Inhibition of longevity regulator PAPP-A modulates tissue homeostasis via restraint of mesenchymal stromal cells.

Pregnancy-associated plasma protein-A (PAPP-A) is a secreted metalloprotease that increases insulin-like growth factor (IGF) availability by cleaving IGF-binding proteins. Reduced IGF signaling extends longevity in multiple species, and consistent with this, PAPP-A deletion extends lifespan and healthspan; however, the mechanism remains unclear. To clarify PAPP-A's role, we developed a PAPP-A neutralizing antibody and treated adult mice with it. Transcriptomic profiling across tissues showed that anti-PAPP-A reduced IGF signaling and extracellular matrix (ECM) gene expression system wide. The greatest reduction in IGF signaling occurred in the bone marrow, where we found reduced bone, marrow adiposity, and myelopoiesis. These diverse effects led us to search for unifying mechanisms. We identified mesenchymal stromal cells (MSCs) as the source of PAPP-A in bone marrow and primary responders to PAPP-A inhibition. Mice treated with anti-PAPP-A had reduced IGF signaling in MSCs and dramatically decreased MSC number. As MSCs are (1) a major source of ECM and the progenitors of ECM-producing fibroblasts, (2) the originating source of adult bone, (3) regulators of marrow adiposity, and (4) an essential component of the hematopoietic niche, our data suggest that PAPP-A modulates bone marrow homeostasis by potentiating the number and activity of MSCs. We found that MSC-like cells are the major source of PAPP-A in other tissues also, suggesting that reduced MSC-like cell activity drives the system-wide reduction in ECM gene expression due to PAPP-A inhibition. Dysregulated ECM production is associated with aging and drives age-related diseases, and thus, this may be a mechanism by which PAPP-A deficiency enhances longevity.

Colonic Inhibition of Phosphatase and Tensin Homolog Increases Colitogenic Bacteria, Causing Development of Colitis in Il10-/- Mice.

Background: Phosphatase and tensin homolog (Pten) is capable of mediating microbe-induced immune responses in the gut. Thus, Pten deficiency in the intestine accelerates colitis development in Il10-/- mice. As some ambient pollutants inhibit Pten function and exposure to ambient pollutants may increase inflammatory bowel disease (IBD) incidence, it is of interest to examine how Pten inhibition could affect colitis development in genetically susceptible hosts. Methods: With human colonic mucosa biopsies from pediatric ulcerative colitis and non-IBD control subjects, we assessed the mRNA levels of the PTEN gene and the gene involved in IL10 responses. The data from the human tissues were corroborated by treating Il10-/-, Il10rb-/-, and wild-type C57BL/6 mice with Pten-specific inhibitor VO-OHpic. We evaluated the severity of mouse colitis by investigating the tissue histology and cytokine production. The gut microbiome was investigated by analyzing the 16S ribosomal RNA gene sequence with mouse fecal samples. Results: PTEN and IL10RB mRNA levels were reduced in the human colonic mucosa of pediatric ulcerative colitis compared with non-IBD subjects. Intracolonic treatment of the Pten inhibitor induced colitis in Il10-/- mice, characterized by reduced body weight, marked colonic damage, and increased production of inflammatory cytokines, whereas Il10rb-/- and wild-type C57BL/6 mice treated with the inhibitor did not develop colitis. Pten inhibitor treatment changed the fecal microbiome, with increased abundance of colitogenic bacteria Bacteroides and Akkermansia in Il10-/- mice. Conclusions: Loss of Pten function increases the levels of colitogenic bacteria in the gut, thereby inducing deleterious colitis in an Il10-deficient condition.

Noncanonical Matrix Metalloprotease 1-Protease-Activated Receptor 1 Signaling Drives Progression of Atherosclerosis.

OBJECTIVE: Protease-activated receptor-1 (PAR1) is classically activated by thrombin and is critical in controlling the balance of hemostasis and thrombosis. More recently, it has been shown that noncanonical activation of PAR1 by matrix metalloprotease-1 (MMP1) contributes to arterial thrombosis. However, the role of PAR1 in long-term development of atherosclerosis is unknown, regardless of the protease agonist. APPROACH AND RESULTS: We found that plasma MMP1 was significantly correlated (R=0.33; P=0.0015) with coronary atherosclerotic burden as determined by angiography in 91 patients with coronary artery disease and acute coronary syndrome undergoing cardiac catheterization or percutaneous coronary intervention. A cell-penetrating PAR1 pepducin, PZ-128, currently being tested as an antithrombotic agent in the acute setting in the TRIP-PCI study (Thrombin Receptor Inhibitory Pepducin-Percutaneous Coronary Intervention), caused a significant decrease in total atherosclerotic burden by 58% to 70% (P<0.05) and reduced plaque macrophage content by 54% (P<0.05) in apolipoprotein E-deficient mice. An MMP1 inhibitor gave similar beneficial effects, in contrast to the thrombin inhibitor bivalirudin that gave no improvement on atherosclerosis end points. Mechanistic studies revealed that inflammatory signaling mediated by MMP1-PAR1 plays a critical role in amplifying tumor necrosis factor α signaling in endothelial cells. CONCLUSIONS: These data suggest that targeting the MMP1-PAR1 system may be effective in tamping down chronic inflammatory signaling in plaques and halting the progression of atherosclerosis.

Identification of Spinal Cord MicroRNA and Gene Signatures in a Model of Chronic Stress-Induced Visceral Hyperalgesia in Rat.

INTRODUCTION: Animal studies have shown that stress could induce epigenetic and transcriptomic alterations essential in determining the balance between adaptive or maladaptive responses to stress. We tested the hypothesis that chronic stress in rats deregulates coding and non-coding gene expression in the spinal cord, which may underline neuroinflammation and nociceptive changes previously observed in this model. METHODS: Male Wistar rats were exposed to daily stress or handled, for 10 days. At day 11, lumbar spinal segments were collected and processed for mRNA/miRNA isolation followed by expression profiling using Agilent SurePrint Rat Exon and Rat miRNA Microarray platforms. Differentially expressed gene lists were generated using the dChip program. Microarrays were analyzed using the Ingenuity Pathways Analysis (IPA) tool from Ingenuity Systems. Multiple methods were used for the analysis of miRNA-mRNA functional modules. Quantitative real time RT-PCR for Interleukin 6 signal transducer (gp130), the Signal Transducer And Activator Of Transcription 3 (STAT3), glial fibrillary acidic protein and mir-17-5p were performed to confirm levels of expression. RESULTS: Gene network analysis revealed that stress deregulated different inflammatory (IL-6, JAK/STAT, TNF) and metabolic (PI3K/AKT) signaling pathways. MicroRNA array analysis revealed a signature of 39 deregulated microRNAs in stressed rats. MicroRNA-gene network analysis showed that microRNAs are regulators of two gene networks relevant to inflammatory processes. Specifically, our analysis of miRNA-mRNA functional modules identified miR-17-5p as an important regulator in our model. We verified miR-17-5p increased expression in stress using qPCR and in situ hybridization. In addition, we observed changes in the expression of gp130 and STAT3 (involved in intracellular signaling cascades in response to gp130 activation), both predicted targets for miR-17-5p. A modulatory role of spinal mir17-5p in the modulation of visceral sensitivity was confirmed in vivo. CONCLUSION: Using an integrative high throughput approach, our findings suggest a link between miR-17-5p increased expression and gp130/STAT3 activation providing new insight into the possible mechanisms mediating the effect of chronic stress on neuroinflammation in the spinal cord.

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, Crohn's 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.

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

PURPOSE OF REVIEW: Ulcerative colitis and Crohn's 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.

MicroRNA-124 regulates STAT3 expression and is down-regulated in colon tissues of pediatric patients with ulcerative colitis.

BACKGROUND & AIMS: Altered levels and functions of microRNAs (miRs) have been associated with inflammatory bowel diseases (IBDs), although little is known about their roles in pediatric IBD. We investigated whether colonic mucosal miRs are altered in children with ulcerative colitis (UC). METHODS: We used a library of 316 miRs to identify those that regulate phosphorylation of signal transducer and activator of transcription 3 (STAT3) in NCM460 human colonocytes incubated with interleukin-6. Levels of miR-124 were measured by real-time polymerase chain reaction analysis of colon biopsies from pediatric and adult patients with UC and patients without IBD (controls), and of HCT-116 colonocytes incubated with 5-aza-2'-deoxycytidine (5-AZA). Methylation of the MIR124 promoter was measured by quantitative methylation-specific polymerase chain reaction. RESULTS: Levels of phosphorylated STAT3 and the genes it regulates (encoding vascular endothelial growth factor (VEGF), BCL2, BCLXL, and matrix metallopeptidase 9 [MMP9]) were increased in pediatric patients with UC compared with control tissues. Overexpression of miR-124, let-7, miR-125, miR-26, or miR-101 reduced STAT3 phosphorylation by ≥ 75% in NCM460 cells; miR-124 had the greatest effect. miR-124 was down-regulated specifically in colon tissues from pediatric patients with UC and directly targeted STAT3 messenger RNA (mRNA). Levels of miR-124 were decreased, whereas levels of STAT3 phosphorylation increased in colon tissues from pediatric patients with active UC compared with those with inactive disease. In addition, levels of miR-124 and STAT3 were inversely correlated in mice with experimental colitis. Down-regulation of miR-124 in tissues from children with UC was attributed to hypermethylation of its promoter region. Incubation of HCT-116 colonocytes with 5-AZA up-regulated miR-124 and reduced levels of STAT3 mRNA. CONCLUSIONS: miR-124 appears to regulate the expression of STAT3. Reduced levels of miR-124 in colon tissues of children with active UC appear to increase expression and activity of STAT3, which could promote inflammation and the pathogenesis of UC in children.

Tumor progression locus 2 mediates signal-induced increases in cytoplasmic calcium and cell migration.

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 Gα(i2)-transduced GPCR signals. Activated Tpl2 promoted the phosphorylation and activation of phospholipase C-ß3 (PLCß(3)); consequently, Tpl2 was required for thrombin-dependent production of inositol 1,4,5-trisphosphate (IP(3)), IP(3)-mediated cytoplasmic calcium ion (Ca(2+)) 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 Ca(2+) signaling pathways. Pharmacological and genetic studies revealed that stimulation of cell migration by Tpl2 depends on both of these pathways. Tpl2 also promoted Ca(2+) signals and cell migration from sphingosine 1-phosphate-responsive GPCRs, which also couple to Gα(i); from Wnt5a; and from the interleukin-1ß (IL-1ß) receptor, a member of the Toll-IL-1R (TIR) domain family. Our data provide new insights into the role of Tpl2 in GPCR-mediated Ca(2+) signaling and cell migration.

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.

Discrete roles of apoA-I and apoE in the biogenesis of HDL species: lessons learned from gene transfer studies in different mouse models.

Using adenovirus-mediated gene transfer in apolipoprotein A-I (apoA-I)-deficient mice, we have established that apoA-I mutations inhibit discrete steps in a pathway that leads to the biogenesis and remodeling of high-density lipoprotein (HDL). To this point, five discrete categories of apoA-I mutants have been characterized that may affect the interactions of apoA-I with ATP-binding cassette superfamily A, member 1 (ABCA1) or lecithin:cholesterol acyl transferase (LCAT) or may influence the plasma phospholipid transfer protein activity or may cause various forms of dyslipidemia. Biogenesis of HDL is not a unique property of apoA-I. Using adenovirus-mediated gene transfer of apoE in apoA-I- or ABCA1-deficient mice, we have established that apolipoprotein E (apoE) also participates in a novel pathway of biogenesis of apoE-containing HDL particles. This process requires the functions of the ABCA1 lipid transporter and LCAT, and it is promoted by substitution of hydrophobic residues in the 261 to 269 region of apoE by Ala. The apoE-containing HDL particles formed in the circulation may have atheroprotective properties. ApoE-containing HDL may also have important biological functions in the brain that confer protection from Alzheimer's disease.

LCAT can rescue the abnormal phenotype produced by the natural ApoA-I mutations (Leu141Arg)Pisa and (Leu159Arg)FIN.

To explain the etiology and find a mode of therapy of genetically determined low levels of high-density lipoprotein (HDL), we have generated recombinant adenoviruses expressing apolipoprotein A-I (apoA-I)(Leu141Arg)Pisa and apoA-I(Leu159Arg)FIN and studied their properties in vitro and in vivo. Both mutants were secreted efficiently from cells but had diminished capacity to activate lecithin/cholesterol acyltransferase (LCAT) in vitro. Adenovirus-mediated gene transfer of either of the two mutants in apoA-I-deficient (apoA-I-/-) mice resulted in greatly decreased total plasma cholesterol, apoA-I, and HDL cholesterol levels. The treatment also decreased the cholesteryl ester to total cholesterol ratio (CE/TC), caused accumulation of prebeta1-HDL and small size alpha4-HDL particles, and generated only few spherical HDL particles, as compared to mice expressing wild-type (WT) apoA-I. Simultaneous treatment of the mice with adenoviruses expressing either of the two mutants and human LCAT normalized the plasma apoA-I, HDL cholesterol levels, and the CE/TC ratio, restored normal prebeta- and alpha-HDL subpopulations, and generated spherical HDL. The study establishes that apoA-I(Leu141Arg)Pisa and apoA-I(Leu159Arg)FIN inhibit an early step in the biogenesis of HDL due to inefficient esterification of the cholesterol of the prebeta1-HDL particles by the endogenous LCAT. Both defects can be corrected by treatment with LCAT.

Naturally occurring and bioengineered apoA-I mutations that inhibit the conversion of discoidal to spherical HDL: the abnormal HDL phenotypes can be corrected by treatment with LCAT.

In the present study we have used adenovirus-mediated gene transfer of apoA-I (apolipoprotein A-I) mutants in apoA-I-/- mice to investigate how structural mutations in apoA-I affect the biogenesis and the plasma levels of HDL (high-density lipoprotein). The natural mutants apoA-I(R151C)Paris, apoA-I(R160L)Oslo and the bioengineered mutant apoA-I(R149A) were secreted efficiently from cells in culture. Their capacity to activate LCAT (lecithin:cholesterol acyltransferase) in vitro was greatly reduced, and their ability to promote ABCA1 (ATP-binding cassette transporter A1)-mediated cholesterol efflux was similar to that of WT (wild-type) apoA-I. Gene transfer of the three mutants in apoA-I-/- mice generated aberrant HDL phenotypes. The total plasma cholesterol of mice expressing the apoA-I(R160L)Oslo, apoA-I(R149A) and apoA-I(R151C)Paris mutants was reduced by 78, 59 and 61% and the apoA-I levels were reduced by 68, 64 and 55% respectively, as compared with mice expressing the WT apoA-I. The CE (cholesteryl ester)/TC (total cholesterol) ratio of HDL was decreased and the apoA-I was distributed in the HDL3 region. apoA-I(R160L)Oslo and apoA-I(R149A) promoted the formation of prebeta1 and alpha4-HDL subpopulations and gave a mixture of discoidal and spherical particles. apoA-I(R151C)Paris generated subpopulations of different sizes that migrate between prebeta and alpha-HDL and formed mostly spherical and a few discoidal particles. Simultaneous treatment of mice with adenovirus expressing any of the three mutants and human LCAT normalized plasma apoA-I, HDL cholesterol levels and the CE/TC ratio. It also led to the formation of spherical HDL particles consisting mostly of alpha-HDL subpopulations of larger size. The correction of the aberrant HDL phenotypes by treatment with LCAT suggests a potential therapeutic intervention for HDL abnormalities that result from specific mutations in apoA-I.

The carboxy-terminal region of apoA-I is required for the ABCA1-dependent formation of alpha-HDL but not prebeta-HDL particles in vivo.

ATP-binding cassette transporter A-1 (ABCA1)-mediated lipid efflux to lipid-poor apolipoprotein A-I (apoA-I) results in the gradual lipidation of apoA-I. This leads to the formation of discoidal high-density lipoproteins (HDL), which are subsequently converted to spherical HDL by the action of lecithin:cholesterol acyltransferase (LCAT). We have investigated the effect of point mutations and deletions in the carboxy-terminal region of apoA-I on the biogenesis of HDL using adenovirus-mediated gene transfer in apoA-I-deficient mice. It was found that the plasma HDL levels were greatly reduced in mice expressing the carboxy-terminal deletion mutants apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)], shown previously to diminish the ABCA1-mediated lipid efflux. The HDL levels were normal in mice expressing the WT apoA-I, the apoA-I[Delta(232-243)] deletion mutant, or the apoA-I[E191A/H193A/K195A] point mutant, which promote normal ABCA1-mediated lipid efflux. Electron microscopy and two-dimensional gel electrophoresis showed that the apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)] mutants formed mainly prebeta-HDL particles and few spherical particles enriched in apoE, while WT apoA-I, apoA-I[Delta(232-243)], and apoA-I[E191A/H193A/K195A] formed spherical alpha-HDL particles. The findings establish that (a) deletions that eliminate the 220-231 region of apoA-I prevent the synthesis of alpha-HDL but allow the synthesis of prebeta-HDL particles in vivo, (b) the amino-terminal segment 1-184 of apoA-I can promote synthesis of prebeta-HDL-type particles in an ABCA1-independent process, and (c) the charged residues in the 191-195 region of apoA-I do not influence the biogenesis of HDL.