Structural insights into IL-6 signaling inhibition by therapeutic antibodies.

Antibody inhibitors of the interleukin-6 (IL-6) signaling pathway, such as tocilizumab and sarilumab, have been used to treat rheumatoid arthritis, chimeric antigen receptor T cell-induced cytokine storm, and severe COVID-19 pneumonia. Here, we solve the cryogenic electron microscopy structures of sarilumab and tocilizumab in complex with IL-6R to resolutions of 3.2 and 3.3 Å, respectively. These structures reveal that both tocilizumab and sarilumab bind to the D3 domain of IL-6R. The binding surfaces of the two antibodies largely overlap, but the detailed interactions are different. Functional studies of various mutants show results consistent with our structural analysis of the antibodies and IL-6R interactions. Structural comparisons with the IL-6/IL-6R/gp130 complex indicate that sarilumab and tocilizumab probably inhibit IL-6/IL-6R signaling by competing for the IL-6 binding site. In summary, this work reveals the antibody-blocking mechanism of the IL-6 signaling pathway and paves the way for future antibody discovery.

Targeted activation of Nrf2/HO-1 pathway by Corynoline alleviates osteoporosis development.

Oxidative stress is preferentially treated as a risk factor for the development and progression of osteoporosis. Corynoline as a component of Corydalis bungeana Turcz presents antioxidative and anti-inflammatory properties. In the present study, the effects of Corynoline on osteoblasts following hydrogen peroxide (H2O2)-induced injury were evaluated accompanied by the investigation of the molecular mechanisms involved. It was found that Corynoline downregulated the intracellular reactive oxygen species (ROS) generation and restored the osteogenic potential of the disrupted osteoblasts by H2O2 exposure. Furthermore, Corynoline was revealed to activate the Nrf2/HO-1 signaling pathway, while ML385 (an Nrf2 inhibitor) would prevent the Corynoline-mediated positive effects on the disrupted osteoblasts. In terms of the animal experiments, Corynoline treatment contributed to a significantly alleviated bone loss. These findings indicate that Corynoline may significantly attenuate the H2O2-induced oxidative damage of osteoblasts via the Nrf2/HO-1 signaling pathway, providing novel insights to the development of treatments for osteoporosis induced by oxidative injury.

Structural basis of vitamin C recognition and transport by mammalian SVCT1 transporter.

Vitamin C (L-ascorbic acid) is an essential nutrient for human health, and its deficiency has long been known to cause scurvy. Sodium-dependent vitamin C transporters (SVCTs) are responsible for vitamin C uptake and tissue distribution in mammals. Here, we present cryogenic electron microscopy structures of mouse SVCT1 in both the apo and substrate-bound states. Mouse SVCT1 forms a homodimer with each protomer containing a core domain and a gate domain. The tightly packed extracellular interfaces between the core domain and gate domain stabilize the protein in an inward-open conformation for both the apo and substrate-bound structures. Vitamin C binds at the core domain of each subunit, and two potential sodium ions are identified near the binding site. The coordination of sodium ions by vitamin C explains their coupling transport. SVCTs probably deliver substrate through an elevator mechanism in combination with local structural arrangements. Altogether, our results reveal the molecular mechanism by which SVCTs recognize vitamin C and lay a foundation for further mechanistic studies on SVCT substrate transport.

A Novel Anti-Osteoporosis Mechanism of VK2: Interfering with Ferroptosis via AMPK/SIRT1 Pathway in Type 2 Diabetic Osteoporosis.

Type 2 diabetic osteoporosis (T2DOP) is a chronic bone metabolic disease. Compared with traditional menopausal osteoporosis, the long-term high glucose (HG) microenvironment increases patients' risk of fracture and osteonecrosis. We were accumulating evidence that implicated ferroptosis as a pivotal mechanism of glucolipotoxicity-mediated death of osteocytes and osteoblast, a novel form of programmed cell death resulting from uncontrolled lipid peroxidation depending on iron. Vitamin K2 (VK2), a fat-soluble vitamin, is clinically applied to prevent osteoporosis and improve coagulation. This study aimed to clarify the role and mechanism of VK2 in HG-mediated ferroptosis. We established the mouse T2DOP model by intraperitoneal injection of streptozotocin solution and a high-fat and high-sugar diet. We also cultured bone marrow mesenchymal stem cells (BMSCs) in HG to simulate the diabetic environment in vitro. Based on our data, VK2 inhibited HG-mediated bone loss and ferroptosis, the latter manifested by decreased levels of mitochondrial reactive oxygen species, lipid peroxidation, and malondialdehyde and increased glutathione in vitro. In addition, VK2 treatment was capable of restoring bone mass and strengthening the expression of SIRT1, GPX4, and osteogenic markers in the distal femurs. As for further mechanism exploration, we found that VK2 could activate AMPK/SIRT1 signaling, and knockdown of SIRT1 by siRNA prevented the VK2-mediated positive effect in HG-cultured BMSCs. Summarily, VK2 could ameliorate T2DOP through the activation of the AMPK/SIRT1 signaling pathway to inhibit ferroptosis.

Cryo-EM structure of the plant nitrate transporter AtCLCa reveals characteristics of the anion-binding site and the ATP-binding pocket.

Nitrate is one of the major nitrogen sources for most plants. Chloride channel (CLC) proteins mediate the transport and vacuole storage of nitrate in plants, but the structural basis of nitrate transport by plant CLC proteins remains unknown. Here, we solved the cryo-EM structure of ATP-bound Arabidopsis thaliana CLCa (AtCLCa) at 2.8 Å resolution. Structural comparison between nitrate-selective AtCLCa and chloride-selective CLC-7 reveals key differences in the central anion-binding site. We observed that the central nitrate is shifted by ∼1.4 Å from chloride, which is likely caused by a weaker interaction between the anion and Pro160; the side chains of aromatic residues around the central binding site are rearranged to accommodate the larger nitrate. Additionally, we identified the ATP-binding pocket of AtCLCa to be located between the cytosolic cystathionine ß-synthase domains and the N-terminus. The N-terminus may mediate the ATP inhibition of AtCLCa by interacting with both ATP and the pore-forming transmembrane helix. Together, our studies provide insights into the nitrate selectivity and ATP regulation of plant CLCs.

Application of interpretable machine learning algorithms to predict distant metastasis in osteosarcoma.

BACKGROUND: Osteosarcoma is well-established as the most common bone cancer in children and adolescents. Patients with localized disease have different prognoses and management than those with metastasis at the time of diagnosis. The purpose of this study was to explore potential risk factors for metastatic disease. METHODS: The Surveillance, Epidemiology, and End Results (SEER) Program database was used to identify patients diagnosed with osteosarcoma between 2004 and 2015. We developed prediction models for distant metastasis using six machine learning (ML) techniques, including logistic regression (LR), support vector machine (SVM), Gaussian Naive Bayes (GaussianNB), Extreme Gradient Boosting (XGBoost), random forest (RF), and k-nearest neighbor algorithm (kNN). The adaptive synthetic (ADASYN) technique was used to deal with imbalanced data. The Shapley Additive Explanation (SHAP) analysis generated visualized explanations for each patient. Finally, the average precision (AP), sensitivity, specificity, accuracy, F1 score, precision-recall curves, calibration plots, and decision curve analysis (DCA) were conducted to evaluate the models' effectiveness. RESULTS: The six machine learning algorithms achieved AP of 0.661-0.781 for predicting distant metastasis. The RF model yielded the best performance with an accuracy of 71.8 percent and an AP of 0.781 and was highly dependent on tumor size, primary surgery, and age. SHAP analysis provided model-independent interpretation, highlighting significant clinical factors associated with the risk of metastasis in osteosarcoma patients. CONCLUSIONS: An accurate machine learning-based prediction model was established for metastasis in osteosarcoma patients to help clinicians during clinical decision-making.

Structure and Function of Plant and Mammalian TPC Channels.

Two-pore channels (TPCs) belong to the family of voltage-gated tetrameric cation channels and are ubiquitously expressed in organelles of animals and plants. These channels are believed to be evolutionary intermediates between homotetrameric voltage-gated potassium/sodium channels and the four-domain, single subunit, voltage-gated sodium/calcium channels. Each TPC subunit contains 12 transmembrane segments that can be divided into two homologous copies of an S1-S6 Shaker-like 6-TM domain. A functional TPC channel assembles as a dimer - the equivalent of a voltage-gated tetrameric cation channel. The plant TPC channel is localized in the vacuolar membrane and is also called the SV channel for generating the slow vacuolar (SV) current observed long before its molecular identification. Three subfamilies of mammalian TPC channels have been defined - TPC1, 2, and 3 - with the first two being ubiquitously expressed in animals and TPC3 being expressed in some animals but not in humans. Mammalian TPC1 and TPC2 are localized to the endolysosomal membrane and their functions are associated with various physiological processes. TPC3 is localized in the plasma membrane and its physiological function is not well defined.

Therapeutic effect of SIRT3 on glucocorticoid-induced osteonecrosis of the femoral head via intracellular oxidative suppression.

Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a serious complication after long-term or excess administration of clinical glucocorticoids intervention, and the pathogenic mechanisms underlying have not been clarified yet. Oxidative stress is considered as a major cause of bone homeostasis disorder. This study is aimed to explore the potential relevance between SIRT3 and GIONFH, as well as the effect of resveratrol, which has been reported for its role in SIRT3 activation, on dexamethasone-induced oxidative stress and mitochondrial compromise in bone marrow stem cells (BMSCs). In this study, our data showed that SIRT3 level was declined in GIONFH rat femoral head, corresponding to a resultant decrease of SIRT3 expression in dexamethasone-treated BMSCs in vitro. We also found that dexamethasone could result in oxidative injury in BMSCs, and resveratrol treatment reduced this deleterious effect via a SIRT3-dependent manner. Moreover, our results demonstrated that rewarding effect of resveratrol on BMSCs osteogenic differentiation was via activation of AMPK/PGC-1α/SIRT3 axis. Meanwhile, resveratrol administration prevented the occurrence of GIONFH, enhanced SIRT3 expression and reduced oxidative level in GIONFH model rats. Therefore, our study provides basic evidence that SIRT3 may be a promising therapeutic target for GIONFH treatment and resveratrol could be an ideal agent for clinical uses.

Apelin-13 induces mitophagy in bone marrow mesenchymal stem cells to suppress intracellular oxidative stress and ameliorate osteoporosis by activation of AMPK signaling pathway.

Osteoporosis is characterized by impaired bone metabolism. Current estimates show that it affects millions of people worldwide and causes a serious socioeconomic burden. Mitophagy plays key roles in bone marrow mesenchymal stem cells (BMSCs) osteoblastic differentiation, mineralization, and survival. Apelin is an endogenous adipokine that participates in bone homeostasis. This study was performed to determine the role of Apelin in the osteoporosis process and whether it affects mitophagy, survival, and osteogenic capacity of BMSCs in in vitro and in vivo models of osteoporosis. Our results demonstrated that Apelin was down-regulated in ovariectomized-induced osteoporosis rats and Apelin-13 treatment activated mitophagy in BMSCs, ameliorating oxidative stress and thereby reviving osteogenic function via AMPK-α phosphorylation. Besides, Apelin-13 administration restored bone mass and microstructure as well as reinstated mitophagy, enhanced osteogenic function in OVX rats. Collectively, our findings reveal the intrinsic mechanisms underlying Apelin-13 regulation in BMSCs and its potential therapeutic values in the treatment of osteoporosis.

LINC01087 is Highly Expressed in Breast Cancer and Regulates the Malignant Behavior of Cancer Cells Through miR-335-5p/Rock1.

PURPOSE: Long non-coding RNA is involved in the genesis and development of various tumors, and it has been found through database screening that LINC01087 is highly expressed in breast cancer (BC), but mechanisms of LINC01087 in BC are still under investigation. Therefore, this study aimed to study relevant mechanisms of LINC01087 in BC to provide potential therapeutic targets for the disease in clinic practice. PATIENTS AND METHODS: The qRT-PCR assay was applied to determine the LINC01087 expression in BC, and the cell counting kit-8 (CCK8) assay, transwell assay, and flow cytometry were used to analyze the proliferation, apoptosis, and invasion of breast cancer cells (BCCs), respectively. The Western blot assay was used to determine the ROCK1 expression, and the luciferase reporter gene assay, RNA-binding protein immunoprecipitation (RIP), and RNA pull-down assays were applied to study the interaction between LINC01087 and miR-335-5p. Moreover, tumor xenotransplantation was conducted in nude mice to explore the effects of LINC01087 on BCCs. RESULTS: The qRT-PCR assay revealed that the LINC01087 expression in BC tissues was higher than that in corresponding tumor-adjacent tissues, and survival analysis revealed an unfavorable prognosis of patients with high expression of LINC01087. Down-regulation of LINC01087 could slow down the proliferation, invasion, and migration of BCCs and accelerate apoptosis of them in vitro. Luciferase reporter gene assay results revealed that LINC01087 enhanced the expression of ROCK1 by regulating miR-335-5p, and LINC01087 could be adopted as a miR-335-5p sponge to inhibit ROCK1 expression. CONCLUSION: LINC01087 is overexpressed in cases with BC, and patients with high expression of it suffer a poor survival. Furthermore, LINC01087 can act as a miR-335-5p sponge to affect the expression of ROCK1 and affect the invasion and migration of BCCs.

Proanthocyanidins-Mediated Nrf2 Activation Ameliorates Glucocorticoid-Induced Oxidative Stress and Mitochondrial Dysfunction in Osteoblasts.

The widespread use of therapeutic glucocorticoids has increased the frequency of glucocorticoid-induced osteoporosis (GIOP). One of the potential pathological processes of GIOP is an increased level of oxidative stress and mitochondrial dysfunction, which eventually leads to osteoblast apoptosis. Proanthocyanidins (PAC) are plant-derived antioxidants that have therapeutic potential against GIOP. In our study, a low dose of PAC was nontoxic to healthy osteoblasts and restored osteogenic function in dexamethasone- (Dex-) treated osteoblasts by suppressing oxidative stress, mitochondrial dysfunction, and apoptosis. Mechanistically, PAC neutralized Dex-induced damage in the osteoblasts by activating the Nrf2 pathway, since silencing Nrf2 partly eliminated the protective effects of PAC. Furthermore, PAC injection restored bone mass and promoted the expression of Nrf2 in the distal femur of Dex-treated osteoporotic rats. In summary, PAC protect osteoblasts against Dex-induced oxidative stress and mitochondrial dysfunction via the Nrf2 pathway activation and may be a promising drug for treating GIOP.

Vitamin K2 Can Rescue the Dexamethasone-Induced Downregulation of Osteoblast Autophagy and Mitophagy Thereby Restoring Osteoblast Function In Vitro and In Vivo.

Chronic long-term glucocorticoids (GC) use is associated with glucocorticoid-induced osteoporosis (GIOP) by inhibiting the survival and impairing the functions of osteoblasts. Autophagy and mitophagy play key roles in osteoblast differentiation, mineralization and survival, and mounting evidence have implicated osteoblast autophagy and mitophagy as a novel mechanism in the pathogenesis of GIOP. Vitamin K2 (VK2) is an essential nutrient supplement that have been shown to exert protective effects against osteoporotic bone loss including GIOP. In this study, we showed that the glucocorticoid dexamethasone (Dex) deregulated osteoblast autophagy and mitophagy by downregulating the expression of autophagic and mitophagic markers LC3-II, PINK1, Parkin. This consequently led to inhibition of osteoblast differentiation and mineralization function in vitro. Interestingly, co-treatment with VK2 significantly attenuated the Dex-induced downregulation of LC3-II, PINK1, Parkin, thereby restoring autophagic and mitophagic processes and normal osteoblastic activity. In addition, using an established rat model of GIOP, we showed that VK2 administration can protect rats against the deleterious effects of Dex on bone by reinstating autophagic and mitophagic activities in bone tissues. Collectively, our results provide new insights into the role of osteoblast autophagy and mitophagy in GIOP. Additionally, the use of VK2 supplementation to augment osteoblast autophagy/mitophagy may significantly improve clinical outcomes of GIOP patients.

Structural insights into the Ca2+-dependent gating of the human mitochondrial calcium uniporter.

Mitochondrial Ca2+ uptake is mediated by an inner mitochondrial membrane protein called the mitochondrial calcium uniporter. In humans, the uniporter functions as a holocomplex consisting of MCU, EMRE, MICU1 and MICU2, among which MCU and EMRE form a subcomplex and function as the conductive channel while MICU1 and MICU2 are EF-hand proteins that regulate the channel activity in a Ca2+-dependent manner. Here, we present the EM structures of the human mitochondrial calcium uniporter holocomplex (uniplex) in the presence and absence of Ca2+, revealing distinct Ca2+ dependent assembly of the uniplex. Our structural observations suggest that Ca2+ changes the dimerization interaction between MICU1 and MICU2, which in turn determines how the MICU1-MICU2 subcomplex interacts with the MCU-EMRE channel and, consequently, changes the distribution of the uniplex assemblies between the blocked and unblocked states.

Aluminosilicate Nanotubes Embedded Polyamide Thin Film Nanocomposite Forward Osmosis Membranes with Simultaneous Enhancement of Water Permeability and Selectivity.

Nanocomposite membranes are strongly desired to break a trade-off between permeability and selectivity. This work reports new thin film nanocomposite (TFN) forward osmosis (FO) membranes by embedding aluminosilicate nanotubes (ANTs) into a polyamide (PA) rejection layer. The surface morphology and structure of the TFN FO membranes were carefully characterized by FTIR, XPS, FESEM and AFM. The ANTs incorporated PA rejection layers exhibited many open and broad "leaf-like" folds with "ridge-and-valley" structures, high surface roughness and relatively low cross-linking degree. Compared with thin film composite (TFC) membrane without ANTs, the TFN membrane with only 0.2 w/v% ANTs loading presented significantly improved FO water permeability, selectivity and reduced structural parameters. This promising performance can be mainly contributed to the special ANTs embedded PA rejection layer, where water molecules preferentially transport through the nanochannels of ANTs. Molecular dynamic simulation further proved that water molecules have much larger flux through the nanotubes of ANTs than sodium and chloride ions, which are attributed to the intrinsic hydrophilicity of ANTs and low external force for water transport. This work shows that these TFN FO membranes with ANTs decorated PA layer are promising in desalination applications due to their simultaneously enhanced permeability and selectivity.

Structural Mechanism of EMRE-Dependent Gating of the Human Mitochondrial Calcium Uniporter.

Mitochondrial calcium uptake is crucial to the regulation of eukaryotic Ca2+ homeostasis and is mediated by the mitochondrial calcium uniporter (MCU). While MCU alone can transport Ca2+ in primitive eukaryotes, metazoans require an essential single membrane-spanning auxiliary component called EMRE to form functional channels; however, the molecular mechanism of EMRE regulation remains elusive. Here, we present the cryo-EM structure of the human MCU-EMRE complex, which defines the interactions between MCU and EMRE as well as pinpoints the juxtamembrane loop of MCU and extended linker of EMRE as the crucial elements in the EMRE-dependent gating mechanism among metazoan MCUs. The structure also features the dimerization of two MCU-EMRE complexes along an interface at the N-terminal domain (NTD) of human MCU that is a hotspot for post-translational modifications. Thus, the human MCU-EMRE complex, which constitutes the minimal channel components among metazoans, provides a framework for future mechanistic studies on MCU.

Structural and functional characterization of an otopetrin family proton channel.

The otopetrin (OTOP) proteins were recently characterized as proton channels. Here we present the cryo-EM structure of OTOP3 from Xenopus tropicalis (XtOTOP3) along with functional characterization of the channel. XtOTOP3 forms a homodimer with each subunit containing 12 transmembrane helices that can be divided into two structurally homologous halves; each half assembles as an α-helical barrel that could potentially serve as a proton conduction pore. Both pores open from the extracellular half before becoming occluded at a central constriction point consisting of three highly conserved residues - Gln232/585-Asp262/Asn623-Tyr322/666 (the constriction triads). Mutagenesis shows that the constriction triad from the second pore is less amenable to perturbation than that of the first pore, suggesting an unequal contribution between the two pores to proton transport. We also identified several key residues at the interface between the two pores that are functionally important, particularly Asp509, which confers intracellular pH-dependent desensitization to OTOP channels.

Structural mechanisms of phospholipid activation of the human TPC2 channel.

Mammalian two-pore channels (TPCs) regulate the physiological functions of the endolysosome. Here we present cryo-EM structures of human TPC2 (HsTPC2), a phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2)-activated, Na+ selective channel, in the ligand-bound and apo states. The apo structure captures the closed conformation, while the ligand-bound form features the channel in both open and closed conformations. Combined with functional analysis, these structures provide insights into the mechanism of PI(3,5)P2-regulated gating of TPC2, which is distinct from that of TPC1. Specifically, the endolysosome-specific PI(3,5)P2 binds at the first 6-TM and activates the channel - independently of the membrane potential - by inducing a structural change at the pore-lining inner helix (IS6), which forms a continuous helix in the open state but breaks into two segments at Gly317 in the closed state. Additionally, structural comparison to the voltage-dependent TPC1 structure allowed us to identify Ile551 as being responsible for the loss of voltage dependence in TPC2.

Ruthenium(II) salicylate complexes inducing ROS-mediated apoptosis by targeting thioredoxin reductase.

Thioredoxin reductase (TrxR), a major component of the thioredoxin system, makes a critical role in regulating cellular redox signaling and is found to be overexpressed in many human cancer cells. TrxR has become an attractive target for anticancer agents. In this work, three Ru(II) complexes with salicylate as ligand, [Ru(phen)2(SA)] (phen = 1,10-phenanthroline, SA = salicylate, 1), [Ru(dmb)2(SA)] (dmb = 4,4'-dimethyl-2,2'-bipyridine, 2) and [Ru(bpy)2(SA)] (bpy = 2,2'-bipyridine, 3), were synthesized and characterized. The anticancer effect exerted by them was evaluated. Complex 1 was found to exhibit obvious anticancer activity, in comparison with cisplatin, against cancer cell lines, while displaying low toxicity to the normal cell line BEAS-2B. The mechanism of complex 1 cancer cell growth suppress was investigated in A549 cells. Complex 1 exerted its anticancer through inducing apoptosis and triggering cell cycle arrest at the G0/G1 phase. Complex 1 can selectively inhibit TrxR activity and thus promote the generation and accumulation of reactive oxygen species (ROS), which subsequently trigger mitochondrial dysfunction and DNA damage, activate oxidative stress-sensitive mitogen activated protein kinase (MAPK), and suppress the protein kinase B (PKB or AKT) signal pathway, resulting in apoptosis in A549 cells.

Combined treatment with vitamin K2 and PTH enhanced bone formation in ovariectomized rats and increased differentiation of osteoblast in vitro.

Osteoporosis is accompanied by insufficient osteogenic capacity. Several lines of evidence suggested that solutions to enhance osteoblastogenesis were important strategies for osteoporotic bone defect repair. This study investigated the effect of combined treatment with vitamin K2 and PTH on bone formation in calvarial bone defect in osteoporotic rats and its influence on osteoblast in vitro. Bilateral ovariectomy was used in SPF Sprague Dawley rats to generate an osteoporosis model. Subsequently, a calvarial defect model was established and all osteoporotic rats were randomly assigned to the following groups: control, VK (vitamin K2, 30 mg/kg everyday), PTH (recombinant human PTH (1-34), 60 µg/kg, three times a week) or VK + PTH (vitamin K2, 30 mg/kg everyday plus PTH, 60 µg/kg three times a week) for 8 weeks. In vitro, bone marrow-derived stem cells (BMSCs) were cultured and treated with vitamin K2, PTH or vitamin K2+PTH. ALP staining and western blot were performed to observe the influence of combined treatment on BMSCs. Bone formation within calvarial defect were assessed by serum γ-carboxylated osteocalcin (Gla-OC), micro-CT, histological and immunofluorescent labeling. In this study, combined treatment of PTH and vitamin K2 showed positive effects on preventing bone loss in femurs in OVX rats. Combined treatment increased serum Gla-OC and promoted bone formation in osteoporotic calvarial bone defects. Immunohistochemistry showed that OCN and RUNX2 were more highly expressed in the VK + PTH group than in the control groups. In vitro studies results suggested that combined treatment with PTH and vitamin K2 increased expression of ALP, BMP2 and RUNX2 in BMSCs. Our data suggested that the combination of vitamin K2 and PTH increased differentiation of osteoblast and had a synergistic effect on bone formation in osteoporotic calvarial bone defect.

Effects of combined menaquinone-4 and PTH1-34 treatment on osetogenesis and angiogenesis in calvarial defect in osteopenic rats.

PURPOSE: The aim of this study was to evaluate the effect of combining human parathyroid hormone (1-34) (PTH1-34; PTH) and menaquinone-4 (MK-4) on calvarial bone defect repair in osteopenic rats. METHODS: Fourteen week olds were subject to craniotomy for the establishment of osteopenic animal models fed through a chronically low-protein diet. After that, critical calvarial defect model was established and all rats were randomly divided into four groups: sham, MK-4, PTH, and PTH + MK-4. The animals received MK-4 (30 mg/kg/day), PTH1-34 (60 µg/kg, three times a week), or PTH1-34 (60 µg/kg, three times a week) plus MK-4 (30 mg/kg/day) for 8 weeks, respectively. Serum γ-carboxylated osteocalcin (Gla-OC) levels, histological and immunofluorescent labeling were employed to evaluate the bone formation and mineralization in calvarial bone defect. In addition, Microfil perfusion, immunohistochemical, and micro-CT suggested enhanced angiogenesis and bone formation in calvarial bone healing. RESULTS: In this study, treatment with either PTH1-34 or MK-4 promoted bone formation and vascular formation in calvarial bone defects compared with the sham group. In addition, combined treatment of PTH1-34 plus MK-4 increased serum level of Gla-OC, improved vascular number and vascular density, and enhanced bone formation in calvarial bone defect in osteopenic conditions as compared with monotherapy. CONCLUSIONS: In summary, this study indicated that PTH1-34 plus MK-4 combination therapy accelerated bone formation and angiogenesis in calvarial bone defects in presence of osteopenia.