The Inflammasome-Dependent Dysfunction and Death of Retinal Ganglion Cells after Repetitive Intraocular Pressure Spikes.

The dysfunction and selective loss of retinal ganglion cells (RGCs) is a known cause of vision loss in glaucoma and other neuropathies, where ocular hypertension (OHT) is the major risk factor. We investigated the impact of transient non-ischemic OHT spikes (spOHT) on RGC function and viability in vivo to identify cellular pathways linking low-grade repetitive mechanical stress to RGC pathology. We found that repetitive spOHT had an unexpectedly high impact on intraocular homeostasis and RGC viability, while exposure to steady OHT (stOHT) of a similar intensity and duration failed to induce pathology. The repetitive spOHT induced the rapid activation of the inflammasome, marked by the upregulation of NLRP1, NLRP3, AIM2, caspases -1, -3/7, -8, and Gasdermin D (GSDMD), and the release of interleukin-1ß (IL-1ß) and other cytokines into the vitreous. Similar effects were also detected after 5 weeks of exposure to chronic OHT in an induced glaucoma model. The onset of these immune responses in both spOHT and glaucoma models preceded a 50% deficit in pattern electroretinogram (PERG) amplitude and a significant loss of RGCs 7 days post-injury. The inactivation of inflammasome complexes in Nlrp1-/-, Casp1-/-, and GsdmD-/- knockout animals significantly suppressed the spOHT-induced inflammatory response and protected RGCs. Our results demonstrate that mechanical stress produced by acute repetitive spOHT or chronic OHT is mechanistically linked to inflammasome activation, which leads to RGC dysfunction and death.

Methotrexate plus electroacupuncture reduces autophagy in ankle synovial tissue in rats with rheumatoid arthritis.

OBJECTIVE: To explore the effect of methotrexate combined with electroacupuncture on autophagy in the ankle synovial tissue in rats modeled with rheumatoid arthritis. METHODS: A rat model of rheumatoid arthritis was constructed by Freund's complete adjuvant injection. The animals were then randomly grouped into the methotrexate + electroacupuncture, methotrexate, electroacupuncture, and model groups. The left hindfoot plantar volume, histopathological morphology of the ankle joint synovium, and autophagy-related genes were detected and compared after the intervention. RESULTS: In comparison with the model group, significantly reduced plantar volume and mRNA and protein levels of autophagy-related gene (Atg) 3, Atg5, Atg12, unc-51-like kinase 1 (ULK1), Beclin1, and light chain 3 (LC3), as well as alleviated synovial hyperplasia were identified in the methotrexate and electroacupuncture groups. The improvement in the above indicators was more pronounced in the methotrexate + electroacupuncture group. CONCLUSIONS: By inhibiting autophagosome formation, both methotrexate and electroacupuncture inhibit synovial cell autophagy, alleviate synovial cell hyperautophagy, and relieve abnormal synovial hyperplasia, thus exerting a protective effect on the joint synovium. The combination of treatment with methotrexate and electroacupuncture works best.

3D-printed composite scaffold with anti-infection and osteogenesis potential against infected bone defects.

In the field of orthopedics, an infected bone defect is a refractory disease accompanied by bone infection and defects as well as aggravated circulation. There are currently no personalized scaffolds that can treat bone infections using local stable and sustained-release antibiotics while providing mechanical support and bone induction to promote bone repair in the process of absorption in vivo. In our previous study, rifampicin/moxifloxacin-poly lactic-co-glycolic acid (PLGA) microspheres were prepared and tested for sustained release and antibacterial activity. The composite scaffold of poly-l-lactic acid (PLLA)/Pearl had a positive effect on mechanics supports and promoted osteogenesis. Therefore, in this study, the personalized scaffolds of PLLA/Pearl were first prepared by 3D printing. Then, rifampicin/moxifloxacin-PLGA (RM-P) microspheres were loaded into the scaffold pores to prepare the PLLA/Pearl/RM-P scaffolds. In this in vitro study, we investigated the structural characteristics and cytocompatibility of 3D-printed composite scaffolds, which indicates the integrity of the components in the scaffolds. The PLLA/Pearl and PLLA/Pearl/RM-P composite scaffolds can promote adhesion, proliferation, and differentiation of human bone marrow mesenchymal stem cells. Moreover, a rabbit model of infected bone defects of the radius was established. PLLA, PLLA/Pearl, and PLLA/Pearl/RM-P scaffolds were implanted into the bone nidus. The therapeutic effect of the three scaffolds on the infected bone defects was evaluated through imaging and microbiological and histological analysis after surgery. Among the three scaffolds, only the PLLA/Pearl/RM-P scaffold had anti-infection and bone defect repair in vivo. 3D printing provides support for personalized scaffold structures, and composite materials ensure that the scaffolds exert anti-infection and bone repair effects. Our study suggests that the PLLA/Pearl/RM-P scaffold is a promising new material in the clinical treatment of infected bone defects.

Platelet-Rich Plasma Combined With Autologous Grafting in the Treatment of Long Bone Delayed Union or Non-union: A Meta-Analysis.

Background: Platelet-rich plasma (PRP) has been suggested as an emerging treatment for bone defects. However, whether PRP could enhance the therapeutic efficacy of autologous bone grafting for long bone delayed union or non-union remains unknown. A meta-analysis of randomized and non-randomized controlled trials (RCT and NRCT) was performed to summarize current evidence. Methods: Relevant RCTs and NRCTs comparing the influences of autologous bone grafting on healing of long bone delayed union or non-union with and without PRP were obtained by searching PubMed, Embase, Cochrane's Library, China National Knowledge Infrastructure, and WanFang databases from inception to September 10, 2020. A random-effect model was applied to pool the results with the incorporation of the potential heterogeneity. Subgroup analysis according to study design was also performed. Results: Six RCTs and two NRCTs with 420 patients were included. Compared to patients allocated to autologous bone grafting alone, those allocated to combined treatment with PRP and autologous bone grafting were not associated with higher rates of radiographic bone healing [risk ratio (RR): 1.06, 95% confidence interval (CI): 0.99-1.13, P = 0.09; I 2 = 24%] or excellent/good posttreatment limb function (RR: 1.14, 95% CI: 0.95-1.37, P = 0.37; I 2 = 0%) but was associated with a shorter healing time (mean difference: -1.35 months, 95% CI: -1.86 to -0.84, P < 0.001; I 2 = 58%). Subgroup analysis according to study design showed similar results for the above outcomes (P-values for subgroup difference all >0.10). Conclusions: Combined treatment with PRP and autologous bone grafting may be effective to accelerate the healing of long bone delayed union or non-union compared to autologous bone grafting alone.

Protection of Icariin Against Hydrogen Peroxide-Induced MC3T3-E1 Cell Oxidative Damage.

OBJECTIVE: The aim of the present study was to evaluate the potential protective mechanism of icariin against oxidative damage caused by hydrogen peroxide in MC3T3-E1 cells. METHODS: MC3T3-E1 cells were treated with different concentrations of icariin to explore the optimal dose of icariin. MC3T3-E1 cells were divided into groups treated with various concentrations of hydrogen peroxide (H2 O2 ; 0, 0.1, 0.2, 0.5, 1, and 2 mM) for 24 h to induce oxidative damage and cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. Then, cells were divided into five groups: control, H2 O2 (0.2 mM), icariin (0.1 µM) and H2 O2 (0.2 mM), + icariin (0.1 µM). Cell viability was detected by CCK-8 assay. In addition, the content of glutathione and superoxide dismutase and the activity level of malondialdehyde in these treatment groups were determined. Alkaline phosphatase (ALP) and alizarin red S (ARS) staining were also performed to measure the early and late osteogenesis, respectively. Protein expression of ß-catenin and cyclin D1 was measured by western blot assay. Then, we used an antagonist of Wnt/ß-catenin signaling pathway (DKK-1) and western blot analysis to further explore potential mechanism. RESULTS: After 24 h of exposure to 0.2 mM H2 O2 , the viability of MC3T3-E1 cells was significantly decreased compared to that of the control cells. We first found that icariin can promote cell proliferation of MC3T3-E1 cells in a dose-dependent manner, with the dosage 0.1 µM showing the best pro-proliferative effect. Furthermore, icariin could promote the protein expression of OSX and RUNX2. The results showed that icariin can reverse the inhibitory osteogenic effects of MC3T3-E1 caused by H2 O2 . In addition, icariin could increase the Wnt-signaling related proteins. The results showed that MC3T3-E1 cells in the H2 O2 (0.2 mM) + icariin (0.1 µM) + Wnt-signaling antagonist (DKK-1) group had weaker ALP and ARS staining compared with that observed in the control and H2 O2 (0.2 mM) + icariin (0.1 µM) groups. The ALP activity and calcium content were decreased in the 0.2 mM H2 O2 + 0.1 µM icariin + DKK-1 group compared to that observed in the 0.2 mM H2 O2 + 0.1 µM icariin group. CONCLUSION: The results showed that icariin can increase the viability of MC3T3-E1 cells, reverse the oxidative stress induced by H2 O2 and protect MC3T3-E1 cells against H2 O2 -induced inhibition of osteogenic differentiation, which may occur through the Wnt/ß-catenin signaling pathway.

[Application of self-made spring plate for treatment of acetabular posterior wall fractures].

OBJECTIVE: To investigate the application and effectiveness of self-made spring plate in surgical treatment of acetabular posterior wall fracturejavascript:void(0)s. METHODS: Between June 2013 and June 2017, 38 patients with acetabular posterior wall fractures were treated. There were 27 males and 11 females with an average age of 53 years (range, 28-68 years). The injury caused by traffic accident in 18 cases, falling from height in 15 cases, and tumble in 5 cases. There were 4 cases of simple posterior wall fracture, 18 cases of posterior wall fracture with posterior dislocation of hip joint, 10 cases of posterior wall fracture with posterior column fracture, and 6 cases of posterior wall fracture with transverse fracture. The time from injury to admission was 1-4 days (mean, 2.5 days). The time from injury to operation was 4-8 days (mean, 5 days). After fracture reduction via the Kocher-Langenbeck approach (35 cases) or the combined ilioinguinal approach (3 cases), the spring plate was used to press the posterior wall fracture, and then the reconstruction plate was pressed against the spring plate and fixed to the posterior column. RESULTS: All the incisions healed by first intention. All patients were followed up 12-36 months (mean, 28 months). Five cases of post-traumatic sciatic nerve injury and 2 cases of sciatic nerve injury caused by traction during operation were fully recovered at 3 months after operation. The imaging examination showed that all the fractures healed. The fracture healing time was 10-16 weeks (mean, 12 weeks). There was no ruptures or failures of internal fixation during the follow-up period. There were 2 cases of femoral head necrosis, 1 case of traumatic arthritis, and 1 case of osteomyositis at last follow-up. The hip joint function was rated as excellent in 27 cases, good in 5 cases, fair in 2 cases, and poor in 4 cases according to the Harris scores at 12 months after operation. CONCLUSION: For the acetabular posterior wall fracture, it has the advantages of easy to use and reliable fixation that the posterior wall fracture is fixed with spring plate firstly, and the spring plate is pressed to fix the posterior column with the reconstruction plate finally.

MicroRNA-106a Inhibits Autophagy Process and Antimicrobial Responses by Targeting ULK1, ATG7, and ATG16L1 During Mycobacterial Infection.

Autophagy is a key element of innate immune response against invading pathogens including Mycobacterium tuberculosis (M. tuberculosis). The emerging roles of microRNAs in regulating host antimicrobial responses against M. tuberculosis have gained widespread attention. However, the process by which miRNAs specifically influence antibacterial autophagy during mycobacterial infection is largely uncharacterized. In this study, we demonstrate a novel role of miR-106a in regulating macrophage autophagy against M. tuberculosis. H37Ra infection leads to downregulation of miR-106a in a time- and dose-dependent manner and concomitant upregulation of its three targets (ULK1, ATG7, and ATG16L1) in THP-1 macrophages. MiR-106a could inhibit autophagy activation and antimicrobial responses to M. tuberculosis by targeting ULK1, ATG7, and ATG16L1. Overexpression of miR-106a dramatically inhibited H37Ra-induced activation of autophagy in human THP-1 macrophages, whereas inhibitors of miR-106a remarkably promoted H37Ra-induced autophagy. The inhibitory effect of miR-106a on autophagy process during mycobacterial infection was also confirmed by Transmission Electron Microscope (TEM) observation. More importantly, forced expression of miR-106a increased mycobacterial survival, while transfection with miR-106a inhibitors attenuated the survival of intracellular mycobacteria. Taken together, these data demonstrated that miR-106a functioned as a negative regulator in autophagy and antimicrobial effects by targeting ULK1, ATG7, and ATG16L1 during M. tuberculosis infection, which may provide a potential target for developing diagnostic reagents or antibacterials against tuberculosis.

Application of a hybrid gravity-driven membrane filtration and dissolved ozone flotation (MDOF) process for wastewater reclamation and membrane fouling mitigation.

This study proposed a novel membrane filtration and dissolved ozone flotation integrated (MDOF) process and tested it at pilot scale. Membrane filtration in the MDOF process was operated in gravity-driven mode, and required no backwashing, flushing, or chemical cleaning. Because ozone was added in the MDOF process, ozonation, coagulation, and membrane filtration could occur in a single reactor. Moreover, in situ ozonation occurred in the MDOF process, which differs from the conventional pre-ozonation membrane filtration process. Significant enhancement of turbidity removal was further achieved through the addition of membrane filtration. Membrane fouling was mitigated in the MDOF process compared to the MDAF process. In situ ozonation in the MDOF process decreased the fluorescence intensity and transformed the high MW dissolved organics into small MW compounds. For the fouling layer, the extracellular polymeric substance (EPS) contents and cake layer morphology were analyzed. The results indicated that the contents of EPS decreased. Furthermore, a thinner and more loosely structured cake layer formed in the MDOF process. Because coagulation and ozonation occurred simultaneously in a single reactor, the generation of hydroxyl radicals was enhanced through the catalytic effect of Al-based coagulants on ozone decomposition, which further alleviated membrane fouling in the MDOF process.

Inflammasome Activation Induces Pyroptosis in the Retina Exposed to Ocular Hypertension Injury.

Mechanical stress and hypoxia during episodes of ocular hypertension (OHT) trigger glial activation and neuroinflammation in the retina. Glial activation and release of pro-inflammatory cytokines TNFα and IL-1ß, complement, and other danger factors was shown to facilitate injury and loss of retinal ganglion cells (RGCs) that send visual information to the brain. However, cellular events linking neuroinflammation and neurotoxicity remain poorly characterized. Several pro-inflammatory and danger signaling pathways, including P2X7 receptors and Pannexin1 (Panx1) channels, are known to activate inflammasome caspases that proteolytically activate gasdermin D channel-formation to export IL-1 cytokines and/or induce pyroptosis. In this work, we used molecular and genetic approaches to map and characterize inflammasome complexes and detect pyroptosis in the OHT-injured retina. Acute activation of distinct inflammasome complexes containing NLRP1, NLRP3 and Aim2 sensor proteins was detected in RGCs, retinal astrocytes and Muller glia of the OHT-challenged retina. Inflammasome-mediated activation of caspases-1 and release of mature IL-1ß were detected within 6 h and peaked at 12-24 h after OHT injury. These coincided with the induction of pyroptotic pore protein gasdermin D in neurons and glia in the ganglion cell layer (GCL) and inner nuclear layer (INL). The OHT-induced release of cytokines and RGC death were significantly decreased in the retinas of Casp1-/-Casp4(11)del, Panx1-/- and in Wild-type (WT) mice treated with the Panx1 inhibitor probenecid. Our results showed a complex spatio-temporal pattern of innate immune responses in the retina. Furthermore, they indicate an active contribution of neuronal NLRP1/NLRP3 inflammasomes and the pro-pyroptotic gasdermin D pathway to pathophysiology of the OHT injury. These results support the feasibility of inflammasome modulation for neuroprotection in OHT-injured retinas.

RPE phagocytic function declines in age-related macular degeneration and is rescued by human umbilical tissue derived cells.

BACKGROUND: Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly characterized by retinal pigment epithelium (RPE) degeneration with accumulation of abnormal intracellular deposits (lipofuscin) and photoreceptor death. RPE is vital for the retina and integrity of photoreceptors through its phagocytic function which is closely linked to formation of lipofuscin through daily phagocytosis of discarded photoreceptor outer segments (POS). Although phagocytosis has been implicated in AMD, it has not been directly shown to be altered in AMD. RPE phagocytic defect was previously shown to be rescued by subretinal injection of human umbilical tissue derived cells (hUTC) in a rodent model of retinal degeneration (RCS rat) through receptor tyrosine kinase (RTK) ligands and bridge molecules. Here, we examined RPE phagocytic function directly in the RPE from AMD patients and the ability and mechanisms of hUTC to affect phagocytosis in the human RPE. METHODS: Human RPE was isolated from the post-mortem eyes of normal and AMD-affected subjects and cultured. RPE phagocytic function was measured in vitro using isolated POS. The effects of hUTC conditioned media, recombinant RTK ligands brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and glial cell-derived neurotrophic factor (GDNF), as well as bridge molecules milk-fat-globule-EGF-factor 8 (MFG-E8), thrombospondin (TSP)-1, and TSP-2 on phagocytosis were also examined in phagocytosis assays using isolated POS. RNA was isolated from normal and AMD RPE treated with hUTC conditioned media and subjected to transcriptome profiling by RNA-Seq and computational analyses. RESULTS: RPE phagocytosis, while showing a moderate decline with age, was significantly reduced in AMD RPE, more than expected for age. hUTC conditioned media stimulated phagocytosis in the normal human RPE and significantly rescued the phagocytic dysfunction in the AMD RPE. RTK ligands and bridge molecules duplicated the rescue effect. Moreover, multiple molecular pathways involving phagocytosis, apoptosis, oxidative stress, inflammation, immune activation, and cholesterol transport were affected by hUTC in the RPE. CONCLUSIONS: We demonstrated for the first time RPE phagocytic dysfunction in AMD, highlighting its likely importance in AMD, and the ability of hUTC to correct this dysfunction, providing insights into the therapeutic potential of hUTC for AMD.

Human umbilical tissue-derived cells rescue retinal pigment epithelium dysfunction in retinal degeneration.

Retinal pigment epithelium (RPE) cells perform many functions crucial for retinal preservation and vision. RPE cell dysfunction results in various retinal degenerative diseases, such as retinitis pigmentosa and age-related macular degeneration (AMD). Currently, there are no effective treatments for retinal degeneration except for a small percentage of individuals with exudative AMD. Cell therapies targeting RPE cells are being developed in the clinic for the treatment of retinal degeneration. Subretinal injection of human umbilical tissue-derived cells (hUTC) in the Royal College of Surgeons (RCS) rat model of retinal degeneration was shown to preserve photoreceptors and visual function. However, the precise mechanism remains unclear. Here, we demonstrate that hUTC rescue phagocytic dysfunction in RCS RPE cells in vitro. hUTC secrete receptor tyrosine kinase (RTK) ligands brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and glial cell-derived neurotrophic factor (GDNF), as well as opsonizing bridge molecules milk-fat-globule-epidermal growth factor 8 (MFG-E8), growth arrest-specific 6 (Gas6), thrombospondin (TSP)-1, and TSP-2. The effect of hUTC on phagocytosis rescue in vitro is mimicked by recombinant human proteins of these factors and is abolished by siRNA-targeted gene silencing in hUTC. The bridge molecules secreted from hUTC bind to the photoreceptor outer segments and facilitate their ingestion by the RPE. This study elucidates novel cellular mechanisms for the repair of RPE function in retinal degeneration through RTK ligands and bridge molecules, and demonstrates the potential of using hUTC for the treatment of retinal degenerative diseases.

[Comparative study on the treatment of acromioclavicular joint dislocation: coracoclavicular ligament reconstruction using lateral half of conjoined tendon or tractusiliotibialis with hook plate fixation].

OBJECTIVE: To explore the clinical outcomes of acromioclavicular joint (ACJ) dislocation treated with coracoclavicular ligament (CCL) reconstruction using lateral half of conjoined tendon and tractusiliotibialis with hook plate fixation. Comparative study on their advantages and disadvantages in order to provide the materials for the clinic. METHODS: From June 2005 to June 2012, the patients with Rockwood type III or severer ACJ dislocation were randomly divided into 2 groups. They underwent CCL reconstruction using lateral half of conjoined tendon (conjoined tendon reconstruction group, n = 36) and tractusiliotibialis (tractusiliotibialis reconstruction group, n = 38) with subsequent fixation of hook plates. During the follow-up, the AC and CC distances were measured on postoperative plain films after a removal of hook plates. And the outcomes were assessed according to Karlsson criteria and Constant-Murley shoulder score. Ranked data were analyzed with the use of χ2 test and measurement date with two-sample t test. Results A total of 74 patients were followed up for an average of 20 (12 - 24) months. No significant inter-group differences existed in age, gender, injured side or classification. And there was no statistical difference in ACor CC distance between two groups within 6 months (P > 0. 05) after a removal of hook plates. The AC and CC distances of conjoined tendon reconstruction group were larger than those of tractusiliotibialis reconstruction group (t = 2. 313, P = 0. 026; t = 2. 114, P = 0. 041) within 12 months. The AC and CC distances of 12 months were both larger than those of 6 months (t =2. 631, P =0. 017; t = 2. 297, P = 0. 032). According to the Constant-Murley shoulder score, conjoined tendon reconstruction group was less than tractusiliotibialis reconstruction group (85. 2 ± 10. 2 vs 93. 1 ± 6. 9, t = 2. 965, P = 0. 006). According to the Karlsson Criteria, the excellent and good rate of functional recovery was 75. 00% in conjoined tendon reconstruction group versus 94. 74% in tractusiliotibialis reconstruction group (χ2 = 8. 111, P = 0. 044). CONCLUSION: The efficacy of Rockwood type III acromioclavicular joint dislocation for reconstructing coracoclavicular ligament using tractusiliotibialis is better than conjoined tendon. The AC and CC distances increase after a removal of hook plates while it is more obvious for conjoined tendon tractusiliotibialis reconstruction.

[Comparative study on the treatment of acromioclavicular joint dislocation: coracoclavicular ligament reconstruction combined with hook plate fixation or suture-anchor fixation].

OBJECTIVE: To investigate the clinical outcomes of acromioclavicula (AC) joint dislocation treated with coracoclavicular (CC) ligament reconstruction and hook plate fixation/suture-anchor fixation. METHODS: There were 105 patients with Rockwood type III or severer AC joint dislocations were randomly divided into two groups from February 2007 to April 2010. They were treated with CC ligament reconstruction using double bundle of Palmaris longus (hook plate fixation group, 54 cases), and subsequently fixed with hook plates or suture-anchors (suture-anchors group, 51 cases). Patients were followed up, and the AC distance and CC distance were measured on the postoperative X-ray films, and the outcomes were assessed according to Karlsson criteria and Constant-Murley shoulder score. Ranked data was analyzed with the use of χ(2) test and measurement data with two sample t test. RESULTS: Eighty-nine patients were followed up for 24-42 months, average 30 months. There were 46 cases in hook plate fixation group and 43 cases in suture-anchor fixation group, without significant difference in age, gender, injured side and Rockwood classification between both groups. Between the two groups, no statistical difference was detected in the AC and CC distance measured within 6 months after operation (P > 0.05). The AC and CC distances of hook plate fixation group measured in 24 months postoperatively were larger than those in suture-anchor fixation group, respectively (F = 1.904 and 1.854, P < 0.05). In hook plate fixation group, the AC and CC distances measured in 24 months postoperatively were larger than those measured in 6 month postoperatively, respectively (F = 1.863 and 1.842, P < 0.05). According to Constant-Murley shoulder score, the average score was 88.5 for hook plate fixation group and 92.7 for suture-anchor fixation group (F = 0.475, P = 0.017). According to Karlsson criteria, the excellent and good rate of the functional recovery was 95.4% in suture-anchor fixation group, better than hook plate fixation group (χ(2) = 4.564, P = 0.033). CONCLUSIONS: The clinical outcomes of AC joint dislocation treated with CC ligament reconstruction and suture-anchor fixation are better than those treated with CC ligament reconstruction and hook plate fixation. The AC and CC distances increase after the removal of hook plate, which may be associated with poor functional recovery.

Inhibition of tumor angiogenesis and melanoma growth by targeting vascular E-selectin.

OBJECTIVES: Aggressive human melanomas express, C-X-C chemokine receptor 4 (CXCR4), the receptor for the chemokine, stromal cell-derived factor-1alpha (SDF-1α). The CXCR4-SDF-1α axis has been postulated to increase melanoma invasiveness. We discovered that SDF-1α specifically upregulates E-selectin on endothelial cells, thus tethering circulating endothelial progenitor cells (EPC) and facilitating homing. We investigated the hypothesis that small interfering ribonucleic acid (siRNA)-mediated E-selectin blockade inhibits melanoma angiogenesis and tumor growth. METHODS: Human melanoma cells overexpressing SDF-1α were xenografted on severe combined immunodeficiency (SCID) mice. SDF-1α expression in cells was measured by enzyme-linked immunosorbent assay (ELISA). In vitro melanoma cell growth was examined by cell proliferation assay. In vivo vascular E-selectin knockdown was achieved by administration of high-volume E-selectin siRNA (100 pmol/180 µL/week × 3 times) and inhibition was validated by immunostaining (N = 6/group, E-Selectin siRNA vs control siRNA). Tumor angiogenesis was quantified (DiI-perfusion and LASER confocal microscopy). EPC homing to tumor vasculature was detected by immunostaining. Explanted in vivo tumor size and weight were measured. RESULTS: Three melanoma cells tested expressed undetectable levels of SDF-1α. Additional enforced overexpression of SDF-1α (by Lenti-SDF-1α) increased melanoma cell growth both in vitro and in vivo, enhanced EPC homing to tumor tissue, and increased tumor angiogenesis. Knocking-down vascular E-selectin significantly inhibited SDF-1α-induced EPC homing, tumor angiogenesis, and decreased melanoma growth in vivo. CONCLUSIONS: Downregulation of vascular E-selectin profoundly inhibits EPC homing, tumor angiogenesis, and tumor growth in human melanoma xenograft murine model, potentially by suppression of E-selectin-mediated EPC-endothelial cells interactions/homing. These findings identify E-selectin as a novel target for inhibition of melanoma angiogenesis and tumor growth.

Xenopus germline nanos1 is translationally repressed by a novel structure-based mechanism.

The translational repressor Nanos is expressed in the germline and stem cell populations of jellyfish as well as humans. Surprisingly, we observed that unlike other mRNAs, synthetic nanos1 RNA translates very poorly if at all after injection into Xenopus oocytes. The current model of simple sequestration of nanos1 within germinal granules is insufficient to explain this observation and suggests that a second level of repression must be operating. We find that an RNA secondary structural element immediately downstream of the AUG start site is both necessary and sufficient to prevent ribosome scanning in the absence of a repressor. Accordingly, repression is relieved by small in-frame insertions before this secondary structure, or translational control element (TCE), that provide the 15 nucleotides required for ribosome entry. nanos1 is translated shortly after fertilization, pointing to the existence of a developmentally regulated activator. Oocyte extracts were rendered fully competent for nanos1 translation after the addition of a small amount of embryo extract, confirming the presence of an activator. Misexpression of Nanos1 in oocytes from unlocalized RNA results in abnormal development, highlighting the importance of TCE-mediated translational repression. Although found in prokaryotes, steric hindrance as a mechanism for negatively regulating translation is novel for a eukaryotic RNA. These observations unravel a new mode of nanos1 regulation at the post-transcriptional level that is essential for normal development.

Identification of E-selectin as a novel target for the regulation of postnatal neovascularization: implications for diabetic wound healing.

OBJECTIVES: We previously reported that stromal cell-derived factor-1α (SDF-1α, a homing signal for recruiting endothelial progenitor cells (EPC) to areas of neovascularization), is down-regulated in diabetic wounds (Gallagher et al, J Clin Invest. 2007;117:1249-1259). We now investigate signals whereby mature endothelial cells (EC) and circulating EPC achieve SDF-1α-mediated EPC homing. METHODS: SDF-1α in diabetic wounds were therapeutically increased by injection of SDF-1α-engineered bone marrow-derived fibroblasts versus control cells (N = 48 [20, non-obese diabetic (NOD)], [28, streptozotocin-C57]). Polymerase chain reaction-array gene expression differences were validated by Western blotting and immunohistochemistry. The role of adhesion molecule(s) in mediating SDF-1α-induced EPC homing, and wound healing was furthered studied using antagonists in vitro and in vivo. RESULTS: Increasing wound SDF-1α via cell-based therapy promotes healing in diabetic mice (∼20% increase in healing rates by day 3, P = 0.006). SDF-1α increased EC-EPC adhesion and specifically upregulated E-selectin expression in human microvascular EC (2.3-fold increase, P < 0.01). This effect was also significant in blood vessels of the experimental mice and resulted in increased wound neovascularization. The regulatory effects of SDF-1α on EC-EPC adhesion and EPC homing were specifically mediated by E-selectin, as the application of E-selectin antagonists significantly inhibited SDF-1α-induced EC-EPC adhesion, EPC homing, wound neovascularization, and wound healing. CONCLUSIONS: SDF-1α-engineered cell-based therapy promotes diabetic wound healing in mice by specifically upregulating E-selectin expression in mature EC leading to increase EC-EPC adhesion, EPC homing, and increased wound neovascularization. These findings provide novel insight into the signals underlying the biological effect of SDF-1α on EPC homing and point to E-selectin as a new potential target for therapeutic manipulation of EPC trafficking in diabetic wound healing.

Rapamycin impairs in vivo proliferation of islet beta-cells.

BACKGROUND: Progressive graft dysfunction is commonly observed in recipients of islet allografts treated with high doses of rapamycin. This study aimed at evaluating the effect of rapamycin on pancreatic islet cell proliferation in vivo. METHODS: The murine pregnancy model was utilized, since a high rate of beta-cell proliferation occurs in a well-defined time frame. Rapamycin (0.2 mg/kg/day) was given to C57BL/6 mice for 5-7 days starting on day 7.5 of pregnancy. Cell proliferation was evaluated by detection of bromodeoxyuridine incorporation by immunohistochemistry. RESULTS: Pregnancy led to increased beta-cell proliferation and islet yield with skewing in islet size distribution as well as higher pancreatic insulin content, when compared to that of nonpregnant females. These effects of pregnancy on beta-cell proliferation and mass were significantly blunted by rapamycin treatment. Minimal effect of rapamycin was observed on islet function both in vivo and in vitro. Rapamycin treatment of islets in vitro resulted in reduced p70s6k phosphorylation, which was paralleled by increased ERK1/2 phosphorylation. CONCLUSIONS: Rapamycin treatment reduces the rate of beta-cell proliferation in vivo. This phenomenon may contribute to impair beta-cell renewal in transplanted patients and to the progressive dysfunction observed in islet graft recipients.