Grancalcin Research Articles

Macrophage-Hepatocyte Circuits Mediated by Grancalcin Aggravate the Progression of Metabolic Dysfunction Associated Steatohepatitis.

The dynamic interplay between parenchymal hepatocytes and non-parenchymal cells (NPCs), such as macrophages, is an important mechanism for liver metabolic homeostasis. Although numerous endeavors have been made to identify the mediators of metabolic dysfunction associated steatohepatitis (MASH), the molecular underpinnings of MASH progression remain poorly understood, and therapies to arrest MASH progression remain elusive. Herein, it is revealed that the expression of grancalcin (GCA) is upregulated in the macrophages of patients and rodents with MASH and correlates with MASH progression. Notably, the administration of recombinant GCA aggravates the development of MASH, whereas, Gca deletion in myeloid cells blunts liver steatosis and inflammation in multiple MASH murine models. Mechanistically, GCA activates macrophages via TLR9-NF-κB signaling, and the activated macrophages promote hepatocyte lipid accumulation and apoptosis via secretion of Interleukin-6(IL-6), Tumor Necrosis Factor α (TNFα), and Interleukin-1β(IL-1β), thereby leading to hepatic steatosis and inflammation. Finally, the therapeutic administration of antibody blocking GCA effectively halts the progression of MASH. Collectively, these findings implicate GCA as a crucial mediator of MASH and clarify a new metabolic signaling axis between the hepatocytes and macrophages, implying that GCA can emerge as a particularly interesting putative therapeutic target for reversing MASH progression.

Targeting Grancalcin Accelerates Wound Healing by Improving Angiogenesis in Diabetes.

Chronic diabetic wounds are a serious complication of diabetes and often result in limb amputations and confer high mortality rates. The proinflammatory secretome in the wound perpetuates defective neovascularization and contributes to dysregulated tissue repair. This study aims to design a gelatin methacrylamide (GelMA) hydrogel to sustained the release of grancalcin-neutralizing antibody (GCA-NAb) and evaluate it as a potential scaffold to promote diabetic wound healing. Results show that the expression of grancalcin(GCA), a protein secreted by bone marrow-derived immune cells, is elevated in the wound sites of individuals and animals with diabetic ulcers. Genetic inhibition of grancalcin expression accelerates vascularization and healing in an animal model. Mechanistic studies show that grancalcin binds to transient receptor potential melastatin 8(TRPM8) and partially inactivates its downstream signaling pathways, thereby impairing angiogenesis in vitro and ex vivo. Systemic or topical administration of a GCA-NAb accelerate wound repair in mice with diabetes. The data suggest that GCA is a potential therapeutic target for the treatment of diabetic ulcers.

Age-related secretion of grancalcin by macrophages induces skeletal stem/progenitor cell senescence during fracture healing

Skeletal stem/progenitor cell (SSPC) senescence is a major cause of decreased bone regenerative potential with aging, but the causes of SSPC senescence remain unclear. In this study, we revealed that macrophages in calluses secrete prosenescent factors, including grancalcin (GCA), during aging, which triggers SSPC senescence and impairs fracture healing. Local injection of human rGCA in young mice induced SSPC senescence and delayed fracture repair. Genetic deletion of Gca in monocytes/macrophages was sufficient to rejuvenate fracture repair in aged mice and alleviate SSPC senescence. Mechanistically, GCA binds to the plexin-B2 receptor and activates Arg2-mediated mitochondrial dysfunction, resulting in cellular senescence. Depletion of Plxnb2 in SSPCs impaired fracture healing. Administration of GCA-neutralizing antibody enhanced fracture healing in aged mice. Thus, our study revealed that senescent macrophages within calluses secrete GCA to trigger SSPC secondary senescence, and GCA neutralization represents a promising therapy for nonunion or delayed union in elderly individuals.

Bone Marrow-Derived GCA+ Immune Cells Drive Alzheimer's Disease Progression.

Alzheimer's disease (AD) is an age-related degenerative disease of the central nervous system (CNS), whereas the role of bone marrow immune cells in the pathogenesis of AD remains unclear. Here, the study reveals that compared to matched healthy individuals, AD patients have higher circulating grancalcin (GCA) levels, which negatively correlate with cognitive function. Bone marrow-derived GCA+ immune cells, which secret abundant GCA and increase during aging, preferentially invaded the hippocampus and cortex of AD mouse model in a C-C Motif Chemokine Receptor 10 (CCR10)-dependent manner. Transplanting GCA+ immune cells or direct stereotaxic injection of recombinant GCA protein intensified amyloid plaque load and aggravated cognitive and memory impairments. In contrast, genetic ablation of GCA in the hematopoietic compartment improves cognitive and memory function. Mechanistically, GCA competitively binds to the low-density lipoprotein receptor-related protein 1 (LRP1) in microglia, thus inhibiting phagocytosis and clearance of Aβ and potentiating neuropathological changes. Importantly, GCA-neutralizing antibody treatment rejuvenated cognitive and memory function and constrained AD progression. Together, the study demonstrates a pathological role of GCA+ immune cells instigating cognitive and memory decline, suggesting that GCA+ immune cells can be a potential target for innovative therapeutic strategies in AD.

What's New in Musculoskeletal Basic Science.

What's New in Musculoskeletal Basic Science.

Molecular modeling, molecular dynamics simulation, and essential dynamics analysis of grancalcin: An upregulated biomarker in experimental autoimmune encephalomyelitis mice

The experimental autoimmune encephalomyelitis mouse model is the most commonly used animal model, and it best represents multiple sclerosis. Grancalcin (GCA) was discovered to be upregulated in EAE mice. GCA comprises 220 amino acids that have been assigned the UniprotKB ID Q8VC88. It is a calcium-binding protein that helps neutrophils adhere to fibronectin and the formation of focal adhesions. However, the protein data bank does not contain the crystal structure of mouse GCA. The current study aims to analyze the structural and physicochemical properties of GCA. Mouse GCA showed a high percentage identity (87%) with the crystal structure of des (1–52) grancalcin with bound calcium (chain A) from Homo sapiens identified by its PDB id 1k94_A. Using the SWISS-MODEL server, we used 1k94_A as a template protein to model the mouse GCA protein. Compared to the template structure 1K94, three potential binding sites for calcium-binding have been proposed, ranging from 13 to 20, 80 to 91, and 109 to 120 amino acids. On an i5 personal computer with 8GB of RAM, GROMACS 2020.1 was utilized to run a 100 ns molecular dynamics (MD) simulation. RMSD, Rg, and RMSF analysis of an MD simulation trajectory indicate a stable and compact state throughout the simulation period of modeled proteins. We found that GCA is primarily alpha helical (Class 1), with eight alpha helices. The essential dynamics analysis captures PCA and SASA, culminating in the biological motions that correspond to the last 1000 frames. These findings will aid the development of potential inhibitors as well as the determination of binding pockets and residues for drug-like molecules.

Grancalcin in Immune Cells is Induced and Regulates Bone-Fat Imbalance During Skeletal Aging

Skeletal aging is characterized by low bone turnover and increased marrow fat accumulation. However, the underlying mechanism remains poorly defined. Here we show that pro-inflammatory and senescent subtypes of immune cells, including macrophages and neutrophils, accumulated in bone marrow and secreted abundant grancalcin (GCA) during aging. Elevation of bone marrow GCA level in young mice leads to premature skeletal aging. Deletion of GCA in neutrophils and macrophages rejuvenates skeletal aging. Mechanistically, we identify that GCA binds to Plexin-b2 receptor and inactivates its downstream pathway. Depletion of plexin-b2 in bone marrow mesenchymal stem cells abrogates the rejuvenated bone phenotype of GCA-knockout mice. Moreover, GCA neutralizing antibody is generated and shows a certain rejuvenating effect on bone. Given that immune cells are involved into diverse pathologic processes, targeting this fundamental mechanism to prevent skeletal aging offers novel treatment strategy not only for osteoporosis, but also for multiple age-related diseases.

Guanylyl Cyclase A/cGMP Signaling Slows Hidden, Age- and Acoustic Trauma-Induced Hearing Loss.

In the inner ear, cyclic guanosine monophosphate (cGMP) signaling has been described as facilitating otoprotection, which was previously observed through elevated cGMP levels achieved by phosphodiesterase 5 inhibition. However, to date, the upstream guanylyl cyclase (GC) subtype eliciting cGMP production is unknown. Here, we show that mice with a genetic disruption of the gene encoding the cGMP generator GC-A, the receptor for atrial and B-type natriuretic peptides, display a greater vulnerability of hair cells to hidden hearing loss and noise- and age-dependent hearing loss. This vulnerability was associated with GC-A expression in spiral ganglia and outer hair cells (OHCs) but not in inner hair cells (IHCs). GC-A knockout mice exhibited elevated hearing thresholds, most pronounced for the detection of high-frequency tones. Deficits in OHC input–output functions in high-frequency regions were already present in young GC-A-deficient mice, with no signs of an accelerated progression of age-related hearing loss or higher vulnerability to acoustic trauma. OHCs in these frequency regions in young GC-A knockout mice exhibited diminished levels of KCNQ4 expression, which is the dominant K+ channel in OHCs, and decreased activation of poly (ADP-ribose) polymerase-1, an enzyme involved in DNA repair. Further, GC-A knockout mice had IHC synapse impairments and reduced amplitudes of auditory brainstem responses that progressed with age and with acoustic trauma, in contrast to OHCs, when compared to GC-A wild-type littermates. We conclude that GC-A/cGMP-dependent signaling pathways have otoprotective functions and GC-A gene disruption differentially contributes to hair-cell damage in a healthy, aged, or injured system. Thus, augmentation of natriuretic peptide GC-A signaling likely has potential to overcome hidden and noise-induced hearing loss, as well as presbycusis.

GCA links TRAF6-ULK1-dependent autophagy activation in resistant chronic myeloid leukemia

ABSTRACTImatinib is the first molecularly targeted compound for chronic myeloid leukemia (CML) capable to inhibit BCR-ABL kinase activity. However, recent clinical evidence indicates that a substantial proportion of CML patients exhibit BCR-ABL-dependent or independent resistance to imatinib. Despite the importance of imatinib resistance in CML, the underlying molecular mechanisms of this resistance are largely unknown. Here, we identified GCA (grancalcin) as a critical regulator of imatinib resistance in chronic phase CML via activation of autophagy. Mechanistically, we demonstrated that GCA activates TRAF6 ubiquitin ligase activity to induce Lys63 ubiquitination of ULK1, a crucial regulator of autophagy, resulting in its stabilization and activation. We also highlighted the role of GCA-TRAF6-ULK1 autophagy regulatory axis in imatinib resistance. Our findings represent the basis for novel therapeutic strategies against CML.Abbreviation: ACTB/β-actin: actin beta; ADM: adrenomedullin; AMBRA1: autophagy and beclin 1 regulator 1; AMPK: AMP-activated protein kinase; ANXA5: annexin A5; CP: cytogenetic response; CML: chronic myeloid leukemia; CUL3: cullin 3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCA: grancalcin; Dx: at diagnosis; E-64-d: (2S,3S)-trans-Epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester; IMres: Imatinib resistance; KLHL20: Kelch-like protein 20; LRMP: lymphoid-restricted membrane protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MMR: major molecular response; NH4Cl: ammonium chloride; PBMCs: peripheral blood mononuclear cells; PTPRC: protein tyrosine phosphatase, receptor type, C; SQSTM1/p62: sequestosome 1; SYK: spleen associated tyrosine kinase; TAP1: transporter 1, ATP binding cassette subfamily B member; TKIs: ABL-specific tyrosine kinase inhibitors; TLR9: toll- like receptor 9; TRAF6: TNF receptor associated factor 6; ULK1: unc-51 like autophagy activating kinase 1

Identification of a Putative Quantitative Trait Gene for Resistance to Obesity in Mice Using Transcriptome Analysis and Causal Inference Tests.

It is still challenging to identify causal genes governing obesity. Pbwg1.5, a quantitative trait locus (QTL) for resistance to obesity, was previously discovered from wild Mus musculus castaneus mice and was fine-mapped to a 2.1-Mb genomic region of mouse chromosome 2, where no known gene with an effect on white adipose tissue (WAT) has been reported. The aim of this study was to identify a strong candidate gene for Pbwg1.5 by an integration approach of transcriptome analysis (RNA-sequencing followed by real-time PCR analysis) and the causal inference test (CIT), a statistical method to infer causal relationships between diplotypes, gene expression and trait values. Body weight, body composition and biochemical traits were measured in F2 mice obtained from an intercross between the C57BL/6JJcl strain and a congenic strain carrying Pbwg1.5 on the C57BL/6JJcl background. The F2 mice showed significant diplotype differences in 12 traits including body weight, WAT weight and serum cholesterol/triglyceride levels. The transcriptome analysis revealed that Ly75, Pla2r1, Fap and Gca genes were differentially expressed in the liver and that Fap, Ifih1 and Grb14 were differentially expressed in WAT. However, CITs indicated statistical evidence that only the liver Ly75 gene mediated between genotype and WAT. Ly75 expression was negatively associated with WAT weight. The results suggested that Ly75 is a putative quantitative trait gene for the obesity-resistant Pbwg1.5 QTL discovered from the wild M. m. castaneus mouse. The finding provides a novel insight into a better understanding of the genetic basis for prevention of obesity.

Transcriptional Regulation of the β-Type Carbonic Anhydrase Gene bca by RamA in Corynebacterium glutamicum.

Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide to bicarbonate and maintains the balance of CO2/HCO3- in the intracellular environment, specifically for carboxylation/decarboxylation reactions. In Corynebacterium glutamicum, two putative genes, namely the bca (cg2954) and gca (cg0155) genes, coding for β-type and γ-type carbonic anhydrase, respectively, have been identified. We here analyze the transcriptional organization of these genes. The transcriptional start site (TSS) of the bca gene was shown to be the first nucleotide “A” of its putative translational start codon (ATG) and thus, bca codes for a leaderless transcript. The TSS of the gca gene was identified as an “A” residue located at position -20 relative to the first nucleotide of the annotated translational start codon of the cg0154 gene, which is located immediately upstream of gca. Comparative expression analysis revealed carbon source-dependent regulation of the bca gene, with 1.5- to 2-fold lower promoter activity in cells grown on acetate as compared to glucose as sole carbon source. Based on higher expression of bca in a mutant deficient of the regulator of acetate metabolism RamA as compared to the wild-type of C. glutamicum and based on the binding of His-tagged RamA protein to the bca promoter region, we here present evidence that RamA negatively regulates expression of bca in C. glutamicum. Functional characterization of a gca deletion mutant of C. glutamicum revealed the same growth characteristics of C. glutamicum ∆gca as that of wild-type C. glutamicum and no effect on expression of the bca gene.

Grancalcin (GCA) modulates Toll‐like receptor 9 (TLR9) mediated signaling through its direct interaction with TLR9

Toll-like receptors (TLRs) are playing important roles in stimulating the innate immune response and intensifying adaptive immune response against invading pathogens. Appropriate regulation of TLR activation is important to maintain a balance between preventing tumor activation and inhibiting autoimmunity. Toll-like receptor 9 (TLR9) senses microbial DNA in the endosomes of plasmacytoid dendritic cells and triggers myeloid differentiation primary response gene 88 (MyD88) dependent nuclear factor kappa B (NF-κB) pathways and type I interferon (IFN) responses. However, mechanisms of how TLR9 signals are mediated and which molecules are involved in controlling TLR9 functions remain poorly understood. Here, we report that penta EF-hand protein grancalcin (GCA) interacts and binds with TLR9 in a yeast two-hybrid system and an overexpression system. Using siRNA-mediated knockdown experiments, we also revealed that GCA positively regulates type I IFN production, cytokine/chemokine production through nuclear localization of interferon regulatory factor 7 (IRF7), NF-κB activation, and mitogen-activated protein kinase (MAPK) activation in plasmacytoid dendritic cells. Our results indicate that heterodimerization of GCA and TLR9 is important for TLR9-mediated downstream signaling and might serve to fine tune processes against viral infection.

Identification of a small molecule targeting annexin A7

Autophagy involves multiple membrane trafficking and fusion events. Annexin A7 (ANXA7) is postulated to play a role in membrane fusion during exocytosis, while the contribution of ANXA7 to autophagy is poorly understood. Our recent studies demonstrated that ABO could promote autophagy via elevation of ANXA7 and triggering ANXA7 subcellular redistribution. However, little is known about the molecular mechanisms how ANXA7 regulates autophagy. As molecular disruption of ANXA7 in mice results in several unwished phenotypes, small molecule modulators may be efficacious in defining the mechanisms of ANXA7 action. However, so far no compounds that selectively target ANXA7 have been identified. So, we hypothesize that ABO might be a potent modulator of ANXA7. We also have detected the colocalization of ANXA7 and microtubule-associated protein 1 light chain 3 (LC3), and ANXA7 was essential for LC3 accumulation in VEC autophagy. As a GTPase, whether ANXA7 affects the phosphorylation of LC3 or other proteins needs further investigation. In this study, we performed site-directed mutagenesis and found that ABO directly bound to Thr286 of ANXA7 and inhibited its phosphorylation. By yeast two-hybrid screening, we found that ANXA7 could interact with grancalcin (GCA). ABO promoted the interaction and inhibited GCA phosphorylation, leading to the decrease of intracellular Ca2+ concentration. At the same time, ABO inhibited the phosphorylation of LC3. Hence, by identifying ABO as an unprecedented modulator of ANXA7 as well as GCA and LC3 as interacting proteins of ANXA7, we demonstrated the possible mechanisms how ANXA7 regulates autophagy for the first time.

OS077. The chromosome 2q22 preeclampsia susceptibility locus reveals shared novel risk factors for CVD

We have previously localized a preeclampsia susceptibility locus on chromosome 2q22 in 34 Australian and New Zealand (AUS/NZL) families. Using an extended number of AUS/NZL families (n=74) we have now performed a comprehensive molecular genetics dissection of this locus. Identify causal genetic risk factors for preeclampsia at the 2q22 risk locus. To prioritize positional candidate genes for analysis we used a combination of bioinformatics, SNPing, whole-genome transcriptional profiling and proximity to the peak linkage signal. Prioritized genes were earmarked for exon-centric re-sequencing in 48 founder individuals from the 74 AUS/NZL families. All identified sequence variants were genotyped back in this extended familial cohort. Variants showing the strongest genetic association were genotyped in independent case-control cohorts from Australia (n=1095), Norway (n=3397) and Finland (n=1519), and in a large cohort of Mexican American families rich in quantitative cardiovascular disease (CVD) risk traits. We interrogated 1598 variants from 52 genes and identified four independent SNPs to be significantly associated with preeclampsia susceptibility in the 74 AUS/NZL families. These four SNPs reside in four novel preeclampsia candidate genes: LCT (rs2322659, p=0.002), LRP1B (rs35821928, p=0.0001), RND3 (rs115015150, p=0.002) and GCA (rs17783344, p=0.002). We could only replicate the LCT SNP association in the Australian case-control population (p=0.04, combined p=0.001). These four SNPs are however, significantly associated with several quantitative CVD risk traits such as oxidative stress indicators, inflammatory biomarkers and obesity risk factors. Previous independent studies have reported significant genetic associations with total cholesterol levels and obesity risk factors for variants within LCT and LRP1B, respectively. RND3 inhibits the biological activity of a downstream effector protein, ROCK, which is known to affect endothelial dysfunction, inflammation, oxidative stress and vascular re-modeling. Grancalcin (GCA) is known to impact the adhesive properties of fibronectin, a marker for endothelial vascular injury. To our knowledge, data from the current study present for the first time empirical evidence of possible shared genetic risk factors underlying both preeclampsia and other CVD-related traits.

OS070. Shared genetic risk factors for preeclampsia and cardiovascular disease

There is compelling evidence to support the hypothesis that a maternal constitutional predisposition to cardiovascular disease (CVD) is a key component in development of preeclampsia. In particular, CVD and preeclampsia share pathological features such as endothelial dysfunction and inflammation, and have several metabolic abnormalities in common. In support of this hypothesis, our recent genetic dissection of the Australian preeclampsia susceptibility locus on chromosome 2q22 revealed shared novel genetic risk factors for preeclampsia and CVD-related traits. To replicate association between our recently reported 2q22 preeclampsia risk variants and CVD-related traits in an independent Australian population based cohort. Four independent SNPs from four genes, rs35821928 (LRP1B), rs17783344 (GCA), rs115015150 (RND3) and rs2322659 (LCT), were recently found to be significantly associated with preeclampsia susceptibility and CVD-related traits. These SNPs were genotyped in a large independent Australian cohort rich in quantitative CVD risk traits; The Western Australian Pregnancy Cohort (Raine) Study. This cohort comprises of blood samples from 1246 mothers and 1461 adolescents and clinical measures such as, but not limited to, anthropometric measures of adiposity and lipid-related measures. Genetic association analyses of these four potential preeclampsia susceptibility SNPs against the CVD-related risk traits were performed using the software package R. All statistical analyses assumed an additive model of gene action. Several significant associations (p<0.05) for all four SNPs with a variety of CVD-related risk traits were detected, both for the mothers and the adolescents. The LRP1B SNP was associated with HDL/cholesterol ratio, LDL cholesterol, triglycerides, skinfold measures and weight. The GCA SNP was associated with total cholesterol, HDL cholesterol, serum insulin, hemoglobin, blood glucose, BMI and skinfold measures. The RND3 SNP was associated with triglycerides and waist-hip ratio. The LCT SNP was associated with hemoglobin, blood glucose and abdominal skinfold. We have recently identified genetic variants within the LRP1B, GCA, RND3 and LCT genes to be significantly associated with preeclampsia susceptibility and CVD-related risk traits. We have now demonstrated thatthese specific genetic variants are associated with CVD-related risk traits in an independent population. Our collective findings provide substantial empirical data to support the hypothesis that genetic risk factors for preeclampsia and CVD are, at least in part, shared.

OS071. Effect of methyldopa on the cerebral circulation in chronic hypertensive pregnancies

Introduction: There is compelling evidence to support the hypothesis that a maternal constitutional predisposition to cardiovascular disease (CVD) is a key component in development of preeclampsia. In particular, CVD and preeclampsia share pathological features such as endothelial dysfunction and inflammation, and have several metabolic abnormalities in common. In support of this hypothesis, our recent genetic dissection of the Australian preeclampsia susceptibility locus on chromosome 2q22 revealed shared novel genetic risk factors for preeclampsia and CVD-related traits. Objectives: To replicate association between our recently reported 2q22 preeclampsia risk variants and CVD-related traits in an independent Australian population based cohort. Methods: Four independent SNPs from four genes, rs35821928 (LRP1B), rs17783344 (GCA), rs115015150 (RND3) and rs2322659 (LCT), were recently found to be significantly associated with preeclampsia susceptibility and CVD-related traits. These SNPs were genotyped in a large independent Australian cohort rich in quantitative CVD risk traits; The Western Australian Pregnancy Cohort (Raine) Study. This cohort comprises of blood samples from 1246 mothers and 1461 adolescents and clinical measures such as, but not limited to, anthropometric measures of adiposity and lipid-related measures. Genetic association analyses of these four potential preeclampsia susceptibility SNPs against the CVD-related risk traits were performed using the software package R. All statistical analyses assumed an additive model of gene action. Results: Several significant associations (p < 0.05) for all four SNPs with a variety of CVD-related risk traits were detected, both for the mothers and the adolescents. The LRP1B SNP was associated with HDL/cholesterol ratio, LDL cholesterol, triglycerides, skinfold measures and weight. The GCA SNP was associated with total cholesterol, HDL cholesterol, serum insulin, hemoglobin, blood glucose, BMI and skinfold measures. The RND3 SNP was associated with triglycerides and waist-hip ratio. The LCT SNP was associated with hemoglobin, blood glucose and abdominal skinfold. Conclusion: We have recently identified genetic variants within the LRP1B, GCA, RND3 and LCT genes to be significantly associated with preeclampsia susceptibility and CVD-related risk traits. We have now demonstrated thatthese specific genetic variants are associated with CVDrelated risk traits in an independent population. Our collective findings provide substantial empirical data to support the hypothesis that genetic risk factors for preeclampsia and CVD are, at least in part, shared.

Endothelial actions of atrial and B‐type natriuretic peptides

The cardiac hormone atrial natriuretic peptide (ANP) is critically involved in the maintenance of arterial blood pressure and intravascular volume homeostasis. Its cGMP-producing GC-A receptor is densely expressed in the microvascular endothelium of the lung and systemic circulation, but the functional relevance is controversial. Some studies reported that ANP stimulates endothelial cell permeability, whereas others described that the peptide attenuates endothelial barrier dysfunction provoked by inflammatory agents such as thrombin or histamine. Many studies in vitro addressed the effects of ANP on endothelial proliferation and migration. Again, both pro- and anti-angiogenic properties were described. To unravel the role of the endothelial actions of ANP in vivo, we inactivated the murine GC-A gene selectively in endothelial cells by homologous loxP/Cre-mediated recombination. Our studies in these mice indicate that ANP, via endothelial GC-A, increases endothelial albumin permeability in the microcirculation of the skin and skeletal muscle. This effect is critically involved in the endocrine hypovolaemic, hypotensive actions of the cardiac hormone. On the other hand the homologous GC-A-activating B-type NP (BNP), which is produced by cardiac myocytes and many other cell types in response to stressors such as hypoxia, possibly exerts more paracrine than endocrine actions. For instance, within the ischaemic skeletal muscle BNP released from activated satellite cells can improve the regeneration of neighbouring endothelia. This review will focus on recent advancements in our understanding of endothelial NP/GC-A signalling in the pulmonary versus systemic circulation. It will discuss possible mechanisms accounting for the discrepant observations made for the endothelial actions of this hormone-receptor system and distinguish between (patho)physiological and pharmacological actions. Lastly it will emphasize the potential therapeutical implications derived from the actions of NPs on endothelial permeability and regeneration.

Natriuretic Peptide Signaling via Guanylyl Cyclase (GC)-A: An Endogenous Protective Mechanism of the Heart

Atrial and brain natriuretic peptides (ANP and BNP, respectively) are cardiac hormones, secretions of which are markedly upregulated during cardiac failure, making their plasma levels clinically useful diagnostic markers. ANP and BNP exert potent diuretic, natriuretic and vasorelaxant effects, which are mediated via their common receptor, guanylyl cyclase (GC)-A (also called natriuretic peptide receptor (NPR)-A). Mice deficient for GC-A are mildly hypertensive and show marked cardiac hypertrophy and fibrosis that is disproportionately severe, given their modestly higher blood pressure. Indeed, the cardiac hypertrophy seen in these mice is enhanced in a blood pressure-independent manner and is suppressed by cardiomyocyte-specific overexpression of GC-A. These results suggest that the actions of a local cardiac ANP/BNP-GC-A system are essential for maintenance of normal cardiac architecture. In addition, GC-A was shown to exert its cardioprotective effects by inhibiting angiotensin II-induced hypertrophic signaling, and recent evidence suggests that regulator of G protein signaling (RGS) subtype 4 is involved in the GC-A-mediated inhibition of Gαq-coupled hypertrophic signal transduction. Furthermore, several different groups have reported that functional mutations in the promoter region of the human GC-A gene are associated with essential hypertension and ventricular hypertrophy. These findings suggest that endogenous GC-A protects the heart from pathological hypertrophic stimuli, and that humans who express only low levels of GC-A are genetically predisposed to cardiac remodeling and hypertension.

Association of CT Dinucleotide Repeat Polymorphism in the 5'-Flanking Region of the Guanylyl Cyclase (GC)-A Gene with Essential Hypertension in the Japanese

Guanylyl cyclase (GC)-A (natriuretic peptide receptor [NPR]-1), the receptor for atrial and brain natriuretic peptide, is important in the regulation of blood pressure in animal models and, possibly, in humans. In this study, we examined the association between dinucleotide repeat polymorphism within the 5'-flanking region of the GC-A gene and essential hypertension in a group of Japanese subjects. By genotyping 177 hypertensive and 170 normotensive subjects, we identified 5 allele types with 6, 9, 10, 11 and 12 CT dinucleotide repeats, respectively, around position -293, upstream of the ATG codon in the human GC-A gene. The frequency of the (CT)n=6 allele was significantly higher among hypertensive than normotensive subjects, while the frequencies of the other allele types did not differ between the two groups. We also examined the linkage between G/A polymorphism at position -77 (rs13306004), downstream of the (CT)n polymorphism, and found that the (CT)n=6 allele was tightly linked to an A at position -77, while all other (CT)n alleles were linked to G. Promoter-reporter analyses carried out in cultured human aortic smooth muscle cells using a luciferase gene fused to the 5'-flanking region of the GC-A gene revealed that the promoter containing (CT)n=6 drove less transcriptional activity than that containing (CT)n=10. Finally, site-directed mutation showed that the (CT)n and G/A polymorphisms act synergistically to affect GC-A promoter activity. Our results thus define the (CT)n polymorphism in the 5'-flanking region of the GC-A gene as a potent and novel susceptibility marker for hypertension.

Association of the IFIH1-GCA-KCNH7 chromosomal region with rheumatoid arthritis

The cause of rheumatoid arthritis (RA) remains unknown, although both genetic and environmental factors are involved. A genome-wide association study of non-synonymous single nucleotide polymorphisms (SNPs) showed the involvement of...