Georgios Koukos

A matrix metalloprotease-PAR1 system regulates vascular integrity, systemic inflammation and death in sepsis.

Sepsis is a deadly disease characterized by the inability to regulate the inflammatory–coagulation response in which the endothelium plays a key role. The cause of this perturbation remains poorly understood and has hampered the development of effective therapeutics. Matrix metalloproteases (MMPs) are involved in the host response to pathogens, but can also cause uncontrolled tissue damage and contribute to mortality. We found that human sepsis patients had markedly elevated plasma proMMP‐1 and active MMP‐1 levels, which correlated with death at 7 and 28 days after diagnosis. Likewise, septic mice had increased plasma levels of the MMP‐1 ortholog, MMP‐1a. We identified mouse MMP‐1a as an agonist of protease‐activated receptor‐1 (PAR1) on endothelial cells. MMP‐1a was released from endothelial cells in septic mice. Blockade of MMP‐1 activity suppressed endothelial barrier disruption, disseminated intravascular coagulation (DIC), lung vascular permeability as well as the cytokine storm and improved survival, which was lost in PAR1‐deficient mice. Infusion of human MMP‐1 increased lung vascular permeability in normal wild‐type mice but not in PAR1‐deficient mice. These findings implicate MMP‐1 as an important activator of PAR1 in sepsis and suggest that therapeutics that target MMP1‐PAR1 may prove beneficial in the treatment of sepsis.

Pharmacology, Biodistribution, and Efficacy of GPCR-Based Pepducins in Disease Models

G protein-coupled receptors (GPCR) are a superfamily of receptors that are vital in a wide array of physiological processes. Modulation of GPCR signaling has been an intensive area of therapeutic study, mainly due to the diverse pathophysiological significance of GPCRs. Pepducins are cell-penetrating lipidated peptides designed to target the intracellular loops of the GPCR of interest. Pepducins can function as agonists or antagonists of their cognate receptor, making them highly useful compounds for the study of GPCR signaling. Pepducins have been used to control platelet-dependent hemostasis and thrombosis, tumor growth, invasion, and angiogenesis, as well as to improve sepsis outcomes in mice. Pepducins have been successfully designed against a wide variety of GPCRs including the protease-activated receptors (PAR1, 2, 4), the chemokine receptors (CXCR1, 2, 4), the sphingosine-1-phosphate receptor (S1P3), the adrenergic receptor (ADRA1B), and have the potential to help reveal the functions of intractable GPCRs. Pharmacokinetic, pharmacodynamic, and biodistribution studies have showed that pepducins are widely distributed throughout the body except the brain and possess appropriate drug-like properties for use in vivo. Here, we discuss the delivery, pharmacology, and biodistribution of pepducins, as well as the effects of pepducins in models of inflammation, cardiovascular disease, cancer, and angiogenesis.

Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1

G protein-coupled receptors (GPCRs) can assume multiple conformations and possess multiple binding sites. Whereas endogenous agonists acting at the orthosteric binding site stabilize the active receptor conformation, small molecules that act at nonorthosteric sites can stabilize alternative conformations. The large majority of these allosteric modulators associate with extracellular loops of GPCRs. The role of intracellular domains in mediating allosteric modulation is largely unknown. In screening a small-molecule library for inhibitors of platelet activation, we identified a family of compounds that modified PAR1-mediated granule secretion. The most potent inhibitory compound, termed JF5, also demonstrated noncompetitive inhibition of the α2A-adrenergic receptor. Aggregation studies using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to GPCRs that possessed a constrained eighth helix, as defined by a C-terminal palmitoylation site and interactions with TM7 and the i1 loop. Inhibition by JF5 was overcome in a PAR1 mutant in which the eighth helix was deleted, confirming a role for helix 8 in JF5 activity. Evaluation of downstream signaling showed that JF5 was selective with regard to G protein coupling, blocking signaling mediated by Gαq but not Gα12. The compound inhibited thrombus formation in vivo following vascular injury with an IC50 of ∼1 mg/kg. These results indicate a role for helix 8 in conferring sensitivity to small molecules, and show that this sensitivity can be exploited to control platelet activation during thrombus formation.

MicroRNA214 Is Associated With Progression of Ulcerative Colitis, and Inhibition Reduces Development of Colitis and Colitis-Associated Cancer in Mice

BACKGROUND & AIMS Persistent activation of the inflammatory response contributes to the development of inflammatory bowel diseases, which increase the risk of colorectal cancer. We aimed to identify microRNAs that regulate inflammation during the development of ulcerative colitis (UC) and progression to colitis-associated colon cancer (CAC). METHODS We performed a quantitative polymerase chain reaction analysis to measure microRNAs in 401 colon specimens from patients with UC, Crohns disease, irritable bowel syndrome, sporadic colorectal cancer, or CAC, as well as subjects without these disorders (controls); levels were correlated with clinical features and disease activity of patients. Colitis was induced in mice by administration of dextran sodium sulfate (DSS), and carcinogenesis was induced by addition of azoxymethane; some mice also were given an inhibitor of microRNA214 (miR214). RESULTS A high-throughput functional screen of the human microRNAome found that miR214 regulated the activity of nuclear factor-κB. Higher levels of miR214 were detected in colon tissues from patients with active UC or CAC than from patients with other disorders or controls and correlated with disease progression. Bioinformatic and genome-wide profile analyses showed that miR214 activates an inflammatory response and is amplified through a feedback loop circuit mediated by phosphatase and tensin homolog (PTEN) and PDZ and LIM domain 2 (PDLIM2). Interleukin-6 induced signal transducer and activator of transcription 3 (STAT3)-mediated transcription of miR214. A miR214 chemical inhibitor blocked this circuit and reduced the severity of DSS-induced colitis in mice, as well as the number and size of tumors that formed in mice given azoxymethane and DSS. In fresh colonic biopsy specimens from patients with active UC, the miR214 inhibitor reduced inflammation by increasing levels of PDLIM2 and PTEN. CONCLUSIONS Interleukin-6 up-regulates STAT3-mediated transcription of miR214 in colon tissues, which reduces levels of PDLIM2 and PTEN, increases phosphorylation of AKT, and activates nuclear factor-κB. The activity of this circuit correlates with disease activity in patients with UC and progression to colorectal cancer.

Protease-Activated Receptor-2 Modulates Protease-Activated Receptor-1–Driven Neointimal Hyperplasia

Objective—Emerging evidence suggests that protease-activated receptors-1 and -2 (PAR1 and PAR2) can signal together in response to proteases found in the rapidly changing microenvironment of damaged blood vessels. However, it is unknown whether PAR1 and PAR2 promote or mitigate the hyperplastic response to arterial injury. Using cell-penetrating PAR1 pepducins and mice deficient in PAR1 or PAR2, we set out to determine the respective contributions of the receptors to hyperplasia and phenotypic modulation of smooth muscle cells (SMCs) in response to arterial injury. Methods and Results—SMCs were strongly activated by PAR1 stimulation, as evidenced by increased mitogenesis, mitochondrial activity, and calcium mobilization. The effects of chronic PAR1 stimulation following vascular injury were studied by performing carotid artery ligations in mice treated with the PAR1 agonist pepducin, P1pal-13. Histological analysis revealed that PAR1 stimulation caused striking hyperplasia, which was ablated in PAR1−/− and, surprisingly, PAR2−/− mice. P1pal-13 treatment yielded an expression pattern consistent with a dedifferentiated phenotype in carotid artery SMCs. Detection of PAR1-PAR2 complexes provided an explanation for the hyperplastic effects of the PAR1 agonist requiring the presence of both receptors. Conclusion—We conclude that PAR2 regulates the PAR1 hyperplastic response to arterial injury leading to stenosis.

Assessment of Circulating MicroRNAs for the Diagnosis and Disease Activity Evaluation in Patients with Ulcerative Colitis by Using the Nanostring Technology

Background:Clinical decision and patient care management in inflammatory bowel diseases is largely based on the assessment of clinical symptoms, while the biomarkers currently in use poorly reflect the actual disease activity. Therefore, the identification of novel biomarkers will serve an unmet clinical need for IBD screening and patient management. We examined the utility of circulating microRNAs for diagnosis and disease activity monitoring in patients with ulcerative colitis (UC). Methods:Blood serum microRNAs were isolated from patients with UC with active and inactive disease and healthy donors. High-throughput microRNA profiling was performed using the Nanostring technology platform. Clinical disease activity was captured by calculating the partial Mayo score. C-reactive protein was measured in patients with UC as part of their clinical monitoring. The profiles of circulating microRNAs and C-reactive protein were correlated with clinical disease indices. Results:We have identified a signature of 12 circulating microRNAs that differentiate patients with UC from control subjects. Moreover, 6 of these microRNAs significantly correlated with UC disease activity. Importantly, a set of 4 microRNAs (hsa-miR-4454, hsa-miR-223-3p, hsa-miR-23a-3p, and hsa-miR-320e), which correlated with UC disease activity were found to have higher sensitivity and specificity values than C-reactive protein. Conclusions:Circulating microRNAs provide a novel diagnostic and prognostic marker for patients with UC. The use of an FDA-approved platform could accelerate the application of microRNA screening in a gastrointenstinal clinical setting. When used in combination with current diagnostic and disease activity assessment modalities, microRNAs could improve both IBD screening and care management.

Systems biology in inflammatory bowel diseases: ready for prime time.

Purpose of review Ulcerative colitis and Crohns disease are the two predominant types of inflammatory bowel disease (IBD), affecting over 1.4 million individuals in the United States. IBD results from complex interactions between pathogenic components, including genetic and epigenetic factors, the immune response, and the microbiome, through an unknown sequence of events. The purpose of this review is to describe a systems biology approach to IBD as a novel and exciting methodology aiming at developing novel IBD therapeutics based on the integration of molecular and cellular ‘omics’ data. Recent findings Recent evidence suggested the presence of genetic, epigenetic, transcriptomic, proteomic, and metabolomic alterations in IBD patients. Furthermore, several studies have shown that different cell types including fibroblasts, epithelial, immune, and endothelial cells together with the intestinal microbiota are involved in IBD pathogenesis. Novel computational methodologies have been developed aiming to integrate high-throughput molecular data. Summary A systems biology approach could potentially identify the central regulators (hubs) in the IBD interactome and improve our understanding of the molecular mechanisms involved in IBD pathogenesis. The future IBD therapeutics should be developed on the basis of targeting the central hubs in the IBD network.

A MicroRNA signature in pediatric ulcerative colitis: Deregulation of the miR-4284/CXCL5 pathway in the intestinal epithelium

Background:Twenty to 25% of the patients with inflammatory bowel disease (IBD) present the disease before the age of 18 to 20, with worse extent and severity, compared with adult-onset IBD. We sought to identify the differential expression of microRNAs in pediatric ulcerative colitis (UC) and their association with different clinical phenotypes. Methods:MicroRNA expression analysis was performed in colonic tissues derived from pediatric patients with UC and controls without IBD. MiR-4284 levels were verified by real-time quantitative polymerase chain reaction in 2 additional cohorts of pediatric patients with UC. Bioinformatics analysis was performed to predict the targets of miR-4284. In vitro experiments using luciferase reporter assays and real-time polymerase chain reaction evaluated the direct effect of miR-4284 on CXCL5 mRNA. In vivo experiments were performed in 2 mouse models of experimental colitis. Results:A 24-microRNA signature was identified in colonic tissues derived from pediatric patients with UC. The most downregulated microRNA in the tissue of pediatric patients UC, relative to non-IBD controls, was miR-4284. In situ hybridization revealed that miR-4284 is present in colonic epithelial cells, and its levels correlate with the disease activity. Furthermore, we found that miR-4284 regulates CXCL5 mRNA expression through binding to its 3′UTR. CXCL5 had increased mRNA levels in colonic tissue from pediatric patients with UC and correlated with disease activity. Furthermore, we found an inverse correlation between miR-4284 and CXCL5 levels in the colonic pediatric UC tissues and in 2 mouse models of experimental colitis. Conclusions:Our data reveal a novel microRNA pediatric UC signature and provide evidence that miR-4284 directly regulates CXCL5 and correlates with the disease activity.

Matrix metalloprotease 1a deficiency suppresses tumor growth and angiogenesis

Matrix metalloprotease-1 (MMP1) is an important mediator of tumorigenesis, inflammation and tissue remodeling through its ability to degrade critical matrix components. Recent studies indicate that stromal-derived MMP1 may exert direct oncogenic activity by signaling through protease-activated receptor-1 (PAR1) in carcinoma cells; however, this has not been established in vivo. We generated an Mmp1a knockout mouse to ascertain whether stromal-derived Mmp1a affects tumor growth. Mmp1a-deficient mice are grossly normal and born in Mendelian ratios; however, deficiency of Mmp1a results in significantly decreased growth and angiogenesis of lung tumors. Coimplantation of lung cancer cells with wild-type Mmp1a+/+ fibroblasts completely restored tumor growth in Mmp1a-deficient animals, highlighting the critical role of stromal-derived Mmp1a. Silencing of PAR1 expression in the lung carcinoma cells phenocopied stromal Mmp1a-deficiency, thus validating tumor-derived PAR1 as an Mmp1a target. Mmp1a secretion is controlled by the ability of its prodomain to facilitate autocleavage, whereas human MMP1 is efficiently secreted because of stable pro- and catalytic domain interactions. Taken together, these data demonstrate that stromal Mmp1a drives in vivo tumorigenesis and provide proof of concept that targeting the MMP1-PAR1 axis may afford effective treatments of lung cancer.

Tumor Progression Locus 2 Mediates Signal-Induced Increases in Cytoplasmic Calcium and Cell Migration

The kinase Tpl2 triggers calcium signaling and cell migration downstream of various receptors implicated in cancer and inflammation. Defining a Migratory Route The serine-threonine kinase Tpl2, a mitogen-activated protein kinase kinase kinase, transduces signals from various plasma membrane receptors and has been implicated in pathways contributing to cancer and inflammation. Building on their earlier work showing that Tpl2 promotes cell migration in response to thrombin activation of the G protein–coupled receptor (GPCR) PAR1, Hatziapostolou et al. now define the signaling pathways through which this occurs. They found that PAR1 coupled to Gαi2 to activate Tpl2 and implicated the subsequent activation of phospholipase C–β3, production of inositol 1,4,5-trisphosphate, and cytoplasmic calcium signals in the migratory response. Moreover, they showed that Tpl2 mediated migratory signals through activation of several receptors other than PAR1. Their data contribute to our understanding of the pathways through which GPCRs and other receptors promote cell migration and may be pertinent to the mechanisms whereby Tpl2 contributes to cancer and inflammation. The mitogen-activated protein kinase kinase kinase (MAPKKK or MAP3K) tumor progression locus 2 (Tpl2) is required for the transduction of signals initiated by the thrombin-activated G protein–coupled receptor (GPCR) protease-activated receptor-1 (PAR1), which promote reorganization of the actin cytoskeleton and cell migration. Here, we show that Tpl2 is activated through Gαi2-transduced GPCR signals. Activated Tpl2 promoted the phosphorylation and activation of phospholipase C–β3 (PLCβ3); consequently, Tpl2 was required for thrombin-dependent production of inositol 1,4,5-trisphosphate (IP3), IP3-mediated cytoplasmic calcium ion (Ca2+) signals, and the activation of classical and novel members of the protein kinase C (PKC) family. A PKC-mediated feedback loop facilitated extracellular signal–regulated kinase (ERK) activation in response to Tpl2 and contributed to the coordinate regulation of the ERK and Ca2+ signaling pathways. Pharmacological and genetic studies revealed that stimulation of cell migration by Tpl2 depends on both of these pathways. Tpl2 also promoted Ca2+ signals and cell migration from sphingosine 1-phosphate–responsive GPCRs, which also couple to Gαi; from Wnt5a; and from the interleukin-1β (IL-1β) receptor, a member of the Toll–IL-1R (TIR) domain family. Our data provide new insights into the role of Tpl2 in GPCR-mediated Ca2+ signaling and cell migration.