumor mass that were distant from stromal fibrinogen/fibrin. By contrast, tumor-associated macrophage but not tumor cell staining was seen in adenocarcinoma and squamous cell carcinoma of the lung, and little or no staining was seen in colon cancer tissue. Negative controls in which either the hirudin probe or its antibody were omitted failed to show staining. These results are in accord with previous findings and suggest that such techniques may be useful for studying the cellular sites of thrombin generation in intact tissues. We postulate that administration of potent and specific thrombin antagonists, such as hirudin, to patients with relevant tumor types might be followed by homing of hirudin to tumor cells in vivo so that effects of local thrombin generation on malignant progression can be determined.

Novel Phytochemical Constituents and their Potential to Manage Diabetes

2020 ◽  
Vol 26 ◽  
Author(s):  
Shaik Ibrahim Khalivulla ◽  
Arifullah Mohammed ◽  
Kokkanti Mallikarjuna
Keyword(s):  
Natural Products ◽  
Large Population ◽  
World Health ◽  
In Vivo Studies ◽  
Antidiabetic Activity ◽  
Therapeutic Drug ◽  
Pharmacological Activities ◽  
Chemical Structures

Background: Diabetes is a chronic disease affecting a large population worldwide and stands as one of the major global health challenges to be tackled. According to World Health Organization, about 400 million are having diabetes worldwide and it is the seventh leading cause of deaths in 2016. Plant based natural products had been in use from ancient time as ethnomedicine for the treatment of several diseases including diabetes. As a result of that, there are several reports on plant based natural products displaying antidiabetic activity. In the current review, such antidiabetic potential compounds reported from all plant sources along with their chemical structures are collected, presented and discussed. This kind of reports are essential to pool the available information to one source followed by statistical analysis and screening to check the efficacy of all known compounds in a comparative sense. This kind of analysis can give rise to few numbers of potential compounds from hundreds, whom can further be screened through in vitro and in vivo studies, and human trails leading to the drug development. Methods: Phytochemicals along with their potential antidiabetic property were classified according to their basic chemical skeleton. The chemical structures of all the compounds with antidiabetic activities were elucidated in the present review. In addition to this, the distribution and their other remarkable pharmacological activities of each species is also included. Results: The scrutiny of literature led to identification of 44 plants with antidiabetic compounds (70) and other pharmacological activities. For the sake of information, the distribution of each species in the world is given. Many plant derivatives may exert antidiabetic properties by improving or mimicking the insulin production or action. Different classes of compounds including sulfur compounds (1-4), alkaloids (5-11), phenolic compounds (12-17), tannins (18-23), phenylpropanoids (24-27), xanthanoids (28-31), amino acid (32), stilbenoid (33), benzofuran (34), coumarin (35), flavonoids (36-49) and terpenoids (50-70) were found to be active potential compounds for antidiabetic activity. Of the 70 listed compounds, majorly 17 compounds are from triterpenoids, 13 flavonoids and 7 are from alkaloids. Among all the 44 plant species, maximum number (7) of compounds are reported from Lagerstroemia speciosa followed by Momordica charantia (6) and S. oblonga with 5 compounds. Conclusion: This is the first paper to summarize the established chemical structures of phytochemicals that have been successfully screened for antidiabetic potential and their mechanisms of inhibition. The reported compounds could be considered as potential lead molecules for the treatment of type-2 diabetes. Further, molecular and clinical trials are required to select and establish the therapeutic drug candidates.

scholarly journals Hydrogel-Forming Microneedle Arrays Allow Detection of Drugs and Glucose In Vivo: Potential for Use in Diagnosis and Therapeutic Drug Monitoring

PLoS ONE ◽  
2015 ◽  
Vol 10 (12) ◽  
pp. e0145644 ◽  
Author(s):  
Ester Caffarel-Salvador ◽  
Aaron J. Brady ◽  
Eyman Eltayib ◽  
Teng Meng ◽  
Ana Alonso-Vicente ◽  
...  
Keyword(s):  
Therapeutic Drug Monitoring ◽  
Drug Monitoring ◽  
Therapeutic Drug ◽  
Microneedle Arrays

scholarly journals N-Terminomics Strategies for Protease Substrates Profiling

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4699
Author(s):  
Mubashir Mintoo ◽  
Amritangshu Chakravarty ◽  
Ronak Tilvawala
Keyword(s):  
Mass Spectrometry ◽  
Protease Activity ◽  
Viral Infections ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modification ◽  
Spectrometry Analysis ◽  
Physiological Conditions ◽  
Inflammatory Conditions ◽  
Biological Mixtures

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.

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