Bone-targeting delivery of platelet lysate exosomes ameliorates glucocorticoid-induced osteoporosis by enhancing bone-vessel coupling.

BACKGROUND: Glucocorticoids (GCs) overuse is associated with decreased bone mass and osseous vasculature destruction, leading to severe osteoporosis. Platelet lysates (PL) as a pool of growth factors (GFs) were widely used in local bone repair by its potent pro-regeneration and pro-angiogenesis. However, it is still seldom applied for treating systemic osteopathia due to the lack of a suitable delivery strategy. The non-targeted distribution of GFs might cause tumorigenesis in other organs. RESULTS: In this study, PL-derived exosomes (PL-exo) were isolated to enrich the platelet-derived GFs, followed by conjugating with alendronate (ALN) grafted PEGylated phospholipid (DSPE-PEG-ALN) to establish a bone-targeting PL-exo (PL-exo-ALN). The in vitro hydroxyapatite binding affinity and in vivo bone targeting aggregation of PL-exo were significantly enhanced after ALN modification. Besides directly modulating the osteogenic and angiogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and endothelial progenitor cells (EPCs), respectively, PL-exo-ALN also facilitate their coupling under GCs' stimulation. Additionally, intravenous injection of PL-exo-ALN could successfully rescue GCs induced osteoporosis (GIOP) in vivo. CONCLUSIONS: PL-exo-ALN may be utilized as a novel nanoplatform for precise infusion of GFs to bone sites and exerts promising therapeutic potential for GIOP.

Inhibition of PLA2G4E/cPLA2 promotes survival of random skin flaps by alleviating Lysosomal membrane permeabilization-Induced necroptosis.

Necrosis that appears at the ischemic distal end of random-pattern skin flaps increases the pain and economic burden of patients. Necroptosis is thought to contribute to flap necrosis. Lysosomal membrane permeabilization (LMP) plays an indispensable role in the regulation of necroptosis. Nonetheless, the mechanisms by which lysosomal membranes become leaky and the relationship between necroptosis and lysosomes are still unclear in ischemic flaps. Based on Western blotting, immunofluorescence, enzyme-linked immunosorbent assay, and liquid chromatography-mass spectrometry (LC-MS) analysis results, we found that LMP was presented in the ischemic distal portion of random-pattern skin flaps, which leads to disruption of lysosomal function and macroautophagic/autophagic flux, increased necroptosis, and aggravated necrosis of the ischemic flaps. Moreover, bioinformatics analysis of the LC-MS results enabled us to focus on the role of PLA2G4E/cPLA2 (phospholipase A2, group IVE) in LMP of the ischemic flaps. In vivo inhibition of PLA2G4E with an adeno-associated virus vector attenuated LMP and necroptosis, and promoted flap survival. In addition, microRNA-seq helped us determine that Mir504-5p was differentially expressed in ischemic flaps. A string of in vitro and in vivo tests was employed to verify the inhibitory effect of Mir504-5p on PLA2G4E, LMP and necroptosis. Finally, we concluded that the inhibition of PLA2G4E by Mir504-5p reduced LMP-induced necroptosis, thereby promoting the survival of random-pattern skin flaps.Abbreviations: AAV: adeno-associated virus; ACTA2/α;-SMA: actin alpha 2, smooth muscle, aorta; ALOX15/12/15-LOX: arachidonate 15- lipoxygenase; c-CASP8: cleaved caspase; c-CASP3: cleaved caspase 3; CTSD: cathepsin D; CTSB: cathepsin B; CTSL: cathepsin L; DMECs: primary mouse dermal microvascular endothelial cells; ELISA: enzyme-linked immunosorbent assay; F-CHP: 5-FAM-conjugated collagen hybridizing peptide; FISH: fluorescence in situ hybridization; HUVECs: human umbilical vein endothelial cells; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; LC-MS: liquid chromatography-mass spectrometry; LDBF: laser doppler blood flow; LMP: lysosomal membrane permeabilization; LPE: lysophosphatidylethanolamine; LPC: lysophosphatidylcholine; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MLKL: mixed lineage kinase domain-like; NDI: N-dodecylimidazole; PECAM1/CD31: platelet/endothelial cell adhesion molecule 1; PLA2G4A/cPLA2: phospholipase A2, group IVA (cytosolic, calcium-dependent); PLA2G4E/cPLA2: phospholipase A2, group IVE; qPCR: quantitative real-time polymerase chain reaction; RIPK1: receptor (TNFRSF)-interacting serine-threonine kinase 1; RIPK3: receptor-interacting serine-threonine kinase 3; RISC: RNA-induced silencing complex; ROS: reactive oxygen species; shRNA: short hairpin RNA; SQSTM1: sequestosome 1; TBHP: tert-butyl hydroperoxide; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labelling.

Wright's Technique with the Addition of Visualized Axial Cortical Windows in Odontoid Fractures.

This study sought to investigate and evaluate a modified axial translaminar screw fixation for treating odontoid fractures. We performed a retrospective study at Wenzhou Medical University Affiliated Second Hospital between March 2016 and June 2018. We retrospectively collected and analyzed the medical records of 23 cases with odontoid fractures. All patients were identified as type II odontoid fractures without neurological deficiency and serious diseases following the classification of Anderson. The average age, gender ratio, and body mass index (BMI) were 54.3 ± 11.1 years, 12 men to 11 women, and 22.6 ± 2.4 kg/m2 , respectively. Patients in this study accepted screw fixation using our modified axial translaminar screw fixation combined with atlas pedicle or lateral mass screw fixation. Within the technique, a small cortical "window" was dug in the middle of the axial contralateral lamina, such that the screws in the lamina were visualized to prevent incorrectly implanting the posterior spinal canal through the visualized "window." A total of 46 bone screws were accurately inserted into the axial lamina without using fluoroscopy. The length of all translaminar screws ranged between 26 and 30 mm, while the diameter was 3.5 mm. During the follow-up survey, the visual analog scale (VAS) and neck disability index (NDI) were measured. We provide a simple modification of Wright's elegant technique with the addition of "visualized windows" at the middle of the axial lamina. In all patients, screws were inserted accurately without bony breach and the screw angle was 56.1 ± 3.0°. Mean operative time was 102 ± 28 min with an average blood loss of 50 ± 25 mL. Postoperative hemoglobin and mean length of hospital stay were 12.0 ± 1.4 g/dL and 10.4 ± 3.4 days, respectively. The average follow-up time of all cases was 14.7 months and no internal fixation displacement, loosening, or breakage was found. All patients with odontoid fractures reported being satisfied with the treatment during the recheck period and good clinical outcomes were observed. At 1, 6, and 12 months, NDI and VAS showed that the symptoms of neck pain and limitations of functional disability improved significantly during follow-up. Our results suggest that the modified translaminar screw fixation technique can efficiently treat Anderson type II odontoid fracture, followed by the benefits of less soft tissue dissection, simple operation, no fluoroscopy, and accurate placement of screws.

Trehalose Augments Neuron Survival and Improves Recovery from Spinal Cord Injury via mTOR-Independent Activation of Autophagy.

Spinal cord injury (SCI) is a major cause of irreversible nerve injury and leads to serious tissue loss and neurological dysfunction. Thorough investigation of cellular mechanisms, such as autophagy, is crucial for developing novel and effective therapeutics. We administered trehalose, an mTOR-independent autophagy agonist, in SCI rats suffering from moderate compression injury to elucidate the relationship between autophagy and SCI and evaluate trehalose's therapeutic potential. 60 rats were divided into 4 groups and were treated with either control vehicle, trehalose, chloroquine, or trehalose + chloroquine 2 weeks prior to administration of moderate spinal cord crush injury. 20 additional sham rats were treated with control vehicle. H&E staining, Nissl staining, western blot, and immunofluorescence studies were conducted to examine nerve morphology and quantify autophagy and mitochondrial-dependent apoptosis at various time points after surgery. Functional recovery was assessed over a period of 4 weeks after surgery. Trehalose promotes autophagosome recruitment via an mTOR-independent pathway, enhances autophagy flux in neurons, inhibits apoptosis via the intrinsic mitochondria-dependent pathway, reduces lesion cavity expansion, decreases neuron loss, and ultimately improves functional recovery following SCI (all p < 0.05). Furthermore, these effects were diminished upon administration of chloroquine, an autophagy flux inhibitor, indicating that trehalose's beneficial effects were due largely to activation of autophagy. This study presents new evidence that autophagy plays a critical neuroprotective and neuroregenerative role in SCI, and that mTOR-independent activation of autophagy with trehalose leads to improved outcomes. Thus, trehalose has great translational potential as a novel therapeutic agent after SCI.

AAV2-mediated and hypoxia response element-directed expression of bFGF in neural stem cells showed therapeutic effects on spinal cord injury in rats.

Neural stem cell (NSCs) transplantation has been one of the hot topics in the repair of spinal cord injury (SCI). Fibroblast growth factor (FGF) is considered a promising nerve injury therapy after SCI. However, owing to a hostile hypoxia condition in SCI, there remains a challenging issue in implementing these tactics to repair SCI. In this report, we used adeno-associated virus 2 (AAV2), a prototype AAV used in clinical trials for human neuron disorders, basic FGF (bFGF) gene under the regulation of hypoxia response element (HRE) was constructed and transduced into NSCs to yield AAV2-5HRE-bFGF-NSCs. Our results showed that its treatment yielded temporally increased expression of bFGF in SCI, and improved scores of functional recovery after SCI compared to vehicle control (AAV2-5HRE-NSCs) based on the analyses of the inclined plane test, Basso-Beattie-Bresnahan (BBB) scale and footprint analysis. Mechanistic studies showed that AAV2-5HRE-bFGF-NSCs treatment increased the expression of neuron-specific neuronal nuclei protein (NeuN), neuromodulin GAP43, and neurofilament protein NF200 while decreased the expression of glial fibrillary acidic protein (GFAP) as compared to the control group. Further, the expressions of autophagy-associated proteins LC3-II and Beclin 1 were decreased, whereas the expression of P62 protein was increased in AAV2-5HRE-bFGF-NSCs treatment group. Taken together, our data indicate that AAV2-5HRE-bFGF-NSCs treatment improved the recovery of SCI rats, which is accompanied by evidence of nerve regeneration, and inhibition of SCI-induced glial scar formation and cell autophagy. Thus, this study represents a step forward towards the potential use of AAV2-5HRE-bFGF-NSCs for future clinical trials of SCI repair.

Molecular structure, gene expression and functional role of WFDC1 in angiogenesis and cancer.

Whey acidic proteins (WAP) perform a diverse range of important biological functions, including proteinase activity, calcium transport and bacterial growth. The WAP four-disulphide core domain protein 1 (WFDC1) gene (also called PS20), encodes the 20 kDa prostate stromal protein (ps20), which is a member of the WAP-type four-disulphide core domain family of proteins, and exhibits characteristics of serine protease inhibitors, such as elafin and secretory leukocyte protease inhibitor. Molecular structural analysis reveals that ps20 consists of four-disulphide bonds formed by eight cysteine residues located at the carboxyl terminus of the protein. Wfdc1-null mice were found to display no overt developmental phenotype, suggesting a dispensable role in organ growth and development. However, WFDC1 was able to mediate endothelial cell migration and pericyte stabilization, which are vital for the formation of functional vascular structures. WFDC1 was also found to be downregulated in cancers and exhibited a regulatory effect on cell proliferation. In addition, it was involved in the modulation of memory T cells during human immunodeficiency virus infection. Gaining a solid understanding of the mechanisms by which WFDC1 regulates tissue homeostasis and disease processes, in a tissue specific manner, will be an important move towards the development of WFDC1/ps20 as potential therapeutic targets.

Exenatide improves random-pattern skin flap survival via TFE3 mediated autophagy augment.

Random-pattern skin flaps are widely applied to rebuild and restore soft-tissue damage in reconstructive surgery; however, ischemia and subsequent ischemia-reperfusion injury lead to flap necrosis and are major complications. Exenatide, a glucagon-like peptide-1 analog, exerts therapeutic benefits for diabetic wounds, cardiac injury, and nonalcoholic fatty liver disease. Furthermore, Exenatide is a known activator of autophagy, which is a complex process of subcellular degradation that may enhance the viability of random skin flaps. In this study, we explored whether exenatide can improve skin flap survival. Our results showed that exenatide augments autophagy, increases flap viability, enhances angiogenesis, reduces oxidative stress, and alleviates pyroptosis. Coadministration of exenatide with 3-methyladenine and chloroquine, potent inhibitors of autophagy, reversed the beneficial effects, suggesting that the therapeutic benefits of exenatide for skin flaps are due largely to autophagy activation. Mechanistically, we identified that exenatide enhanced activation and nuclear translocation of TFE3, which leads to autophagy activation. Furthermore, we found that exenatide activates the AMPK-SKP2-CARM1 and AMPK-mTOR signaling pathways, which likely lead to exenatide's effects on activating TFE3. Overall, our findings suggest that exenatide may be a potent therapy to prevent flap necrosis, and we also reveal novel mechanistic insight into exenatide's effect on flap survival.

Molecular structure and function of microfibrillar-associated proteins in skeletal and metabolic disorders and cancers.

Microfibrillar-associated proteins (MFAPs) are extracellular matrix glycoproteins, which play a role in microfibril assembly, elastinogenesis, and tissue homeostasis. MFAPs consist of five subfamily members, including MFAP1, MFAP2, MFAP3, MFAP4, and MFAP5. Among these, MFAP2 and MFAP5 are most closely related, and exhibit very limited amino acid sequence homology with MFAP1, MFAP3, and MFAP4. Gene expression profiling analysis reveals that MFAP2, MFAP5, and MFAP4 are specifically expressed in osteoblastic like cells, whereas MFAP1 and MFAP3 are more ubiquitously expressed, indicative of their diverse role in the tropism of tissues. Molecular structural analysis shows that each MFAP family member has distinct features, and functional evidence reveals discrete purposes of individual MFAPs. Animal studies indicate that MFAP2-deficient mice exhibit progressive osteopenia with elevated receptor activator of NF-κB ligand (RANKL) expression, whereas MFAP5-deficient mice are neutropenic, and MFAP4-deficient mice displayed emphysema-like pathology and the impaired formation of neointimal hyperplasia. Emerging data also suggest that MFAPs are involved in cancer progression and fat metabolism. Further understanding of tissue-specific pathophysiology of MFAPs might offer potential novel therapeutic targets for related diseases, such as skeletal and metabolic disorders, and cancers.

TFE3, a potential therapeutic target for Spinal Cord Injury via augmenting autophagy flux and alleviating ER stress.

Background and Aim: Increasing evidence suggests that spinal cord injury (SCI)-induced defects in autophagic flux may contribute to an impaired ability for neurological repair following injury. Transcription factor E3 (TFE3) plays a crucial role in oxidative metabolism, lysosomal homeostasis, and autophagy induction. Here, we investigated the role of TFE3 in modulating autophagy following SCI and explored its impact on neurological recovery. Methods: Histological analysis via HE, Nissl and Mason staining, survival rate analysis, and behavioral testing via BMS and footprint analysis were used to determine functional recovery after SCI. Quantitative real-time polymerase chain reaction, Western blotting, immunofluorescence, TUNEL staining, enzyme-linked immunosorbent assays, and immunoprecipitation were applied to examine levels of autophagy flux, ER-stress-induced apoptosis, oxidative stress, and AMPK related signaling pathways. In vitro studies using PC12 cells were performed to discern the relationship between ROS accumulation and autophagy flux blockade. Results: Our results showed that in SCI, defects in autophagy flux contributes to ER stress, leading to neuronal death. Furthermore, SCI enhances the production of reactive oxygen species (ROS) that induce lysosomal dysfunction to impair autophagy flux. We also showed that TFE3 levels are inversely correlated with ROS levels, and increased TFE3 levels can lead to improved outcomes. Finally, we showed that activation of TFE3 after SCI is partly regulated by AMPK-mTOR and AMPK-SKP2-CARM1 signaling pathways. Conclusions: TFE3 is an important regulator in ROS-mediated autophagy dysfunction following SCI, and TFE3 may serve as a promising target for developing treatments for SCI.

Endothelial cells produce angiocrine factors to regulate bone and cartilage via versatile mechanisms.

Blood vessels are conduits distributed throughout the body, supporting tissue growth and homeostasis by the transport of cells, oxygen and nutrients. Endothelial cells (ECs) form the linings of the blood vessels, and together with pericytes, are essential for organ development and tissue homeostasis through producing paracrine signalling molecules, called angiocrine factors. In the skeletal system, ECs - derived angiocrine factors, combined with bone cells-released angiogenic factors, orchestrate intercellular crosstalk of the bone microenvironment, and the coupling of angiogenesis-to-osteogenesis. Whilst the involvement of angiogenic factors and the blood vessels of the skeleton is relatively well established, the impact of ECs -derived angiocrine factors on bone and cartilage homeostasis is gradually emerging. In this review, we survey ECs - derived angiocrine factors, which are released by endothelial cells of the local microenvironment and by distal organs, and act specifically as regulators of skeletal growth and homeostasis. These may potentially include angiocrine factors with osteogenic property, such as Hedgehog, Notch, WNT, bone morphogenetic protein (BMP), fibroblast growth factor (FGF), insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF). Understanding the versatile mechanisms by which ECs-derived angiocrine factors orchestrate bone and cartilage homeostasis, and pathogenesis, is an important step towards the development of therapeutic potential for skeletal diseases.

The role of glial cell line-derived neurotrophic factor family member artemin in neurological disorders and cancers.

Artemin (ARTN) is a member of the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), which encompasses family members, GDNF, neurturin (NRTN) and persephin (PSPN). ARTN is also referred to as Enovin or Neublastin, and bears structural characteristics of the TGF-ß superfamily. ARTN contains a dibasic cleavage site (RXXR) that is predicted to be cleaved by furin to yield a carboxy-terminal 113 amino acid mature form. ARTN binds preferentially to receptor GFRα3, coupled to a receptor tyrosine kinase RET, forming a signalling complex for the regulation of intracellular pathways that affect diverse outcomes of nervous system development and homoeostasis. Standard signalling cascades activated by GFLs via RET include the phosphorylation of mitogen-activated protein kinase or MAPK (p-ERK, p-p38 and p-JNK), PI3K-AKT and Src. Neural cell adhesion molecule (NCAM) is an alternative signalling receptor for ARTN in the presence of GFRα1, leading to activation of Fyn and FAK. Further, ARTN also interacts with heparan sulphate proteoglycan syndecan-3 and mediates non-RET signalling via activation of Src kinases. This review discusses the role of ARTN in spinal cord injury, neuropathic pain and other neurological disorders. Additionally, ARTN plays a role in non-neuron tissues, such as the formation of Peyer's patch-like structures in the lymphoid tissue of the gut. The emerging role of ARTN in cancers and therapeutic resistance to cancers is also explored. Further research is necessary to determine the function of ARTN in a tissue-specific manner, including its signalling mechanisms, in order to improve the therapeutic potential of ARTN in human diseases.

The repair and autophagy mechanisms of hypoxia-regulated bFGF-modified primary embryonic neural stem cells in spinal cord injury.

There is no effective strategy for the treatment of spinal cord injury (SCI), a devastating condition characterized by severe hypoxia and ischemic insults. In this study, we investigated the histology and pathophysiology of the SCI milieu in a rat model and found that areas of hypoxia were unevenly interspersed in compressed SCI. With this new knowledge, we generated embryonic neural stem cells (NSCs) expressing basic fibroblast growth factor (bFGF) under the regulation of five hypoxia-responsive elements (5HRE) using a lentiviral vector (LV-5HRE-bFGF-NSCs) to specifically target these hypoxic loci. SCI models treated with bFGF expressed by the LV-5HRE-bFGF-NSCs viral vector demonstrated improved recovery, increased neuronal survival, and inhibited autophagy in spinal cord lesions in the rat model due to the reversal of hypoxic conditions at day 42 after injury. Furthermore, improved functional restoration of SCI with neuron regeneration was achieved in vivo, accompanied by glial scar inhibition and the evidence of axon regeneration across the scar boundary. This is the first study to illustrate the presence of hypoxic clusters throughout the injury site of compressed SCI and the first to show that the transplantation of LV-5HRE-bFGF-NSCs to target this hypoxic microenvironment enhanced the recovery of neurological function after SCI in rats; LV-5HRE-bFGF-NSCs may therefore be a good candidate to evaluate cellular SCI therapy in humans.

Metformin Promotes Axon Regeneration after Spinal Cord Injury through Inhibiting Oxidative Stress and Stabilizing Microtubule.

Spinal cord injury (SCI) is a devastating disease that may lead to lifelong disability. Thus, seeking for valid drugs that are beneficial to promoting axonal regrowth and elongation after SCI has gained wide attention. Metformin, a glucose-lowering agent, has been demonstrated to play roles in various central nervous system (CNS) disorders. However, the potential protective effect of metformin on nerve regeneration after SCI is still unclear. In this study, we found that the administration of metformin improved functional recovery after SCI through reducing neuronal cell apoptosis and repairing neurites by stabilizing microtubules via PI3K/Akt signaling pathway. Inhibiting the PI3K/Akt pathway with LY294002 partly reversed the therapeutic effects of metformin on SCI in vitro and vivo. Furthermore, metformin treatment weakened the excessive activation of oxidative stress and improved the mitochondrial function by activating the nuclear factor erythroid-related factor 2 (Nrf2) transcription and binding to the antioxidant response element (ARE). Moreover, treatment with Nrf2 inhibitor ML385 partially abolished its antioxidant effect. We also found that the Nrf2 transcription was partially reduced by LY294002 in vitro. Taken together, these results revealed that the role of metformin in nerve regeneration after SCI was probably related to stabilization of microtubules and inhibition of the excessive activation of Akt-mediated Nrf2/ARE pathway-regulated oxidative stress and mitochondrial dysfunction. Overall, our present study suggests that metformin administration may provide a potential therapy for SCI.

Molecular structure and the role of high-temperature requirement protein 1 in skeletal disorders and cancers.

Human high-temperature requirement protein 1 (HTRA1) is a member of serine proteases and consists of four well-defined domains-an IGFBP domain, a Kazal domain, a protease domain and a PDZ domain. HTRA1 is a secretory protein and also present intracellularly and associated with microtubules. HTRA1 regulates a broad range of physiological processes via its proteolytic activity. This review examines the role of HTRA1 in bone biology, osteoarthritis, intervertebral disc (IVD) degeneration and tumorigenesis. HTRA1 mediates diverse pathological processes via a variety of signalling pathways, such as TGF-ß and NF-κB. The expression of HTRA1 is increased in arthritis and IVD degeneration, suggesting that HTRA1 protein is attributed to cartilage degeneration and disease progression. Emerging evidence also suggests that HTRA1 has a role in tumorigenesis. Further understanding the mechanisms by which HTRA1 displays as an extrinsic and intrinsic regulator in a cell type-specific manner will be important for the development of HTRA1 as a therapeutic target.

Astilbin prevents bone loss in ovariectomized mice through the inhibition of RANKL-induced osteoclastogenesis.

Osteoporosis is the most common osteolytic disease characterized by excessive osteoclast formation and resultant bone loss, which afflicts millions of patients around the world. Astilbin, a traditional herb, is known to have anti-inflammatory, antioxidant and antihepatic properties, but its role in osteoporosis treatment has not yet been confirmed. In our study, astilbin was found to have an inhibitory effect on the RANKL-induced formation and function of OCs in a dose-dependent manner without cytotoxicity. These effects were attributed to its ability to suppress the activity of two transcription factors (NFATc1 and c-Fos) indispensable for osteoclast formation, followed by inhibition of the expression of bone resorption-related genes and proteins (Acp5/TRAcP, CTSK, V-ATPase-d2 and integrin ß3). Furthermore, we examined the underlying mechanisms and found that astilbin repressed osteoclastogenesis by blocking Ca2+ oscillations and the NF-κB and MAPK pathways. In addition, the therapeutic effect of MA on preventing bone loss in vivo was further confirmed in an ovariectomized mouse model. Therefore, considering its ability to inhibit RANKL-mediated osteoclastogenesis and the underlying mechanisms, astilbin might be a potential candidate for treating osteolytic bone diseases.

Betulinic Acid Enhances the Viability of Random-Pattern Skin Flaps by Activating Autophagy.

Random-pattern skin flap replantation is commonly used to repair skin defects during plastic and reconstructive surgery. However, flap necrosis due to ischemia and ischemia-reperfusion injury limits clinical applications. Betulinic acid, a plant-derived pentacyclic triterpene, may facilitate flap survival. In the present study, the effects of betulinic acid on flap survival and the underlying mechanisms were assessed. Fifty-four mice with a dorsal random flap model were randomly divided into the control, betulinic acid group, and the betulinic acid + 3-methyladenine group. These groups were treated with dimethyl sulfoxide, betulinic acid, and betulinic acid plus 3-methyladenine, respectively. Flap tissues were acquired on postoperative day 7 to assess angiogenesis, apoptosis, oxidative stress, and autophagy. Betulinic acid promoted survival of the skin flap area, reduced tissue edema, and enhanced the number of microvessels. It also enhanced angiogenesis, attenuated apoptosis, alleviated oxidative stress, and activated autophagy. However, its effects on flap viability and angiogenesis, apoptosis, and oxidative stress were reversed by the autophagy inhibitor 3-methyladenine. Our findings reveal that betulinic acid improves survival of random-pattern skin flaps by promoting angiogenesis, dampening apoptosis, and alleviating oxidative stress, which mediates activation of autophagy.

Trehalose promotes the survival of random-pattern skin flaps by TFEB mediated autophagy enhancement.

Random-pattern skin flaps are commonly used and valuable tools in reconstructive surgery, however, post-operative random skin flap necrosis remains a major and common complication. Previous studies have suggested that activating autophagy, a major pathway for degradation of intracellular waste, may improve flap survival. In this study, we investigated whether trehalose, a novel and potent autophagy activator, improves random skin flap viability. Our results demonstrated that trehalose significantly improves viability, augments blood flow, and decreases tissue edema. Furthermore, we found that trehalose leads to increased angiogenesis, decreased apoptosis, and reduced oxidative stress. Using immunohistochestry and western blot, we demonstrated that trehalose augments autophagy, and that inhibition of autophagy augmentation using 3MA significantly blunted the aforementioned benefits of trehalose therapy. Mechanistically, we showed that trehalose's autophagy augmentation is mediated by activation and nuclear translocation of TFEB, which may be due to inhibition of Akt and activation of the AMPK-SKP2-CARM1 signaling pathway. Altogether, our results established that trehalose is a potent agent capable for significantly increasing random-pattern skin flap survival by augmenting autophagy and subsequently promoting angiogenesis, reducing oxidative stress, and inhibiting cell death.

Chondromodulin-1 in health, osteoarthritis, cancer, and heart disease.

The human chondromodulin-1 (Chm-1, Chm-I, CNMD, or Lect1) gene encodes a 334 amino acid type II transmembrane glycoprotein protein with characteristics of a furin cleavage site and a putative glycosylation site. Chm-1 is expressed most predominantly in healthy and developing avascular cartilage, and healthy cardiac valves. Chm-1 plays a vital role during endochondral ossification by the regulation of angiogenesis. The anti-angiogenic and chondrogenic properties of Chm-1 are attributed to its role in tissue development, homeostasis, repair and regeneration, and disease prevention. Chm-1 promotes chondrocyte differentiation, and is regulated by versatile transcription factors, such as Sox9, Sp3, YY1, p300, Pax1, and Nkx3.2. Decreased expression of Chm-1 is implicated in the onset and progression of osteoarthritis and infective endocarditis. Chm-1 appears to attenuate osteoarthritis progression by inhibiting catabolic activity, and to mediate anti-inflammatory effects. In this review, we present the molecular structure and expression profiling of Chm-1. In addition, we bring a summary to the potential role of Chm-1 in cartilage development and homeostasis, osteoarthritis onset and progression, and to the pathogenic role of Chm-1 in infective endocarditis and cancers. To date, knowledge of the Chm-1 receptor, cellular signalling, and the molecular mechanisms of Chm-1 is rudimentary. Advancing our understanding the role of Chm-1 and its mechanisms of action will pave the way for the development of Chm-1 as a therapeutic target for the treatment of diseases, such as osteoarthritis, infective endocarditis, and cancer, and for potential tissue regenerative bioengineering applications.

Protein Cytl1: its role in chondrogenesis, cartilage homeostasis, and disease.

Cytokine-like protein 1 (Cytl1), also named Protein C17 or C4orf4 is located on human chromosome 4p15-p16 and encodes a polypeptide of 126 amino acid residues that displays characteristics of a secretory protein. Cytl1 is expressed by a sub-population of CD34+ human mononuclear cells from bone marrow and cord blood, and by chondrocytes (cartilage-forming cells). In this review, we explore evidence suggesting that Cytl1 may be involved in the regulation of chondrogenesis, cartilage homeostasis and osteoarthritis progression, accompanied by the modulation of Sox9 and insulin-like growth factor 1 expression. In addition, Cytl1 exhibits chemotactic and pro-angiogenic biological effects. Interestingly, CCR2 (C-C chemokine receptor type 2) has been identified as a likely receptor for Cytl1, which mediates the ERK signalling pathway. Cytl1 also appears to mediate the TGF-beta-Smad signalling pathway, which is hypothetically independent of the CCR2 receptor. More recently, studies have also potentially linked Cytl1 with a variety of conditions including cardiac fibrosis, smoking, alcohol dependence risk, and tumours such as benign prostatic hypertrophy, lung squamous cell carcinoma, neuroblastoma and familial colorectal cancer. Defining the molecular structure of Cytl1 and its role in disease pathogenesis will help us to design therapeutic approaches for Cytl1-associated pathological conditions.

Therapeutic potential of pravastatin for random skin flaps necrosis: involvement of promoting angiogenesis and inhibiting apoptosis and oxidative stress.

Background: Random skin flap is frequently used in plastic and reconstructive surgery, but its distal part often occurs ischemia and necrosis. Pravastatin (Prava) with bioactivities of pro-angiogenesis, anti-apoptosis and anti-oxidative stress, may be beneficial for flap survival. Materials and methods: A modified McFarlane flap model was performed in Sprague-Dawley rats. The animals were divided into the Control and Prava groups and treated as follows: the Prava group was injected intraperitoneally with 2 mg/kg Prava for consecutive 7 days, and the Control group received an equal volume of vehicle daily. On day 7, the necrosis skin flaps were observed, while visualization of blood flow below the tissue surface was performed by Laser Doppler blood flow imaging (LDBFI). Then animals were euthanized, and levels of angiogenesis, apoptosis and oxidative stress were analyzed. Results: Prava decreased necrosis and edema of skin flaps compared with the Control group, with more blood flow in the flap under LDBFI. Prava treatment increased the mean vessels density, elevated the expression levels of angiogenic proteins (matrix metallopeptidase 9, vascular endothelial growth factor, Cadherin5) and antioxidant proteins (superoxide dismutase 1 (SOD1), endothelial nitric oxide synthase, heme oxygenase), and decreased the expression of apoptotic factors (BAX, CYC, Caspase3). In addition, malondialdehyde content was reduced, and glutathione level and SOD activity were increased in the skin flaps after treatment with Prava. Conclusion: Prava promotes survival of random skin flap through induction of angiogenesis, and inhibition of apoptosis and oxidative stress.