Macrophage lineages in heart valve development and disease.

Heterogeneous macrophage lineages are present in the aortic and mitral valves of the heart during development and disease. These populations include resident macrophages of embryonic origins and recruited monocyte-derived macrophages prevalent in disease. Soon after birth, macrophages from haematopoietic lineages are recruited to the heart valves, and bone marrow transplantation studies in mice demonstrate that haematopoietic-derived macrophages continue to invest adult valves. During myxomatous heart valve disease, monocyte-derived macrophages are recruited to the heart valves and they contribute to valve degeneration in a mouse model of Marfan syndrome. Here, we review recent studies of macrophage lineages in heart valve development and disease with discussion of clinical significance and therapeutic applications.

Deficiency of Circulating Monocytes Ameliorates the Progression of Myxomatous Valve Degeneration in Marfan Syndrome.

BACKGROUND: Myxomatous valve degeneration (MVD) involves the progressive thickening and degeneration of the heart valves, leading to valve prolapse, regurgitant blood flow, and impaired cardiac function. Leukocytes composed primarily of macrophages have recently been detected in myxomatous valves, but the timing of the presence and the contributions of these cells in MVD progression are not known. METHODS: We examined MVD progression, macrophages, and the valve microenvironment in the context of Marfan syndrome (MFS) using mitral valves from MFS mice (Fbn1C1039G/+), gene-edited MFS pigs (FBN1Glu433AsnfsX98/+), and patients with MFS. Additional histological and transcriptomic evaluation was performed by using nonsyndromic human and canine myxomatous valves, respectively. Macrophage ontogeny was determined using MFS mice transplanted with mTomato+ bone marrow or MFS mice harboring RFP (red fluorescent protein)-tagged C-C chemokine receptor type 2 (CCR2) monocytes. Mice deficient in recruited macrophages (Fbn1C1039G/+;Ccr2RFP/RFP) were generated to determine the requirements of recruited macrophages to MVD progression. RESULTS: MFS mice recapitulated histopathological features of myxomatous valve disease by 2 months of age, including mitral valve thickening, increased leaflet cellularity, and extracellular matrix abnormalities characterized by proteoglycan accumulation and collagen fragmentation. Diseased mitral valves of MFS mice concurrently exhibited a marked increase of infiltrating (MHCII+, CCR2+) and resident macrophages (CD206+, CCR2-), along with increased chemokine activity and inflammatory extracellular matrix modification. Likewise, mitral valve specimens obtained from gene-edited MFS pigs and human patients with MFS exhibited increased monocytes and macrophages (CD14+, CD64+, CD68+, CD163+) detected by immunofluorescence. In addition, comparative transcriptomic evaluation of both genetic (MFS mice) and acquired forms of MVD (humans and dogs) unveiled a shared upregulated inflammatory response in diseased valves. Remarkably, the deficiency of monocytes was protective against MVD progression, resulting in a significant reduction of MHCII macrophages, minimal leaflet thickening, and preserved mitral valve integrity. CONCLUSIONS: All together, our results suggest sterile inflammation as a novel paradigm to disease progression, and we identify, for the first time, monocytes as a viable candidate for targeted therapy in MVD.

Mutations in RABL3 alter KRAS prenylation and are associated with hereditary pancreatic cancer.

Pancreatic ductal adenocarcinoma is an aggressive cancer with limited treatment options1. Approximately 10% of cases exhibit familial predisposition, but causative genes are not known in most families2. We perform whole-genome sequence analysis in a family with multiple cases of pancreatic ductal adenocarcinoma and identify a germline truncating mutation in the member of the RAS oncogene family-like 3 (RABL3) gene. Heterozygous rabl3 mutant zebrafish show increased susceptibility to cancer formation. Transcriptomic and mass spectrometry approaches implicate RABL3 in RAS pathway regulation and identify an interaction with RAP1GDS1 (SmgGDS), a chaperone regulating prenylation of RAS GTPases3. Indeed, the truncated mutant RABL3 protein accelerates KRAS prenylation and requires RAS proteins to promote cell proliferation. Finally, evidence in patient cohorts with developmental disorders implicates germline RABL3 mutations in RASopathy syndromes. Our studies identify RABL3 mutations as a target for genetic testing in cancer families and uncover a mechanism for dysregulated RAS activity in development and cancer.

Endothelial Cell Lineage Analysis Does Not Provide Evidence for EMT in Adult Valve Homeostasis and Disease.

Epithelial-to-mesenchymal transition (EMT) enables stationary epithelial cells to exhibit migratory behavior and is the key step that initiates heart valve development. Recent studies suggest that EMT is reactivated in the pathogenesis of myxomatous valve disease (MVD), a condition that involves the progressive degeneration and thickening of valve leaflets. These studies have been limited to in vitro experimentation and reliance on histologic costaining of epithelial and mesenchymal markers as evidence of EMT in mouse and sheep models of valve disease. However, longitudinal analysis of cell lineage origins and potential pathogenic or reparative contributions of newly generated mesenchymal cells have not been reported previously. In this study, a genetic lineage tracing strategy was pursued by irreversibly labeling valve endothelial cells in the Osteogenesis imperfecta and Marfan syndrome mouse models to determine whether they undergo EMT during valve disease. Tie2-CreER T2 and Cdh5(PAC)-CreER T2 mouse lines were used in combination with colorimetric and fluorescent reporters for longitudinal assessment of endothelial cells. These lineage tracing experiments showed no evidence of EMT during adult valve homeostasis or valve pathogenesis. Additionally, CD31 and smooth muscle α-actin (αSMA) double-positive cells, used as an indicator of EMT, were not detected, and levels of EMT transcription factors were not altered. Interestingly, contrary to the endothelial cell-specific Cdh5(PAC)-CreER T2 driver line, Tie2-CreER T2 lineage-derived cells in diseased heart valves also included CD45+ leukocytes. Altogether, our data indicate that EMT is not a feature of valve homeostasis and disease but that increased immune cells may contribute to MVD. Anat Rec, 302:125-135, 2019. © 2018 Wiley Periodicals, Inc.

Macrophage Transitions in Heart Valve Development and Myxomatous Valve Disease.

OBJECTIVE: Hematopoietic-derived cells have been reported in heart valves but remain poorly characterized. Interestingly, recent studies reveal infiltration of leukocytes and increased macrophages in human myxomatous mitral valves. Nevertheless, timing and contribution of macrophages in normal valves and myxomatous valve disease are still unknown. The objective is to characterize leukocytes during postnatal heart valve maturation and identify macrophage subsets in myxomatous valve disease. APPROACH AND RESULTS: Leukocytes are detected in heart valves after birth, and their numbers increase during postnatal valve development. Flow cytometry and immunostaining analysis indicate that almost all valve leukocytes are myeloid cells, consisting of at least 2 differentially localized macrophage subsets and dendritic cells. Beginning a week after birth, increased numbers of CCR2+ (C-C chemokine receptor type 2) macrophages are present, consistent with infiltrating populations of monocytes, and macrophages are localized in regions of biomechanical stress in the valve leaflets. Valve leukocytes maintain expression of CD (cluster of differentiation) 45 and do not contribute to significant numbers of endothelial or interstitial cells. Macrophage lineages were examined in aortic and mitral valves of Axin2 KO (knockout) mice that exhibit myxomatous features. Infiltrating CCR2+ monocytes and expansion of CD206-expressing macrophages are localized in regions where modified heavy chain hyaluronan is observed in myxomatous valve leaflets. Similar colocalization of modified hyaluronan and increased numbers of macrophages were observed in human myxomatous valve disease. CONCLUSIONS: Our study demonstrates the heterogeneity of myeloid cells in heart valves and highlights an alteration of macrophage subpopulations, notably an increased presence of infiltrating CCR2+ monocytes and CD206+ macrophages, in myxomatous valve disease.

MRI supersedes ictal EEG when other presurgical data are concordant.

PURPOSE: When ictal EEG is discordant with MRI and other presurgical data, our group has sometimes discounted the ictal findings and proceeded with epilepsy surgical resection based on MRI. We aimed to evaluate the prudence of such practice by comparing the outcome of MRI-lesional epilepsy surgery patients with discordant ictal EEG with those with concordant ictal EEG. METHOD: We retrospectively studied 115 children with epilepsy who underwent surgical resection of an MRI lesion that was corroborated as the epileptogenic focus by other presurgical findings. Ictal findings on video-EEG were categorized as: "positive ictal EEG" if the ictal onset localization was concordant with MRI and other presurgical data; "negative ictal EEG" if the ictus was discordant with them. Seizure-free outcome at 2 years was compared between the "positive" and the "negative" ictal EEG groups. RESULTS: Seizure-free outcome did not differ between children with positive ictal EEG (73%) and those with negative ictal EEG (80%). Positive ictal EEG did not result in better outcome regardless of the location of the surgery or the pathology of the lesion. Ictal EEG with 73% positive predictive value provided no added benefit in this cohort whose seizure-free outcome was of 77% irrespective of ictal EEG findings. CONCLUSIONS: In our selected cohort of pediatric epilepsy surgery patients with an epileptogenic lesion on MRI and concordant other data, ictal EEG had limited predictive value. This calls into question the additive role of ictal recordings in patients with an MRI lesion and concordant other presurgical data.

Seizure Outcomes in Children Following Electrocorticography-Guided Single-Stage Surgical Resection.

BACKGROUND: In children with abnormal imaging, single-stage epilepsy surgery is an attractive alternative to the two-stage approach that relies on invasive recording of seizures. Implanted electrodes carry risks of their own and extend hospitalization, but the efficacy of one-stage resections in a variety of pathologies and cerebral locations is not well established. We report our center's experience with single-stage epilepsy surgery guided by intraoperative electrocorticography (ECoG). METHODS: We retrospectively analyzed 130 consecutive patients who underwent single-stage epilepsy surgery before age 19 years and had at least a two-year follow-up. Intraoperative ECoG was available for review in 113. Patients were considered seizure-free if they were continuously Engel Class I up to the two-year postoperative mark. ECoG findings were classified according to the presence of interictal attenuation, spikes, both, or neither. Complications and hospital length of stay were evaluated. RESULTS: Eighty percent of 130 patients were seizure-free at two years. All but one had an abnormal MRI. Patients with tumor had a better seizure outcome than patients with cortical malformation. Frontal resections had worse outcome, especially among tumors. Intraoperative ECoG revealed both attenuation and spikes in 48%, attenuation only in 23%, spikes only in 20%, and neither in 9%. The complication rate was 6.9%, with no major neurological complications. The average length of stay was 5.7 nights. CONCLUSIONS: With ECoG-guided single-stage surgery, we achieved results comparable with other pediatric surgical series and with a low complication rate. An extensive two-stage approach may not be required when there is a lesion on imaging and other information is concordant, even when the MRI abnormality is subtle and unclearly delineated. Frontal foci may present a challenge because of their proximity to "eloquent" nonresectable cortex or critical structures.

Selenoprotein H is an essential regulator of redox homeostasis that cooperates with p53 in development and tumorigenesis.

Selenium, an essential micronutrient known for its cancer prevention properties, is incorporated into a class of selenocysteine-containing proteins (selenoproteins). Selenoprotein H (SepH) is a recently identified nucleolar oxidoreductase whose function is not well understood. Here we report that seph is an essential gene regulating organ development in zebrafish. Metabolite profiling by targeted LC-MS/MS demonstrated that SepH deficiency impairs redox balance by reducing the levels of ascorbate and methionine, while increasing methionine sulfoxide. Transcriptome analysis revealed that SepH deficiency induces an inflammatory response and activates the p53 pathway. Consequently, loss of seph renders larvae susceptible to oxidative stress and DNA damage. Finally, we demonstrate that seph interacts with p53 deficiency in adulthood to accelerate gastrointestinal tumor development. Overall, our findings establish that seph regulates redox homeostasis and suppresses DNA damage. We hypothesize that SepH deficiency may contribute to the increased cancer risk observed in cohorts with low selenium levels.

Cannabinoid receptor signaling regulates liver development and metabolism.

Endocannabinoid (EC) signaling mediates psychotropic effects and regulates appetite. By contrast, potential roles in organ development and embryonic energy consumption remain unknown. Here, we demonstrate that genetic or chemical inhibition of cannabinoid receptor (Cnr) activity disrupts liver development and metabolic function in zebrafish (Danio rerio), impacting hepatic differentiation, but not endodermal specification: loss of cannabinoid receptor 1 (cnr1) and cnr2 activity leads to smaller livers with fewer hepatocytes, reduced liver-specific gene expression and proliferation. Functional assays reveal abnormal biliary anatomy and lipid handling. Adult cnr2 mutants are susceptible to hepatic steatosis. Metabolomic analysis reveals reduced methionine content in Cnr mutants. Methionine supplementation rescues developmental and metabolic defects in Cnr mutant livers, suggesting a causal relationship between EC signaling, methionine deficiency and impaired liver development. The effect of Cnr on methionine metabolism is regulated by sterol regulatory element-binding transcription factors (Srebfs), as their overexpression rescues Cnr mutant liver phenotypes in a methionine-dependent manner. Our work describes a novel developmental role for EC signaling, whereby Cnr-mediated regulation of Srebfs and methionine metabolism impacts liver development and function.

Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis.

NCOA4 is a selective cargo receptor for the autophagic turnover of ferritin, a process critical for regulation of intracellular iron bioavailability. However, how ferritinophagy flux is controlled and the roles of NCOA4 in iron-dependent processes are poorly understood. Through analysis of the NCOA4-FTH1 interaction, we demonstrate that direct association via a key surface arginine in FTH1 and a C-terminal element in NCOA4 is required for delivery of ferritin to the lysosome via autophagosomes. Moreover, NCOA4 abundance is under dual control via autophagy and the ubiquitin proteasome system. Ubiquitin-dependent NCOA4 turnover is promoted by excess iron and involves an iron-dependent interaction between NCOA4 and the HERC2 ubiquitin ligase. In zebrafish and cultured cells, NCOA4 plays an essential role in erythroid differentiation. This work reveals the molecular nature of the NCOA4-ferritin complex and explains how intracellular iron levels modulate NCOA4-mediated ferritinophagy in cells and in an iron-dependent physiological setting.

Interictal attenuation in pediatric electrocorticography can be reliably detected by EEG readers.

Intraoperative electrocorticography (ECoG) helps to demarcate epileptogenic cortex, but a commonly observed feature, interictal attenuation, has received little attention. This may limit its use in the determination of the resection margin. In order to test how reliably EEGers can discern attenuation, we assessed how well EEGers agree with each other and with an objective, quantified measure of attenuation. ECoG segments (n=34) were evaluated for attenuation by two EEGers independently and in consensus, and by an amplitude spectral analysis-based quantitative method. A third EEGer divided the 34 ECoG segments into 3 subgroups-physiologic field present, physiologic field uncertain, and physiologic field absent-based on the clustering patterns of the attenuated electrodes. Inter-rater agreement between two independent EEGers (kappa=0.56) was moderate, and between consensus EEGers and the quantitative method (kappa=0.71) was substantial. These agreements were especially good among the physiologic field present subgroup where the attenuation clearly involved contiguous electrodes, and thus, more likely pathologic (kappa=0.64 for two independent EEGers and kappa=0.78 for consensus EEGers and quantitative method). Our results suggest that interictal attenuation, especially when involving contiguous electrodes, is an ECoG marker that can be consistently and reliably discerned by trained EEGers.

Identification of retinol binding protein 1 promoter hypermethylation in isocitrate dehydrogenase 1 and 2 mutant gliomas.

BACKGROUND: Mutations in isocitrate dehydrogenase 1 (IDH1) and associated CpG island hypermethylation represent early events in the development of low-grade gliomas and secondary glioblastomas. To identify candidate tumor suppressor genes whose promoter methylation may contribute to gliomagenesis, we compared methylation profiles of IDH1 mutant (MUT) and IDH1 wild-type (WT) tumors using massively parallel reduced representation bisulfite sequencing. METHODS: Reduced representation bisulfite sequencing was performed on ten pathologically matched WT and MUT glioma samples and compared with data from a methylation-sensitive restriction enzyme technique and data from The Cancer Genome Atlas (TCGA). Methylation in the gene retinol-binding protein 1 (RBP1) was identified in IDH1 mutant tumors and further analyzed with primer-based bisulfite sequencing. Correlation between IDH1/IDH2 mutation status and RBP1 methylation was evaluated with Spearman correlation. Survival data were collected retrospectively and analyzed with Kaplan-Meier and Cox proportional hazards analysis. All statistical tests were two-sided. RESULTS: Methylome analysis identified coordinated CpG island hypermethylation in IDH1 MUT gliomas, consistent with previous reports. RBP1, important in retinoic acid metabolism, was found to be hypermethylated in 76 of 79 IDH1 MUT, 3 of 3 IDH2 MUT, and 0 of 116 IDH1/IDH2 WT tumors. IDH1/IDH2 mutation was highly correlated with RBP1 hypermethylation (n = 198; Spearman R = 0.94, 95% confidence interval = 0.92 to 0.95, P < .001). The Cancer Genome Atlas showed IDH1 MUT tumors (n = 23) to be RBP1-hypermethylated with decreased RBP1 expression compared with WT tumors (n = 124). Among patients with primary glioblastoma, patients with RBP1-unmethylated tumors (n = 102) had decreased median overall survival compared with patients with RBP1-methylated tumors (n = 22) (20.3 months vs 36.8 months, respectively; hazard ratio of death = 2.48, 95% confidence interval = 1.30 to 4.75, P = .006). CONCLUSION: RBP1 promoter hypermethylation is found in nearly all IDH1 and IDH2 mutant gliomas and is associated with improved patient survival. Because RBP1 is involved in retinoic acid synthesis, our results suggest that dysregulation of retinoic acid metabolism may contribute to glioma formation along the IDH1/IDH2-mutant pathway.

Chronic cadmium exposure in vitro causes acquisition of multiple tumor cell characteristics in human pancreatic epithelial cells.

BACKGROUND: Cancer may be a stem cell (SC)-based disease involving formation of cancer SCs (CSCs) potentially arising from transformation of normal SCs. Cadmium has been linked to human pancreatic cancer. OBJECTIVE: We studied cadmium exposure of human pancreatic ductal epithelial (HPDE) cells and whether SCs may be targeted in this process. METHODS: We chronically exposed HPDE cells to low level cadmium (1 µM) for ≤ 29 weeks. Nonadherent spheroid formation was used to indicate CSC-like cell production, and we assessed tumor cell characteristics in such spheres. Assessed tumor cell characteristics including secretion of matrix metalloproteinase-9 (MMP-9), invasion, and colony formation were fortified by evaluating expression of relevant genes by real-time reverse transcription polymerase chain reaction and by Western blot. RESULTS: Increased MMP-9 secretion and overexpression of the pancreatic cancer marker S100P occurred in chronic (29 weeks of exposure) cadmium-exposed (CCE) cells. CCE cells also showed markedly higher colony formation and invasion, typical of cancer cells. Floating "spheres" of viable cells, known to contain an abundance of normal SCs or CSCs, form in vitro with many cell types. CCE cells produced 3-fold more spheres than control cells and were more invasive, secreted more MMP-9, and overexpressed markers for pancreatic SCs/CSCs (i.e., CXCR4, OCT4, CD44) and S100P, a marker for pancreatic cancer. CCE-derived spheres rapidly produced aggressive, highly branched, and poorly differentiated glandular-like structures in Matrigel. CONCLUSIONS: Chronic cadmium exposure produced multiple tumor cell characteristics in HPDE cells and CCE cell-derived spheres. These data support the plausibility of cadmium as a human pancreatic carcinogen.