Stromal androgen and hedgehog signaling regulates stem cell niches in pubertal prostate development.

Stromal androgen-receptor (AR) action is essential for prostate development, morphogenesis and regeneration. However, mechanisms underlying how stromal AR maintains the cell niche in support of pubertal prostatic epithelial growth are unknown. Here, using advanced mouse genetic tools, we demonstrate that selective deletion of stromal AR expression in prepubescent Shh-responsive Gli1-expressing cells significantly impedes pubertal prostate epithelial growth and development. Single-cell transcriptomic analyses showed that AR loss in these prepubescent Gli1-expressing cells dysregulates androgen signaling-initiated stromal-epithelial paracrine interactions, leading to growth retardation of pubertal prostate epithelia and significant development defects. Specifically, AR loss elevates Shh-signaling activation in both prostatic stromal and adjacent epithelial cells, directly inhibiting prostatic epithelial growth. Single-cell trajectory analyses further identified aberrant differentiation fates of prostatic epithelial cells directly altered by stromal AR deletion. In vivo recombination of AR-deficient stromal Gli1-lineage cells with wild-type prostatic epithelial cells failed to develop normal prostatic epithelia. These data demonstrate previously unidentified mechanisms underlying how stromal AR-signaling facilitates Shh-mediated cell niches in pubertal prostatic epithelial growth and development.

Antagonizing CD105 and androgen receptor to target stromal-epithelial interactions for clinical benefit.

Androgen receptor signaling inhibitors (ARSIs) are standard of care for advanced prostate cancer (PCa) patients. Eventual resistance to ARSIs can include the expression of androgen receptor (AR) splice variant, AR-V7, expression as a recognized means of ligand-independent androgen signaling. We demonstrated that interleukin (IL)-6-mediated AR-V7 expression requires bone morphogenic protein (BMP) and CD105 receptor activity in both PCa and associated fibroblasts. Chromatin immunoprecipitation supported CD105-dependent ID1- and E2F-mediated expression of RBM38. Further, RNA immune precipitation demonstrated RBM38 binds the AR-cryptic exon 3 to enable AR-V7 generation. The forced expression of AR-V7 by primary prostatic fibroblasts diminished PCa sensitivity to ARSI. Conversely, downregulation of AR-V7 expression in cancer epithelia and associated fibroblasts was achieved by a CD105-neutralizing antibody, carotuximab. These compelling pre-clinical findings initiated an interventional study in PCa patients developing ARSI resistance. The combination of carotuximab and ARSI (i.e., enzalutamide or abiraterone) provided disease stabilization in four of nine assessable ARSI-refractory patients. Circulating tumor cell evaluation showed AR-V7 downregulation in the responsive subjects on combination treatment and revealed a three-gene panel that was predictive of response. The systemic antagonism of BMP/CD105 signaling can support ARSI re-sensitization in pre-clinical models and subjects that have otherwise developed resistance due to AR-V7 expression.

CX3CR1+ Macrophage Facilitates the Resolution of Allergic Lung Inflammation via Interacting CCL26.

Rationale: The resolution of inflammation is an active process coordinated by mediators and immune cells to restore tissue homeostasis. However, the mechanisms for resolving eosinophilic allergic lung inflammation triggered by inhaled allergens have not been fully elucidated. Objectives: Our objectives were to investigate the cellular mechanism of tissue-resident macrophages involved in the resolution process of eosinophilic lung inflammation. Methods: For the study, we used the institutional review board-approved protocol for human subsegmental bronchoprovocation with allergen, mouse models for allergic lung inflammation, and novel transgenic mice, including a conditional CCL26 knockout. The samples were analyzed using mass cytometry, single-cell RNA sequencing, and biophysical and immunological analyses. Measurements and Main Results: We compared alveolar macrophage (AM) subsets in the BAL before and after allergen provocation. In response to provocation with inhaled allergens, the subsets of AMs are dynamically changed in humans and mice. In the steady state, the AM subset expressing CX3CR1 is a relatively small fraction in bronchoalveolar space and lung tissue but drastically increases after allergen challenges. This subset presents unique patterns of gene expression compared with classical AMs, expressing high C1q family genes. CX3CR1+ macrophages are activated by airway epithelial cell-derived CCL26 via a receptor-ligand interaction. The binding of CCL26 to the CX3CR1+ receptor induces CX3CR1+ macrophages to secrete C1q, subsequently facilitating the clearance of eosinophils. Furthermore, the depletion of CX3CR1 macrophages or CCL26 in airway epithelial cells delays the resolution of allergic lung inflammation displaying prolonged tissue eosinophilia. Conclusions: These findings indicate that the CCL26-CX3CR1 pathway is pivotal in resolving eosinophilic allergic lung inflammation.

Complement C1q essential for aeroallergen sensitization via CSF1R+ conventional dendritic cells type 2.

BACKGROUND: Dendritic cells (DCs) are heterogeneous, comprising multiple subsets with unique functional specifications. Our previous work has demonstrated that the specific conventional type 2 DC subset, CSF1R+cDC2s, plays a critical role in sensing aeroallergens. OBJECTIVE: It remains to be understood how CSF1R+cDC2s recognize inhaled allergens. We sought to elucidate the transcriptomic programs and receptor-ligand interactions essential for function of this subset in allergen sensitization. METHODS: We applied single-cell RNA sequencing to mouse lung DCs. Conventional DC-selective knockout mouse models were employed, and mice were subjected to inhaled allergen sensitization with multiple readouts of asthma pathology. Under the clinical arm of this work, human lung transcriptomic data were integrated with mouse data, and bronchoalveolar lavage (BAL) specimens were collected from subjects undergoing allergen provocation, with samples assayed for C1q. RESULTS: We found that C1q is selectively enriched in lung CSF1R+cDC2s, but not in other lung cDC2 or cDC1 subsets. Depletion of C1q in conventional DCs significantly attenuates allergen sensing and features of asthma. Additionally, we found that C1q binds directly to human dust mite allergen, and the C1q receptor CD91 (LRP1) is required for lung CSF1R+cDC2s to recognize the C1q-allergen complex and induce allergic lung inflammation. Lastly, C1q is enriched in human BAL samples following subsegmental allergen challenge, and human RNA sequencing data demonstrate close homology between lung IGSF21+DCs and mouse CSF1R+cDC2s. CONCLUSIONS: C1q is secreted from the CSF1R+cDC2 subset among conventional DCs. Our data indicate that the C1q-LRP1 axis represents a candidate for translational therapeutics in the prevention and suppression of allergic lung inflammation.

Carboplatin enhances lymphocyte-endothelial interactions to promote CD8+ T cell trafficking into the ovarian tumor microenvironment.

OBJECTIVES: Standard chemotherapy agents, including carboplatin, have known immunogenic properties. We sought to determine how carboplatin may influence lymphocyte trafficking to tumor sites. METHODS: Murine models of ovarian cancer were utilized to examine lymphocyte trafficking with common clinically used agents including carboplatin, anti-PD-1 antibody, or anti-VEGFR-2 antibody. Adhesion interactions of lymphocytes with tumor vasculature were measured using intravital microscopy, lymphocyte homing with immunohistochemistry, and treatment groups followed for overall survival. RESULTS: Carboplatin chemotherapy profoundly alters the tumor microenvironment to promote lymphocyte adhesive interactions with tumor vasculature and resultant improvement in lymphocyte trafficking. The measured results seen with carboplatin in the tumor microenvironment were superior to anti-PD-1 treatment or anti-VEGFR-2 which may have contributed to increased overall survival in carboplatin treated groups. CONCLUSIONS: These novel findings suggest a role for chemotherapeutic agents to broadly influence anti-tumor immune responses beyond the induction of immunogenic tumor cell death.

Aging gene signature of memory CD8+ T cells is associated with neurocognitive functioning in Alzheimer's disease.

BACKGROUND: Memory CD8+ T cells expand with age. We previously demonstrated an age-associated expansion of effector memory (EM) CD8+ T cells expressing low levels of IL-7 receptor alpha (IL-7Rαlow) and the presence of its gene signature (i.e., IL-7Rαlow aging genes) in peripheral blood of older adults without Alzheimer's disease (AD). Considering age as the strongest risk factor for AD and the recent finding of EM CD8+ T cell expansion, mostly IL-7Rαlow cells, in AD, we investigated whether subjects with AD have alterations in IL-7Rαlow aging gene signature, especially in relation to genes possibly associated with AD and disease severity. RESULTS: We identified a set of 29 candidate genes (i.e., putative AD genes) which could be differentially expressed in peripheral blood of patients with AD through the systematic search of publicly available datasets. Of the 29 putative AD genes, 9 genes (31%) were IL-7Rαlow aging genes (P < 0.001), suggesting the possible implication of IL-7Rαlow aging genes in AD. These findings were validated by RT-qPCR analysis of 40 genes, including 29 putative AD genes, additional 9 top IL-7R⍺low aging but not the putative AD genes, and 2 inflammatory control genes in peripheral blood of cognitively normal persons (CN, 38 subjects) and patients with AD (40 mild cognitive impairment and 43 dementia subjects). The RT-qPCR results showed 8 differentially expressed genes between AD and CN groups; five (62.5%) of which were top IL-7Rαlow aging genes (FGFBP2, GZMH, NUAK1, PRSS23, TGFBR3) not previously reported to be altered in AD. Unbiased clustering analysis revealed 3 clusters of dementia patients with distinct expression levels of the 40 analyzed genes, including IL-7Rαlow aging genes, which were associated with neurocognitive function as determined by MoCA, CDRsob and neuropsychological testing. CONCLUSIONS: We report differential expression of "normal" aging genes associated with IL-7Rαlow EM CD8+ T cells in peripheral blood of patients with AD, and the significance of such gene expression in clustering subjects with dementia due to AD into groups with different levels of cognitive functioning. These results provide a platform for studies investigating the possible implications of age-related immune changes, including those associated with CD8+ T cells, in AD.

Risk stratification models for predicting preventable hospitalization in commercially insured late middle-aged adults with depression.

BACKGROUND: A significant number of late middle-aged adults with depression have a high illness burden resulting from chronic conditions which put them at high risk of hospitalization. Many late middle-aged adults are covered by commercial health insurance, but such insurance claims have not been used to identify the risk of hospitalization in individuals with depression. In the present study, we developed and validated a non-proprietary model to identify late middle-aged adults with depression at risk for hospitalization, using machine learning methods. METHODS: This retrospective cohort study involved 71,682 commercially insured older adults aged 55-64 years diagnosed with depression. National health insurance claims were used to capture demographics, health care utilization, and health status during the base year. Health status was captured using 70 chronic health conditions, and 46 mental health conditions. The outcomes were 1- and 2-year preventable hospitalization. For each of our two outcomes, we evaluated seven modelling approaches: four prediction models utilized logistic regression with different combinations of predictors to evaluate the relative contribution of each group of variables, and three prediction models utilized machine learning approaches - logistic regression with LASSO penalty, random forests (RF), and gradient boosting machine (GBM). RESULTS: Our predictive model for 1-year hospitalization achieved an AUC of 0.803, with a sensitivity of 72% and a specificity of 76% under the optimum threshold of 0.463, and our predictive model for 2-year hospitalization achieved an AUC of 0.793, with a sensitivity of 76% and a specificity of 71% under the optimum threshold of 0.452. For predicting both 1-year and 2-year risk of preventable hospitalization, our best performing models utilized the machine learning approach of logistic regression with LASSO penalty which outperformed more black-box machine learning models like RF and GBM. CONCLUSIONS: Our study demonstrates the feasibility of identifying depressed middle-aged adults at higher risk of future hospitalization due to burden of chronic illnesses using basic demographic information and diagnosis codes recorded in health insurance claims. Identifying this population may assist health care planners in developing effective screening strategies and management approaches and in efficient allocation of public healthcare resources as this population transitions to publicly funded healthcare programs, e.g., Medicare in the US.

Stratifying non-small cell lung cancer patients using an inverse of the treatment decision rules: validation using electronic health records with application to an administrative database.

BACKGROUND: To validate a stratification method using an inverse of treatment decision rules that can classify non-small cell lung cancer (NSCLC) patients in real-world treatment records. METHODS: (1) To validate the index classifier against the TNM 7th edition, we analyzed electronic health records of NSCLC patients diagnosed from 2011 to 2015 in a tertiary referral hospital in Seoul, Korea. Predictive accuracy, stage-specific sensitivity, specificity, positive predictive value, negative predictive value, F1 score, and c-statistic were measured. (2) To apply the index classifier in an administrative database, we analyzed NSCLC patients in Korean National Health Insurance Database, 2002-2013. Differential survival rates among the classes were examined with the log-rank test, and class-specific survival rates were compared with the reference survival rates. RESULTS: (1) In the validation study (N = 1375), the overall accuracy was 93.8% (95% CI: 92.5-95.0%). Stage-specific c-statistic was the highest for stage I (0.97, 95% CI: 0.96-0.98) and the lowest for stage III (0.82, 95% CI: 0.77-0.87). (2) In the application study (N = 71,593), the index classifier showed a tendency for differentiating survival probabilities among classes. Compared to the reference TNM survival rates, the index classification under-estimated the survival probability for stages IA, IIIB, and IV, and over-estimated it for stages IIA and IIB. CONCLUSION: The inverse of the treatment decision rules has a potential to supplement a routinely collected database with information encoded in the treatment decision rules to classify NSCLC patients. It requires further validation and replication in multiple clinical settings.

Circulating Tumor Cell-Based Messenger RNA Scoring System for Prognostication of Hepatocellular Carcinoma: Translating Tissue-Based Messenger RNA Profiling Into a Noninvasive Setting.

Numerous studies in hepatocellular carcinoma (HCC) have proposed tissue-based gene signatures for individualized prognostic assessments. Here, we develop a novel circulating tumor cell (CTC)-based transcriptomic profiling assay to translate tissue-based messenger RNA (mRNA) signatures into a liquid biopsy setting for noninvasive HCC prognostication. The HCC-CTC mRNA scoring system combines the NanoVelcro CTC Assay for enriching HCC CTCs and the NanoString nCounter platform for quantifying the HCC-CTC Risk Score (RS) panel in enriched HCC CTCs. The prognostic role of the HCC-CTC RS was assessed in The Cancer Genome Atlas (TCGA) HCC cohort (n = 362) and validated in an independent clinical CTC cohort (n = 40). The HCC-CTC RS panel was developed through our integrated data analysis framework of 8 HCC tissue-based gene signatures and identified the top 10 prognostic genes (discoidin domain receptor tyrosine kinase 1 [DDR1], enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase [EHHADH], androgen receptor [AR], lumican [LUM], hydroxysteroid 17-beta dehydrogenase 6[HSD17B6], prostate transmembrane protein, androgen induced 1 [PMEPA1], tsukushi, small leucine rich proteoglycan [TSKU], N-terminal EF-hand calcium binding protein 2 [NECAB2], ladinin 1 [LAD1], solute carrier family 27 member 5 [SLC27A5]) highly expressed in HCC with low expressions in white blood cells. The panel accurately discriminated overall survival in TCGA HCC cohort (hazard ratio [HR], 2.0; 95% confidence interval [CI], 1.4-2.9). The combined use of the scoring system and HCC-CTC RS panel successfully distinguished artificial blood samples spiked with an aggressive HCC cell type, SNU-387, from those spiked with PLC/PRF/5 cells (P = 0.02). In the CTC validation cohort (n = 40), HCC-CTC RS remained an independent predictor of survival (HR, 5.7; 95% CI, 1.5-21.3; P = 0.009) after controlling for Model for End-Stage Liver Disease score, Barcelona Clinic Liver Cancer stage, and CTC enumeration count. Our study demonstrates a novel interdisciplinary approach to translate tissue-based gene signatures into a liquid biopsy setting. This noninvasive approach will allow real-time disease profiling and dynamic prognostication of HCC.

Chromosomal instability in untreated primary prostate cancer as an indicator of metastatic potential.

BACKGROUND: Metastatic prostate cancer (PC) is highly lethal. The ability to identify primary tumors capable of dissemination is an unmet need in the quest to understand lethal biology and improve patient outcomes. Previous studies have linked chromosomal instability (CIN), which generates aneuploidy following chromosomal missegregation during mitosis, to PC progression. Evidence of CIN includes broad copy number alterations (CNAs) spanning > 300 base pairs of DNA, which may also be measured via RNA expression signatures associated with CNA frequency. Signatures of CIN in metastatic PC, however, have not been interrogated or well defined. We examined a published 70-gene CIN signature (CIN70) in untreated and castration-resistant prostate cancer (CRPC) cohorts from The Cancer Genome Atlas (TCGA) and previously published reports. We also performed transcriptome and CNA analysis in a unique cohort of untreated primary tumors collected from diagnostic prostate needle biopsies (PNBX) of localized (M0) and metastatic (M1) cases to determine if CIN was linked to clinical stage and outcome. METHODS: PNBX were collected from 99 patients treated in the VA Greater Los Angeles (GLA-VA) Healthcare System between 2000 and 2016. Total RNA was extracted from high-grade cancer areas in PNBX cores, followed by RNA sequencing and/or copy number analysis using OncoScan. Multivariate logistic regression analyses permitted calculation of odds ratios for CIN status (high versus low) in an expanded GLA-VA PNBX cohort (n = 121). RESULTS: The CIN70 signature was significantly enriched in primary tumors and CRPC metastases from M1 PC cases. An intersection of gene signatures comprised of differentially expressed genes (DEGs) generated through comparison of M1 versus M0 PNBX and primary CRPC tumors versus metastases revealed a 157-gene "metastasis" signature that was further distilled to 7-genes (PC-CIN) regulating centrosomes, chromosomal segregation, and mitotic spindle assembly. High PC-CIN scores correlated with CRPC, PC-death and all-cause mortality in the expanded GLA-VA PNBX cohort. Interestingly, approximately 1/3 of M1 PNBX cases exhibited low CIN, illuminating differential pathways of lethal PC progression. CONCLUSIONS: Measuring CIN in PNBX by transcriptome profiling is feasible, and the PC-CIN signature may identify patients with a high risk of lethal progression at the time of diagnosis.

A Translational Hepatic Artery Infusion (HAI) Model for Hepatocellular Carcinoma in Woodchucks.

BACKGROUND: Woodchucks (Marmota monax) are a well-accepted animal model for the investigation of spontaneous hepatocellular carcinoma (HCC). As HCC tumors obtain nutrient blood supply exclusively from the hepatic artery, hepatic artery infusion (HAI) has been applied to HCC. However, there is a scarcity of experimental animal models to standardize drug regimens and examine novel agents. The purpose of this study was to establish an HAI model in woodchucks. MATERIALS AND METHODS: HAI ports were placed in the gastroduodenal artery (GDA) of 11 woodchucks. The ports were infused with either a vehicle (dextrose 5% in water) or an experimental drug, CBL0137, once a week for 3 wk. Technical success rates, anatomical variation, morbidity and mortality, and tumor responses between groups were analyzed. RESULTS: The GDA access was feasible and reproducible in all woodchucks (11/11). The average operation time was 95 ± 20 min with no increase in the levels of liver enzymes detected from either infusate. The most common morbidity of CBL0137 therapy was anorexia after surgery. One woodchuck died due to hemorrhage at the gallbladder removal site from hepatic coagulopathy. Significantly higher CBL0137 concentrations were measured in the liver compared with blood after each HAI. Tumor growth was suppressed after multiple CBL0137 HAI treatments which corresponded to greater T cell infiltration and increased tumor cell apoptosis. CONCLUSIONS: HAI via GDA was a feasible and reproducible approach with low morbidity and mortality in woodchucks. The described techniques serve as a reliable platform for the identification and characterization of therapeutics for HCC.

Downregulation of CENPF Remodels Prostate Cancer Cells and Alters Cellular Metabolism.

Metabolic alterations in prostate cancer (PC) are associated with progression and aggressiveness. However, the underlying mechanisms behind PC metabolic functions are unknown. The authors' group recently reported on the important role of centromere protein F (CENPF), a protein associated with the centromere-kinetochore complex and chromosomal segregation during mitosis, in PC MRI visibility. This study focuses on discerning the role of CENPF in metabolic perturbation in human PC3 cells. A series of bioinformatics analyses shows that CENPF is one gene that is strongly associated with aggressive PC and that its expression is positively correlated with metastasis. By identifying and reconstructing the CENPF network, additional associations with lipid regulation are found. Further untargeted metabolomics analysis using gas chromatography-time-of-flight-mass spectrometry reveals that silencing of CENPF alters the global metabolic profiles of PC cells and inhibits cell proliferation, which suggests that CENPF may be a critical regulator of PC metabolism. These findings provide useful scientific insights that can be applied in future studies investigating potential targets for PC treatment.

Quantitative Proteomic Analysis Reveals Caffeine-Perturbed Proteomic Profiles in Normal Bladder Epithelial Cells.

Lower urinary tract symptoms (LUTSs) are highly prevalent among the elderly and negatively impact quality of life. Since caffeinated beverages are enjoyed worldwide and the relationship between LUTS and caffeine is still not fully understood, it would be of particular interest to examine the underlying mechanisms that drive caffeine's influence on LUTS development and progression. The aim of this study is to characterize the effects of caffeine on hTert-immortalized normal bladder epithelial cells by investigating whether exposure to caffeine can cause potential changes in the bladder proteome and/or biological pathways. In labeled LC-MS/MS proteomic analysis, 57 proteins are found as being differentially expressed in caffeine-treated bladder epithelial cells, compared to controls; this included 32 upregulated and 25 downregulated proteins. Further functional gene enrichment analysis reveals that caffeine affects major biological pathways, including those for "muscle contraction" and "chromatin assembly." These findings provide new scientific insights that may be useful in future studies investigating the role of caffeine in bladder dysfunctions.

Cumulative Dose Threshold for the Chemopreventive Effect of Aspirin Against Gastric Cancer.

OBJECTIVES: Many studies have found evidence that aspirin has protective effects against certain cancers, but quantitative dose-response data have been available only on a limited basis. This study aimed to confirm the dose-response relationship of aspirin usage and gastric cancer and to estimate the cumulative dose threshold of aspirin to achieve protective effects against gastric cancer in the general population. METHODS: A total of 461,489 individuals in a population-based longitudinal cohort provided by the National Health Insurance Services (NHIS) in the Republic of Korea were observed from 2007 to 2012 to identify gastric cancer incident cases. The pharmacy claims data of these individuals from 2002 to 2006 were reviewed to assess cumulative medication exposure using the defined daily dose (DDD) system. Hazard ratios (HRs) of aspirin use for gastric cancer were estimated using multivariate Cox Proportional Hazard regression. Sensitivity analyses, including propensity-score matching and a nested case-control design, were performed to evaluate the variability caused by study design. RESULTS: A total of 5674 incident gastric cancers were identified from 2,965,500 person-years of follow-up observation, giving an overall incidence rate of 191.00 gastric cancers per 100,000 person-years. Compared to non-users, those with aspirin use of ≥3 DDD-years showed a statistically significant protective effect of aspirin use against gastric cancer; the adjusted HR (95% confidence intervals) were 0.79 (0.63-0.98) and 0.63 (0.48-0.83) for those with aspirin use of 3-4 DDD-years and 4-5 DDD-years, respectively (P for trend < 0.001). Sensitivity analyses using propensity-score matching and a nested case-control design consistently showed a chemopreventive effect of aspirin. CONCLUSION: Long-term aspirin use was associated with reduced gastric cancer incidence in the general population of South Korea when the cumulative dose was >3 DDD-years.

Association networks in a matched case-control design - Co-occurrence patterns of preexisting chronic medical conditions in patients with major depression versus their matched controls.

OBJECTIVE: We present a method for comparing association networks in a matched case-control design, which provides a high-level comparison of co-occurrence patterns of features after adjusting for confounding factors. We demonstrate this approach by examining the differential distribution of chronic medical conditions in patients with major depressive disorder (MDD) compared to the distribution of these conditions in their matched controls. MATERIALS AND METHODS: Newly diagnosed MDD patients were matched to controls based on their demographic characteristics, socioeconomic status, place of residence, and healthcare service utilization in the Korean National Health Insurance Service's National Sample Cohort. Differences in the networks of chronic medical conditions in newly diagnosed MDD cases treated with antidepressants, and their matched controls, were prioritized with a permutation test accounting for the false discovery rate. Sensitivity analyses for the associations between prioritized pairs of chronic medical conditions and new MDD diagnosis were performed with regression modeling. RESULTS: By comparing the association networks of chronic medical conditions in newly diagnosed depression patients and their matched controls, five pairs of such conditions were prioritized among 105 possible pairs after controlling the false discovery rate at 5%. In sensitivity analyses using regression modeling, four out of the five prioritized pairs were statistically significant for the interaction terms. CONCLUSION: Association networks in a matched case-control design can provide a high-level comparison of comorbid features after adjusting for confounding factors, thereby supplementing traditional clinical study approaches. We demonstrate the differential co-occurrence pattern of chronic medical conditions in patients with MDD and prioritize the chronic conditions that have statistically significant interactions in regression models for depression.

Negative regulation of hypoxic responses via induced Reptin methylation.

Lysine methylation within histones is crucial for transcriptional regulation and thus links chromatin states to biological outcomes. Although recent studies have extended lysine methylation to nonhistone proteins, underlying molecular mechanisms such as the upstream signaling cascade that induces lysine methylation and downstream target genes modulated by this modification have not been elucidated. Here, we show that Reptin, a chromatin-remodeling factor, is methylated at lysine 67 in hypoxic conditions by the methyltransferase G9a. Methylated Reptin binds to the promoters of a subset of hypoxia-responsive genes and negatively regulates transcription of these genes to modulate cellular responses to hypoxia.

Novel mouse models of hepatic artery infusion.

BACKGROUND: The liver has unique anatomy in that most blood flow to normal hepatocytes is derived from the portal venous system, whereas liver tumors obtain their nutrient blood supply exclusively from the hepatic artery. The focused arterial delivery of anticancer agents to liver tumors has been performed for decades; however, preclinical models to standardize drug regimens and examine novel agents have been lacking. The purpose of this study was to establish preclinical hepatic artery infusion (HAI) models in a mouse and to evaluate the safety and delivery capability of the models. MATERIAL AND METHODS: C57BL/6 and BALB/c mice were used to develop models of HAI via the hepatic artery (HA), superior pancreaticoduodenal artery (SPDA), or lienogastric artery (LGA). Success rates, distribution of perfusion, and associated morbidity and mortality were analyzed between groups. RESULTS: All three models were feasible and reproducible in mice, and there was no statistical difference on body weight change between models. The HA model had a 13.3% mortality from acute liver failure, and the SPDA model demonstrated duodenal and pancreatic toxicity. SPDA and LGA routes had the highest success rates (96.7% and 91.4%, respectively) with low mortality, better drug delivery, and preserved physiologic liver function compared with the HA model. CONCLUSIONS: The optimal route of HAI was mouse breed specific; SPDA access in BALB/c mice, and the LGA access in C57BL/6 mice. The described techniques serve as a reproducible platform for the identification and characterization of therapeutics for diverse metastatic liver tumors.

ADP-ribosylation factor 6 (ARF6) bidirectionally regulates dendritic spine formation depending on neuronal maturation and activity.

Recent studies have reported conflicting results regarding the role of ARF6 in dendritic spine development, but no clear answer for the controversy has been suggested. We found that ADP-ribosylation factor 6 (ARF6) either positively or negatively regulates dendritic spine formation depending on neuronal maturation and activity. ARF6 activation increased the spine formation in developing neurons, whereas it decreased spine density in mature neurons. Genome-wide microarray analysis revealed that ARF6 activation in each stage leads to opposite patterns of expression of a subset of genes that are involved in neuronal morphology. ARF6-mediated Rac1 activation via the phospholipase D pathway is the coincident factor in both stages, but the antagonistic RhoA pathway becomes involved in the mature stage. Furthermore, blocking neuronal activity in developing neurons using tetrodotoxin or enhancing the activity in mature neurons using picrotoxin or chemical long term potentiation reversed the effect of ARF6 on each stage. Thus, activity-dependent dynamic changes in ARF6-mediated spine structures may play a role in structural plasticity of mature neurons.

A murine model of targeted infusion for intracranial tumors.

Historically, intra-arterial (IA) drug administration for malignant brain tumors including glioblastoma multiforme (GBM) was performed as an attempt to improve drug delivery. With the advent of percutaneous neuorovascular techniques and modern microcatheters, intracranial drug delivery is readily feasible; however, the question remains whether IA administration is safe and more effective compared to other delivery modalities such as intravenous (IV) or oral administrations. Preclinical large animal models allow for comparisons between treatment routes and to test novel agents, but can be expensive and difficult to generate large numbers and rapid results. Accordingly, we developed a murine model of IA drug delivery for GBM that is reproducible with clear readouts of tumor response and neurotoxicities. Herein, we describe a novel mouse model of IA drug delivery accessing the internal carotid artery to treat ipsilateral implanted GBM tumors that is consistent and reproducible with minimal experience. The intent of establishing this unique platform is to efficiently interrogate targeted anti-tumor agents that may be designed to take advantage of a directed, regional therapy approach for brain tumors.

BMP9 Induces Cord Blood-Derived Endothelial Progenitor Cell Differentiation and Ischemic Neovascularization via ALK1.

OBJECTIVE: Modulating endothelial progenitor cells (EPCs) is essential for therapeutic angiogenesis, and thus various clinical trials involving EPCs are ongoing. However, the identification of environmental conditions and development of optimal methods are required to accelerate EPC-driven vasculogenesis. APPROACH AND RESULTS: We evaluated gene expression profiles of cord blood-derived EPCs and endothelial cells to identify the key factors in EPC→endothelial cell differentiation and to show that transforming growth factor-ß family members contribute to EPC differentiation. The expression levels of activin receptor-like kinase 1 (ALK1) and its high-affinity ligand, bone morphogenetic protein 9 (BMP9) were markedly changed in EPC→endothelial cell differentiation. Interestingly, BMP9 induced EPC→endothelial cell differentiation and EPC incorporation into vessel-like structures by acting on ALK1 expressed on EPCs in vitro. BMP9 also induced neovascularization in mice with hindlimb ischemia by increasing vessel formation and the incorporation of EPCs into vessels. Conversely, neovascularization was impaired when ALK1 signaling was blocked. Furthermore, EPCs exposed to either short- or long-term BMP9 stimulation demonstrated these functions in EPC-mediated neovascularization. CONCLUSIONS: Collectively, our results indicated that BMP9/ALK1 augmented vasculogenesis and angiogenesis, and thereby enhanced neovascularization. Thus, we suggest that BMP9/ALK1 may improve the efficacy of EPC-based therapies for treating ischemic diseases.