Up-regulation of the human-specific CHRFAM7A gene protects against renal fibrosis in mice with obstructive nephropathy.

Renal fibrosis is a major factor in the progression of chronic kidney diseases. Obstructive nephropathy is a common cause of renal fibrosis, which is also accompanied by inflammation. To explore the effect of human-specific CHRFAM7A expression, an inflammation-related gene, on renal fibrosis during obstructive nephropathy, we studied CHRFAM7A transgenic mice and wild type mice that underwent unilateral ureteral obstruction (UUO) injury. Transgenic overexpression of CHRFAM7A gene inhibited UUO-induced renal fibrosis, which was demonstrated by decreased fibrotic gene expression and collagen deposition. Furthermore, kidneys from transgenic mice had reduced TGF-ß1 and Smad2/3 expression following UUO compared with those from wild type mice with UUO. In addition, the overexpression of CHRFAM7A decreased release of inflammatory cytokines in the kidneys of UUO-injured mice. In vitro, the overexpression of CHRFAM7A inhibited TGF-ß1-induced increase in expression of fibrosis-related genes in human renal tubular epithelial cells (HK-2 cells). Additionally, up-regulated expression of CHRFAM7A in HK-2 cells decreased TGF-ß1-induced epithelial-mesenchymal transition (EMT) and inhibited activation f TGF-ß1/Smad2/3 signalling pathways. Collectively, our findings demonstrate that overexpression of the human-specific CHRFAM7A gene can reduce UUO-induced renal fibrosis by inhibiting TGF-ß1/Smad2/3 signalling pathway to reduce inflammatory reactions and EMT of renal tubular epithelial cells.

Human-specific CHRFAM7A primes macrophages for a heightened pro-inflammatory response at the earlier stage of inflammation.

α7-Nicotinic acetylcholine receptor (α7-nAChR) is the key effector molecule of the cholinergic anti-inflammatory pathway. Evolution has evolved a uniquely human α7-nAChR encoded by CHRFAM7A. It has been demonstrated that CHRFAM7A dominant negatively regulates the functions of α7-nAChR. However, its role in inflammation remains to be fully characterized. CHRFAM7A transgenic (Tg) mice were phenotypically normal and their peritoneal macrophages exhibited decreased ligand-binding capability and, importantly, an activated gene expression profile of pro-inflammatory cytokines. Surprisingly, when challenged with sepsis, the Tg mice showed no survival disadvantage relative to their wild-type (Wt) counterparts. Further analysis showed that the complete blood count and serum levels of pro-inflammatory cytokines were comparable at resting state, but the degrees of leukocyte mobilization and the increase of pro-inflammatory cytokines were significantly higher in Tg than Wt mice at the early stage of sepsis. In vitro, peritoneal macrophages of the Tg mice exhibited an exaggerated response to lipopolysaccharides (LPSs), especially at the earlier time points and at lower dosages of LPS. Remarkably, monocytes from CHRFAM7A-carrier showed similar dynamic changes of the pro-inflammatory cytokines to that observed in the Tg mice upon LPS challenge. Our results suggest that CHRFAM7A increases the mobilization of leukocytes and primes macrophages that confer an enhanced immune response at the early stage of inflammation, which may lead to prompt pathogen clearance, an evolutionary advantage in less severe inflammatory conditions.

Heart-targeting exosomes from human cardiosphere-derived cells improve the therapeutic effect on cardiac hypertrophy.

Exosomes of human cardiosphere-derived cells (CDCs) are very promising for treating cardiovascular disorders. However, the current challenge is inconvenient delivery methods of exosomes for clinical application. The present study aims to explore the potential to enhance the therapeutic effect of exosome (EXO) from human CDCs to myocardial hypertrophy. A heart homing peptide (HHP) was displayed on the surface of exosomes derived from CDCs that were forced to express the HHP fused on the N-terminus of the lysosomal-associated membrane protein 2b (LAMP2b). The cardiomyocyte-targeting capability of exosomes were analyzed and their therapeutic effects were evaluated in a mouse model of myocardial hypertrophy induced by transverse aorta constriction (TAC). The molecular mechanisms of the therapeutic effects were dissected in angiotensin II-induced neonatal rat cardiomyocyte (NRCMs) hypertrophy model using a combination of biochemistry, immunohistochemistry and molecular biology techniques. We found that HHP-exosomes (HHP-EXO) accumulated more in mouse hearts after intravenous delivery and in cultured NRCMs than control exosomes (CON-EXO). Cardiac function of TAC mice was significantly improved with intravenous HHP-EXO administration. Left ventricular hypertrophy was reduced more by HHP-EXO than CON-EXO via inhibition of ß-MHC, BNP, GP130, p-STAT3, p-ERK1/2, and p-AKT. Similar results were obtained in angiotensin II-induced hypertrophy of NRCMs, in which the beneficial effects of HHP-EXO were abolished by miRNA-148a inhibition. Our results indicate that HHP-EXO preferentially target the heart and improve the therapeutic effect of CDCs-exosomes on cardiac hypertrophy. The beneficial therapeutic effect is most likely attributed to miRNA-148a-mediated suppression of GP130, which in turn inhibits STAT3/ERK1/2/AKT signaling pathway, leading to improved cardiac function and remodeling.

Alteration of purinergic signaling in diabetes: Focus on vascular function.

Diabetes is an important risk factor for the development of cardiovascular disease including atherosclerosis and ischemic heart disease. Vascular complications including macro- and micro-vascular dysfunction are the leading causes of morbidity and mortality in diabetes. Disease mechanisms at present are unclear and no ideal therapies are available, which urgently calls for the identification of novel therapeutic targets/agents. An altered nucleotide- and nucleoside-mediated purinergic signaling has been implicated to cause diabetes-associated vascular dysfunction in major organs. Alteration of both purinergic P1 and P2 receptor sensitivity rather than the changes in receptor expression accounts for vascular dysfunction in diabetes. Activation of P2X7 receptors plays a crucial role in diabetes-induced retinal microvascular dysfunction. Recent findings have revealed that both ecto-nucleotidase CD39, a key enzyme hydrolyzing ATP, and CD73, an enzyme regulating adenosine turnover, are involved in the renal vascular injury in diabetes. Interestingly, erythrocyte dysfunction in diabetes by decreasing ATP release in response to physiological stimuli may serve as an important trigger to induce vascular dysfunction. Nucleot(s)ide-mediated purinergic activation also exerts long-term actions including inflammatory and atherogenic effects in hyperglycemic and diabetic conditions. This review highlights the current knowledge regarding the altered nucleot(s)ide-mediated purinergic signaling as an important disease mechanism for the diabetes-associated vascular complications. Better understanding the role of key receptor-mediated signaling in diabetes will provide more insights into their potential as targets for the treatment.

Open reading frame mining identifies a TLR4 binding domain in the primary sequence of ECRG4.

The embedding of small peptide ligands within large inactive pre-pro-precursor proteins encoded by orphan open reading frames (ORFs) makes them difficult to identify and study. To address this problem, we generated oligonucleotide (< 100-400 base pair) combinatorial libraries from either the epidermal growth factor (EGF) ORF that encodes the > 1200 amino acid EGF precursor protein or the orphan ECRG4 ORF, that encodes a 148 amino acid Esophageal Cancer Related Gene 4 (ECRG4), a putative cytokine precursor protein of up to eight ligands. After phage display and 3-4 rounds of biopanning for phage internalization into prostate cancer epithelial cells, sequencing identified the 53-amino acid EGF ligand encoded by the 5' region of the EGF ORF and three distinct domains within the primary sequence of ECRG4: its membrane targeting hydrophobic signal peptide, an unanticipated amino terminus domain at ECRG437-63 and a C-terminus ECRG4133-148 domain. Using HEK-blue cells transfected with the innate immunity receptor complex, we show that both ECRG437-63 and ECRG4133-148 enter cells by interaction with the TLR4 immune complex but neither stimulate NFkB. Taken together, the results help establish that phage display can be used to identify cryptic domains within ORFs of the human secretome and identify a novel TLR4-targeted internalization domain in the amino terminus of ECRG4 that may contribute to its effects on cell migration, immune cell activation and tumor suppression.

miR-499 released during myocardial infarction causes endothelial injury by targeting α7-nAchR.

The surged systemic vascular inflammation after acute myocardial infarction (AMI) aggravates the atherosclerotic endothelial injury. To explore roles of miR-499 released from cardiomyocytes during AMI in endothelial injury. Using qPCR and ELISA, we discovered that patients with AMI had significantly increased plasma miR-499, which was directly correlated with serum thrombomodulin, a marker for endothelial injury. Plasma of AMI patients, when incubated with human umbilical vein endothelial cells (HUVECs), significantly increased the expression of endothelial injury markers, which could be abrogated by antagomiR-499. In vitro, neonatal rat cardiomyocytes subjected to hypoxia/reoxygenation (HX/R) released miR-499 that could be internalized into rat pulmonary microvascular endothelial cells (RPMECs), worsening the high glucose-induced injury. In silico analysis demonstrated that CHRNA7 encoding α7-nAchR is a target of miR-499, which was validated in cell lines expressing endogenous α7-nAchR. In high glucose-induced RPMECs injury model, miR-499 aggravated, whereas forced CHRNA7 expression ameliorated the injury. Moreover, the perfusate from Langendorff perfused rat heart subjected to HX/R contained higher level of miR-499 that significantly impaired the Bradykinin-mediated endothelium-dependent relaxation in both conduit and resistance arteries, which could be partially abrogated by antagomiR-499. Finally, the correlation between plasma miR-499 and endothelial injury was further confirmed in another cohort of AMI patients. We conclude that miR-499 released from injured cardiomyocytes contributes to the endothelial injury by targeting α7-nAchR. This study implies that miR-499 may serve as a potential target for the treatment of the surged vascular inflammation post-AMI.

The Response to Burn Injury in Mice With Human Hematolymphoid Systems.

OBJECTIVE: To develop an animal model of injury that more closely represents the human inflammatory cell response to injury. BACKGROUND: Because the mouse inflammatory response to burn injury cannot account for the contribution of human-specific genes, animal models are needed to more closely recapitulate the human inflammatory response and improve the translational impact of injury research. To this end, we hypothesized that the human inflammatory cell response to injury could be selectively assessed after severe burn injury using humanized mice. METHODS: NOD-Scid-IL2Rγ null mice were transplanted with human hematopoietic CD34+ progenitor cells; their engraftment confirmed and then subjected to 30% total body surface area steam burn injury. Blood, bone marrow, and lung tissue were collected 4 hours after injury and human inflammatory cell mobilization analyzed using flow cytometry and immunohistochemistry. RESULTS: Burn injury caused mobilization of human inflammatory cells into the systemic circulation. Next, burn injury was accompanied by evidence of histologic lung injury and concomitant mobilization of human CD45+ immune cells into the lung that were associated with increased trafficking of human CD11b+ myeloid cells. CONCLUSIONS: These experiments are the first to demonstrate the suitability of humanized mice for injury research. They offer the possibility to address very specific research questions that are not amenable to traditional mouse models of injury, for example, the emerging role of certain human-specific genes that are either unrepresented or totally absent, from the mouse genome.

CHRFAM7A: a human-specific α7-nicotinic acetylcholine receptor gene shows differential responsiveness of human intestinal epithelial cells to LPS.

The human genome contains a unique, distinct, and human-specific α7-nicotinic acetylcholine receptor (α7nAChR) gene [CHRNA7 (gene-encoding α7-nicotinic acetylcholine receptor)] called CHRFAM7A (gene-encoding dup-α7-nicotinic acetylcholine receptor) on a locus of chromosome 15 associated with mental illness, including schizophrenia. Located 5' upstream from the "wild-type" CHRNA7 gene that is found in other vertebrates, we demonstrate CHRFAM7A expression in a broad range of epithelial cells and sequenced the CHRFAM7A transcript found in normal human fetal small intestine epithelial (FHs) cells to prove its identity. We then compared its expression to CHRNA7 in 11 gut epithelial cell lines, showed that there is a differential response to LPS when compared to CHRNA7, and characterized the CHRFAM7A promoter. We report that both CHRFAM7A and CHRNA7 gene expression are widely distributed in human epithelial cell lines but that the levels of CHRFAM7A gene expression vary up to 5000-fold between different gut epithelial cells. A 3-hour treatment of epithelial cells with 100 ng/ml LPS increased CHRFAM7A gene expression by almost 1000-fold but had little effect on CHRNA7 gene expression. Mapping the regulatory elements responsible for CHRFAM7A gene expression identifies a 1 kb sequence in the UTR of the CHRFAM7A gene that is modulated by LPS. Taken together, these data establish the presence, identity, and differential regulation of the human-specific CHRFAM7A gene in human gut epithelial cells. In light of the fact that CHRFAM7A expression is reported to modulate ligand binding to, and alter the activity of, the wild-type α7nAChR ligand-gated pentameric ion channel, the findings point to the existence of a species-specific α7nAChR response that might regulate gut epithelial function in a human-specific fashion.

Mice engrafted with human hematopoietic stem cells support a human myeloid cell inflammatory response in vivo.

Mice engrafted with human CD34+ hematopoietic stem and progenitor cells (CD34+ -HSPCs) have been used to study human infection, diabetes, sepsis, and burn, suggesting that they could be highly amenable to characterizing the human inflammatory response to injury. To this end, human leukocytes infiltrating subcutaneous implants of polyvinyl alcohol (PVA) sponges were analyzed in immunodeficient NSG mice reconstituted with CD34+ -HSPCs. It was reported that human CD45+ (hCD45+ ) leukocytes were present in PVA sponges 3 and 7 days postimplantation and could be localized within the sponges by immunohistochemistry. The different CD45+ subtypes were characterized by flow cytometry and the profile of human cytokines they secreted into PVA wound fluid was assessed using a human-specific multiplex bead analyses of human IL-12p70, TNFα, IL-10, IL-6, IL1ß, and IL-8. This enabled tracking the functional contributions of HLA-DR+ , CD33+ , CD19+ , CD62L+ , CD11b+ , or CX3CR1+ hCD45+ infiltrating inflammatory leukocytes. PCR of cDNA prepared from these cells enabled the assessment and differentiation of human, mouse, and uniquely human genes. These findings support the hypothesis that mice engrafted with CD34+ -HSPCs can be deployed as precision avatars to study the human inflammatory response to injury.

Atrial-selective block of sodium channels by acehytisine in rabbit myocardium.

Acehytisine, a multi-ion channel blocker, can markedly inhibit INa, ICa, IKur, If at various concentrations and effectively terminate and prevent atrial fibrillation (AF) in patients and animal models, but the molecular mechanism underlying its blockage remains elusive. In this study, we investigated the effects of acehytisine on action potentials and sodium channels of atrial and ventricular myocytes isolated from rabbit, using whole-cell recording system. We found that acehytisine exerted stronger blocking effects on sodium channels in atria than in ventricles, especially at depolarization (IC50: 48.48 ± 7.75 µmol/L in atria vs. 560.17 ± 63.98 µmol/L in ventricles). It also significantly shifted steady state inactivation curves toward negative potentials in atrial myocytes, without affecting the recovery kinetics from inactivation of sodium channels in the same cells. In addition, acehytisine inhibited INa in a use-dependent manner and regulated slow inactivation kinetics by different gating configurations. These findings indicate that acehytisine selectively blocks atrial sodium channels and possesses affinity to sodium channel in certain states, which provides additional evidence for the anti-AF of acehytisine.

A Human-Specific α7-Nicotinic Acetylcholine Receptor Gene in Human Leukocytes: Identification, Regulation and the Consequences of CHRFAM7A Expression.

The human genome contains a variant form of the α7-nicotinic acetylcholine receptor (α7nAChR) gene that is uniquely human. This CHRFAM7A gene arose during human speciation and recent data suggests that its expression alters ligand tropism of the normally homopentameric human α7-AChR ligand-gated cell surface ion channel that is found on the surface of many different cell types. To understand its possible significance in regulating inflammation in humans, we investigated its expression in normal human leukocytes and leukocyte cell lines, compared CHRFAM7A expression to that of the CHRNA7 gene, mapped its promoter and characterized the effects of stable CHRFAM7A overexpression. We report here that CHRFAM7A is highly expressed in human leukocytes but that the levels of both CHRFAM7A and CHRNA7 mRNAs were independent and varied widely. To this end, mapping of the CHRFAM7A promoter in its 5'-untranslated region (UTR) identified a unique 1-kb sequence that independently regulates CHRFAM7A gene expression. Because overexpression of CHRFAM7A in THP1 cells altered the cell phenotype and modified the expression of genes associated with focal adhesion (for example, FAK, P13K, Akt, rho, GEF, Elk1, CycD), leukocyte transepithelial migration (Nox, ITG, MMPs, PKC) and cancer (kit, kitL, ras, cFos cyclinD1, Frizzled and GPCR), we conclude that CHRFAM7A is biologically active. Most surprisingly however, stable CHRFAM7A overexpression in THP1 cells upregulated CHRNA7, which, in turn, led to increased binding of the specific α7nAChR ligand, bungarotoxin, on the THP1 cell surface. Taken together, these data confirm the close association between CHRFAM7A and CHRNA7 expression, establish a biological consequence to CHRFAM7A expression in human leukocytes and support the possibility that this human-specific gene might contribute to, and/or gauge, a human-specific response to inflammation.

Esophageal cancer-related gene-4 (ECRG4) interactions with the innate immunity receptor complex.

OBJECTIVE AND DESIGN: The human c2orf40 gene encodes a tumor suppressor gene called esophageal cancer-related gene-4 (ECRG4) with pro- and anti-inflammatory activities that depend on cell surface processing. Here, we investigated its physical and functional association with the innate immunity receptor complex. METHODS: Interactions between ECRG4 and the innate immunity receptor complex were assessed by flow cytometry, immunohistochemistry, confocal microscopy, and co-immunoprecipitation. Phage display was used for ligand targeting to cells that overexpress the TLR4-MD2-CD14. RESULTS: Immunoprecipitation and immunohistochemical studies demonstrate a physical interaction between ECRG4 and TLR4-MD2-CD14 on human granulocytes. Flow cytometry shows ECRG4 on the cell surface of a subset of CD14(+) and CD16(+) leukocytes. In a cohort of trauma patients, the C-terminal 16 amino acid domain of ECRG4 (ECRG4(133-148)) appears to be processed and shed, presumably at a thrombin-like consensus sequence. Phage targeting this putative ligand shows that this peptide sequence internalizes into cells through the TLR4/CD14/MD2 complex, but modulates inflammation through non-canonical, NFκB signal transduction. CONCLUSIONS: ECRG4 is present on the surface of human monocytes and granulocytes. Its interaction with the human innate immunity receptor complex supports a role for cell surface activation of ECRG4 during inflammation and implicates this receptor in its mechanism of action.

Pulmonary preconditioning, injury, and inflammation modulate expression of the candidate tumor suppressor gene ECRG4 in lung.

PURPOSE: The human c2orf40 gene encodes a candidate tumor suppressor called Esophageal Cancer-Related Gene-4 (ECRG4) that is a cytokine-like epigenetically-regulated protein that is characteristically downregulated in cancer, injury, inflammation, and infection. Here, we asked whether ECRG4 gene expression is detectable in lung epithelial cells and if its expression changes with inflammation, infection, and/or protective preconditioning. MATERIALS AND METHODS: We used immunoblotting, PCR, and quantitative PCR to measure ECRG4 and either inhalation anesthesia preconditioning, lipopolysaccharide injection, or laparotomy to modulate lung inflammation. RESULTS: Immunoblotting establishes the presence of the full-length 14 kDa ECRG4 peptide in mouse lung. Immunohistochemistry localizes ECRG4 to type l alveolar epithelial cells. Basal ECRG4 mRNA is greater than TNF-α, IL-1ß, and IL-6 but following inflammatory lung injury, TNF-α, IL-1ß, IL-6, and IL-10 are upregulated while ECRG4 gene expression is decreased. Similar findings are observed after an intravenous administration of lipopolysaccharide. In contrast, lung preconditioning with isoflurane anesthesia increases lung ECRG4 gene expression. Over-expression of ECRG4 in human lung epithelial cells in vitro decreases cell proliferation implying that a loss of ECRG4 in vivo would be permissive to cell growth. CONCLUSIONS: This study supports the hypothesis that ECRG4 acts as a sentinel growth inhibitor in lung alveolar epithelial cells. Its downregulation by injury, infection, and inflammation and upregulation by preconditioning supports a role for ECRG4 in regulating the alveolar epithelium response to injury and inflammation. By extension, the findings support a functional consequence to its inhibition by promoter hypermethylation (i.e. lung cancer) and suggest potential benefits to its upregulation.

A potential role of human esophageal cancer-related gene-4 in cardiovascular homeostasis.

Human esophageal cancer related gene-4 (ECRG-4) encodes a 148-aminoacid pre-pro-peptide that can be processed tissue-dependently into multiple small peptides possessing multiple functions distinct from, similar to, or opposite to the tumor suppressor function of the full-length Ecrg4. Ecrg-4 is covalently bound to the cell surface through its signal peptide, colocalized with the innate immunity complex (TLR4-CD14-MD2), and functions as a 'sentinel' molecule in the maintenance of epithelium and leukocyte homeostasis, meaning that the presence of Ecrg-4 on the cell surface signals the maintained homeostasis, whereas the loss of Ecrg-4 due to tissue injury activates pro-inflammatory and tissue proliferative responses, and the level of Ecrg-4 gradually returns to its pre-injury level upon wound healing. Interestingly, Ecrg-4 is also highly expressed in the heart and its conduction system, endothelial cells, and vascular smooth muscle cells. Accumulating evidence has shown that Ecrg-4 is involved in cardiac rate/rhythm control, the development of atrial fibrillation, doxorubicin-induced cardiotoxicity, the ischemic response of the heart and hypoxic response in the carotid body, the pathogenesis of atherosclerosis, and likely the endemic incidence of idiopathic dilated cardiomyopathy. These preliminary discoveries suggest that Ecrg-4 may function as a 'sentinel' molecule in cardiovascular system as well. Here, we briefly review the basic characteristics of ECRG-4 as a tumor suppressor gene and its regulatory functions on inflammation and apoptosis; summarize the discoveries about its distribution in cardiovascular system and involvement in the development of CVDs, and discuss its potential as a novel therapeutic target for the maintenance of cardiovascular system homeostasis.

Cell-specific processing and release of the hormone-like precursor and candidate tumor suppressor gene product, Ecrg4.

The human open reading frame C2orf40 encodes esophageal cancer-related gene-4 (Ecrg4), a newly recognized neuropeptide-like precursor protein whose gene expression by cells in vitro, over-expression in mice in vivo, and knock-down in zebrafish affects cell proliferation, migration and senescence, progenitor cell survival and differentiation, and inflammatory function. Unlike traditionally secreted neuropeptide precursors, however, we find that Ecrg4 localizes to the epithelial cell surface and remains tethered after secretion. Here, we used cell surface biotinylation to establish that 14-kDa Ecrg4 localizes to the cell surface of prostate (PC3) or kidney (HEK) epithelial cells after transfection. Accordingly, this Ecrg4 is resistant to washing cells with neutral, high salt (2 M NaCl), acidic (50 mM glycine, pH 2.8), or basic (100 mM Na(2)CO(3), pH 11) buffers. Mutagenesis of Ecrg4 established that cell tethering was mediated by an NH(2)-terminus hydrophobic leader sequence that enabled both trafficking to the surface and tethering. Immunoblotting analyses, however, showed that different cells process Ecrg4 differently. Whereas PC3 cells release cell surface Ecrg4 to generate soluble Ecrg4 peptides of 6-14 kDa, HEK cells do neither, and the 14-kDa precursor resembles a sentinel attached to the cell surface. Because a phorbol ester treatment of PC3 cells stimulated Ecrg4 release from, and processing at, the cell surface, these data are consistent with a multifunctional role for Ecrg4 that is dependent on its cell of origin and the molecular form produced.

The emergence of the uniquely human α7 nicotinic acetylcholine receptor gene and its roles in inflammation.

The uniquely human CHRFAM7A gene is evolved from the fusion of two partially duplicated genes, ULK4 and CHRNA7. Transcription of CHRFAM7A gene produces a 1256-bp open reading frame (ORF) that encodes duplicate α7-nAChR (dup-α7-nAChR), in which a 27-aminoacid peptide derived from ULK4 gene replaces the 146-aminoacid N-terminal extracellular domain of α7-nAChR, and the rest protein domains are exactly the same as those of α7-nAChR. In vitro, dup-α7-nAChR has been shown to form hetero-pentamer with α7-nAChR and dominant-negatively inhibits the channel functions of the latter. α7-nAChR has been shown to participate in many pathophysiological processes such as cognition, memory, neuronal degenerative disease, psychological disease, and inflammatory diseases, among others, and thus has been extensively exploited as potential therapeutic targets for many diseases. Unfortunately, many lead compounds that showed potent therapeutic effect in preclinical animal models failed clinical trials, suggesting the possibility that the contribution of the uniquely human CHRFAM7A gene may not be accounted for in the preclinical research. Here, we review the emergence of CHRFAM7A gene and its transcriptional regulation, the regulatory roles of CHRFAM7A gene in α7-nAChR-mediated cholinergic anti-inflammatory pathway, and the potential implications of CHRFAM7A gene in translational research and drug discovery.

The Biphasic Effect of Retinoic Acid Signaling Pathway on the Biased Differentiation of Atrial-like and Sinoatrial Node-like Cells from hiPSC.

Background and Objectives: Although human-induced pluripotent stem cells (hiPSC) can be efficiently differentiated into cardiomyocytes (CMs), the heterogeneity of the hiPSC-CMs hampers their applications in research and regenerative medicine. Retinoic acid (RA)-mediated signaling pathway has been proved indispensable in cardiac development and differentiation of hiPSC toward atrial CMs. This study was aimed to test whether RA signaling pathway can be manipulated to direct the differentiation into sinoatrial node (SAN) CMs. Methods and Results: Using the well-characterized GiWi protocol that cardiomyocytes are generated from hiPSC via temporal modulation of Wnt signaling pathway by small molecules, RA signaling pathway was manipulated during the differentiation of hiPSC-CMs on day 5 post-differentiation, a crucial time point equivalent to the transition from cardiac mesoderm to cardiac progenitor cells in cardiac development. The resultant CMs were characterized at mRNA, protein and electrophysiology levels by a combination of qPCR, immunofluorescence, flow cytometry, and whole-cell patch clamp. The results showed that activation of the RA signaling pathway biased the differentiation of atrial CMs, whereas inhibition of the signaling pathway biased the differentiation of sinoatrial node-like cells (SANLCs). Conclusions: Our study not only provides a novel and simple strategy to enrich SANLCs but also improves our understanding of the importance of RA signaling in the differentiation of hiPSC-CMs.

Cardiac Expression of Esophageal Cancer-Related Gene-4 is Regulated by Sp1 and is a Potential Early Target of Doxorubicin-Induced Cardiotoxicity.

Esophageal Cancer-Related Gene 4 (Ecrg4) expressed in cardiomyocytes and the cardiac conduction system is downregulated during cardiac ischemia and atrial fibrillation. To explore whether Ecrg4 plays any role in doxorubicin (DOX)-induced cardiotoxicity. Rats and neonatal rat cardiomyocytes (NRCMs) were employed to study the effect of DOX on Ecrg4 transcription. Bioinformatics combined with promoter analysis were used to map the rat Ecrg4 promoter. ChIP assay was used to evaluate the binding of Sp1 to the Ecrg4 promoter. Transient transfection was used to study the effect of Sp1 on the expression of endogenous Ecrg4. DOX decreased endogenous Ecrg4 gene expression in the heart and cultured NRCMs. In silico analysis showed that the 5'UTR immediately upstream of the start codon ATG, harbors a putative promoter that is GC-rich, and contains CpG islands, multiple overlapping Sp1sites. Transcription is initiated mainly on the 'C' at - 15. Serial 5'-deletion combined with dual-luciferase assays showed that the rat Ecrg4 core promoter resides at - 1/- 800. Sp1 transactivated Ecrg4 gene, which was almost abolished by DOX. Furthermore, ChIP assay showed that Sp1 specifically bound to the Ecrg4 promoter was interrupted by DOX. Finally, DOX suppressed Sp1 protein expression, and restoration of Sp1 increased Ecrg4 expression that was resistant to DOX-induced Ecrg4 downregulation. Importantly, cardiomyocyte-specific loss of Ecrg4 significantly enriched the differentially expressed proteins in the signaling pathways commonly involved in DOX-induced cardiotoxicity. Our results indicate that Sp1 mediates DOX-induced suppression of Ecrg4, which may contribute indirectly to its cardiotoxicity.

TBX3 induces biased differentiation of human induced pluripotent stem cells into cardiac pacemaker-like cells.

BACKGROUND: TBX3 plays a critical role in the formation of the sinoatrial node (SAN) during embryonic heart development. However, the contribution of TBX3 in driving the differentiation of human induced pluripotent stem cells (hiPSC)into pacemaker cells remains to be explored. RESULTS: Using the pan-cardiomyocyte differentiation protocol of human induced pluripotent stem cells (hiPSC)，TBX3 gene was introduced into the differentiating hiPSC on day 5 post-differentiation, and the differentiation of pacemaker-like cardiomyocytes was evaluated on day 21. The results showed that TBX3 significantly induced biased differentiation of hiPSC into pacemaker-like cells as judged by significantly increased expression of SAN-specific marker gene, SHOX2, and slightly decreased expression of SAN-detrimental transcription factor, NKX2-5. CONCLUSION: Our results suggest that TBX3 plays an important role in driving the differentiation of hiPSC into pacemaker-like cells, and manipulation of TBX3 expression during pan-cardiomyocyte differentiation may lead to the development of therapeutic pacemaker cells.

Genetically engineered exosomes display RVG peptide and selectively enrich a neprilysin variant: a potential formulation for the treatment of Alzheimer's disease.

Exosome is a promising next generation nano-based drug delivery vehicle. However, the unknown molecular mechanisms underlying its natural tissue tropism and the relatively low quantity of naturally enriched molecules of therapeutic value hamper exosome's clinical application. The aim of the research was to create a targeted and highly efficacious exosome formulation for the treatment of Alzheimer's disease (AD). Genetic engineering techniques combined with co-transfection of parental cells were employed to create an exosome formulation that displays RVG peptide on its surface targeting α7-nAChR and simultaneously enriches a neprilysin variant with increased specificity and efficacy in degrading ß amyloid peptide (Aß). The exosome formulation was preferentially internalised into cell lines in an α7-nAChR expression level-dependent manner. When incubated with Aß-producing N2a cells, it significantly decreased intracellular and secreted Aß40 levels, a potency that is superior to exosomes derived from adipose-derived stem cell. When systemically administered into mice, the exosome formulation was preferentially targeted to the hippocampus region of the brain and significantly decreased the expression of proinflammatory genes, IL1α, TNFα and NF-κB, and simultaneously increased the expression of anti-inflammatory gene, IL10. Our exosome formulation may be explored as an over-the-counter treatment for AD.