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Our overall research aim is to statically derive execution cost and other metrics from program code written in the P4 programming language. For this purpose, we extract a detailed control ow graph (CFG) from the code, that can be turned into a full, formal model of execution, to extract properties - such as execution cost - from the model. While CFG extraction and analysis is well researched area, details are dependent on code representation and therefore application of textbook algorithms (often defined over unstructured code listings) to real programming languages is often non-trivial. Our aim is to present an algorithm for CFG extraction over P4 abstract syntax trees (AST). During the extraction we create direct links between nodes of the CFG and the P4 AST: this way we can access all information in the P4 AST during CFG traversal. We are utilizing Gremlin, a graph query language to take advantage of graph databases, but also for compactness and to formally prove algorithm correctness. [ABSTRACT FROM AUTHOR]

Type 1 alveolar epithelial cells (AT1s) and type 2 alveolar epithelial cells (AT2s) regulate the structural integrity and function of alveoli. AT1s mediate gas exchange, whereas AT2s serve multiple functions, including surfactant secretion and alveolar repair through proliferation and differentiation into AT1s as progenitors. However, mechanisms regulating AT2 proliferation and differentiation remain unclear. Here we demonstrate that Gremlin, an intrinsic inhibitor of bone morphogenetic protein (BMP), induces AT2 proliferation and differentiation. Transient overexpression of Gremlin in rat lungs by adenovirus vector delivery suppressed BMP signaling, induced proliferation of AT2s and the production of Bmp2, which in turn led to the recovery of BMP signaling and induced AT2 differentiation into AT1s. Bleomycin-induced lung injury upregulated Gremlin and showed a similar time course of biomarker expression comparable to the adenovirus model. TGF-β and IL-1β induced Gremlin expression in fibroblasts. Taken together, our findings implicate that Gremlin expression during lung injury leads to precisely timed inhibition of BMP signaling and activates AT2s, leading to alveolar repair. • Regulation mechanisms of type 2 alveolar epithelial cells (AT2) are unclear. • Gremlin, an intrinsic BMP inhibitor, leads to AT2 proliferation and differentiation. • Gremlin expression upregulated in lung injury, leading to AT2 activation. • TGF-beta and IL-1beta induce Gremlin expression in fibroblasts. • Findings implicate Gremlin as key player in lung injury repair. [ABSTRACT FROM AUTHOR]

This study assessed the novel concept that osteoclast-derived Grem1 has regulatory functions in the skeletal response to calcium stress using an osteoclastic Grem1 conditional knockout (cKO) mouse model. The calcium stress was initiated by feeding cKO mutants and wildtype (WT) littermates a calcium-deficient diet for 2 weeks. Deletion of Grem1 in mature osteoclasts did not affect developmental bone growth nor basal bone turnover. In response to calcium depletion, male cKO mutants showed greater increases in osteoclastic resorption and trabecular bone loss than male WT littermates, indicating an enhanced skeletal sensitivity to calcium depletion in male mutants. The enhanced sensitivity to calcium depletion was sex-dependent, as female cKO mutants showed lower increases in osteoclastic resorption and bone loss than female WT littermates as well as male cKO mutants. The sex disparity in osteoclastic resorption response to calcium stress was intrinsic to osteoclasts since osteoclasts of male but not female cKO mutants showed greater in vitro bone resorption activity than osteoclasts of WT littermates of respective sex. Male cKO mutants displayed smaller bone formation response to calcium depletion than male WT littermates, while female mutants showed bigger bone formation response than female WT littermates, indicating that cKO mutants also displayed sex disparity in bone formation response. The sex disparity in bone formation response was not caused by intrinsic differences in osteoblasts but might be due to sex-dependent differential osteoclastic release of osteogenic factors. In summary, osteoclast-derived gremlin-1 has complicated and sex-dependent regulatory roles in skeletal response to calcium stress., Competing Interests: Declarations. Conflict of Interest: All authors (Matilda H.-C. Sheng, Charles H. Rundle, David J. Baylink, and Kin-Hing William Lau) state that they have no conflicts of interest.                                                           Human and Animal Rights and Informed Consent: The animal protocol (protocol #1298) was reviewed and approved by the Animal Care and Use Committee (IACUC) of VA Loma Linda Healthcare System as well as by the IACUC of the Loma Linda University and the Animal Care and Use Review Office (ACURO) of the US Army Medical Research and Materiel Command of the Department of Defense. In conducting research using animals, the investigators adhered to the Animal Welfare Act Regulations and other federal statures relating to animals and animal experimentation and the principles set forth in the current version of the National Institutes of Health (NIH) Guidelines for Research Involving Recombinant DNA Molecules. Disclaimers: The views, opinions and/or findings contained in this report are those of the authors and should not be construed as an official position, policy or decision of the US Army or the United States government, unless so designated by other documentation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., (© 2025. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Due to the differences between the storage mode of relational database and graph database,during the process of transforming data in relational database to graph database,it is necessary to solve the main problems of edge definition,vertex uniqueness and retention of original database constraint information.To solve the above problems,a method of transforming relational database to graph database is proposed.Firstly,by customizing the existing primary key,combined with the uniqueness of the table name,the problem of ensuring the uniqueness of the vertex is solved;through different configuration schemes,the constraint information of the original relational database can be maximized.Then,the edge definition method based on configuration and intermediate table (EDCIT) method is proposed,it provides different edge mapping solutions for multiple types of databases and solves the definition of edges during the transformation.Finally,through experiments on multiple data sets,and using Gremlin statement to test the transformed data,it verifies the integrity and reliability of the transformed data.

Background & objectives: The present study proposed a series of computational techniques such as homology modelling, molecular simulation, and molecular docking to be performed to explore the structural features and binding mechanism of Cytochrome c oxidase subunit I (COX1) protein with known inhibitors. Methods: Elucidation of the three-dimensional structure of COX1 protein was carried out by using MODELLER software. The modelled protein was validated using GROMACS, structural qualitative tools and web servers. Finally the model was docked with carbon monoxide (CO) and nitric oxide (NO) using Auto Dock Tools. Results: The three-dimensional structure of mitochondrial transmembrane protein COX1 was built using homology modelling based on high-resolution crystal structures of Bos taurus. Followed by inserting the lipid bilayer, molecular dynamics simulation was performed on the modelled protein structure. The modelled protein was validated using qualitative structural indices. Known inhibitors such as carbon monoxide (CO) and nitric oxide (NO) inhibit their active binding sites of mitochondrial COX1 and the inhibitors were docked into the active site of attained model. A structure-based virtual screening was performed on the basis of the active site inhibition with best scoring hits. The COX1 model was submitted and can be accessible from the Model Archive site through the following link https://www.modelarchive.org/doi/10.5452/ma-at44v. Interpretation & conclusion: Structural characterization and active site identification can be further used as target for the planning of potent mosquitocidal compounds, thereby assisting the information in the field of research.

The DAN gene family (DAN, Differential screening-selected gene Aberrant in Neuroblastoma) is a group of genes that is expressed during development and plays fundamental roles in limb bud formation and digitation, kidney formation and morphogenesis and left-right axis specification. During adulthood the expression of these genes are associated with diseases, including cancer. Although most of the attention to this group of genes has been dedicated to understanding its role in physiology and development, its evolutionary history remains poorly understood. Thus, the goal of this study is to investigate the evolutionary history of the DAN gene family in vertebrates, with the objective of complementing the already abundant physiological information with an evolutionary context. Our results recovered the monophyly of all DAN gene family members and divide them into five main groups. In addition to the well-known DAN genes, our phylogenetic results revealed the presence of two new DAN gene lineages; one is only retained in cephalochordates, whereas the other one (GREM3) was only identified in cartilaginous fish, holostean fish, and coelacanth. According to the phyletic distribution of the genes, the ancestor of gnathostomes possessed a repertoire of eight DAN genes, and during the radiation of the group GREM1, GREM2, SOST, SOSTDC1, and NBL1 were retained in all major groups, whereas, GREM3, CER1, and DAND5 were differentially lost. [Display omitted] • We recovered the monophyly of all DAN paralogs and divided them into five groups. • We described two new DAN gene lineages. • The ancestor of gnathostomes possessed a repertoire of eight DAN genes. [ABSTRACT FROM AUTHOR]

Kako smo unatrag nekoliko godina dobili određena sredstva (tzv. sveučilišne potpore) kojima smo istraživali određene aspekte grafovskih baza podataka, odlučili smo rezultate istraživanja predstaviti i hrvatskoj akademskoj i stručnoj zajednici. Smatramo kako su istraživanja bila kvalitetna i da rezultati istih imaju potencijal za širu primjenu i implementaciju u dostupnim sustavima za upravljanje grafovskim bazama podataka. Također, postoje i određene mogućnosti za proširenje implementiranih rješenja, kao i prostor za dodatna istraživanja i preslikavanja relacijskih u koncepte grafovskih baza podataka.

Background: Mechanisms regulating BMP and Wnt pathways and their interactions are not well studied in Hydra. Results: We report identification of BMP inhibitor gremlin, comparison of its expression with that of noggin and possible antagonism between Wnt and BMP signaling in Hydra. Gremlin is expressed in body column with high levels in budding region and in early buds. Noggin, on the other hand, is expressed in the hypostome, base of tentacles, lower body column, and basal disc. During budding, noggin is expressed at the sites of tentacle emergence. This was confirmed in ectopic tentacles in polyps treated with alsterpaullone (ALP), a GSK‐3β inhibitor that leads to upregulation of Wnt pathway. RT‐PCR data show that upregulation of Wnt is accompanied by downregulation of bmp 5‐8b though noggin and gremlin remain unaltered till 24 hours. Conclusions: Different expression patterns of gremlin and noggin suggest their roles in budding and patterning of tentacles, respectively. Further, bmp 5‐8b inhibition by activated Wnt signaling does not directly involve noggin and gremlin in Hydra. Our data suggest that Wnt/BMP antagonism may have evolved early for defining the oral‐aboral axis, while the involvement of BMP antagonists during axial patterning is a recent evolutionary acquisition within the Bilateria lineage. Key Findings: Gremlin is predominantly expressed during budding.Noggin expression is specifically associated with tentacle formation.Wnt activation leads to bmp5‐8b downregulation in hydra.Noggin and gremlin are not directly involved in bmp5‐8b inhibition by activated Wnt signalling. [ABSTRACT FROM AUTHOR]

Proliferative vitreoretinopathy (PVR) is characterised by the contraction and growth of fibrotic membranes on the retina and within the vitreous body. Retinal pigment epithelial (RPE) cells, a major cellular component of the fibrotic membrane, is one of the cell types that have been previously reported to associate with PVR pathogenesis. During PVR, RPE cells undergo increased cell proliferation, migration and the secretion of extracellular matrix molecules, such as fibronectin and type I collagen. A variety of cytokines and growth factors are involved in the formation of the fibrotic membrane. Although gremlin has been reported to serve an important role in the regulation of epithelial-to-mesenchymal transition in PVR, the relationship between gremlin and the expression of profibrogenic factors in human RPE cells remains unclear. In the present study, gremlin promoted RPE cell proliferation and the expression of type I collagen and fibronectin. In addition, knocking down gremlin expression by siRNA significantly suppressed the transforming growth factor (TGF)-β1- and TGF-β2-induced expression of type I collagen and fibronectin in RPE cells. These findings suggest that gremlin may serve an important role in the development of PVR. [ABSTRACT FROM AUTHOR]

Crescentic glomerulonephritis is a devastating autoimmune disease that without early and properly treatment may rapidly progress to end-stage renal disease and death. Current immunosuppressive treatment provides limited efficacy and an important burden of adverse events. Epigenetic drugs are a source of novel therapeutic tools. Among them, bromodomain and extraterminal domain (BET) inhibitors (iBETs) block the interaction between bromodomains and acetylated proteins, including histones and transcription factors. iBETs have demonstrated protective effects on malignancy, inflammatory disorders and experimental kidney disease. Recently, Gremlin-1 was proposed as a urinary biomarker of disease progression in human anti-neutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis. We have now evaluated whether iBETs could regulate Gremlin-1 in experimental anti-glomerular basement membrane nephritis induced by nephrotoxic serum (NTS) in mice, a model resembling human crescentic glomerulonephritis. In NTS-injected mice, the iBET JQ1 inhibited renal Gremlin-1 overexpression and diminished glomerular damage, restoring podocyte numbers. Chromatin immunoprecipitation assay demonstrated BRD4 enrichment of the Grem-1 gene promoter in injured kidneys, consistent with Gremlin-1 epigenetic regulation. Moreover, JQ1 blocked BRD4 binding and inhibited Grem-1 gene transcription. The beneficial effect of iBETs was also mediated by modulation of NOTCH pathway. JQ1 inhibited the gene expression of the NOTCH effectors Hes-1 and Hey-1 in NTS-injured kidneys. Our results further support the role for epigenetic drugs, such as iBETs, in the treatment of rapidly progressive crescentic glomerulonephritis.

Objective: The present study was conducted to determine the role of gremlin during the development of posterior capsular opacification (PCO) via in vitro and in vivo experiments. Methods: The activation, roles and relationships of the BMPs/Smad1/5, MAPK, FAK and AKT signaling pathways in human lens epithelial cells (HLECs) after gremlin induction were detected by western blotting and real-time PCR. Wound-healing, transwell, capsular bag models and rat PCO models assays were used to test the effects of gremlin on HLECs' migration, proliferation, EMT-specific protein α-smooth muscle actin（α-SMA）and development of PCO in rats. Results: Our data showed that knockdown of the gremlin inhibited the development of PCO and reduced expression of α-SMA in rats. While gremlin did not alter the migration of HLECs, it increased the expression of p-ERK and p-AKT. Knockout of Smad2 or Smad3 inhibited the expression of p-ERK and p-AKT proteins induced by gremlin. Gremlin also reduced BMP4-induced expression of the p-Smad1/5 protein. Finally, knockout of Smad1/5 increased gremlin-induced expression of α-SMA, fibronectin and type I collagen (COL-1) in HLECs. Conclusion: These results suggested that gremlin contributed to the development of PCO by promoting LEC proliferation, activation of TGF-β/Smad, ERK and AKT signaling and inhibition of BMPs/Smad1/5 signaling. Furthermore, inhibiting gremlin effectively impaired both PCO development in rats and EMT in the lens capsule. Thus, our data suggest that gremlin might be a potential target for PCO. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) is emerging as an important health problem due to the increase number of CKD patients and the absence of an effective curative treatment. Gremlin has been proposed as a novel therapeutic target for renal inflammatory diseases, acting via Vascular Endothelial Growth Factor Receptor-2 (VEGFR2). Although many evidences suggest that Gremlin could regulate renal fibrosis, the receptor involved has not been yet clarified. Gremlin, as other TGF-β superfamily members, regulates tubular epithelial to mesenchymal transition (EMT) and, therefore, could contribute to renal fibrosis. In cultured tubular epithelial cells Gremlin binding to VEGFR2 is linked to proinflammatory responses. Now, we have found out that in these cells VEGFR2 is also involved in the profibrotic actions of Gremlin. VEGFR2 blockade by a pharmacological kinase inhibitor or gene silencing diminished Gremlin-mediated gene upregulation of profibrotic factors and restored changes in EMT-related genes. Moreover, VEGFR2 inhibition blocked EMT phenotypic changes and dampened the rate of wound healing in response to Gremlin. The role of VEGFR2 in experimental fibrosis was evaluated in experimental unilateral ureteral obstruction. VEFGR2 inhibition diminished the upregulation of profibrotic genes and EMT changes, as well as the accumulation of extracellular matrix proteins, such as fibronectin and collagens in the obstructed kidneys. Notch pathway activation participates in renal damage progression by regulating cell growth/proliferation, regeneration and inflammation. In cultured tubular epithelial cells, Notch inhibition markedly downregulated Gremlin-induced EMT changes and wound healing speed. These results show that Gremlin regulates the EMT process via VEGFR2 and Notch pathway activation, suggesting that the Gremlin/VEGFR2 axis could be a potential therapeutic target for CKD.

Idiopathic pulmonary fibrosis (IPF), a chronic progressive interstitial pneumonia, is characterized by excessive fibroproliferation. Key effector cells in IPF are myofibroblasts that are recruited from three potential sources: resident fibroblasts, fibrocytes, and epithelial cells. We hypothesized that IPF myofibroblasts from different sources display unique gene expression differences and distinct functional characteristics. Primary human pulmonary fibroblasts (normal and IPF), fibrocytes, and epithelial cells were activated using the profibrotic factors TGF-β and TNF-α. The resulting myofibroblasts were characterized using cell proliferation, soluble collagen, and contractility assays, ELISA, and human fibrosis PCR arrays. Genes of significance in human whole lung were validated by immunohistochemistry on human lung sections. Fibroblast-derived myofibroblasts exhibited the greatest increase in expression of profibrotic genes and genes involved in extracellular matrix remodeling and signal transduction. Functional studies demonstrated that myofibroblasts derived from fibrocytes expressed mostly soluble collagen and chemokine (C-C) motif ligand (CCL) 18 but were the least proliferative of the myofibroblast progeny. Activated IPF fibroblasts displayed the highest levels of contractility and CCL2 production. This study identified novel differences in gene expression and functional characteristics of different myofibroblast populations. Further investigation into the myofibroblast phenotype may lead to potential therapeutic targets in future IPF research. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) is emerging as an important health problem due to the increase number of CKD patients and the absence of an effective curative treatment. Gremlin has been proposed as a novel therapeutic target for renal inflammatory diseases, acting via Vascular Endothelial Growth Factor Receptor-2 (VEGFR2). Although many evidences suggest that Gremlin could regulate renal fibrosis, the receptor involved has not been yet clarified. Gremlin, as other TGF-β superfamily members, regulates tubular epithelial to mesenchymal transition (EMT) and, therefore, could contribute to renal fibrosis. In cultured tubular epithelial cells Gremlin binding to VEGFR2 is linked to proinflammatory responses. Now, we have found out that in these cells VEGFR2 is also involved in the profibrotic actions of Gremlin. VEGFR2 blockade by a pharmacological kinase inhibitor or gene silencing diminished Gremlin-mediated gene upregulation of profibrotic factors and restored changes in EMT-related genes. Moreover, VEGFR2 inhibition blocked EMT phenotypic changes and dampened the rate of wound healing in response to Gremlin. The role of VEGFR2 in experimental fibrosis was evaluated in experimental unilateral ureteral obstruction. VEFGR2 inhibition diminished the upregulation of profibrotic genes and EMT changes, as well as the accumulation of extracellular matrix proteins, such as fibronectin and collagens in the obstructed kidneys. Notch pathway activation participates in renal damage progression by regulating cell growth/proliferation, regeneration and inflammation. In cultured tubular epithelial cells, Notch inhibition markedly downregulated Gremlin-induced EMT changes and wound healing speed. These results show that Gremlin regulates the EMT process via VEGFR2 and Notch pathway activation, suggesting that the Gremlin/VEGFR2 axis could be a potential therapeutic target for CKD. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the expression of bone morphogenetic protein-7 (BMP-7), gremlin, and p-Smad/1/5/8 in carcinomatous and para-carcinoma tissue specimens from patients with hepatocellular carcinoma (HCC). The association of serum BMP-7 levels with clinicopathological parameters was examined to assess its relevance as a clinical biomarker for HCC. A total of 27 patients with HCC and 7 healthy subjects were included. Gene expression levels of BMP-7 and p-Smad1/5/8 were examined by reverse transcription-quantitative polymerase chain reaction. Immunohistochemical and western blot analysis were performed to determine the protein expression of target genes. The serum levels of BMP-7 were assessed by enzyme linked immunosorbent assay. The mRNA and protein expression of BMP-7 and gremlin were significantly down- and upregulated in HCC tumor tissues, respectively, compared with para-carcinoma tissues (P<0.05). The association of BMP-7 and gremlin expression with the differentiation status of HCC was also analyzed. There was a relatively higher level of BMP-7 and a lower level of gremlin expression in tumor tissues from patients with highly differentiated HCC when compared with poorly or moderately differentiated HCC (BMP-7, F=42.29, P<0.01; gremlin, F=37.93, P<0.01). Furthermore, the level of BMP-7 and p-Smad1/5/8 was decreased in patients with advanced stages of HCC, when compared with stage I HCC. The findings from the present study suggest that the BMP-7/p-Smad signaling pathway may be involved in the pathogenesis of HCC. The serum levels of BMP-7 may serve as a potential biomarker for HCC. [ABSTRACT FROM AUTHOR]

Objective To investigate the effects of Gremlin on the proliferation, apoptosis, and extracellular matrix secretion of primary hepatic stellate cells (HSCs) in mice. Methods Primary HSCs were isolated from mouse liver by two-step enzyme perfusion method. HSCs were randomly divided into the control group, DMSO group, and low-dose and high-dose Gremlin groups. The cells in the control group and the DMSO group were added with the DMEM complete medium and the DMEM medium containing 0. 05% DMSO, respectively. The cells in the low-dose and high-dose Gremlin groups were added with 100 and 200 μg/L Gremlin DMEM medium，respectively. The cell proliferation rate and apoptosis rate of each group were detected by CCK-8 and flow cytometry. The expression levels of Collagen I，Collagen III，a-SMA mRNA and protein were detected by RT-qPCR and Western blotting. Results The proliferation rate of low-dose and high- dose group Gremlin groups was higher than that of control group and DMSO group， and the apoptotic rate was lower than that of control group and DMSO group ( both P < 0. 05 ). There was no significant difference in the cell proliferation rate and apoptosis rate between low-dose group and high-dose Gremlin group and between the control group and DMSO group （all P > 0. 05 ) . The relative mRNA expression of Collagen I， Collagen III， and a-SMA in the low-dose and high-dose Gremlin groups were higher than those in the control group and DMSO group ( all P <0.05 )，and it was higher in the high- dose Gremlin group than in the low-dose Gremlin group ( P < 0. 05 )， but there was no significant difference between the control group and the DMSO group (P >0. 05). The relative protein expression levels of Collagen I，Collagen III，and a- SMA in the high-dose Gremlin group were higher than those in the low-dose Gremlin group， control group and DMSO group (all P <0.05). There was no significant difference between the low-dose Gremlin group, the control group, and the DMSO group (P > 0. 05 ) . Conclusion Gremlin can induce the activation of primary HSCs and increase the secretion of extracellular matrix, thereby participating in the occurrence and development of liver fibrosis. [ABSTRACT FROM AUTHOR]

The current therapeutic strategy for the treatment of chronic kidney diseases only ameliorates disease progression. During renal injury, developmental genes are re-expressed and could be potential therapeutic targets. Among those genes reactivated in the adult damaged kidney, Gremlin is of particular relevance since recent data suggest that it could be a mediator of diabetic nephropathy and other progressive renal diseases. Earlier studies have shown that Gremlin is upregulated in trans-differentiated renal proximal tubular cells and in several chronic kidney diseases associated with fibrosis. However, not much was known about the mechanisms by which Gremlin acts in renal pathophysiology. The role of Gremlin as a bone morphogenetic protein antagonist has clearly been demonstrated in organogenesis and in fibrotic-related disorders. Gremlin binds to vascular endothelial growth factor receptor 2 (VEGFR2) in endothelial and tubular epithelial cells. Activation of the Gremlin-VEGFR2 axis was found in several human nephropathies. We have recently described that Gremlin activates the VEGFR2 signaling pathway in the kidney, eliciting a downstream mechanism linked to renal inflammatory response. Gremlin deletion improves experimental renal damage, diminishing fibrosis. Overall, the available data identify the Gremlin-VEGFR2 axis as a novel therapeutic target for kidney inflammation and fibrosis and provide a rationale for unveiling new concepts to investigate in several clinical conditions. [ABSTRACT FROM AUTHOR]

Gremlin1 (Grem1), an antagonist of bone morphogenetic proteins, plays a key role in embryogenesis. A highly specific temporospatial gradient of Grem1 and bone morphogenetic protein signaling is critical to normal lung, kidney, and limb development. Grem1 levels are increased in renal fibrotic conditions, including acute kidney injury, diabetic nephropathy, chronic allograft nephropathy, and immune glomerulonephritis. We demonstrate that a small number of grem1-/- whole body knockout mice on a mixed genetic background (8%) are viable, with a single, enlarged left kidney and grossly normal histology. The grem1-/- mice displayed mild renal dysfunction at 4 wk, which recovered by 16 wk. Tubular epithelial cell-specific targeted deletion of Grem1 (TEC-grem1-cKO) mice displayed a milder response in the acute injury and recovery phases of the folic acid model. Increases in indexes of kidney damage were smaller in TEC-grem1-cKO than wild-type mice. In the recovery phase of the folic acid model, associated with renal fibrosis, TEC-grem1-cKO mice displayed reduced histological damage and an attenuated fibrotic gene response compared with wild-type controls. Together, these data demonstrate that Grem1 expression in the tubular epithelial compartment plays a significant role in the fibrotic response to renal injury in vivo. [ABSTRACT FROM AUTHOR]

Of the circa 40 cytokines of the TGF-β superfamily, around a third are currently known to bind to heparin and heparan sulphate. This includes TGF-β1, TGF-β2, certain bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs), as well as GDNF and two of its close homologues. Experimental studies of their heparin/HS binding sites reveal a diversity of locations around the shared cystine-knot protein fold. The activities of the TGF-β cytokines in controlling proliferation, differentiation and survival in a range of cell types are in part regulated by a number of specific, secreted BMP antagonist proteins. These vary in structure but seven belong to the CAN or DAN family, which shares the TGF-β type cystine-knot domain. Other antagonists are more distant members of the TGF-β superfamily. It is emerging that the majority, but not all, of the antagonists are also heparin binding proteins. Any future exploitation of the TGF-β cytokines in the therapy of chronic diseases will need to fully consider their interactions with glycosaminoglycans and the implications of this in terms of their bioavailability and biological activity. [ABSTRACT FROM AUTHOR]

Gremlin-1, a bone morphogenetic protein (BMP) antagonist, has essential roles in kidney and limb bone development, and is important in chronic diseases including tissue fibrosis. It also functions as an activating ligand of the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2), and binds strongly to the sulfated polysaccharide, heparin. Here we investigated the extent to which gremlin binds to the related polysaccharide heparan sulfate (HS), which unlike heparin is widely distributed spread within tissues. We determined that both highly sulfated HS and kidney HS are able to partially compete for the binding of heparin to gremlin, whereas low sulfated HS is a poor competitor. In further investigations of the interaction between gremlin and HS, we found that wild-type gremlin is able to bind broadly across the various regions of kidney in an HS-dependent manner, with particularly intense binding to tubular structures in the renal cortex. In a model of chronic kidney disease, fibrotic changes in the kidney result in a loss of gremlin binding sites. Gremlin mutants with reduced affinity for heparin showed negligible binding under the same conditions. These mutants nonetheless remain functional as BMP antagonists on C2C12 myoblastic cells transfected with a Smad 1 reporter gene construct. Overall our findings indicate that on secretion, gremlin will bind to HS structures on the cell surface and in the extracellular matrix, thus providing for a localised reservoir which can modulate BMP activity in a temporospatially restricted manner. Although binding of heparin/HS to gremlin has been shown elsewhere to be necessary for gremlin activation of VEGFR2, this does not appear to be essential for BMP antagonism by gremlin. Thus these sulfated polysaccharides differentially regulate the activities of gremlin. [ABSTRACT FROM AUTHOR]

En enfermedad renal crónica (ERC) el riesgo aumentado de muerte por causa cardiovascular se relaciona con calcificaciones vasculares precoces y progresivas. Estas calcificaciones ocurren por un proceso altamente regulado. En los vasos de los pacientes con ERC coexisten calcificaciones en la íntima vascular asociadas a ateroesclerosis, pero además hay calcificaciones en la túnica media del vaso. Estos dos tipos de calcificaciones tienen distintas consecuencias fisiopatológicas cardiovasculares. Los principales componentes que interactúan para producir calcificación de la matriz extracelular son la célula muscular lisa vascular (CMLV), factores que inician el proceso de transformación osteocondrogénica, inductores e inhibidores de la calcificación, factores que regulan a estos últimos. Como respuesta al proceso de calcificación, el vaso expresa inhibidores que disminuyen la diferenciación osteoblástica, amplificando la lesión ósea adinámica. Así, el conocimiento del proceso patológico bidireccional del eje Hueso-Vaso ayuda a explicar la estrecha correlación entre pérdida de masa ósea y calcificación vascular.

Bone morphogenetic protein 2 (BMP-2) is a member of the transforming growth factor-β (TGF-β) signalling family and has a very broad biological role in development. Its signalling is regulated by many effectors: transmembrane proteins, membraneattached proteins and soluble secreted antagonists such as Gremlin-1. Very little is known about themolecularmechanism by which Gremlin-1 and other DAN (differential screening-selected gene aberrative in neuroblastoma) family proteins inhibit BMP signalling. We analysed the interaction of Gremlin-1 with BMP-2 using a range of biophysical techniques, and used mutagenesis to map the binding site on BMP-2. We have also determined the crystal structure of Gremlin-1, revealing a similar conserved dimeric structure to that seen in other DAN family inhibitors. Measurements using biolayer interferometry (BLI) indicate that Gremlin-1 and BMP-2 can form larger complexes, beyond the expected 1:1 stoichiometry of dimers, forming oligomers that assemble in alternating fashion. These results suggest that inhibition of BMP-2 by Gremlin-1 occurs by a mechanism that is distinct from other known inhibitors such as Noggin and Chordin and we propose a novel model of BMP-2-Gremlin-1 interaction yet not seen among any BMP antagonists, and cannot rule out that several different oligomeric states could be found, depending on the concentration of the two proteins. [ABSTRACT FROM AUTHOR]

Lung cancer is the major cause of cancer-related death worldwide, and 80 % of them are non-small cell lung cancer (NSCLC) cases. Gremlin, a bone morphogenetic protein (BMP) antagonist, is overexpressed in various cancerous tissues; however, little is known about the roles of Gremlin in lung carcinogenesis, and it remains unclear whether Gremlin expression may associate with EGFR-TKI resistance. In this study, expression of Gremlin mRNA and protein in matched tumor and normal lung specimens are quantified by quantitative real-time PCR and western blot. The functional role of Gremlin in NSCLC cells was evaluated by interference RNA (siRNA). The effects of Silenced Gremlin on the resistant PC-9/GR cell line were investigated by proliferation and apoptosis analysis compared with control PC-9 cells. Our results found that Gremlin expression levels were higher in NSCLC tissues, and Gremlin was more highly expressed in PC-9/GR cells compared to PC-9 cells. Knocking down of Gremlin in PC-9/GR cells decreased cell proliferation and increased the expression of BMP7 protein. In addition, Gremlin silencing significantly potentiated apoptosis induced by gefitinib in PC-9/GR with Gremlin knockdown compared to PC-9 transfected with control shRNA, suggesting Gremlin contributes to gefitinib resistance in NSCLC. Gremlin might be explored as a candidate of therapeutic target for modulating EGFR-TKI sensitivity in NSCLC. [ABSTRACT FROM AUTHOR]

Of the circa 40 cytokines of the TGF-β superfamily, around a third are currently known to bind to heparin and heparan sulphate. This includes TGF-β1, TGF-β2, certain bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs), as well as GDNF and two of its close homologues. Experimental studies of their heparin/HS binding sites reveal a diversity of locations around the shared cystine-knot protein fold. The activities of the TGF-β cytokines in controlling proliferation, differentiation and survival in a range of cell types are in part regulated by a number of specific, secreted BMP antagonist proteins. These vary in structure but seven belong to the CAN or DAN family, which shares the TGF-β type cystine-knot domain. Other antagonists are more distant members of the TGF-β superfamily. It is emerging that the majority, but not all, of the antagonists are also heparin binding proteins. Any future exploitation of the TGF-β cytokines in the therapy of chronic diseases will need to fully consider their interactions with glycosaminoglycans and the implications of this in terms of their bioavailability and biological activity.

The current study aims to identify the pro-fibrogenic role of Gremlin, an endogenous antagonist of bone morphogenetic proteins (BMPs) in chronic pancreatitis (CP). CP is a highly debilitating disease characterized by progressive pancreatic inflammation and fibrosis that ultimately leads to exocrine and endocrine dysfunction. While transforming growth factor (TGF)-β is a known key pro-fibrogenic factor in CP, the TGF-β superfamily member BMPs exert an anti-fibrogenic function in CP as reported by our group recently. To investigate how BMP signaling is regulated in CP by BMP antagonists, the mouse CP model induced by cerulein was used. During CP induction, TGF-β 1 messenger RNA (mRNA) increased 156-fold in 2 weeks, a BMP antagonist Gremlin 1 ( Grem1) mRNA levels increased 145-fold at 3 weeks, and increases in Grem1 protein levels correlated with increases in collagen deposition. Increased Grem1 was also observed in human CP pancreata compared to normal. Grem1 knockout in Grem1 mice revealed a 33.2 % reduction in pancreatic fibrosis in CP compared to wild-type littermates. In vitro in isolated pancreatic stellate cells, TGF-β induced Grem1 expression. Addition of the recombinant mouse Grem1 protein blocked BMP2-induced Smad1/5 phosphorylation and abolished BMP2's suppression effects on TGF-β-induced collagen expression. Evidences presented herein demonstrate that Grem1, induced by TGF-β, is pro-fibrogenic by antagonizing BMP activity in CP. Key messages: [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN) is currently a leading cause of end-stage renal failure worldwide. Gremlin was identified as a gene differentially expressed in mesangial cells exposed to high glucose and in experimental diabetic kidneys. We have described that Gremlin is highly expressed in biopsies from patients with diabetic nephropathy, predominantly in areas of tubulointerstitial fibrosis. In streptozotocin (STZ)-induced experimental diabetes, Gremlin deletion using Grem1 heterozygous knockout mice or by gene silencing, ameliorates renal damage. To study the in vivo role of Gremlin in renal damage, we developed a diabetic model induced by STZ in transgenic (TG) mice expressing human Gremlin in proximal tubular epithelial cells. The albuminuria/ creatinuria ratio, determined at week 20 after treatment, was significantly increased in diabetic mice but with no significant differences between transgenic (TG/STZ) and wild-type mice (WT/STZ). To assess the level of renal damage, kidney tissue was analyzed by light microscopy (periodic acid-Schiff and Masson staining), electron microscopy, and quantitative PCR. TG/STZ mice had significantly greater thickening of the glomerular basement membrane, increased mesangial matrix, and podocytopenia vs. WT/STZ. At the tubuloin-terstitial level, TG/STZ showed increased cell infiltration and mild interstitial fibrosis. In addition, we observed a decreased expression of podocin and overexpression of monocyte chemoattractant protein-1 and fibrotic-related markers, including transforming growth factor-1, Col1a1, and-smooth muscle actin. Together, these results show that TG mice overexpressing Gremlin in renal tubules develop greater glomerular and tubulointerstitial injury in response to diabetic-mediated damage and support the involvement of Gremlin in diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Gremlin is a member of the CAN (cerberus and DAN) family of secreted BMP (bone morphogenetic protein) antagonists and also an agonist of VEGF (vascular endothelial growth factor) receptor-2. It is critical in limb skeleton and kidney development and is re-expressed during tissue fibrosis. Gremlin binds strongly to heparin and heparan sulfate and, in the present study, we sought to investigate its heparin-binding site. In order to explore a putative non-contiguous binding site predicted by computational molecular modelling, we substituted a total of 11 key arginines and lysines located in three basic residue sequence clusters with homologous sequences from cerberus and DAN (differential screening selected gene abberative in neuroblastoma), CAN proteins which lack basic residues in these positions. A panel of six Myc-tagged gremlin mutants, MGR- 1-MGR-6 (MGR, mutant gremlin), each containing different combinations of targeted substitutions, all showed markedly reduced affinity for heparin as demonstrated by their NaCl elution on heparin affinity chromatography, thus verifying our predictions. Both MGR-5 and MGR-6 retained BMP-4-binding activity comparable to that of wild-type gremlin. Low-molecularmass heparin neither promoted nor inhibited BMP-4 binding. Finally, glutaraldehyde cross-linking demonstrated that gremlin forms non-covalent dimers, similar behaviour to that of DAN and also PRDC (protein related to cerberus and DAN), another CAN protein. The resulting dimer would possess two heparin-binding sites, each running along an exposed surface on the second β-strand finger loop of one of the monomers. [ABSTRACT FROM AUTHOR]

Published recently in the Journal of Pathology, Lavoz et al. show that Gremlin promotes renal inflammation directly via VEGFR2. As Gremlin has been implicated in many other diseases, such as heart, lung and liver fibrosis, osteogenesis, angiogenesis and cancer, the new findings provide a rationale for novel concepts to investigate and potentially treat several pathologies. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Inflammation is a main feature of progressive kidney disease. Gremlin binds to bone morphogenetic proteins ( BMPs), acting as an antagonist and regulating nephrogenesis and fibrosis among other processes. Gremlin also binds to vascular endothelial growth factor receptor-2 ( VEGFR2) in endothelial cells to induce angiogenesis. In renal cells, gremlin regulates proliferation and fibrosis, but there are no data about inflammatory-related events. We have investigated the direct effects of gremlin in the kidney, evaluating whether VEGFR2 is a functional gremlin receptor. Administration of recombinant gremlin to murine kidneys induced rapid and sustained activation of VEGFR2 signalling, located in proximal tubular epithelial cells. Gremlin bound to VEGFR2 in these cells in vitro, activating this signalling pathway independently of its action as an antagonist of BMPs. In vivo, gremlin caused early renal damage, characterized by activation of the nuclear factor ( NF)- κB pathway linked to up-regulation of pro-inflammatory factors and infiltration of immune inflammatory cells. VEGFR2 blockade diminished gremlin-induced renal inflammatory responses. The link between gremlin/ VEGFR2 and NF-κB/inflammation was confirmed in vitro. Gremlin overexpression was associated with VEGFR2 activation in human renal disease and in the unilateral ureteral obstruction experimental model, where VEGFR2 kinase inhibition diminished renal inflammation. Our data show that a gremlin/ VEGFR2 axis participates in renal inflammation and could be a novel target for kidney disease. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Bone morphogenetic proteins (BMPs) are secreted extracellular matrix (ECM)-associated proteins that regulate a wide range of developmental processes, including limb and kidney formation. A critical element of BMP regulation is the presence of secreted antagonists that bind and inhibit BMP binding to their cognate Ser/Thr kinase receptors at the plasma membrane. Antagonists such as Noggin, Chordin, Gremlin (Grem1), and twisted gastrulation-1 (Twsg1) have been shown to inhibit BMP action in a range of different cell types and developmental stage-specific contexts. Here we review new developments in the field of BMP and BMP antagonist biology during mammalian development and suggest strategies for targeting these proteins in human disease. [ABSTRACT FROM AUTHOR]

Background Endothelin receptor antagonists improve pulmonary arterial hypertension (PAH). Mutations in the bone morphogenetic protein (BMP) type 2 receptor (BMPR2) predispose to PAH. Here, we sought to determine whether there might exist interactions between these 2 signaling pathways and their effect on the acquisition of the altered phenotype of pulmonary artery smooth muscle cells (PA-SMCs) observed in PAH. Methods Expression of BMPR2, of the BMP agonist BMP4, and of the BMP antagonists gremlin1 and gremlin2 was evaluated in lungs and in PA-SMCs from 6 PAH patients and 14 controls treated with endothelin-1. Endothelin-1 pre-treated PA-SMCs were assessed for proliferation, apoptosis, and downstream signaling activation of Smad1/5/8 and p38 mitogen-activated protein kinase (p38 MAPK ) after BMP2 treatment. Results In PA-SMCs from PAH patients, expression of BMPR2 and BMP4 decreased, whereas expression of gremlin1 and gremlin2 increased compared with controls. Treatment of control PA-SMCs with endothelin-1 induced a dose-dependent increase in gremlin1 and gremlin2, whereas BMPR2 and BMP4 expression decreased, reaching similar levels as those observed in PAH cells. In control PA-SMCs, endothelin-1 pre-treatment reduced inhibitor of DNA binding 1 (Id1) expression and Smad1/5/8 activation induced by BMP2, whereas it enhanced p38 MAPK activation. Moreover, BMP2 decreased serum-induced proliferation and increased the pro-apoptotic Bax/Bcl-2 ratio. These effects were attenuated by endothelin-1 pre-treatment. Endothelin-1 did not alter BMPR2 signaling in PA-SMCs from PAH patients. Conclusions Endothelin-1 downregulates canonical BMPR2 signaling. This is related to decreased BMPR2 and increased anti-BMP gremlin expression associated with increased activation of p38 MAPK and results in PA-SMC proliferation. [ABSTRACT FROM AUTHOR]

Gremlin (Grem1) is a member of the DAN family of secreted bone morphogenetic protein (BMP) antagonists. Bone morphogenetic protein-7 (BMP-7) mediates protective effects during renal fibrosis associated with diabetes and other renal diseases. The pathogenic mechanism of Grem1 during diabetic nephropathy (DN) has been suggested to be binding and inhibition of BMP-7. However, the precise interactions between Grem1, BMP-7 and other BMPs have not been accurately defined. In the present study, we show the affinity of Grem1 for BMP-7 is lower than that of BMP-2 and BMP-4, using a combination of surface plasmon resonance and cell culture techniques. Using kidney proximal tubule cells and HEK (human embryonic kidney)-293 cell Smad1/5/8 phosphorylation and BMP-dependent gene expression as readouts, Grem1 consistently demonstrated a higher affinity for BMP-2>BMP-4>BMP-7. Cell-associated Grem1 did not inhibit BMP-2- or BMP-4-mediated signalling, suggesting that Grem1-BMP-2 binding occurred in solution, preventing BMP receptor activation. These data suggest that Grem1 preferentially binds to BMP-2 and this may be the dominant complex in a disease situation where levels of Grem1 and BMPs are elevated. [ABSTRACT FROM AUTHOR]

GREMLIN 2 (GREM2)—formerly, protein related to Dan and cerberus (PRDC)—is a potent antagonist of the bone morphogenetic proteins 2 and 4, but little else in known about its functions. We found that Grem2-/- mice developed small deformed mandibular and maxillary incisors, indicating that GREMLIN2 is required for normal tooth morphogenesis. Although DEXA scans suggested that bone mineral density might be increased in Grem2-/- mice, histology did not reveal any evident bone phenotype. Grem2-/- mice did not display any other notable phenotypes evaluated in a high-throughput screening process that encompassed a range of immunologic, metabolic, ophthalmic, and behavioral parameters. Our findings indicate that Grem2 can be added to the growing list of genes that affect tooth development in mice. [ABSTRACT FROM PUBLISHER]

In this work, the role of shock wave-induced increase of bone morphogenetic proteins in modulating the osteogenic properties of osteoblast-like cells seeded on a bioactive scaffold was investigated using gremlin as a bone morphogenetic protein antagonist. Bone-like glass-ceramic scaffolds, based on a silicate experimental bioactive glass developed at the Politecnico di Torino, were produced by the sponge replication method and used as porous substrates for cell culture. Human MG-63 cells, exposed to shock waves and seeded on the scaffolds, were treated with gremlin every two days and analysed after 20 days for the expression of osteoblast differentiation markers. Shock waves have been shown to induce osteogenic activity mediated by increased expression of alkaline phosphatase, osteocalcin, type I collagen, BMP-4 and BMP-7. Cells exposed to shock waves plus gremlin showed increased growth in comparison with cells treated with shock waves alone and, conversely, mRNA contents of alkaline phosphatase and osteocalcin were significantly lower. Therefore, the shock wave-mediated increased expression of bone morphogenetic protein in MG-63 cells seeded on the scaffolds is essential in improving osteogenic activity; blocking bone morphogenetic protein via gremlin completely prevents the increase of alkaline phosphatase and osteocalcin. The results confirmed that the combination of glass-ceramic scaffolds and shock waves exposure could be used to significantly improve osteogenesis opening new perspectives for bone regenerative medicine. [ABSTRACT FROM AUTHOR]

Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin , which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9 -expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis. [ABSTRACT FROM AUTHOR]

Bone morphogenetic proteins ( BMPs) are members of the TGFβ superfamily of secreted cysteine knot proteins that includes TGFβ1, nodal, activins and inhibins. BMPs were first discovered by Urist in the 1960s when he showed that implantation of demineralized bone into intramuscular tissue of rabbits induced bone and cartilage formation. Since this seminal discovery, BMPs have also been shown to play key roles in several other biological processes, including limb, kidney, skin, hair and neuronal development, as well as maintaining vascular homeostasis. The multifunctional effects of BMPs make them attractive targets for the treatment of several pathologies, including bone disorders, kidney and lung fibrosis, and cancer. This review will summarize current knowledge on the BMP signalling pathway and critically evaluate the potential of recombinant BMPs as pharmacological agents for the treatment of bone repair and tissue fibrosis in patients. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Monocytes and macrophages, together with vascular smooth muscle cells (VSMCs), play key roles at all stages of atherogenesis. There is also growing evidence that BMP signaling is involved in vascular diseases, including atherosclerosis. Here we evaluate the role played by the BMP agonist/antagonist axis in monocyte recruitment during atherogenesis. Methods and results: Using ApoE−/− mice and BMPs, Gremlin and BMPRII siRNAs we show that BMPs (2 and 4) and their antagonist Gremlin are co-expressed in murine and human atherosclerotic vessels. Additionally, those genes are co-expressed and upregulated in cultured vascular smooth muscle cells early in atherosclerosis formation in ApoE−/− mice. Furthermore, we demonstrate that BMP-2 and -4 produced in atherosclerotic VSMCs promote, whereas Gremlin inhibits, monocyte chemoattraction. Finally, we demonstrate that chemotaxis induction occurs through direct BMP receptor II (BMPRII) activation. Conclusion: These findings suggest that the balance between BMPs (2 and 4) and Gremlin levels modulate crosstalk processes between vascular and immune cells and ultimately the homeostasis in normal vasculature. They also indicate that under pro-atherogenic conditions, BMP signaling prevails, favoring monocyte recruitment and inflammation. Manipulation of BMP signaling may enable the identification of novel molecular approaches for preventing, stabilizing, and reverting atherosclerosis. [Copyright &y& Elsevier]

Highlights: [•] TGF-β2, CTGF and gremlin can induce HLECs expression of EMT and ECM. [•] Silencing gremlin and CTGF effectively block gremlin and CTGF-induced EMT and ECM. [•] TGF-β2, gremlin and CTGF can activate Smad signaling pathway in HLECs. [•] Silencing gremlin or CTGF effectively inhibit the TGF-β2/Smad signaling pathway. [•] Blockade of Smad2 and Smad3 can inhibit gremlin and CTGF-induced EMT and ECM. [ABSTRACT FROM AUTHOR]