Matrix metalloproteinases’s association of polymorphisms of MMP3 and MMP9 with development of genital endometriosis

We present a comparative analysis of the allelic polymorphisms of the matrix metalloproteinase (MMP) gene family, including MMP3 (rs3025058), MMP7 (rs11568818), MMP9 (rs17576, rs2250889), MMP12 (rs2276109), and MMP13 (rs2252070), in patients with external genital endometriosis (EGE) and in a control group of healthy women proven to be free of disease by laparoscopic inspection. We found significant differences in the incidence of particular MMP3 and MMP9 alleles, which substantiate the role of matrix metalloproteinases in EGE pathogenesis. We used the Multifactor Dimensionality Reduction (MDR) analysis to show that 14 allelic combinations of the MMP containing MMP3 (rs3025058) × MMP7 (rs11568818) × MMP9 (rs17576) alleles showed a statistically significant association with an increased risk of EGE, while 10 other combinations correlated with a reduced risk of the disease. MDR analysis produced two statistically significant models for MMP allelic combinations involved in EGE progression, both with 100% penetrance and 83 and 78% accuracy.     

MMP1 and MMP7 as potential peripheral blood biomarkers in idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disease associated with substantial morbidity and mortality. The objective of this study was to determine whether there is a peripheral blood protein signature in IPF and whether components of this signature may serve as biomarkers for disease presence and progression.

Methods and Findings

We analyzed the concentrations of 49 proteins in the plasma of 74 patients with IPF and in the plasma of 53 control individuals. We identified a combinatorial signature of five proteins—MMP7, MMP1, MMP8, IGFBP1, and TNFRSF1A—that was sufficient to distinguish patients from controls with a sensitivity of 98.6% (95% confidence interval [CI] 92.7%–100%) and specificity of 98.1% (95% CI 89.9%–100%). Increases in MMP1 and MMP7 were also observed in lung tissue and bronchoalveolar lavage fluid obtained from IPF patients. MMP7 and MMP1 plasma concentrations were not increased in patients with chronic obstructive pulmonary disease or sarcoidosis and distinguished IPF compared to subacute/chronic hypersensitivity pneumonitis, a disease that may mimic IPF, with a sensitivity of 96.3% (95% CI 81.0%–100%) and specificity of 87.2% (95% CI 72.6%–95.7%). We verified our results in an independent validation cohort composed of patients with IPF, familial pulmonary fibrosis, subclinical interstitial lung disease (ILD), as well as with control individuals. MMP7 and MMP1 concentrations were significantly higher in IPF patients compared to controls in this cohort. Furthermore, MMP7 concentrations were elevated in patients with subclinical ILD and negatively correlated with percent predicted forced vital capacity (FVC%) and percent predicted carbon monoxide diffusing capacity (DL_CO%).

Conclusions

Our experiments provide the first evidence for a peripheral blood protein signature in IPF to our knowledge. The two main components of this signature, MMP7 and MMP1, are overexpressed in the lung microenvironment and distinguish IPF from other chronic lung diseases. Additionally, increased MMP7 concentration may be indicative of asymptomatic ILD and reflect disease progression.     

A promoter SNP rs4073T>A in the common allele of the interleukin 8 gene is associated with the development of idiopathic pulmonary fibrosis via the IL-8 protein enhancing mode

Background

Interleukin-8 (IL-8) is a potent chemo-attractant cytokine responsible for neutrophil infiltration in lungs with idiopathic pulmonary fibrosis (IPF). The IL-8 protein and mRNA expression are increased in the lung with IPF. We evaluated the effect of single nucleotide polymorphisms (SNPs) of the IL-8 gene on the risk of IPF.

Methods

One promoter (rs4073T>A) and two intronic SNPs (rs2227307T>G and rs2227306C>T) of the IL-8 genes were genotyped in 237 subjects with IPF and 456 normal controls. Logistic regression analysis was applied to evaluate the association of these SNPs with IPF. IL-8 in BAL fluids was measured using a quantitative sandwich enzyme immunoassay, and promoter activity was assessed using the luciferase reporter assay.

Results

The minor allele frequencies of rs4073T>A and rs2227307T>G were significantly lower in the 162 subjects with surgical biopsy-proven IPF and 75 subjects with clinical IPF compared with normal controls in the recessive model (OR = 0.46 and 0.48, p = 0.006 and 0.007, respectively). The IL-8 protein concentration in BAL fluids significantly increased in 24 subjects with IPF compared with 14 controls (p = 0.009). Nine IPF subjects homozygous for the rs4073 T>A common allele exhibited higher levels of the IL-8 protein compared with six subjects homozygous for the minor allele (p = 0.024). The luciferase activity of the rs4073T>A common allele was significantly higher than that of the rs4073T>A minor allele (p = 0.002).

Conclusion

The common allele of a promoter SNP, rs4073T>A, may increase susceptibility to the development of IPF via up-regulation of IL-8.     

Small cell lung cancer: Circulating tumor cells of extended stage patients express a mesenchymal-epithelial transition phenotype

Small cell lung cancer (SCLC) is distinguished by aggressive growth, early dissemination and a poor prognosis at advanced stage. The remarkably high count of circulating tumor cells (CTCs) of SCLC allowed for the establishment of permanent CTC cultures at our institution for the first time. CTCs are assumed to have characteristics of cancer stem cells (CSCs) and an epithelial-mesenchymal transition (EMT) phenotype, but extravasation of tumors at distal sites is marked by epithelial features. Two SCLC CTC cell lines, namely BHGc7 and BHGc10, as well as SCLC cell lines derived from primary tumors and metastases were analyzed for the expression of pluripotent stem cell markers and growth factors. Expression of E-cadherin and β-Catenin were determined by flow cytometry. Stem cell-associated markers SOX17, α-fetoprotein, OCT-3/4, KDR, Otx2, GATA-4, Nanog, HCG, TP63 and Goosecoid were not expressed in the 2 CTC lines. In contrast, high expression was found for HNF-3β/FOXA2, SOX2, PDX-1/IPF1 and E-cadherin. E-cadherin expression was restricted to the 2 CTCs and 2 cell lines derived from pleural effusion (SCLC26A) and bone metastases (NCI-H526), respectively. Thus, these SCLC CTCs established from extended disease SCLC patients lack expression of stem cell markers which suppress the epithelial phenotype. Instead they express high levels of E-cadherin consistent with a mesenchymal-epithelial transition (MET or EMrT) and form large tumorospheres possibly in response to the selection pressure of first-line chemotherapy. HNF-3β/FOXA2 and PDX-1/IPF1 expression seem to be related to growth factor dependence on insulin/IGF-1 receptors and IGF-binding proteins.     

Matrix metalloproteinase gene polymorphisms and bronchopulmonary dysplasia: identification of MMP16 as a new player in lung development

Backgound

Alveolarization requires coordinated extracellular matrix remodeling, a process in which matrix metalloproteinases (MMPs) play an important role. We postulated that polymorphisms in MMP genes might affect MMP function in preterm lungs and thus influence the risk of bronchopulmonary dysplasia (BPD).

Methods and Findings

Two hundred and eighty-four consecutive neonates with a gestational age of <28 weeks were included in this prospective study. Forty-five neonates developed BPD. Nine single-nucleotide polymorphisms (SNPs) were sought in the MMP2, MMP14 and MMP16 genes. After adjustment for birth weight and ethnic origin, the TT genotype of MMP16 C/T (rs2664352) and the GG genotype of MMP16 A/G (rs2664349) were found to protect from BPD. These genotypes were also associated with a smaller active fraction of MMP2 and with a 3-fold-lower MMP16 protein level in tracheal aspirates collected within 3 days after birth. Further evaluation of MMP16 expression during the course of normal human and rat lung development showed relatively low expression during the canalicular and saccular stages and a clear increase in both mRNA and protein levels during the alveolar stage. In two newborn rat models of arrested alveolarization the lung MMP16 mRNA level was less than 50% of normal.

Conclusions

MMP16 may be involved in the development of lung alveoli. MMP16 polymorphisms appear to influence not only the pulmonary expression and function of MMP16 but also the risk of BPD in premature infants.     

IL1RN genetic variations and risk of IPF: a meta-analysis and mRNA expression study

Idiopathic pulmonary fibrosis (IPF) is a rare and devastating lung disease of unknown aetiology. Genetic variations in the IL1RN gene, encoding the interleukin-1 receptor antagonist (IL-1Ra), have been associated with IPF susceptibility. Several studies investigated the variable number tandem repeat (VNTR) or single nucleotide polymorphisms rs408392, rs419598 and rs2637988, with variable results. The aim of this study was to elucidate the influence of polymorphisms in IL1RN on IPF susceptibility and mRNA expression. We performed a meta-analysis of the five case–control studies that investigated an IL1RN polymorphism in IPF in a Caucasian population. In addition, we investigated whether IL1RN mRNA expression was influenced by IL1RN polymorphisms. The VNTR, rs408392 and rs419598 were in tight linkage disequilibrium, with D′ > 0.99. Furthermore, rs2637988 was in linkage disequilibrium with the VNTR (D′ = 0.90). A haploblock of VNTR*2 and the minor alleles of rs408392and rs419598 was constructed. Meta-analysis revealed that this VNTR*2 haploblock is associated with IPF susceptibility both with an allelic model (odds ratio = 1.42, p = 0.002) and a carriership model (odds ratio = 1.60, p = 0.002). IL1RN mRNA expression was significantly influenced by rs2637988, with lower levels found in carriers of the (minor) GG genotype (p < 0.001). From this meta-analysis, we conclude that the VNTR*2 haploblock is associated with susceptibility to IPF. In addition, polymorphisms in IL1RN influence IL-1Ra mRNA expression, suggesting that lower levels of IL-1Ra predispose to developing IPF. Together these findings demonstrate that the cytokine IL-1Ra plays a role in IPF pathogenesis.     

Genetic Polymorphisms at TIMP3 Are Associated with Survival of Adenocarcinoma of the Gastroesophageal Junction

The poor survival of adenocarcinomas of the gastroesophageal junction (GEJ) makes them clinically important. Discovery of host genetic factors that affect outcome may guide more individualized treatment. This study tests whether constitutional genetic variants in matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) genes are associated with outcome of GEJ adenocarcinoma. Single nucleotide polymorphisms (SNPs) at four TIMP (TIMP1-4) and three MMP genes (MMP2, MMP7 and MMP9) were genotyped in DNA samples from a prospective cohort of patients with primary adenocarcinoma of the GEJ admitted to the British Columbia Cancer Agency. Cox proportional hazards regression, with adjustment for patient, disease and treatment variables, was used to estimate the association of SNPs with survival. Genotypes for 85 samples and 48 SNPs were analyzed. Four SNPs across TIMP3, (rs130274, rs715572, rs1962223 and rs5754312) were associated with survival. Interaction analyses revealed that the survival associations with rs715572 and rs5754312 are specific and significant for 5FU+cisplatin treated patients. Sanger sequencing of the TIMP3 coding and promoter regions revealed an additional SNP, rs9862, also associated with survival. TIMP3 genetic variants are associated with survival and may be potentially useful in optimizing treatment strategies for individual patients.     

Genome-wide study identifies two loci associated with lung function decline in mild to moderate COPD

Accelerated lung function decline is a key COPD phenotype; however, its genetic control remains largely unknown. We performed a genome-wide association study using the Illumina Human660W-Quad v.1_A BeadChip. Generalized estimation equations were used to assess genetic contributions to lung function decline over a 5-year period in 4,048 European American Lung Health Study participants with largely mild COPD. Genotype imputation was performed using reference HapMap II data. To validate regions meeting genome-wide significance, replication of top SNPs was attempted in independent cohorts. Three genes (TMEM26, ANK3 and FOXA1) within the regions of interest were selected for tissue expression studies using immunohistochemistry. Two intergenic SNPs (rs10761570, rs7911302) on chromosome 10 and one SNP on chromosome 14 (rs177852) met genome-wide significance after Bonferroni. Further support for the chromosome 10 region was obtained by imputation, the most significantly associated imputed SNPs (rs10761571, rs7896712) being flanked by observed markers rs10761570 and rs7911302. Results were not replicated in four general population cohorts or a smaller cohort of subjects with moderate to severe COPD; however, we show novel expression of genes near regions of significantly associated SNPS, including TMEM26 and FOXA1 in airway epithelium and lung parenchyma, and ANK3 in alveolar macrophages. Levels of expression were associated with lung function and COPD status. We identified two novel regions associated with lung function decline in mild COPD. Genes within these regions were expressed in relevant lung cells and their expression related to airflow limitation suggesting they may represent novel candidate genes for COPD susceptibility.     

The MUC5B Variant Is Associated with Idiopathic Pulmonary Fibrosis but Not with Systemic Sclerosis Interstitial Lung Disease in the European Caucasian Population

A polymorphism on the MUC5B promoter (rs35705950) has been associated with idiopathic pulmonary fibrosis (IPF) but not with systemic sclerosis (SSc) with interstitial lung disease (ILD). We genotyped the MUC5B promoter in the first 142 patients of the French national prospective cohort of IPF, in 981 French patients with SSc (346 ILD), 598 Italian patients with SSc (207 ILD), 1383 French controls and 494 Italian controls. A meta-analysis was performed including all American data available. The T risk allele was present in 41.9% of the IPF patients, 10.8% of the controls (P = 2×10^–44), OR 6.3 [4.6–8.7] for heterozygous patients and OR 21.7 [10.4–45.3] for homozygous patients. Prevalence of the T allele was not modified according to age, gender, smoking in IPF patients. However, none of the black patients with IPF presented the T allele. The prevalence of the T risk allele was similar between French (10%) and Italian (12%) cohorts of SSc whatever the presence of an ILD (11.1% and 13.5%, respectively). Meta-analysis confirmed the similarity between French, Italian and American cohorts of IPF or SSc-ILD. This study confirms 1) an association between the T allele risk and IPF, 2) an absence of association with SSc-ILD, suggesting different pathophysiology.     

Chemokine (C-X-C motif) ligand 14 contributes to lipopolysaccharide-induced fibrogenesis in mouse L929 fibroblasts via modulating PPM1A

Herein, we found that serum chemokine ligand 14 (CXCL14) was significantly enhanced in patients with idiopathic pulmonary fibrosis (IPF). In our current study, mouse L929 fibroblasts were stimulated with lipopolysaccharide (LPS) (100 ng/mL). Cell proliferation, the levels of matrix metalloproteinase 2 (MMP2) and MMP9, as well as extracellular matrix (ECM) content were assessed to evaluate the fibrogenesis of L929 cells. Proliferating cell nuclear antigen and cell viability were assessed to evaluate cell proliferation. Hydroxyproline (Hyp), collagen I/III, connective tissue growth factor (CTGF), and phosphorylated Smad2/3 (p-Smad2/3) were assessed to evaluate ECM secretion and deposition. α-Smooth muscle actin (α-SMA) was used to measure the occurrence of differentiation from fibroblast toward myofibroblast. Our data suggested that knockdown of CXCL14 prevented LPS-induced fibrogenesis of L929 cells through inhibiting cell proliferation and decreasing the expression of MMP2/9, Hyp, collagen I/III, CTGF, p-Smad2/3, and α-SMA. Notably, upregulation of protein phosphatase magnesium-dependent 1A (PPM1A) was involved in this process. On the contrary, recombinant CXCL14 protein led to an opposite effect. We first suggested that overexpression of PPM1A ameliorated LPS-induced fibrogenesis. Furthermore, we substantiated that knockdown of CXCL14 exerted an antifibrotic effect in IPF in vitro probably via the upregulation of PPM1A. Besides, evidently enhanced CXCL14, yet reduced PPM1A, was found in bleomycin-induced rat pulmonary fibrosis, confirming the roles of CXCL14 and its potential association with PPM1A in IPF in vivo. In conclusion, CXCL14 could be considered as a therapeutic target for preventing fibrogenesis of mouse L929 fibroblasts.     

Genome Sequencing of Idiopathic Pulmonary Fibrosis in Conjunction with a Medical School Human Anatomy Course

Even in cases where there is no obvious family history of disease, genome sequencing may contribute to clinical diagnosis and management. Clinical application of the genome has not yet become routine, however, in part because physicians are still learning how best to utilize such information. As an educational research exercise performed in conjunction with our medical school human anatomy course, we explored the potential utility of determining the whole genome sequence of a patient who had died following a clinical diagnosis of idiopathic pulmonary fibrosis (IPF). Medical students performed dissection and whole genome sequencing of the cadaver. Gross and microscopic findings were more consistent with the fibrosing variant of nonspecific interstitial pneumonia (NSIP), as opposed to IPF per se. Variants in genes causing Mendelian disorders predisposing to IPF were not detected. However, whole genome sequencing identified several common variants associated with IPF, including a single nucleotide polymorphism (SNP), rs35705950, located in the promoter region of the gene encoding mucin glycoprotein MUC5B. The MUC5B promoter polymorphism was recently found to markedly elevate risk for IPF, though a particular association with NSIP has not been previously reported, nor has its contribution to disease risk previously been evaluated in the genome-wide context of all genetic variants. We did not identify additional predicted functional variants in a region of linkage disequilibrium (LD) adjacent to MUC5B, nor did we discover other likely risk-contributing variants elsewhere in the genome. Whole genome sequencing thus corroborates the association of rs35705950 with MUC5B dysregulation and interstitial lung disease. This novel exercise additionally served a unique mission in bridging clinical and basic science education.     

Association between variations in cell cycle genes and idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating and progressive lung disease. Its aetiology is thought to involve damage to the epithelium and abnormal repair. Alveolar epithelial cells near areas of remodelling show an increased expression of proapoptotic molecules. Therefore, we investigated the role of genes involved in cell cycle control in IPF. Genotypes for five single nucleotide polymorphisms (SNPs) in the tumour protein 53 (TP53) gene and four SNPs in cyclin-dependent kinase inhibitor 1A (CDKN1A), the gene encoding p21, were determined in 77 IPF patients and 353 controls. In peripheral blood mononuclear cells (PBMC) from 16 healthy controls mRNA expression of TP53 and CDKN1A was determined.Rs12951053 and rs12602273, in TP53, were significantly associated with survival in IPF patients. Carriers of a minor allele had a 4-year survival of 22% versus 57% in the non-carrier group (p = 0.006). Rs2395655 and rs733590, in CDKN1A, were associated with an increased risk of developing IPF. In addition, the rs2395655 G allele correlated with progression of the disease as it increased the risk of a rapid decline in lung function. Functional experiments showed that rs733590 correlated significantly with CDKN1A mRNA expression levels in healthy controls.This is the first study to show that genetic variations in the cell cycle genes encoding p53 and p21 are associated with IPF disease development and progression. These findings support the idea that cell cycle control plays a role in the pathology of IPF. Variations in TP53 and CDKN1A can impair the response to cell damage and increase the loss of alveolar epithelial cells.