Wnt signaling has emerged as a central regulator of skeletal modeling and remodeling. Loss- or gain-of-function mutations in two Wnt co-receptors, Lrp5 and (more recently) Lrp6, have drawn attention to the importance of the Wnt pathway in bone biology. This review summarizes our current understanding of how the Wnt pathway operates on bone and the implications this has for skeletal physiology and drug discovery. Over the past 9 yr, rapid advances have been made in our understanding of the cellular targets for Wnt signaling and of the important regulatory molecules in this metabolic pathway. Both canonical and noncanonical signaling pathways seem to be important for mediating the effects of Wnt in bone. A rapidly expanding catalog of genetically engineered mice has been used to establish the importance of downstream effector molecules (such as β-catenin) in the Wnt pathway, as well as the critical role of endogenous inhibitors of Wnt signaling (such as Dkk1 and sclerostin) in bone metabolism. Indeed, regulation of sclerostin in osteocytes is emerging as an important final pathway for regulating bone anabolism in response to diverse trophic stimuli, from mechnotransduction to the anabolic actions of PTH. From the outset, it had been assumed that the effects of Wnt signaling in bone were caused by direct actions in osteoblast precursors, osteoblasts, and osteocytes. However, startling recent findings have challenged this view and suggest that a key target, at least in mice, is the duodenal enterochromaffin cell. There, Wnt signaling transduced by Lrp5 regulates serotonin synthesis, which acts in an endocrine fashion to regulate bone cell metabolism. It will take time to reconcile this new information with the considerable body of information we already have regarding the actions of Wnt in bone. The Wnt pathway has rapidly emerged as a therapeutic target for drug discovery. Neutralizing antibodies and small-molecule inhibitors of endogenous Wnt inhibitors have shown early promise as bone anabolic agents. However, given the central role of the Wnt pathway in regulating growth and development in extraskeletal tissues, as well as our still rudimentary understanding of how this signaling cascade actually affects bone metabolism, considerable work will be needed to ensure the safety of these new therapies. Lrp5 and Lrp6) genes were cloned in 1998 based on their homology with the low-density lipoprotein receptor (LDLR),, it wouldf cancer, congenitLrp5 and Lrp6 are 71% homologous and form a subclass of the LDLR family. They shaLrp5-deficient mice cytoplasmic domain. The most obvious motifs in the cytoplasmic domain are a series of highly conserved PPPS/TP motifs. The serine or threonine residues in these proline-rich regions are phosphorylated on activation by Wnts or other ligands such as PTH (see below). These phospho-serine or phospho-threonine residues serve as binding sites for the axin proteins, resulting in stabilization of β-catenin. In additLRP5 may also play a role in regulating the actions of this co-receptor. One of the first reports on Lrp5  described an alternatively sized mRNA expressed during embryogenesis and in the testis., a step required for nuclear localization of β-catenin.Activation of the pathway can also be caused by inactivating mutations in genes encoding proteins needed for normal targeting of β-catenin for degradation or activating mutations in the β-catenin gene itself. These inWu et al. have repq/11 and targets the novel protein kinase C family member, PKCδ, which drives osteoblast differentiation. family. Dkks are encoded by a four-member gene family in vertebrates, of which three members  are reported to interact with Lrp5 and/or Lrp6. The protG171V gain-of-function mutation (see below). Cranial nerve root entrapment leading to loss of hearing and vision, as well as trigeminal neuralgia, can also occur in this disease. The expression of sclerostin is restricted to osteocytes, making it an appealing therapeutic target for drug therapy of low bone mass (see below). As an example, overexpression of a constitutively active PTH receptor in osteocytes results in inhibition of sclerostin and a striking increase in bone mass, consistent with the hypothesis that some of the anabolic actions of PTH may be mediated by suppressing sclerostin expression. bind and regulate the activity of Lrp5 and Lrp6. Sclerostpression. Finally,pression.Osteoporosis pseudoglioma (OPPG) is a rare syndrome associated with a dramatic reduction in skeletal mass, evident in infancy, that results in devastating fragility fractures. Limited histologic data indicate that bone formation rates are reduced in this disease. OPPG is accompanied by progressive blindness also beginning in childhood. In 2001, inactivating mutations in LRP5 were identified as the causative genetic basis for OPPG. AnalysisG171V mutations have normal lifespans, rarely fracture, and have little skeletal morbidity, although one of us has cared for a patient who developed thoracic compression myelopathy caused by bony overgrowth of his vertebral body .Additional support for the role of LRP5 in bone growth was provided when two groups independently reported that a point mutation in LRP5 (G171V) was present in affected individuals of families showing an autosomally dominant high bone mass trait.,46 TheseLrp5 have been identified and correlated with changes in bone mass. of LRP5 polymorphisms with bone mass at selected skeletal sites in adults and children. However, two large, recent, genome-wide association studies have firmly established an association between allelic variants in LRP5 and both bone mass and fracture risk. caused by an R886W point mutation.. Dietary restriction of tryptophan resulted in reduced circulating levels of serotonin in Lrp5-deficient mice and normalized the suppressed bone formation parameters in these animals, suggesting that the increase in serotonin production might be responsible for this effect. Consistent with this idea, restricted expression of Lrp5G171V in the duodenum resulted in a high bone mass phenotype. These investigators went on to show that the serotonin receptor Htr1b mediates the actions of serotonin in osteoblasts, because selective deletion of this receptor in osteoblasts resulted in a high bone mass phenotype. Interestingly, using a genetic approach they provide evidence that the mechanism by which Lrp5 regulates Tph1 activity in the duodenum is not through a β-catenin pathway. These data suggest that a new endocrine loop, from the gut to bone in which serotonin is the effector hormone, may play a central and hitherto unappreciated role on bone metabolism. They also call into question the role of Lrp5 in bone cells. Recent clinical evidence has suggested an important role for the serotonergic system in regulating bone mass, with reports of reduced bone mass and an increased risk of fracture in patients taking selective serotonin reuptake inhibitors (SSRIs)..–76 SSRIsLRP5 result in a high bone mass phenotype in which the quality of the bone is good and fracture risk is very low, there is considerable interest in attempting to therapeutically manipulate Wnt signaling in patients with low bone mass. Initial efforts have focused on suppressing endogenous inhibitors of the Wnt signaling system.Because gain-of-function mutations in Sclerostin has emerged as an attractive therapeutic target because its expression is restricted to osteocytes. A neutralizing antibody to sclerostin has shown promise in initial preclinical and early clinical trials.,78 Exper–responsive reporter construct in vitro and that PTH induces interaction of the PTH receptor, PTHR1, with LRP6.(Because PTH is the only available anabolic agent for bone, the relationship between its use for that purpose and Wnt-mediated bone anabolism has been an area of considerable investigative interest. A number of studies suggest that these two signaling pathways do indeed overlap. Recent examples include preliminary reports that PTH is able to stimulate a β-cateninith LRP6. The formith LRP6.,82 will The past 7 yr has seen an explosion in our understanding of the mechanisms underlying the role of Wnt signaling in the regulation of bone mass. The recent work of Yadav et al. that indicates a central role for circulating serotonin in regulating bone formation suggests that inhibiting the actions of serotonin in bone or inhibiting its duodenal synthesis may be new therapeutic targets for treating low bone mass conditions. These observations will undoubtedly stimulate a flurry of activity directed at whether and how the effects of serotonin and the documented role of Wnt/β-catenin signaling within osteoblasts interact to control bone development and homeostasis.Finally, Lee et al. have als


MAP17 is a small 17 kDa non-glycosylated membrane protein previously identified as being overexpressed in carcinomas. Breast tumor cells that overexpress MAP17 show an increased tumoral phenotype with enhanced proliferative capabilities both in the presence or the absence of contact inhibition, decreased apoptotic sensitivity, and increased migration. MAP17-expressing clones also grow better in nude mice. The increased malignant cell behavior induced by MAP17 is associated with an increase in reactive oxygen species (ROS) production, and the treatment of MAP17-expressing cells with antioxidants results in a reduction in the tumorigenic properties of these cells. The MAP17-dependent increase in ROS and tumorigenesis relies on its PDZ-binding domain because disruption of this sequence by point mutations abolishes the ability of MAP17 to enhance ROS production and tumorigenesis. MAP17 is overexpressed in a great variety of human carcinomas, including breast tumors. Immunohistochemical analysis of MAP17 during cancer progression demonstrates that overexpression of the protein strongly correlates with tumoral progression. Generalized MAP17 overexpression in human carcinomas indicates that MAP17 can be a good marker for tumorigenesis and, especially, for malignant progression. The complex physiology of vertebrates requires the continuous renewal of most tissues, which may become damaged either by external agents or by the toxic byproducts of their own metabolism, to maintain homeostasis. Cancer arises as a consequence of genetic changes that deregulate the mechanisms that control the renewal process, either by activation of the pathways that promote survival and proliferation, or through inactivation of growth suppression pathways. In order for cancer cells to grow and metastasize, they must overcome additional barriers to their expansion by promoting angiogenesis, acquiring characteristics that allow them to survive in organs different from their origin or by evading immune surveillance mechanisms , 2011.Figure 1). MAP17 overexpression in carcinomas was first described by using the technique of differential display . Therefore, resistance to TNF may provide cancer cells with a selective advantage against host elimination. Ectopic expression of MAP17 in tumor cells prevents TNF-induced G1 arrest by impairing p21waf1 induction. However, expression of MAP17 does not inhibit TNF-induced apoptosis in Me180-sensitive tumor cells. The inhibition of TNF is specific because MAP17 does not alter the response to other cytokines such as IFN-α. As described in the MAP17 overexpression in carcinomas occurs mostly through mRNA amplification, but promoter activation has also been observed by some oncogenes . ImmunohFigure 2). We have found that the MAP17 protein is overexpressed in a large percentage of the tumors analyzed and is significantly correlated with the tumor grade in ovarian, breast, and prostate carcinomas . These expression patterns provide a mechanistic insight and a possible target for future therapies (AKT inhibition).MAP17 is highly expressed in renal proximal tubular cells and has been previously described to be associated with carcinomas , 1996. Wrcinomas , 2012. T+-coupled co-transporters leads to an inhibition of a ROS increase and a decrease in the malignant cell behavior in MAP17-expressing clones  production, and the treatment of MAP17-expressing cells with antioxidants results in a reduction in the tumorigenic properties of these cells. Treatment of breast cells with inhibitors of Nag clones . Finallyg clones . Furtherg clones , which sFigure 3). Importantly, modulation of ROS by antioxidant treatment prevented activation of AKT, thus, restoring the level of apoptosis in serum starved Rat1/c-Myc fibroblasts  through its PDZ-binding motif . NHeRFs MAP17 form complexes with PDZK1 and NHe3 contributing to basal and calcium inhibition of NH3 activity . RecentlFigure 4).On the other hand, MAP17 has been shown also to increase glucose uptake  thus enhIn summary, MAP17 overexpression in human breast carcinomas indicates that MAP17 can be a good marker for tumorigenesis and for malignant progression. Our results indicate that this protein is likely to play an important role in carcinogenesis.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Migraine is a common, recurrent, and disabling primary headache disorder with a genetic component which affects up to 20% of the population. One third of all patients with migraine experiences aura, a focal neurological disturbance that manifests itself as visual, sensitive or motor symptoms preceding the headache. In the pathophysiology of migraine with aura, activation of the trigeminovascular system from the meningeal vessels mediates migraine pain via the brainstem and projections ascend to the thalamus and cortex. Cortical spreading depression (CSD) was proposed to trigger migraine aura and to activate perivascular trigeminal nerves in the cortex. Quinine, quinidine and the derivative mefloquine are able to inhibit CSD suggesting an involvement of neuronal connexin36 channels in CSD propagation. More recently, CSD was shown to induce headache by activating the trigeminovascular system through the opening of stressed neuronal Pannexin1 channels. A novel benzopyran compound, tonabersat, was selected for clinical trial on the basis of its inhibitory activity on CSD and neurogenic inflammation in animal models of migraine. Interestingly, in the time course of animal model trials, tonabersat was shown to inhibit trigeminal ganglion (TGG) neuronal-glial cell gap junctions, suggesting that this compound could prevent peripheral sensitization within the ganglion. Three clinical trials aimed at investigating the effectiveness of tonabersat as a preventive drug were negative, and conflicting results were obtained in other trials concerning its ability to relieve attacks. In contrast, in another clinical trial, tonabersat showed a preventive effect on attacks of migraine with aura but had no efficacy on non-aura attacks. Gap junction channels seem to be involved in several ways in the pathophysiology of migraine with aura and emerge as a new promising putative target in treatment of this disorder. Migraine is a common, recurrent, and disabling primary headache disorder which affects up to 20% of the population -3,4-dihydro-2,2-dimethyl-2H-benzo[b]pyrane-3S-ol , was selected for clinical trials as an anti-migraine agent. Preclinical studies showed that tonabersat inhibited cortical spreading depression (CSD) and neurogenic inflammation in animal models of migraine . Each hemichannel (or connexon) is an oligomerized hexamer of proteins called connexins (Cxs), surrounding an aqueous pore (1–1.5 nm diameter). To date, 20 members of the Cx family have been identified in mice and 21 in humans  of 2–3 nm, where transmembrane channels are clustered in microdomains. Gap junction channels assemble from the docking of two hemichannels, each of them originating from cells in contact. These channels connect the cytoplasms of the adjacent cells and allow the intercellular passage  of small molecules up to approximately 1 kDa . Some of them are expressed in the same cell type, forming intercellular channels with different structural combinations and properties whose physiological significance remains largely unknown , pH, metabolites (nitric oxide (NO), arachidonic acid), and neurotransmitter (glutamate) concentrations , which, in turn, sensitize and increase neural activity in the ipsilateral TGG and in the caudal portion of the trigeminal nucleus (TGN) in the brainstem . In the same time, collateral axons of activated neurons in the TGG release proinflammatory peptides  in the dura mater, leading to a local sterile inflammatory reaction  and then to headache. Moreover, a central trigeminal-parasympathetic reflex produces vasodilatation of dura mater (and pia mater) vessels. For this, the activation of the TGN, in turn, also induces a stimulation of the superior salivary nucleus (SSN) of the brainstem and perivascular parasympathetic efferents of the dura mater via the sphenopalatine ganglia (SPG). Postganglionic parasympathetic efferents release vasoactive molecules  into the dura mater, promote vasodilatation and hyperemia and then could also explain autonomic symptoms that frequently occur during migraine attack. The perception of pain is mediated by rostral projections from the TGN to brain structures. It was proposed that in the attack of migraine with aura, nociception almost originated from pial perivascular trigeminal afferents with a later contribution from dural perivascular ones  receptor antagonist, MK-801, blocks CSD but, unlike the gap junction blocker carbenoxolone, does not inhibit transmission of Ca2+ waves  and known to be involved in pain pathways, has been linked to migraine with aura in a single family  encodes a member of the transient receptor potential (TRP) superfamily of channels that is involved in neuropathic pain; LRP1 (12q13.3) encodes a receptor that could modulate glutamatergic synaptic transmission; PRDM16 (1q36.32) which potential role in migraine is unclear  allowed the identification of various susceptibility loci for common forms of migraine. The minor allele of Then, in genetically susceptible patients, mutations in these genes may reduce the threshold for CSD. Triggers of migraine attacks would induce CSD in such a hyper-excitable cortex by modifying the activity of ionic channels and then by altering ionic flow across cell membrane  or reducing the frequency of occurrence of attacks, respectively Figure . The chrPainful symptoms, arising from vasodilatation of cerebral vessels and inflammatory processes, require medication with powerful and fast action. The effectiveness of an acute therapy of migraine mainly depends on its earliness.1A,1B,1D receptors, are also dopaminergic and adrenergic antagonists. Only two molecules are indicated in migraine attacks: ergotamine (exclusive use) and dihydroergotamine also used for prophylaxis. For all these drugs, there are oral, nasal and injectable forms, suitable for emergency treatment. Triptans cause vasoconstriction of the meningeal vessels by their agonist effect on the serotonin receptor 5-HT1B. They also induce an inhibition of pro-inflammatory neuropeptide release by trigeminal nerve endings through the activation of 5-HT1D receptors. However, the effectiveness of triptan treatment during the aura phase remains controversial  in acute migraine attacks is recommended because these drugs can improve the absorption of co-administered analgesics  and/or intensity of the symptoms, as well as the duration of the aura. To reduce these parameters , a variety of drugs (sometimes off-label) acting through various mechanisms can be used.Some of migraine drugs act through the serotonin receptors , calcium and sodium voltage-gated channels  or the glutamatergic neurotransmission (lamotrigine). Besides, topiramate and valproate are also GABA receptor agonists. Lamotrigine is an anti-epileptic drug that would specifically prevent migraine with aura  have been shown to experimentally suppress CSD susceptibility Figure . Acute aIn conclusion, if synapses, ionic channels, neurotransmitter receptors of neurons are numerous targets for such prophylactic treatment, the role of gap junction channels in CSD propagation, activation of the trigeminovascular system and peripheral  sensitization opens new perspectives of research.cis benzopyran, on the basis of its high efficacy at inhibiting neuronal hyperexcitability and trigeminal nerve stimulated neurogenic inflammation in rodent models  potently inhibited NO release following a cortical KCl stimulus in anaesthetized cats , tonabersat seemed to be devoid of adverse cardiovascular side-effects in isolated human tissues  produced 4 h later a maximal inhibition of the neurovascular reflex (30% compared to control) whereas an intraduodenal administration (10 mg·kg−1) produced it (55% inhibition) 2 h later  inhibited neuronal-satellite cell GJIC in TGG likely through a decrease of Cx26 expression since tonabersat treatment reduced Cx26 staining and number of junctional plaques .Carbenoxolone, a potent blocker of both Cx43-made gap junctions and hemichannels and purinergic P2X7 receptor-associated Panx1 hemichannels completely suppressed central sensitization in an 143 channels (where the sequence of the first extracellular loop of Cx32 is replaced by that of Cx43) are not affected by probenecid , phase II study further investigated tonabersat as a putative migraine prophylactic agent  involved approximately 500 patients with episodic migraine with or without aura. The TEMPUS study was significantly longer than the 3 months treatment period in the earlier phase IIa trial since the earlier trial showed that tonabersat was more effective toward the end of the 3 months period. The fact that tonabersat was very well tolerated in the previous study, allowed to explore the effects of increasing the dose , randomized, double-blind crossover, placebo-controlled phase II clinical trial. In contrast to the former, this study only included patients having migraine with aura exhibiting frequent aura attacks  suffering migraines with or without aura for at least 1 year before the trial, with 1–6 attacks each month. In the first study evaluating tonabersat as an acute treatment of migraine, the clinical trial was conducted in 53 centers in 12 countries  and G protein-coupled receptors (Sandilos and Bayliss, + into the extracellular environment (Martins-Ferreira et al., +, could result in a reduced Panx1 channel activity. In the same way, inhibition of CSD, by reducing stressed neuronal Panx1 channel activity and, in turn, iNOS induction in astrocytes forming the glia limitans, could explain the potent inhibition of NO release observed following tonabersat pre-treatment in the CSD-anaesthetized cat model (Read et al., Tonabersat, was selected for clinical trial on the basis of its inhibitory activity on CSD and neurogenic inflammation in animal models of migraine. Moreover, in preclinical studies, tonabersat was shown to inhibit GJIC between neurons and satellite glial cells within the TGG (Durham and Garrett, The extent to which migraine with aura and migraine without aura are different disorders has been the subject of much debate. Tonabersat, having a preventive effect in migraine with aura but no efficacy on non-aura attacks, suggests that an asymptomatic CSD is not involved in attacks of migraine without aura.Preclinical studies and clinical trials also showed that tonabersat was well tolerated, with no effects on arterial blood pressure and heart rate. Moreover, the pharmacokinetic characteristics of tonabersat indicate that it is more suitable as a prophylactic therapy for migraine attacks. Comparing with the potential targets of currently utilized preventive drugs, tonabersat has a unique mechanism of action without vasoconstrictive adverse effect and might represent an interesting future possibility for the prophylaxis of this disease. It is estimated that about a third of migraineurs experiences aura. So, even if tonabersat benefits only to patients suffering migraine with aura, a substantial number of them could benefit from this drug. Moreover, the risk of ischemic stroke is doubled in migraine with aura, particularly in young women. The risk is greater for those with a higher frequency of migraine attacks (Kurth et al., Epilepsy and migraine are chronic neurological disorders with episodic manifestations that frequently occur together. Neuronal hyperexcitability is an underlying mechanistic similarity common to both disorders. Some antiepileptic drugs have a preventive effect in migraine without aura and possibly in migraine with aura (Haut et al., The pathophysiology of migraine with aura involves GJIC between neurons and glial cells in the neocortex and TGG. Tonabersat treatment reduced Cx26 staining and number of junctional plaques in TGG cells. However, given the large number of Cxs expressed in nervous system, it is probable that other Cx proteins may also be regulated by tonabersat. Moreover, Panx1 channel activity at the plasma membrane being modulated by trafficking dynamics, a potential modulation of Panx1 trafficking by tonabersat requires experimental testing (Sandilos and Bayliss, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ankrd1 gene mutations in cardiomyopathy patients, this suggests their important roles in cardiac development, function, and disease. To determine the functional role of MARPs in vivo, we studied knockout (KO) mice of each of the three family members. Single KO mice were viable and had no apparent cardiac phenotype. We therefore hypothesized that the three highly homologous MARP proteins may have redundant functions in the heart and studied double and triple MARP KO mice. Unexpectedly, MARP triple KO mice were viable and had normal cardiac function both at basal levels and in response to mechanical pressure overload induced by transverse aortic constriction as assessed by echocardiography and hemodynamic studies. Thus, CARP, Ankrd2, and DARP are not essential for normal cardiac development and function at basal conditions and in response to mechanical pressure overload.Ankrd1/CARP, Ankrd2/Arpp, and Ankrd23/DARP belong to a family of stress inducible ankyrin repeat proteins expressed in striated muscle (MARPs). The MARPs are homologous in structure and localized in the nucleus where they negatively regulate gene expression as well as in the sarcomeric I-band, where they are thought to be involved in mechanosensing. Together with their strong induction during cardiac disease and the identification of causative  In cardiac and skeletal muscle, a family of muscle specific ankyrin repeat proteins (MARPs) includes cardiac ankyrin repeat protein (CARP/Ankrd1) Ankrd1 gene were recently shown to be causative for human dilated and hypertrophic cardiomyopathy in vitro studies, CARP has been shown to bind to the ubiquitous Y-box transcription factor 1 (YB-1) and act as a nuclear transcriptional cofactor by negatively regulating the expression of cardiac genes, including the ventricular specific myosin light chain-2v, atrial natriuretic factor, and cardiac troponin C Cardiac ankyrin repeat protein (CARP) was first identified as a cytokine-inducible gene in microvascular endothelial cells Ankyrin repeat protein 2 (stretch responsive-muscle) (Ankrd2)/Ankyrin Repeat protein with PEST and Proline-rich region (Arpp) was first described by Kemp as a striated muscle specific gene induced by mechanical stretch and involved in mechanically-induced skeletal muscle hypertrophy Diabetes-related ankyrin repeat protein (DARP) is expressed in heart, skeletal muscle, and brown adipose tissue in vivo remains unknown. To address this, we studied knockout (KO) mice for each of the three MARP family members. All three KO mouse models are viable and have no apparent cardiac phenotype, which we hypothesized might be due to redundant functions of the highly homologous MARP proteins. We therefore studied double  and triple (CARP/Ankrd2/DARP) MARP KO mice, but to our surprise both double and triple KO mice were viable and extensive analyses up to 17 months of age did not reveal any abnormalities in cardiac development or function, either at baseline or in response to biomechanical stress induced by pressure overload hypertrophy. Thus, our results suggest that CARP, Ankrd2, and DARP are not required for normal cardiac development and function at basal conditions and in response to mechanical pressure overload in vivo.Taken together, several lines of evidence suggest that the MARPs belong to a family of stress responsive proteins, which plays critical roles in the heart. However, although the MARPs have been extensively studied, their functional role in the heart All animal procedures were approved by the University of California San Diego Animal Care and Use Committee  and performed in full compliance with the guidelines of the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . Special attention was paid to animal welfare and to minimize the number of animals used and their suffering.CARP, Ankrd2, and DARP, respectively. The first 2–3 exons of each gene were replaced by cDNA encoding lacZ and a pGK neo cassette. In this manner, the β-galactosidase cDNA was brought under the control of the endogenous promoters, while also ablating the endogenous CARP, Ankrd2, and DARP genes, respectively. The generation of Ankrd2 KO mice has been described previously et alATAGACTCACGGCTGCCAACAT; reverse: CTCCATTTCTGAACTCCCCAGG), mutant ; Ankrd2/Arpp WT , mutant ; DARP WT , mutant . Double and triple MARP KO mice were generated by interbreeding of single KO mice. To ensure that WT control mice had the same genetic background as double and triple MARP KO mice, also WT mice were generated from the crosses.Although the analysis of the skeletal muscle phenotype of MARP triple KO mice has been published et al. Animals were anaesthetized by intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (5 mg/kg) and sacrificed by cervical dislocation before removal of tissues. Total protein extracts were prepared from heart and skeletal muscle Dissected mouse hearts were fixed with 4% paraformaldehyde followed by dehydration and paraffin embedding. 10 µm sections were stained with hematoxylin and eosin or by Masson’s trichrome method.For transthoracic echocardiography, mice were anesthetized with 1% isoflurane and imaged as previously described Data are indicated as average ± standard deviation. Statistical comparisons between WT and KO mice were done using the unpaired Students t-test. A P value of <0.05 is considered significant.in vivo, mice deficient for each of the three family members, CARP, Ankrd2, and DARP KO mice were generated. The generation of Ankrd2 KO mice has been described elsewhere et alet alet alet alet alTo study the functional role of the muscle ankyrin repeat family et alCARP, Ankrd2, and DARP KO mice were born at expected Mendelian ratios, were fertile, and survived till adulthood. KO mice were indistinguishable from wildtype (WT) littermate control mice and detailed analyses revealed normal cardiac morphology and physiology and no significant differences in heart/body weight ratios (see below). Since the three proteins are highly homologous in structure and are all expressed in the heart, we hypothesized that they may have overlapping functions and generated double and triple MARP KO mice by intercrossing single KO mice. WT mice generated from these crosses were used as controls to ensure that KO and WT control mice had the same genetic background. The successful ablation of all three proteins was confirmed by PCR as well as Northern and Western blot analyses et al.MARP triple KO mice were born at expected Mendelian ratios and were indistinguishable from WT control mice. No statistical difference in heart/body weight ratios between MARP triple KO and WT mice was observed and cardiac histological analysis by hematoxylin and eosin and Masson’s trichrome staining showed no signs of hypertrophy, infarction, fibrosis, necrosis, calcification, or fat infiltration in MARP triple KO mice up to 17 months of age (data not shown).Cardiac function was evaluated noninvasively by echocardiography at 4 and 17 months of age. As shown in It is well known that CARP, Ankrd2, and DARP are upregulated in response to stress such as cardiac hypertrophy in vivo, we generated CARP, Ankrd2, and DARP KO mice. Since no phenotype was found in any of the individual KO mice, we hypothesized that the three highly homologous proteins have overlapping functions in the heart and generated MARP triple KO mice. However, in our detailed analyses, we were unable to detect any significant abnormalities in cardiac development and basal cardiac function up to 17 months of age and the hypertrophic response in response to acute pressure overload induced by TAC was similar between WT and triple KO mice. On the other hand, transgenic mice with cardiac specific overexpression of CARP were recently found to show attenuated cardiac hypertrophy and fibrosis in response to pressure overload and isoproterenol infusion, although the they were not protected against heart failure in vivo. Nevertheless, in light of the strong induction of MARPs in the heart in response to by various forms of stress, including cardiac hypertrophy and cardiac disease, as well as an accumulating body of evidence suggesting important roles of the MARP protein family in cardiogenesis, gene regulation, sarcomeric stress/stretch sensing, and disease, it is very surprising that the absence of all three MARP family members does not appear to have any effect on cardiac function in vivo, in particular since an increasing number of mutations in the Ankrd1 gene have been identified in patients with dilated and hypertrophic cardiomyopathy To study the functional role of the MARP family members in vivo.The lack of a cardiac phenotype in MARP triple KO mice following 14 days of TAC does not exclude that the MARP KO mice may show a phenotype after long-term TAC or in response to other types of stress conditions, such as cardiac ischemia, myocarditis, and chronic β-adrenergic stimulation. Also, it is possible that MARPs might play a role in other organs, such as in skeletal muscle and the vasculature. We previously studied skeletal muscle function in MARP triple KO mice and found a significant increase in resting sarcomere length associated with the expression of longer titin isoforms


Rattus norvegicus. Here, we summarize the advances in our understanding of the signaling pathways regulating mES cell self-renewal that led to the first derivation of rat ES cells and highlight the new opportunities presented for transgenic modeling on diverse genetic backgrounds. We also comment on the implications of this work for our understanding of pluripotent stem cells across mammalian species.Mouse embryonic stem (ES) cells are defined by their capacity to self-renew and their ability to differentiate into all adult tissues including the germ line. Along with efficient clonal propagation, these properties have made them an unparalleled tool for manipulation of the mouse genome. Traditionally, mouse ES (mES) cells have been isolated and cultured in complex, poorly defined conditions that only permit efficient derivation from the 129 mouse strain; genuine ES cells have not been isolated from another species in these conditions. Recently, use of small molecule inhibitors of glycogen synthase kinase 3 (Gsk3) and the Fgf-MAPK signaling cascade has permitted efficient derivation of ES cells from all tested mouse strains. Subsequently, the first verified ES cells were established from a non-mouse species,  Embryonic stem (ES) cells were first isolated in 1981 by Martin in California Hopes that other animals would yield ES cells, facilitating genetic manipulation in diverse species, met with frustration post-implantation mouse epiblast; these were named epiblast stem cells (EpiSCs) In the late 1990s, pluripotent cell lines were derived from non-human primate and human blastocysts and deemed to be ES cells EpiSCs and mES cells are considered to represent the developmental stages from which they are derived: the post- and the pre-implantation epiblast, respectively  [18]. ByOur limited ability to capture naïve pluripotent stem cells has been a barrier to efficient genetic manipulation in non 129-strain mice and other species. ES cells also have been widely used as a model to study early development and lineage commitment  Classical culture conditions employed serum-containing media and a layer of mitotically inactivated fibroblasts (feeder) cells Stat3-null ES cells can be derived using Gsk3 and Fgf-MAPK pathway inhibitors (2i), formally proving that extrinsic LIF-STAT3 signaling is not necessary for ES cell self-renewal in these conditions The apparent dependence of ES cells upon growth factors underpinned a belief that exogenous signals drive ES cell maintenance. Downstream of LIF, Stat3 was identified as a major functional mediator of self-renewal In 1997, Brook and Gardner noted a “persisting ignorance about the genetic basis of permissivity” While the susceptibility of the 129 strain to ES cell derivation may be related to its propensity to develop gonadal teratomas  Indeed, serum-free 2i culture condition has overcome the limitations imposed by mouse genetic background on ES cell derivation and maintenance. In striking contrast to conventional culture, 2i+LIF yields stable ES cell lines from any tested strain of mouse with high efficiency Soon after the first mES cells were derived, work began to isolate ES cells from the rat However, utilizing 2i, LIF, and fibroblast feeder cells, cells with the morphology and gene expression pattern of ES cells, and most importantly, the ability to contribute to chimeras and make functional germ cells, were derived from the SD p53 locus in rat ES cell by homologous recombination p53 knockout through the germline failed, with the authors citing >65% polyploidy of the injected line as a likely cause. Only through sub-cloning and careful morphological selection during passaging was a karyotypically normal, germline-competent line maintained Qi-Long Ying's group recently reported successful targeting of the For the full potential of rat ES cells for genetic research to be realized, cell lines and culture methods should be further optimized. It should be remembered that before optimization and standardization of culture conditions throughout the mES cell field, it was widely considered that only early passage cells were germline competent and useful for gene targeting Human and non-human primate ES cells were originally derived by Thomson and colleagues from pre-implantation blastocysts using serum and feeders As previously noted, the primed, EpiSC-like state of hES cells is accompanied by characteristics such as low viability after single cell dissociation EpiSCs can be reprogrammed to an ES cell state by over-expression of the pluripotency transcription factors Nanog Advances in our understanding of ES cell biology in the mouse have led to the development of a culture condition for self-renewal based on small molecule inhibitors of Fgf-MAPK signaling and Gsk3. Employing these inhibitors, naïve pluripotent ES cells have been derived from multiple strains of mouse and rat and are presenting new opportunities for genetic intervention in these species. It is plausible that since rodents have a capacity for developmental suspension at the blastocyst stage, a phenomenon known as diapause, that the ability to capture the naïve ES cell state may be specific to the limited number of species with this ability (discussed in


Endothelial dysfunction is a key event in the development of vascular disease, and it precedes clinically obvious vascular pathology. Abnormal activation of the RhoA/Rho kinase (ROCK) pathway has been found to elevate vascular tone through unbalancing the production of vasodilating and vasoconstricting substances. Inhibition of the RhoA/ROCK pathway can prevent endothelial dysfunction in a variety of pathological conditions. This review, based on recent molecular, cellular, and animal studies, focuses on the current understanding of the ROCK pathway and its roles in endothelial dysfunction. Vascular disease, particularly atherosclerosis is a major cause of disability and death in patients with diabetes mellitus. The pathophysiology of vascular disease in diabetes involves abnormal function of the vascular endothelial and smooth muscle cells (SMC) as well as platelets. Endothelial dysfunction may be a critical and initiating factor in the development of diabetic vascular disease.2 The broThe small GTPase RhoA and its downstream target Rho kinase (ROCK) regulate cellular adherence, migration, and proliferation through control of the actin–cytoskeletal assembly and cell contraction. Since thRhoA/ROCK is also involved in endothelial NO synthase (eNOS) function, as their activation decreases eNOS expression by reducing the eNOS mRNA stability. Also, usROCK is a serine/threonine kinase with a molecular mass of ~160 kDa, which has been identified as the first downstream target of the small GTP-binding protein RhoA.15 ROCK mTwo ROCK isoforms have been identified in the mammalian system. ROCK1 (ROKβ or p160ROCK) is located on chromosome 18 and encodes a 1354-amino acid protein.20 ROCK2 1421ROCK1 and ROCK2 are ubiquitously expressed in murine tissues from early embryonic development to adulthood. ROCK1 is widely and highly expressed in most tissues except in the brain and muscle, whereas ROCK2 is most highly expressed in muscle, brain, heart, lung, and placenta tissues.2224 Both22Although ROCK1 and ROCK2 are ubiquitously expressed and highly homologous, several mechanisms have been reported that differentially regulate ROCK isoform activities. For example, the overexpression of ROCK1 and ROCK2 can both increase MLC phosphorylation, but through different mechanisms. ROCK2, bA hallmark of endothelial dysfunction is reduced bioavailability of NO, which may be caused by reduced expression of eNOS, impairment of eNOS activation, or inactivation of NO by oxidative stress. Accumulating evidence indicates that the expression and activity of eNOS is regulated by the RhoA/ROCK pathway. For example, activation of the RhoA/ROCK pathway significantly inhibits endothelial NO synthase expression and phosphorylation (Ser1177) in the mesenteric arteries of hypertensive profilin1 transgenic mice. ThrombinThere are many signaling molecules involved in the pathogenesis of endothelial dysfunction via impairment of NO bioavailability. Some of the signaling molecules have been reported to have a link with the RhoA/ROCK pathway. These are phosphoinositide 3-kinase (PI3K)/Akt, reactive oxygen species (ROS), and arginase.Akt (protein kinase B) is a serine/threonine protein kinase, which is the key downstream effector of PI3K. PI3K-dependent Akt activation can be regulated through the tumor suppressor phosphatase and tensin homolog (PTEN), which works essentially as the opposite of PI3K. Akt can directly phosphorylate eNOS on serine 1179  and activate the enzyme, leading to NO production. Studies 3941It is well known that ROS reduce the bioavailability of NO. The reaction between superoxide and NO forms peroxynitrite, which oxidizes and decreases the level of tetrahydrobiopterin (BH4), a cofactor required for eNOS activity and NO synthesis. Moreover, increased peroxynitrite positively correlates with a significant upregulation of the active RhoA in models of experimental diabetes. RhoA pla44In addition, elevated arginase activity also limits NO availability. Arginase is a hydrolytic enzyme that converts L-arginine into urea and ornithine. Thus, enhanced arginase activity can decrease the tissue and cellular L-arginine availability to eNOS, which le432627In vascular diseases, endothelial dysfunction is also due in part to the release of EDCF, which counteracts the vasodilator action of NO or PGI2. The vessel contraction mediated by EDCF is widely called endothelium-dependent contraction in the scientific literature. Although this term is somewhat imprecise, it has become widely used.In blood vessels, endothelium-dependent contraction to Ach is not observed under normal physiological conditions. However, it is observed under pathological conditions, such as in hypertension and in diabetes, in which endothelial function is markedly impaired.–54 TxA2 5361In the presence of a pathologic vascular endothelial layer, EDCFs may prevail over EDRFs, subsequently inducing activation of the RhoA/ROCK pathway in the VSM layer, resulting in enhanced vasocontractile activity. In clinical studies, the intraarterial infusion of the ROCK inhibitor fasudil lowers blood pressure, and this decrement is higher in hypertensive patients than in normotensive subjects, suggesting that ROCK contributes to endothelium-dependent contraction to Ach. In carotAlthough an increasing number of reports show that ROCK plays an important role in endothelial dysfunction, more insights into the molecular mechanisms that contribute to increased ROCK activity or the downstream targets for ROCK are needed. Determination of the precise role of the two ROCK isoforms is limited by the lack of specific and selective pharmacological inhibitors currently available. Statins are indirect inhibitors of the RhoA/ROCK pathway, which act by decreasing the synthesis of isoprenoids. An intravenous injection of pravastatin prevents impaired NO-dependent vasodilation by blocking the full activation of unprocessed RhoA and Rac1 and the downregulation of Akt/eNOS pathways in Wistar and SHR.66 Fasudi67There is no doubt that ROCK1 and ROCK2 knockout mice are the most accurate and specific way to investigate the in vivo distribution/function of ROCK isoforms. Complete loss of ROCK1 in mice results in the eyelids being open at birth and an intestinal protrusion phenotype, whereas There is growing evidence that the RhoA/ROCK pathway has an important pathophysiological role in vascular endothelial dysfunction. Inhibition of ROCK may be an attractive therapeutic target for preventing endothelial dysfunction . However


Contrast-induced nephropathy (CIN) is the third leading cause of acute renal failure in hospitalized patients. Endothelial dysfunction, renal medullary ischemia, and tubular toxicity are regarded as the most important factors in the pathogenesis of CIN. Mannose-binding lectin (MBL), a pattern recognition protein of the lectin pathway of complement, has been found to aggravate and mediate tissue damage during experimental renal ischemia/reperfusion (I/R) injury which was alleviated by inhibition with C1 inhibitor, a potent MBL, and lectin pathway inhibitor. In this paper, we highlight the potential role of MBL in the pathogenesis of human CIN. In experimental I/R models, MBL was previously found to induce tubular cell death independent of the complement system. In addition, after binding to vascular endothelial cells, MBL and its associated serine proteases were able to trigger a proinflammatory reaction and contribute to endothelial dysfunction. In humans, urinary MBL was increased after administration of contrast media and in individuals with CIN. Moreover, individuals with normal/high MBL levels were at increased risk to develop radiocontrast-induced renal dysfunction. Hence, MBL and the lectin pathway seem to be a promising target given that a licensed, powerful, human recombinant inhibitor exits to be added to the scarce armamentarium currently available for prophylaxis of CIN. Despite numerous attempts, preventive strategies are largely confined to intensive hydration with sodium chloride [Iodinated contrast media (CM) are an essential component of contemporary imaging and interventional studies, and its use is steadily increasing as a consequence of the exponential growth of contrast studies over the past decade . Althougchloride  and potechloride , which mchloride .Evidence from numerous studies suggests that a combination of several mechanisms is responsible for the development of CIN , 11 inclIn this paper, we will discuss mannose-binding lectin (MBL), a complement protein which has been implicated in apoptosis and ischemia/reperfusion (IR) injury in various organs, as a mediator/aggravator of contrast-induced local renal damage.MBL2 gene negatively influence the concentration of circulating functional MBL multimers [MBL2 gene is located on chromosome 10q21.1 and at least 6 single nucleotide polymorphisms in the promoter and exon 1 regions segregate under linkage disequilibrium to produce 7 common haplotypes of MBL. In the literature, exon 1 variant alleles are often collectively designated as O and the wild-type gene as A, and the most influential promoter variant allele and the wild-type gene designated as X and Y, respectively [MBL2 genotypes, respectively [Mannose-binding lectin (MBL) is a circulating innate pattern-recognition protein of the complement system that is primarily synthesized in the liver and rarely detected in other organs in the absence of inflammation . Its carultimers . The MBLectively  and growectively  have beeectively . Overallectively . The sigectively , 27) or ectively –30).More recently, MBL has been implicated in apoptosis and ischemia/reperfusion (IR) injury, two proposed main mechanisms in CIN .in vitro models. Hence, we will discuss the relevance of MBL and the lectin pathway in different pathophysiologic mechanisms implicated in CIN assuming analogy.There are scarce data on the role of MBL and the lectin pathway of complement in CIN, essentially limited to two human studies , 33 withLocal I/R is regarded as being at least in part responsible for the development of CIN. Conversely, plentiful rodent –37 and hHow does MBL inflict renal damage after I/R injury? While activation of the complement system after binding of MBL to hypoxic cells with subsequent killing and opsonophagocytosis of these cells might seem plausible, a recent study has shed light on a new mechanism independent of complement activation . In a raHow does MBL bind to reperfused renal parenchymal cells? Circulating natural IgMs have long been implicated in reperfusion-mediated damage by binding to ischemia-conditioned tissue and activating the complement cascade as shown in mice totally deficient in immunoglobulins , 46 or cIn vivo, MBL was found to colocalize with IgM and complement C3 on the endothelial surface of ischemic brain vessels but not in vessels of non-ischemic ipsilateral brain territories or the contralateral hemisphere [in vitro [in vivo [in vivo thrombogenesis [MBL does not only seem to aggravate reperfusion damage by impacting on renal parenchymal cells but also via its binding to endothelial cells. Over a decade ago, Collard et al. demonstrated binding of MBL to endothelial cells immediately after oxidative stress with subsequent endothelial C3 deposition, which could be prevented by anti-MBL monoclonal antibodies . Furthermisphere . Similarmisphere  and compmisphere . Indepenmisphere . Of notemisphere  which inin vitro , 58 and [in vivo . In factogenesis . Of note2) who were part of a randomized controlled trial examining three different prophylactic regimens. Interestingly, the incidence of CIN as defined by the standard definition (increase in serum creatinine concentration of more than 44 μmol/L or 25% within 48 hours after exposure to CM) was only 6.5% in the whole cohort. MBL levels in patients who experienced an acute deterioration of renal function did not differ significantly from patients that showed stable creatinine concentrations. Similarly, the incidence of MBL deficiency (defined as MBL levels < 500 ng/mL) was nearly identical in the two groups. However, analysis of cystatin C kinetics, a more sensitive and rapid indicator of changes in GFR than serum creatinine [After reviewing the impact of MBL and the lectin pathway in renal I/R injury in general, we will discuss human studies that have examined this pathway in CIN. Wang et al. analyzed urine samples from 12 patients undergoing diagnostic or therapeutic cardiac catheterization with two-dimensional fluorescence differential gel electrophoresis followed by liquid chromatography mass spectrometry . Among teatinine , 60, revIn contrast to clinical scenarios of acute ischemia  where detrimental MBL binding and activation of the complement cascade has already occurred before pharmacological inhibition is feasible, CIN does offer the unique opportunity to attenuate renal injury by interfering with contributing pathways before they become activated. Indeed, treatment with anti-MBL monoclonal antibodies or inhibitors of MASP-1/-2 might be possible in advance of CM administration similar to hydration with sodium chloride, even in the setting of an acute ischemic event. Additionally, inhibition of MBL and the lectin pathway in the latter settings might even “kill two birds with one stone” by ameliorating not only CIN but also tissue damage caused by the initial ischemic event and subsequent reperfusion via therapeutic revascularization. However, clinical data supporting a role of MBL in CIN are still very limited, and there is clearly a need for further studies in humans.Given the fact that renal injury after I/R seems to be mediated independently by MBL or via MASP-1/-2 and activation of the lectin pathway, pharmacological candidates should ideally interfere with MBL and the lectin pathway as far upstream as possible. Hence, antibodies against MBL or inhibition of MASP-1/-2 seem to be preferable compared to inhibition of C3 and C5 or even further downstream proteins. Indeed, transient inhibition of the lectin pathway by systemic pretreatment with inhibitory MASP-2-specific monoclonal antibodies was effective in significantly ameliorating gastrointestinal I/R in a rodent model . RecombiCIN is still a frequent complication of diagnostic and interventional contrast studies and associated with significant morbidity and long-term mortality. After decades of research, prophylactic treatment is essentially still limited to hydration with normal saline and potentially the use of sodium bicarbonate, an unsatisfactory fact. Fortunately, over the last decade our knowledge about the development of CIN has steadily increased and led to the identification of I/R injury as the culprit event in the complex pathophysiology of CIN. Similarly, ample evidence suggests that MBL and the lectin pathway of complement aggravate the course of I/R in various organs including the kidneys. Hence, it seems plausible that MBL might also impact on the severity of CIN in patients at risk. Evidence from renal I/R injury models imply that the detrimental effects of MBL are propagated by induction of endothelial cell dysfunction, tubular cell apoptosis, activation of the complement cascade, and recruitment of neutrophils. Preliminary evidence from two human studies suggests that the harmful effects of contrast media are at least partly mediated by MBL and the lectin pathway. As effective inhibitors of MBL and the lectin pathway are already available for prophylactic treatment of CIN in humans, there is a need of additional animal and human studies to fully elucidate the role of MBL in the pathogenesis of CIN. In the context of a predicted further increase in diagnostic and interventional contrast studies and a still scarce choice of prophylactic treatment options, research regarding MBL and the lectin pathway in CIN seems to be promising in our opinion.


Since mitochondria play a central role in apoptosis, modulation of their ion channels by pharmacological means may lead to death of cancer cells. The nuclear potassium channel Kv10.1 and the nuclear chloride channel CLIC4 as well as the endoplasmatic reticulum (ER)-located inositol 1,4,5-trisphosphate (IP3) receptor, the ER-located Ca2+ depletion sensor STIM1 , a component of the store-operated Ca2+ channel and the ER-resident TRPM8 are also mentioned. Furthermore, pharmacological tools affecting organellar channels and modulating cancer cell survival are discussed. The channels described in this review are summarized on Figure Several types of channels play a role in the maintenance of ion homeostasis in subcellular organelles including endoplasmatic reticulum, nucleus, lysosome, endosome, and mitochondria. Here we give a brief overview of the contribution of various mitochondrial and other organellar channels to cancer cell proliferation or death. Much attention is focused on channels involved in intracellular calcium signaling and on ion fluxes in the ATP-producing organelle mitochondria. Mitochondrial K ROS are involved in the regulation of physiological processes, but may also be harmful if produced excessively. Mitochondria are the checkpoint of the intrinsic pathway of apoptosis: the release of caspase cofactors, such as cytochrome c (cyt c) and SMAC/Diablo, results in the assembly of the apoptosome and in commitment of the cell to apoptosis. In cancer cells mitochondrial metabolism is deregulated to optimize the production of glycolytic intermediates for anabolic reactions. Much effort has been devoted to discover drugs inducing cancer cell death by targeting tumor-specific alterations of mitochondrial metabolism or by stimulating OMM permeabilization and thus, allowing the release of apoptotic co-factors  allows the formation of an electrochemical proton gradient which drives the aerobic synthesis of ATP. The “semipermeable” outer membrane (OMM) encloses a periplasmic space where proteins with fundamental roles in cell death are stored until a sufficiently strong pro-apoptotic signal arrives. Mitochondria have assumed a peculiar role in cancer cell physiology , considered as a final common pathway of cell death  amino) phenylarsenoxide are currently being evaluated in clinical trials for cancer treatment of refractory tumors   has been revealed also in intracellular membranes, including nuclear membrane, ER, Golgi and mitochondria , selectively inhibited by clotrimazole and TRAM-34, has been recorded from the inner mitochondrial membranes of human cancer cells , a two-pore potassium channel, was identified in mitochondria of melanoma and keratinocyte , which mediates proton leak , responsible for the low-affinity uptake of calcium into the mitochondrial matrix . For example the calcium-permeable ion channel TRPM8, overexpressed in several tumors, has been located to the ER (Zhang and Barritt, 2+] and increased resistance to apoptosis (Bidaux et al., 3 receptors acting as Ca2+ store release channels is altered in glioblastoma (Kang et al., 3- mediated Ca2+ elevation by Bcl-2 has been proposed to contribute to the pathophysiology of chronic lymphocytic leukemia (Zhong et al., Finally, we should briefly mention other intracellular channels involved in Cain vivo studies targeting these channels underline the importance of pursuing this line of research.In summary, while considerable further work is required to clarify the mechanisms by which intracellular channels contribute to tumorigenesis and tumor progression, or intervene in cell death, a few The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The significant impact of microRNAs (miRNAs) on disease pathology is becoming increasingly evident. These small non-coding RNAs have the ability to post-transcriptionally silence the expression of thousands of genes. Therefore, dysregulation of even a single miRNA could confer a large polygenic effect. Schizophrenia is a genetically complex illness thought to involve multiple genes each contributing a small risk. Large genome-wide association studies identified miR-137, a miRNA shown to be involved in neuronal maturation, as one of the top risk genes. To assess the potential mechanism of impact of miR-137 in this disorder and identify its targets, we used a combination of literature searches, ingenuity pathway analysis (IPA), and freely accessible bioinformatics resources. Using TargetScan and the schizophrenia gene resource (SZGR) database, we found that in addition to CSMD1, C10orf26, CACNA1C, TCF4, and ZNF804A, five schizophrenia risk genes whose transcripts are also validated miR-137 targets, there are other schizophrenia-associated genes that may be targets of miR-137, including ERBB4, GABRA1, GRIN2A, GRM5, GSK3B, NRG2, and HTR2C. IPA analyses of all the potential targets identified several nervous system (NS) functions as the top canonical pathways including synaptic long-term potentiation, a process implicated in learning and memory mechanisms and recently shown to be altered in patients with schizophrenia. Among the subset of targets involved in NS development and function, the top scoring pathways were ephrin receptor signaling and axonal guidance, processes that are critical for proper circuitry formation and were shown to be disrupted in schizophrenia. These results suggest that miR-137 may indeed play a substantial role in the genetic etiology of schizophrenia by regulating networks involved in neural development and brain function. C. elegans  are small non-coding RNAs with the ability to silence the expression of multiple target genes by binding to specific sequences of mRNAs. A single miRNA can impact hundreds to thousands of targets and can affect pathways controlling a large variety of processes, from normal development to oncogenesis. Pairing is primarily based on sequence complementarity of a “seed” sequence within the miRNA to a binding site of the mRNA, generally in the 3′ UTR of the mRNA being suppressed Bartel, . The mectrans-activation response RNA binding protein (TRBP) and the protein activator of the interferon induced protein kinase (PACT) into an approximately 22 nucleotide long double stranded RNA  can also modify and regulate miRNA function, both in the nucleus and cytoplasm Figure . AdenosiSchizophrenia is a severe mental illness that has an average lifetime development risk of 0.7%, with an average annual incidence rate of 15 per 100,000  Consortium , a database of a variety of schizophrenia related gene lists  and the National Institute of Mental Health (NIMH)  generating a canonical pathway analysis and related network analyses for each level. The networks are given a score based on the probability of inclusion of the number of molecules in the generated networks over the probability of a network being generated by chance with random molecules. This score is generated as a negative log p value based on a right tailed Fisher’s exact test   in all tissues, (b) only in the nervous system (NS), or (c) those associated with NS development and function are shown in Figure The biological significance of the molecules included in these canonical pathways is best illustrated by their associated interactive networks Figures –7. As shThere is evidence that schizophrenia is a highly complex polygenic disorder with multiple genes contributing only a small risk. Given that individual miRNAs can affect the expression of up to thousands of genes post-transcriptionally, and that differential expression of miRNAs between patients with schizophrenia and controls has been shown in many different studies; it seems likely that miRNAs may play a role in the etiology of the disease. miR-137 in particular was shown to have a SNP (rs1625579) with the highest association with schizophrenia in the largest schizophrenia GWAS study performed to date  receptor independent form of LTP  was identified as a hub in all three tiers of IPA network analyses. While the normal function of this protein is still largely unknown, it is possible that HTT has been included in the IPA database as having many biological relationships largely because it is well studied. However, recent evidence suggests that HTT is involved highly in neuronal development, playing a role in early neuronal survival, regulation of axonal transport, regulation of BDNF production particularly in the cortex, and controlling synaptic activity  canonical pathway. Figure shows the synaptic LTP pathway from IPA with the symbols for miR-137 putative and verified targets shown in orange.Click here for additional data file.Supplementary Figure S4miR-137 targets in ephrin receptor signaling canonical pathway. Figure shows the ephrin receptor signaling pathway from IPA with the symbols for all miR-137 putative and verified targets shown in orange.Click here for additional data file.Supplementary Figure S5miR-137 targets in axonal guidance canonical pathway. Figure shows the axonal guidance pathway from IPA with the symbols for all miR-137 putative and verified targets shown in orange.Click here for additional data file.


EHP 121(3):359–366; Chamorro-Garcia et al.].The term “obesogen” describes a chemical that promotes excessive weight gain by increasing adipocyte (fat cell) size or number, changing metabolism to favor fat storage, or altering the control of appetite and satiety. Previous studies have suggested that prenatal exposure to obesogens can alter metabolism in mice and predispose multipotent mesenchymal stem cells (MSCs) to become fat cells, but whether these changes last through subsequent generations was unknown. A team of researchers investigating this question exposed pregnant mice to tributyltin (TBT) and found impacts on adipogenesis through the third generation [TBT, which has been used as a biocide in marine paints and in textile products such as carpets, is a persistent organic pollutant that is ubiquitous in the environment. Exposure to TBT is thought to occur through many sources, including food, house dust, and consumer products.In the current study, male offspring  of mice exposed to TBT during pregnancy showed marked increases in the number and size of white fat cells and significant but less pronounced increases in the weight of white fat deposits (or “depots”) around the kidney and under the skin. The two subsequent generations (F2 and F3) also showed increases in fat cell size and number and, at the two higher TBT doses, in fat depot weight. Females showed more modest differences following TBT prenatal exposure, including both increases and decreases in cell numbers, depending on location.The team also found that F3 animals in the TBT group showed substantially increased expression of adipogenic markers in bone marrow–derived MSCs and decreased expression of osteogenic (bone lineage) markers—in other words, the stem cell population appeared reprogrammed to create fat instead of bone. The shift was more pronounced in males in all generations, and especially in the F3 generation.When the team looked at accumulation of lipids in the livers of each generation, they found that TBT-exposed F1 males and females showed marked increases in lipid accumulation; F2 and F3 generations also showed less pronounced increases in liver lipids. The investigators also found significant increases in the expression of hepatic genes related to fat creation and storage in males and females from all three generations of mice.F1 animals were exposed to TBT as fetuses, and F2 animals were potentially exposed as germ cells within the F1 fetuses. F3 animals were not exposed to TBT, yet they exhibited signs characteristic of exposure. This is the first known study to show that prenatal exposure to low, environmentally relevant concentrations of an obesogen led to transgenerational effects on white fat volume, altered stem cell programming, and liver fat accumulation in multiple generations of mice.


There is a growing body of evidence to support a connection between diabetes (predominantly type 2), obesity and cancer. Multiple meta-analyses of epidemiological data show that people with diabetes are at increased risk of developing many different types of cancers, along with an increased risk of cancer mortality. Several pathophysiological mechanisms for this relationship have been postulated, including insulin resistance and hyperinsulinaemia, enhanced inflammatory processes, dysregulation of sex hormone production and hyperglycaemia. In addition to these potential mechanisms, a number of common risk factors, including obesity, may be behind the association between diabetes and cancer. Indeed, obesity is associated with an increased risk of cancer and diabetes. Abdominal adiposity has been shown to play a role in creating a systemic pro-inflammatory environment, which could result in the development of both diabetes and cancer. Here, we examine the relationship between diabetes, obesity and cancer, and investigate the potential underlying causes of increased cancer risk in individuals with diabetes. Current treatment recommendations for reducing the overall disease burden are also explored and possible areas for future research are considered. Worldwide, cancer is the 2nd and diabetes the 12th leading cause of death The connection between diabetes and cancer was first postulated more than 75 years ago. Although these two conditions share many common risk factors, there continues to be a lack of understanding of the biological relationship between them, Alternatively, the relationship may be due, wholly or in part, to the sharing of common predisposing conditions, such as obesity Unravelling the pathology behind the link between these diseases may be further complicated by the multidrug therapy required for the treatment of diabetes, obesity and cancer Individually, the health consequences and economic costs of diabetes, obesity and cancer are high, and their impact is exacerbated when all three conditions co-present. In order to address some of the mounting concerns regarding the management of diabetes and cancer, the American Diabetes Association and American Cancer Society have published a consensus report providing recommendations for clinical care The aims of this review article are to further examine the association between diabetes, obesity and cancer, discuss the potential underlying causes and explore the current treatment recommendations for reducing the overall burden associated with these conditions.Numerous clinical studies have suggested a link between diabetes and a variety of different types of cancer, including breast Owing to the co-morbidities frequently associated with diabetes, as well as the complex nature of T2DM itself, each site-specific cancer needs to be evaluated individually in order to differentiate between risk factors associated with cancers in general and those that influence specific cancers AR, SRD5A2 and VDR genotypes which are associated with prostate cancer risk A negative association has been shown between diabetes and the risk of prostate cancer Table, with thAnother possible biological mechanism linking diabetes with a protective effect on prostate cancer risk is the link between hyperinsulinaemia and prostate cell growth. Insulin is positively associated with the growth of both normal and cancerous prostate cells, and, therefore, decreased insulin production, as observed in people with diabetes, may inhibit cell growth There is an added level of complexity in the association between diabetes and pancreatic cancer, as both diseases involve the same organ, with studies suggesting that diabetes could be both an early manifestation of pancreatic cancer and an aetiological factor In addition, it has been found that individuals who develop exocrine pancreatic cancer tend to have moderate increases in glycated haemoglobin (HbA1c) levels, relatively independent of obesity and insulin resistance Mortality rates in diabetes, obesity and cancer populations are high, with both T2DM and obesity being independently associated with an increased risk of cancer-related mortality The Health, Eating, Activity, and Lifestyle (HEAL) study of women diagnosed with stage I-IIIA breast cancer found that increasing insulin resistance and beta-cell dysfunction were associated with reduced breast cancer survival  and reduced all-cause survival  There are a number of possible reasons for this elevated mortality. These include presentation of more advanced disease at diagnosis in people with diabetes Several diabetes-related pathophysiological mechanisms, including hyperinsulinaemia, hyperglycaemia and inflammation, have been implicated in increasing cancer risk via their influence on neoplastic processes  4.Insulin resistance, hyperinsulinaemia (either endogenous due to insulin resistance or exogenous due to administered insulin or insulin secretagogues) and elevated levels of IGF-1 reduce apoptosis and increase cell proliferation in target cells, leading to tumour development The effects of insulin and hyperinsulinaemia on tumorigenesis are thought to be mediated by the insulin receptor, which is expressed in both normal tissues and tumours Production of the sex hormone oestrogen can also be modulated by insulin and hyperinsulinaemia, and this is thought to play a role in tumorigenesis α, is also thought to mediate an increase in the dysregulation of metabolic pathways leading to insulin resistance, which in turn leads to an increase in insulin levels, thus further increasing the inflammatory response The production of pro-inflammatory cytokines, such as interleukin (IL)-6 and tumour necrosis factor (TNF)-Elevated fasting serum glucose levels have been shown to be an independent risk factor for certain cancers, and the risk increases with rising glucose levels, as does cancer-related mortality In vitro studies involving breast cancer cell lines have shown that hyperglycaemia directly impacts these cells 50 by 1.5- and 1.3-fold, respectively Hyperglycaemia may impact on the outcome of chemotherapy for cancer Obesity is a major confounding factor in studies of diabetes and cancer as the risk of developing T2DM increases with growing body mass Body fat distribution can also be a marker of future risk, with abdominal adiposity being a potential mechanism of cancer development owing to increased levels of free fatty acids and cytokines, leading to insulin resistance and increased IGF availability Observations of increased risk of premenopausal breast cancer in obese individuals have been linked to altered regulation of inflammatory mediators and adipokine levels α, IL-6, IL-1, C-reactive protein, plasminogen activator inhibitor-1 and fibrinogen, and tissue-specific hormone activity mediated through the activation of nuclear factor kappa-light-chain-enhancer of activated B cells and peroxisome proliferator-activated receptor gamma pathways appear to be mechanistically linked with obesity Several mechanisms have been proposed for obesity-induced cancers, including abnormalities in adipokine secretion, including hyperleptinaemia, by central adipose tissue, which plays a role in various stages of obesity-induced carcinogenesis Obese people have also been shown to have poorer outcomes from treatment. For example, obese people undergoing surgical resection for colorectal cancer have been reported to have significantly longer operating times for laparoscopic surgery, as well as a greater likelihood of conversion from laparoscopic to open surgery Current diabetes treatments have been implicated in the development of cancer. Consequently, cancer risk may now play a part in the risk–benefit analysis that a physician undertakes when prescribing diabetes medication. The potential increased risk of cancer associated with diabetes medications may be a result of direct or indirect effects on insulin resistance and levels of circulating insulin or other mechanisms in vitro research has also shown that metformin may selectively kill cancer stem cells and enhance the effectiveness of breast cancer treatment regimens in vitro and in vivo studies, Kourelis et al. postulated that there are at least seven mechanisms that could account for the anticancer effects of metformin. These include: (i) activation of the liver kinase B1/AMPK pathway, in which metformin activates the AMPK pathways that inhibit the mammalian target of rapamycin pathway via phosphorylation, stabilizing TSC2, a tumour suppressor gene Metformin, the most commonly used therapy in people with T2DM A recent meta-analysis of 12 randomized controlled trials which compared metformin with other glucose-lowering therapies or placebo/usual care found no significant difference between the groups in terms of cancer risk The protective effect of metformin on cancer seen in epidemiological studies is so strong that metformin is now being investigated as an adjuvant therapy for cancer in three ongoing prospective trials. The Effect of Metformin on Breast Cancer Metabolism Study (NCT01266486) is investigating biomarkers that indicate a change in biological response in S6 kinase, 4E binding protein 1 and AMPK over a 2-week period. This study in metformin-naïve women with breast cancer aims to assess any metformin-induced phosphorylation of S6 kinase, 4E binding protein 1 and AMPK via immunohistochemical analysis, as well as investigating whether metformin could be used as a cancer therapy Diabetologia examined a possible link between insulin glargine monotherapy and all cancers or breast cancer, with conflicting results Diabetologia, the most recent, large clinical studies have focused on insulin glargine and these are discussed below.There have been suggestions of a link between insulin analogues and cancer owing to their altered affinity for the insulin receptor The 6-year, international Outcome Reduction with Initial Glargine Intervention (ORIGIN) Study, designed primarily to determine whether insulin glargine and/or omega-3 fatty acids, could reduce cardiovascular events in 12 537 people with elevated blood glucose levels, had cancer incidence as one of its secondary outcomes. The study compared treatment with insulin glargine with standard care and found, over a median of 6.2 years, no significant difference between study arms in the incidence of all cancers combined ; site-specific cancers, including lung cancer , colon cancer , breast cancer , prostate cancer , melanoma  and other cancers ; or cancer mortality  between the insulin glargine and standard of care arms To further investigate the potential link between insulin analogues and cancer, an updated meta-analysis of the Northern European Database Study was performed. This included data from epidemiological studies involving 907 008 subjects. A total of 2 597 602 person-years of observation found no increased risk of developing cancer with insulin glargine versus all other insulins  Data from two other large database studies  were presented at the 2012 American Diabetes Association Scientific Sessions. It was reported that there was no connection between insulin glargine and cancer, and publications on these studies are eagerly awaited Clinical studies have associated sulfonylureas with an increased risk of cancer In vitro and in vivo studies on thiazolidinediones have produced inconclusive results; it is possible that these drugs may increase, decrease or have a neutral effect on the risk of cancer or cancer progression Dipeptidyl peptidase-4 (DPP-4) inhibitors indirectly increase the level of glucagon-like peptide (GLP)-1 in the body by inhibiting its degradation It has been suggested that some GLP-1 based therapies, GLP-1 receptor agonists and DPP-4 inhibitors, could increase the risk of pancreatitis, with a number of cases being reported in the literature. In response to these case reports, analyses have been published in the literature examining the currently available data on incretin drugs. These analyses have reported conflicting results and there is no clear evidence that either DPP-4 inhibitors or GLP-1 receptor agonists are associated with an increased risk for any pancreatic disorder; prospective long-term studies are needed to properly quantify these risks. A number of these analyses are discussed below.A study investigating GLP-1 based drugs that analysed the US Food and Drug Administration's (FDA) adverse event data reported a sixfold increased risk of pancreatic cancer, as well as an increased risk of thyroid cancer, in people treated with exenatide or sitagliptin (a DPP-4 inhibitor) I2 = 30%] or liraglutide  was associated with an increased risk of acute pancreatitis. In addition, neither drug was associated with an increased risk of cancer, including thyroid cancer In order to address whether sitagliptin use increased the risk for pancreatitis, a pooled analysis including 19 double blind, randomized controlled trials of sitagliptin, which involved 10 246 people with T2DM, was performed, with the analysis adjusted to take into account the different exposures A retrospective, observational database study compared the risk for acute pancreatitis between the following subpopulations: people with diabetes versus people without diabetes; and between people who had used exenatide, sitagliptin and other antidiabetic therapies post hoc database study and only a small number of the people included had received exenatide or sitagliptin . In addition, a report has suggested that based upon results in mice prone to pancreatic cancer, the use of GLP-1-based therapy may be linked to the progression and transformation of premalignant pancreatic intraepithelial neoplasia However, a recent, population-based, case–control study involving 2538 adults with T2DM reported that treatment with exenatide and sitagliptin was associated with increased odds of hospitalization for acute pancreatitis Owing to the conflicting results from different trials, the risk of pancreatitis at present can be neither proposed nor excluded. Prospective long-term studies are needed to properly quantify these risks.Similar concerns have been raised for the GLP-1 receptor agonist liraglutide and the risk for thyroid cancer Using multidisciplinary treatment strategies that reduce diabetes, obesity and cancer will probably have a greater impact on mortality than tackling each disease individually. Primary prevention should target improvements in lifestyle factors such as smoking cessation and weight management, while patient and physician education on the benefits of these approaches are important. Maintenance of a low waist circumference is a promising way to support cancer prevention and extend life expectancy. Secondary prevention would aim to treat cancer aggressively, manage complications and effectively optimize diabetes management. In general, the American Diabetes Association/American Cancer Society consensus panel recommends that physicians promote a healthy diet, physical activity and weight management for all individuals and routinely screen people with diabetes for cancer. In addition, for the majority of this population, cancer risk should not be a key factor in choosing antidiabetic treatment Diabetes, diabetes risk factors and some diabetes treatments may be associated with cancer, with certain cancers developing more commonly in people with T2DM. Strong and plausible evidence exists to suggest links between diabetes, obesity and cancer; however, the underlying mechanisms remain unclear and there is little clinical evidence to guide the appropriate management of people presenting with these diseases concurrently. Joint management and reduction of diabetes, cancer and obesity is likely to result in greater improvements in mortality than treating these diseases separately. Consequently, a multidisciplinary approach is needed to uncover the mechanisms underlying the associations between these diseases and, ultimately, improve clinical outcomes.


Ligands of peroxisome-proliferator activated receptors (PPARs), such as non-esterified fatty acids (NEFAs), induce expression of angiopoietin-like protein 4 (ANGPTL4). Recently ANGPTL4 has been reported to be a mediator of intracellular adipose lipolysis induced by glucocorticoids.To determine the concentrations of ANGPTL4 in cord serum of neonates born by spontaneous vaginal delivery (SVD) and by pre-labor cesarean section (CS) from healthy women, and to relate them to parameters of neonatal lipolytic activity at birth.In 54 neonates born by SVD and in 56 neonates born by CS, arterial cord blood was drawn to determine insulin, cortisol, triacylglycerols (TAGs), glycerol, non-esterified fatty acids (NEFAs), individual fatty acids, ANGPTL4, adiponectin, retinol binding protein 4 (RBP4) and leptin.Birth weight and neonatal fat mass in SVD and CS showed no difference, but the concentrations of glycerol, adiponectin, RBP4, NEFAs and most individual fatty acids were higher in cord serum of neonates born by SVD compared to CS, indicating a higher adipose tissue breakdown in the SVD group. The concentrations of TAG and cortisol were also higher and that of insulin was lower in cord serum of SVD compared to the CS group. However, the concentration in cord serum of ANGPTL4 did not differ between the two groups and no positive correlation with either NEFA or glycerol concentrations were detected.ANGPTL4 is known to stimulate lipolysis in adults, but does not appear to mediate the increased activity in SVD, indicating the presence of different regulatory inputs. Basal and hormone-stimulated triacylglycerol (TAG) lipolysis and efflux of fatty acids from adipose tissue is a complex process involving multiple regulatory factors −/− mice, the concentrations of cAMP and the absolute abundance of phosphorylated forms of hormone sensitive lipase (HSL) and perilipin were lower than in wild type controls ANGPTL4 is synthesized primarily in liver and adipose tissue ANGPTL4 gene expression is regulated by several signals, including fasting and glucocorticoids Physiologically, spontaneous delivery is a condition where there is a decline in circulating insulin and an increase in serum catecholamines and cortisol concentrations in the newborn The neonates from 110 uncomplicated pregnancies were studied. Fifty four were born by spontaneous vaginal delivery (SVD) and fifty six by scheduled pre-labor cesarean section (CS). Indications for elective CS were previous cesareans, suspected macrosomia, suspected feto-pelvic disproportion, breech presentation, suspected intrauterine growth retardation with or without suspected fetal Doppler indices, and maternal request. Both populations were recruited from women who gave birth at the Department of Obstetrics of the Vivantes Medical Center in Berlin. All the women showed normal 75 g oral glucose tolerance test (OGTT) according to Carpenter & Coustan criteria (90/180/155 mg/dl) Cord blood samples from one of the umbilical arteries were obtained from a segment of the cord immediately after delivery. Blood samples were centrifuged (1500×g at 4°C for 25 min) and serum was divided into small aliquots and immediately stored at −80°C until analysis. None of the samples used in the study were hemolysed.Serum cholesterol, TAG , glycerol and NEFA  were determined enzymatically using commercial kits. HDL-cholesterol (HDL-c) was measured after precipitation of apoB-containing lipoproteins with phosphotungstic acid and magnesium . LDL-cholesterol (LDL-c) was calculated according Friedewald's formula Serum cortisol, ANGPTL4, insulin, leptin, adiponectin and retinol-binding protein 4 (RBP4) concentrations were measured using ELISA kits according to the manufacturer's instructions. Specifically, cortisol was measured using a competitive ELISA kit , ANGPTL4 were determined using sandwich ELISA kit , with intra-assay variations of <10% and inter-assay variation of <12%, insulin, adiponectin and leptin were measured using sandwich ELISA kits  and RBP4 was measured using a sandwich ELISA kit .For the analysis of fatty acid profiles, serum lipids were extracted in chloroform/methanol (2∶1) Results are expressed as means ± SEM. Statistical differences between groups were determined by analysis of variance (ANOVA) after adjusting for maternal pre-pregnancy body mass index (BMI), gestational age, sex and neonatal fat mass. When differences were statistically significant, multiple comparisons were performed using Tukey's post hoc test. Given their skewed distributions, concentrations of cortisol, insulin, TAG, NEFA, glycerol, adiponectin, RBP4, leptin, ANGPTL4 and polyunsaturated fatty acids (PUFAs) were log-transformed before statistical comparison. Correlations were tested by Pearson's method using the log-transformed data as indicated. To ascertain the independent predictors of NEFA, stepwise multiple regressions with backward selection were performed. All statistical analysis was performed using a computer software package .As shown in The concentrations of several metabolic parameters in cord serum after adjusting for pre-pregnancy BMI, gestational weeks at birth, sex and neonatal fat mass, are shown in When the concentrations of individual fatty acids were determined in umbilical arterial serum , it was In order to test the hypothesis that a relationship between serum ANGPTL4 and NEFA or glycerol concentrations and its ratio exists, simple regression analysis was performed in spontaneous delivery and cesarean section groups separately. As shown in The study reported herein showed that the concentration of ANGPTL4 in umbilical artery serum of neonates born by spontaneous delivery was similar to those found in neonates delivered by pre-labor cesarean section, in spite of the fact that the concentrations of NEFA, glycerol, and individual fatty acids as well as the NEFA/glycerol ratio were higher in the spontaneous delivery group. This finding indicated that, at least in gestations resulting in spontaneous vaginal delivery, there was no association of neonatal ANGPTL4 with the higher lipolytic products in adipose tissue that accompanies the onset of labor, delivery and the initial stages of neonatal adaptation.It has been proposed that ANGPTL4 could act as a signal to prevent fat storage and stimulate intracellular adipose tissue lipolysis, especially in the situations of high energy demand, when ANGPTL4 could develop a key role in increasing the availability of fatty acids for β-oxidation We found here that in umbilical arterial serum of neonates born by spontaneous delivery, the concentration of cortisol was almost double that present in cord serum of neonates delivered by caesarean section. Accordingly, it was also found that the concentration of NEFA, glycerol and the ratio NEFA/glycerol were higher in umbilical arterial serum of neonates born by spontaneous delivery, indicating increased lipolysis of TAG, without differences in other lipid fractions such as total serum cholesterol, HDL-cholesterol or LDL-cholesterol. Following the same trend, the concentrations of adiponectin and RBP4, two of the most abundant adipocytokines produced by adipose tissue, were higher in cord serum of neonates born by spontaneous delivery. However, we did not find any difference in the concentrations of leptin in cord serum probably because the neonatal fat mass was similar in both groups, confirming the relationship between these two parameters A major limitation of this study is the inability to measure directly the lipolytic activity of adipose tissue and therefore, to ensure that what we are considering as products of adipose tissue lipolysis, not actually come from the placenta. However, using the serum from the umbilical artery we minimize this possibility, since this mainly transport fetal metabolism products to the placenta The absence of differences in the concentrations of ANGPTL4 between the groups was unexpected for several reasons. On one hand, ANGPTL4 is a downstream target of the PPARα, PPARγ and PPARδ/β, so that its expression is induced by PPAR agonists in several types of cells and animal models Although our results appear to contradict the current wisdom on the relationship between ANGPTL4 and NEFA and glucocorticoids, it should be noted that plasma ANGPTL4 concentrations in humans are only increased by conditions associated with extreme elevated concentrations of NEFA, including intravenous infusion of lipid emulsion In conclusion, in spite of the remarkable lipolytic activity in adipose tissue in neonates born by spontaneous delivery, the concentration of ANGPTL4 remained unchanged compared to that found in neonates born by pre-labor cesarean section. The higher concentrations of cortisol and especially of NEFA observed in arterial cord serum of neonates born by spontaneous delivery did not induce the increase in the levels of ANGPTL4 reported to be present in those conditions of adults that are accompanied by an acute raise of NEFA. These results support the hypothesis that the regulation of circulating ANGPTL4 concentrations in vivo is more complex than is currently understood, with potential involvement of additional regulatory inputs.


Long predicted from studies of model vertebrates, the first human example of abnormal patterning of the early neural tube leading to underdevelopment of the cerebellum has been demonstrated. Related research article Yu T, Meiners LC, Danielsen K, Wong MTY, Bowler T, Reinberg D, Scambler PJ, van Ravenswaaij-Arts CMA, Basson MA. 2013. Deregulated FGF and homeotic gene expression underlies cerebellar vermis hypoplasia in CHARGE syndrome. eLife2:e01305. doi: 10.7554/eLife.01305Image MRI scan of a patient with CHARGE syndromecoloboma (a hole in the eye), heart defects, choanal atresia , retarded growth and development, genital abnormalities, and ear anomalies  and co-workers—including Tim Yu of KCL as first author—report that underdevelopment of a region of the cerebellum called the vermis is also associated with CHARGE syndrome  have shown that if the Isthmic Organizer fails to develop, there is dramatic and rapid cell death in adjacent regions of the neural tube. Mutant mice in which the Isthmic Organizer cannot express Fgf8 at all do not survive post-natally . HoweverWith recent developments in neuroimaging, neuropathology and neurogenetics, many developmental disorders of the cerebellum, including cerebellar vermis hypoplasia, have emerged as causes of neurodevelopmental dysfunction . TogetheCHD7; Previously several clinical reports had noted cerebellar deficits in a few CHARGE syndrome patients. Now Yu, Basson and co-workers—who are based in London, Groningen and New York—report the results of MRI scans of a large cohort of 20 patients with CHARGE syndrome  is altered in mice lacking both working copies of the Chd7 gene. Chromatin immunoprecipitation assays subsequently revealed that Chd7 is associated with enhancer elements belonging to the Otx2 and Gbx2 genes, which suggests that Chd7 has a direct role in regulating these essential Isthmic Organizer genes.Going one step further, Yu et al. next demonstrated that the expression of two genes that regulate Fgf8 expression at the Isthmic Organizer  are still likely to be incompatible with human life, this study implies that genes which regulate the expression of central Isthmic Organizer genes may cause cerebellar malformation when mutated.Together these results provide substantial evidence that cerebellar vermis hypoplasia in CHARGE syndrome is caused by dysfunction of the Isthmic Organizer in the early embryo. Further, the work of Basson, Yu and co-workers suggests that Isthmic Organizer disruption may be a more common cause of human cerebellar malformation than previously thought. Although complete loss-of-function mutations in genes that are central to the formation of the Isthmic Organizer (such as Chd7 disrupts Isthmic Organizer Fgf8 expression, Fgf8 expression is normal in the adjacent pharyngeal arches of the embryo. One possibility is that Cdh7 has different binding partners in different tissues.CHD7 is expressed in a wide variety of tissues during development, and CHARGE syndrome phenotypes indicate that it has tissue-specific and developmental stage-specific roles. A difficult question remains as to how CHD7 achieves different functions in different tissues. For example although loss of Intriguingly, it has recently been shown that Cdh7 interacts with a small number of other transcription factors in neural stem cells, and that mutations in these other factors underlie a number of human malformation disorders . One exc