Kindlin-2 controls angiogenesis through modulating Notch1 signaling.

Kindlin-2 is critical for development and homeostasis of key organs, including skeleton, liver, islet, etc., yet its role in modulating angiogenesis is unknown. Here, we report that sufficient KINDLIN-2 is extremely important for NOTCH-mediated physiological angiogenesis. The expression of KINDLIN-2 in HUVECs is significantly modulated by angiogenic factors such as vascular endothelial growth factor A or tumor necrosis factor α. A strong co-localization of CD31 and Kindlin-2 in tissue sections is demonstrated by immunofluorescence staining. Endothelial-cell-specific Kindlin-2 deletion embryos die on E10.5 due to hemorrhage caused by the impaired physiological angiogenesis. Experiments in vitro show that vascular endothelial growth factor A-induced multiple functions of endothelial cells, including migration, matrix proteolysis, morphogenesis and sprouting, are all strengthened by KINDLIN-2 overexpression and severely impaired in the absence of KINDLIN-2. Mechanistically, we demonstrate that KINDLIN-2 inhibits the release of Notch intracellular domain through binding to and maintaining the integrity of NOTCH1. The impaired angiogenesis and avascular retinas caused by KINDLIN-2 deficiency can be rescued by DAPT, an inhibitor of γ-secretase which releases the intracellular domain from NOTCH1. Moreover, we demonstrate that high glucose stimulated hyperactive angiogenesis by increasing KINDLIN-2 expression could be prevented by KINDLIN-2 knockdown, indicating Kindlin-2 as a potential therapeutic target in treatment of diabetic retinopathy. Our study for the first time demonstrates the significance of Kindlin-2 in determining Notch-mediated angiogenesis during development and highlights Kindlin-2 as the potential therapeutic target in angiogenic diseases, such as diabetic retinopathy.

Multifunctional Nanoengineered Hydrogels Consisting of Black Phosphorus Nanosheets Upregulate Bone Formation.

Tissue-engineered hydrogels have received extensive attention as their mechanical properties, chemical compositions, and biological signals can be dynamically modified for mimicking extracellular matrices (ECM). Herein, the synthesis of novel double network (DN) hydrogels with tunable mechanical properties using combinatorial screening methods is reported. Furthermore, nanoengineered (NE) hydrogels are constructed by addition of ultrathin 2D black phosphorus (BP) nanosheets to the DN hydrogels with multiple functions for mimicking the ECM microenvironment to induce tissue regeneration. Notably, it is found that the BP nanosheets exhibit intrinsic properties for induced CaP crystal particle formation and therefore improve the mineralization ability of NE hydrogels. Finally, in vitro and in vivo data demonstrate that the BP nanosheets, mineralized CaP crystal nanoparticles, and excellent mechanical properties provide a favorable ECM microenvironment to mediate greater osteogenic cell differentiation and bone regeneration. Consequently, the combination of bioactive chemical materials and excellent mechanical stimuli of NE hydrogels inspire novel engineering strategies for bone-tissue regeneration.

Adenylyl cyclase 1 as a major isoform to generate cAMP signaling for apoA-1-mediated cholesterol efflux pathway.

HDL apoA-1-mediated cholesterol efflux pathway requires multiple cellular proteins and signal transduction processes, including adenylyl cyclase (AC)/cAMP signaling. Due to the existence of multiple transmembrane AC isoforms, it was not known how many AC isoforms are expressed and which ones are essential for cholesterol efflux in macrophage foam cells. These questions were investigated in THP-1 macrophages in this study. Quantitative RT-PCR detected mRNAs for all nine transmembrane AC isoforms, but only the mRNA and protein of the AC1 isoform were consistently upregulated by cholesterol loading and apoA-1. AC1 shRNA interference decreased AC1 mRNA and protein levels, resulting in reduction of apoA-1-mediated cAMP production and cholesterol efflux, while the intracellular cholesterol levels remained high. Confocal microscopy showed that apoA-1 promoted translocation of cholesterol and formation of cholesterol-apoA-1 complexes (protrusions) on the cholesterol-loaded macrophage surface. AC1 shRNA-interfered macrophages showed no translocation of cholesterol to the cell surface. AC1 shRNA interference also disrupted cellular localization of the intracellular cholesterol indicator protein adipophillin, and the expression as well as surface translocation of ABCA1. Together, our results show that AC1 is a major isoform for apoA-1-activated cAMP signaling to promote cholesterol transport and exocytosis to the surface of THP-1 macrophage foam cells.

A novel sprayable thermosensitive hydrogel coupled with zinc modified metformin promotes the healing of skin wound.

A novel sprayable adhesive is established (ZnMet-PF127) by the combination of a thermosensitive hydrogel (Pluronic F127, PF127) and a coordination complex of zinc and metformin (ZnMet). Here we demonstrate that ZnMet-PF127 potently promotes the healing of traumatic skin defect and burn skin injury by promoting cell proliferation, angiogenesis, collagen formation. Furthermore, we find that ZnMet could inhibit reactive oxygen species (ROS) production through activation of autophagy, thereby protecting cell from oxidative stress induced damage and promoting healing of skin wound. ZnMet complex exerts better effects on promoting skin wound healing than ZnCl2 or metformin alone. ZnMet complex also displays excellent antibacterial activity against Staphylococcus aureus or Escherichia coli, which could reduce the incidence of skin wound infections. Collectively, we demonstrate that sprayable PF127 could be used as a new drug delivery system for treatment of skin injury. The advantages of this sprayable system are obvious: (1) It is convenient to use; (2) The hydrogel can cover irregular skin defect sites evenly in a liquid state. In combination with this system, we establish a novel sprayable adhesive (ZnMet-PF127) and demonstrate that it is a potential clinical treatment for traumatic skin defect and burn skin injury.

Protective effects of methyl protodioscin against lipid disorders and liver injury in hyperlipidemic gerbils.

Methyl protodioscin (MPD) is the main component of total diosgenin, which was reported to reduce cholesterol and triglyceride levels potentially. This study aimed to investigate the beneficial effects of MPD against lipid disorder in hyperlipidemic gerbils induced by a high-fat diet (HFD). Hyperlipidemia was induced in gerbils by feeding them with HFD for six weeks, and a daily oral dose of MPD solution (25 and 50 mg/kg/day) was administered. This study investigated blood lipid levels and hepatic lipid accumulation in hyperlipidemic gerbils. The potential mechanism of MPD was explored by detecting the expression level of genes, including SREBPs, ACC, FASN, HMGCR, PCSK9, and LDL-R. The results showed that MPD treatment decreased the body weight, the relative weight of the liver, blood lipid, and hepatic lipid levels of gerbils fed with HFD. The administration of MPD alleviates liver steatosis and injury in gerbils fed with an HFD. MPD treatment reduced the expression of HMGCR, increased the expression of LDL-R, and decreased the expression of PCSK9 for cholesterol reduction. Additionally, MPD treatment reduced the expression of hepatic ACC and FASN for triglycerides reduction. The underlying mechanisms for these effects are attributed to MPD-induced inhibition of protein expression of LXR, SREBP1, and SREBP2. This study demonstrates that MPD protects gerbils against lipid disorders and liver injury by suppressing hepatic SREBPs expression.

Targeting Kindlin-2 in adipocytes increases bone mass through inhibiting FAS/PPARγ/FABP4 signaling in mice.

Osteoporosis (OP) is a systemic skeletal disease that primarily affects the elderly population, which greatly increases the risk of fractures. Here we report that Kindlin-2 expression in adipose tissue increases during aging and high-fat diet fed and is accompanied by decreased bone mass. Kindlin-2 specific deletion (K2KO) controlled by Adipoq-Cre mice or adipose tissue-targeting AAV (AAV-Rec2-CasRx-sgK2) significantly increases bone mass. Mechanistically, Kindlin-2 promotes peroxisome proliferator-activated receptor gamma (PPARγ) activation and downstream fatty acid binding protein 4 (FABP4) expression through stabilizing fatty acid synthase (FAS), and increased FABP4 inhibits insulin expression and decreases bone mass. Kindlin-2 inhibition results in accelerated FAS degradation, decreased PPARγ activation and FABP4 expression, and therefore increased insulin expression and bone mass. Interestingly, we find that FABP4 is increased while insulin is decreased in serum of OP patients. Increased FABP4 expression through PPARγ activation by rosiglitazone reverses the high bone mass phenotype of K2KO mice. Inhibition of FAS by C75 phenocopies the high bone mass phenotype of K2KO mice. Collectively, our study establishes a novel Kindlin-2/FAS/PPARγ/FABP4/insulin axis in adipose tissue modulating bone mass and strongly indicates that FAS and Kindlin-2 are new potential targets and C75 or AAV-Rec2-CasRx-sgK2 treatment are potential strategies for OP treatment.

Talin-1 inhibits Smurf1-mediated Stat3 degradation to modulate ß-cell proliferation and mass in mice.

Insufficient pancreatic ß-cell mass and reduced insulin expression are key events in the pathogenesis of diabetes mellitus (DM). Here we demonstrate the high expression of Talin-1 in ß-cells and that deficiency of Talin-1 reduces ß-cell proliferation, which leads to reduced ß-cell mass and insulin expression, thus causing glucose intolerance without affecting peripheral insulin sensitivity in mice. High-fat diet fed exerbates these phenotypes. Mechanistically, Talin-1 interacts with the E3 ligase smad ubiquitination regulatory factor 1 (Smurf1), which prohibits ubiquitination of the signal transducer and activator of transcription 3 (Stat3) mediated by Smurf1, and ablation of Talin-1 enhances Smurf1-mediated ubiquitination of Stat3, leading to decreased ß-cell proliferation and mass. Furthermore, haploinsufficiency of Talin-1 and Stat3 genes, but not that of either gene, in ß-cell in mice significantly impairs glucose tolerance and insulin expression, indicating that both factors indeed function in the same genetic pathway. Finally, inducible deletion Talin-1 in ß-cell causes glucose intolerance in adult mice. Collectively, our findings reveal that Talin-1 functions as a crucial regulator of ß-cell mass, and highlight its potential as a therapeutic target for DM patients.

Circular RNA circStag1 promotes bone regeneration by interacting with HuR.

Postmenopausal osteoporosis is a common bone metabolic disorder characterized by deterioration of the bone microarchitecture, leading to an increased risk of fractures. Recently, circular RNAs (circRNAs) have been demonstrated to play pivotal roles in regulating bone metabolism. However, the underlying functions of circRNAs in bone metabolism in postmenopausal osteoporosis remain obscure. Here, we report that circStag1 is a critical osteoporosis-related circRNA that shows significantly downregulated expression in osteoporotic bone marrow mesenchymal stem cells (BMSCs) and clinical bone tissue samples from patients with osteoporosis. Overexpression of circStag1 significantly promoted the osteogenic capability of BMSCs. Mechanistically, we found that circStag1 interacts with human antigen R (HuR), an RNA-binding protein, and promotes the translocation of HuR into the cytoplasm. A high cytoplasmic level of HuR led to the activation of the Wnt signaling pathway by stabilizing and enhancing low-density lipoprotein receptor-related protein 5/6 (Lrp5/6) and ß-catenin expression, thereby stimulating the osteogenic differentiation of BMSCs. Furthermore, overexpression of circStag1 in vivo by circStag1-loaded adeno-associated virus (circStag1-AAV) promoted new bone formation, thereby preventing bone loss in ovariectomized rats. Collectively, we show that circStag1 plays a pivotal role in promoting the regeneration of bone tissue via HuR/Wnt signaling, which may provide new strategies to prevent bone metabolic disorders such as postmenopausal osteoporosis.

Promising diagnostic and therapeutic circRNAs for skeletal and chondral disorders.

Circular RNAs (circRNAs) belong to a highly conserved subtype of non-coding RNAs, produced by the back-splicing of specific regions of pre-mRNA. CircRNAs have wide-ranging effects on eukaryotic physiology and pathology by acting as transcription regulators, miRNA sponges, protein sponges, and templates for translation. Skeletal and chondral disorders are the leading causes of pain and disability, especially for elders, affecting hundreds of millions of people worldwide. Plenty of evidence have shown that circRNAs are dysregulated and play vital roles in the occurrence and progression of skeletal and chondral disorders. Herein, we systematically summarize the emerging roles and underlying molecular mechanisms of hub circRNAs in the pathogenesis of several representative skeletal and chondral disorders. Our findings may provide further insight into the mechanistic details of the role of circRNA in bone or cartilage metabolism, and highlight the promising application of circRNAs in serving as potential diagnostic or therapeutic targets for the prevention and treatment of skeletal and chondral disorders.

Robust Underwater Adhesives Based on Dynamic Hydrophilic and Hydrophobic Moieties to Diverse Surfaces.

Underwater adhesives (UAs) have promising applications in diverse areas. However, traditional UAs have several drawbacks such as weak and irreversible adhesion behaviors as well as poor performance in biological environments. To address these challenges, we engineered a novel synthetic adhesive based on dynamic hydrophilic and hydrophobic moieties, which shows very strong underwater adhesion strength (30-110 kPa) and debonding energy (20-100 J/m2) to diverse substrates. Interestingly, the UAs could also be switched reversibly and repeatedly by the dynamic exchange of hydrophilic and hydrophobic moieties under alternating temperatures. We also demonstrate the versatile functions and practical value of the UAs for clinical applications as tissue sealants and hemostatic dressing in emergency rescue operations. This general and efficient strategy may be generalized to develop additional next generation UAs for many emerging technological and medical applications.

PiRNA-63049 inhibits bone formation through Wnt/ß-catenin signaling pathway.

Bone remodeling is a dynamic process between bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts. Disrupted bone remodeling is a key factor in postmenopausal osteoporosis, a metabolic disorder characterized by deteriorated bone microarchitecture and increased risk of fracture. Recent studies have shown that piwi-binding RNA (piRNA) is involved in the pathogenesis of certain diseases at the post-transcriptional level. Here, we analyzed piRNA-63049 (piR-63049), which may play an essential role in bone remodeling. The expression of piR-63049 significantly increased in both bone tissues and plasma of osteoporotic rats and postmenopausal osteoporotic patients. Overexpressing piR-63049 could inhibit the osteoblastogenesis of bone marrow stromal cells (BMSCs) while knocking down piR-63049 could promote the osteoblastogenesis of BMSCs through the Wnt2b/ß-catenin signaling pathway. Moreover, knocking-down piR-63049 (piR-63049-antagonist) in vivo could attenuate the bone loss in ovariectomized rats by promoting bone formation. Taken together, the current study shows that piR-63049 inhibits bone formation through the Wnt2b/ß-catenin signaling pathway. This novel piRNA may be a potential target to increase bone formation in bone loss disorders such as postmenopausal osteoporosis.

Pinch Loss Ameliorates Obesity, Glucose Intolerance, and Fatty Liver by Modulating Adipocyte Apoptosis in Mice.

The mammalian focal adhesion proteins Pinch1/2 activate integrins and promote cell-extracellular matrix adhesion and migration; however, their roles in adipose tissue and metabolism are unclear. Here we find that high-fat diet (HFD) feeding dramatically increases expression of Pinch1/2 proteins in white adipose tissue (WAT) in mice. Furthermore, expression of Pinch1 is largely upregulated in WAT in leptin-deficient ob/ob type 2 diabetic mice and obese humans. While mice with loss of Pinch1 in adipocytes or global Pinch2 do not display any notable phenotypes, deleting Pinch1 in adipocytes and Pinch2 globally significantly decreases body weight and WAT mass, but not brown adipose tissue mass, in HFD-fed, but not normal chow diet-fed, mice. Pinch loss ameliorates HFD-induced glucose intolerance and fatty liver. After HFD challenge, Pinch loss slightly but significantly accelerates energy expenditure. While Pinch loss decreases adipocyte size and alters adipocyte size distribution, it greatly accelerates cell apoptosis primarily in epididymal WAT and to a lesser extent in subcutaneous WAT. In vitro studies demonstrate that Pinch loss accelerates adipocyte apoptosis by activating the Bim/Caspase-8 pathway. In vivo, genetic ablation of Caspase-8 expression in adipocytes essentially abolishes the ameliorating effects of Pinch deficiency on obesity, glucose intolerance, and fatty liver in mice. Thus, we demonstrate a previously unknown function of Pinch in control of adipose mass, glucose, and fat metabolism via modulation of adipocyte apoptosis. We may define a novel target for the prevention and treatment of metabolic diseases, such as obesity and diabetes.

Rationally designed protein cross-linked hydrogel for bone regeneration via synergistic release of magnesium and zinc ions.

The development of recombinant protein cross-linked injectable hydrogels with good mechanical strength and effective drug loading capacity for bone regeneration is extremely attractive and rarely reported. Here, we report the fabrication of a smart hydrogel delivery system by incorporating a rationally designed T4 lysozyme mutant (T4M) to mediate the localized delivery and synergistic release of Mg2+ and Zn2+ for bone repair. Apart from its intrinsic antibacterial properties, T4M bears abundant free amine groups on its surface to function as effective covalent crosslinkers to strengthen the hydrogel network as well as exhibits specific binding affinity to multivalent cations such as Zn2+. Moreover, the integrin receptor-binding Arg-Gly-Asp (RGD) sequence was introduced onto the C-terminus of T4 lysozyme to improve its cellular affinity and further facilitate rapid tissue regeneration. The final composite hydrogel displays excellent injectability, improved mechanical properties, antibacterial activity, and unique bioactivities. The effective loading of Mg2+/Zn2+ in the hydrogels could mediate the sequential and sustained release of Mg2+ and Zn2+, thereby resulting in synergistic enhancement on bone regeneration through modulation of the MAPK signaling pathway. We believe that the strategy proposed in this paper opens up a new route for developing protein cross-linked smart delivery systems for tissue regeneration.