Chromatin is the physical substrate of the genome that carries the DNA sequence and ensures its proper functions and regulation in the cell nucleus. While a lot is known about the dynamics of chromatin during programmed cellular processes such as development, the role of chromatin in experience-dependent functions remains not well defined. Accumulating evidence suggests that in brain cells, environmental stimuli can trigger long-lasting changes in chromatin structure and tri-dimensional (3D) organization that can influence future transcriptional programs. This review describes recent findings suggesting that chromatin plays an important role in cellular memory, particularly in the maintenance of traces of prior activity in the brain. Inspired by findings in immune and epithelial cells, we discuss the underlying mechanisms and the implications for experience-dependent transcriptional regulation in health and disease. We conclude by presenting a holistic view of chromatin as potential molecular substrate for the integration and assimilation of environmental information that may constitute a conceptual basis for future research. The mammalian brain is a complex organ whose functions are strongly influenced by experience. Across life, the brain needs to assimilate and integrate information about the environment, and use this information to adapt and direct actions and behavior. These biological functions are fundamental for animals and human beings and have a social, ecological and evolutionary impact –3. EarlyIn the cell nucleus, genome organization relies on chromosomal interactions and on associations between chromatin and the nuclear lamina at the periphery of the nucleus , 27. ChrRegulatory elements can physically interact via long-range interactions and create chromatin loops . At a hicis-regulatory elements, particularly enhancers and promoters, and modulate gene transcription, for instance in differentiating stem cells, during circadian cycle and even during activation of brain cells [c-Fos and Arc, which leads to gene transcription [The 3D organization of the genome is an essential component of transcriptional regulation in mammals and undergoes major changes during development and cellular activity , 41, 42.in cells –45. Chroin cells , 19, 46.cription , 47. Impcription –50.Mechanistically, the establishment and stabilization of long-range interactions and chromatin loops primarily involve the activity of the architectural proteins cohesin and binding of the CCCTC-binding factor (CTCF) , 52. TheChromatin-based mechanisms that control gene expression contribute to cell differentiation during brain development and to higher-order functions in the adult. Early postnatal life is a time of intense epigenome remodeling in brain cells. In the maturing brain, enhancer–promoter interactions undergo global rewiring, and TADs and chromosomal compartments are reorganized within neuronal and glial cells nuclei , 64. At Chromatin states are also modified by neuronal activity, which involves activation of enhancers . SeveralOverall, changes in chromatin structure and 3D organization have been associated with neurodevelopmental processes and higher-order brain functions in adulthood, but emerging evidence suggests that they can also be implicated in experience-dependent regulatory memory in brain cells. Below we review current evidence supporting a role for chromatin in the maintenance of traces of prior activity in brain cells and we describe potential mechanisms of establishment and functional implications in health and disease.In mammals, neuronal activation induces the expression of genes necessary for neuronal plasticity, a cellular property essential for learning, memory and cognition . At the Unlike transcription, changes in the structure, composition or 3D organization of chromatin that occur after IEGs activation are not transient and chromatin is not necessarily re-established at pre-stimulation states. For instance, neuronal activation by electroconvulsive stimulation increases chromatin accessibility at thousands of genomic sites after one hour in hippocampal slices . But altKainic acid, frequently used to induce seizures in animal models, also persistently affects chromatin in neurons. A single injection is sufficient to strongly activate neurons and increase transcription in excitatory neurons of the adult hippocampus. It extensively changes chromatin accessibility and long-range 3D interactions between regulatory elements . Like wiRemarkably, genomic regions with lasting changes in chromatin accessibility are also engaged in persistent long-range interactions that are independent of CTCF binding. These regions are enriched for the binding motif of the family of TFs AP-1, suggesting a role for AP-1 TFs in the establishment and maintenance of chromatin states (see Mechanisms). Sites with stable changes in occupancy are also located at the promoter or enhancer of genes involved in axonal growth or protein aggregation , suggestEnvironmental exposure and salient experiences, i.e., experiences that stand out, are extreme and emotionally charged, can have long-lasting effects on chromatin in brain cells. Their effects can vary depending on the time window of exposure (whether before or after birth or in adulthood), but also its type  and duration (acute or chronic). While the adult brain is susceptible to changes, the developing brain is probably even more sensitive and may be more profoundly affected since many developmental processes such as cell division and differentiation in prenatal life and cell migration, synaptogenesis and myelination after birth are ongoing . FurtherDot1l and Kdm2b, which are H3K79 methyltransferase and demethylase respectively, in dopaminergic neurons of NAc [Dot1l dysregulation occurs only in early adulthood, the observed changes in H3K79me1/2 are not an immediate consequence of stress at the time of exposure but rather emerge later in development, suggesting a form of molecular memory of prior stress.Early life conditions such as stress before or after birth can have effects on physiology, behavior and cognition that can last until adulthood . These es of NAc . Althougs of NAc  and enhas of NAc . Since DWhile transcriptional and epigenetic changes have been well studied in animal models and humans, changes at the level of chromatin itself remain not well characterized in the brain. Most evidence for changes in chromatin by stress is in immune cells. Perinatal glucocorticoids have been shown to favor chromatin accessibility in cytotoxic T cells at genes carrying glucocorticoid receptor binding sites necessary for interferon production . The effFurther to stress, exposure to chemicals early in life can also affect behavior and cognition in mammals and involve lasting changes in chromatin structure and organization affecting gene expression in the brain. Repeated exposure to midazolam, an anesthetic frequently used in children, can cause neurological abnormalities. In mouse, midazolam exposure from PND10 to PND12 impairs hippocampus-dependent object memory on a novel location recognition task as well as fear memory on a contextual fear conditioning test . At the Memory formation has been associated with long-lasting changes in chromatin accessibility. Neuronal activation by fear conditioning rapidly increases chromatin accessibility at thousands of genomic loci in hippocampal neurons (within 1.5 h). Some of the changes persist for at least five days, suggesting lasting chromatin states . ConditiΔFosB promoter and enhancer elements have increased chromatin accessibility upon acute cocaine administration that persists during prolonged withdrawal and upon relapse [Further to memory, addiction has also been associated with lasting changes in chromatin. Addiction is a pathological dysfunction characterized by a loss of self-control over one’s action that can be harmful. One of the detrimental features associated with addiction is relapse, that can occur even after prolonged abstinence and is caused by “drug” memory. In mice, chronic exposure to cocaine followed by 30-day withdrawal increases chromatin accessibility at thousands of genomic regions including potential enhancer elements, in medium spiny neurons of NAc expressing the D1 dopamine receptor (cocaine target) . Some of relapse . ImportaFurther to neuronal cells, immune cells in the adult brain can also acquire lasting changes in chromatin composition. In adult mice, peripheral inflammation induced by lipopolysaccharides injection causes differential deposition of H3K4me1 and H3K27ac at enhancer elements that persist for at least 6 months in microglia, brain resident macrophages . EnhanceOne of the consequences of the genomic redistribution of DNA binding proteins such as TFs are changes in chromatin accessibility. Because TFs can remain bound to DNA, they have been proposed to regulate the establishment and maintenance of chromatin states –96. ConsAn epigenetic mechanism for dynamic changes in gene expression mediated by lasting modifications of chromatin accessibility has also been proposed in the context of neuronal activation . IncreasWhile still limited in brain cells, there is evidence that TFs play a role in the establishment, maintenance and reactivation of chromatin states in the context of inflammatory memory in adult stem cells , 104. InA hit-and-run mechanism mediated by constitutive and environmentally induced TFs has also been described in adult hematopoietic stem cells to explain long-lasting changes in chromatin accessibility induced by lipopolysaccharides . In thesGenomic regions with chromatin memory in brain cells are also differentially enriched in histone post-translational modifications such as H3K4me1, H3K27ac, H3K79me2/3 among others , 12. ThiAn alternative scenario in which histone modifications may directly influence chromatin memory is via changes in 3D genome organization. Such changes may affect the segregation of genomic regions carrying the modifications into separate compartments (A or B) leading to their isolation and stabilization within the nucleus . Such seH2afz and enhanced association of the H2A.Z chaperone ANP32E to chromatin [H2afz gene itself remains significantly downregulated after chronic cocaine and during withdrawal, H2A.Z incorporation is reduced at promoter regions and in NAc-specific enhancers of genes such as FosB [1165,97. This suggests that the reduced genomic binding of H2A.Z during withdrawal might result from lower H2afz transcription and increased H2A.Z removal, potentially affecting chromatin accessibility genome-wide.Another mechanism that contributes to the maintenance of chromatin states is the replacement of histone variants. H2A.Z is one of the variants known to be reduced by chronic cocaine administration in the rodent brain and that remains low for up to 30 days after drug withdrawal . H2A.Z ihromatin . Interes as FosB . However as FosB , it may The establishment and maintenance of chromatin states induced by exposure are largely influenced by the developmental time window and duration of exposure. While long-lasting changes in chromatin structure can be potentially established any time during development, the molecular mechanisms allowing their stabilization and their cellular and functional consequences on the brain largely depend on the time of exposure. In particular, postnatal stages of brain development are periods of increased susceptibility because NSCs proliferate and differentiate into post-mitotic neurons that no longer divide nor undergo molecular “renewal” by replication. This means that epigenetic modifications acquired at that time may remain associated to the genome and constitute a form of chromatin memory in newborn neurons , 102. FuIn brain cells, changes in chromatin accessibility have been associated with differential gene expression. Exposure of NSCs to midazolam results in transcriptional upregulation of quiescence genes which correlates with increased chromatin accessibility at the promoter of these genes that persists in adult cells . Howeverc-Fos expression associated with a lasting gain in chromatin accessibility at Jdp2, a gene located downstream of c-Fos that encodes an AP-1 repressor [Pathological neuronal activation such as during epileptic seizure (or status epilepticus) can also result in long-lasting changes in chromatin accessibility at key regulatory elements and potentially affect transcriptional programs. Mice subjected to seizure by kainic acid have memory deficits and blunted epressor . Changesepressor . OverallExperiences are major determinants of brain functions that shape developmental and molecular trajectories of brain cells and have lasting effects influencing neural responses and behavior in adulthood. While synaptic processes are classically thought to be the main site for neural stability and plasticity, new findings indicate that chromatin also has these properties. Its composition, structure and 3D organization within the cell nucleus can contribute to transcriptional programs activated by experience and act as mediator of information assimilation in the genome in the form of chromatin memory.Mechanistically, the establishment and maintenance of chromatin memory rely on the activity of TFs that bind to genomic sites upon stimulation, and on the stabilization of activated chromatin states. Such mechanisms have been implicated in inflammatory memory in immune cells and epithelial stem cells, and we postulate that they are also at play in brain cells. We propose that experiences can lead to persistent changes in chromatin structure and organization via differential activity of AP-1 TFs. Since these TFs have been implicated in signal-induced long-range interactions between regulatory elements , they maWhile important progress has been made in the understanding of chromatin regulation in the brain, several major questions about the mechanisms of chromatin memory and their implication in brain functions and diseases remain open and need experimental work to be addressed. In particular, studies on the formation of chromatin memory in brain cells with a better temporal resolution (from minutes/hours to days or months after exposure) coupled with genome-wide characterization of binding profiles of AP-1 TFs, histone modifications and cell-type specific and constitutive candidate TFs are needed. Such experiments should ideally be conducted in vivo but also in vitro using primary cultures, differentiated ESCs or even brain organoids, and analysis may focus on activated cells to maximize the detection of effects. Experiments designed to identify and characterize the temporal dynamics of chromatin memory formation and maintenance should also help clarify the stability of changes. It is also still unclear how long after exposure changes in chromatin can be considered stable or still a remnant of regulatory dynamics operating after stimulation. Further, studies characterizing the acquisition of chromatin memory in brain cells in response to the same stimulus but at different period of life  should inform on the effect that time of exposure can have on the vulnerability or resilience of the genome to lasting changes in chromatin and whether this correlates with cognitive and behavioral alterations.The available evidence at the moment suggests that chromatin memory is a more widespread phenomenon than previously anticipated. Therefore, its study in the context of brain functions should not be limited to neurons, unlike done so far, but should also include glial cells such as microglia, and even epithelial cells, since environmental exposures are likely to affect all cell types in the brain. Further, multiple brain regions in addition to classical hippocampus, such as the medial pre-frontal cortex should be examined. Critically, the functional consequences of chromatin memory in the brain remain to be clarified but likely involve transcriptional programs that affect brain cells physiology and therefore, behavior and cognition. The manipulation of regulatory sequences with chromatin memory in vitro or in vivo could inform on their contribution to transcriptional programs after experience and to changes in behavior and cognition.Finally, while accumulating evidence strongly supports the existence of chromatin memory in brain cells, this evidence is mostly derived from animal models, rodents in particular. Therefore, there is also a need to address the existence of chromatin memory, its mechanisms and potential functional implications in human brain cells. In this regard, the use of neurons and glia derived from induced pluripotent stem cells (iPSCs) in culture or in the form or organoids coupled with different types of exposure should provide valuable models to characterize chromatin memory in human brain cells. We anticipate that mechanisms of chromatin memory identified in rodents also operate in human cells, however, direct experimental evidence is needed to support such claim. We postulate that lasting changes in chromatin structure and organization in human cells, acquired during our life time, might not just influence cognition and behavior but also the likelihood for the development of psychiatric and mood disorders as well as the origin and evolution of neurodegenerative diseases.


The non-vesicular transport of lipids between organelles mediated by lipid transportproteins (LTPs) is a key determinant of organelle biogenesis and function. Despiteperforming a vital function in organelle homeostasis, none of the LTP-encoding genesidentified so far are truly essential, even in the simple genome of yeast, suggestingwidespread redundancy. In line with this fact, it has been found that a number of LTPshave overlapping functions, making it challenging to assign unique roles for an individualLTP in lipid distribution. In our genetic screens under stringent conditions in which thedistinct function of an LTP might become essential, we stumbled upon Csf1, a highlyconserved protein with a Chorein-N motif found in other lipid transporters and unraveled anew function for Csf1 in lipid remodeling and homeoviscous adaptation of the lipidome.Here, we further speculate on the potential mechanisms of how the putative function ofCsf1 in lipid transport could be intimately connected to its role in lipid remodelingacross organelles. Lipid transport is a critical cellular process that underlies the biogenesis and distinctfunctions of organelles. The distribution of lipids from their site of synthesis, mainly theendoplasmic reticulum (ER), to a destination organelle can be mediated by vesicles, or bylipid transport proteins (LTPs) in a non-vesicular fashion. LTPs, which operate at sites ofclose contact between organelle membranes, are thought to mediate the bulk of lipidtransport by solubilizing lipids from a donor membrane and transferring them to an acceptormembrane .There aIn our recent study , we re-eCSF1 becameessential in an artificial condition in which phosphatidylethanolamine (PE) was synthesizedexclusively in the mitochondrial inner membrane and phosphatidylcholine (PC) in peroxisomes.While Csf1's absence did not cause a defect in PE and PC biosynthesis even in theseartificial conditions, a cue to its role came from a previous finding that it is requiredfor growth in cold temperature  . One posThe recent elucidations and predictions of 3D structures for several Chorein-Nmotif-containing proteins like Vps13 and Atg2 has revealed a hydrophobic groove fold thatcan facilitate bulk transport of lipid molecules between membranes . As coldWith multiple observations pointing to a very likely role for Csf1 in lipid transport, ourdiscovery of its requirement in homeoviscous adaptation has opened new avenues to understandthe molecular principles of how cells coordinate lipid transport and lipid remodeling. Ifindeed Csf1 turns out to be an authentic LTP, understanding its regulation in vivo duringnormal and cold temperatures, the functional significance of its interaction withmitochondria and PM, the selective nature of the hydrophobic groove as well as thedirectionality of lipid movement will significantly enhance our knowledge on lipid transportmechanisms at membrane contact sites.


KCNQ1 in two patients  (8%). Variants of uncertain significance were identified in SCN5A, KCNJ5, AKAP9 and ANK2 in four patients (16%). Although the results are limited by the small number of patients included in the study, they highlight a minor contribution of LQTS genes for QTc prolongation in HCM patients. The screening for ion channel genes mutations may be considered in HCM patients with prolonged QTc unexplained by any other cause. This in-depth molecular diagnosis may contribute to improve risk stratification and treatment planning.Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease, characterized by the presence of unexplained left ventricular hypertrophy. This condition is often associated with electrocardiographic abnormalities including QTc prolongation occurring in 13% of patients. The main explanation for prolonged QTc in HCM is myocardial hypertrophy and the related structural damage. However, other mechanisms, including long QT syndrome (LQTS) genes mutations, may be involved. In the present study we explored the hypothesis of a distinct genetic basis underlying QTc prolongation in HCM by investigating the potential co-inheritance of pathogenic gene variants associated with LQTS and HCM. For this purpose, starting from a cohort of 150 HCM patients carrying pathogenic variants in sarcomere genes, we selected 25 patients carrying a QTc prolongation unexplained by any other cause. The QTc was considered prolonged if greater than 450 ms in males and greater than 470 ms in females. The NGS analysis was performed with Illumina TrueSight Cardio panel genes on Illumina MiniSeq platform. We identified pathogenic/likely pathogenic variants in the  Hypertrophic cardiomyopathy (HCM) is a condition characterized by the presence of left ventricular asymmetric hypertrophy and is one of the most common cardiovascular genetic diseases, inherited with autosomal pattern and incomplete penetrance . This coTo explain the prolonged QTc in HCM, the role of gene mutations involved in the LQTS syndrome cannot be excluded. In fact, the additive effect of 2 disease-causing mutations has been reported in other pathological conditions . MoreoveIn the present study we investigated the possible co-inheritance of pathogenic variants associated with LQTS and HCM, exploring the hypothesis of a distinct genetic basis underlying QTc prolongation in HCM.For this study we considered a prospectively collected database, including 150 consecutive HCM outpatients carrying pathogenic variants in sarcomere genes, based on previous genetic screenings, referred to the tertiary HCM Center of Sant’Andrea Hospital, Sapienza University of Rome between January 2014 and June 2021.Each patient was evaluated considering the HCM echocardiographic findings detected by transthoracic Doppler echocardiography , the electrocardiographic findings, the clinical history and New York Heart Association (NYHA) classification, the pedigree analysis. The following echocardiographic parameters were considered: Left Ventricular (LV) end-diastolic diameter , LV maximal wall thickness , left atrial diameter , the highest maximal LV outflow tract gradient among those measured at rest, in the orthostatic position and after the Valsalva maneuver , LV ejection fraction assessed with Simpson's biplane methods . The diagnosis of HCM was based on a MWT of 15 mm unexplained by abnormal loading conditions or in accordance with published criteria for the diagnosis of disease in relatives of patients with unequivocal disease .The QTc was considered prolonged if greater than 450 ms in males and greater than 470 ms in females and it was unexplained by any other cause . ExclusiAccordingly, a total of 25 HCM patients carrying pathogenic variants in sarcomere genes and QTc prolongation not explained by any other cause was selected for the study purpose.The study complied with the ethical standards of the Declaration of Helsinki and was reviewed and approved by the institutional ethics committee of our center. Written informed consent was obtained from all participants.After a 5-min rest in the supine position, each HCM patient underwent 12-lead surface ECG at 25 mm/s. The latter was used to manually measure each interval length in every lead with a digital caliper . The following intervals were obtained: R-R, Q-R-S, Q-Tend , Q-T peak , Tpeak-Tend , J-Tend , J-Tpeak . The QT interval was measured from the onset of the QRS complex to the end of the T wave. To minimize the possible confounder of different heart rate values, each interval was also corrected according to Bazett's classic formula. In patients with more than one ECG with QTcBazett ≥ 450 ms during the study period, only the first ECG was chosen.AKAP9, ANK2, CACNA1C, CAV3, KCNE1, KCNE2, KCNH2, KCNJ2, KCNJ5, KCNQ1, SCN4B, SCN5A, SNTA1, TRDN). The known variants identified were classified according to the American College of Medical Genetics and Genomics criteria  to evaluate the presence of pathogenetic variants in LQTS related genes. Genomic DNA included was extracted from peripheral whole blood of the patients and used for the preparation of libraries with the TruSight Cardio Panel Kit (Illumina) and subsequently analyzed on the Illumina MiniSeq Sequencer. The TruSight Cardio Panel kit exploiting the NGS methodology provides complete coverage of 174 genes associated with known hereditary heart diseases, including 14 related to monogenic LQT syndromes  in two patients (8%). Moreover, few uncertain significant variants were identified in SCN5A, KCNJ5, AKAP9 and ANK2 in four patients (16%).We identified pathogenic/likely pathogenic variants in the LQTS related gene NOS1AP on QTc interval duration (TNNI3 and KCNQ1 genes (de novo CALM2 mutation with LQTS and HCM (The presence of long QTc is found in about 13% of HCM patients  and is cduration . MoreoveQ1 genes . Another and HCM . TherefoKCNQ1 in two patients. We also identified uncertain significant variants in SCN5A, KCNJ5, AKAP9 and ANK2 in four patients. Therefore, the results of our study highlight a minor contribution of LQTS genes in HCM patients carrying prolonged QTc, and they suggest that other mechanisms should be taken into consideration to explain the electrocardiographic abnormality of HCM patients.In the present study, performed in highly selected HCM patients to further explore the existence of a distinct genetic origin of prolonged QTc, we identified pathogenic/likely pathogenic variants in the LQTS related gene Cardiac myocytes are well coupled electrically by gap junctions in normal hearts, resulting in a rapid propagation of the electrical impulse and repolarization. Experimental models reveal that cardiac pathologies induce heterogeneous changes in myocyte electrophysiology, which primarily affect the repolarization phase of the action potential, leading to increased transmural dispersion of ventricular repolarization .The lengthening of the QT interval is mostly linked to mechanical overload and to the ensuing compensatory hypertrophy. Notably, the acquired lengthening of the QT interval is not associated with cardiac hypertrophy of any other origin than mechanical overload, such as cardiac hypertrophy due to thyrotoxicosis. By contrast, the inherited long-QT, and the drug- or metabolic-induced acquired long-QT are not merely associated with cardiac hypertrophy.The possible mechanisms underlying increased QT dispersion in HCM are ion channels abnormalities  and gap The small number of patients enrolled in the study, selected at one single tertiary HCM center, represents an obvious limitation. Further studies in larger HCM cohorts are warranted to reinforce the current evidence.Although the results of our study are limited by the small number of patients included in the analysis, they highlight a minor contribution of LQTS genes mutations in HCM patients carrying a QTc prolongation and they suggest that other causes could be involved. On the other hand, it is known that patients with HCM-related genes mutations may exhibit impaired QT variables even in the absence of ventricular hypertrophy , 24.Based on the results of the present analysis, and consistently with recent evidence obtained through a comprehensive genetic testing for cardiomyopathies and arrhythmias , we sugg


First described in the early 20th century, diurnal oscillations in stem cell proliferation exist in multiple internal epithelia, including in the gastrointestinal tract, and in the epidermis. In the mouse epidermis, 3- to 4-fold more stem cells are in S-phase during the night than during the day. More recent work showed that an intact circadian clock intrinsic to keratinocytes is required for these oscillations in epidermal stem cell proliferation. The circadian clock also regulates DNA excision repair and DNA damage in epidermal stem cells in response to ultraviolet B radiation. During skin inflammation, epidermal stem cell proliferation is increased and diurnal oscillations are suspended. Here we discuss possible reasons for the evolution of this stem cell phenomenon. We argue that the circadian clock coordinates intermediary metabolism and the cell cycle in epidermal stem cells to minimize the accumulation of DNA damage from metabolism-generated reactive oxygen species. Circadian disruption, common in modern society, leads to asynchrony between metabolism and the cell cycle, and we speculate this will lead to oxidative DNA damage, dysfunction of epidermal stem cells, and skin aging.  This Concise Review describes how the circadian clock regulates proliferation of stem cells. Disruption of the circadian clock, which is common in modern society, may influence the function of adult stem cells, contributing to diseases such as aging and impaired regeneration.1 including hematopoietic stem cells,2 intestinal stem cells,3 and various populations of skin stem cells.5 This Concise Review focuses on the role of the circadian clock in regulating the proliferation of interfollicular epidermal (IFE) stem cells, an important model for understanding how and why the circadian clock regulates adult epithelial stem cells.Work over the last 15 years has revealed a role for the circadian clock in regulating properties and function of adult stem cells,6 In addition to maintaining tissue homeostasis, epithelial stem cells are activated to coordinate tissue repair after injury and wounding.7 Skin contains a number of different types of stem cells.8 These include quiescent hair follicle bulge stem cells, which are intermittently activated to supply cells for the growing hair follicles, as well as actively proliferating Lgr5-positive stem- and progenitor cells of the secondary hair germ , but later work revealed that all or nearly all cells of the body contain an active circadian clock. The SCN clock, which is set by light signals from the retina, synchronizes clocks in peripheral tissues through hormonal and direct neuronal mechanisms  and REV-ERB , which activate and inhibit, respectively, expression of BMAL1. This core transcriptional network also creates diurnal oscillations in the expression of approximately 10% of the transcriptome in each tissue, often affecting genes that participate in the characteristic physiological function of each organ.27At a molecular level, the clock is created by a negative feed-back loop where heterodimeric bHLH-PAS transcription factors, BMAL1 and CLOCK, drive the expression of their own inhibitors, PER  and CRY (CRY1 and 2) .26 BMAL128 Systematic analysis across 32 human cancers revealed widespread clock gene alterations and disrupted circadian rhythms that correlated with oncogenic pathways and survival.29 Consistent with these findings, studies in mice have shown that jet lag paradigms or genetic circadian disruption accelerate lung30 and intestinal32 tumorigenesis.Epidemiological studies have linked shift work and circadian disruption to a number of diseases that involve epithelial stem cells, including skin diseases and cancer.33Although the circadian clock has important roles in adult epithelial stem cells, there is little evidence for its importance in epithelial stem cells during embryonic development. Mice with mutations in core clock genes are born with normal skin and gastrointestinal system and the circadian clock does not seem to be fully functional in the skin until postnatally.34 Thus, for the slow-cycling hair follicle stem cells, the circadian clock may help select cells for activation during hair growth. MPZL3, a nuclear-encoded, mitochondrially localized, immunoglobulin-like V-type protein, has also been proposed as a link between the circadian clock and hair follicle cycling.35 In the hair follicle bulge, the clock contributes to the heterogeneity of quiescent stem cells by regulating the sensitivity to inhibitory (TGFβ) and activating (Wnt) signals.36 In mice deleted for Bmal1 globally, the secondary hair germ cells downregulate REV-ERBα, which leads to upregulation of the G1 cell cycle inhibitor p21; this may arrest the secondary hair germ cells at the G1-S cell cycle stage and delay initiation of hair growth by several days.37 Loss of PTEN, a major regulator of the PI3K-Akt pathway, leads to upregulation of BMAL1 and increased number of hair follicle stem cells, which expand in a BMAL1-dependent manner.38As hair growth is initiated, the circadian clock genes are expressed to high levels in the secondary hair germ of the early anagen follicle, which is composed of actively cycling stem- and progenitor cells.Bmal1 was specifically deleted in keratinocytes, no delay in hair growth was observed,39 suggesting that the clock modulates hair growth through non-keratinocyte skin cells or through systemic effects.40 Another study pointing to indirect regulation of hair follicle stem cells showed that light stimulus to the eyes activates retinal ganglion cells, which project to the SCN. The SCN then activates sympathetic nerves projecting to skin where they promote the proliferation of hair follicle stem cells via hedgehog signaling.41Later studies showed that when 43 Later studies characterized the daily cycling of epithelial stem cells in multiple epithelial organs including the gut44 and human skin.45 In mice, the proliferation of IFE stem cells oscillates over the day with maximum fraction of stem cells in S-phase at night between 11 PM and 3 AM (ZT17-21) and minimum fraction of cells in S-phase during the day between 11 AM and 3 PM (ZT5-9)  .39 Maxim-phase46 , indicat47 Circadian S-phase in IFE stem cells is obliterated with deletion of both Cry genes47 or Bmal1, either globally or specifically in keratinocytes39  response and UVB-induced DNA damage in IFE stem cells. When UVB is applied to mouse skin at night, there is a greater accumulation of cyclobutane pyrimidine dimers, 6-4 photoproducts, and double-stranded DNA breaks in IFE stem cells than when UVB is applied during the day.39 a cell cycle stage sensitive to UVB-induced DNA damage. In addition, the clock controls the expression of XPA, a rate-limiting enzyme in DNA excision repair, which is lowest during the night, indicating that DNA excision repair is less efficient at night.60 Daytime-restricted feeding decreases the expression of Xpa and reverses the daily rhythms in sensitivity to UVB-induced DNA damage.20 One of the consequences of this diurnal variation in UVB-induced DNA damage is that more skin tumors form in mice when UVB is applied at night than during the day.47The enhanced sensitivity to UVB-induced DNA damage at night may link to the circadian clock-regulation of IFE stem cell proliferation because at night most cells are in S-phase,Why mammals have evolved diurnal variation in epithelial stem cell proliferation remains an area of active research. It is hard to appreciate how diurnal variation in stem cell proliferation during homeostasis would be beneficial for the formation of epithelial barriers, which are formed over the course of several days during differentiation. Here, we review 3 of the hypotheses that have been proposed to explain the circadian clock-controlled oscillations in epidermal stem cell proliferation.61 Consistent with this idea, the DNA repair system is more active during the day than at night in such organisms. Because skin among other mammalian organs is uniquely exposed to UV radiation, this possibility deserves consideration for the mammalian skin clock. In particular, as previously discussed, mouse IFE stem cells have the lowest fraction of cells in S-phase and most active DNA excision repair during the day, which would fit with the solar exposure model.47Studies in simpler organisms, which out in nature are fully exposed to solar radiation during the day, suggest that the clock has evolved to better deal with solar-induced DNA damage.46 and diurnal humans62 are antiphasic. So, while mice may be better protected during the day, clock-regulation of IFE stem cells may make humans more sensitive to UVB-induced DNA damage during the day than the night.Yet, there are several reasons why adaptation to solar exposure is an unlikely explanation for the diurnal rhythms in mammalian epithelial stem cell DNA replication. First, in the wild, mice and other rodents are well protected from sunlight by their fur, and being nocturnal, they stay out of the sunlight during the day. Second, the diurnal rhythms in DNA replication and repair are not limited to epidermal stem cells but are also found in other epithelia that are not exposed to UV. Third, the diurnal cell division cycles in the epidermis of nocturnal rodents33The epidermis is a barrier protecting us from harmful environmental effects. In addition to protection against solar radiation, the epidermis wards off pathogenic microorganisms, allergens, toxic pollutants, and physical injury. Upon injury, the regenerative response features multiple cell types, including hair follicle and IFE stem cells, fibroblasts, and immune cells, all of which are activated with increased proliferation and migration toward the injury site. Multiple aspects of the skin immune and injury response are under the influence of the circadian clock.64 and in mice lacking BMAL1,65 possibly because of increased oxidative stress.66 The interferon response to a TLR7 agonist is highly diurnal and regulated by BMAL1 in the skin.52 Epidermal keratinocytes produce the chemokine CXCL14 in a circadian pattern, which suppresses skin bacterial proliferation in a diurnal manner through the regulation of the innate immune system.67 The migration of dermal dendritic cells into lymphatic vessels is under the control of the chemokine CCL21 and adhesion molecules, which in turn are directly regulated by the circadian clock.68Skin wound healing is impaired in animals with circadian disruption69 and studies have shown that wounds incurred during the active phase heal faster than wounds incurred during the rest phase.70 It is, therefore, possible that an increased proportion of IFE stem cells in S-phase during the active phase has evolved to facilitate repair and regeneration in response to wounds and other cellular stressors. During homeostasis, the daily oscillations could be important as constant high activity of stress response pathways, immunity, and DNA synthesis, could increase the propensity to diseases such as autoimmunity and cancer.The likelihood of encountering harmful external stressors is greater during the active phase than during sleep72 In mammals, the metabolic environment of day and night varies drastically, in part due to alterations in food intake and physical activity.Another hypothesis is that the daily rhythms in IFE stem cell proliferation have evolved to adapt to the internal environment of the body. This concept is derived in part from experiments in budding yeast showing that metabolic cycles are temporally compartmentalized and synchronized with other cellular functions.73 and over an organism’s lifetime may lead to accumulation of cellular damage, including DNA mutations. The reliance of cancer cells and other highly proliferative cells on glycolysis may be to minimize cell-damaging oxidative stress.75Such a model is also more consistent with the antiphasic relationship between the behavior of IFE stem cells in nocturnal and diurnal animals than the solar exposure model. In particular, the pattern of DNA synthesis and repair in IFE stem cells could decrease accumulating DNA damage from oxidative metabolism. Reactive oxygen species (ROS) are a normal byproduct of mitochondrial respiration39 Furthermore, the expression of many OXPHOS genes in skin is BMAL1-regulated, and in Bmal1-mutant mice, ROS levels rise and become invariant over the day.39As S-phase is a cell cycle phase that is sensitive to ROS-mediated damage, there would be an advantage to having the fewest cells in S-phase during the time of maximum oxidative stress. Consistent with this model, the expression of OXPHOS genes and skin levels of reactive oxygen species (ROS) are antiphasic to the peak in IFE stem cell S-phase.76 These experiments showed that IFE stem cells favor glycolysis during the night when the proportion of S-phase cells is highest and OXPHOS during the day when the proportion of S-phase cells is lowest. These daily fluctuations in the NADH/NAD+ redox ratio depend on BMAL1 . Initial observations of this phenomenon go back 100 years, long before the discovery of the molecular circadian clock mechanism. Subsequent work in the late 20th century characterized these daily cell-cycle oscillations in a variety of epithelial tissues and species at a phenomenological level. But it was not until 10 years ago when it was shown that the intact circadian clock is required for these cell cycle oscillations in epidermal stem cells. Although the circadian clock within keratinocytes is required for these stem cell oscillations, it is not known whether the underlying mechanisms represent direct control of cell cycle regulator expression by the circadian clock or oscillating external signals emanating from keratinocytes and acting on IFE stem cells. Epidermal stem cell proliferation oscillations are dampened when proliferation rate is increased, such as in skin inflammation. Presumably, the circadian clock-mediated dampening in S-phase during the rest phase is suspended when there is an increased need for stem cell proliferation. In vivo studies indicate that the circadian clock coordinates intermediary metabolism and the cell cycle in epidermal stem cells such that glycolysis dominates over oxidative phosphorylation when most cells are in S-phase. This clock-regulated coordination between metabolism and the cell cycle may shield S-phase DNA from damage caused by oxidative metabolism-generated ROS. This hypothesis implies that long-term asynchrony between metabolism and the cell cycle—which can occur during jet lag or when feeding occurs at unusual times of the day—may accelerate epidermal stem cell dysfunction and skin aging.


Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung with poor survival. The incidence and mortality of IPF are rising, but treatment remains limited. Currently, two drugs can slow the scarring process but often at the expense of intolerable side effects, and without substantially changing overall survival. A better understanding of mechanisms underlying IPF is likely to lead to improved therapies. The current paradigm proposes that repetitive alveolar epithelial injury from noxious stimuli in a genetically primed individual is followed by abnormal wound healing, including aberrant activity of extracellular matrix–secreting cells, with resultant tissue fibrosis and parenchymal damage. However, this may underplay the importance of the vascular contribution to fibrogenesis. The lungs receive 100% of the cardiac output, and vascular abnormalities in IPF include (a) heterogeneous vessel formation throughout fibrotic lung, including the development of abnormal dilated vessels and anastomoses; (b) abnormal spatially distributed populations of endothelial cells (ECs); (c) dysregulation of endothelial protective pathways such as prostacyclin signaling; and (d) an increased frequency of common vascular and metabolic comorbidities. Here, we propose that vascular and EC abnormalities are both causal and consequential in the pathobiology of IPF and that fuller evaluation of dysregulated pathways may lead to effective therapies and a cure for this devastating disease. Idiopathic pulmonary fibrosis (IPF)  is a proThe pathogenesis of IPF is considered to result from injury to the alveolar epithelium by a range of insults including cigarette smoke, gastric acid, air pollution, and viruses in a genetically primed individual, driving aberrant activity of mesenchymal cell populations . SubsequVascular abnormalities in IPF include the development of vascular connections known as pulmonary-bronchial artery anastomoses and are accompanied by abnormal populations of endothelial cells (ECs) and vascular comorbidities such as pulmonary hypertension (PH) and coronary artery disease  Figure . HoweverMargaret Turner-Warwick described the expansion of the vasculature with numerous pulmonary-bronchial arterial anastomoses in diffuse pulmonary fibrosis as early as 1963  Figure . These aIt is, therefore, unclear whether the VEGF-inhibiting effects of nintedanib are undermining or augmenting its antifibrotic effects. In advanced disease states, the overall effect is a net reduction in pulmonary vascular surface area, which, in part, explains the increased frequency of PH. This loss of pulmonary vascular surface area is associated with distinct cellular changes in ECs, vascular smooth muscle cells (VSMCs), and pericytes, all of which may directly contribute to the pathogenesis of IPF .Alveolar injury is likely to directly injure ECs . ECs shaIn IPF, an abnormal, ectopic population of COL151A-expressing ECs have been identified using single-cell RNA sequencing . These CNumerous animal models have shed light on the role of the endothelium in pulmonary fibrosis. In one mouse model, pulmonary capillary ECs have been shown to support epithelial cell proliferation and alveolar regeneration through the secretion of factors including MMP-14, a process that is dependent on the activation of VEGF receptors present on ECs . BleomycECs may also be directly involved in fibrogenesis through endothelial-mesenchymal transition (EndMT) whereby they develop a contractile, α-smooth muscle actin–expressing phenotype . This efThese data suggest that following lung injury, damaged ECs secrete profibrotic mediators and are capable of migrating to the parenchymal regions of lung and transdifferentiating into pathogenic myofibroblasts. This model implies that ECs play a fundamental role in pathogenesis, serving as much more than mere bystander cells.VSMCs are highly contractile cells that constitute the structural form of blood vessels. There is abnormal proliferation and distribution of smooth muscle cells in IPF tissue , and undVSMCs isolated from patients with IPF show a hyperproliferative state and produce more collagen I via induction of reactive oxygen species compared with cells from control donors. This effect can be blunted by the antifibrotic therapeutic pirfenidone . The proAdenoviral overexpression of TGF-β1 in the lungs of mice leads to signaling crosstalk between ECs, VSMCs, and fibroblasts, which not only results in increased rates of apoptosis of ECs but also induces activation and proliferation of VSMCs. Importantly, defective bone morphogenetic protein receptor 2 (BMPR2) signaling has been implicated in VSMC activation and proliferation . In partPericytes are mesenchymal cells, closely related to fibroblasts, that form extensive physical contacts with ECs  with which they share a basement membrane . PericytThe alveolar epithelial-capillary basement membrane (BM) is a vital scaffold structure that supports normal repair of the alveolar parenchyma, and loss of BM integrity has been known for many years to be a fundamental pathogenic change in IPF . Loss of1 and α2 collagen IV chains. This change in BM composition limits myofibroblast migration and promotes their survival, which could contribute to myofibroblast persistence and sustained activity in fibroblastic foci  is expressed in vascular ECs and produces NO via a calcium/calmodulin pathway. NO subsequently activates soluble guanylyl cyclase (sGC) to generate cGMP, which in turn activates protein kinase G (PKG), leading to modulation/reduction of intracellular calcium concentration, leading to EC permeability, smooth muscle relaxation, and the inhibition of platelet aggregation . When eNThe sGC stimulator riociguat targets the NO/cGMP pathway and is licensed for use specifically in chronic thromboembolic pulmonary hypertension and also has demonstrable antifibrotic effects in animal models , 66. In Inhaled NO gas has recently been shown to improve exercise capacity (as assessed by ability to undertake moderate to vigorous activities) and was well tolerated in patients with ILD and PH (ILD-PH) where IPF represented the largest subgroup of ILD in a phase IIb RCT . HoweverEndothelin (ET) is a potent vasoconstrictor with vascular remodeling properties; therefore, ET antagonists are used in the treatment of idiopathic PAH. ETA and ETB receptors are expressed on alveolar epithelial cells and fibroblasts, both of which, under certain disease conditions, are capable themselves of ET synthesis . ET can The dual ETA and ETB receptor antagonist bosentan was compared with placebo in 158 patients with IPF in the BUILD-1 study. Bosentan was well tolerated; however, the trial did not meet its primary endpoint of improvement in 6-minute walk distance (6MWD) . HowevercAMP is an intracellular messenger formed by adenylate cyclase. In the endothelium, cAMP functions to maintain barrier junction integrity and permeability (through a combined effect with Rho) and vascular smooth muscle tone . In humaThe nonselective PDE4 inhibitor roflumilast is currently in use in chronic obstructive pulmonary disease and has proven effective in bleomycin mouse models in limiting fibrosis and vascular remodeling . HowevercGMP is a parallel intracellular second messenger to cAMP with similar functional effects in some systems. cGMP is catabolized by a number of PDE enzymes, with PDE5 being the most therapeutically valuable and a standard target in the treatment of PAH. Inhibition of PDE5 in in vitro models with sildenafil prevents TGF-β–induced EndoMT and VSMC-mesenchymal transition through downstream signaling on ERK1/2 and SMAD, providing further evidence of a beneficial antifibrotic effect of these pathways .In a small open-label study, beneficial effects of sildenafil on 6MWD observed in patients with IPF-PH suggested that enhancing NO/cGMP pathways has therapeutic potential in this condition . Subsequ2 (PGE2), PGI2 (prostacyclin), and PGD2, all formed from arachidonic acid (2 and converted to PGH2 by the action of cyclooxygenase 1 (COX-1) (constitutive) or COX-2 (inducible). PGH2 is converted to its respective prostanoid by the action of the site-specific synthase. In the vasculature, prostacyclin is the primary prostanoid produced, largely because of coexpression of COX-1 and prostacyclin synthase by ECs  (Prostanoids have long been investigated as possible antifibrotic mediators. Reduced levels of PGEwith IPF . In genewith IPF . This efF (CTGF) . cAMP alF (CTGF) . This syF (CTGF) . In VSMCF (CTGF) . In addiF (CTGF) .2 is a central component of the inflammatory response in humans. While a multitude of cells release the wider range of prostanoids, PGE2 is considered a critical regulator of inflammation, and inhibition of PGE2 at the site of inflammation explains much of the therapeutic benefit of NSAIDs. Animal models and in vitro experiments on human lung fibroblasts have demonstrated reduced production of COX-2–dependent PGE2, which may be explained by epigenetic changes in patients with IPF and the milieu of chemokines such as CCL2, which inhibits PGE2 release  expression in fibrotic tissue. PGE2 can inhibit fibroblast proliferation and ECM production through the EP2 and EP4 receptors in a cAMP/PKA-dependent manner; however, higher PGE2 concentrations can have a profibrotic effect via the EP1 receptor (through downregulation of cAMP) and the EP3 receptor . PGE2 can also inhibit the effect of TGF-β and the SMAD pathway. Administration of exogenous PGE2 in mice has a protective effect against bleomycin-induced fibrosis , leading to phosphorylation of myosin light chains to reorganize the actin cytoskeleton, promoting cell contraction, motility, and adhesion in inflammatory cells, smooth muscle cells (notably VSMCs that are important for regulating vascular tone), and platelets , 113. TwA mechanistic link has been demonstrated in shared pathways in RhoA activation and PDE4 via A-kinase anchoring protein 13 (AKAP13) . AKAP13 In animal models of fibrosis, inhibition of ROCK using fasudil  and the experimental compound Y-27632 attenuates fibrosis and vascular remodeling , 121. InThere are a number of potential mechanisms through which fibrosis and abnormal clotting may occur. Tissue factor initiates the extrinsic coagulation pathway, is highly expressed by alveolar epithelial cells in patients with IPF, and generates lung-tissue fibrin deposits, which serve as a platform for inflammatory cells and profibrotic cytokines, enhancing their accumulation at sites of injury . This envβ6 integrin  with placebo, demonstrated harm, and this detriment was due to accelerated fibrosis rather than bleeding complications . Recent ne model .vβ1 and αvβ6, which are implicated in PF and have important effects on EC function (Other EC-relevant pathways that have been investigated in PF include (a) autotaxin, the enzyme responsible for generation of the profibrotic lipid mediator LPA, which also has phosphodiesterase activity and is highly expressed by ECs; (b) the integrins, including αfunction ; (d) CTGfunction . Specififunction .It is evident from cell and molecular biology that the vasculature, and related signaling pathways, have an important role in the development of IPF. If the vasculature is playing a prominent role in the pathogenesis of IPF, one would hypothesize that there would be substantial systemic disease associated with IPF. While PH is a well-recognized complication of IPF, other comorbidities are gaining prominence. Indeed, systemic hypertension and diabetes are common comorbidities in IPF , while cThe epithelium — the primary site of initial insult in IPF — sits in close proximity to the endothelial layer in the alveolus, separated only by a thin basement membrane, which itself is abnormal in the condition. Injury to the former will undeniably affect the latter. There is compelling evidence from various in vivo, in vitro, and ex vivo models that aberrant endothelial responses occur with resultant loss of integrity of the basement membrane, vascular remodeling, and the generation of vascular signals further driving ECM deposition, fibrosis, and parenchymal lung damage. Whether it is the epithelial signal to the endothelium that initiates this process in its entirety, or whether the endothelium is the primary culprit, remains an area for development in the field of fibrosis research. However, given the findings, one cannot ignore the potential role of the circulation in the pathobiology of IPF.The numerous disrupted vascular-relevant signaling pathways present in IPF are now being explored in greater detail and mapped more clearly. The specific targeting of these pathways — especially with prostanoid agents and phosphodiesterase inhibitors — represents an expanding chapter in the treatment of this challenging disease. As closer associations between IPF and numerous more common cardiovascular and metabolic conditions are made, a deeper understanding of the disease and how to treat it is likely to ensue. Therefore, strategies that target endothelial repair by focusing on reprogramming abnormal metabolic responses may ultimately provide an opportunity to prevent the progression of, or potentially even reverse, the fibrotic response.


Ion channels are key signaling proteins found throughout the body; they are critical in many, wide-ranging physiological processes, from gene expression, sensory perception and processing to the cardiac action potential. When ion channel activity goes awry, for example, via mutation, damage or disrupted homeostasis, the outcome can result in causation, development and/or maintenance of disease. Ion channel dependent diseases have been dubbed channelopathies. Recent studies on the role of ion channels in cancer biology suggest that cancer is one such channelopathy. Many ion channels have now been implicated in the cellular processes that are affected in a multitude of cancers. In the last two decades, the field of ion channel and cancer research has been growing exponentially: a combination of developments in molecular biology, genetics, electrophysiology and automation have driven an explosion in our capabilities to interrogate ion channel pathways; how, why and where they go wrong and therapeutic interventions to correct their pathophysiology in cancer. A review of this vast and rapidly developing field would require a titanic tome to merely dimple the surface of research that has ballooned recently. In lieu of that huge undertaking—for the benefit of both authors and readers - this review discusses select examples of primary, applied and clinical research, aiming to shine a light on some of the more innovative and novel findings that this exciting field is excavating. Ion channels and transporters are proteins involved in bioelectric properties of living cells and some of them are also able to modulate the intracellular ionic concentrations. The human genome includes at least 400 genes encoding for ion channels which are selectively expressed in the cellular compartments of all cells. Although ion channels have been traditionally studied for their role in controlling excitability in terminally differentiated cells such as neurons and cardiac myocytes, it is well understood that this class of proteins is involved in a large variety of biological processes ranging from cell proliferation, to metabolism and cell death. Alterations of biochemical signaling underlying these cellular processes are significant factors in carcinogenesis, thus ion channels are key factors in cancer biology. Nevertheless, research on the role of specific ion channels in controlling hallmarks of cancer has been sparse. Remarkably, in recent years, improvements of both conceptual framework and technologies have driven an exponential growth in the interest and understanding of roles of ion channels and cancer see .For example, in the now seminal work by Hanahan and Weinberg  “hallmarUsing selected hallmarks of cancer, the current review aims to capture some of the innovative technologies, novel molecules and pathways that have been uncovered during the recent years, highlighting key advances in our understanding of ion channels, their role, involvement and identification as potential therapeutic targets in cancer. With such a vast vista of new findings to cover the authors have selected examples in each of primary, applied and clinical studies to highlight particular developments in the field.Wingless-related integration site (Wnt) peptide ligands are the signaling molecules in Wnt pathways: signaling cascades shown to be critical in embryonic development, controlling processes like cell proliferation, differentiation and apoptosis . When WnIndeed, now for over three decades, Wnt peptides have been shown to be involved in cancer (for a review see 2+ gradients (possibly upon opening of the TRPM8 Ca2+ channel).Thus, in a study by n), but also low volume, rapidly exchanged and steady-state applications of scarce Wnt peptide directly at the planar patch clamp recording site. Each recording site is fed by a microfluidic channel to apply low volumes (µLs) of recording solutions directly to the recording site micro-environment . Using this novel application of APC technology, the authors showed that even low concentrations (2 nM) of Wnt peptides had subtle, slow-developing ion channel activating effects that initiated the Wnt signaling cascade in cancer cell lines, effects they argued would likely be missed using MPC due to the low number of biological repeats that can be feasibly obtained with this approach . APC notment see .The schematic shows the microfluidics inlets and throughflow waste reservoir for application of cell suspensions (orange spheres), intracellular (wavy blue pattern) and extracellular solutions (solid light blue) to the patch clamp recording site. In addition to the microfluidic flow channels, each recording well also houses the reference (labelled red rectangle) and measuring electrodes (labelled red rectangle), completing the electrical circuit between cell and feedback amplifier. This is one of 16 or 48 recording wells in a planar array in a recording plate (QPlate) used on the QPatch, an APC made by Sophion Bioscience.Intracellular organelles—the endoplasmic reticulum (ER), Golgi apparatus, nucleus, endosomes, lysosomes and mitochondria - have several key cellular activities, like gene expression, cell metabolism, motility and apoptosis. Cancer cells often “hijack” such cellular processes, leading to dysfunctional metabolism, cell proliferation, apoptosis and ultimately tumorigenesis.Several ion channels are found in organelles and their dysfunction can lead to disease, including metabolic and neurodegenerative diseases and cancers . Table 1inside the cell require highly developed and adapted manual patch clamp (MPC) techniques (Our capabilities to record organelle ion channel (OIC) currents has made significant technological progress in recent years: unlike typical cell plasma membrane channel recordings, organelle ion channel recordings on membranes chniques . Anotherchniques ; or, viachniques  that canV1.3 ion channels - found in the Golgi apparatus, nucleus and mitochondria - studies have shown the increasing importance of KV1.3 channel involvement in cancer tumorigenesis, cell proliferation and apoptosis (With these improved techniques numerous organellar ion channels have been identified as key elements in cellular dysregulation leading to cancers. Focusing on Kpoptosis .V1.3. It has long been known that cell membrane located KV1.3 plays a key role in the activation and proliferation of immune cells (leukocytes and T cells). Conversely, mitochondrial KV1.3 has been shown to promote cell death and apoptosis in cancer cells  revealing that cancer cells/tumors exposed to Kv11.1 agonists activated a senescent-like phenotype without generating significant side effects. Although these cells are metabolically active, cellular senescence is characterized by a permanent loss of the proliferative ability thus it is considered a powerful anticancer mechanism  signaling pathways [reviewed in V1.7 for non-opioid, non-addictive chronic pain medications (The voltage gated sodium channel (VGSC) Naications .V1.7 primary and applied research, V1.7 was selectively over-expressed in medullary thyroid cancer (MTC) but was not found in non-cancerous thyroid tissue. The group used both immortalized MTC cell lines and tissue from cancer patients to confirm this overexpression.Building on this wealth of Nav1.5 in breast cancer cells  is an activator of Kir6/SUR2. By binding specifically to SUR2, Minoxidil causes hyperpolarization of smooth muscle cells resulting in vasodilation by reducing Ca2+ influx through voltage-gated Ca2+ channels (®).Kir6.1 or Kir6.2 are the functional units for which either SUR1 or SUR2 are necessary activating subunits. The selective expression of the sulphonylurea receptor (SUR) subunits makes the channel relevant for specific physiological functions. The Ki6/SUR1 complex is best known for its involvement in insulin secretion while Kir6/SUR2 activity plays a role in regulation of the vascular tone . For thechannels . Later, 2+ entry resulting in a deregulation of metabolism and increased mitochondria-dependent reactive oxygen species (ROS) production . Elevated TRPV6 has been seen in several cancers  and its activity is linked to the calcium-dependent transcription of NFAT which has been identified as a key pathway in these cancers .Blarina brevicauda), shortened to the C-terminal 13mer peptide (SOR-C13), was shown to be a potent TRPV6 blocker (IC50 = 14 nM). Further, SOR-C13 binding and blocking of TRPV6 channels inhibited tumors in ovarian and breast cancer xenograft models . VGSCs have also been found to be overexpressed in several cancer cells, and their activity was linked to cell migration and invasion  and promote proton extrusion. This process aids acidic cysteine cathepsins (Cath), released by cancer cells, which in turn leads to ECM degradation  led by Dr. Rajendra A. Badwe recently evaluated the effect of inhibiting VGSCs during surgery in patients with operable breast cancer on the recurrence of cancer. The study aimed to repurpose the VGSC inhibitor, Lidocaine, that has been universally used as local anesthetic since its approval by the FDA in 1948. During the surgery, 0.5% lidocaine was injected peritumoral prior to excision. The phase 3 trial enrolled 1,600 patients and results were published in April 2023  and overall survival (OS). The relative risk reductions associated with this therapeutic intervention were 26% and 29% for DFS and OS, respectively, when compared to the control group. Whilst this benefit was moderate, the results are clinically meaningful and open the possibility for further developments in the future. An important aspect of the study is that the intervention provides a low-cost, easily implementable, one-time procedure that can be practiced in nearly all parts of the world. A further simplifying factor is that no subgroups were identified wherein the results are significantly different from those of the full study population, suggesting a general benefit to all patients with operable breast cancer.Ion channels have critical and multi-faceted roles in cellular signaling pathways in key processes like cell division and cell death; consequently, they are inextricably linked to cancer. The large and growing body of evidence defining ion channels as critical factors in all of the “hallmarks of cancer,” coupled with novel developments in ion channel techniques and technologies, has led to an explosion of research in the last decade see , 2.With such vast vistas of research, even the most voracious and fervent students of cancer ion channel studies would not be able to cover the field adequately. This review - via select examples of primary, applied and clinical research - attempts to give the reader a scintilla of the field, by covering some of the more interesting and innovative studies that have been done in recent years.Thus, the authors’ aim was to provide a guide to exemplar key developments in our understanding and the latest tools used to prosecute ion channel targets, pathways, causes and maintenance of cancer. The proof is in the pudding, namely, the drug discovery and development pipeline: the three examples discussed here , alongside several other companies developing ion channel cancer therapeutics . The picture painted by all these extensive research endeavours is promising, and surely a life-saving ion channel cancer drug or therapy is on the horizon.


Rheumatoid arthritis (RA) is characterised by increased rates of cardiovascular disease (CVD), which represents the leading cause of death. Patients with RA presents increased prevalence of hypertension, which substantially contributes to the increased CVD burden associated with the disease. A solid pathophysiological background supports the presence of microvascular dysfunction in RA even in the absence of established CVD, while macrovascular dysfunction in the form of large artery stiffening has been further described. Janus kinase (JAK) inhibitors constitute a novel class of disease-modifying anti-rheumatic drugs (DMARDs) for the management of rheumatoid arthritis (RA). However, the vascular effects of JAK inhibitors in RA patients remain largely understudied. More recent evidence suggests higher risk of major adverse cardiovascular events with JAK inhibition compared to treatment with a TNF inhibitor, and calls for more careful consideration of potential negative effects on the cardiovascular system. The present prospective observational cohort study aims to investigate the impact of JAK inhibitors on ambulatory blood pressure and haemodynamic profile, as well as markers of micro- and macrovasculopathy among patients with RA. A solid pathophysiological background supports the presence of vascular dysfunction in RA, which is detected even in the absence of established CVDs.5 Dysregulation of autoimmunity and cumulative inflammatory load in RA leads to overexpression of adhesion molecules, pro-thrombotic, and pro-inflammatory factors, which in turn increase vascular smooth muscle cell proliferation and tone and promote the development of atherosclerosis.6 Indeed, patients with RA show impaired vascular biomarkers such as flow-mediated dilation (FMD) of the brachial artery.7 Furthermore, elevated levels of circulating biomarkers of thromboinflammation and endothelial dysfunction in patients with autoimmune inflammatory diseases suggest that an ongoing vasculodestructive process is already underway despite the absence of clinical evidence of CVD.8Rheumatoid arthritis (RA) is the most prevalent autoimmune rheumatic disorder characterised by increased rates of cardiovascular complications, which represent the leading cause of death in these patients.9 Among these patients, hypertension represents the most prevalent CVD risk factor and significantly aggravates cardiovascular prognosis.10 Despite major implications in terms of CVD development and progression, hypertension remains largely underdiagnosed and undertreated in patients with RA.11 Furthermore, the impact of anti-rheumatic treatment is often neglected and needs to be further taken into consideration. Antirheumatic treatment modalities exert divergent effects on the vasculature, with positive, neutral, or even negative effects which further contribute to the CVD complications associated with RA. While these effects have been well-described or are being intensively investigated for cornerstone treatments such as corticosteroids or biologics, the vascular effects of novel antirheumatic treatments, such Janus kinase (JAK) inhibitors, remain in comparison understudied.13 Ultimately, chronic inflammation, traditional CVD risk factors and, to a different extent, specific antirheumatic treatment modalities act synergistically to form a prothrombotic microenvironment and trigger the degeneration and remodelling of the vascular architecture.While microvascular endothelial dysfunction is considered inherent to the disease, patients with RA present increased prevalence of traditional CVD risk factors.14 In the 2019 updated European League Against Rheumatism (EULAR) recommendations for the management of RA with synthetic and biological disease-modifying antirheumatic drugs (DMARDs), it is recommended to add any biological DMARD or JAK inhibitor on top of conventional synthetic DMARDs upon presence of poor prognostic factors . Potential safety concerns secondary to JAK inhibition include a higher risk of herpes zoster infections and venous thromboembolic events .15 With regard to the latter, the effects of JAK inhibitors on the cardiovascular system are being thoroughly investigated and have not yet been specified in detail. A systematic review and meta-analysis of randomised controlled trials indicated no significant change in cardiovascular risk for patients with RA treated with JAK inhibitors at least in a short-term perspective.16 Nevertheless, the recent ORAL Surveillance trial comparing the combined tofacitinib doses with a TNF inhibitor in a cardiovascular risk–enriched population with RA showed higher risks of major adverse cardiovascular events and cancer with the JAK inhibitor than with a TNF inhibitor.17 No apparent mechanism of action has been proposed or proven currently for this safety signal.18 Despite their efficacy in the suppression of RA-related inflammation, treatment with JAK inhibitors may negatively affect CVD risk factors, such as platelet count and lipidemic profile.19 By contrast, data remain scarce regarding the impact of JAK inhibitors on blood pressure, especially ambulatory blood pressure, haemodynamic profile, and markers of micro- and macrovasculopathy among patients with RA. Notably, both the macrovasculature and the microvascular network in RA can be easily assessed by use of non-invasive techniques that are being widely applied in the field of CVDs, such as assessment of arterial stiffness and nailfold videocapillaroscopy (NVC), respectively.JAK inhibitors are the newest drug class of disease-modifying medication that have been approved for the treatment of RA. Their mechanism of action is based on the modification of the signal transducer and activator of transcription (STAT) constituents in cytokine signalling, which is implicated in the pathogenesis of RA. As a targeted low molecular mass drug that can pass the lipid bilayer of the cellular membrane, they offer the first long-term oral biologic option in RA.20 Its predictive value in terms of cardiovascular health is superior to the conventional office blood pressure measurements, as stated in current hypertension guidelines.21 Patients with RA present increased prevalence of hypertension, which along with smoking confers the highest CVD risk of all traditional CVD risk factors in this specific group of patients.9 They additionally present disturbed abnormal blood pressure rhythm, with increased prevalence of nocturnal hypertension and disturbed nocturnal blood pressure drop (non-dipping).22 Remarkably, the newest devices for ambulatory blood pressure monitoring can simultaneously record haemodynamic parameters throughout the whole 24h period, including central (aortic) blood pressure, wave reflection, total vascular resistance, as well as markers of arterial stiffness such as augmentation index (AIx) and pulse wave velocity (PWV).24h ambulatory blood pressure is a powerful predictor of CVD morbidity and mortality in the general population and in high CVD risk populations.23 Other markers of arterial stiffness include the assessment of AIx from the analysis of the wave reflections, and pulse pressure calculated by subtracting diastolic blood pressure from systolic blood pressure.24 Data from tenths of thousands of individuals have established the prognostic value of arterial stiffness in terms of CVD risk prediction.25 Arterial stiffness is closely associated with traditional CVD risk factors, predominantly age and hypertension.24 Increased PWV is an independent predictor of CVD morbidity and mortality in the general population and high risk patients such as those with hypertension, type 2 diabetes mellitus, end-stage renal disease, and elderly individuals.26 Its predictive value not only remains significant even after adjustment for Framingham risk score,27 but also enhances the utility of current risk prediction models when added on top of classical CVD risk factors.28 Arterial stiffness in RA has been addressed in several studies, with the majority concluding that markers of arterial stiffness are increased in RA individuals. However, whether and to which extent increased arterial stiffness in RA reflects the increased prevalence of traditional CVD risk factors or the consequences of chronic inflammation remain under investigation.29 Furthermore, the impact of novel antirheumatic treatments including JAK inhibitors on arterial stiffness remains unclear.Arterial stiffness is defined as the loss of arterial elasticity and compliance subsequent to mechanical and structural changes of the vascular wall. The gold-standard marker of arterial stiffness is measurement of PWV, which corresponds to the rate at which pressure waves travel downstream through the arterial wall.30 and correlate with the presence of pulmonary arterial hypertension31 and as markers of CVD risk, such as arterial stiffness33 and increased levels of uric acid.34 Capillaroscopic microvascular abnormalities are also common in patients with CVDs. Evaluation of NVC in autoimmune rheumatic diseases has been proposed as a potential tool for the prediction of microvascular heart involvement,35 while preliminary data have shown that capillary abnormalities correlate with CVD risk, either as an individual marker in high-risk patients36 or in combination with other microvascular indices in low-to-moderate risk hypertensive patients.37 Several patterns of capillaroscopic abnormalities have been previously described with NVC in patients with diabetes mellitus including capillary dilatation, avascular zones and tortuous capillaries,38 while presence of diabetes mellitus further aggravates structural and functional capillary density in patients with chronic kidney disease.39 Capillary rarefaction is particularly common in patients with hypertension, both as a cause and consequence of high blood pressure.40 Last but not least altered capillaroscopic parameters indicative of peripheral microangiopathy have been described in patients with pulmonary arterial hypertension associated with various autoimmune diseases41 or due to congenital heart disease,42 and chronic thromboembolic pulmonary hypertension.43 Dermal capillary alterations detected with NVC and their clinical significance are being thoroughly investigated in patients with RA and have been summarized in detail elsewhere.44The dermal capillary network represents an “open window” for the study of the microcirculation. NVC has been widely applied as a bedside aid in rheumatology, especially in the diagnosis and follow-up of patients with systemic sclerosis. In these patients, peripheral microvascular abnormalities detected with NVC reflect the severity of visceral organ involvement24h BP and its circadian rhythmArterial stiffness, specifically AIx and PWV which represent the most widely used markers of large artery stiffening.Microangiopathy of the nailfold capillaries.Patients’ haemodynamic profile throughout a whole 24h period Prothrombotic markersEstimated cardiovascular riskThe present study aims to provide further insight on the effects of treatment with JAK inhibitors on blood pressure and haemodynamic parameters, microvascular function, and subclinical macrovascular injury among patients with RA. More specifically, we aim to investigate whether treatment with JAK inhibitors in patients with RA induces alterations in:This is a prospective, observational cohort study, that will be conducted by the Fourth Internal Medicine Department at Hippokrateion General Hospital of Thessaloniki in collaboration with the Second Medical Department of the same hospital.45 who are eligible for treatment with JAK inhibitors  for first time, irrespective of previous administration of conventional or biologic DMARDs based on international recommendations (EULAR 2020).15This study enrols adult patients with an established diagnosis of RA according to the American Rheumatism Association 1987 Revised Criteria,46Exclusion criteria include: age <18 years, inability to comprehend and sign the informed consent, previous exposure to JAK inhibitors, concomitant active malignancy or other disease with poor prognosis, recent major adverse cardiovascular event  within the past 6 months, and stage III–IV heart failure according to the New York Heart Association (NYHA) criteria.Recording of medical history and current medical treatmentAnthropometric measurements, clinical examination and assessment of disease severity with calculation of DAS28 (Disease Activity Score in 28 joints)Evaluation of functional ability and quality of life through specifically designed questionnairesOffice blood pressure recordingEstimation of 10-year cardiovascular riskVideocapillaroscopy of the nailfold capillary networkAssessment of 24h blood pressure, its day- to night-time variation and haemodynamic parameters with the Mobil-O-Graph deviceBlood sampling, centrifugation and thawing of the supernatantRA patients eligible for treatment with JAK inhibitors who will be selected on the basis of inclusion and exclusion criteria to participate in the research protocol will undergo the following procedures, before the initiation of JAK inhibitors:All patients will be re-evaluated and the above procedures will be repeated at 3 and 6 months following the baseline visit, whilst the patients are under treatment with JAK inhibitors.21 The Mobil-O-Graph  device will be applied to all participants, which has been approved by Food and Drug Administration (FDA) and the European Union and has been validated according to the protocols of the European and the British Society of Hypertension48. Automatic BP readings will be performed at 20-min intervals during the day and at 30-min intervals during the night. Only ABPM reports with a minimum of 70% successful readings will be regarded as technically sufficient. Mean 24h, day- and night-time BP will be obtained, as well as the nocturnal fall in BP (%), also defined as “% dipping”. According to the dipping values, individuals will be classified as “dippers” (≥10%) and “non-dippers” (<10%).24h blood pressure recording is recommended as the ideal method of BP monitoring according to current hypertension guidelines.Moreover, arterial stiffness indices  and haemodynamic parameters  are simultaneously obtained at each blood pressure measurement. The mean values of the above markers will be included in the analysis.NVC is an established non-invasive method to assess the capillary vasculature of the digital arteries, which provides information on the structural and functional abnormalities of the capillaries. It is performed at room temperature (22 °C–23 °C) with the patient seated and resting for 15 minutes. NVC will be performed at the Fourth Internal Medicine Department at Hippokrateion Hospital using an Optilia Digital Capillaroscope. Pictures and videos of 15 seconds duration will be captured using the 200x magnification video camera from the 2nd to the 5th finger of each hand. The images will be analysed with Optipix capillaroscopy software 1.7.x on a computer. The NVC parameters to be measured are: capillary density, defined as the number of capillaries in the first row in 1mm (capillaries / mm), capillary width and length (μm), the presence of micro-bleeding, oedema, thrombi, and the presence of any abnormalities in the morphology or disorder of capillary architecture. These parameters will be determined on each nail and the average of all measurements for each patient will be calculated at the end. In addition to quantitative and qualitative parameters, a semi-quantitative score will be established to assess the severity of peripheral microangiopathy.Routine blood tests will be performed for the evaluation of haematological and biochemical parameters , erythrocyte sedimentation rate, and C-reactive protein.In addition, blood samples will be collected, centrifuged and frozen at −80°C. Levels of the following prothrombotic markers will be simultaneously measured with ELISA (enzyme-linked immunosorbent assay) using commercially available kits: von Willebrand Factor antigen (vWF:Ag) and activity (vWF:RCo), thrombomodulin and ADAMTS13 activity.49. In our study, the relevant algorithm from the Framingham Heart Study will be used to calculate 10-year risk of general cardiovascular disease.50Cardiovascular risk calculators specifically developed for RA patients have not been proved to be superior to the general population cardiovascular risk calculators, including Framingham Risk ScoreTo determine potential differences at 3 and 6 months following treatment with JAK inhibitors compared to baseline, in the following parameters: a) 24h BP, b) arterial stiffness, c) microangiopathy of dermal capillary network, d) prothrombotic markers.- To determine potential differences in the haemodynamic profile and the estimated 10-year cardiovascular risk of patients with RA at 3 and 6 months post-treatment with JAK inhibitors, compared to the baseline visit.- To detect potential associations of prothrombotic markers with 24h BP, arterial stiffness and microangiopathy of the dermal capillary network.A total of 30–50 patients will be studied.Data analysis will be performed using SPSS  software, version 22. Results will be expressed as frequencies for qualitative variables, and as mean±standard deviation (m±SD) or median (interquartile range) for continuous variables. Comparisons of variables will be performed between the baseline and the follow-up visits. Comparison of frequencies will be performed by Pearson chi-square test. Comparison of differences between mean values will be performed with Student’s t-test or Mann–Whitney test. Correlations between the main continuous variables will be assessed using the parametric Pearson or the nonparametric Spearman’s Rho correlation coefficient. Linear regression and logistic regression analysis will be performed to investigate whether treatment with JAK inhibitors is associated with NVC parameters and parameters obtained from 24h ambulatory blood pressure recording after adjustment for other variables. A probability value of p≤0.05 will be considered statistically significant.19 More recent evidence shows an increased risk of major adverse cardiovascular events associated with JAK inhibitors in RA, although the underlying mechanisms remain unclear.17 It has been argued that the patients’ aggravated cardiovascular profile with presence of multiple CVD risk factors may account, at least partially, for this observation.18 Hypertension is highly prevalent in RA and contributes substantially to the increased CVD burden associated with the disease,11 while ambulatory blood pressure monitoring is superior to conventional blood pressure measurements in terms of CVD risk prediction.21 Furthermore, subclinical microvascular injury is present in patients of RA before the establishment of overt CVD51 and is considered as an intermediate pathophysiological process towards the development of cardiovascular complications.6 At the same time, large artery stiffening represents a surrogate marker of CVD with incremental predictive value beyond traditional cardiovascular risk factors.28 However, the impact of JAK inhibition on blood pressure including its 24h variation, and markers of micro- and macrovascular injury has not been investigated so far in patients with RA.Despite their clinical efficacy in alleviating symptoms and progression of RA, cardiometabolic effects of JAK inhibitors in patients with RA remain in comparison underinvestigated, especially in the long-term. JAK inhibitors may confer increased risk for circulatory disorders, specifically thromboembolic events, and adversely affect lipids and platelet count.Therefore, shedding light on the impact of JAK inhibitors on the above critical parameters  and the prothrombotic markers that will be assessed, will add substantial information to the available literature regarding cardiovascular aspects of JAK inhibitors in RA. It is anticipated that our findings will have direct clinical implications. They will either highlight potential pleiotropic beneficial effects of JAK inhibitors on the cardiovascular system, or by contrast detect and highlight potential negative aspects which should be taken into consideration by treating physicians, in order to take actions targeting at their proper monitoring and management.Besides, evaluation of changes in patients’ haemodynamic profile and circulating biomarkers of thrombotic predisposition will point towards underlying pathophysiological mechanisms mediating cardiovascular effects of JAK inhibitors in RA. Considering the innovative study design, our results will most probably have an international impact following their dissemination in conferences and the press and their publication in peer-reviewed journals.