Blood vessels expand via sprouting angiogenesis, and this process involves numerous endothelial cell behaviors, such as collective migration, proliferation, cell–cell junction rearrangements, and anastomosis and lumen formation. Subsequently, blood vessels remodel to form a hierarchical network that circulates blood and delivers oxygen and nutrients to tissue. During this time, endothelial cells become quiescent and form a barrier between blood and tissues that regulates transport of liquids and solutes. Bone morphogenetic protein (BMP) signaling regulates both proangiogenic and homeostatic endothelial cell behaviors as blood vessels form and mature. Almost 30 years ago, human pedigrees linked BMP signaling to diseases associated with blood vessel hemorrhage and shunts, and recent work greatly expanded our knowledge of the players and the effects of vascular BMP signaling. Despite these gains, there remain paradoxes and questions, especially with respect to how and where the different and opposing BMP signaling outputs are regulated. This review examines endothelial cell BMP signaling in vitro and in vivo and discusses the paradox of BMP signals that both destabilize and stabilize endothelial cell behaviors. Blood vessels form during embryonic development in response to signals that originate from developing tissues and organs, and many of these same pathways remain engaged as blood vessels remodel and transition to homeostasis. Among these signaling pathways are some dedicated to vessel formation and maintenance, while other pathways are also utilized in multiple developmental programs. BMP (bone morphogenetic protein) is an example of a signal that is used iteratively throughout metazoan development. Our understanding of how BMP signaling affects vascular development and function initially lagged behind that of signals more dedicated to blood vessel functions such as VEGFA, as rigorous examination of BMP pathway function in vivo required the ability to manipulate signaling both spatially and temporally. An understanding of vascular BMP signaling has proven particularly elusive, as numerous pathway components show complex and context-dependent vascular phenotypes when manipulated. For example, global genetic deletion of BMP/TGFβ pathway components in mice yielded complex phenotypes, suggesting an important role for the TGFβ superfamily (that includes BMP) in cardiovascular development, but early lethality and the co-occurrence of both cardiac and vascular defects made these phenotypes difficult to interpret . These cDrosophila via invertebrate Decapentaplegic (DPP) [The BMP pathway is part of the larger transforming growth factor β (TGF β) pathway family, first identified as regulators of wing patterning in ic (DPP) , 3. BMP ic (DPP) . BMP alsic (DPP) , 6. ThesEvidence that BMP signaling regulates vascular development and function initially came from two lines of experimental evidence. First, cell-based studies showed that endothelial cells migrate and proliferate in response to BMP signals . These eThis review will describe the BMP pathway components most relevant to blood vessel development and function (for more comprehensive pathway reviews see –15). BecCanonical BMP signaling occurs via a core set of molecular events in all cell types Fig.  , such as betaglycan and endoglin (ENG), are membrane-localized BMP pathway components that lack intrinsic kinase activity but instead enhance signaling via receptor interactions. Two inhibitory SMADs (i-SMADs), SMAD6 and SMAD7, negatively regulate BMP signaling in a cell-intrinsic manner, likely through interactions with Type I receptors and the R-SMADs. BMPER  is another cell-intrinsic BMP pathway regulator that acts both negatively and positively on BMP signaling, depending on the context. Other negative regulators of BMP signaling are primarily cell-extrinsic, including Noggin, Chordin, Follistatin, and Gremlin; these antagonists primarily bind BMP ligands in the extracellular space, preventing their binding to receptors.BMP signaling is complex, as several different ligands, receptors, and co-receptors are often involved, even in a single cell type. Although the binding preferences of different BMP ligands for receptor complexes are thought to primarily result from differences in the Type I receptors, these data are largely derived from biochemical experiments in vitro, and how these preferences translate to in vivo situations is unclear. It is also not well-understood how co-receptors influence ligand binding to surface receptors, how competition among different BMP receptor complexes plays out in cells, and how ligands are presented to receptors . This variety and complexity likely contribute to the disparate cellular phenotypes that confound generation of simple molecular models of BMP function. BMP signaling complexity is even more relevant in the endothelial cells of blood vessels, where canonical BMP signaling as described above leads to opposite cellular phenotypes—either proangiogenic or homeostatic (anti-angiogenic) cell behaviors, in ways that are poorly understood. Below we describe the BMP pathway components thought to be most relevant to BMP signaling in endothelial cells.Bmp9 and/or Bmp10 [Bmp4 failed to differentiate mesoderm and died shortly after gastrulation, while mice globally lacking Bmp6 were viable with mild metabolic abnormalities [Numerous BMP ligands share a general structure Fig. A but difor Bmp10 –30; howemalities –33.Fig. BMP ligands primarily signal to endothelial cells in a paracrine manner, although some ligands circulate at physiological levels in the bloodstream, while others are expressed by endothelial cells and may provide autocrine signaling . BMP ligType I BMP receptors are membrane-localized serine/threonine kinases that share significant overall homology. However, these receptors diverge substantially in their ligand-binding domains, leading to different affinities for BMP ligands and potentially different signaling outcomes. Type I receptors have non-redundant functions in endothelial cells, as evidenced by in vitro studies and the different vascular phenotypes of mouse loss-of-function mutations for the Type I receptors  in this region: ALK1/ALK2 share 88%, and ALK3/ALK6 share 95% kinase domain amino acid homology, while all other Type 1 receptor pairings exhibit between 77 and 79% homology. Similarly, ALK1/ALK2 share the high homology in the GS domain (87%), while ALK3/ALK6 share only 54% GS domain homology. The BMP Type 1 receptors are the least homologous in the ligand-binding domain, with any two ALKs sharing merely 22–34% homology, although this divergence most often leads to binding preferences rather than rigorous ligand–receptor binding partners. The amino acid residues that differ in the ligand-binding domain of the ALKs create changes in the ligand-receptor interfaces that facilitate different stabilizing interactions .Type I receptors provide significant selectivity for ligand binding in the heterotetrameric complex with Type II receptors –67. CurrBMP Type II receptors are transmembrane serine/threonine kinases that efficiently phosphorylate Type I receptors once the heterotetrameric receptor–ligand complex is formed. They are structurally related to Type I receptors in that they include an N-terminal extracellular ligand-binding domain, a membrane-spanning α-helical domain, and a C-terminal cytoplasmic portion that includes the kinase domain  Fig. 2CC2C. HoweThe BMP arm of the TGFβ superfamily has three Type II receptors, BMPR2, ACVR2A (ActRII) and ACVR2B (ActRIIB); of these, BMPR2 is thought to be most relevant to endothelial cell signaling, although ACVR2A functions to balance BMP and TGFβ signaling in pulmonary endothelial cells . BMPR2 iBMP co-receptors, also called Type III receptors, enhance the responses of receptor complexes to BMP ligands. Co-receptors have an extensive extracellular domain that interacts with and modulates the affinity of BMP ligands for Type I and II receptors, but they lack a substantial intracellular domain and do not signal on their own Fig. D. MolecuENG strongly binds BMP9 and ALK1, potentiating BMP signaling through ALK1 , 92, 93.A soluble form of ENG (sENG) is produced in some situations. Matrix metalloproteinases can cleave the extracellular domain, producing sENG which downregulates pro-angiogenic proteins in human and mouse endothelial cells and inhibits angiogenesis, sprouting, and tube formation , 97, 98.After BMP ligands bind receptors on endothelial cells, canonical BMP signaling proceeds via phosphorylation of effector R-SMADs; phosphorylation changes R-SMAD conformation and allows for binding to the Co-SMAD, SMAD4. The R-SMADs involved in BMP signaling include SMAD1, 5, and 9 (SMAD9 was formerly referred to as SMAD8) . The R-SSMAD4 is the common effector SMAD of both BMP and TGFβ signaling, and it transduces both proangiogenic and homeostatic BMP signaling in endothelial cells. The MH1 and MH2 domains of SMAD4 are highly homologous to those of the R-SMADs  are a class of SMADs that negatively regulate BMP signaling in a cell-intrinsic manner; SMAD6 is relatively selective for BMP signaling in its effects, while SMAD7 is thought to more broadly affect BMP/TGFβ signaling , 107. BoBmper levels decreased endothelial cell barrier function, further cementing its role as an endothelial regulator [As with other aspects of BMP signaling, the context and concentration of an antagonist may alter outcomes. For example, BMPER is synthesized in endothelial cells and secreted into the extracellular matrix, where it binds BMP ligands to regulate their function. Its role as either agonist or antagonist of vascular BMP signaling is dose-dependent: at low concentrations, BMPER promoted sprouting and vessel formation, while at high concentrations it repressed these activities –123. In egulator .Further fine-tuning of BMP signaling is achieved through extracellular antagonists, including Chordin, Noggin, Gremlin, and MGP (matrix gamma-carboxyglutamate protein). These proteins are secreted by non-endothelial cells in tissues, and in general they bind BMP ligands and prevent ligand access to BMP receptors . ChordiTissues cannot survive without a proper supply of oxygen and nutrients. The formation of a mature vascular network is complex and includes numerous processes, such as vasculogenesis, sprouting angiogenesis, branching, lumenization, remodeling and homeostasis , 135. VaBMP pathway members have been implicated in each of the steps required for new blood vessel formation. Numerous cell-based assays in two or three dimensions have assessed BMP signaling involvement in specific endothelial cell behaviors and overall sprouting. These assays recapitulate aspects of the complex in vivo expansion of vessel networks in experimentally tractable systems that are often better suited to identification of specific effects, and for unraveling mechanisms and epistatic relationships.A first step in sprouting angiogenesis involves dynamic rearrangement of endothelial cell–cell adherens junctions. Angiogenic sprouting proceeds by a form of collective migration, whereby endothelial cells maintain connections with other endothelial cells, but remodel junctions to change spatial relationships as sprouting proceeds . EndotheTip cells are defined as the endothelial cells at the front of a sprout, and they guide sprout extension, usually by extending filopodia that sense the micro-environment . Stalk cAlk2/3 and Bmpr2. Alk2, Alk3 and Bmpr2 enhance vascular density, and Alk3 and Bmpr2 augment endothelial cell sprouting at the vascular front, while Alk1 prevents excessive vessel density at the front [bmp2b in zebrafish embryos caused ectopic sprouting from the axial vein but not the dorsal aorta [Endothelial cell behaviors linked to sprouting angiogenesis are often studied in vivo using the early postnatal retina , withouthe front . Gain-ofal aorta , and BMPal aorta . HoweverHow BMP9/10 and ALK1 affect angiogenic sprouting is complex. Early experiments involving the BMP9/ALK1 signaling axis demonstrated increased proliferation and migration of mouse endothelial cells upon stimulation with TGFβ or BMP9 or expression of constitutively active ALK1 , 155. Thpotential of endothelial cells to respond to proangiogenic signals. This is important because pathological situations may tip the balance to a proangiogenic phenotype in vivo that is more than just the absence of a homeostatic signal—for example, BMP ligands are often elevated in tumors [Many studies investigate proangiogenic BMP signaling in cultured primary endothelial cells. The phenotypes of genetic manipulations in animal models are complex, and they suggest overall that BMP signaling does not produce a strong unilateral proangiogenic signal in vivo, but rather a balance between proangiogenic and homeostatic BMP signaling that is important for formation and function of blood vessels. We posit that BMP signaling outputs differentially affect sprouting angiogenesis in vitro vs. in vivo because of a unique role for BMP signaling downstream of flow-mediated mechanotransduction in vascular endothelial cells. In this scenario, the outputs documented for proangiogenic signaling in vitro provide a picture of the n tumors  and accoWhy is BMP signaling not more prominent in vascular sprouting in vivo? This might reflect the fact that the components of proangiogenic BMP signaling are used iteratively during development in numerous tissues and organs. As discussed, BMP signaling is important for a wide variety of critical developmental processes, including embryonic patterning, lung morphogenesis, and bone formation. It is conceivable that the lack of specialization of this pathway, especially in comparison to pathways such as VEGFA whose expression and effects are much more tissue-restricted, makes it a difficult pathway to co-op purely for angiogenesis except in certain contexts. It might also result from the evolutionary addition of a BMP signaling module that functions in homeostasis in endothelial cells. Since embryonic vessels in vivo likely balance proangiogenic sprouting with homeostatic outputs, and BMP-induced homeostasis appears to be dominant under normal developmental conditions, this suggests that proangiogenic BMP signaling is not active developmentally in the vasculature, or that proangiogenic and homeostatic BMP signaling balance in complex ways in vivo.Once a primitive vascular plexus forms via sprouting angiogenesis, flow-mediated signals lead to vessel remodeling. This involves the pruning of unnecessary conduits, regression of vessels no longer under flow, and expansion of other vessels to accommodate increased flow, sometimes with additional recruitment of smooth muscle cells and fibroblasts , 162. InSmad1 and Smad5 in the postnatal retina was accompanied by reduced vessel regression [Smad4 in postnatal retinas increased vascular density and branchpoints, although this was attributed to increased endothelial cell proliferation [Smad4 and Smad1/Smad5 function is important in remodeling to form a hierarchically branched vasculature. It should be noted that SMAD4 is common to both the TGFβ and BMP signaling pathways, while SMAD1/5 signals predominantly downstream of the BMP signaling arm, although the similarity in the deletion phenotypes suggests that BMP signaling regulates vascular remodeling in the early postnatal retina. Endothelial cell-selective deletion of Alk1 in postnatal retinas also caused vascular hyper-branching and an increase in filopodial density, indicating that ALK1 is important for vascular pruning and regression [Eng, a co-receptor of ALK1 in transducing endothelial cell flow-mediated responses, caused a similar hyper-proliferative vascular phenotype in retinas: veins were enlarged due to increased endothelial cell proliferation and peripheral vessel branching was increased [The increased vascular density induced by concurrent endothelial cell-selective deletion of both gression . Similarferation . Since igression , 168. Enncreased , 170. TaBMPR2 mutations in patients, and mice lacking one copy of Bmpr2 had mild pulmonary hypertension and impaired pulmonary vascular remodeling under hypoxic conditions, a phenotype reminiscent of human PAH [Bmpr2 using an ALK1-L1-Cre driver that is expressed in E9.5 extraembryonic vessels and is predominant in E13.5 lung endothelial cells, resulted in a predisposition for adult mice to develop PAH [GDF2) associated with PAH patients and animal studies [Vessel remodeling also occurs in pathologies. For example, Pulmonary Arterial Hypertension (PAH) results in abnormal vascular remodeling and hypertension and is linked to uman PAH . Moreoveelop PAH , 173. BM studies , 175. ThEng in endothelial cells led to ectopic expression of the smooth muscle marker αSMA on retinal veins, consistent with loss of arteriovenous identity [Smad4 also led to arteriovenous identity perturbations in retinal vessels, while earlier deletion caused dysfunctional venous structures with reduced venous marker expression in embryos [Alk3 developmentally resulted in stunted embryos that made arteries but not veins; the early lethality suggests that the deletion affects primary artery–vein identity rather than flow-mediated maintenance of artery–vein identity [Arteries and veins have different functions, and they maintain unique identities to form a circuit delivering oxygenated blood to peripheral tissues and returning deoxygenated blood to the heart. Differences between arteries and veins include vessel-specific gene expression, the composition and thickness of surrounding mural cells and extracellular matrix, and vessel diameter (for detailed reviews see –179). Heidentity . Notch sidentity . Postnat embryos , 183–185ENG, ALK1, SMAD4 and GDF2 (Bmp9) are linked to human diseases involving AVM formation [Arteriovenous malformations (AVMs) are aberrant connections between arteries and veins that shunt blood directly between the large vessels, bypassing normal capillary beds. The exact mechanisms of AVM formation are still unknown, but AVMs are associated with dysregulation of all the functions discussed above: vascular remodeling, vessel caliber, endothelial barrier function, and arteriovenous identity. Evidence supports a multi-hit model of AVM formation that involves both genetic and environmental changes. First, heterozygosity for various BMP pathway components is followed by mosaic loss-of-heterozygosity to generate patches of mutant endothelial cells in the vasculature . Along w GDF2 Bmp are linkormation , 189. Suormation .Eng deletion in postnatal retinas led to 70% of animals developing AVMs, and 20% of these AVMs led to significant bleeding [Alk1 either globally in embryos or postnatally in endothelial cells led to AVM formation, and in retinas the AVMs formed only in areas of high blood flow [Alk1 in adult mice did not obviously perturb the vasculature but prevented AVM formation caused by endothelial cell loss of Alk1 in retinas and wounded skin [alk1 loss-of-function embryos developed dilated high-flow cranial AVMs [. Additionally, alk1 disruption via morpholino knockdown led to cerebral AVM formation and dilated cranial vessels [Smad4 global knockout mice developed AVMs in the brain, intestine, nose, and retinas, while adults formed AVMs in the gastrointestinal tract and skin following wounding [Smad4 deletion led to AVM formation in retinal vessels [Alk1, Eng, and Smad4, all genes that are linked to AVMs in humans.AVM formation is a hallmark of mutations in BMP pathway genes in mice as well, although not all BMP mutations lead to AVMs, and AVM formation is not exclusive to BMP pathway mutations  . For example, FK506 is a compound that prevents FKBP12 inhibition of BMPR2 signaling in blood vessels, and the restoration of BMP signaling afforded by the drug ameliorates symptoms in mouse models of PAH . As our


RAR transcripts in hESCs revealed that RARα in particular was essential for proper decidualization. These data provide direct functional evidence that uterine RAR-mediated RA signaling was crucial for mammalian embryo implantation, and its disruption led to failure of uterine receptivity and decidualization, resulting in severely compromised fertility.Retinoic acid (RA) signaling has long been speculated to regulate embryo implantation, because many enzymes and proteins responsible for maintaining RA homeostasis and transducing RA signals are tightly regulated in the endometrium during this critical period. However, due to a lack of genetic data, it was unclear whether RA signaling is truly required for implantation and which specific RA signaling cascades are at play. Herein we utilize a genetic murine model that expresses a dominant-negative form of RA receptor (RAR) specifically in female reproductive organs to show that functional RA signaling is fundamental to female fertility, particularly implantation and decidualization. Reduction in RA signaling activity severely affects the ability of the uterus to achieve receptive status and decidualize, partially through dampening follistatin expression and downstream activin B/bone morphogenetic protein 2 signaling. To confirm translational relevance of these findings to humans, human endometrial stromal cells (hESCs) were treated with a pan-RAR antagonist to show that in vitro decidualization is impaired. RNA interference perturbation of individual  During early pregnancy, the mammalian endometrium responds to changing ovarian hormones and signaling cues from embryos. The endometrium undergoes extensive growth and differentiation to become appropriately receptive to the incoming blastocysts for implantation , 2. CommRetinoic acid (RA), a physiologically active metabolite of its inactive precursor retinol (vitamin A), is essential for many biological processes, including cell survival, differentiation, and apoptosis . VitaminPrevious studies have implicated RA signaling in regulating female fertility. In both the rodent and human endometrium, expressions of RA-synthesizing  and -metabolizing enzymes (CYP26), as well as RA-binding proteins that regulate its bioavailability (CRBPs/CRABPs/FABP5), are temporally and spatially controlled during early pregnancy –17, suggRaraT403 truncated form of human RARα knocked in to the Rosa26 locus to dissect the role of RA/RAR signaling in embryo implantation , and subsequent inhibition of endogenous RAR-mediated transcriptional regulation of target genes. We showed that functional RAR signaling is required for mammalian uterine receptivity and decidualization both in the mouse model and in cultured hESCs.In the current study, we utilized a previously characterized mouse strain that carries the antation . Cre-medRaraDNfl/+ mice were mated to Pgr-Cre mice, resulting in offspring females carrying both alleles  and littermate controls . Pgr-Cre–mediated gene recombination in the uterus is first detected in the luminal and glandular epithelia starting at 2 weeks of age, and gradually expands to the stroma and myometrium . Reduction in expression of the majority of the Hox genes in the RaraDNPgr uteri demonstrates that RAR signaling was successfully disrupted in these animals. Female fertility was evaluated by breeding test of WT males with either RaraDNPgr or CTRL females and tracking the number of litters and pups produced by each female for 200 consecutive days. Of the RaraDNPgr females tested, 6 of 7 were completely sterile, producing 0 litters over the span of 7 months . No apparent developmental and behavioral abnormalities were observed in the 2 pups born to the RaraDNPgr sterility escapee. Vaginal plugs were consistently observed in the RaraDNPgr females, ruling out the possibility of behavioral issues preventing mating. As these females never presented palpable pregnancies, early-stage pregnancy defects were suspected.To generate mice with disrupted RA signaling in the female reproductive tracts, ometrium . To asse7 months . The remng setup . RaraDNPn = 3) but were completely absent in the RaraDNPgr uteri .Mouse embryo implantation occurs between 3.5 and 4.5 dpc, when blastocysts attach to the LE, which in turn triggers the underlying stromal cells to undergo decidualization. Successful implantation is accompanied by increased local vascular permeability, which can be visualized by tail vein injection of Chicago blue dye. Distinct blue dots indicating implantation sites were easily detectable along the uterine horns of CTRL mice on 4.5 dpc , indicating normal fertilization rates. In addition, evaluation of serum ovarian hormone levels at this stage by ELISA revealed no significant differences , a member of the epidermal growth factor family, is upregulated exclusively in the uterine epithelium on 3.5 dpc surrounding the embedding embryos in a P4-dependent manner (RaraDNPgr mutant (Egr1), a zinc finger transcription factor that is crucial for cell proliferation and angiogenesis, was previously reported to be induced in the subluminal stroma surrounding the blastocysts (Ccnd1), as well as facilitating its nuclear translocation  was performed to further interrogate expression of genes involved in uterine receptivity. Amphiregulin  plays a critical role in uterine receptivity and decidualization in the mouse, and its expression is induced in endometrial stromal cells starting on 3.5 dpc and increases over time  failed to be induced in the mutant uteri  in the RaraDNPgr on 3.5 dpc , components of activin B and downstream target of FST signaling, was significantly upregulated  in hESCs. In particular, knocking down RARA significantly inhibited in vitro decidualization of the hESCs, evidenced by a decreased expression of decidualization markers IGFBP1 and PRL  in the developing mouse ovary does not affect ovary differentiation or ovarian function  and silencing mediator of retinoic acid (SMRT), to prevent transcription –50. The eceptors . Dose-deeceptors , 47, 58;RaraDNPgr uterus were partially caused by loss of Fst expression. FST, also known as activin-binding protein, is a glycoprotein that regulates TGF-β superfamily signaling, primarily through binding to activin  and littermate CTRLs (RaraDNfl/+). Artificial decidualization and tail vein injection were performed following standard procedures as previously described , cut into 2 to 3 mm pieces, and digested in 1% trypsin (MilliporeSigma) in HBSS for 1 hour at room temperature with gentle shaking. After incubation, LE of each uterine segment was gently squeezed out using fine forceps along the longitudinal axis of the uterus. The remaining uterine tissues were transferred to a fresh tube, further digested in 0.25% trypsin and 1 mg/mL collagenase (MilliporeSigma) in HBSS for 30 minutes at 37°C with gentle shaking, and dissociated by pipetting several times after incubation. Cell suspension and tissue remnants were filtered through a 70 μm nylon filter, and stromal cells were resuspended in hESC culture media . Stromal cells were seeded in 12-well plates, and follistatin  was added to the culture media at indicated concentrations. For organ culture, 2.5 dpc uteri were cut into 2 to 3 mm segments and placed on the membrane of multiwall inserts. The inserts were then placed into 12-well culture plates containing 0.5 mL culture medium, and 10 agarose beads soaked in 100 ng/μL follistatin  or BSA were transferred into the lumen of each segment . Both pr2. To induce decidualization in vitro, culture medium was replaced with induction medium , and hESCs were allowed to decidualize for 96 hours before RNA extraction. Gene knockdown experiments were performed using DharmaFECT4 transfection reagent  and Silencer Select Validated siRNAs  following the manufacturer’s instructions. The morning after transfection, 10× induction cocktail topper was added to the culture to a final concentration of 1×, and cells were cultured for an additional 72 hours before harvest. For RAR antagonist treatment, hESCs were treated with induction medium with or without AGN194310 (MilliporeSigma) at indicated concentrations for 72 hours, before harvest for RNA extraction.Immortalized hESCs were previously characterized  and purcRpl7). Primers are listed in RNA was extracted in RNA STAT-60 reagent following the manufacturer’s instructions (Tel Test Inc). Reverse transcription was performed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems), and qPCR was performed on ViiA 7 Real-Time PCR System (Applied Biosystems) using PowerUp SYBR Green Master Mix (Applied Biosystems). All results were repeated in 3 biological replicates unless specified and relative gene expression changes were determined by ΔΔ Ct method  for color development. Antibody catalog numbers are listed in Tissues fixed in Bouin’s fixative were processed for serial dehydration and embedding at the Developmental Biology Histology Core at Washington University School of Medicine. Eight-micrometer paraffin sections were used for H&E staining and immunofluorescence . All antg for 10 minutes at 4°C, and supernatant (serum) aliquoted and stored at –80°C until use. Cayman Chemical ELISA kits for detection of E2 (501890) and P4 (582601) were used to determine serum hormone levels following the manufacturer’s instructions. Plates were read on a Bio-Rad 3550 microplate reader at the wavelength of 405 nm, and data were processed in Microsoft Excel and visualized in GraphPad Prism. Four biological replicates were tested and presented for each genotype.Whole blood was collected by cardiac puncture and allowed to coagulate in 1.5 mL Eppendorf tubes at room temperature for 20 minutes. The blood samples were centrifuged at 800Bmp2, catalog 406661; probe-Mm-Fst, catalog 454331), and detailed in situ procedure has been previously described . Gene-specific double-“Z” oligo probes compatible with the kit were ordered from Advanced Cell Diagnostics (probe-Mm-escribed .t test assuming unequal variance was performed to compare means of the experimental groups. For dose response experiments, 1-way ANOVA with post hoc Tukey’s honestly significant difference test was performed. Data are presented as mean ± SD, with raw individual experimental data displayed as dot plot overlay, and a P value of less than 0.05 was considered statistically significant.All experimental groups contained 3 biological replicates, if not specified otherwise. Two-tailed Student’s The animal studies included herein were reviewed and approved by the IACUC of Washington University. All studies were performed to the current standards of the American Association for Laboratory Animal Science so as to minimize pain, suffering, and total animals necessary for conclusive findings.YY, MEH, and LM designed the study. YY, MEH, and SBC conducted experiments. YY, MEH, SBC, RK, and LM analyzed data and wrote the manuscript.


This work aimed to improve the functional properties of soybean protein isolate (SPI) by high hydrostatic pressure (HHP) and develop SPI incorporated yogurt. Response surface methodology (RSM) was used to optimize the HHP treatment parameters, including pressure, holding time, and the ratio of SPI/water. Water holding capacity, emulsifying activity index, solubility, and hardness of SPI gels were evaluated as response variables. The optimized HPP treatment conditions were 281 MPa of pressure, 18.92 min of holding time, and 1:8.33 of SPI/water ratio. Water and oil holding capacity, emulsifying activity, and stability of SPI at different pH were improved. Additionally, relative lipoxygenase (LOX) activity of HHP treated SPI (HHP-SPI) was decreased 67.55 ± 5.73%, but sulphydryl group content of HHP-SPI was increased 12.77%, respectively. When incorporating 8% of SPI and HHP-SPI into yogurt, the water holding capacity and rheological properties of yogurt were improved in comparison with yogurt made of milk powders. Moreover, HHP-SPI incorporated yogurt appeared better color and flavor. Soy is a source of the predominant vegetable proteins. Soy protein isolates (SPI) are generally produced from soybean by being collected as precipitated curd in the acidic condition , and SPIHigh hydrostatic pressure (HHP) technology is a non-thermal food-processing method showing potential for the development of new food products with additional functional and health benefit . Under HPlant protein replaced food products have received great attention lately due to the increasing consumer awareness about the impacts of animal-based food production on the environment and health benefits of plant protein over animal protein, e.g., lowering blood cholesterol level ,13. SoybThe main purpose of this work is to use HHP treatment to improve the functional properties of SPI and apply it in producing soy-based yogurt. Response surface methodology was used to optimize the HHP process to modify the functional properties of SPI, including solubility, water holding capacity (WHC), and emulsifying activity index (EAI). A SPI-milk mixed yogurt was then produced, and its physicochemical and rheological properties, as well as the volatile flavor compounds characteristics, were evaluated.Soy protein isolate (SPI) was provided by Yihai Kerry Group  and protein content (dry basis) was 85.1%, respectively. Whole milk powder  was purchased from Fonterra Co-operative Group . 1-Anilino-8-naphthalenesulfonate (ANS) and 5, 5-dithiobis (2-bitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich .HHP treatments were performed using a High Pressure Iso-Lab System  and a hydraulic type cell with an inner capacity of 1 L and a water jacket for temperature control. A mixture of propylene glycol and water (30:70) was used as pressure-transmitting medium. SPI solution  was prepared by dissolving the powder in distilled water and stirring for 2 h. Then, 360 g SPI solution is vacuum-packed and subjected to HHP treatments at pressure of 200 ℃ 400 MPa, holding time of 10–30 min. The processing temperature was set at 25 ℃ . After HX1), holding time  and material-liquid ratio (X3)) on three response variables , emulsifying activity index , and solubility  were evaluated using Box–Behnken design (BBD), X1, X2, and X3 on the response value R2 were evaluated using the empiric second-order polynomial regression model as Equation (1):RSM is useful for optimizing, designing, developing, and improving processes where the responses are affected by several variables. Moreover, interactions between variables can be identified and quantified by RSM and its widely used in food industry processes for optimizations. The effects of three independent variables . The protein solubility was calculated as Equation (2):P was the mass of resolved protein in the supernatants and totalP was the total protein content.The solubility of SPI was determined according to the method of Condés et al. with minor modifications . In brieg for 10 min at 25 ℃. The supernatant was then removed and the residue was weighted. WHC was calculated as Equation (3):W2 was the weight of centrifuge tube and precipitated protein after absorbing water; W1 was the weight of centrifuge tube and protein sample; W0 was the weight of protein.The water holding capacity of SPI was determined using the method described by Ogunwolu et al. with minor modifications : 1.5 g ow/v). The absorbance of the produced emulsions was measured at 500 nm using a spectrophotometer  Co., Ltd.). EAI and ESI were calculated by the following Equations (4) and (5), respectively:c was the initial concentration of protein, φ was the fraction of oil used to form the emulsion, DF was the dilution factor, and A0 and A10 were the absorbance of the diluted emulsions at 0 and 10 min, respectively.The EAI and emulsifying stability index (ESI) of SPI were determined according to the method of Pearce and Kinsella with some modifications, respectively : 2 g/L SThe surface hydrophobicity was analyzed according to the method of Yang et al. using 1-anilino-8-naphthalene-sulfonate (ANS) as a fluorescence probe . SPI disc was the initial concentration of protein, DF was the dilution factor, and A412 was the absorbance of the diluted emulsions, respectively.Sulfhydryl group content of soybean proteins was measured according to the method of Beveridge et al. using Ellman’s reagent . Ellman’g and 4 ℃ for 20 min. The supernatant was then reacted with an enzyme substrate prepared previously. Right after mixing, the absorbance was measured at 234 nm and 25 ℃ by a UV spectrophotometer. The residual activity (RA) of LOX was calculated as Equation (7):A was the LOX activity of SPI after HHP treatment, and A0 was the initial LOX activity before treatment.LOX activity of SPI was measured according to the method of Li et al., with minor modifications . Briefly2/d mol).A protein concentration of 0.1 mg/mL was selected for CD spectra analysis . The samw/v]) and untreated or optimized HHP treated SPI (8% [w/v]) in distilled water under continuous stirring for 30 min and then homogenized in high-speed homogenizer at 10,000 rpm. The mixtures were heated at 95 ℃ for 10 min, then cooled down to 42 ℃, and finally inoculated with 0.2% (w/v) deep frozen commercial cultures  of Lactobacillus bulgaricus and Streptococcus thermophilus in water bath (42 ℃) for 7 h. The yogurt was then stored at 4 ℃ for 24 h for quality evaluation. The yogurt made from milk powder (15% [w/v]) using the same procedure was used as a control.The yogurt was prepared by reconstituting milk powder . The titration acidity (TA) was measured according to the titration method of Silva et al. . WHC wasThe color of samples was determined using a colorimeter , in which CIEL * a * b * system. L *, a *, and b * were recorded, and eference .−1. The Ostwald deWaele model was used to fit the experiment data, and was represented by the Equation (8):aη was the apparent viscosity (Pa·s), τ was the shear stress (Pa), γ was the shear rate (s−1), k was the consistency index (Pa·sn), and n was the flow behavior index.The rheological properties of SPI-milk yogurts were evaluated at 25 ± 0.1 ℃ according to the method described by Mei et al., using a Haake RheoStress 6000 rheometer  . A cylin−1, and then held at 250 ℃ for 5 min. The total run time was 50 min. The NIST 2011 mass spectral library  was used to identify the volatile compounds.Flavor is one of the most important properties of yogurt products, and volatile components leading to off-flavors can cause the product to be unsatisfactory for the tastes of consumers . The yogp < 0.05.Data were analyzed using SPSS  following an analysis of variance (ANOVA) one-way linear model and reported as mean values and standard deviations. Mean comparisons were performed using the Duncan test, and the significance level was established for R2 values of all models were higher than 0.9, indicating that more than 90% of the changes could be expressed by these models. The coefficients of the models are shown in 1), EAI (Y2), and solubility (Y3) in coded variables are given in (Equations (9)–(11)), respectively. The p-values of all models were less than 0.01  of SPI reached the highest point when treated at 500 MPa, but decreased when pressure further increased [ncreased .1), time (X2), and SPI/water ratio (X3) on emulsifying activity index (EAI) were illustrated through response surfaces and contour plots in 1) and time (X2) due to the partial or total denaturation of SPI enhancing the surface activity. Low ratio of SPI/water (X3) during the HHP treatment also reduced EAI, probably owing to the interaction between the subunits of protein. Molina et al. suggested that 10% (w/v) 11S globulins of SPI showed the optimum value of EAI after treatment at 200 MPa for 15 min, while 7S globulins and SPI at the same concentration showed the highest EAI after treating at 400 MPa for 15 min [The effects of pressure (Xr 15 min .1), time (X2), and SPI/water ratio (X3) on SPI solubility. At the beginning, when the pressure and holding time increased, the solubility increased to around 30% due to the change of SPI tertiary structure enhancing the protein–solvent interactions. As the pressure and holding time continued to rise, the solubility went down in all the trials. 1) = 281.09 MPa, time (X2) = 18.92 min, and SPI/water ratio (X3) = 1:8.33. Under these conditions, the maximum WHC, EAI, and solubility were 5.19 g/g, 27.06 m2/g, and 31.6%, respectively. In order to validate the reasonability of the model equations, a treatment of SPI was carried out in triplicate under the optimal conditions. The WHC, EAI, solubility, and hardness of gel were 5.35 ± 0.04 g/g, 28.11 ± 1.67 m2/g, and 33.7 ± 0.22%, respectively, which was in good agreement with the predicted data.The optimization of the HHP treatment was chosen based on the prerequisite of fulfilling the weight of four response values as 1:1:1:1 by applying the optimum formulation point of numerical method generated by Design Expert software. The optimum conditions for the HHP treatments were calculated to be at pressure  residual activity, and circular dichroism (CD) spectra of the untreated and HPP treated (281 MPa) SPI were shown in Circular dichroism (CD) spectra of untreated and HHP treated SPI were shown in The functional properties of the untreated and HHP treatment (281 MPa) SPI, including water holding capacity, solubility, and emulsifying properties in different pH were shown in The change of emulsifying activity index (EAI) and emulsifying stability index (ESI) of SPI produced by HHP treatment showed significant difference with different pH conditions C,D. EAI Physicochemical properties, including final pH, titration acidity (TTA), WHC, and color, of three kinds of yogurt samples were reported in A darker and yellower color of the yogurt was clearly observed by the naked eye in SPI and HHP-SPI yogurt samples. Correspondingly, the L * (lightness) values were lower in those two samples compared with control. However, HHP-SPI yogurt had higher L * value than SPI yogurt, which might be explained by the particle dispersion after HHP that result in light reflection on the yogurt gel surface . The b *0 and SH content that formed stronger gels. The curves were fitted to the Ostwald deWaele model and the values of the flow behavior index (n) and the consistency index (k) were obtained. The flow behavior index (n) was a measure of deviation of shear thinning fluids from Newtonian flow; meanwhile, the consistency index (k) was believed to be related to product acceptability for yogurt [R2 values generally above 0.98. Low n values (n < 1) were recorded in all samples, and the addition of SPI decreased the n value, indicating an increase of the pseudoplastic behavior of yogurt. It was regarded that higher pseudoplasticity could lead to better consumer acceptance of the product [r yogurt . The exp product . FurtherThe volatile compounds of three yogurt samples were shown in This study demonstrated that the functional properties of SPI could be significantly improved by HHP treatments. The identified optimal HHP conditions were 281 MPa pressure, 19 min holding time, and 1:8.3 of SPI/water ratio based on the response surface methodology. The HHP treated SPI exhibited higher surface hydrophobicity and sulphydryl groups content, and lower LOX activity. Water holding capacity and emulsifying activity index at pH 3 of SPI were significantly improved by HHP treatment. The yogurt with HHP-SPI displayed better water holding capacity and lighter color comparing with SPI incorporated one. In addition, the content of five volatile components relating to the beany flavor decreased in HHP-SPI yogurt, demonstrating the superior flavor. HPP is a promising technology to improve the functionality of SPI, which could be a superior plant protein for diary product.


Antidepressants are well-known drugs to treat depression and major depressive disorder for humans. However, the misuse and abuse of antidepressants keep increasing with several side effects reported. The aim of this study was to assess the potential adverse effects of 18 antidepressants by monitoring zebrafish larval locomotor activity performance based on the total distance traveled, burst movement count, and total rotation count at four dark-light intercalated phases. In general, zebrafish larvae displayed sedative effects after antidepressant exposure by showing a significant reduction in all of the locomotor activity-related endpoints. However, three antidepressants i.e., amitriptyline, amoxapine, and sertraline were able to trigger a significantly high locomotor activity in zebrafish larvae during the light cycle. These differences might be due to the pharmacologic differences among the antidepressants. In addition, since each antidepressant possesses a different dosage range from the other, overdoses of these antidepressants might also be the causes of these differences. Furthermore, based on these results, a further study was conducted to observe the effect of these three antidepressants in lower concentrations. From the results, biphasic effects in terms of zebrafish larval locomotor activity were demonstrated by these drugs. Even though further studies are still required to validate the mechanism, these findings indicate that these antidepressants might share a common mechanism responsible for their effects on zebrafish larval locomotor activity although there were some differences in potency of these effects.  Depression is a common mental health condition associated with a state of sadness, hopelessness, and grief that may affect the thinking process, behavior, and feelings. The changes in mood, emotion, and behavior are due to chemical imbalances of neurotransmitters in the brain . DepressDanio rerio) is a well-accepted animal model in various research fields including genetic studies, drug discovery, and behavioral analysis. In addition, it is frequently used for pharmacology safety investigations since it possesses several advantages over other animal models [Nowadays, zebrafish -96 h of selective serotonin reuptake inhibitor (SSRI) exposure in fish was within 3.29–6.34 mg/L [50 of amitriptyline exposure in zebrafish embryos is within mg/L concentration (2–100 mg/L). However, 1 mg/L concentration is still deemed to be safe as the mortality rate was below 25% [The present study aimed to generate systematical data for a deeper understanding of the adverse effects of various types of antidepressants based on animal behavior of zebrafish larvae. In this study, the zebrafish larval photomotor responses to an intense light–dark transition exposure were assessed since the previous study in amphipods, crustaceans, and fish found that antidepressant drugs induced the phototaxis behavior . Behavio.34 mg/L . In addielow 25% . TherefoEighteen different antidepressants were used for this experiment  zebrafish aged 4–6 months were used for the breeding process. Afterward, the embryos were collected and placed into a 9 cm petri dish, filled with a mixture of E3 medium and methylene blue, which was used to prevent fungal infections, until a concentration of 0.1 mL/L was reached following our previous protocols ,65. ZebrThe antidepressants were diluted from the stock solution to working concentration (1 mg/L). Meanwhile, for further experiments, several lower concentrations  of selected antidepressants, which were AMY, AMO, and SRT were administered to the zebrafish larvae by immersion. Later, after 4 days post fertilization (dpf), zebrafish larvae were randomly selected for the treatment and control groups. For the control group, the sample of zebrafish was placed into a 9 cm petri dish filled with a 40 mL mixture of E3 medium and methylene blue. Meanwhile, for the treatment group, the zebrafish were placed into a 9 cm petri dish supplemented with a 40 mL mixture of E3 medium, methylene blue, and 1 mg/L antidepressant. Both the control and treatment groups were incubated at 28 °C with pH 6.8–7.5 for 24 h  . After 3To provide a better resolution for the adverse effects of antidepressants, the phenomic approach analysis was used to combine data collected from zebrafish locomotion alteration  , a scienp < 0.0001), except MRT, which was not significantly different compared to the control group (p = 0.4438) (p = 0.0004); DOX, SEG, MCP, VEN, BUP, MIA, and TRA treated groups (p < 0.0001) (p < 0.0001); FLX (p = 0.0113); DLX (p = 0.0240); MRT (p = 0.0011) displayed an elevated locomotor activity and these increments were observed in several antidepressant-treated groups. In addition, no significant difference in locomotor activity was observed in the group treated with ESC (p = 0.8121), PAR (p = 0.4985), and ATM (p = 0.1652)-treated groups during the light cycle  B. Meanwh 0.0001) A. On theht cycle A. p < 0.0001); IMP (p = 0.0002)-treated groups. Furthermore, it is interesting to find that AMY, AMO, and SRT significantly increased the burst movement activity in zebrafish larvae during the light cycle (p < 0.0001) (p = 0.6044) compared to the control group in this cycle. Similar to the light cycle, almost all burst movement activity from antidepressants-treated zebrafish was reduced during the dark cycle (p < 0.0001) while no significant difference was shown in AMY (p = 0.4524) and FLX (p = 0.2383)-treated groups compared to control. On the contrary, a significant change in locomotor activity was exhibited by three different antidepressant groups; AMO, MIA, and TRA (p < 0.0001)  C. On therk cycle D. These  0.0001) D. Taken p < 0.0001) , respectively (p = 0.7292), AMO (p = 0.5334), and ESC (p = 0.7079)-treated groups during the light cycle regarding their movement orientation. The details of the two-way ANOVA test results from these experiments can be found in Finally, the potential effect of antidepressants on movement orientation was investigated by calculating the rotation movement counted. Overall, average rotation movement in antidepressant treated groups was significantly reduced in both light and dark cycles ( 0.0001) , except ectively E,F. HoweTo evaluate the behavioral endpoint similarities triggered by different antidepressants, PCA and hierarchical clustering with information collected from the locomotor activity tests were performed. From the hierarchical clustering result, three major clusters were generated. BUP, MIA, TRA, FLX, MCP, and SEG were found to be grouped in a single major cluster while ATM, MRT, and ESC belonged to another major cluster with the control group in it. Lastly, another cluster, which contained most of the groups, consisted of DLX, MEM, SRT, IMP, PAR, DOX, VEN, AMY, and AMO A,B. The Based on the locomotor activity test results above, the three antidepressants, which were AMY, AMO, and SRT, caused distinct effects in zebrafish larvae locomotor activity compared to most of antidepressants tested. While other antidepressants caused hypoactivity in both cycles, these antidepressants led to hyperactivity in zebrafish larvae during the light cycle even though suppressed locomotion was still observed in the dark cycle. Thus, to do a further examination of their effects in different doses, zebrafish larvae were exposed with the other three lower concentrations and locomotor activity of zebrafish larvae was again conducted. Overall, both AMY and AMO at low concentrations, especially 1 and 10 ppb, caused hyperactivity in zebrafish larvae during both light and dark cycles. These phenomena were indicated by the significantly high levels of several behavior endpoints, especially total distance traveled . InteresZebrafish is well known as a sensitive bioindicator since it is able to respond to a large variety of chemicals . FurtherAs expected, after exposure to the antidepressants, decrements in locomotion in zebrafish larvae were observed almost in all treated groups. This phenomenon is plausible since antidepressants work by targeting neurotransmitters in regions of the brain involved in neuroadaptive changes, which regulate the neurotransmission signaling cascades . These nPimephales promelas) after exposure to SRT, one of the SSRI [However, while most antidepressants led to changes in animal behavior  and prodthe SSRI . Here, Sthe SSRI . In linethe SSRI  and reduthe SSRI . Some prthe SSRI ,101. Furthe SSRI . Moreovethe SSRI . In addithe SSRI . These athe SSRI . Two posthe SSRI .Interestingly, after the further investigation of these three antidepressants at lower concentrations, it was found that they displayed NMDR on locomotor activity in zebrafish larvae, that is, they can decrease and increase zebrafish larval locomotion resulting in a biphasic curve. In fact, even though they were infrequently observed, the biphasic effects of some antidepressants had been demonstrated in several prior studies. The biphasic effects of these antidepressant drugs have been clearly demonstrated on producing the spike activity in perfused guinea pig hippocampal slices with some drugs displaying an opposite effect to each other. These results indicated that some antidepressant drugs may exhibit both anticonvulsant and convulsant effects . FurtherAs mentioned above, different effects between each antidepressant were also demonstrated in the current study. These phenomena likely occurred due to the pharmacological differences among the antidepressants and overdoses of these antidepressants since each of them has its own dosage range  and thusIn addition, based on the heatmap clustering and PCA results, several antidepressants were grouped together with the control group in a single major cluster. This phenomenon indicated that these antidepressants, which were ESC, ATM, and MRT, caused less severe behavioral effects to zebrafish larvae than other antidepressants. In humans, the dosage ranges of these antidepressants are relatively lower than the other antidepressants, such as AMO, MEM, and BUP . Thus, tTaken together, this is the first study to address the adverse effects of multiple antidepressants based on locomotor activity and phototaxis behavior in zebrafish larvae by using three different endpoints: total distance traveled, burst, and rotation movement. This study proves that the behavioral approach is useful as an early screening tool to assess antidepressants. Overall, the results indicated that even though the majority of antidepressants produce anxiolytic-like effect in locomotion assay, there were several antidepressants that caused an anxiety-like behavior in zebrafish larvae, displayed by abnormal behavior with hyperactivity and altered phototaxis behavior. All of the behavior alterations caused by some antidepressants might be due to the pharmacologic differences among the antidepressants. In addition, the present study also found that several antidepressants, which were AMY, AMO, and SRT, exhibited biphasic effects, indicating that they can increase and decrease zebrafish larval locomotion in different concentrations. However, the results of this study do not rule out that other antidepressants might also have a biphasic effect since each antidepressant might have a specific range of effective concentration. The biphasic effect demonstrated by these three antidepressants might be related to the noradrenaline released from the adrenergic nerve and the induction of stereotypy by those drugs. Thus, further studies are required to investigate the specific mechanisms of antidepressant effects in different life stages of zebrafish.


Caenorhabditis elegans and mammalian species, i.e., reduced activity of this pathway extends lifespan, whereas increased activity accelerates the aging process. The insulin/IGF-1 pathway controls protein and energy metabolism as well as the proliferation and differentiation of insulin/IGF-1-responsive cells. Insulin/IGF-1 signaling also regulates the functions of the innate and adaptive immune systems. The purpose of this review was to elucidate whether insulin/IGF-1 signaling is linked to immunosuppressive STAT3 signaling which is known to promote the aging process.The insulin/IGF-1 signaling pathway has a major role in the regulation of longevity both in Original and review articles encompassing the connections between insulin/IGF-1 and STAT3 signaling were examined from major databases including Pubmed, Scopus, and Google Scholar.The activation of insulin/IGF-1 receptors stimulates STAT3 signaling through the JAK and AKT-driven signaling pathways. STAT3 signaling is a major activator of immunosuppressive cells which are able to counteract the chronic low-grade inflammation associated with the aging process. However, the activation of STAT3 signaling stimulates a negative feedback response through the induction of SOCS factors which not only inhibit the activity of insulin/IGF-1 receptors but also that of many cytokine receptors. The inhibition of insulin/IGF-1 signaling evokes insulin resistance, a condition known to be increased with aging. STAT3 signaling also triggers the senescence of both non-immune and immune cells, especially through the activation of p53 signaling.Given that cellular senescence, inflammaging, and counteracting immune suppression increase with aging, this might explain why excessive insulin/IGF-1 signaling promotes the aging process. The aging process is associated with chronic low-grade inflammation.C. elegans and mammalian species.Insulin/IGF-1 signaling regulates the longevity of Insulin/IGF-1 signaling stimulates STAT3 signaling through several pathways.STAT3 signaling evokes anti-inflammatory/immunosuppressive responsesInsulin/IGF-1 signaling promotes the aging process through STAT3 signaling. Caenorhabditis elegans and mammalian species [Several aging studies have revealed that the insulin/IGF-1 signaling pathway controls the lifespan of both  species . There i species . With re species , 5  gene, are typical long-lived mouse models. These mice display a reduced level of GH/IGF-1 hormones and have a low stature, thus they are called dwarfs. Interestingly, these dwarf mice live 20–70% longer than their wild-type control counterparts. Accordingly, Ortega-Molina et al. [GH-R gene leading to the deficiency in the level of IGF-1, is characterized by obesity, resistance against tumors, and extended lifespan [Currently, it is known that several signaling networks co-operate in the regulation of the aging process. Interestingly, the insulin/IGF-1 pathway displays an antagonistic pleiotropy with aging, i.e., during development insulin/IGF-1 signaling has fundamental functions in cellular differentiation and the growth of animals, whereas later in the life, it regulates metabolism but its activity clearly accelerates the aging process . Given ta et al.  reporteda et al. . In humalifespan . Moreovelifespan  demonstrCaenorhabditis elegans, the Daf-2 pathway, an ortholog of the mammalian insulin/IGF-1 signaling, also displays the antagonistic pleiotropy enhancing the aging process. The loss-of-function mutations of several daf (DAuer Formation) genes, e.g., daf-2 (InsR/IGF-1R), age-1 (PI3K), and daf-16 (FoxO), significantly extend the lifespan of C. elegans and can induce the dauer phenotype [C. elegans [daf-18 gene with the human Pten gene induced a long-lived mutant phenotype of C. elegans. It seems that the Daf-16/FoxO transcription factor is the major enhancer of the aging process in C. elegans, whereas mTOR (CeTor/Let 363 in C. elegans) is a potent promoter of the aging process in rodents [The insulin/IGF-1 signaling pathway seems to be an evolutionarily well-conserved mechanism involved in the regulation of longevity in species ranging from metazoans to mammals . In Caenhenotype , 15. Acc elegans  demonstr rodents , 5, 15.  rodents . It is kThere is convincing evidence that insulin/IGF-1 signaling inhibits inflammatory responses not only by reducing pro-inflammatory hyperglycemia but also by modulating the immune system and decreasing the generation of inflammatory mediators . However+ effector T cells in vitro and the adoptive transfer of IL-10-primed monocytes into mice suppressed the inflammatory state in experimental colitis. There are several studies indicating that IGF-1 treatment promotes the polarization of the pro-inflammatory M1 macrophages toward the anti-inflammatory M2 macrophages [Insulin/IGF-1 signaling controls the phenotype and functional activities of immune cells. Ge et al.  reportedrophages , 32. Sparophages  demonstrrophages , 34. Bilrophages  demonstrrophages . TGF-β irophages . WhetherThe InsR and IGF-1R proteins are receptor tyrosine kinases (RTK) and thus they are members of the tyrosine kinase superfamily. The receptors of several growth factors, cytokines, and hormones are RTKs which bind to and phosphorylate different signaling transducers, e.g., members of the JAK family . JAKs acFurthermore, Zhang et al.  demonstrThe PI3K/AKT axis, downstream from the InsR/IGF-1R complex, has several upstream and downstream signaling connections Fig. . There iPyruvate kinase isozyme M2 (PKM2) is one of the enzymes which are activated by the IGF-1/AKT axis , 55 , i.e., mTORC1 and mTORC2, are important downstream targets of PI3K/AKT signaling , especially FoxO1 and FoxO3a, have crucial functions in T cell biology, e.g., they regulate the differentiation and survival of T cells and the development of immunosuppressive Tregs . For insm et al.  demonstrm et al.  demonstrh et al.  also repThe NF-κB system is the hub of upstream and downstream signaling connections which regulate innate and adaptive immunity as well as the functions associated with cell growth and death. The NF-κB system acts in close co-operation with several signaling pathways, e.g., those of JAK-STAT3, SMAD3, and insulin/IGF-1 signaling Fig. . There iThere is close crosstalk between IKKα/β and mTOR in the activation of NF-κB signaling Fig. . IKKα/β The JAK-STAT3 pathway transduces signals from the cell surface receptors of distinct cytokines, growth factors, and hormones into the nuclei. For instance, the receptors of IL-6, IL-10, IL-23, interferons, EGF, and Ins/IGF-1 as well as certain G-protein coupled receptors (GPCR) are linked to the activation of JAK1/JAK2 which subsequently recruit STAT3 proteins to the receptor complex and phosphorylate STAT3 at Tyr705 . The phoThere is convincing evidence that STAT3 signaling has a crucial role in the generation of an immunosuppressive microenvironment around tumor sites . STAT3 iThe immunosuppressive network refers to the immune cells which express the regulatory, immune suppressive activities; these cells include MDSCs, Tregs, Bregs, M2 macrophages (Mreg), and the regulatory subsets of dendritic cells (DCreg), NK cells (NKreg), and type II natural killer T cells (NKT) . This isDrosophila and mammalian species [Drosophila, insulin/TOR signaling regulates the size and differentiation of the hematopoietic niche [Ikaros gene to induce the commitment of hemotopoietic progenitors to the lymphoid lineage. There is convincing evidence that the aging process affects the differentiation of hematopoietic lineages, i.e., myelopoiesis is clearly upregulated with aging [Inflammatory mediators released from peripheral tissues enhance myelopoiesis, especially the generation of immunosuppressive, immature MDSCs, mostly in bone marrow (BM). Zhang et al.  reported species –100. In ic niche . Duan etic niche  demonstrth aging .IL-10 gene in human breast cancer-associated Treg cells. There is a robust evidence that IGF-1 stimulates the proliferation and increases the immunosuppressive activity of FoxP3-positive Tregs in mouse and human cells [Immunosuppressive Treg cells have crucial functions in the maintenance of self-tolerance and immune homeostasis in diverse pathological conditions. Tregs not only suppress the functions of effector T cells but they can also inhibit the activities of innate immunity, e.g., that of NK cells . In addian cells , 28, 110an cells  revealedStat3 gene increased the level of two cytolytic enzymes perforin and granzyme B in mouse NK cells and accordingly a lack of STAT3 signaling enhanced the NK cell-dependent tumor surveillance [Macrophages and NK cells are major players in the maintenance of innate immune defense. There is substantial evidence that the STAT3-dependent signaling induces the polarization of macrophages toward the immunosuppressive M2 phenotype . Intereseillance . It is keillance . Moreoveeillance , 121. Iteillance .STAT3, NF-κB, and SMAD3 transcription factors are the main regulators of immune functions involving both the differentiation of immune cells and the modification of their different phenotypes and properties. The crosstalk between these factors is mediated through their mutual interaction or their abilities to control the expression of compounds which affect the coordination of their activities Fig. . For insIL-6 and RANTES genes, in human HME1 cells. They revealed that U-STAT3 interacted with the NF-κB complex in cytoplasm, competing with the binding of IκB proteins which are specific inhibitors of NF-κB activation. The U-STAT3/NF-κB complex translocated into nuclei exploiting the nuclear localizing sequence (NLS) of STAT3 protein and targeted to a subset of NF-κB-dependent genes. Lee et al. [IDO gene in human MDSCs. They revealed that STAT3 signaling induced the expression of IDO through the activation of noncanonical NF-κB signaling through the binding of the RelB/p52 complex to the promoter of IDO gene. These studies indicate that STAT3 signaling can utilize NF-κB components to evoke an immunosuppressive state in host tissues.There is also an active co-operation between STAT3 and NF-κB signaling driving either pro-inflammatory or anti-inflammatory responses in a context-dependent manner. As described earlier, the connections between AKT/mTOR/IKK signaling hub is able to control the activity of NF-κB signaling through different pathways Fig. . Interese et al.  reportede et al.  revealede et al.  demonstrCurrently, it is known that a low-grade inflammatory process is associated with a compensatory anti-inflammatory state which involves the activation of the immunosuppressive network. Recently, we have reviewed in detail the immunosuppressive remodeling of the immune system in conjunction with aging . In brieThere are many clinical observations indicating that the aging process is associated with a profound remodeling of the immune system. The inflammaging process involving the accumulation of pro-inflammatory senescent cells triggers immunosuppressive mechanisms to maintain tissue homeostasis . For insThere is clear evidence that insulin/IGF-1 exposure can induce/augment the cellular senescence of different non-immune cells in several experimental conditions –147. It Caenorhabditis elegans and Drosophila [C. elegans [The aging process is associated with a significant remodeling of both the innate and adaptive immune systems. A chronic low-grade inflammaging process is accompanied by immunosenescence, i.e., a gradual deterioration of the immune system with aging, especially the functions of adaptive immunity decline , 151. Imosophila , 153. Cuosophila . The halosophila –156. Intosophila , 155. Th elegans , 158. It elegans , 159. Th elegans  demonstr elegans  reported elegans , 19.The signaling pathways of several cytokines and growth factors, such as insulin/IGF-1, converges at the activation of STAT3 factor Figs. , 3 whichIn the mid of 1980’s, it was observed that insulin resistance (IR) was associated with the aging process in human tissues , 168. CuThe antagonistic pleiotropy in the function of the insulin/IGF-1 pathway reveals a paradox of the aging process since insulin/IGF-1 signaling is an important signaling pathway of growth and differentiation during development but its function accelerates the aging process later in the lifespan. mTOR signaling, a downstream target of the insulin/IGF-1 pathway, is a well-known enhancer of the anabolic processes required during the growth phase when its capacity to inhibit autophagy might be beneficial but later in the lifespan, its inhibition promotes the degeneration of aging tissues. Interestingly, insulin/IGF-1 signaling is also able to control immunosuppression and cellular senescence through its several connections to STAT3 signaling. For instance, STAT3 signaling is a potent enhancer of immunosuppressive properties in MDSCs, Tregs, and M2 macrophages. The increase in the numbers of these cells with aging not only counteracts chronic age-related inflammation but also disturbs the maintenance of tissue homeostasis, enhancing cellular senescence and tissue degeneration . For ins


Toll-like receptors (TLRs) are critical sensors for the detection of potentially harmful microbes. They are instrumental in initiating innate and adaptive immune responses against pathogenic organisms. However, exaggerated activation of TLR receptor signaling can also be responsible for the onset of autoimmune and inflammatory diseases. While positive regulators of TLR signaling, such as protein serine/threonine kinases, have been studied intensively, only little is known about phosphatases, which counterbalance and limit TLR signaling. In this review, we summarize protein phosphorylation events and their roles in the TLR pathway and highlight the involvement of protein phosphatases as negative regulators at specific steps along the TLR-initiated signaling cascade. Then, we focus on individual phosphatase families, specify the function of individual enzymes in TLR signaling in more detail and give perspectives for future research. A better understanding of phosphatase-mediated regulation of TLR signaling could provide novel access points to mitigate excessive immune activation and to modulate innate immune signaling.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-021-00722-1. Toll like receptors (TLRs) sense pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) by innate immune cells, but also by various other cell types . Upon liBalancing phosphorylation and dephosphorylation of proteins is pivotal to initiate, drive, and terminate TLR signaling. Right from the initial events at the plasma membrane, cytoplasmic protein kinases with an activity directed against serine or threonine residues in their substrates (Ser/Thr protein kinases) are key drivers of TLR signaling. Representatives of this group of enzymes can be found in complex with the receptor at the plasma membrane, in a large, multi-component signalosome in the cytosol, and as regulators of gene expression in the nucleus. In fact, three main Ser/Thr protein kinase families are conserved elements of TLR signaling: Interleukin-1 receptor-associated kinases (IRAKs), the transforming growth factor-β-activated kinase 1 (TAK-1), and the IκB kinase (IKK) complex. However, additional serine/threonine kinases such as NIK, MST4, TBK-1, TANK, and RIPK also contribute to TLR-initiated processes , 10–12IRAK family members  play a central role in TLR/IL-1 signaling. Upon ligand binding to the TLR and association with the adapter molecule MyD88, IRAKs are promptly recruited to MyD88 through death-domain interactions . IRAK4 iSignal propagation by poly-ubiquitinated TRAF6 occurs through association with the Transforming Growth Factor-β-activated kinase 1 (TAK1) in complex with TAK1 binding protein-1/2 (TAB1/2). The TAK1/TAB complex is activated by binding to K63 poly-ubiquitin chains, such as those attached to TRAF6, and by phosphorylation at Thr184, Thr187 and Ser412 in the TAK1 kinase domain –28. ActiAlready this short summary of canonical TLR-initiated processes illustrates the multitude of phosphorylation events, which govern this particular signaling pathway inside cells and which are summarized in Fig. Negative regulation of TLR signaling is mediated by several mechanisms. Besides inhibition of kinase activity and poly-ubiquitin-initiated protein degradation, the dephosphorylation by protein phosphatases is a rapid and reversible means to limit TLR pathway output. Therefore, there is growing interest in understanding the role of different protein phosphatases in TLR signaling. Protein serine/threonine phosphatases (PSPs) in general are subdivided into three families based on structural homology: phospho-protein phosphatases , metal-dependent protein phosphatases  and aspartate-based phosphatases  , 38, 39.2+-calmodulin (CaM) binding motif (CBD) and an autoinhibitory sequence in its C-terminal region and two divergent regions in the catalytic domain. PP5 contains in its N-terminal domain three tetratricopeptide repeats (TPR). Finally, PP7 differs from all the other phosphatases in that it contains EF-hand motifs in the C-terminal domain and a large insert within the catalytic core domain [Human PPP-family serine/threonine protein phosphatases include PP1, PP2A, PP2B , PP4, PP5, PP6 and PP7. In general, these enzymes contain a highly conserved catalytic core domain, which can combine with a variety of regulatory subunits to determine substrate specificity and subcellular localization . PP1, PPe domain , 39.Fig.PP1 is a ubiquitously expressed protein serine/threonine phosphatase with three homologous variants of the catalytic subunit  encoded in the human genome. A single catalytic PP1 subunit combines with one out of nearly 200 regulatory subunits to determine substrate specificity and/or sub-cellular localization , 41. In 408, RVFF422) located at the N-terminal and C-terminal ends of the DNA-binding domain of human IRF7, which match canonical PP1-binding motifs. Indeed, PP1 and IRF7 physically interact and PP1 targets four IRF7 key phosphorylation sites  [Focusing on PP1 as a negative regulator of the TLR-mediated inflammatory response, the PP1-GADD34-CUE domain–containing 2 (CUEDC2) complex sequesters IKK-α and IKK-β in the cytoplasm and maintains them in a non-phosphorylated, inactive form. Upon stimulation with a TLR ligand (e.g. LPS), IKK is ‘recruited’ away from the PP1-GADD34-CUEDC2 module by TRAF2/TRAF6 and then is phosphorylated and activated by the TNF receptor signaling complex. Subsequently, when active IKK is released from the TNF receptor signaling complex, IKKs kinase domain binds to the carboxyl terminus of CUEDC2, presumably via TRAF2/6 , 43. Via7, S479) .Surprisingly, PP1γ has also been described as a positive regulator of NF-κB activation. Upon TLR-initiated MyD88 signaling, PP1γ associates with TRAF6 and promotes oligomerization of this ubiquitin E3 ligase . It is sSimilar to PP1, two variants of the catalytic subunit of the serine/threonine phosphatase PP2A, termed PP2Acα and PP2Acβ, are encoded in the human genome. Either of the catalytic subunits forms the central enzyme, which combines with the scaffold subunit (PP2Aa⍺ or PP2Aaβ) and one out of 26 different regulatory subunits to build the holoenzyme complex. Again, the regulatory subunits determine the spatio-temporal specificity of PP2A enzyme activity –54. A neOne negative feed-back-loop that involves PP2A and that dampens TLR-initiated signaling is based on the lipid ceramide, which can be produced from membrane sphingolipids during inflammation , 63. CerInterestingly, PP2Ac⍺ can also associate with and de-phosphorylate serine residues in the TIR domain of MyD88 . Phospho2+/calmodulin-dependent serine/threonine phosphatase, which is also known as Calcineurin. In vertebrates, three genes  encode three related catalytic subunits termed Calcineurin A or PP2BC. The genes PPP3CA and PPP3CB are ubiquitously expressed, while PPP3CC transcripts are only found in testis and brain [2+-binding EF-hand domains, while the catalytic subunit Calcineurin A contains two Calmodulin-binding sites, which are involved in regulating PP2B phosphatase activity in a Ca2+/Calmodulin-dependent manner [PP2B is a Cand brain . In addind brain . The regt manner . Interest manner . Nevertht manner –74. In Tt manner . In addit manner , 77. Thet manner . PP2B alFor instance, PP2B acts as a negative regulator of the TLR signaling pathway in human and murine macrophages, astrocytes, and fibroblasts . In macrIn contrast to these negative regulatory effects of PP2B on TLR signaling and NF-κB mediated gene expression events, several studies have indicated a positive contribution of PP2B to this pathway, which would be analogous to the situation in T-cell receptor signaling. For example, primary macrophages lacking a negative regulator of calcineurin-1 termed RCAN-1 show elevated inflammation in response to bacterial LPS due to enhanced TLR4-MyD88-NF-kB signaling . FurtherTogether, these investigations do not draw a clear picture of PP2B’s involvement in TLR signaling. One should bear in mind that several of these studies to a large extend rely on prolonged use of Calcineurin inhibitors such as Tacrolimus or cyclophilin A. Importantly, knock-out macrophages with genetic ablation of the regulatory subunit Calcineurin B1 indicate that such pharmacological inhibitors can have effects, even when the primary target molecule is not present . TherefoPP4 is a conserved protein serine/threonine phosphatase, which is expressed in all cell types and which is found throughout the cytoplasm and the nucleus, showing enrichment around the centrosome and microtubule nucleation points –90. PP4 However, the role of PP4 in TLR signaling appears to be complex, as numerous examples of PP4′s negative regulatory function in the TLR signaling pathway became apparent. For example, the regulatory subunit R1 of PP4 (PP4R1) physically interacts with TRAF2 and TRAF6 upon LPS stimulation in murine macrophages , 94. PP4PP6, closely related to PP2A and PP4, is a Ser/Thr phosphatase with a bimetallic catalytic center and is expressed in most human cells and tissues , 101. PPProteomic and gene depletion analyses revealed that PP6 specifically down-regulates TAK1 activity by dephosphorylating the phospho-Thr-187 residue in the activation loop of the kinase . As phos2+/Mn2+ dependent phosphatases with a highly conserved core catalytic domain. Importantly, they generally differ from the PPP phosphatases by not forming holoenzymes. Instead, all PPM members possess N-terminal or C-terminal non-enzymatic domains, which contribute to substrate specificity and/or structural stability  and most of them are widely expressed in human tissues , 106. Stity Fig.  [38, 106Generally, PPM phosphatases are involved in diverse cellular processes such as cell proliferation, growth, survival, apoptosis, regulation of metabolism, and stress signaling , 112–116PPM1A is ubiquitously expressed in human tissues and, together with PPM1B, is the PPM family member, which has been studied most intensively with respect to innate immunity.  In the cMycobacterium tuberculosis infection are reduced in PPM1A-overexpressing THP-1 monocytic cells [Likewise, PPM1A also limits TLR-initiated responses, as cytokine release upon LPS stimulation or upon ic cells . In contic cells . These ric cells .The ability of PPM1A to dampen TLR signaling appears to hinge on the dephosphorylation of IKKβ at residues p-Ser-177 and p-Ser-181 . PPM1A aPPM1B is structurally similar to PPM1A and its genetic deletion abrogates embryonic development . PPM1B pPPM1D) is a well-studied oncogenic member of the PPM family. It is rapidly and transiently expressed upon DNA-damaging agents and ionizing or UV irradiation in a p53-dependent manner [The wildtype p53-induced phosphatase 1  . SimilarPPM1M accumulates in the nucleus and its function correlates with other members of the PPM family such as PPM1A and B in selectively acting on IKKβ downstream of TAK1 to suppress IL-1-induced NF-κB activation . AccordiB. abortus infection [PPM1N is one of the least studied members of the PPM family. However, genome-wide investigations revealed that PPM1N expression in bone marrow macrophages was up-regulated upon nfection , suggestBordetella pertussis to block iNOS expression and NO production in TLR-activated macrophages and also exploited by Neisseria gonorrhoeae or Moraxella catarrhalis to block IL-1β production via regulation of the SYK-TLR4-CEACAM1 complex [Listeria monocytogenes directly hijacks PPM1A and PPM1B to dephosphorylate and translocate SIRT2 into the nucleus to promote effective infection [Identifying the involved protein phosphatases and their molecular targets in TLR signaling provides a rational basis to expand existing therapeutic strategies in acute and chronic inflammation as well as autoimmune diseases . A detai complex , 165. Sinfection . Since tIn general, pinpointing the contribution of a particular protein phosphatase to specific cellular events remains a challenge for several reasons: (i) in contrast to protein kinases there is a lack of potent and selective phosphatase inhibitors; (ii) the action of a phosphatase can in many cases only be revealed upon a positive stimulus, hampering the direct evaluation of phosphatase action; (iii) the low complexity and essential function of catalytic subunits, especially in the case of PPPs, often leads to lethal phenotypes in knock-out approaches; and (iv) redundancy between closely related enzymes, as seen for PPMs, often results in similar phenotypes upon overexpression.The absence of highly specific enzyme inhibitors is a major obstacle for the phosphatase field in general. In the past, inhibitors such as ocadaic acid or microcystins have been widely used, but the explanatory power of such findings is often limited as PP1, PP2A, PP4, and PP6, with their extensive homology, are all sensitive to the same class of inhibitors. Thus, it is desirable, but also more demanding, to complement such pharmacologial approaches with genetic manipulation of individual phosphatases. However, one has to be aware that genetic approaches requiring prolonged selection, e.g. generation of clonal knock-out or knock-down cell lines, can trigger compensatory mechanisms that entail altered expression of related enzymes. Such compensatory activity by other, related phosphatases with overlapping substrate spectrum can easily mask a phenotype. Therefore, novel small molecule inhibitors or activators of specific phosphatases that allosterically interfere with enzyme activity of the catalytic subunit are welcome additions to the investigators toolbox , 168. FuGiven the prominent role of protein phosphorylation as a regulatory principle in TLR signaling, it is obvious, that manipulation of phosphatase activity represents a promising strategy to control innate immune responses. As we have discussed, there are numerous examples of protein phosphatases exerting negative regulatory roles in TLR signaling, but the opposite situation, a positive contribution to NF-κB activation, is also known for some phosphatase enzymes. Accordingly, a suite of small molecule stimulators as well as inhibitors of defined protein phosphatases would be ideally suited to allow a transient tuning of the TLR pathway in either direction: towards increased signaling output, when the ultimate goal is to limit infectious agents, or towards dampened responses in a situation of exaggerated or chronic inflammation. Though such approaches are still in their infancy, it is about time to unleash the translational potential of protein phosphatases in innate immunity.


To identify medial open-wedge high tibial osteotomy (MOWHTO) prognostic factors with wedge-shaped spacer implantation (spacer-type MOWHTO) for varus medial compartment knee osteoarthritis.Patients who underwent spacer-type MOWHTO between August 2018 and September 2019 were prospectively enrolled in this study. Patients were divided into effective group and invalid group based on the Western Ontario and McMasters University Osteoarthritis Index (WOMAC) score one year postoperatively. The variables assessed at baseline and one year postoperatively including age, sex, body mass index (BMI), Kellgren–Lawrence (K–L) grade, hip–knee–ankle angle (HKAA), medial proximal tibial angle (MPTA), posterior tibial slope angle (PTSA), Blackburn–Peel index (BPI), duration of symptoms, and WOMAC score were compared. Prognostic factors were analyzed using logistic regression, and the corresponding odds ratios were also calculated.2, K–L grade IV, and pre-HKAA > 10° . Multivariable logistic regression analysis revealed that age > 70 (OR = 4.861) and K–L grade IV (OR = 6.590) were significantly associated with the higher WOMAC score at one year postoperatively.A total of 104 patients were enrolled in the study protocol at one year postoperatively. The WOMAC score decreased from 72.39 ± 12.95 at baseline to 20.06 ± 12.96 at one year postoperatively. Univariate analysis revealed that the significant predictors of the WOMAC score were age > 70 years, BMI > 30 kg/mSpacer-type MOWHTO is an effective treatment for osteoarthritis with varus deformity. The prognostic factors for spacer-type MOWHTO are age and K–L grade. Osteoarthritis (OA) is one of the major causes of mobility disorders and disability and affects over 10% of men and 13% of women older than 60 years . OA is aMOWHTO is generally performed through an oblique incision on the medial side of the tibia while preserving the lateral tibial cortex , 17. ZhaConsidering the advantages of this novel osteotomy method, we hypothesized that the prognostic factors for MOWHTO with spacer implantation (spacer-type MOWHTO) would differ from those for conventional MOWHTO with internal fixation. Thus, spacer-type MOWHTO may have more limited and strict indications than conventional MOWHTO. Additionally, it is unclear whether the results of spacer-type MOWHTO are significantly influenced by the pre- and postoperative radiographic axes. Therefore, it is necessary to define clear prognostic factors for evaluation prior to spacer-type MOWHTO. The objective of this prospective study was to identify the prognostic factors for successful spacer-type MOWHTO.Study approval was obtained from the research ethics committee (QYFYWZLL26146). From August 2018 to September 2019, a total of 111 spacer-type MOWHTO surgeries were performed by a single surgeon. Patient characteristics and surgical data were collected. All patients were asked for their consent to participate in the study before surgery. The inclusion criteria were as follows: (1) spacer-type MOWHTO performed during the aforementioned study period, (2) no history of knee surgery or trauma, (3) unilateral medial compartment knee OA with < 15° varus malalignment on weight-bearing full-leg anteroposterior radiographs, (4) K–L grade II–IV, (5) knee range of motion of more than 20° in extension and 100° in flexion, and (6) minimum follow-up of one year with clinical outcome evaluation. We excluded seven patients. Two patients required a plate to stabilize the tibia during the surgical procedures because of an unstable hinge fracture. Two patients needed reoperation because of tibial fractures due to a fall during the rehabilitation process. In addition, three patients were excluded due to being considered lost to follow-up.The same doctor performed the operation under general anesthesia. The surgical procedure was composed of two main steps: proximal fibular osteotomy and high tibial osteotomy. Proximal fibular osteotomy: A 3-cm longitudinal incision was made approximately four fingerbreadths below the head of the fibula to avoid iatrogenic injury to the peroneal nerve. The proximal fibular shaft was exposed by splitting the septum between the soleus and peroneus. Then, the fibula with a length of approximately 2 cm was cut using a saw blade, and the peroneal nerve was protected from damage by a retractor Fig. a, b. FinPreoperative physical preparation: All patients completed a 3-week preoperative high-intensity isokinetic resistance training program focusing on quadriceps and hamstring strength. Postoperative rehabilitation: Antibiotics were administered within 24 h after surgery. Patients were encouraged to exercise joint function on the bed to avoid joint stiffness and deep vein thrombosis. The isokinetic resistance training program focusing on the quadriceps and hamstring strength was performed on the duration before full weight-bearing was allowed. Partial weight-bearing with crutches was initiated at 6 weeks postoperatively. Full weight-bearing was allowed when there was evidence of bone union at the osteotomy site   score, which consisted of three subscales: pain, function, and stiffness. The WOMAC scores were evaluated preoperatively and one year postoperatively by two orthopedic surgeons who did not participate in the surgery. According to the postoperative total WOMAC scores, patients were divided into two groups: The patients who had a total WOMAC score ≤ 40 points were classified into the effective group and patients who had a total WOMAC score > 40 points were classified into the invalid group. The method and classification criteria have been described in previous research , 21.A radiological evaluation was performed preoperatively and one year postoperatively Fig. . All radt tests. Analysis of categorical or dichotomized data utilized the chi-squared test. Multiple logistic regression analysis was performed for the variables with a P < 0.1 in the univariate analyses. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Intraclass correlation coefficients (ICCs) with 95% CIs were used to evaluate the reproducibility or radiographic measurements, and ICCs > 0.75 were considered to represent excellent agreement. All computations were performed using standard software ; P < 0.05 was considered statistically significant.Normally distributed variables are expressed as the means and standard deviations, and categorical variables are presented as frequencies. The Kolmogorov–Smirnov normality test was employed before statistical analysis to determine whether parametric tests were used. The radiologic measurements between the two groups were compared with independent A total of 104 patients  with a mean age of 61.67 ± 7.71 years (range 46–78 years) were eligible for our study protocol. Further details of the characteristics of the study cohort and the study protocol are shown in Table P < 0.001). According to the classification criteria, 16 (15.4%) patients were classified into the invalid group, and 88 (84.6%) were classified into the effective group. The postoperative HKAA and MPTA were significantly corrected compared with preoperative indicators. All preoperative varus deformities were corrected to a slight valgus or to a neutral position, causing a significant decrease in the varus angle .The WOMAC scores were significantly decreased at one year after surgery compared with the preoperative baseline values   Table . Multival) Table .Table 3CMOWHTO reportedly achieves significant improvements in pain and function, slows down the progression of OA, and postpones the need for TKA , 14, 18.Because of the differences between surgical procedures, the prognostic factors of spacer-type MOWHTO may differ from those of conventional MOWHTO with internal fixation . In our Although the incidence of knee OA was significantly higher in women than in men, our study found that sex was not a significant prognostic factor for spacer-type MOWHTO , 10. FurAge was a significant predictive factor of a poor outcome after spacer-type MOWHTO. For patients older than 70 years, it is difficult to achieve ideal knee functional recovery after surgery. Long-term incomplete weight-bearing (approximately three months) and osteoporosis may be the main causes of muscular atrophy and decreased physical activity , 32, 33.In our study, the outcome of spacer-type MOWHTO was much worse for patients with severe knee OA with a K–L grade of IV than for patients with a K–L grade of II-III. This suggests that the outcome of spacer-type MOWHTO was affected by degeneration of the knee joint. Severe knee lesions such as patellofemoral arthrosis , synovitAlthough BMI was not considered to be an independent risk factor for spacer-type MOWHTO, we thought that patients with a high BMI had a higher risk of surgical complications. The incidence of complications such as loss of valgus correction angle due to loosening and prolapse of the implantation, hinge fracture, persistent postoperative pain and numbness, and reoperation was higher in patients with a high BMI. However, for conventional MOWHTO, the locking compression plate provides reliable stability that enables the cut tibia to bear body weight . Due to Compared with traditional MOWHTO, this novel method has many advantages, such as a lower cost, avoiding the need for secondary surgery for fixation removal, maintaining the patellar height to avoid degeneration of the patellofemoral joint , 38–40, Our study had several limitations that may have influenced the results. First, the follow-up duration was relatively short, with a maximum of 1 year. Thus, the follow-up duration was too short to enable the accurate evaluation of the outcome of the spacer-type MOWHTO but can be used to evaluate the short- or medium-term curative effect. Long-term follow-up studies are needed to confirm the predictive factors affecting the clinical outcomes of spacer-type MOWHTO. Second, fibular osteotomy was performed during spacer-type MOWHTO, which may have impacted the clinical outcomes. However, recent studies have reported good outcomes after fibular osteotomy.The results of our study showed that age and K–L grade are the main prognostic factors associated with postoperative outcomes following spacer-type MOWHTO, which supports our hypothesis. Spacer-type MOWHTO seems most suitable for younger patients with less severe OA of the medial compartment. Therefore, to ensure appropriate patient expectations regarding surgical outcomes, these factors should be considered when choosing between spacer-type MOWHTO or conventional MOWHTO. The patients should be fully informed about the advantages and disadvantages of the two surgical procedures.