In a prospective study of 66 subjects with or without post- acute coronary syndrome (ACS), plasma concentrations of bioactive CNP and bio-inactive aminoterminal proCNP (NTproCNP), ET-1, B-type Natriuretic Peptide (BNP) and high sensitivity C Reactive Protein (hsCRP) were measured together with lipids before and at intervals of 1, 2 and 7 days after commencing atorvastatin 40 mg/day - and for a further period of 6months in those with ACS. Plasma lipids fell significantly in all subjects but plasma CNP, NTproCNP and ET-1 were unchanged by atorvastatin. In ACS, baseline hsCRP, BNP and CNP but not NTproCNP or ET-1 were significantly raised compared to values in age-matched controls. The ratio of NTproCNP to CNP was significantly lower in ACS throughout the study and was unaffected by statin therapy. We conclude that conventional doses of atorvastatin do not affect plasma CNP products or ET-1. Elevated CNP after cardiac injury likely results from regulated changes in clearance, not enhanced production.C-type Natriuretic Peptide (CNP) and Endothelin-1 (ET-1) have reciprocal roles in maintaining vascular homeostasis and are acutely modulated by statins in human cultured endothelial cells. Whether these actions of statins  In vitro studies suggest that statins may have important beneficial effects on important regulators of vascular structure and function. Among these are the endothelial peptides, C-type Natriuretic Peptide (CNP) and Endothelin-1 (ET-1) both of which are modulated by statins in studies using human vascular endothelial cells in tissue culture3.Statin therapy is widely employed to prevent coronary artery disease and stroke. Statins reduce serum cholesterol and have contributed to reduced morbidity and mortality of cardiovascular disease in the last three decades. Independent of actions on lipids, numerous studies show potentially beneficial (pleiotropic) effects of statins – including anti-inflammatory actions within vascular tissues4. While evidence supports a largely paracrine mode of action of CNP, clinical studies show that products of proCNP in plasma  are increased in settings of vascular stress in both young adults5 and at mid-life6 – possibly as an adaptive response to inflammation and/or shear stress8. These results together with other findings suggest that tissue changes in CNP or ET-1 production9 may be captured by concurrent changes in plasma if studied under well standardised conditions. Since the CNP gene (NPPC) expression is upregulated by statins in at least three different tissues – human umbilical vein endothelial cells10, porcine aortic valve interstitial cells11 and hepatic endothelial cells cultured from cirrhotic rats12, and some of the benefits of statins are lost in the absence of the CNP receptor11, further study of interactions of statins with CNP secretion in humans is clearly needed. Endothelin, a pro-inflammatory product of the vascular endothelium, concomitantly inhibited as NPPC is upregulated by statins3, also deserves closer study especially as the effect of statins on plasma ET-1 is controversial15.CNP is a growth factor expressed in the vascular endothelium wherein anti-inflammatory, anti-proliferative and vaso-dilator actions of the mature peptide have vasoprotective roles by reducing intimal injury16, we hypothesised that in healthy young adults, statins will acutely increase plasma CNP products whereas responses in statin naive older subjects with overt CAD will differ from age-matched controls without cardiovascular disease. We further posited that the responses of CNP (increase) and ET (decline) to statins would be reciprocal.We have examined possible modulation of CNP peptides and ET-1 by statin therapy, as evidenced by change in their circulating levels in a carefully planned prospective study of i) young and older statin naïve subjects without evidence of renal or cardiovascular disease, and ii) older statin naïve subjects with history of a recent coronary occlusive event. Because CNP production from healthy (atheroma protected) endothelial cells in response to shear stress is much greater than from atheroma prone arterial endothelial cellsThe study was completed without any adverse events. Clinical and biochemical data (medians and inter quartile range) at baseline and day 7 are detailed in Table In Group 3 subjects Table , baselinPlasma hsCRP, higher in older than in young healthy subjects (p = 0.02), did not change in either Group 1 or 2 during statin treatment Fig. . Values Examining relationships between CNP products and ET-1, in all subjects at baseline Table  an inverin-vivo study examining the impact of statin therapy on CNP products in any species. In this study circulating levels of CNP products were not significantly modulated by lipid lowering doses of atorvastatin. These findings need to be seen in the context of the many factors contributing to the concentration of plasma CNP products, and the disparate conditions underlying in vitro cellular responses from those acting in vivo. Compelling evidence from cultured human umbilical vein endothelial cells shows that the transcription factor KLF2, upregulated in response to laminar shear stress16 or inflammation1, potently stimulates a range of proangiogenic factors including endothelial NO synthase (eNOS) and CNP while inhibiting endothelin-1 expression3. In cell culture studies over expressing KLF2, CNP was one of the most strongly upregulated genes, and associated with dramatically increased CNP protein levels in the supernatant3. Aligning with these findings, and other evidence of CNP’s athero-protective role17, athero-prone and athero-protective regions of human carotid arterial wall endothelial cells exhibit differential responses to shear stress: Klf2 expression is more than 5 fold and Nppc more than 13 fold enhanced in athero protective regions16. Since many of the pleiotropic effects of statins – including anti-inflammatory actions and upregulation of eNOS gene expression18 – mimic actions of KLF2 in endothelial cells, formal studies of statin effects have been examined using human umbilical vein endothelial cells10. In these studies, dose responsive upregulation of Klf2 by a range of statin drugs was observed after 8 hr and sustained for at least 24 hr. Of the recognised downstream targets of Klf2, upregulation of Nppc was not only the most sensitive (increased at 8 hr) but also the most prominent at 24 hr. Inhibiting Klf2 by siRNA nullified the Nppc response to statin – suggesting that changes in CNP gene expression induced by statin were Klf2 dependent. Collectively these in vitro findings suggest that circulating levels of CNP products may be acutely modulated by statins in humans – and therefore could have applications in drug monitoring as well as providing insight to the integrity of the arterial vasculature10. On the basis of the positive link of Klf2/CNP responses in athero protective tissues, compared to the relative lack of response in athero prone tissue, we further posited that the CNP response to statin would be age dependent, and greater in those free of arterial disease compared to those with a recent coronary ischaemic event. Our findings that circulating concentrations of the stable product of proCNP (NTproCNP) – nor the concentration of authentic CNP – did not change during standard (lipid reducing) doses of atorvastatin for periods of 7 days in healthy subjects, and beyond during an extended period of 6 months in those with CAD, lead us to conclude that in males, doses of the drug used in routine clinical practice do not affect plasma CNP products. Exactly the same conclusion can be drawn from the observations that plasma ET-1 is similarly unchanged by statins. Although responses of lipids to atorvastatin are similar in both sexes19, whether similar findings apply in statin naïve treated females remain to be studied.This is the first First, findings from in vitro cell studies  may not recapitulate physiology20. Although in vivo studies of the molecular events underpinning vascular homeostasis continue to support the important role of Klf220 and/or its downstream targets21, with one exception21 none have explored the in vivo effects of statins on these molecular pathways in arterial tissues from humans2. In experimental animals, two studies have specifically addressed the role of CNP in statin’s actions in vivo. Using a rat atherosclerosis model of carotid artery intimal thickening22, Li et al. showed that atorvastatin (10 mg/k/d for 14 days), in reducing intimal thickening, not only increased the arterial tissue content of CNP but also upregulated Nppc and its downstream target (cGPK) when compared to vehicle administration. In another setting – hepatic cirrhosis in rats – simvastatin (25 mg/k/d for 3 days) increased Klf2, eNOS and Nppc gene expression in hepatic sinusoidal endothelial cells12. Collectively these results suggest that in vitro findings may be reproduced in vivo – at least during exceptionally high dose statin administration. Second, drug concentration in endothelial tissue and type of statin are likely to be important. Concentrations effective in stimulating Klf2 and Nppc gene expression in vitro approximate 100–1000 nmol/L – much higher than mean (24 hr) plasma concentrations (4–33 nmol/L) found during conventional statin therapy24. However in a pilot study undertaken by us in Sprague Dawley male rats aged 6 weeks (n = 2 for each study), plasma CNP and NTproCNP levels did not differ from vehicle treated controls after receiving simvastatin 10 mg/kg/day by gavage for 24 hr, or 40 mg/kg/day for 7 days. Type of statin also could be important10 depending on lipophilicity and membrane permeability. Although a complex issue, evidence suggests that atorvastatin has comparable ability to penetrate cells25 when compared to other statins found effective in vitro. Third, the extent to which changes in endothelial CNP products in tissues affect plasma concentrations is an important issue – particularly in light of the paracrine role of the peptide and the rapid degradation of bioactive CNP at source. Although the bio inactive product NTproCNP is not likely to be degraded at source, whether efflux from extracellular fluids is sufficient to affect plasma concentrations is unclear. While difficult to rigorously assess in humans, our findings from veno-arterial sampling across organs27, and recent findings linking higher values of plasma NTproCNP  with vascular stress in adults without history of heart disease6, support the view that vascular endothelial proCNP does contribute to circulating levels in some settings. Furthermore, in endothelial cell-specific CNP knock out mice, plasma CNP is significantly reduced29. On the basis of these reports we conclude that if concentrations in tissues do indeed increase during statin therapy, any such increase is insufficient to affect plasma levels during the initiation of conventional doses of atorvastatin. Findings relating to plasma ET-1 concentrations are similarly affected by issues 1–3 as discussed above. Although up regulation of endothelin in tissues was reflected by increased plasma ET-1 in mice studies9, the degree to which tissue levels impact on plasma ET-1 remains uncertain30. Uncertainty is increased by the inconsistent effects of statins on plasma ET-1 levels in humans. A small but significant reduction (mean 0.3 pg/mL corresponding to 0.13 pmol/L) was found in a meta-analysis of 15 controlled clinical trials of subjects – many of whom had serious co morbidities13. At least three other studies have found no change in plasma ET-1 levels during statin therapy31. However, none of these studies examined plasma levels within days of commencing therapy. The significant decline at day 7 noted in Group 3 of the current study, and associated fall in hsCRP presumably relates to reduced inflammation post ictus33. Values after 7 days remained unchanged despite a further fall in hsCRP suggesting that only severe inflammation affects ET-1 expression and circulating levels in this age group. Absence of any change in these endothelial products in plasma during statin therapy, in the face of compelling evidence of their actions in vitro, now calls for studies using conventional or higher doses of statin in which tissue protein and gene expression in arteries from experimental animals (or from humans undergoing vascular resections) are made. This is important as current recommended doses of statin, as pointed out by others1, have targeted effects on lipids and are not optimised for targeting putative pleiotropic effects such as Klf2 dependent biomarkers which are highly relevant to vascular biology and progression of atherosclerosis2. Findings from such studies could be important in selecting type of statin and appropriate dose in clinical practice.Several factors contributing to the lack of effect of atorvastatin on plasma CNP peptides need consideration. 34. In Group 3 males, compared to other groups, higher levels at baseline of plasma CNP (but not NTproCNP), which are maintained on day 7 and subsequently, is an important finding which resonates with previous results from sequential sampling in subjects with acute coronary syndrome35. Similar disproportionate increase in CNP compared to NTproCNP was noted in subjects with heart failure36, and in subjects undergoing cardiac catheterisation for acute chest pain27. The anomaly has been attributed to competitive interactions among natriuretic peptides in clearance pathways38 when cardiac secretion of ANP and BNP are increased by cardiac stress. However this explanation seems unlikely in the current study especially in light of the relatively modest elevations in plasma BNP in Group 3 subjects. Provided renal function is normal, NTproCNP concentration will reflect proCNP production in tissues whereas CNP concentrations in plasma – rapidly cleared at source4 and from plasma39 – are at the limits of detection in healthy adults. Comparing Group 3 levels of CNP products with age matched controls, notably NTproCNP does not differ but CNP is raised in Group 3 post ictus, and the ratio NTproCNP/CNP is much reduced throughout the 6 months of study. In contrast, plasma BNP significantly declines. These findings indicate that post ictus CNP production does not change – higher values resulting from reduced CNP clearance. The fact that BNP declines whereas CNP does not is consistent with the much lower affinity of human BNP for NPR340 but whether BNP clearance is affected after cardiac injury requires concurrent measurement of both bio and bio-inactive forms. Recent work showing that the clearance receptor NPR3 is down regulated after hypoxic or ischaemic insults in a wide range of cardiac tissues, and remains reduced in established heart failure41, provides a plausible mechanism. Alternatively upregulation of osteocrin – a peptide expressed and secreted by the failing human heart42 and which has anti-inflammatory actions reducing cardiac remodelling in experimental animals43 – could mediate the changes by displacing CNP from NPR344 as shown recently in transgenic mice45. Future study of temporal responses of plasma osteocrin in subjects presenting with ACS, and plasma levels of microRNA-100 and miRNA-143  militate against a different outcome had these additional interventions been made. No dose-response study in humans was done in light of the lack of effect of very high doses in pilot studies of rats. Conceivably higher dose atorvastatin (80 mg/day) achieving reductions in LDL cholesterol >50% in humans may increase CNP products in plasma. However study of effects of conventional doses used in clinical practice – known to be effective in prevention of cardiovascular disease – was our primary objective. By limiting confounders affecting plasma CNP peptides , variable renal function (correcting for serum creatinine), and variable cardiac status in subjects with overt CAD (measuring plasma BNP and correcting for BNP cross reactivity in the CNP assay), the findings clearly set a limit on the putative actions of statins on CNP production within the healthy and diseased vasculature.st and 2nd degree relatives) of coronary artery disease (CAD), stroke or other arterial disease, were non-smokers and had no personal history of high blood pressure, diabetes, renal disease or lipid disorder. None was receiving medication. Body mass index (18–25), systolic (<140 mmHg) and diastolic (<90 mmHg) blood pressure were normal. All had a normal blood lipid profile. Group 2 (21 subjects) were aged 40–60 yr and met the same criteria as detailed for Group 1. Neither group had previously received statin therapy. Group 3  were enrolled soon after discharge from hospital once stabilised after an acute coronary event established by coronary angiography. None had previously received statin drugs. Subjects with heart failure, diabetes, metabolic bone disease or recent fracture, or with raised serum creatinine (>110 umol/L) were excluded.Participants: Lack of females presenting with CAD restricted the study to males. Three groups of male subjects were enrolled for the study. Group 1 (23 subjects) comprised healthy young subjects (age 20–25 yr) without family history  for measurement of CNP products, B-type Natriuretic peptide (BNP), ET-1, lipids , high sensitive C Reactive Peptide (hsCRP) and serum creatinine. In Group 3 participants, atorvastatin 40 mg was administered daily for a period of 6 months. Venous blood was sampled (0800 hr fasting) for the same analytes at baseline, day 7 and then at intervals of one month.This study was approved by the New Zealand Northern B Health and Disability Ethics Committee (13/NTB/67) and all participants provided written informed consent. All procedures were performed under protocols approved by the New Zealand Northern B Health and Disability Ethics Committee and conducted in accordance with the Declaration of Helsinki. The study is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12613000525785, registration date 13/5/2013.49. Because there could be significant cross reactivity from BNP in the CNP assay50, BNP concentrations were also measured and CNP concentration adjusted accordingly27. Only the corrected CNP values are reported here. The molar ratio of NTproCNP to CNP (NTproCNP/CNP) was calculated for each sample by dividing the former by the latter. Intra- and inter-assay CV for CNP and NTproCNP were (4.4% and 8.3% at 1.8 pmol/L) and (6.4% and 8.9% at 18 pmol/L) respectively. Intra- and inter-assay CV for ET-1 and BNP were (6.1% and 9.6% at 1.3 pmol/L) and (5.4% and 8.3% at 9.7 pmol/L) respectively. Standard methods were used to measure plasma lipids, serum creatinine and CRP .Assays: Plasma CNP, NTproCNP and ET-1 following extraction over Sep Pac C18 cartridges were measured by radioimmunoassay as previously describedStatistical analyses: Data are presented as mean ± sem unless otherwise stated. Repeated measures ANOVA was used to assess changes in plasma markers among groups and across time. Where significant changes were observed with ANOVA, Bonferroni post hoc analysis was used to detect differences from baseline time 0 values and inter group time-matched data as appropriate. BNP and hsCRP data were log10-transformed to satisfy parametric assumptions. Spearman correlation coefficients were used to test the associations between plasma CNP products with other indices. All tests were two sided and statistical significance was assumed when P < 0.05.


Memory B cell (mBC) induction and maintenance is one of the keys to long-term protective humoral immunity. MBCs are fundamental to successful medical interventions such as vaccinations and therapy in autoimmunity. However, their lifestyle and anatomic residence remain enigmatic in humans. Extrapolation from animal studies serves as a conceptual basis but might be misleading due to major anatomical distinctions between species.Multicolor immunofluorescence stainings on fixed and unfixed frozen tissue sections were established using primary antibodies coupled to haptens and secondary signal amplification. The simultaneous detection of five different fluorescence signals enabled the localization and characterization of human CD27+CD20+Ki67- mBCs for the first time within one section using laser scanning microscopy. As a result, human tonsillar mBCs were initially identified within their complex microenvironment and their relative location to naïve B cells, plasma cells and T cells could be directly studied and compared to the human splenic mBC niche. In all investigated tonsils (n = 15), mBCs appeared to be not only located in a so far subepithelial defined area but were also follicle associated with a previous undescribed gradual decline towards the follicular mantle comparable to human spleen. However, mBC areas around secondary follicles with large germinal centers (GCs) in tonsils showed interruptions and a general widening towards the epithelium while in spleen the mBC-containing marginal zones (MZ) around smaller GCs were relatively broad and symmetrical. Considerably fewer IgM+IgD+/- pre-switch compared to IgA+ or IgG+ post-switch mBCs were detected in tonsils in contrast to spleen.This study extends existing insights into the anatomic residence of human mBCs showing structural similarities of the superficial follicular area in human spleen and tonsil. Our data support the debate of renaming the human splenic MZ to ‘superficial zone‘ in order to be aware of the differences in rodents and, moreover, to consider this term equally for the human palatine tonsil. Long-lived memory lymphocytes are key to lifelong immunity against encountered pathogens. Their activation and differentiation takes place in secondary lymphatic organs (SLOs), which in humans include the lymph nodes, the spleen as well as the mucosa-associated lymphatic tissues (MALT) such as tonsils. One special feature of the organization of all SLOs is that the T and B lymphocytes are largely segregated into different anatomical compartments, a process shown to be directed to a great extent by chemokines and adhesion molecules.  The paraA large proportion of human mBCs appears to recirculate, however, the majority of human mBCs resides within lymphatic tissues and share many phenotypic and clonal similarities. , 6 OrganThis study aimed to extend the existing investigations in humans to delineate the residence of mBCs by establishing and applying five-color immunofluorescence stainings on frozen tissue sections of palatine tonsil and spleen. The complex staining enabled unambiguous identification of resting mBCs by labelling them as CD27+CD20+Ki67- cells. We found that mBCs colonized a superficial follicular zone in both spleen and tonsil, formerly referred to as human MZ. Consistently, the data support a nomenclature change of tissue resident mBC structures from “MZ-like” towards ‘superficial zone’ as proposed before , 11, whiInitially, seven spleen specimens S1-S7  served fOf particular note, the samples S8 and S9 , obtaineWritten informed consent was obtained in all cases, in accordance with the local ethics’ committee of the Charité University Medicine Berlin (EA1/303/12).3 native tissue samples were briefly immersed in PBS to wash off blood residue, then rested for 5 min in excess Tissue-Tek O.C.T. Compound , transferred to a Tissue-Tek cryomold (Sakura Finetek) and subsequently frozen in liquid nitrogen for 30 sec and stored at -80°C until tissue sections were prepared.Fresh 1 cmEach tissue was cut into 8 μm sections using a freezing microtome. Sections were placed onto SuperFrost Plus Slides  and dried at room temperature (RT) for 30 min before subsequent usage or storage at -80°C.For immunofluorescence staining, sections were blocked for 5 min with 1x PBS buffer / 3% BSA . After careful washing in PBS, sections were incubated with primary antibodies  for five-color fluorescence detection  with theFor reliable histological detection, we defined mBCs as resting, antigen-experienced B cells as described previously , 16. BasInitial detection of human splenic MZ mBCs served as method verification and validation. The following evaluations refer to secondary follicles, containing GC, and PALS in the white pulp of spleen samples identified in S8 and S9. We excluded follicles without definable GC, which was the case for all follicles of samples S1 –S7. Of note, classical primary follicles containing only naïve B cells were not found in any of the examined sections of the samples S1 –S9.In the white pulp of human spleen, Ki67-CD27-CD20+CD3- naïve B cells, Ki67-CD27+CD20+CD3- mBCs and Ki67-CD27++CD20-CD3+ T cells could be identified . In accoKi67+ proliferating cells were largely limited to the GC itself, where they were distributed homogeneously over the entire GC. Although we cannot assess how this arrangement was influenced by cutting of individual sections, we detected many circular GCs (n>30) with such non-polarized distribution. We henceforth termed such secondary follicles ‘symmetrical’ . In contThe main population in the area surrounding the GC and FM, referred to as the MZ, were mBCs. Correlating with identification of symmetrical secondary follicles were T cells, which appeared organized in a band within the MZ center . This T In the iMZ, the majority of mBCs expressed IgM with low and partially no surface IgD, whereas oMZ mBCs largely co-expressed IgM with IgD . The denNaïve B cells preferentially populated densely within the FM. Notably, they were also detected at the oMZ red pulp transition with a more loose organization and increased IgM and IgD expression . They paIn summary, the established stainings enabled a detailed assessment of mBC organization within the human MZ. This follicle associated MZ could be subdivided into an oMZ and an iMZ with a mixed population of mBCs gradually declinig towards the follicular mantle.Subsequent studies examined the organization of mBCs in human palatine tonsils. This is the first detailed histological description of CD27 expressing tonsillar B and T lymphocyte localization. The following section will address the evaluated subpopulations by assessing the expression intensity. All 15 tonsil specimens exhibited multiple asymmetrical secondary follicles with DZ and LZ, with an average diameter twice as large as found in splenic secondary follicles . Ten tonIn the tonsil, the strongest CD27 signal was detected on PCs , area 1.As in spleens, tonsillar T cells showed an increased CD27 signal compared to mBCs , area 2.In the SE and superficial follicular area, a variable mBC compartment was found to form a patchy hem around the entire secondary follicle in all specimens , area 3.The follicular area (GC and FM) within tonsils showed a diminished signal for CD27 compared to the rest of the tissue analyzed  were not primarily influenced by immunological disease patterns and since secondary follicles with degenerating GCs represent the typical stage of human spleen in adulthood , it was The density of IgM-IgD- post-switch mBCs increased towards the direction of the red pulp but was generally diminished compared to IgM+IgD+/- mBCs. However, since the cells are very close to each other in direction to the FM, the use of an indirect definition appears to be less reliable. We analyzed the four predominating mBC Ig isotypes for the first time within one frozen section. In agreement with serial paraffin sections each stained for IgM, IgD and IgA by Steiniger , respectThe role of the human splenic MZ for recirculating IgM+IgD+/- MZ mBCs is not fully understood but appears to depend on the presence of the spleen.  SplenectIn summary, we here show for the first time an innovative five color staining of human lymphoid tissue sections applicable for native antigen detection. The data on mBC aggregation support to rename the human splenic marginal zone to ‘superficial zone‘ that also applies to the human palatine tonsil. Regarding the observed differences in mBC area symmetries including their Ig isotype proportion, the significance of B cells interacting with follicular structures for immune memory require further consideration.S1 FigA: Secondary follicle that appears to dissolve with lifted tissue architecture (exemplarily marked in a circle) and perforated FM with complete interruptions (arrows). * Crypt lumen. B: Kidney-shaped constricted follicular formation (GC 1 / FM 1), which could not be explained by cutting. Follicle GC 2 / FM 2 might be cut like marked by the white dotted line.. (PDF)Click here for additional data file.


The choroid, which provides vascular supply to the outer retina, demonstrates progressive degeneration in aging and age-related macular degeneration (AMD). However mechanisms that maintain or compromise choroidal homeostasis are obscure. We discovered that the ablation of choroidal macrophages via CSF1R blockade was associated with choroidal vascular atrophy and retinal pigment epithelial (RPE) changes including structural disruption, downregulation of visual cycle genes, and altered angiogenic factor expression. Suspending CSF1R blockade following ablation enabled spontaneous macrophage regeneration, which fully restored original macrophage distributions and morphologies. Macrophage regeneration was accompanied by arrested vascular degeneration and ameliorated pathological RPE alterations. These findings suggest that choroidal macrophages play a previously unappreciated trophic role in maintaining choroidal vasculature and RPE cells, implicating insufficiency in choroidal macrophage function as a factor in aging- and AMD-associated pathology. Modulating macrophage function may constitute a strategy for the therapeutic preservation of the choroid and RPE in age-related retinal disorders. The choroid of the mammalian eye comprises a dense vascular network located external to the retina and provides necessary material transport to the outer retina across the retinal pigment epithelial (RPE) layer constituting the outer blood retinal barrier . The funLike in vascular networks located at border regions surrounding the brain within the dura mater, subdural meninges and choroid plexus , the choDysfunction in choroidal macrophages has been linked to choroidal vascular alterations detected in aging and in AMD. Altered macrophage morphology and distribution have been correlated with attenuation of the choriocapillaris in the aged mouse choroid , and incWe investigated here the homeostatic relationship that choroidal macrophages have with choroidal vasculature and RPE cells. We found that pharmacological CSF1R inhibition, which has been found to ablate tissue-resident innate immune cells in different CNS compartments, but relatively sparing circulating monocytes, induced the ablation of choroidal macrophages in a sustained manner. We discovered using in vivo structural imaging and histopathological and molecular analyses that choroidal macrophage ablation was associated with progressive vascular atrophy and with multiple structural and functional changes in the RPE layer, in ways resembling those observed in the aging and AMD eyes. We also discovered that residual choroidal macrophages, following depletion, can undergo regeneration upon suspension of CSF1R inhibition. Although normally non-proliferative in nature, residual macrophages following depletion proliferated in situ and underwent morphological maturation to fully reconstitute features of the endogenous macrophage population. These regenerated macrophages appear to restore endogenous macrophage homeostatic functions, as their repopulation of the choroid was associated with the arrest of vascular atrophy and the amelioration of pathological RPE alterations.Taken together, our findings implicate a previously unappreciated constitutive function for resident choroidal macrophages in maintaining structural and functional homeostasis of choroidal vasculature and RPE cells and the ability of macrophages to regenerate the full complement of cells following depletion. These suggest a potential etiological mechanism of macrophage insufficiency for pathological choroidal vascular atrophy and highlight choroidal macrophage renewal as a phenomenon that may be potentially harnessed in regenerative strategies in the outer retina. Modulating choroidal macrophage presence and phenotype may be therapeutically effective in maintaining choroidal vasculature and consequently delaying AMD progression.Aging has previously been associated with negative changes in the human choroid, including reductions in choroidal thickness on in vivo imaging  and decrIba1 mRNA in sclerochoroidal tissue was correspondingly decreased from baseline levels at week 1 and week 7 of PLX5622 administration , RPE differentiation (Mitf), and tight junction structure (Tjp1/Zo1) (2)  . Electrojp1/Zo1) . In deplZo1) (2) . EnlargeZo1) (2) . While tZo1) (2) . TogetheVegfa and Vegfc (Serpinf1 (Pedf) mRNA and PEDF protein levels , for homeostatic maintenance , we invend Vegfc  and protnd Vegfc  were signd Vegfc . We alsond Vegfc . We founn levels , as welln levels . In contn levels , were non levels . These dn levels , occurs Tgfb1 mRNA levels and TGFB1 immunopositivity were reduced in the choroid following 7 weeks of macrophage depletion  animals that were subjected to macrophage depletion for 3 weeks and then allowed to repopulate for another 4 weeks (the depletion/repopulation group) with (2) animals subjected to continuous depletion for a total of 7 weeks (the continuous depletion group) . We founWe similarly assessed the function of repopulating macrophages in the support of RPE layer in these two experimental groups . In the Lrat and Rlbp1), Mitf, and Tjp1/Zo1 (Vegfa and Vegfc mRNA and VEGF protein expression were largely reversed with repopulation (Serpinf1 (Pedf) mRNA and PEDF protein expression  animal facility under a 12 hr light/12 hr dark cycle with food ad libitum.Adult 3 month-old female wild type BALB/cJ mice  and 3 month-old albino transgenic mice, containing a transgene with a α-smooth muscle actin (αSMA) promoter driving the expression of green fluorescent protein in perivascular cells in the choroid , were usWe employed a model for macrophage depletion in the adult mouse choroid which involved the dietary administration of PLX5622 , a potent and selective inhibitor of the CSF1R that we previously demonstrated to deplete microglia in the retina . Animals2 inhalation. Enucleated eyes were dissected to form posterior segment eyecups which were then fixed in 4% paraformaldehyde in PBS for 1 hr at room temperature. Eyecups were either processed for vibratome sectioning  or dissected to obtain sclerochoroidal flatmounts, which were then immersed for 1 hr in a blocking buffer  at room temperature. Primary antibodies, which included IBA1 , MHC-II , RPE65 , VEGFA , or PEDF  were diluted in blocking buffer and added to sections/flat-mounts to incubate for 24 hr at 4°C. After washing in 1 × PBS with 0.2% Triton X-100, sections/flat-mounts were then incubated overnight with secondary antibodies . Phalloidin  was added and incubated for 4 hr label F-actin that enables visualization of RPE cell morphology. DAPI  was added to label cell nuclei. Stained samples were imaged with confocal microscopy (Zeiss LSM700). To visualize macrophage distributions over the entire choroid, tiled images of sclerochoroidal flatmounts were obtained under a 10x objective and stitched together with image analysis software . For visualization of RPE monolayer, multiplane z-series were captured under a 40x oil-immersion objective. Resulting images were subjected to computer-assisted analysis ; the density of choroidal macrophages was assessed across the entire choroid and expressed as number per unit area. The number of nuclei per RPE cells were manually assessed from high-magnification images of RPE flat-mounts captured at or near the midpoint between the optic nerve and the peripheral edge of the flat-mount.Mice were euthanized by COSclerochoroidal flatmounts immunolabeled with RPE65 and phalloidin were imaged with confocal microscopy (Zeiss LSM880) and multiplane z-series images were captured under a 40x oil-immersion objective. Morphological analyses of RPE mosaics were performed in an automated manner using REShAPE software, as previously described . BrieflyMice were anesthetized with intraperitoneal ketamine (90 mg/kg) and xylazine (8 mg/kg) and their pupils were dilated. Choroidal and retinal structure was assessed using OCT imaging  in anesthetized animals through a dilated pupil. OCT images in the enhanced depth imaging (EDI)-mode were captured longitudinally in individual experimental animals in the same matched fundus location (with images captured using ‘follow-up’ mode in the instrument software). The imaging was performed using a 30x objective (Heidelberg) and scan fields, measuring 6.5 by 4.4 mm and centered on the optic nerve  were captured in each imaging session. Repeat OCT imaging was performed at 1, 3, 5, and 7 weeks following the initiation of PLX5622. Thickness measurements in OCT images were made using the instrument manufacturer software; choroidal thickness measurements  were measured at 300 µm temporal and nasal from the optic nerve on the horizontal meridian, and average retinal thickness measurements per eye  were made at 300 µm temporal and nasal from the optic nerve in corresponding locations.Choroidal vessels are labeled by cardiac perfusion of an aqueous solution containing 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine perchlorate , a lipophilic dye that incorporates into membranes of endothelial cells revealing the structure of choroidal vessels as previously described . Brieflyst strand cDNA synthesis kit (TaKaRa Bio) per manufacturers’ instructions. cDNA in 1 µL volume was used for real-time PCR analysis using SYBR Green PCR Mastermix (Affymetrix) on the CFX96 Real-Time System (Bio-Rad). Levels of mRNA expression were normalized to those in controls as determined using the comparative CT (2ΔΔCT) method. GAPDH was used as an internal control. Tgfb1 primer was purchased from Bio-Rad (Assay ID qMmuCID0017320), and other oligonucleotide primer pairs used are listed in the Key Resources Table.Quantitative reverse transcription-PCR analysis of isolated mouse choroid was performed to identify the molecular changes after macrophage depletion. RPE-sclerochoroidal tissue were acutely isolated from euthanized animals and total RNA was extracted using a RNeasy Mini kit (Qiagen) and amplified to cDNA using the PrimeScript 1Eyes from control and 7 weeks PLX5622-treated animals were isolated and fixed for 30 min in 4% glutaraldehyde followed by 10% formalin for at least 24 hr. Fixed eyes were embedded in methacrylate, serially sectioned and stained with hematoxylin and eosin. Images were taken with a Zeiss microscope and mean thickness of the choroid from temporal and nasal quadrants and mean cross-sectional area of vascular lumina were measured using ImageJ software (NIH).The eyes from control and PLX5622-treated mice were isolated and fixed in 2.5% glutaraldehyde. The specimens were prepared for transmission electron microscopy (TEM) as previously described . BrieflyMouse RPE-choroidal samples were isolated from the following groups: untreated control group, group treated with 7 weeks of PLX5622, group treated with 3 weeks of PLX5622 and switched to regular diet for another 4 weeks to allow macrophage repopulation. Four biological replicates were obtained for each group. Pigment epithelium-derived factor (PEDF) protein levels were measured in the samples using the Mouse SERPINF1/PEDF ELISA kit . Protein levels of VEGF, PDGF-AA and PDGF-BB were analyzed using a Magnetic Luminex Assay kit .−4 to 10 cd·s/m2 under dark-adapted conditions, and 0.3 to 100 cd·s/m2 under a background light that saturates rod function. The stimulus interval between flashes varied from 5 s at the lowest stimulus strengths to 60 s at the highest ones. Two to ten responses were averaged depending on flash intensity. ERG signals were sampled at 1 kHz and recorded with 0.3 Hz low-frequency and 300 Hz high-frequency cutoffs. Analysis of a-wave and b-wave amplitudes was performed using customized Espion ERG Data Analyzer software (v2.2) that digitally filters out high-frequency oscillatory potential wavelets. The a-wave amplitude was measured from the baseline to the negative peak and the b-wave was measured from the a-wave trough to the maximum positive peak. ERGs were recorded before and after macrophage depletion. The a- and b-wave measurements of animals with or without macrophage depletion were compared using a two-way ANOVA.ERGs were recorded using an Espion E2 system (Diagnosys). Mice were dark-adapted for 12 hr and their pupils were dilated prior to ERG assessment. Mice were anesthetized and flash ERG recordings were obtained simultaneously from both eyes using gold wire loop electrodes, with the reference electrode placed in the animal’s mouth and the ground subdermal electrode at the tail. ERG responses were obtained at increasing light intensities over the ranges of 1 × 10Statistical analyses were performed using Prism 7.0d (GraphPad). For comparisons involving two data columns, t tests (paired or unpaired) or nonparametric tests (Mann–Whitney U) were used, depending on whether the data followed a Gaussian distribution as determined by normality tests. A normality test (D’Agostino and Pearson) was used to analyze the distribution of all datasets. For comparisons involving three or more data columns, a one-way ANOVA (with Dunnett’s multiple-comparisons test) was used if the data in an experimental group were found in a Gaussian distribution, and a nonparametric Kruskal–Wallis test (with Dunn’s multiple-comparisons test) was used otherwise. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Mechanisms that maintain choroidal homeostasis are not well understood, yet are important in aging and age-related macular degeneration (AMD). The authors report that ablation of choroidal macrophages via CSF1R blockade is associated with choroidal atrophy and retinal pigment epithelial (RPE) changes, decrease in visual cycle gene expression, and altered angiogenic factor expression. Suspending CSF1R blockade allowed macrophage regeneration, which arrested vascular degeneration and reversed RPE alterations. The study suggests that choroidal macrophages help maintain choroidal vasculature and RPE cells, and macrophages may be a factor in AMD pathology.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Lois Smith as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Tadatsugu Taniguchi as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Ye Sun (Reviewer #2).Thank you for submitting your article "CSF1R blockade induces macrophage ablation and results in vascular atrophy and RPE disorganization in the mouse choroid" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The choroid degenerates in aging and age-related macular degeneration (AMD). Mechanisms that maintain choroidal homeostasis are not well understood. The authors report that the ablation of choroidal macrophages via CSF1R blockade was associated with choroidal vascular atrophy and retinal pigment epithelial (RPE) structural disruption, down regulation of visual cycle genes, and altered angiogenic factor expression. Suspending CSF1R blockade then enabled spontaneous macrophage regeneration, which fully restored original macrophage distributions and morphologies. Macrophage regeneration arrested vascular degeneration and reversed RPE alterations. These findings suggest that choroidal macrophages help maintain choroidal vasculature and RPE cells, implicating choroidal macrophage depletion or malfunction as a factor in aging- and AMD-associated pathology.Essential revisions:1) Please add the number of mice and eyes assessed in each sub figure.2) The purpose of this manuscript is to show a relationship with macrophages and visual function, if there is experimental recovery of macrophages is there recover of visual function? This significantly strengthens the conclusions.3) Were the mice perfused before the eyes were collected. How much of the observed staining is intravascular cells? It is difficult with the whole mounts to determine if the decrease in vascular structure is because of lost functional choroidal cells, or because of reduced number of white blood cells in the blood circulation causing the vessels to look smaller and less stained.4) None of the figures have staining controls presented. Secondary antibody staining controls should be provided, at least as a supplement. Essential revisions:1) Please add the number of mice and eyes assessed in each sub figure.We apologize for the omission. The number of mice and eyes assessed in the experiments have been added to the figure legend in all the supplementary figures.2) The purpose of this manuscript is to show a relationship with macrophages and visual function, if there is experimental recovery of macrophages is there recover of visual function? This significantly strengthens the conclusions.We appreciate the reviewer’s query. While we have associated the depletion of choroidal macrophages with degenerative changes in choroidal and RPE structure, as well as decreases in ERG physiological function, we found that the repopulation of choroidal macrophages was associated with the recovery of some but not all of these phenotypes. Phenotypes such as choroidal thickness were stabilized upon macrophage repopulation but did not recover back to baseline levels.With regards to the potential recovery of ERG function with macrophage repopulation, we have now added Figure 8—figure supplement 2 to describe the changes in ERG function. This experiment involved the analysis of two separate groups of age-matched animals: (1) the continuous depletion group which was administered a PLX5622 diet continuously over 7 weeks and assessed with ERG at baseline, 3 weeks and 7 weeks, and (2) the depletion-repopulation group which was administered PLX5622 for only the first 3 weeks and resumed on a regular diet thereafter; these animals were also assessed at baseline, 3 weeks and 7 weeks. We found that in the continuous depletion group, the scotopic a- and b-wave amplitudes decreased significantly from baseline at 3 weeks of depletion. and then decreased only a small amount further at 7 weeks of depletion. In the depletion-repopulation group, a similar decrease in the scotopic a- and b-wave amplitudes were observed at 3 weeks of depletion, but the scotopic a- and b-wave amplitudes stabilized and were unchanged between 3 to 7 weeks during macrophage repopulation. These changes in ERG changes upon macrophage repopulation resembled the changes in choroidal thickness which stabilized but did not recover after macrophage repopulation.In the Results section, we have added the following description:“In addition to the RPE morphology and gene expression changes, the scotopic a- and b-wave amplitudes decreased significantly at 3 weeks of depletion in the continuous depletion group and decreased slightly further in the scotopic b-wave amplitude at 7 weeks of depletion while the scotopic a-wave amplitude remained stable. However, in the depletion-repopulation group, while similar decrease in the scotopic a- and b-wave amplitudes were observed at 3 weeks of depletion, the scotopic a- and b-wave amplitudes were unchanged between 3 to 7 weeks during macrophage repopulation .”“Taken together, these data demonstrate that structural and gene expression changes induced in the RPE-choroid complex upon macrophage depletion were significantly ameliorated or stabilized upon macrophage repopulation, supporting the notion of a homeostatic supportive function for choroidal macrophages.”The following figure legend was added to Figure 8—figure supplement 2:“Figure 8—figure supplement 2. Changes in ERG amplitudes during macrophage depletion and subsequent macrophage repopulation. […] n = 10 eyes from 5 animals in each treatment group.”3) Were the mice perfused before the eyes were collected. How much of the observed staining is intravascular cells? It is difficult with the whole mounts to determine if the decrease in vascular structure is because of lost functional choroidal cells, or because of reduced number of white blood cells in the blood circulation causing the vessels to look smaller and less stained.All experimental mice referenced in this section were perfused prior to enucleation with DiI-containing perfusate; the incorporation of DI into endothelial cell membranes enabled labeling of the choroidal vascular structure. It is possible that the removal of intravascular cells may have introduced some alterations in the vascular structure of choroidal vessels, however because all animals were similarly perfused, the detected vascular structural differences between depleted and non-depleted animals do not arise from this potential influence. These differences between the DiI-labelled vascular structures of depleted vs. non-depleted animals were corroborated by comparisons using H&E-stained choroidal sections and in vivo OCT imaging – techniques that do not involve vascular perfusion. As such, we are of the opinion that vascular perfusion did not constitute a confounding factor in our study.For clarification, we added the sentence “to visualize the choriocapillaris, vessels were perfused and labeled with Dil lipophilic dye”in the Results section.4) None of the figures have staining controls presented. Secondary antibody staining controls should be provided, at least as a supplement.We had used the same secondary antibody in the immunohistochemical analysis for VEGF, PEDF and TGFβ1 in the vibratome sections. Image panels showing secondary antibody staining controls for VEGF, PEDF and TGFβ1 in vibratome sections are now added as the Figure 5—figure supplement 2; these showed no positive staining in the absence of the primary antibody.The following figure legend was added to Supplementary file 3. “Figure 5—figure supplement 2. Secondary antibody staining control. […] n = 3 animals in each treatment group.”


MCM2 gene as a methylation-driven gene of which the representative epigenetic markers could serve both as predictive and prognostic markers. Briefly, our work provides several potential HCC progression epigenetic biomarkers as well as a new signature that may enhance patients surveillance and advances in personalized treatment.Although extensive advancements have been made in treatment against hepatocellular carcinoma (HCC), the prognosis of HCC patients remains unsatisfied. It is now clearly established that extensive epigenetic changes act as a driver in human tumors. This study exploits HCC epigenetic deregulation to define a novel prognostic model for monitoring the progression of HCC. We analyzed the genome-wide DNA methylation profile of 374 primary tumor specimens using the Illumina 450 K array data from The Cancer Genome Atlas. We initially used a novel combination of Machine Learning algorithms  to capture early tumor progression features. The subsets of probes obtained were used to train and validate Random Forest models to predict a Progression Free Survival greater or less than 6 months. The model based on 34 epigenetic probes showed the best performance, scoring 0.80 accuracy and 0.51 Matthews Correlation Coefficient on testset. Then, we generated and validated a progression signature based on 4 methylation probes capable of stratifying HCC patients at high and low risk of progression. Survival analysis showed that high risk patients are characterized by a poorer progression free survival compared to low risk patients. Moreover, decision curve analysis confirmed the strength of this predictive tool over conventional clinical parameters. Functional enrichment analysis highlighted that high risk patients differentiated themselves by the upregulation of proliferative pathways. Ultimately, we propose the oncogenic  Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths worldwide. According to the 2018 statistical report of global cancer burden (GLOBOCAN), HCC is the sixth for incidence and the fourth for mortality cancer, accounting for 841,080 new cases and 781,631 deaths per year worldwide [With its consistent immune/inflammatory pathogenesis, HCC appears as a strong candidate for the application of immune-based therapies; however, this strategy has been shown to be only partially successful . CurrentDespite the extensive advancement in earlier diagnosis, therapy decision-making and interdisciplinary evaluation, the prognosis of HCC patients remains poor. Ongoing prognostic models integrate tumor node metastasis (TNM) staging, liver function, comorbidities and other parameters to predict HCC progression and prognosis ; howeverThe use of whole gene expression and methylation analysis of tumors have proved that it is possible to highlight patterns and signatures related to prognosis, tumor classification and response to treatment ,12. MeanIn this study, we aimed to build and evaluate a predictive model able to classify HCC patients with a progression-free survival (PFS) time greater or less than six months by using their methylation profiles. HCC patients were from a large dataset within the Liver Hepatocellular Carcinoma (LIHC) project of The Cancer Genome Atlas (TCGA). We initially used a Machine Learning algorithm (Random Forest) combined with different features selection algorithms to select the best prediction subset of methylation probes on cytosine-phosphate-guanine dinucleotides (CpGs), resulting in a final 34 CpGs-based model for PFS prediction. Then, starting from these final 34 markers, we performed a univariate Cox regression analysis to select PFS relevant CpG probes followed by the construction of a CpGs-based prognostic signature using a stepwise model selection. A four-CpGs-based risk model was successfully built, validated and used to stratify the patients in high risk and low risk for an early cancer progression. Finally, we conducted an analysis of differentially expressed genes (DEGs) followed by a functional enrichment analysis to gain more insights into the biological differences between high risk and low risk patients as well as into the processes involved. This prognostic signature could be useful in the HCC patient administration, by providing a stratification system that reliably separates patients with respect to the progression prognosis, ultimately impacting both therapy and clinical decision-making.portal.gdc.cancer.gov).The transcriptome data (HTSeq raw read counts), methylation data , survival information and clinicopathological information within the Liver Hepatocellular Carcinoma (LIHC) project of The Cancer Genome Atlas (TCGA) were downloaded from the GDC data repository  includes the analysis of 374 primary tumor specimens. The genomic annotation of each CpG probe was accomplished using the omosomes ; third, omosomes ; fourth,omosomes ; fifth, omosomes .The workflow used to develop and assess the Machine Learning (ML) model for patient classification, with respect to a PFS greater or less than six months, is reported in IVF) (basic filtering + variance filtering) and a basic filtered dataset (ALL). Then both datasets were split into 80% training and 20% test partitions in a stratified manner, making sure that the pair of classes were present in both partitions and in equal proportion. The training set was used in the model construction step while the testset was kept aside to perform the model validation.Methylation data were processed and filtered as described in the datasets section. To test whether reducing the number of variables in an unsupervised manner would have improved the classification performance, we started with an independent variance filtered dataset (ALL), following a backward features selection  (RFE), following an all relevant features analysis (Boruta), and on the overlapping features extracted from these two selection techniques (RFE∩Boruta + RF). The RFE features selection was achieved using the rfe function of “caret” package (v6.0-86) [Within the model construction step, we evaluated the performance of a supervised learning algorithm namely Random Forest (RF), using the “ranger” package (v0.12.1) . Differev6.0-86) , within v6.0-86) , within train function of caret package; again, the fold assignment was stratified to avoid class imbalances. The hyperparameters optimization was performed within the CV passing a custom grid parameter to the train function.In order to forcefully assess the pipelines performance, the algorithms were implemented within a 10 × 5 CV scheme, using the To avoid overoptimistic inflated results, especially on imbalanced datasets, performance was assessed both in terms of accuracy (ACC) and Matthews Correlation Coefficient (MCC) . The ovep-value < 0.05 [The CpG sites’ methylation effect on cis regulated gene expression was assessed using Pearson’s correlation (r) test between the CpGs ng genes . The thre < 0.05 .p-value adjusted BH < 0.05) CpGs were used to construct a multivariate Cox regression model using a stepwise model selection in both direction, using the stepAIC function from “MASS” package (v7.3-51.6) [Univariate Cox proportional hazards regression analysis was performed using the “survival” package (v3.2-3)  to scree.3-51.6) . The cordca available at http://www.decisioncurveanalysis.org. PFS at 6 months was defined as binary outcome variable  and, as predictors, we selected tumor features, biomarkers and the risk score predicted by our CpGs-based prognostic signature.To assess the adequacy of our CpGs-based prognostic signature and the possible clinical advantage over currently used parameters, we performed a decision curve analysis (DCA)  using thp-value . Gene expression between the two groups was considered deregulated with a FDR < 0.05 and logFC ≥ 1 or ≤−1, the latter indicating up- and downregulated expression in the high risk group, respectively.To identify DEGs between high risk and low risk groups we used the “DESeq2” package (v1.26.0) . The DEGp-value  < 0.05 was considered as the threshold level of statistical significance.Different enrichment methods were used to analyze the functional characteristics of DEGs between the two groups. Gene Ontology (GO) annotation was performed using “goseq” package (v1.38.0) , DiseaseAll the analyses were performed in R environment (v3.6.3)  on a 12-In The study included 374 primary tumor cases of HCC from The Cancer Genome Atlas (TCGA) cohort. The main clinical and etiological features of cohort used in this study are summarized in The CpG beta values from 450k DNA methylation microarray analysis, consisted of 485,577 CpG methylation probes, that were pre-processed by applying different basic filters to remove: probes containing missing values ; highly correlated probes ; probes residing on X and Y chromosomes ; and probes containing single-nucleotide polymorphisms (n = 4798). By using this approach, we obtained a final series of 323,564 probes for the subsequent analyses. We then performed an unsupervised feature selection by an independent variance filtering (IVF) on the half most variable probes  .The methylation filtered data were split into training (300 patients) and test (74 patients) sets. The training set was used for the model development and optimization within a 10 × 5 CV and the testset was used for assessing the model performance. In parallel, within the model development step, we used two feature selection methods called RFE and Boruta to reduce the number of features and to possibly identify PFS-specific methylation markers. By using RFE, we got a set of 415 CpG probes that led to the RF algorithm with the highest MCC value. With Boruta analysis, we found that the best subset of probes was the first 200 CpG ranked by importance. We also took in consideration the overlapping probes from the two methods  Table A.ALL + RF), independent variance filtered probes (IVF + RF), probes selected using RFE method (RFE + RF), probes selected using Boruta method (Boruta + RF) and overlapping probes from RFE and Boruta (RFE∩Boruta + RF). In ALL + RF) and (IVF + RF) achieved a trainset mean cross-validation MCC of 0.127 and 0.162, respectively. In contrast, (RFE + RF), (Boruta + RF) and (RFE∩Boruta + RF) achieved a trainset mean cross-validation MCC of 0.467, 0.485 and 0.533, respectively, that were significantly higher than RF models trained with thousands of features. The trend of increased performance due to dimensional reduction was also seen in the testset partition (The subsets of probes obtained were used to train RF models following the approach illustrated in artition .RFE∩Boruta + RF) showed the best performance metrics and 34 CpG probes  and 2 CpG sites showed a moderate significant correlation  [DNA methylation is an epigenetic mechanism that involves the addition of a methyl group to a DNA cytosine and occurs mainly at CpG dinucleotide sequences in mammals. The degree of methylation at CpG sites but also the balancing of methylated and unmethylated CpGs over the genome control several biological functions such as gene expression regulation, cellular differentiation and mammalian development . Aberran < 0.05)   .MCM2) and SPG20 were the genes that showed a moderate negative correlation with their matched CpG probe. The SPG20 gene encodes a protein called Spartin that has been found to be involved in intracellular epidermal growth factor receptor trafficking; SPG20 promoter has been found to be hypermethylated in colorectal cancer, resulting in gene silencing and cytokinesis arrest [SPG20 promoter hypermethylation was also validated as a novel noninvasive biomarker [MCM2 is one of six highly conserved proteins recruited to form the MCM protein complex, a cell ubiquitous hexamer that works as molecular motor performing DNA duplex unwinding and fork progression during DNA replication [MCM2 has been found to be overexpressed in several cancers such as oral, gastric, colon, lung and breast cancer. High MCM2 expression in cancers was associated with higher grades, more advanced stages and poor prognosis [Minichromosome maintenance complex component 2  , cg11889692 , cg12961607(SRSF7), cg22539431 (SND1). Three CpG sites  were characterized by a negative coefficient that in this case implies a higher PFS for patients with higher beta values of these CpG sites; conversely one site (cg11889692) with a positive coefficient confers a lower PFS for patients with higher beta values of this CpG site. Importantly, lower values of cg08889930 were associated with a worse PFS and this is in keeping with the evidence that we reported that its value was negatively correlated with MCM2 expression. The hypomethylation of cg08889930 and the resultant overexpression of MCM2 were in keeping with what was previous reported [To study the possible prognostic impact of the selected CpG probes we used Cox regression analysis to assess which of the 34 final CpG probes were associated with PFS time in the trainset. Eleven CpG probes were significantly correlated with the PFS (d <0.05) . Afterwad <0.05) : cg08889Taking into account the estimated Cox regression coefficients, we then constructed a prognostic risk model described by the formula as follows: The corresponding risk scores of each trainset patient were calculated using the formula and the samples were stratified into high-score and low-score groups based on the risk score = 1 as cutoff. Next, we used Kaplan–Meier survival analysis to assess the prognostic impact of the model in the risk stratified trainset. The relationship between PFS time and risk score was statistically significant, with patients at high risk of progression (n = 137) showing a considerable adverse PFS with respect to the patients at low risk of progression (n = 163). This trend appeared as particularly visible during the first 12 months A. PatienTo validate the risk model, we also tested the established model in the testset. The risk scores were calculated using the aforementioned formula and the patients were stratified in patients at high risk of progression (n = 29) and patients at low risk of progression (n = 45). Low risk patients showed a significant advantage in PFS time compared to high risk patients B. These p-value = Alpha-fetoprotein (AFP) is the first introduced and most extensively utilized marker for diagnosis, prognosis and monitoring of HCC . To testl of HCC .Decision curve analysis (DCA) is a method that can be used to assess the value of prognostic models . In brieA number of models have been developed to calculate the tumor stage and prognosis. Since the important role of the liver, HCC evaluation includes not only tumor features but also liver function parameters as key prognostic factors for survival and progression. Different staging algorithms are currently in use , they usWe analyzed the predicted probability of PFS at six months using the risk calculated by our 4 CpGs model and by common in-use clinical parameters, to assess the net benefit that patients could obtain. As shown in With the aim to understand the functional significance of the proposed epigenetic 4 CpGs score, we evaluated the putative gene expression modulation between the two epigenetic risk categories. To do this, we performed a DEG analysis. Patients at low risk of progression were used as reference. A total of 850 DEGs were identified, consisting of 619 upregulated genes (FDR < 0.05 and logFC ≥ 1) and 231 downregulated genes (FDR < 0.05 and logFC ≤ 1). In In order to gain more insights into the biological function of the risk prediction signature, we applied GO terms and KEGG pathway analyses to identify associated biological terms and pathways from DEG profiles of risk stratified HCC patients. The results of the GO analysis revealed that downregulated DEGs in patients at high risk of progression were significantly enriched in biological processes including ‘hormone metabolic process’, ‘lipid metabolic process’, ‘xenobiotic metabolic process’ and ‘cellular response to xenobiotic stimulus’ A. The upKEGG pathway analysis revealed that downregulated DEGs in high risk patients were associated with pathways including ‘Retinol metabolism’, ‘Chemical carcinogenesis’ and ‘Drug metabolism—cytochrome P450 ’ B. The upADH) genes that are involved in alcohol metabolism, uridine diphosphate glucuronyl transferase (UGT) genes which play roles in phase II drug metabolism and cytochromes (CYPs) P450 genes that catalyze the oxidation and metabolism of a large number of xenobiotics and endogenous compounds. Within downregulated genes, we also found solute carriers (SLC) and ATP-binding cassette (ABC) proteins that play an essential role in the uptake and in the export of a large variety of anti-tumor drugs, respectively.In E2F2, cyclin-dependent kinase 1 (CDK1) and MCM2 but also cytokine gene network with crucial effects on inflammation and tumor immunology as well. Within upregulated genes we found surface markers that are expressed by stage- and lineage-specific hematopoietic cell; as an example, overexpression of CD19, CD24 and CD38 could indicate an higher infiltration level of B regulatory cells (Bregs) with a Upregulated genes included several genes involved in the control of the cell cycle like ding HCC ,48,49.HCC remains one of the most frequent malignancies and a leading challenge for public health worldwide. Even after the application of curative treatments, such as resection, local ablation and liver transplantation, and the extensive advancement in earlier diagnosis, staging systems and therapy decision-making, the long-term prognosis of HCC remains poor. The evaluation of conventional parameters such as proper liver function, vessel invasion, tumor staging and biomarker levels are commonly used in HCC prognosis prediction; nevertheless, their performance is still not satisfactory and this could be due to the high degree of heterogeneity among HCC cases. Therefore, the recognition of novel prognostic biomarkers from large scale omics data and the establishment of more accurate prognostic models could dynamically recapitulate the biological progression of HCC and may have a superior predictive accuracy than conventional ongoing parameters. Recently, numerous studies with gene expression, microRNAs and methylation profiling have shown great potential in prognosis prediction and staging systems of HCC ,52,53,54The present study aimed to identify efficient prognostic markers to stratify HCC patients according to their epigenetic features involved in tumor pathogenesis and progression. We applied a combination of Machine Learning algorithms that performed a supervised features selection  on methylation data to increase the chances of catching CpG markers related to the PFS ,56. The One of the advantages of RF is the built-in variable importance measure that ranks the features with respect to their relevance for prediction. This is performed calculating the Gini Importance (Mean Decrease in Impurity—MDI)  or the PConsequently, to evaluate the size and direction of the relationship between the 34 CpG features and the PFS, but also to investigate their prognostic impact, we firstly performed the univariate Cox regression analysis of the final 34 markers and then, we established a 4-CpG-based prognostic model for HCC. The signature was validated using the internal left-out testing set, indicating the stability of this model in terms of ability to stratify HCC patients into high and low risk groups for early cancer progression. To assess the adequacy and the clinical advantages of our signature over currently used parameters, we performed a decision curve analysis proving that our method showed a significantly improved performance when compared to in-use conventional clinical parameters , thus indicating a more powerful and dynamic reflection of HCC heterogeneity. This was true either if the 4 CpG signature was considered alone or in combination with well-recognized prognosis parameters. The dataset considered in this analysis is the most comprehensive collection of multi-omic data from HCC cases . UnfortuMCM2 expression  protein complex which is involved in the initiation of DNA replication and DNA unwinding [MCM2 is a subunit/component of the hexameric protein complex that consists of MCM2-7 and directly interacts with MCM5 [MCM2 silencing inhibits cell proliferation by affecting the G1/S transition and conversely the overexpression of MCM2 promotes cell proliferation in lung cancer cells [MCM2 knockdown inhibits cell migration in lung cancer cells. Notably, during the last few years, several studies have evaluated the action of in use treatments against MCM2. In particular, MCM2 is a therapeutic target of Trichostatin A in colon cancer cells [MCM2 has been proposed as therapeutic target of lovastatin in human non-small cell lung carcinomas [MCM2 silencing and carboplatin treatment may represent a novel therapeutic strategy to treat ovarian cancer [MCM2 gene and consequently the expression level of MCM2. Its mehylation level could serve not only as prognostic marker to evaluate HCC patient progression but also as predictive marker to evaluate the efficacy of a therapeutic intervention. Another important implication is that the MCM complex offers a fascinating target for drug development in HCC, since it is an essential replication factor that couples DNA replication to both cell cycle progression and checkpoint regulation [Our CpGs signature comprises the methylation level of 4 CpG sites , of whicpression ; this sirognosis . Aberranrognosis ,62. Our nwinding . MCM2 isith MCM5 . In vitrer cells ; moreoveer cells , MCM2 harcinomas , the comn cancer . Here wegulation .MCM2 is upregulated in patients belonging to the high risk group and this could be at least in part recapitulated by the enrichment of genes involved in the control of DNA replication, cell division and cell cycle (CDKs) and several modulators [CDK1 is the only CDK that is essential for cell cycle progression [CDK1 network and that are usually upregulated in cancer  [ITGB8) [LAMA1) [One of the major constituents of the environment is the extracellular matrix (ECM). The ECM supervises crucial processes like intratumoral signaling, transport mechanisms, metabolisms and immunogenicity. For its activity, the ECM has been associated with tumor establishment, disease progression and therapy resistance in several tumors . The ECM, FRAS1) ,78, drug [ITGB8)  and prol [LAMA1) .CXCL5, CXCL17 and IL20RA  and this communication is guaranteed by the release of key mediators such as cytokines and chemokines. These signaling molecules and their receptors affect multiple processes including tumor cell proliferation, invasion and metastasis, tumor immune response and angiogenesis . In thisd IL20RA C. CXCL5 ltration . Moreovepatients .SLC22A1, SLCO1B3, ABCG2, CYP3A4). SLC22A1 is a member of the solute carriers (SLC) family, a collection of proteins that play an essential role in the uptake of anticancer drugs. Loss of SLC22A1 in the plasma membrane of tumor cells results in a reduction of sorafenib uptake, that has been correlated with less favorable prognosis of HCC patients treated with this drug [SLC22A1 expression results in an improved receptivity of sorafenib in HCC cells [SLCO1B3, a component of a family of genes that plays a role in the transport of TKIs drugs. SLCO1B3 accomplishes the uptake of cabozantinib [ABCG2 plays a crucial role in sorafenib efflux and higher ABCG2 expression has also been correlated with chemoresistance in HCC and reduced overall survival in HCC patients [ABCG2, so in this case the higher expression level could mainly affect the sorafenib behavior in low risk HCC patients. Several CYPs genes are deregulated in high risk patients including crucial genes such as CYP3A4, CYP2C9, CYP1A2 (CYP3A4 through an oxidation process [CYP3A4 in HCC microsomes leads to a significantly altered sorafenib metabolism in the liver tumor tissue of HCC patients [The downregulated genes in high risk HCC patients include several metabolic genes, drug metabolism genes, transporters and carriers genes . This schis drug . FurtherCC cells . Anotherzantinib , clears zantinib  and its zantinib . The ATPpatients . In our , CYP1A2 C. Deregu process ,92,93. Dpatients .MCM2 gene as a methylation-driven gene of which the representative CpG site cg08889930 could serve as a predictive marker of therapeutic interventions. Finally, we provided evidence that our model is capable to classify HCC patients into high and low risk for progression groups. Thus, this predictive tool may enhance the management of patients at high risk of progression and the development of personalized treatment for HCC patients.In conclusion, starting from the methylation profile of primary HCC specimens, we used a novel combination of Machine Learning algorithms to capture early tumor progression features and to focus on relevant CpG sites. These final features laid the foundations for the development of a prognostic model for early HCC progression based on 4 CpG sites that showed a significantly improved performance over conventional clinical parameters. Notably, we proposed the oncogenic


PurposeRadiotherapy to the pelvis and especially the anal sphincters can result in long-term faecal incontinence. The aim of this study was to assess the effectiveness of biofeedback anal strengthening exercises in radiotherapy-related faecal incontinence.MethodsA retrospective cohort study was performed on seven patients with radiotherapy-related faecal incontinence. Education and biofeedback based anal strengthening exercises were provided. Baseline and follow-up measurements were performed assessing anal sphincter fatigue time, resting, squeeze, and cough pressure. Continence scores and quality of life measures were assessed. Patients were followed up at five months.ResultsThe radiotherapy target varied between prostate, uterus, and rectum. Four of the seven patients were female. Pescatori (0-10) and St Mark’s (0-10) continence scores had a median improvement of 2 (range 0-5) and 1 (range 0-8) respectively. Self-completed patient continence scores (0-10mm) had a median improvement of 2mm (1-6mm). Anal fatigue time measurements (Isotonic Fatigue Time (seconds) and Isometric Fatigue Time (Cycles)) showed a median improvement of three seconds (-4 - 36 seconds) and three seconds (-1 - 6 seconds), respectively. Resting Pressure (mmHg), Squeeze Pressure (mmHg) and Cough Pressure (mmHg) showed median improvement of 10mmHg (-10-21mmHg), 15mmHg (-16 - 100mmHg) and 21mmHg (-3 - 53mmHg), respectively. Patient-defined quality of life (QOL) measures showed a median change of 0.2 (range 0-0.5).ConclusionPatients in this pilot study with radiotherapy-related faecal incontinence had improved anal pressure metrics, continence, and QOL following biofeedback based anal strengthening exercises. These early results suggest a benefit for anal strengthening in patients undergoing pelvic radiotherapy. Faecal incontinence is the involuntary loss of solid or liquid faeces. Individuals with this condition suffer significant embarrassment and morbidity and it places significant burden on carers and health services . In AustBiofeedback retraining involves the use of mechanical or electrical devices to increase the awareness of a biological response  facilitating patient learning and improved control . It provBiofeedback retraining is an established treatment modality for faecal incontinence  however This project was presented as a poster at the Royal Australasian College of Surgeons 88th Annual Scientific Congress in 2019.MethodologyPatients who were referred for treatment for incontinence after pelvic radiotherapy were retrospectively identified. Patients were included in the study if they had faecal incontinence following radiotherapy and were engaged in the biofeedback retraining program. Participants were excluded if they underwent a surgical procedure that removed the anus  or they had a de-functioning stoma. Biofeedback is a type of reconditioning therapy with the aim of increasing a patient’s conscious control of the pathways controlling defecation. Various modalities have been described including balloon systems to facilitate patient awareness of rectal distension, ultrasound visualisation of the anal sphincter and demonstrating the effects of a patient's exercises and the use of anal manometric probes to teach the patient how to exercise the anal sphincter . Our proOur biofeedback retraining program initially takes 90 minutes with education and teaching of anal strengthening exercises. An anal manometer records pressure measurements including resting, cough, and squeeze pressure in addition to the length of time the patient can hold a squeeze without muscle fatigue. This is displayed on a visible screen so the patient receives immediate visual feedback on their actions Figure . This miPatients in the study received face to face education and biofeedback-based anal strengthening exercises. Baseline and follow-up anal pressure measurements were performed assessing anal sphincter fatigue time, resting, squeeze, and cough pressure. Continence scores (Pescatori and St Mark’s) ,11 and pFollowing approval by our local Human Research Ethics Committee  the prospectively collected data was retrospectively collated using Research Electronic Data Capture software (REDCap). Statistical analysis was performed using Excel with the Data Analysis Toolpak. Descriptive statistics were calculated including mean, median, standard deviation and range. Given the small sample size, we used non-parametric data tests. Statistical significance of median differences across variables was calculated using the Wilcoxon Matched Pairs Signed Rank Test. The P-value is given as a range, using the critical values in appendix B12 of Zar, as there are fewer than 100 matched pairs .Seven patients with radiotherapy-related faecal incontinence were identified. The radiotherapy target varied from prostate 4/7), uterus 2/7) and rectum (1/7). Two patients underwent a hysterectomy and one had an ultralow anterior resection. Three patients were male and four were female 57%). Pescatori 0-10) and St Mark’s (0-10) continence scores had a median improvement of 2 (range: 0-5 p<0.001) and 1 range: 0-8 p<0.001) respectively  and no improvement in the quality of life. This may be due to the proximity of the surgical procedure to the anal sphincter complex. We would argue that the continence scores and quality of life measures are important patient-perceived outcomes, perhaps more relevant than the objective measures obtained from the manometer. Patients reported improvement in key areas such as "reducing leakage", "decreasing accidents" and improving "waiting time". Patients generally have a keen interest in maintaining faecal continence and their engagement enables accurate assessment of their own function. The major limitation of this study is the small sample size. Although we included all patients that have undergone this treatment, the study still has a small heterogeneous group. We will address this shortfall in our future prospective study. There were three patients identified during the data collection phase that had not completed follow-up, so these patients were unable to be included in the study. Another limitation is that the biofeedback program we deliver is coordinated by an unblinded single nurse practitioner. This may introduce an unintended bias in the way in which the patient data has been initially collected. This was unavoidable with the design of the study however it would have been minimised by the use of our standardised data collection forms. An integral part of our retraining program is the inclusion of dietary advice around fibre and water intake, exercise guidance, education around functional toileting and psychological support. This may indicate that anal strengthening is not the sole contributor to continence improvement in our patient group however there is biological plausibility that a strong, intact anal sphincter would assist with maintaining continence in the patients. Studies suggest that biofeedback is effective however it is often delivered as a package of care rather than an isolated entity . Many ofThese early promising results suggest improved faecal continence in patients with radiotherapy related faecal incontinence and we are currently developing a prospective study to further evaluate the role of biofeedback training in this patient population.