Nathaniel M. Weathington

A novel peptide CXCR ligand derived from extracellular matrix degradation during airway inflammation

We describe the tripeptide neutrophil chemoattractant N-acetyl Pro-Gly-Pro (PGP), derived from the breakdown of extracellular matrix (ECM), which shares sequence and structural homology with an important domain on alpha chemokines. PGP caused chemotaxis and production of superoxide through CXC receptors, and administration of peptide caused recruitment of neutrophils (PMNs) into lungs of control, but not CXCR2-deficient mice. PGP was generated in mouse lung after exposure to lipopolysaccharide, and in vivo and in vitro blockade of PGP with monoclonal antibody suppressed PMN responses as much as chemokine-specific monoclonal antibody. Extended PGP treatment caused alveolar enlargement and right ventricular hypertrophy in mice. PGP was detectable in substantial concentrations in a majority of bronchoalveolar lavage samples from individuals with chronic obstructive pulmonary disease, but not control individuals. Thus, PGPs activity links degradation of ECM with neutrophil recruitment in airway inflammation, and PGP may be a biomarker and therapeutic target for neutrophilic inflammatory diseases.

Estrogen Modulates Inflammatory Mediator Expression and Neutrophil Chemotaxis in Injured Arteries

Background—We have previously shown that estrogen (17&bgr;-estradiol; E2) inhibits neointima formation and migration of leukocytes, particularly neutrophils, into rat carotid arteries after acute endoluminal injury. This study tested the hypothesis that E2 inhibits expression of adhesion molecules, chemokines, and proinflammatory cytokines in rat carotid arteries in the early hours after balloon injury, thus attenuating the stimulus for leukocyte entry and negatively modulating the injury response. Methods and Results—Ovariectomized (OVX) rats were randomly assigned to treatment with E2 or vehicle (V) and subjected to balloon injury of the right carotid artery. After 2, 6, and 24 hours, rats were euthanized, and both carotid arteries were processed for real-time reverse transcription–polymerase chain reaction (2 and 24 hours), ELISA (6 hours), or neutrophil chemotaxis assay (24 hours). Expression of mRNA for adhesion molecules (P-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1), chemoattractants (cytokine-induced neutrophil chemoattractant [CINC]-2&bgr; and monocyte chemoattractant protein [MCP]-1), and proinflammatory cytokines (interleukin [IL]-1 and IL-6) was markedly increased (2 to 5000 times) in injured arteries of OVX+V rats at 2 hours and was reduced by 24 hours. E2 significantly attenuated expression of the proinflammatory mediators (by 60% to 80%) at 2 hours. ELISA confirmed injury-induced upregulation of neutrophil and monocyte/macrophage chemoattractants (CINC-2&agr;, MCP-1) in OVX+V arteries and E2-induced inhibition of CINC-2&agr; expression. E2 significantly (by 65%) inhibited neutrophil chemotactic activity of arterial homogenates. Conclusions—E2 attenuates the early vascular injury response, at least in part, by negatively modulating proinflammatory mediator expression and the resultant chemotactic activity of injured vessels for neutrophils.

Induction of lung emphysema is prevented by L-arginine-threonine-arginine

In patients with chronic obstructive pulmonary disease (COPD), an inflammatory process is ongoing in the lungs, with concomitant damage of the alveolar structures and loss of airway function. In this inflammatory process, extracellular matrix degradation is observed. During this lung matrix degradation, small peptide fragments consisting of proline and glycine repeats generated from collagen fibers are liberated from the matrix by matrix metalloproteinases. Chemotactic activities of these collagen‐derived peptides such as N‐acetyl‐proline‐glycine‐proline (PGP) via CXCR1 and CXCR2 have been reported. We show here that PGP induces neutrophil migration in vivo, which is dose dependent. Moreover, PGP is involved in the development of emphysema‐like changes in the airways. The complementary peptide, l‐arginine‐threonine‐arginine (RTR), has been shown to bind to PGP sequences and inhibit neutrophil infiltration. We show that RTR impedes both PGP‐ and interleukin‐8‐induced chemotaxis in vitro. In vivo, RTR prevents both migration and activation of neutrophils induced by PGP. Furthermore, RTR completely inhibits PGP‐induced lung emphysema, assessed by changes in alveolar enlargement and right ventricular hypertrophy. In conclusion, these data indicate that collagen breakdown products, especially PGP, are important in the pathogenesis of COPD and that PGP antagonism via RTR ameliorates lung emphysema.—van Houwelingen, A. H., Weathington, N. M., Verweij, V., Blalock, J. E., Nijkamp, F. P., Folkerts, G. Induction of lung emphysema is prevented by L‐arginine‐threonine‐arginine. FASEB J. 22, 3403–3408 (2008)

Emerging therapies targeting the ubiquitin proteasome system in cancer

The ubiquitin proteasome system (UPS) is an essential metabolic constituent of cellular physiology that tightly regulates cellular protein concentrations with specificity and precision to optimize cellular function. Inhibition of the proteasome has proven very effective in the treatment of multiple myeloma, and this approach is being tested for utility in other malignancies. New pharmaceuticals targeting the proteasome itself or specific proximal pathways of the UPS are in development as antiproliferatives or immunomodulatory agents. In this article, we discuss the biology of UPS-targeting drugs, their use as therapy for neoplasia, and the state of clinical and preclinical development for emerging therapeutics.

Human Neutrophil Elastase-Mediated Cleavage Sites of MMP-9 and TIMP-1: Implications to Cystic Fibrosis Proteolytic Dysfunction

Cystic fibrosis (CF) is a lethal genetic disorder characterized by airway remodeling and inflammation, leading to premature death. Recent evidence suggests the importance of protease activity in CF pathogenesis. One prominent protease, matrix metalloprotease (MMP)-9, demonstrates increased activity in CF individuals undergoing acute pulmonary exacerbation. This is thought to be mediated by both direct MMP-9 activation and the degradation of its natural inhibitor, tissue inhibitor of metalloprotease-1 (TIMP-1). To examine if this relationship exists in nonexacerbating CF individuals, we examined protease activity in sputum from these individuals compared with nondisease controls. We demonstrated increased gelatinolytic activity in CF sputum. These samples had elevated human neutrophil elastase (HNE) levels which correlated with an increased MMP-9/TIMP-1 ratio. To determine if HNE could discretely cleave and activate MMP-9, these enzymes were coincubated and two specific cleavage sites, between Valine38 and Alanine39, and between Alanine 39 and glutamic acid40 were observed. These sites corresponded with appropriate molecular weight for the activated MMP-9 isoform in CF sputum. Using N-terminal sequencing of cleavage fragments obtained with TIMP-1 incubation with HNE, we confirmed the TIMP-1 cleavage site for HNE is at Valine69-Cysteine70. We also show for the first time that human neutrophils were capable of degrading TIMP-1 exvivo and that a 16 kDa TIMP-1 fragment was identified in CF sputum, consistent with the expected cleavage of TIMP-1 by HNE. These results demonstrate increased MMP-9 activity in stable CF lung disease, and the presence of specific protease products in CF sputum highlights that HNE-mediated activity plays a role in this dysregulation.

Attacking the multi-tiered proteolytic pathology of COPD: new insights from basic and translational studies.

Protease activity in inflammation is complex. Proteases released by cells in response to infection, cytokines, or environmental triggers like cigarette smoking cause breakdown of the extracellular matrix (ECM). In chronic inflammatory diseases like chronic obstructive pulmonary disease (COPD), current findings indicate that pathology and morbidity are driven by dysregulation of protease activity, either through hyperactivity of proteases or deficiency or dysfunction their antiprotease regulators. Animal studies demonstrate the accuracy of this hypothesis through genetic and pharmacologic tools. New work shows that ECM destruction generates peptide fragments active on leukocytes via neutrophil or macrophage chemotaxis towards collagen and elastin derived peptides respectively. Such fragments now have been isolated and characterized in vivo in each case. Collectively, this describes a biochemical circuit in which protease activity leads to activation of local immunocytes, which in turn release cytokines and more proteases, leading to further leukocyte infiltration and cyclical disease progression that is chronic. This circuit concept is well known, and is intrinsic to the protease-antiprotease hypothesis; recently analytic techniques have become sensitive enough to establish fundamental mechanisms of this hypothesis, and basic and clinical data now implicate protease activity and peptide signaling as pathologically significant pharmacologic targets. This review discusses targeting protease activity for chronic inflammatory disease with special attention to COPD, covering important basic and clinical findings in the field; novel therapeutic strategies in animal or human studies; and a perspective on the successes and failures of agents with a focus on clinical potential in human disease.

Bioactive extracellular matrix fragments in lung health and disease

The extracellular matrix (ECM) is the noncellular component critical in the maintenance of organ structure and the regulation of tissue development, organ structure, and cellular signaling. The ECM is a dynamic entity that undergoes continuous degradation and resynthesis. In addition to compromising structure, degradation of the ECM can liberate bioactive fragments that cause cellular activation and chemotaxis of a variety of cells. These fragments are termed matrikines, and their cellular activities are sentinel in the development and progression of tissue injury seen in chronic lung disease. Here, we discuss the matrikines that are known to be active in lung biology and their roles in lung disease. We also consider the use of matrikines as disease markers and potential therapeutic targets in lung disease.

Targeting F Box Protein Fbxo3 To Control Cytokine-Driven Inflammation

Cytokine-driven inflammation underlies the pathobiology of a wide array of infectious and immune-related disorders. The TNFR-associated factor (TRAF) proteins have a vital role in innate immunity by conveying signals from cell surface receptors to elicit transcriptional activation of genes encoding proinflammatory cytokines. We discovered that a ubiquitin E3 ligase F box component, termed Fbxo3, potently stimulates cytokine secretion from human inflammatory cells by mediating the degradation of the TRAF inhibitory protein, Fbxl2. Analysis of the Fbxo3 C-terminal structure revealed that the bacterial-like ApaG molecular signature was indispensible for mediating Fbxl2 disposal and stimulating cytokine secretion. By targeting this ApaG motif, we developed a highly unique, selective genus of small-molecule Fbxo3 inhibitors that by reducing TRAF protein levels, potently inhibited cytokine release from human blood mononuclear cells. The Fbxo3 inhibitors effectively lessened the severity of viral pneumonia, septic shock, colitis, and cytokine-driven inflammation systemically in murine models. Thus, pharmacological targeting of Fbxo3 might be a promising strategy for immune-related disorders characterized by a heightened host inflammatory response.

Rational design of peptide vaccines for autoimmune disease: harnessing molecular recognition to fix a broken network

Autoreactive T-cells and antibodies are found at low levels in normal individuals and are thought to be kept at bay by regulatory T-cells and a network of idiotypic and anti-idiotype-bearing antigen receptors on lymphocytes as well as idiotypic anti-idiotypic antibodies. Disruption of this network by genetic, environmental and unknown factors is thought to result in autoimmune diseases. An obvious, ideal and specific therapy for such disorders would be to harness this regulatory network to re-establish immunologic homeostasis. In practice, however, this is not an easy task as most autoimmune diseases involve polyclonal responses to self antigen. Thus, we are faced with the conundrum of not knowing which autoreactive idiotype-bearing antibody or antigen receptor(s) to target in order to restore or induce network regulatory function. The thesis of this review is that understanding a fundamental property governing peptide/protein shape can be used in part to circumvent the problems of self reactivity and polyclonality in autoimmune disorders. More specifically, an algorithm has been developed to design peptide vaccines with shapes that are thought to be complementary in contour to self epitopes which seem to be the focus of autoimmunity. In theory, such complementary shapes should be engendered in certain autoreactive antigen receptors – these complementary constructs consequently represent receptor mimetics. By targeting an immune response against such mimetics, one generates a polyclonal anti-idiotype response that matches the complexity of the autoimmune response itself. This article will describe the algorithm for vaccine design, summarize the in vitro and in vivo evidence for its efficacy and discuss possible therapeutic utility in human autoimmune diseases.

The Emerging Role of the Ubiquitin Proteasome in Pulmonary Biology and Disease

Derangements in normal cellular homeostasis at the protein level can cause or be the consequence of initiation and progression of pulmonary diseases related to genotype, infection, injury, smoking, toxin exposure, or neoplasm. We discuss one of the fundamental mechanisms of protein homeostasis, the ubiquitin proteasome system (UPS), as it relates to lung disease. The UPS effects selective degradation of ubiquitinated target proteins via ubiquitin ligase activity. Important pathobiological mechanisms relating to the UPS and lung disease have been the focus of research, with inappropriate cellular proteolysis now a validated therapeutic target. We review the contributions of this system in various lung diseases, and discuss the exciting area of UPS-targeting drug development for pulmonary disease.