The Role of SOX9 in IGF-II-Mediated Pulmonary Fibrosis.

Pulmonary fibrosis (PF) associated with systemic sclerosis (SSc) results in significant morbidity and mortality. We previously reported that insulin-like growth factor-II (IGF-II) is overexpressed in lung tissues and fibroblasts from SSc patients, and IGF-II fosters fibrosis by upregulating collagen type I, fibronectin, and TGFß. We now show that IGF-II augments mRNA levels of profibrotic signaling molecules TGFß2 (p ≤ 0.01) and TGFß3 (p ≤ 0.05), collagen type III (p ≤ 0.01), and the collagen posttranslational modification enzymes P4HA2 (p ≤ 0.05), P3H2 (p ≤ 0.05), LOX (p = 0.065), LOXL2 (p ≤ 0.05), LOXL4 (p ≤ 0.05) in primary human lung fibroblasts. IGF-II increases protein levels of TGFß2 (p ≤ 0.01), as well as COL3A1, P4HA2, P4Hß, and LOXL4 (p ≤ 0.05). In contrast, IGF-II decreases mRNA levels of the collagen degradation enzymes cathepsin (CTS) K, CTSB, and CTSL and protein levels of CTSK (p ≤ 0.05). The SRY-box transcription factor 9 (SOX9) is overexpressed in SSc lung tissues at the mRNA (p ≤ 0.05) and protein (p ≤ 0.01) levels compared to healthy controls. IGF-II induces SOX9 in lung fibroblasts (p ≤ 0.05) via the IGF1R/IR hybrid receptor, and SOX9 regulates TGFß2 (p ≤ 0.05), TGFß3 (p ≤ 0.05), COL3A1 (p ≤ 0.01), and P4HA2 (p ≤ 0.001) downstream of IGF-II. Our results identify a novel IGF-II signaling axis and downstream targets that are regulated in a SOX9-dependent and -independent manner. Our findings provide novel insights on the role of IGF-II in promoting pulmonary fibrosis.

Ameliorating Fibrosis in Murine and Human Tissues with END55, an Endostatin-Derived Fusion Protein Made in Plants.

Organ fibrosis, particularly of the lungs, causes significant morbidity and mortality. Effective treatments are needed to reduce the health burden. A fragment of the carboxyl-terminal end of collagen XVIII/endostatin reduces skin and lung fibrosis. This fragment was modified to facilitate its production in plants, which resulted in the recombinant fusion protein, END55. We found that expression of END55 had significant anti-fibrotic effects on the treatment and prevention of skin and lung fibrosis in a bleomycin mouse model. We validated these effects in a second mouse model of pulmonary fibrosis involving inducible, lung-targeted expression of transforming growth factor ß1. END55 also exerted anti-fibrotic effects in human lung and skin tissues maintained in organ culture in which fibrosis was experimentally induced. The anti-fibrotic effect of END55 was mediated by a decrease in the expression of extracellular matrix genes and an increase in the levels of matrix-degrading enzymes. Finally, END55 reduced fibrosis in the lungs of patients with systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF) who underwent lung transplantation due to the severity of their lung disease, displaying efficacy in human tissues directly relevant to human disease. These findings demonstrate that END55 is an effective anti-fibrotic therapy in different organs.

Pan-cancer analysis of prognostic metastatic phenotypes.

Although cancer is highly heterogeneous, all metastatic cancer is considered American Joint Committee on Cancer (AJCC) Stage IV disease. The purpose of this project was to redefine staging of metastatic cancer. Internal validation of nationally representative patient data from the National Cancer Database (n = 461 357; 2010-2013), and external validation using the Surveillance, Epidemiology and End Results database (n = 106 595; 2014-2015) were assessed using the concordance index for evaluation of survival prediction. A Cox proportional hazards model was used for overall survival by considering identified phenotypes (latent classes) and other confounding variables. Latent class analysis was performed for phenotype identification, where Bayesian information criterion (BIC) and sample-size-adjusted BIC were used to select the optimal number of distinct clusters. Kappa coefficients assessed external cluster validation. Latent class analysis identified five metastatic phenotypes with differences in overall survival (P < .0001): (Stage IVA) nearly exclusive bone-only metastases (n = 59 049, 12.8%; median survival 12.7 months; common in lung, breast and prostate cancers); (IVB) predominant lung metastases (n = 62 491, 13.5%; 11.4 months; common in breast, stomach, kidney, ovary, uterus, thyroid, cervix and soft tissue cancers); (IVC) predominant liver/lung metastases (n = 130 014, 28.2%; 7.0 months; common in colorectum, pancreatic, lung, esophagus and stomach cancers); (IVD) bone/liver/lung metastases predominant over brain (n = 61 004, 13.2%; 5.9 months; common in lung and breast cancers); and (IVE) brain/lung metastases predominant over bone/liver (n = 148 799, 32.3%; 5.7 months; lung cancer and melanoma). Long-term survivors were identified, particularly in Stages IVA-B. A pan-cancer nomogram model to predict survival (STARS: site, tumor, age, race, sex) was created, validated and provides 13% better prognostication than AJCC: 1-month concordance index of 0.67 (95% confidence interval [CI]: 0.66-0.67) vs 0.61 (95% CI: 0.60-0.61). STARS is simple, uses easily accessible variables, better prognosticates survival outcomes and provides a platform to develop novel metastasis-directed clinical trials.

Insulin-like growth factor (IGF)-II- mediated fibrosis in pathogenic lung conditions.

Type 2 insulin-like growth factor (IGF-II) levels are increased in fibrosing lung diseases such as idiopathic pulmonary fibrosis (IPF) and scleroderma/systemic sclerosis-associated pulmonary fibrosis (SSc). Our goal was to investigate the contribution of IGF receptors to IGF-II-mediated fibrosis in these diseases and identify other potential mechanisms key to the fibrotic process. Cognate receptor gene and protein expression were analyzed with qRT-PCR and immunoblot in primary fibroblasts derived from lung tissues of normal donors (NL) and patients with IPF or SSc. Compared to NL, steady-state receptor gene expression was decreased in SSc but not in IPF. IGF-II stimulation differentially decreased receptor mRNA and protein levels in NL, IPF, and SSc fibroblasts. Neutralizing antibody, siRNA, and receptor inhibition targeting endogenous IGF-II and its primary receptors, type 1 IGF receptor (IGF1R), IGF2R, and insulin receptor (IR) resulted in loss of the IGF-II response. IGF-II tipped the TIMP:MMP balance, promoting a fibrotic environment both intracellularly and extracellularly. Differentiation of fibroblasts into myofibroblasts by IGF-II was blocked with a TGFß1 receptor inhibitor. IGF-II also increased TGFß2 and TGFß3 expression, with subsequent activation of canonical SMAD2/3 signaling. Therefore, IGF-II promoted fibrosis through IGF1R, IR, and IGF1R/IR, differentiated fibroblasts into myofibroblasts, decreased protease production and extracellular matrix degradation, and stimulated expression of two TGFß isoforms, suggesting that IGF-II exerts pro-fibrotic effects via multiple mechanisms.

Induction of a Th17 Phenotype in Human Skin-A Mimic of Dermal Inflammatory Diseases.

Th17 cells are a subset of effector T helper cells that produce interleukin (IL)-17A, IL-17F, IL-22, and IL-26, which can promote tissue inflammation and contribute to the pathogenesis of rheumatic, fibrosing, and other diseases. Research into these diseases is often limited by a lack of an animal model that closely mimics human disease and the paucity of patient clinical tissues. Therefore, the development of relevant experimental models is crucial. Three media formulations of Th17-skewing cocktail (CT) were evaluated for the ability to induce a Th17 signature in an ex vivo human skin model: CT9 contained αCD3, αCD28, IL-23, IL-1ß, IFNγ, IL-4, IL-6, IL-21, and TGFß; CT8 lacked IL-1ß; and CT4 only contained αCD3, αCD28, IL-23, and IL-1ß. Healthy donor skin was defatted, distributed as 3 mm punch biopsies, and incubated with one of the cocktail formulations or vehicle for 48 h. All of the cocktail formulations independently significantly stimulated the expression of each gene examined. CT4 induced IL-17A expression 1024-fold, significantly higher than CT9 and CT8. IL-17F was robustly stimulated by CT4 (1557-fold), CT9 (622-fold), and CT8 (111-fold), with significant differences between the CT groups. All of the formulations significantly induced IL-22 (16-42-fold). CT9 stimulated the highest IL-26 response (41-fold), which was significantly higher than CT4 and CT8. IL-10 was stimulated significantly higher with CT8 (10-fold) than CT4 or CT9. The secretion of IL-17A was significantly elevated with all cocktail formulations. Robust IL-17A/IL-17F cytokine induction was preferentially mediated by CT4, which suggested that its components are the minimal constituents necessary for the full induction of these genes in this human skin explant model, while the downstream cytokines were preferentially upregulated by CT4 (IL-22), CT9 (IL-26), or CT8 (IL-10). In summary, our findings suggest that the induction of a Th17 phenotype in human skin is feasible and can be used as a model for rheumatic and fibrosing diseases where Th17 skewing is observed.

NADPH oxidase-mediated induction of reactive oxygen species and extracellular matrix deposition by insulin-like growth factor binding protein-5.

Insulin-like growth factor binding protein-5 (IGFBP-5) induces production of the extracellular matrix (ECM) components collagen and fibronectin both in vitro and in vivo and is overexpressed in patients with fibrosing lung diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). However, the mechanism by which IGFBP-5 exerts its fibrotic effect is incompletely understood. Recent reports have shown a substantial role of reactive oxygen species (ROS) in fibrosis; thus we hypothesized that IGFBP-5 induces production of ROS to mediate the profibrotic process. In vitro analyses revealed that ROS production was induced by recombinant and adenoviral vector-mediated IGFBP-5 (AdBP5) in a dose- and time-dependent manner, regulated through MEK/ERK and JNK signaling, and primarily mediated by NADPH oxidase (Nox). Silencing IGFBP-5 in SSc and IPF fibroblasts reduced ROS production. The antioxidants diphenyleneiodonium and N-acetylcysteine blocked IGFBP-5-stimulated ECM production in normal, SSc, and IPF human primary lung fibroblasts. In murine fibroblasts lacking critical components of the Nox machinery, AdBP5-stimulated ROS production and fibronectin expression were reduced compared with wild-type fibroblasts. IGFBP-5 stimulated transcriptional expression of Nox3 in human fibroblasts while selective knockdown of Nox3 reduced ROS production by IGFBP-5. Thus IGFBP-5 mediates fibrosis through production of ROS in a Nox-dependent manner.

Analysis of longitudinal semicontinuous data using marginalized two-part model.

BACKGROUND: Connective tissue growth factor (CTGF), is a secreted matricellular factor that has been linked to increased risk of cardiovascular disease in diabetic subjects. Despite the biological role of CTGF in diabetes, it still remains unclear how CTGF expression is regulated. In this study, we aim to identify the clinical parameters that modulate plasma CTGF levels measured longitudinally in type 1 diabetic patients over a period of 10 years. A number of patients had negligible measured values of plasma CTGF that formed a point mass at zero, whereas others had high positive values of CTGF that were measured on a continuous scale. The observed combination of excessive zero and continuous positively distributed non-zero values in the CTGF outcome is referred to as semicontinuous data. METHODS: We propose a novel application of a marginalized two-part model (mTP) extended to accommodate longitudinal semicontinuous data in which the marginal mean is expressed in terms of the covariates and estimates of their effect on the mean responses are generated. The continuous component is assumed to follow distributions that stem from the generalized gamma family whereas the binary measure is analyzed using logistic model and both have correlated random effects. Other approaches including the one- and two-part with uncorrelated and correlated random effects models were also applied and their estimates were all compared. RESULTS: Our results using the mTP model identified intensive glucose control treatment and smoking as clinical factors that were associated with decreased and increased odds of observing non-zero CTGF values respectively. In addition, hemoglobin A1c, systolic blood pressure, and high density lipoprotein were all shown to be significant risk factors that contribute to increasing CTGF levels. These findings were consistently observed under the mTP model but varied with the distributions for the other models. Accuracy and precision of the mTP model was further validated using simulation studies. CONCLUSION: The mTP model identified new clinical determinants that modulate the levels of CTGF in diabetic subjects. Applicability of this approach can be extended to other biomarkers measured in patient populations that display a combination of negligible zero and non-zero values.

Plasma Connective Tissue Growth Factor (CTGF/CCN2) Levels Predict Myocardial Infarction in the Veterans Affairs Diabetes Trial (VADT) Cohort.

OBJECTIVE: Connective tissue growth factor (CTGF), also known as CCN2, is a potent chemotactic and extracellular matrix-inducing matricellular protein that has been implicated in progression of inflammatory and fibroproliferative disorders. An emerging role of CTGF/CCN2 is that of a prosclerotic factor implicated in the development of cardiac disease. Our objective was to determine the role of CTGF/CCN2 as a predictor of cardiovascular events in type 2 diabetes in the Veterans Affairs Diabetes Trial (VADT) cohort. RESEARCH DESIGN AND METHODS: Levels of CTGF/CCN2 were measured in 952 VADT patients a median of 1.9 years after entry into the study. Participants were followed for an average of 3.3 years for vascular outcomes. CTGF/CCN2 categories were defined as below the detectable limit (referent, 54.5%), lower half of detectable values (22.8%), and upper half of detectable values (22.7%). Hazard ratios (HRs) for cardiovascular end points in relation to CTGF/CCN2 categories were calculated by Cox proportional hazards models. RESULTS: During follow-up, 4.8% had a myocardial infarction (MI), 6.9% had an MI or cardiovascular death, and 6.9% died. After adjustments by conventional risk factors, individuals in the highest category of CTGF/CCN2 were at higher risk of MI (HR 2.43 [95% CI 1.15, 5.14]), MI or cardiovascular death (HR 2.71 [95% CI 1.44, 5.08]), and all-cause mortality (HR 2.70 [95% CI 1.43, 5.08]) relative to individuals with CTGF below the detectable limit. CONCLUSIONS: Our study indicates that high levels of CTGF/CCN2 predict future MI and cardiovascular death in patients with type 2 diabetes.

Identification of dual mechanisms mediating 5-hydroxytryptamine receptor 1F-induced mitochondrial biogenesis.

Our laboratory recently made the novel observation that 5-hydroxytryptamine 1F (5-HT1F) receptor activation induces mitochondrial biogenesis (MB), the production of new, functional mitochondria, in vitro and in vivo. We sought to determine the mechanism linking the 5-HT1F receptor to MB in renal proximal tubule cells. Using LY344864 , a selective 5-HT1F receptor agonist, we determined that the 5-HT1F receptor is coupled to Gαi/o and induces MB through Gßγ-dependent activation of Akt, endothelial nitric oxide synthase (eNOS), cyclic guanosine-monophosphate (cGMP), protein kinase G (PKG), and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). We also report that the 5-HT1F receptor signals through a second, Gßγ-dependent pathway that is linked by Akt phosphorylation of Raf. In contrast to the activated Akt pathway, Raf phosphorylation reduced extracellular signal regulated kinases (ERK1/2) and foxhead box O3a (FOXO3a) phosphorylation, suppressing an inhibitory MB pathway. These results demonstrate that the 5-HT1F receptor regulates MB through Gßγ-dependent dual mechanisms that activate a stimulatory MB pathway, Akt/eNOS/cGMP/PKG/PGC-1α, while simultaneously repressing an inhibitory MB pathway, Raf/MEK/ERK/FOXO3a. Novel mechanisms of MB provide the foundation for new chemicals that induce MB to treat acute and chronic organ injuries.

The mighty fibroblast and its utility in scleroderma research.

Fibroblasts are the effector cells of fibrosis characteristic of systemic sclerosis (SSc, scleroderma) and other fibrosing conditions. The excess production of extracellular matrix (ECM) proteins is the hallmark of fibrosis in different organs, such as skin and lung. Experiments designed to assess the pro-fibrotic capacity of factors, their signaling pathways, and potential inhibitors of their effects that are conducted in fibroblasts have paved the way for planning clinical trials in SSc. As such, fibroblasts have proven to be valuable tools in the search for effective anti-fibrotic therapies for fibrosis. Herein we highlight the characteristics of fibroblasts, their role in the etiology of fibrosis, utility in experimental assays, and contribution to drug development and clinical trials in SSc.

Inhibition of Sphingosine Kinase 1 Ameliorates Angiotensin II-Induced Hypertension and Inhibits Transmembrane Calcium Entry via Store-Operated Calcium Channel.

Angiotensin II (AngII) plays a critical role in the regulation of vascular tone and blood pressure mainly via regulation of Ca(2+) mobilization. Several reports have implicated sphingosine kinase 1 (SK1)/sphingosine 1-phosphate (S1P) in the mobilization of intracellular Ca(2+) through a yet-undefined mechanism. Here we demonstrate that AngII-induces biphasic calcium entry in vascular smooth muscle cells, consisting of an immediate peak due to inositol tris-phosphate-dependent release of intracellular calcium, followed by a sustained transmembrane Ca(2+) influx through store-operated calcium channels (SOCs). Inhibition of SK1 attenuates the second phase of transmembrane Ca(2+) influx, suggesting a role for SK1 in AngII-dependent activation of SOC. Intracellular S1P triggers SOC-dependent Ca(2+) influx independent of S1P receptors, whereas external application of S1P stimulated S1P receptor-dependent Ca(2+) influx that is insensitive to inhibitors of SOCs, suggesting that the SK1/S1P axis regulates store-operated calcium entry via intracellular rather than extracellular actions. Genetic deletion of SK1 significantly inhibits both the acute hypertensive response to AngII in anaesthetized SK1 knockout mice and the sustained hypertensive response to continuous infusion of AngII in conscious animals. Collectively these data implicate SK1 as the missing link that connects the angiotensin AT1A receptor to transmembrane Ca(2+) influx and identify SOCs as a potential intracellular target for SK1.

Agonism of the 5-hydroxytryptamine 1F receptor promotes mitochondrial biogenesis and recovery from acute kidney injury.

Many acute and chronic conditions, such as acute kidney injury, chronic kidney disease, heart failure, and liver disease, involve mitochondrial dysfunction. Although we have provided evidence that drug-induced stimulation of mitochondrial biogenesis (MB) accelerates mitochondrial and cellular repair, leading to recovery of organ function, only a limited number of chemicals have been identified that induce MB. The goal of this study was to assess the role of the 5-hydroxytryptamine 1F (5-HT1F) receptor in MB. Immunoblot and quantitative polymerase chain reaction analyses revealed 5-HT1F receptor expression in renal proximal tubule cells (RPTC). A MB screening assay demonstrated that two selective 5-HT1F receptor agonists, LY334370 (4-fluoro-N-[3-(1-methyl-4-piperidinyl)-1H-indol-5-yl]benzamide) and LY344864 (N-[(3R)-3-(dimethylamino)-2,3,4,9-tetrahydro-1H-carbazol-6-yl]-4-fluorobenzamide; 1-100 nM) increased carbonylcyanide-p-trifluoromethoxyphenylhydrazone-uncoupled oxygen consumption in RPTC, and validation studies confirmed both agonists increased mitochondrial proteins [e.g., ATP synthase ß, cytochrome c oxidase 1 (Cox1), and NADH dehydrogenase (ubiquinone) 1ß subcomplex subunit 8 (NDUFB8)] in vitro. Small interfering RNA knockdown of the 5-HT1F receptor blocked agonist-induced MB. Furthermore, LY344864 increased peroxisome proliferator-activated receptor coactivator 1-α, Cox1, and NDUFB8 transcript levels and mitochondrial DNA (mtDNA) copy number in murine renal cortex, heart, and liver. Finally, LY344864 accelerated recovery of renal function, as indicated by decreased blood urea nitrogen and kidney injury molecule 1 and increased mtDNA copy number following ischemia/reperfusion-induced acute kidney injury (AKI). In summary, these studies reveal that the 5-HT1F receptor is linked to MB, 5-HT1F receptor agonism promotes MB in vitro and in vivo, and 5-HT1F receptor agonism promotes recovery from AKI injury. Induction of MB through 5-HT1F receptor agonism represents a new target and approach to treat mitochondrial organ dysfunction.