Variable CGRP family peptide signaling durations and the structural determinants thereof.

Calcitonin gene-related peptides alpha and beta (αCGRP, ßCGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) function in pain signaling, neuroimmune communication, and regulation of the cardiovascular and lymphatic systems by activating either of two class B GPCRs, CLR and CTR, in complex with a RAMP1, -2, or -3 modulatory subunit. Inspired by our recent discovery that AM2/IMD(1-47) activation of CLR-RAMP3 elicits long duration cAMP signaling, here we used a live-cell cAMP biosensor assay to characterize the signaling kinetics of the two CGRP peptides and several bioactive AM and AM2/IMD fragments with variable N-terminal extensions. Remarkably, AM2/IMD(8-47) and AM2/IMD-53 exhibited even longer duration signaling than the 1-47 fragment. AM2/IMD(8-47) was a striking 8-fold longer acting than AM(13-52) at CLR-RAMP3. In contrast, the N-terminal extension of AM had no effect on signaling duration. AM(1-52) and (13-52) were equally short-acting. Analysis of AM2/IMD-AM mid-region chimeras and AM2/IMD R23 and R33 point mutants showed the importance of these residues for long-duration signaling and identified AM2/IMD peptides that exhibited up to 17-fold diminished signaling duration at CLR-RAMP3, while retaining near wildtype signaling potencies. ßCGRP was âˆ¼ 3-fold longer acting than αCGRP at the CGRP (CLR-RAMP1) and the amylin1 (CTR-RAMP1) receptors. Chimeric CGRP peptides showed that the single residue difference near the N-terminus, and the two differences in the mid-region, equally contributed to the longer duration of ßCGRP signaling. This work uncovers key temporal differences in cAMP signaling among the CGRP family peptides, elucidates the structural bases thereof, and provides pharmacological tools for studying long-duration AM2/IMD signaling.

Identification of hypoxic macrophages in glioblastoma with therapeutic potential for vasculature normalization.

Monocyte-derived tumor-associated macrophages (Mo-TAMs) intensively infiltrate diffuse gliomas with remarkable heterogeneity. Using single-cell transcriptomics, we chart a spatially resolved transcriptional landscape of Mo-TAMs across 51 patients with isocitrate dehydrogenase (IDH)-wild-type glioblastomas or IDH-mutant gliomas. We characterize a Mo-TAM subset that is localized to the peri-necrotic niche and skewed by hypoxic niche cues to acquire a hypoxia response signature. Hypoxia-TAM destabilizes endothelial adherens junctions by activating adrenomedullin paracrine signaling, thereby stimulating a hyperpermeable neovasculature that hampers drug delivery in glioblastoma xenografts. Accordingly, genetic ablation or pharmacological blockade of adrenomedullin produced by Hypoxia-TAM restores vascular integrity, improves intratumoral concentration of the anti-tumor agent dabrafenib, and achieves combinatorial therapeutic benefits. Increased proportion of Hypoxia-TAM or adrenomedullin expression is predictive of tumor vessel hyperpermeability and a worse prognosis of glioblastoma. Our findings highlight Mo-TAM diversity and spatial niche-steered Mo-TAM reprogramming in diffuse gliomas and indicate potential therapeutics targeting Hypoxia-TAM to normalize tumor vasculature.

Adrenomedullin, transcriptionally regulated by vitamin D receptors, alleviates atherosclerosis in mice through suppressing AMPK-mediated endothelial ferroptosis.

PURPOSE: Vitamin D receptors (VDR) play important roles in cardiovascular, immune, metabolic and other functions. Activation of VDR may help improve endothelial dysfunction, atherosclerosis, vascular calcification, and cardiac hypertrophy. However, the specific target genes and mechanisms of VDR in improving Human Umbilical Vein Endothelial Cell (HUVEC) functions remain unclear. This study aims to investigate the function and mechanism of VDR in HUVECs. METHODS: Endothelial dysfunction cell model was constructed by oxidized low-density lipoprotein (ox-LDL). An animal model of atherosclerosis was established in male homozygous Apoe-/- mice (6 weeks) on a high fat diet for 6 weeks. The relationship between VDR and adrenomedullin (ADM) was studied by bioinformatics analysis, ChIP, and luciferase reporter gene analysis. Endothelial cell function was evaluated by Transwell migration and Tube Formation tests. Ferroptosis was detected by measuring intracellular iron content, levels of oxidative stress markers, and ferroptosis related proteins. RESULTS: Overexpression of VDR in HUVECs inhibits ox-LDL-induced endothelial dysfunction and ferroptosis. VDR binds to the ADM promoter sequence and regulates the transcription of ADM. Inhibition of ADM promotes ox-LDL-induced endothelial dysfunction and ferroptosis. ADM regulates ox-LDL-induced endothelial dysfunction and ferroptosis through the AMPK signaling pathway. Overexpression of VDR in Apoe-/- mice inhibited lipid deposition and plaque area in atherosclerotic mice. CONCLUSION: VDR inhibits ox-LDL-induced endothelial dysfunction and ferroptosis by regulating ADM transcription and acting on AMPK signaling pathway. Overexpression of VDR in Apoe-/- mice reduced lipid deposition and plaque area in the thoracic aorta of atherosclerotic mice.

Receptor activity-modifying proteins of adrenomedullin (RAMP2/3): Roles in the pathogenesis of ARDS.

Acute respiratory distress syndrome (ARDS) is a life-threatening lung condition characterized by widespread inflammation and pulmonary edema. Adrenomedullin (AM), a bioactive peptide with various functions, is expected to be applied in treating ARDS. Its functions are regulated primarily by two receptor activity-modifying proteins, RAMP2 and RAMP3, which bind to the AM receptor calcitonin receptor-like receptor (CLR). However, the roles of RAMP2 and RAMP3 in ARDS remain unclear. We generated a mouse model of ARDS via intratracheal administration of lipopolysaccharide (LPS), and analyzed the pathophysiological significance of RAMP2 and RAMP3. RAMP2 expression declined with LPS administration, whereas RAMP3 expression increased at low doses and decreased at high doses of LPS. After LPS administration, drug-inducible vascular endothelial cell-specific RAMP2 knockout mice (DI-E-RAMP2-/-) showed reduced survival, increased lung weight, and had more apoptotic cells in the lungs. DI-E-RAMP2-/- mice exhibited reduced expression of Epac1 (which regulates vascular endothelial cell barrier function), while RAMP3 was upregulated in compensation. In contrast, after LPS administration, RAMP3-/- mice showed no significant changes in survival, lung weight, or lung pathology, although they exhibited significant downregulation of iNOS, TNF-α, and NLRP3 during the later stages of inflammation. Based on transcriptomic analysis, RAMP2 contributed more to the circulation-regulating effects of AM, whereas RAMP3 contributed more to its inflammation-regulating effects. These findings indicate that, while both RAMP2 and RAMP3 participate in ARDS pathogenesis, their functions differ distinctly. Further elucidation of the pathophysiological significance and functional differences between RAMP2 and RAMP3 is critical for the future therapeutic application of AM in ARDS.

Adrenomedullin/FOXO3 enhances sunitinib resistance in clear cell renal cell carcinoma by inhibiting FDX1 expression and cuproptosis.

Cuproptosis, a new type of copper-induced cell death, is involved in the antitumor activity and resistance of multiple chemotherapeutic drugs. Our previous study revealed that adrenomedullin (ADM) was engaged in sunitinib resistance in clear cell renal cell carcinoma (ccRCC). However, it has yet to be investigated whether and how ADM regulates sunitinib resistance by cuproptosis. This study found that the ADM expression was elevated in sunitinib-resistant ccRCC tissues and cells. Furthermore, the upregulation of ADM significantly enhanced the chemoresistance of sunitinib compared with their respective control. Moreover, cuproptosis was involved in ADM-regulated sunitinib resistance by inhibiting mammalian ferredoxin 1 (FDX1) expression. Mechanically, the upregulated ADM activates the p38/MAPK signaling pathway to promote Forkhead box O3 (FOXO3) phosphorylation and its entry into the nucleus. Consequently, the increased FOXO3 in the nucleus inhibited FDX1 transcription and cell cuproptosis, promoting chemoresistance. Collectively, cuproptosis has a critical effector role in ccRCC progress and chemoresistance and thus is a relevant target to eradicate the cell population of sunitinib resistance.

Rational design of highly stabilized and selective adrenomedullin analogs.

The peptide hormone adrenomedullin (ADM) consists of 52 amino acids with a disulfide bond and an amidated C-terminus. Due to the vasodilatory and cardioprotective effects, the agonistic activity of the peptide on the adrenomedullin 1 receptor (AM1 R) is of high pharmacological interest. However, the wild-type peptide shows low metabolic stability leading to rapid degradation in the cardiovascular system. Previous work by our group has identified proteolytic cleavage sites and demonstrated stabilization of ADM by lipidation, cyclization, and N-methylation. Nevertheless, these ADM analogs showed reduced activity and subtype selectivity toward the closely related calcitonin gene-related peptide receptor (CGRPR). Here, we report on the rational development of ADM derivatives with increased proteolytic stability and high receptor selectivity. Stabilizing motifs, including lactamization and lipidation, were evaluated regarding AM1 R and CGRPR activation. Furthermore, the central DKDK motif of the peptide was replaced by oligoethylene glycol linkers. The modified peptides were synthesized by Fmoc/t-Bu solid-phase peptide synthesis and receptor activation of AM1 R and CGRPR was measured by cAMP reporter gene assay. Peptide stability was tested in human blood plasma and porcine liver homogenate and analyzed by RP-HPLC and MALDI-ToF mass spectrometry. Combination of the favorable lactam, lipidation, ethylene glycol linker, and previously described disulfide mimetic resulted in highly stabilized analogs with a plasma half-life of more than 144 h. The compounds display excellent AM1 R activity and wild-type-like selectivity toward CGRPR. Additionally, dose-dependent vasodilatory effects of the ADM derivatives lasted for several hours in rodents. Thus, we successfully developed an ADM analog with long-term in vivo activity.

Anti-apoptotic effect of adrenomedullin gene delivery on Leydig cells by suppressing TGF-ß1 via the Hippo signaling pathway.

This study aims to establish whether adrenomedullin (ADM) is capable to restore the steroidogenic functions of Leydig cells by suppressing transforming growth factor-ß1 (TGF-ß1) through Hippo signaling. Primary Leydig cells were treated with lipopolysaccharide (LPS), an adeno-associated virus vector that expressed ADM (Ad-ADM) or sh-RNA of TGF-ß1 (Ad-sh-TGF-ß1). The cell viability and medium concentrations of testosterone were detected. Gene expression and protein levels were determined for steroidogenic enzymes, TGF-ß1, RhoA, YAP, TAZ and TEAD1. The role of Ad-ADM in the regulation of TGF-ß1 promoter was confirmed by ChIP and Co-IP. Similar to Ad-sh-TGF-ß1, Ad-ADM mitigated the decline in the number of Leydig cells and plasma concentrations of testosterone by restoring the gene and protein levels of SF-1, LRH1, NUR77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD. Similar to Ad-sh-TGF-ß1, Ad-ADM not only inhibited the LPS-induced cytotoxicity and cell apoptosis but also restored the gene and protein levels of SF-1, LRH1, NUR77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD, along with the medium concentrations of testosterone in LPS-induced Leydig cells. Like Ad-sh-TGF-ß1, Ad-ADM improved LPS-induced TGF-ß1 expression. In addition, Ad-ADM suppressed RhoA activation, enhanced the phosphorylation of YAP and TAZ, reduced the expression of TEAD1 which interacted with HDAC5 and then bound to TGF-ß1 gene promoter in LPS-exposed Leydig cells. It is thus suspected that ADM can exert anti-apoptotic effect to restore the steroidogenic functions of Leydig cells by suppressing TGF-ß1 through Hippo signaling.

Intermedin (adrenomedullin 2) plays a protective role in sepsis by regulating T- and B-cell proliferation and activity.

BACKGROUND: Sepsis is the major cause of death in intensive care units. We previously found that intermedin (IMD), a calcitonin family peptide, can protect against sepsis by dynamically repairing vascular endothelial junctions and can ameliorate the inflammatory response by inhibiting the infiltration of macrophages in peripheral tissues. The effects of IMD on inflammatory and immune responses indicate that IMD may play a role in immunity. However, whether IMD affects immune cell development, differentiation and response to infection remains unclear. METHODS: IMD-knockout (Adm2-/-) mice were generated in our previous work. Wild-type and IMD-KO mice were subjected to sham or cecal ligation and puncture (CLP) surgery, and bone marrow cells were obtained for RNA sequencing (RNA-Seq) analysis. The RNA-Seq results were verified by real-time RT-PCR. The effect of IMD KO or IMD rescue on the septic mice was explored using mild and severe infection models induced by CLP surgery at different levels of severity, and the survival outcomes were analyzed using Kaplan-Meier curves and the log-rank test. The mechanism underlying the effects of IMD in T/B cell proliferation and differentiation were investigated by PCR, Western blot (WB), and cell proliferation assays and flow cytometry analysis. RESULTS: RNA-Seq showed that IMD-KO mice exhibited a primary immunosuppression phenotype characterized by a marked decrease in the expression of T- and B-cell function-related genes. This immunosuppression made the IMD-KO mice vulnerable to pathogenic invasion, and even mild infection killed nearly half of the IMD-KO mice. Supplementation with the IMD peptide restored the expression of T/B-cell-related genes and significantly reduced the mortality rate of the IMD-KO mice. IMD is likely to directly promote T- and B-cell proliferation through ERK1/2 phosphorylation, stimulate T-cell differentiation via Ilr7/Rag1/2-controled T cell receptor (TCR) recombination, and activate B cells via Pax5, a transcription factor that activates at least 170 genes needed for B-cell functions. CONCLUSION: Together with previous findings, our results indicate that IMD may play a protective role in sepsis via three mechanisms: protecting the vascular endothelium, reducing the inflammatory response, and activating T/B-cell proliferation and differentiation. Our study may provide the first identification of IMD as a calcitonin peptide that plays an important role in the adaptive immune response by activating T/B cells and provides translational opportunities for the design of immunotherapies for sepsis and other diseases associated with primary immunodeficiency.

Reassessing the adrenomedullin scavenging function of ACKR3 in lymphatic endothelial cells.

Atypical chemokine receptor 3 (ACKR3) is a scavenger of the chemokines CXCL11 and CXCL12 and of several opioid peptides. Additional evidence indicates that ACKR3 binds two other non-chemokine ligands, namely the peptide hormone adrenomedullin (AM) and derivatives of the proadrenomedullin N-terminal 20 peptide (PAMP). AM exhibits multiple functions in the cardiovascular system and is essential for embryonic lymphangiogenesis in mice. Interestingly, AM-overexpressing and ACKR3-deficient mouse embryos both display lymphatic hyperplasia. Moreover, in vitro evidence suggested that lymphatic endothelial cells (LECs), which express ACKR3, scavenge AM and thereby reduce AM-induced lymphangiogenic responses. Together, these observations have led to the conclusion that ACKR3-mediated AM scavenging by LECs serves to prevent overshooting AM-induced lymphangiogenesis and lymphatic hyperplasia. Here, we further investigated AM scavenging by ACKR3 in HEK293 cells and in human primary dermal LECs obtained from three different sources in vitro. LECs efficiently bound and scavenged fluorescent CXCL12 or a CXCL11/12 chimeric chemokine in an ACKR3-dependent manner. Conversely, addition of AM induced LEC proliferation but AM internalization was found to be independent of ACKR3. Similarly, ectopic expression of ACKR3 in HEK293 cells did not result in AM internalization, but the latter was avidly induced upon co-transfecting HEK293 cells with the canonical AM receptors, consisting of calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying protein (RAMP)2 or RAMP3. Together, these findings indicate that ACKR3-dependent scavenging of AM by human LECs does not occur at ligand concentrations sufficient to trigger AM-induced responses mediated by canonical AM receptors.

Clinical Potential of Adrenomedullin Signaling in the Cardiovascular System.

Numerous clinical studies have revealed the utility of circulating AM (adrenomedullin) or MR-proAM (mid-regional proAM 45-92) as an effective prognostic and diagnostic biomarker for a variety of cardiovascular-related pathophysiologies. Thus, there is strong supporting evidence encouraging the exploration of the AM-CLR (calcitonin receptor-like receptor) signaling pathway as a therapeutic target. This is further bolstered because several drugs targeting the shared CGRP (calcitonin gene-related peptide)-CLR pathway are already Food and Drug Administration-approved and on the market for the treatment of migraine. In this review, we summarize the AM-CLR signaling pathway and its modulatory mechanisms and provide an overview of the current understanding of the physiological and pathological roles of AM-CLR signaling and the yet untapped potentials of AM as a biomarker or therapeutic target in cardiac and vascular diseases and provide an outlook on the recently emerged strategies that may provide further boost to the possible clinical applications of AM signaling.

Role of Adrenomedullin 2/Intermedin in the Pathogenesis of Neovascular Age-Related Macular Degeneration.

Adrenomedullin 2 (AM2; also known as intermedin) is a member of the adrenomedullin (AM) peptide family. Similarly to AM, AM2 partakes in a variety of physiological activities. AM2 has been reported to exert protective effects on various organ disorders; however, its significance in the eye is unknown. We investigated the role of AM2 in ocular diseases. The receptor system of AM2 was expressed more abundantly in the choroid than in the retina. In an oxygen-induced retinopathy model, physiological and pathologic retinal angiogenesis did not differ between AM2-knockout (AM2-/-) and wild-type mice. In contrast, in laser-induced choroidal neovascularization, a model of neovascular age-related macular degeneration, AM2-/- mice had enlarged and leakier choroidal neovascularization lesions, with exacerbated subretinal fibrosis and macrophage infiltration. Contrary to this, exogenous administration of AM2 ameliorated the laser-induced choroidal neovascularization-associated pathology and suppressed gene expression associated with inflammation, fibrosis, and oxidative stress, including that of VEGF-A, VEGFR-2, CD68, CTGF, and p22-phox. The stimulation of human adult retinal pigment epithelial (ARPE) cell line 19 cells with TGF-ß2 and TNF-α induced epithelial-to-mesenchymal transition (EMT), whereas AM2 expression was also elevated. The induction of EMT was suppressed when the ARPE-19 cells were pretreated with AM2. A transcriptome analysis identified 15 genes, including mesenchyme homeobox 2 (Meox2), whose expression was significantly altered in the AM2-treated group compared with that in the control group. The expression of Meox2, a transcription factor that inhibits inflammation and fibrosis, was enhanced by AM2 treatment and attenuated by endogenous AM2 knockout in the early phase after laser irradiation. The AM2 treatment of endothelial cells inhibited endothelial to mesenchymal transition and NF-κB activation; however, this effect tended to be canceled following Meox2 gene knockdown. These results indicate that AM2 suppresses the neovascular age-related macular degeneration-related pathologies partially via the upregulation of Meox2. Thus, AM2 may be a promising therapeutic target for ocular vascular diseases.

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.

Overexpression of adrenomedullin (ADM) alleviates the senescence of human dental pulp stem cells by regulating the miR-152/CCNA2 pathway.

The limitation of human dental pulp stem cells (DPSCs), which have potential application value in regenerative medicine, is that they are prone to age in vitro. Studies have shown adrenomedullin (ADM) is believed to promote the proliferation of human DPSCs, but whether it can also affect aging remains to be investigated. A lentivirus vector was used to construct human DPSCs overexpressing ADM. Senescence tests were carried out on cells of the 7th and 15th passage. Transcriptome analysis was conducted to analyze microRNA expression regulation changes after human DPSCs overexpressed ADM. H2O2 induced the aging model of human DPSCs, and we examined the mechanism of recovery of aging through transfection experiments with miR-152 mimic, pCDH-CCNA2, and CCNA2 siRNA. Overexpression of ADM significantly upregulated the G2/M phase ratio of human DPSCs in natural passage culture (P = 0.001) and inhibited the expression of p53 (P = 0.014), P21 WAF1 (P = 0.015), and P16 INK4A (P = 0.001). Decreased ROS accumulation was observed in human DPSCs during long-term natural passage (P = 0.022). Transcriptome analysis showed that miR-152 was significantly upregulated during human DPSC senescence (P = 0.001) and could induce cell senescence by directly targeting CCNA2. Transfection with miR-152 mimic significantly reversed the inhibitory effect of ADM overexpression on p53 (P = 0.006), P21 WAF1 (P = 0.012), and P16 INK4A (P = 0.01) proteins in human DPSCs (H2O2-induced). In contrast, pCDH-CCNA2 weakened the effect of the miR-152 mimic, thus promoting cell proliferation and antiaging. ADM-overexpressing human DPSCs promote cell cycle progression and resist cellular senescence through CCNA2 expression promotion by inhibiting miR-152.

miR-1297 sensitizes glioma cells to temozolomide (TMZ) treatment through targeting adrenomedullin (ADM).

BACKGROUND: Gliomas account for about 80% of all malignant brain and other central nervous system (CNS) tumors. Temozolomide (TMZ) resistance represents a major treatment hurdle. Adrenomedullin (ADM) has been reported to induce glioblastoma cell growth. METHODS: Cell viability was measured using the CCK-8 assay. The apoptosis analysis was performed using the Annexin V-FITC Apoptosis Detection Kit. The mitochondrial membrane potential was determined by JC-1 staining. A nude mouse tumor assay was used to detect tumor formation. Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were performed in tissue sections. Activation of Akt and Erk and expression of apoptosis-related proteins were determined by immunoblotting. RESULTS: ADM expression has been found upregulated in TMZ -resistant glioma samples based on bioinformatics and experimental analyses. Knocking down ADM in glioma cells enhanced the suppressive effects of TMZ on glioma cell viability, promotive effects on cell apoptosis, and inhibitory effects on mitochondrial membrane potential. Moreover, ADM knockdown also enhanced TMZ effects on Bax/Bcl-2, Akt phosphorylation, and Erk1/2 phosphorylation. Bioinformatics and experimental investigation indicated that miR-1297 directly targeted ADM and inhibited ADM expression. miR-1297 overexpression exerted similar effects to ADM knockdown on TMZ-treated glioma cells. More importantly, under TMZ treatment, inhibition of miR-1297 attenuated TMZ treatment on glioma cells; ADM knockdown partially attenuated the effects of miR-1297 inhibition on TMZ-treated glioma cells. CONCLUSIONS: miR-1297 sensitizes glioma cells to TMZ treatment through targeting ADM. The Bax/Bcl-2, Akt, and Erk1/2 signaling pathways, as well as mitochondrial functions might be involved.

Local adrenomedullin gene delivery inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo.

Adrenomedullin (ADM) has beneficial effects on Leydig cells under pathological conditions, including lipopolysaccharide (LPS)-induced orchitis. Our previous studies demonstrated that ADM exerts a restorative effect on steroidogenesis in LPS-treated primary rat Leydig cells by attenuating oxidative stress, inflammation and apoptosis. In this study, we aim to investigate whether ADM inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo. Rats were administered with LPS and injected with Ad-ADM, an adeno-associated virus vector that expressed ADM. Then, rat testes were collected for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) immunofluorescence staining. Steroidogenic enzymes or steroidogenic regulatory factors or protein, including steroidogenic factor-1 (SF-1), liver receptor homologue-1 (LRH1), Nur77, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc), 3ß-HSD, cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17) and 17ß-hydroxysteroid dehydrogenase (17ß-HSD), were detected via gene expression profiling and western blot analysis. Plasma testosterone concentrations were measured. Results showed that ADM may inhibit Leydig cell dysfunction by rescuing steroidogenic enzymes and steroidogenic regulatory factors in vivo. The reduction in the number of Leydig cells after LPS exposure was reversed by ADM. ADM rescued the gene or protein levels of SF-1, LRH1, Nur77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD and plasma testosterone concentrations. To summarize ADM could rescue some important steroidogenic enzymes, steroidogenic regulatory factors and testosterone production in Leydig cells in vivo.

Adrenomedullin is an Important Pathological Mediator in Progression of Chronic Neuropathic Pain.

BACKGROUND: The characterization of neuropathic pain is maladaptive plasticity within the nociceptive system. Multiple alterations contribute to complex pain phenotypes. Adrenomedullin (AM) has been documented to be a pain mediator. However, its involvement in pathological pain is poorly understood. We studied the contribution of AM to chronic neuropathic pain in the spinal nerve ligation (SNL) model. METHODS: Daily injection of the AM receptor antagonist AM22-52 (10 nmol) via an intrathecal (i.t.) route after SNL inhibited mechanical allodynia starting on day 6. Single administration of AM22-52 produced an immediate attenuation on pain hypersensitivity on day 2 or 10 post-SNL. Protein and mRNA levels were assayed by immunofluorescent staining and qRT-PCR, respectively, on days 1, 3, 7 and 15 post-SNL. RESULTS: The results showed that AM at both protein and mRNA levels was increased in both injured (L5) and adjacent uninjured (L4) nerves starting on day 3 post-SNL. In dorsal root ganglion (DRG) at L5, AM was increase on days 1-7 at mRNA level but only on day 7 at protein level. However, AM was increase at mRNA level on days 1-7 and at protein level on days 3-15 in L4 DRG. AM mRNA expression was upregulated on days 1-7 in the spinal cord. Expression of receptor activity-modifying protein 2 (RAMP2), an essential AM1 receptor component, was upregulated in small and medium-diameter neurons on days 1-15 in both L5 and L4 DRG. Furthermore, single administration of AM22-52 suppressed the increase of nNOS in DRG induced by SNL and daily injection of AM22-52 for 7 days inhibited SNL-induced increase of CGRP mRNA in the spinal dorsal horn. CONCLUSIONS: This study indicates that the increased AM bioactivity in injured and uninjured peripheral nerves, uninjured adjacent DRG and the spinal dorsal horn play a critical role mainly in the late-phase development of neuropathic pain. The mechanism may involve the recruitment of nNOS and CGRP.

Adrenomedullin Expression Is Associated With the Severity and Poor Prognosis of Interstitial Lung Disease in Dermatomyositis Patients.

Objective: To evaluate adrenomedullin mRNA levels in the peripheral blood mononuclear cells (PBMCs) of patients with dermatomyositis (DM) as well as their correlation with the severity of interstitial lung disease (ILD). Methods: A total of 41 DM patients and seven immune-mediated necrotizing myopathy (IMNM) patients were recruited, in addition to 21 healthy controls (HCs). The adrenomedullin mRNA levels in PBMCs were measured via quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR). The associations between adrenomedullin expression levels and major clinical, laboratory, pulmonary function parameters and the prognosis of patients with DM-related ILD (DM-ILD) were analyzed. Immunohistochemical analysis was performed on lung tissues of DM-ILD patients to determine adrenomedullin expression. Results: Adrenomedullin mRNA levels in PBMCs were significantly higher in DM patients than in IMNM patients and HCs (p = 0.022 and p<0.001, respectively). Among DM patients, the levels were significantly higher in those with rapidly progressive ILD (RP-ILD) than in those with chronic ILD (p = 0.002) or without ILD (p < 0.001). The adrenomedullin mRNA levels in DM-ILD were positively correlated with serum ferritin (r =0.507, p =0.002), lactate dehydrogenase (LDH) (r =0.350, p =0.045), and lung visual analog scale (VAS) (r=0.392, p=0.021) and were negatively correlated with pulmonary function test parameters, including predicted forced vital capacity (FVC)% (r = -0.523, p = 0.025), forced expiratory volume in 1 s (FEV1)% (r = -0.539, p = 0.020), and diffusing capacity of carbon monoxide (DLco)% (r = -0.495, p = 0.036). Immunohistochemical analysis of adrenomedullin confirmed higher expression in the alveolar epithelial cells and macrophages of DM patients with RP-ILD. Among the DM patients with ILD, the six decedents exhibited higher adrenomedullin levels than the 28 survivors (p = 0.042). The cumulative survival rate was significantly lower (62.5% vs. 100%, P = 0.005) in patients with an adrenomedullin level > 0.053 than in those with a level <0.053. Conclusions: Adrenomedullin levels are upregulated in DM patients with RP-ILD and are associated with ILD severity and poor prognosis. Adrenomedullin may be a potential prognostic biomarker in DM patients with ILD, although need further investigation.

Plasma Adrenomedullin, Allelic Variations in the ADM Gene, and Risk for Lower-Limb Amputation in People With Type 2 Diabetes.

OBJECTIVE: Patients with diabetes have an increased risk for lower-limb amputation (LLA), but biomarkers to assess risk of LLA are lacking. Adrenomedullin (ADM) is a vasodilator peptide that also plays a role in fluid and electrolyte homeostasis in the kidney, increasing natriuresis and diuresis. ADM was shown to be associated with cardiovascular and renal events in diabetes, but it was not investigated in terms of LLA risk. We investigated the hypothesis that ADM is associated with LLA in people with type 2 diabetes. RESEARCH DESIGN AND METHODS: We studied 4,375 participants in the DIABHYCAR and SURDIAGENE cohorts (men, 68%; mean 66 years of age; mean duration of diabetes 12 years; and median follow-up 5.3 years). Plasma midregional proadrenomedullin (MR-proADM; a surrogate for ADM) was measured by immunofluorescence. Five single nucleotide polymorphisms (SNPs) in the ADM gene region were genotyped. RESULTS: LLA requirement during follow-up by increasing tertiles of plasma MR-proADM distribution was 1.0% (tertile 1 [T1]), 2.3% (T2), and 4.4% (T3) (P < 0.0001). In Cox multivariate analysis, the adjusted hazard ratio (95% CI) for LLA was 4.40 (2.30-8.88) (P < 0.0001) for T3 versus T1. Moreover, MR-proADM significantly improved indices for risk stratification of LLA. Four SNPs were associated with plasma MR-proADM concentration at baseline and with LLA during follow-up. Alleles associated with higher MR-proADM were associated with increased LLA risk. CONCLUSIONS: We observed associations of plasma MR-proADM with LLA and of ADM SNPs with plasma MR-proADM and with LLA in people with type 2 diabetes. This pattern of Mendelian randomization supports the causality of the association of ADM with LLA.

Adrenomedullin and its receptors are expressed in mouse pancreatic ß-cells and suppresses insulin synthesis and secretion.

Gestational diabetes mellitus (GDM) is associated with defective pancreatic ß-cell adaptation in pregnancy, but the underlying mechanism remains obscure. Our previous studies demonstrated that GDM women display increased plasma adrenomedullin (ADM) levels, and non-obese GDM mice show decreased serum concentrations of insulin and the number of ß-cells in pancreas islets. The aims of this study is to examine if ADM and its receptors are expressed in female mouse pancreas, and if so, whether insulin secretion is regulated by ADM in mouse ß-cell line, NIT-1 cells and isolated mouse pancreatic islets. Present study shows that ADM and its receptor components CRLR, RAMPs are present in mouse pancreatic islets and co-localized with insulin. The expressions of ADM, CRLR and RAMP2 in islets from pregnant mice are reduced compared to that of non-pregnant mice. NIT-1-ß cells express ADM and its receptor mRNA, and glucose dose-dependently stimulates expressions. Furthermore, ADM inhibits NIT-1-ß cell growth, and this inhibition is reversed by ADM antagonist, ADM22-52. The glucose-induced insulin secretion was suppressed by ADM in NIT-1-ß cells and isolated pancreatic islets from pregnant mice. These inhibitory effects are accompanied by upregulation of endoplasmic reticulum (ER) stress biomarker genes in NIT-1-ß cells. This study unveils that reduced ADM and its receptors may play a role in ß-cell adaptation during pregnancy, while increased plasma ADM in GDM may contribute to the ß-cells dysfunction, and blockade of ADM may reverse ß-cell insulin production.

Therapeutic Targets for Heart Failure Identified Using Proteomics and Mendelian Randomization.

BACKGROUND: Heart failure (HF) is a highly prevalent disorder for which disease mechanisms are incompletely understood. The discovery of disease-associated proteins with causal genetic evidence provides an opportunity to identify new therapeutic targets. METHODS: We investigated the observational and causal associations of 90 cardiovascular proteins, which were measured using affinity-based proteomic assays. First, we estimated the associations of 90 cardiovascular proteins with incident heart failure by means of a fixed-effect meta-analysis of 4 population-based studies, composed of a total of 3019 participants with 732 HF events. The causal effects of HF-associated proteins were then investigated by Mendelian randomization, using cis-protein quantitative loci genetic instruments identified from genomewide association studies in more than 30 000 individuals. To improve the precision of causal estimates, we implemented an Mendelian randomization model that accounted for linkage disequilibrium between instruments and tested the robustness of causal estimates through a multiverse sensitivity analysis that included up to 120 combinations of instrument selection parameters and Mendelian randomization models per protein. The druggability of candidate proteins was surveyed, and mechanism of action and potential on-target side effects were explored with cross-trait Mendelian randomization analysis. RESULTS: Forty-four of ninety proteins were positively associated with risk of incident HF (P<6.0×10-4). Among these, 8 proteins had evidence of a causal association with HF that was robust to multiverse sensitivity analysis: higher CSF-1 (macrophage colony-stimulating factor 1), Gal-3 (galectin-3) and KIM-1 (kidney injury molecule 1) were positively associated with risk of HF, whereas higher ADM (adrenomedullin), CHI3L1 (chitinase-3-like protein 1), CTSL1 (cathepsin L1), FGF-23 (fibroblast growth factor 23), and MMP-12 (matrix metalloproteinase-12) were protective. Therapeutics targeting ADM and Gal-3 are currently under evaluation in clinical trials, and all the remaining proteins were considered druggable, except KIM-1. CONCLUSIONS: We identified 44 circulating proteins that were associated with incident HF, of which 8 showed evidence of a causal relationship and 7 were druggable, including adrenomedullin, which represents a particularly promising drug target. Our approach demonstrates a tractable roadmap for the triangulation of population genomic and proteomic data for the prioritization of therapeutic targets for complex human diseases.