Expression of Calcitonin Gene-Related Peptide and Calcitonin Receptor-like Receptor in Colorectal Adenocarcinoma.

Colorectal cancer is one of the most widespread types of cancer that still causes many deaths worldwide. The development of new diagnostic and prognostic markers, as well as new therapeutic methods, is necessary. The calcitonin gene-related peptide (CGRP) neuropeptide alongside its receptor calcitonin receptor-like receptor (CRLR) could represent future biomarkers and a potential therapeutic target. Increased levels of CGRP have been demonstrated in thyroid, prostate, lung, and breast cancers and may also have a role in colorectal cancer. At the tumor level, it acts through different mechanisms, such as the angiogenesis, migration, and proliferation of tumor cells. The aim of this study was to measure the level of CGRP in colorectal cancer patients' serum by enzyme-linked immunosorbent assay (ELISA) and determine the level of CGRP and CRLR at the tumor level after histopathological (HP) and immunohistochemical (IHC) analysis, and then to correlate them with the TNM stage and with different tumoral characteristics. A total of 54 patients with newly diagnosed colorectal adenocarcinoma were evaluated. We showed that serum levels of CGRP, as well as CGRP and CRLR tumor level expression, correlate with the TNM stage, with local tumor extension, the presence of lymph node metastasis, and distant metastasis, and also with the tumor differentiation degree. CGRP is present in colorectal cancer from the incipient TNM stage, with levels increasing with the stage, and can be used as a diagnostic and prognostic marker and may also represent a potentially new therapeutic target.

Coronary Artery Disease Risk Variant Dampens the Expression of CALCRL by Reducing HSF Binding to Shear Stress Responsive Enhancer in Endothelial Cells In Vitro.

BACKGROUND: CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells. METHODS: To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq [transposase-accessible chromatin with sequencing], chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells. RESULTS: We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production. CONCLUSIONS: Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.

CALCRL induces resistance to daunorubicin in acute myeloid leukemia cells through upregulation of XRCC5/TYK2/JAK1 pathway.

Chemotherapy is the main treatment option for acute myeloid leukemia (AML), but acquired resistance of leukemic cells to chemotherapeutic agents often leads to difficulties in AML treatment and disease relapse. High calcitonin receptor-like (CALCRL) expression is closely associated with poorer prognosis in AML patients. Therefore, this study was performed by performing CALCRL overexpression constructs in AML cell lines HL-60 and Molm-13 with low CALCRL expression. The results showed that overexpression of CALCRL in HL-60 and Molm-13 could confer resistance properties to AML cells and reduce the DNA damage and cell cycle G0/G1 phase blocking effects caused by daunorubicin (DNR) and others. Overexpression of CALCRL also reduced DNR-induced apoptosis. Mechanistically, the Cancer Clinical Research Database analyzed a significant positive correlation between XRCC5 and CALCRL in AML patients. Therefore, the combination of RT-PCR and Western blot studies further confirmed that the expression levels of XRCC5 and PDK1 genes and proteins were significantly upregulated after overexpression of CALCRL. In contrast, the phosphorylation levels of AKT/PKCε protein, a downstream pathway of XRCC5/PDK1, were significantly upregulated. In the response study, transfection of overexpressed CALCRL cells with XRCC5 siRNA significantly upregulated the drug sensitivity of AML to DNR. The expression levels of PDK1 protein and AKT/PKCε phosphorylated protein in the downstream pathway were inhibited considerably, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were upregulated. Animal experiments showed that the inhibitory effect of DNR on the growth of HL-60 cells and the number of bone marrow invasions were significantly reversed after overexpression of CALCRL in nude mice. However, infection of XCRR5 shRNA lentivirus in HL-60 cells with CALCRL overexpression attenuated the effect of CALCRL overexpression and upregulated the expression of apoptosis-related proteins induced by DNR. This study provides a preliminary explanation for the relationship between high CALCRL expression and poor prognosis of chemotherapy in AML patients. It offers a more experimental basis for DNR combined with molecular targets for precise treatment in subsequent studies.

Adrenomedullin 2/intermedin is a slow off-rate, long-acting endogenous agonist of the adrenomedullin2 G protein-coupled receptor.

Adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and calcitonin gene-related peptide (CGRP) have functions in the cardiovascular, lymphatic, and nervous systems by activating three heterodimeric receptors comprising the class B GPCR CLR and a RAMP1, -2, or -3 modulatory subunit. CGRP and AM prefer the RAMP1 and RAMP2/3 complexes, respectively, whereas AM2/IMD is thought to be relatively nonselective. Accordingly, AM2/IMD exhibits overlapping actions with CGRP and AM, so the rationale for this third agonist for the CLR-RAMP complexes is unclear. Here, we report that AM2/IMD is kinetically selective for CLR-RAMP3, known as the AM2R, and we define the structural basis for its distinct kinetics. In live cell biosensor assays, AM2/IMD-AM2R elicited longer-duration cAMP signaling than the other peptide-receptor combinations. AM2/IMD and AM bound the AM2R with similar equilibrium affinities, but AM2/IMD had a slower off-rate and longer receptor residence time, thus explaining its prolonged signaling capacity. Peptide and receptor chimeras and mutagenesis were used to map the regions responsible for the distinct binding and signaling kinetics to the AM2/IMD mid-region and the RAMP3 extracellular domain (ECD). Molecular dynamics simulations revealed how the former forms stable interactions at the CLR ECD-transmembrane domain interface and how the latter augments the CLR ECD binding pocket to anchor the AM2/IMD C terminus. These strong binding components only combine in the AM2R. Our findings uncover AM2/IMD-AM2R as a cognate pair with unique temporal features, reveal how AM2/IMD and RAMP3 collaborate to shape CLR signaling, and have significant implications for AM2/IMD biology.

Expression profile of adrenomedullin and its specific receptors in liver tissues from patients with hepatocellular carcinoma and in tumorigenic cell line-secreted extracellular vesicles.

The transcriptional profile of adrenomedullin (AM), a new metastasis-related factor involved in hepatocellular carcinoma (HCC), and its specific receptors (CLR, RAMP1, RAMP3) were evaluated in liver tissues of HCV-positive HCC subjects undergoing liver transplantation (LR) and in donors (LD). AM and its specific receptor expression were also assessed in extracellular vesicles (EVs) secreted by tumorigenic (HepG2) and non-tumorigenic (WRL68) cells by Real-Time PCR. AM expression resulted significantly elevated in LR concerning LD (p = 0.0038) and, for the first time, significantly higher levels in HCC patients as a function of clinical severity (MELD score), were observed. RAMP3 and CLR expression increased in LR as a function of clinical severity while RAMP1 decreased. Positive correlations were found among AM, its receptors, and apoptotic markers. No AM mRNA expression difference was observed between HepG2 and WRL68 EVs. RAMP1 and RAMP3 resulted lower in HepG2 concerning WRL68 while significantly higher levels were observed for CLR. While results at tissue level characterize AM as a regulator of carcinogenesis-tumor progression, those obtained in EVs do not indicate AM as a target candidate, neither as a pathological biomarker nor as a marker involved in cancer therapy.

Schwann cell endosome CGRP signals elicit periorbital mechanical allodynia in mice.

Efficacy of monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor (calcitonin receptor-like receptor/receptor activity modifying protein-1, CLR/RAMP1) implicates peripherally-released CGRP in migraine pain. However, the site and mechanism of CGRP-evoked peripheral pain remain unclear. By cell-selective RAMP1 gene deletion, we reveal that CGRP released from mouse cutaneous trigeminal fibers targets CLR/RAMP1 on surrounding Schwann cells to evoke periorbital mechanical allodynia. CLR/RAMP1 activation in human and mouse Schwann cells generates long-lasting signals from endosomes that evoke cAMP-dependent formation of NO. NO, by gating Schwann cell transient receptor potential ankyrin 1 (TRPA1), releases ROS, which in a feed-forward manner sustain allodynia via nociceptor TRPA1. When encapsulated into nanoparticles that release cargo in acidified endosomes, a CLR/RAMP1 antagonist provides superior inhibition of CGRP signaling and allodynia in mice. Our data suggest that the CGRP-mediated neuronal/Schwann cell pathway mediates allodynia associated with neurogenic inflammation, contributing to the algesic action of CGRP in mice.

Peptide ligand interaction with maltose-binding protein tagged to the calcitonin gene-related peptide receptor: The inhibitory role of receptor N-glycosylation.

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are peptide hormones and their receptors play a critical role in migraine progression and blood pressure control, respectively. CGRP and AM receptors are structurally related since they are the complex of the calcitonin receptor-like receptor (CLR) with the different types of receptor activity-modifying protein (RAMP). Several crystal structures of the CGRP and AM receptor extracellular domain (ECD) used maltose-binding protein (MBP) as a tag protein to facilitate crystallization. Unexpectedly, the recent crystal structures of CGRP receptor ECD showed that the N-terminal tag MBP located in proximity of bound/mutated peptide ligands. This study provided evidence that MBP N-terminally tagged to the CGRP receptor ECD formed chemical interaction with the mutated peptide ligands. Interestingly, N-glycosylation of the CGRP receptor ECD was predicted to prevent MBP docking to the mutated peptide ligands. I found that the N-glycosylation of CLR ECD N123 was the most critical for inhibiting MBP interaction with the mutated peptide ligands. The MBP tag protein interaction was also dependent on the sequence of the peptide ligands. In contrast to the CGRP receptor, the MBP tag was not involved in peptide ligand binding at AM receptor ECD. Here, I provided evidence that N-glycosylation of the CGRP receptor ECD inhibited the tag protein interaction suggesting an additional function of N-glycosylation in the MBP-fused CGRP receptor ECD. This study reveals the importance of using tag protein-free versions of the CGRP receptor for the accurate assessment of peptide binding affinity.

Identification and validation of CALCRL-associated prognostic genes in acute myeloid leukemia.

In the past few decades, several advances have been made in the field of acute myeloid leukemia (AML), especially in the development of novel drugs. However, the overall survival rate remains particularly disappointing due to a high rate of chemotherapy resistance and relapse. The calcitonin receptor-like receptor (CALCRL) is a novel promising therapeutic target of AML and has been indicated to be strongly correlated with chemotherapy resistance and relapse driven by leukemic stem cells. Nevertheless, the CALCRL downstream genes associated with the drug resistance and relapse of AML remain to be elucidated. Within this study, we used multiple gene expression datasets from the Gene Expression Omnibus (GEO) database and cBioPortal to explore the candidate CALCRL-associated genes that could potentially mediate the chemoresistance and relapse of AML. Then, we investigated the prognostic value, coexpression relationship with CALCRL, and expression characteristics of these genes using independent data from The Cancer Genome Atlas (TCGA). Eventually, three genes were screened out as CALCRL-associated prognostic genes. The expression of AGTPBP1 and LYST was negatively correlated with CALCRL, high expression of which was associated with favorable prognosis in AML. In contrast, the expression of ETS2 was positively correlated with CALCRL, high expression of which was associated with poor prognosis in AML. The results indicated that the three prognostic genes are potential CALCRL downstream genes that mediate drug resistance and relapse in AML. This study helps to further explore the role and molecular pathways of CALCRL in mediating drug resistance and relapse of AML.

Molecular simulations reveal the impact of RAMP1 on ligand binding and dynamics of calcitonin gene-related peptide receptor (CGRPR) heterodimer.

Calcitonin gene-related peptide receptor (CGRPR) is a heterodimer protein complex consisting of a class-B G protein-coupled receptor (GPCR) named calcitonin receptor-like receptor (CLR) and an accessory protein, receptor activity modifying protein type 1 (RAMP1). Here in this study, with several molecular modeling approaches and molecular dynamics (MD) simulations, the structural and dynamical effects of RAMP1 on the binding of small molecule CGRPR inhibitors (namely rimegepant and telcagepant) to the CGRPR extracellular ectodomain complex site (site 1) and transmembrane binding site (site 2) are investigated. Results showed that although these molecules stay stable at site 1, they can also bind to site 2, which may be interpreted as non-specificity of the ligands, however, most of these interactions at transmembrane binding site are not sustainable or are weak. Furthermore, to examine the site 2 for gepant binding, different in silico experiments (i.e., alanine scanning mutagenesis, SiteMap, ligand decomposition binding free energy analyses) are also conducted and the results confirmed the putative binding pocket (site 2) of the gepants at the CGRPRs.

CALCR knockdown inhibits the development and progression of non-small-cell lung cancer.

G-protein-coupled receptors (GPCRs) have been reported to participate in the occurrence and development of a variety of human cancers. CALCR is one of the hundreds of GPCRs, but its expression level and functional importance have never been investigated in non-small-cell lung cancer (NSCLC). In the present study, the protein expression level of CALCR was detected by immunohistochemical staining and western blot analysis. The Celigo cell counting assay was used to assess cell proliferation. Both the wound-healing assay and the transwell assay were performed to evaluate cell migration. Flow cytometric analysis was utilized to detect cell apoptosis and cell cycle. A mouse xenograft model was constructed to conduct the in vivo experiments. The results indicated that the CALCR expression was abundantly up-regulated in NSCLC and positively related to tumor infiltrate. Besides, CALCR knockdown could significantly suppress cell proliferation, migration, enhance apoptosis and arrest cell cycle. The in vivo study verified the inhibitory effects of CALCR knockdown on NSCLC tumorigenesis. The abovementioned results provided a reference for the treatment of NSCLC, that was, CALCR knockdown might be a considerable therapeutic strategy.

A genetic map of the mouse dorsal vagal complex and its role in obesity.

The brainstem dorsal vagal complex (DVC) is known to regulate energy balance and is the target of appetite-suppressing hormones, such as glucagon-like peptide 1 (GLP-1). Here we provide a comprehensive genetic map of the DVC and identify neuronal populations that control feeding. Combining bulk and single-nucleus gene expression and chromatin profiling of DVC cells, we reveal 25 neuronal populations with unique transcriptional and chromatin accessibility landscapes and peptide receptor expression profiles. GLP-1 receptor (GLP-1R) agonist administration induces gene expression alterations specific to two distinct sets of Glp1r neurons-one population in the area postrema and one in the nucleus of the solitary tract that also expresses calcitonin receptor (Calcr). Transcripts and regions of accessible chromatin near obesity-associated genetic variants are enriched in the area postrema and the nucleus of the solitary tract neurons that express Glp1r and/or Calcr, and activating several of these neuronal populations decreases feeding in rodents. Thus, DVC neuronal populations associated with obesity predisposition suppress feeding and may represent therapeutic targets for obesity.

Adrenomedullin 2 and 5 activate the calcitonin receptor-like receptor (clr) - Receptor activity-modifying protein 3 (ramp3) receptor complex in Xenopus tropicalis.

The adrenomedullin (AM) family is involved in diverse biological functions, including cardiovascular regulation and body fluid homeostasis, in multiple vertebrate lineages. The AM family consists of AM1, AM2, and AM5 in tetrapods, and the receptor for mammalian AMs has been identified as the complex of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2) or RAMP3. However, the receptors for AM in amphibians have not been identified. In this study, we identified the cDNAs encoding calcrl (clr), ramp2, and ramp3 receptor components from the western clawed frog (Xenopus tropicalis). Messenger RNAs of amphibian clr and ramp2 were highly expressed in the heart, whereas that of ramp3 was highly expressed in the whole blood. In HEK293T cells expressing clr-ramp2, cAMP response element luciferase (CRE-Luc) reporter activity was activated by am1. In HEK293T cells expressing clr-ramp3, CRE-Luc reporter activity was increased by the treatment with am2 at the lowest dose, but with am5 and am1 at higher dose. Our results provided new insights into the roles of AM family peptides through CLR-RAMP receptor complexes in the tetrapods.

Role of calcitonin gene-related peptide in pain regulation in the parabrachial nucleus of naive rats and rats with neuropathic pain.

Researches have shown that calcitonin gene-related peptide (CGRP) plays a pivotal role in pain modulation. Nociceptive information from the periphery is relayed from parabrachial nucleus (PBN) to brain regions implicated involved in pain. This study investigated the effects and mechanisms of CGRP and CGRP receptors in pain regulation in the PBN of naive and neuropathic pain rats. Chronic sciatic nerve ligation was used to model neuropathic pain, CGRP and CGRP 8-37 were injected into the PBN of the rats, and calcitonin receptor-like receptor (CLR), a main structure of CGRP receptor, was knocked down by lentivirus-coated CLR siRNA. The hot plate test (HPT) and the Randall Selitto Test (RST) was used to determine the latency of the rat hindpaw response. The expression of CLR was detected with RT-PCR and western blotting. We found that intra-PBN injecting of CGRP induced an obvious anti-nociceptive effect in naive and neuropathic pain rats in a dose-dependent manner, the CGRP-induced antinociception was significantly reduced after injection of CGRP 8-37, Moreover, the mRNA and protein levels of CLR, in PBN decreased significantly and the antinociception CGRP-induced was also significantly lower in neuropathic pain rats than that in naive rats. Knockdown CLR in PBN decreased the expression of CLR and the antinociception induced by CGRP was observably decreased. Our results demonstrate that CGRP induced antinociception in PBN of naive or neuropathic pain rats, CGRP receptor mediates this effect. Neuropathic pain induced decreases in the expression of CGRP receptor, as well as in CGRP-induced antinociception in PBN.

Adrenomedullin-CALCRL axis controls relapse-initiating drug tolerant acute myeloid leukemia cells.

Drug tolerant/resistant leukemic stem cell (LSC) subpopulations may explain frequent relapses in acute myeloid leukemia (AML), suggesting that these relapse-initiating cells (RICs) persistent after chemotherapy represent bona fide targets to prevent drug resistance and relapse. We uncover that calcitonin receptor-like receptor (CALCRL) is expressed in RICs, and that the overexpression of CALCRL and/or of its ligand adrenomedullin (ADM), and not CGRP, correlates to adverse outcome in AML. CALCRL knockdown impairs leukemic growth, decreases LSC frequency, and sensitizes to cytarabine in patient-derived xenograft models. Mechanistically, the ADM-CALCRL axis drives cell cycle, DNA repair, and mitochondrial OxPHOS function of AML blasts dependent on E2F1 and BCL2. Finally, CALCRL depletion reduces LSC frequency of RICs post-chemotherapy in vivo. In summary, our data highlight a critical role of ADM-CALCRL in post-chemotherapy persistence of these cells, and disclose a promising therapeutic target to prevent relapse in AML.

Short tandem repeat near hypoxia response element (HRE) instead of HRE genetic variants in promoter calcitonin receptor-like receptor (CRLR) gene as risk factor in severe preeclampsia: a preliminary study.

OBJECTIVE: Calcitonin receptor-like receptor (CRLR) regulates vasoconstriction and dilatation; the expression increases during hypoxia via activation of hypoxia response element (HRE) in CRLR gene promoter region. Variant in HRE, as well short tandem repeat (STR) variants near HRE in CRLR alters the gene expression. This study focused on a case-control study to investigate the expression of genetic typing CLRL promoter variant in pregnant women with severe preeclampsia and normal pregnancies, we also tried to describe interesting findings of the genetic expression in anemic patients in the severe preeclampsia group. Our aimed to observe the correlation of CRLR gene promoter variant and anemia in severe preeclampsia. RESULTS: There was no nucleotide variant in HRE; CACA box prior to HRE varied in length (15-24); CACA box with length > 20 was used as cut off point. Hb was lower in CACA box length ≥ 21 (10.33 ± 1.57) vs. < 21 (11.01 ± 1.67; p = 0.391). CACA box polymorphism and anemia were correlated in severe preeclampsia (p = 0.005) OR 0.038 (CI 0.003-0.544); not in normal (p = 0.069).

Identifying a novel 5-gene signature predicting clinical outcomes in acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is the most common type of acute leukemia and biologically heterogeneous diseases with poor prognosis. Thus, we aimed to identify prognostic markers to effectively predict the prognosis of AML patients and eventually guide treatment. METHODS: Prognosis-associated genes were determined by Kaplan-Meier and multivariate analyses using the expression and clinical data of 173 AML patients from The Cancer Genome Atlas database and validated in an independent Oregon Health and Science University dataset. A prognostic risk score was computed based on a linear combination of 5-gene expression levels using the regression coefficients derived from the multivariate logistic regression model. The classification of AML was established by unsupervised hierarchical clustering of CALCRL, DOCK1, PLA2G4A, FCHO2 and LRCH4 expression levels. RESULTS: High FCHO2 and LRCH4 expression was related to decreased mortality. While high CALCRL, DOCK1, PLA2G4A expression was associated with increased mortality. The risk score was predictive of increased mortality rate in AML patients. Hierarchical clustering analysis of the five genes discovered three clusters of AML patients. The cluster1 AML patients were associated with lower cytogenetics risk than cluster2 or 3 patients, and better prognosis than cluster3 patients (P values < 0.05 for all cases, fisher exact test or log-rank test). CONCLUSION: The gene panel comprising CALCRL, DOCK1, PLA2G4A, FCHO2 and LRCH4 as well as the risk score may offer novel prognostic biomarkers and classification of AML patients to significantly improve outcome prediction.

Area Postrema Cell Types that Mediate Nausea-Associated Behaviors.

Nausea, the unpleasant sensation of visceral malaise, remains a mysterious process. The area postrema is implicated in some nausea responses and is anatomically privileged to detect blood-borne signals. To investigate nausea mechanisms, we built an area postrema cell atlas through single-nucleus RNA sequencing, revealing a few neuron types. Using mouse genetic tools for cell-specific manipulation, we discovered excitatory neurons that induce nausea-related behaviors, with one neuron type mediating aversion imposed by multiple poisons. Nausea-associated responses to agonists of identified area postrema receptors were observed and suppressed by targeted cell ablation and/or gene knockout. Anatomical mapping revealed a distributed network of long-range excitatory but not inhibitory projections with subtype-specific patterning. These studies reveal the basic organization of area postrema nausea circuitry and provide a framework toward understanding and therapeutically controlling nausea.

Mechanism of miRNA-based Aconitum leucostomum Worosch. Monomer inhibition of bone marrow-derived dendritic cell maturation.

Delvestidine (DLTD) is a monomeric compound isolated from Aconitum leucostomum Worosch, a widely used medicine for local treatment of rheumatoid arthritis (RA). Studies have shown that Aconitum leucostomum Worosch. can inhibit maturation of bone marrow-derived dendritic cells (BMDCs). Further, microRNAs (miRNAs) have regulatory effects on DC maturity and function. However, the mechanism underlying DLTD effects on DC maturity and RA remains to be elucidated. This study investigated whether DLTD-mediated inhibition of DC maturation is regulated by miRNAs. LPS-induced mature BMDCs were treated with DLTD for 48 h. CD80 and CD86 expression on BMDCs was detected by flow cytometry, and levels of inflammatory factors IL-6, IL-23, IL-1ß, and TNF-α were detected by ELISA and PCR. Further, gene expression and miRNA expression profiles were investigated by bioinformatics analysis and verified by PCR. DLTD was found to inhibit CD80 and CD86 expression on the surface of BMDCs and secretion of inflammatory factors IL-6, IL-23, IL-1ß, and TNF-α. In total, 54 differentially expressed miRNAs were detected, including 29 up-regulated and 25 down-regulated miRNAs after DLTD treatment. Analysis of biological information revealed that the differentially expressed target genes mainly regulated biological processes, including cell differentiation, cell cycle, and protein kinase complexes. Additionally, miR-511-3p downstream targets Calcr, Fzd10, and Eps8, were closely related to BMDCs maturation. DLTD may induce BMDCs maturity through regulation of miRNAs that affect Calcr, Fzd10, and Eps8 gene signals.

Adrenomedullin - Current perspective on a peptide hormone with significant therapeutic potential.

The peptide hormone adrenomedullin (ADM) consists of 52 amino acids and plays a pivotal role in the regulation of many physiological processes, particularly those of the cardiovascular and lymphatic system. Like calcitonin (CT), calcitonin gene-related peptide (CGRP), intermedin (IMD) and amylin (AMY), it belongs to the CT/CGRP family of peptide hormones, which despite their low little sequence identity share certain characteristic structural features as well as a complex multicomponent receptor system. ADM, IMD and CGRP exert their biological effects by activation of the calcitonin receptor-like receptor (CLR) as a complex with one of three receptor activity-modifying proteins (RAMP), which alter the ligand affinity. Selectivity within the receptor system is largely mediated by the amidated C-terminus of the peptide hormones, which bind to the extracellular domains of the receptors. This enables their N-terminus consisting of a disulfide-bonded ring structure and a helical segment to bind within the transmembrane region and to induce an active receptor confirmation. ADM is expressed in a variety of tissues in the human body and is fundamentally involved in multitude biological processes. Thus, it is of interest as a diagnostic marker and a promising candidate for therapeutic interventions. In order to fully exploit the potential of ADM, it is necessary to improve its pharmacological profile by increasing the metabolic stability and, ideally, creating receptor subtype-selective analogs. While several successful attempts to prolong the half-life of ADM were recently reported, improving or even retaining receptor selectivity remains challenging.

Investigation of the therapeutic mechanism of subthreshold micropulse laser irradiation in retina.

PURPOSE: Subthreshold micropulse laser irradiation has been used for the treatment of retinal edema; however, there are few reports about the mechanism of its therapeutic effect. In this study, we compared threshold short pulse and subthreshold micropulse laser irradiation in mice and investigated their mechanism. METHODS: Nine to 12-week-old male C57BL/6J mice were used in this study. After general anesthesia, threshold short pulse or subthreshold micropulse laser irradiation was performed on the right eye using IQ577. Enucleation was performed 24 h after the laser irradiation, and histological and gene expression analyses were carried out. RESULTS: Coagulation spots and atrophy of the retinal pigment epithelium were observed after threshold short pulse laser irradiation but not after subthreshold micropulse laser irradiation. Twenty-four hours after laser, aquaporin (AQP) 1, 2, 7, and 11 levels were significantly elevated by 1.7- to 3-fold in the threshold short pulse laser group compared with non-treated control group. AQP 3 was increased significantly and prominently by 100-fold. VEGF-A and VEGFR2 were upregulated 1.5- and 2.3-fold, respectively. In the subthreshold micropulse laser group, AQP 3 was increased by 6-fold compared with the non-treated control group. Angiopoietin-1 and the adrenomedullin (AM) receptor CLR were decreased by 0.6-fold and 0.5-fold, respectively. CONCLUSION: Threshold short pulse laser irradiation caused retinal damage and prominent changes in the expression of various genes. Contrarily, subthreshold micropulse laser irradiation did not induce retinal damage; it upregulated AQP 3, which might have improved retinal edema by drainage of subretinal fluid.