Extracellular Vesicles Delivered by Injectable Collagen Promote Bone-Tendon Interface Healing and Prevent Fatty Degeneration of Rotator Cuff Muscle.

PURPOSE: This study aimed (1) to confirm the maintenance of the extracellular vesicles (EVs) delivered via injectable collagen at the application site, and (2) to evaluate the effect of EVs derived from the human umbilical cord-derived mesenchymal stem cells and loaded in an injectable collagen gel after rotator cuff repair (RCR). METHODS: Rabbits (n = 20) were assigned to normal (N), repair-only (R), and those administered with injectable collagen after repair (RC), and EV-laden injectable collagen after repair (RCE) groups. The EVs isolated by ultra-centrifugation from the human umbilical cord-derived mesenchymal stem cells spent medium were mixed with collagen and administered accordingly. After 12 weeks, the rabbits were sacrificed to evaluate the healing of the bone-to-tendon junction and the fatty degeneration of muscle. Histomorphometric scoring for bone-tendon interface, fatty infiltration (%), and biomechanical tests were performed. Separately, groups of 3 rabbits were assigned to 3 different time points to evaluate maintenance of green fluorescence-labeled EVs with injectable collagen via tracking on the bursal side of the rotator cuff (3 groups: 3 days, 2, and 4 weeks). RESULTS: The EVs delivered by injectable collagen remained until 4 weeks at the bursal side of the cuff tissue. The RCE group showed a significantly greater histomorphometric total score (P < .001, and P = .013, respectively) and significantly lower fatty degeneration than the RC and R groups (P = .001, and P = .013, respectively). The biomechanical tests revealed significant growing trends in load-to-failure and stiffness (P = .002, and P = .013, respectively), in the R, RC, RCE, and N groups. CONCLUSIONS: EVs mounted in injectable collagen remained at the repair site for at least 4 weeks after application. Furthermore, they effectively promote bone-to-tendon healing via collagen maturation in bone-tendon interface and preventing fatty degeneration of rotator muscle after RCR as compared with collagen-only or repair-only groups. CLINICAL RELEVANCE: The combination of collagen with EVs significantly promotes rotator cuff healing demonstrating potential clinical application during partial rotator cuff tear or after RCR.

Decellularized Bovine Pericardial Patch Loaded With Mesenchymal Stromal Cells Enhance the Mechanical Strength and Biological Healing of Large-to-Massive Rotator Cuff Tear in a Rat Model.

PURPOSE: The purpose of this study was to determine whether the addition of decellularized bovine pericardial patch loaded with mesenchymal stromal cells enhanced bone-to-tendon healing and improved the biomechanical strength of large-to-massive rotator cuff tears in a small animal model. METHODS: Adipose-derived mesenchymal stromal cells (MSCs) from rat inguinal fat were isolated, cultured, and loaded onto decellularized bovine pericardium patches. To simulate large-to-massive tears, rats were managed with free cage activity for 6 weeks after tear creation. A total of 18 rats were randomly allocated to repair-only (control), repair with pericardial patch augmentation (patch), or repair with MSC loaded pericardial patch augmentation (patch-MSC). Each group had 6 rats (one shoulder of each rat was used for histological evaluation and another for biomechanical evaluation). MSCs seeded on the pericardial patches were traced on four shoulders from 2 other rats at 4 weeks after surgery. Histological evaluation for bone-to-tendon healing and biomechanical testing was carried out at 8 weeks after repair. RESULTS: MSCs tagged with a green fluorescent protein were observed in the repair site 4 weeks after the repair. One shoulder each in the control and patch groups showed complete discontinuity between the bone and tendon. One shoulder in the control group showed attenuation with only a tenuous connection. Fibrocartilage and tidemark formation at the bone-to-tendon interface (P = .002) and collagen fiber density (P = .040) and orientation (P = .003) were better in the patch-MSC group than in the control or patch group. Load-to-failure in the patch-MSC and patch groups was higher than that in the control group (P = .001 and .009, respectively). CONCLUSION: Decellularized bovine pericardial patches loaded with adipose-derived and cultured mesenchymal stromal cells enhanced healing in terms of both histology and mechanical strength at 8 weeks following rotator cuff repair in a rat model. CLINICAL RELEVANCE: Large-to-massive rotator tears need a strategy to prevent retear and enhance healing. The addition of decellularized bovine pericardial patch loaded with MSCs can enhance bone-to-tendon healing and improve biomechanical healing of large-to-massive rotator cuff tears following repair.

Retear bigger than preoperative tear size would lead to treatment failure after rotator cuff repair.

BACKGROUND: This study aimed to (1) define treatment failure using the referred patient acceptable symptomatic state (PASS) values for pain visual analog scale (PVAS), American Shoulder and Elbow Surgeons Standardized Shoulder Assessment Form (ASES), and Single Assessment Numeric Evaluation (SANE) clinical scores and (2) identify the factors that lead to patient dissatisfaction after arthroscopic rotator cuff repair (ARCR). METHODS: We analyzed the arthroscopic rotator cuff surgery registry data from January 2015 to December 2016. Patients were followed for ≥2 years and categorized as dissatisfied or satisfied based on our own definition of treatment failure at 2 years postoperatively. For defining treatment failure, the referred PASS values for the PVAS, ASES, and SANE scores were used. Patients who failed to attain the PASS value for the PVAS, ASES, or SANE score were categorized into the dissatisfied group. Pre- and postoperative imaging and basic demographic data were compared between groups. Univariable and multivariable logistic regression analyses were performed to identify the factors affecting patient satisfaction at 2 years after rotator cuff repair. RESULTS: Of 117 patients, 30 (25.6%) were defined as the dissatisfied group (mean follow-up period, 37.5 months). Seventeen patients (14.5%) had confirmed retear on follow-up magnetic resonance imaging. In the univariate analysis, sex significantly differed between the groups (female, satisfied vs. dissatisfied groups: 39 [44.8%] vs. 22 [73.3%]; P = .010). Retear alone did not affect patient satisfaction in the univariate analysis (P = .11). Progressed retear size featured a significantly higher risk of patient dissatisfaction (P = .024; odds ratio 6.430, 95% confidence interval 1.270-32.541) in the multivariable analysis using symptom duration, sex, preoperative ASES score, preoperative tear size, retear, and progressed retear size as variables. Moreover, female sex had an increased odds for dissatisfaction (odds ratio 4.646, 95% confidence interval 1.590-13.578; P = .005). CONCLUSION: Two years after ARCR, most patients (74.4%) reported satisfaction with their outcomes. However, satisfaction levels can be altered by female sex or progressed retear size compared with the preoperative state.

Postoperative Pain Control After Arthroscopic Rotator Cuff Repair: Arthroscopy-Guided Continuous Suprascapular Nerve Block Versus Ultrasound-Guided Continuous Interscalene Block.

PURPOSE: To compare the clinical efficacy and safety of arthroscopy-guided continuous suprascapular nerve block and ultrasound-guided continuous interscalene block in postoperative analgesia in patients undergoing arthroscopic rotator cuff repair. METHODS: A prospective study was performed between March and November 2020. In total, 76 patients were enrolled and divided into 2 groups: in the 38 patients of group 1 (arthroscopy-guided continuous suprascapular nerve block), an indwelling catheter was introduced via the Neviaser portal under arthroscopic view before closing the portal at the end of the surgery; and in the 38 patients of group 2 (ultrasound-guided continuous interscalene block), an indwelling catheter was inserted and directed toward the interscalene brachial plexus prior to the surgery under ultrasound guidance. The primary outcome was the pain score measured by the visual analog scale at postoperative 24 hours during admission. Comparisons were conducted at different time points (postoperative 4, 8, 24, and 48 hours). The secondary outcome was any of these events: neurologic complications, such as sensory/motor change in the upper extremities; hemidiaphragmatic paresis; dyspnea; dysphonia; and Horner's syndrome. Opioid usage until postoperative 3 weeks was compared between the groups. RESULTS: The visual analog scale scores in groups 1 and 2 were comparable at each postoperative time point (analysis of variance, P = .919; trends, P = .132). Neurologic deficits were more common in group 2 than in group 1 (8 vs 32 patients, P < .001). Decreased excursion of the diaphragm was more common in group 2 (partial or complete paresis of the hemidiaphragm: 1 vs 29 patients, P < .001). Opioid consumption was similar in both groups (morphine milligram equivalents per kilogram; 1.75 vs 1.55, P = .195). CONCLUSIONS: Our findings show that arthroscopy-guided continuous suprascapular nerve block is not inferior to ultrasound-guided continuous interscalene block for postoperative pain control after arthroscopic rotator cuff repair while showing fewer temporary neurologic complications. LEVEL OF EVIDENCE: Level II, prospective cohort study, interventional study.

Is the Chain of Oxidation and Reduction Process Reversible in Luminescent Graphene Quantum Dots?

Graphene-based quantum dots (QDs) have received a tremendous amount of attention as a new type of light-emitting materials. However, their luminescence origins remain controversial due to extrinsic states of the impurities and disorder structures. Especially, the function of oxygen-contents should be understood and controlled as a crucial element for tuning the optical properties of graphene-based QDs. Herein, a series of graphene oxide QDs (GOQDs) with different amounts of oxygen-contents are first synthesized via a direct oxidation route of graphite nanoparticle and thoroughly compared with a series of reduced GOQDs (rGOQDs) prepared by the conventional chemical reduction. Irreversible emission and different carrier dynamics are observed between the GOQDs and rGOQDs, although both routes show a similar tendency with regard to the variation of oxygen-functional components. Their luminescence mechanisms are closely associated with different atomic structures. The mechanism for the rGOQDs can be associated with a formation of small sp(2) nanodomains as luminescent centers, whereas those of GOQDs may be composed of oxygen-islands with difference sizes depending on oxidation conditions surrounded by a large area of sp(2) bonding. Important insights for understanding the optical properties of graphene-based QDs and how they are affected by oxygen-functional groups are shown.

Thermoelectric imaging of structural disorder in epitaxial graphene.

Heat is a familiar form of energy transported from a hot side to a colder side of an object, but not a notion associated with microscopic measurements of electronic properties. A temperature difference within a material causes charge carriers, electrons or holes to diffuse along the temperature gradient inducing a thermoelectric voltage. Here we show that local thermoelectric measurements can yield high-sensitivity imaging of structural disorder on the atomic and nanometre scales. The thermopower measurement acts to amplify the variations in the local density of states at the Fermi level, giving high differential contrast in thermoelectric signals. Using this imaging technique, we uncovered point defects in the first layer of epitaxial graphene, which generate soliton-like domain-wall line patterns separating regions of the different interlayer stacking of the second graphene layer.

Seebeck effect at the atomic scale.

The atomic variations of electronic wave functions at the surface and electron scattering near a defect have been detected unprecedentedly by tracing thermoelectric voltages given a temperature bias [Cho et al., Nat. Mater. 12, 913 (2013)]. Because thermoelectricity, or the Seebeck effect, is associated with heat-induced electron diffusion, how the thermoelectric signal is related to the atomic-scale wave functions and what the role of the temperature is at such a length scale remain very unclear. Here we show that coherent electron and heat transport through a pointlike contact produces an atomic Seebeck effect, which is described by the mesoscopic Seebeck coefficient multiplied by an effective temperature drop at the interface. The mesoscopic Seebeck coefficient is approximately proportional to the logarithmic energy derivative of local density of states at the Fermi energy. We deduced that the effective temperature drop at the tip-sample junction could vary at a subangstrom scale depending on atom-to-atom interaction at the interface. A computer-based simulation method of thermoelectric images is proposed, and a point defect in graphene was identified by comparing experiment and the simulation of thermoelectric imaging.

Finite-temperature hydrogen adsorption and desorption thermodynamics driven by soft vibration modes.

It has been widely accepted that enhanced dihydrogen adsorption is required for room-temperature hydrogen storage on nanostructured porous materials. Here we report, based on results of first-principles total energy and vibrational spectrum calculations, finite-temperature adsorption and desorption thermodynamics of hydrogen molecules that are adsorbed on the metal center of metal-porphyrin-incorporated graphene. We have revealed that the room-temperature hydrogen storage is achievable not only with the enhanced adsorption enthalpy, but also with soft-mode driven vibrational entropy of the adsorbed dihydrogen molecule. The soft vibration modes mostly result from multiple orbital coupling between the hydrogen molecule and the buckled metal center, for example, in Ca-porphyrin-incorporated graphene. Our study suggests that the current design strategy for room-temperature hydrogen storage materials should be modified with explicitly taking the finite-temperature vibration thermodynamics into account.

Can deep learning reduce the time and effort required for manual segmentation in 3D reconstruction of MRI in rotator cuff tears?

BACKGROUND/PURPOSE: The use of MRI as a diagnostic tool has gained popularity in the field of orthopedics. Although 3-dimensional (3D) MRI offers more intuitive visualization and can better facilitate treatment planning than 2-dimensional (2D) MRI, manual segmentation for 3D visualization is time-consuming and lacks reproducibility. Recent advancements in deep learning may provide a solution to this problem through the process of automatic segmentation. The purpose of this study was to develop automated semantic segmentation on 2D MRI images of rotator cuff tears by using a convolutional neural network to visualize 3D models of related anatomic structures. METHODS: MRI scans from 56 patients with rotator cuff tears (T2 Linear Coronal MRI; 3.0T, 512 mm × 512 mm, and 2.5-mm slice thickness) were collected. Segmentation masks for the cuff tendon, muscle, bone, and cartilage were obtained by four orthopedic shoulder surgeons, and these data were revised by a shoulder surgeon with more than 20 years' experience. We performed 2D and 3D segmentation using nnU-Net with secondary labels for reducing false positives. Final validation was performed in an external T2 MRI dataset (10 cases) acquired from other institutions. The Dice Similarity Coefficient (DSC) was used to validate segmentation quality. RESULTS: The use of 3D nnU-Net with secondary labels to reduce false positives achieved satisfactory results, even with a limited amount of data. The DSCs (mean ± SD) of the cuff tendon, muscle, bone, and cartilage in the internal test set were 80.7% ± 9.7%, 85.8% ± 8.6%, 97.8% ± 0.6%, and 80.8% ± 15.1%, respectively. In external validation, the DSC of the tendon segmentation was 82.74±5.2%. CONCLUSION: Automated segmentation using 3D U-Net produced acceptable accuracy and reproducibility. This method could provide rapid, intuitive visualization that can significantly facilitate the diagnosis and treatment planning in patients with rotator cuff tears.

Do Nonsteroidal Anti-Inflammatory or COX-2 Inhibitor Drugs Increase the Nonunion or Delayed Union Rates After Fracture Surgery?: A Propensity-Score-Matched Study.

BACKGROUND: The effects of nonsteroidal anti-inflammatory drugs (NSAIDs)/cyclooxygenase (COX)-2 inhibitors on postoperative fracture-healing are controversial. Thus, we investigated the association between NSAID/COX-2 inhibitor administration and postoperative nonunion or delayed union of fractures. We aimed to determine the effects of NSAID/COX-2 inhibitor administration on postoperative fracture-healing with use of a common data model. METHODS: Patients who underwent operative treatment of a fracture between 1998 and 2018 were included. To determine the effects of NSAID/COX-2 inhibitor administration on fracture-healing, postoperative NSAID/COX-2 inhibitor users were compared and 1:1 matched to nonusers, with 3,264 patients matched. The effect of each agent on bone-healing was determined on the basis of the primary outcome (nonunion/delayed union), defined as having a diagnosis code for nonunion or delayed union ≥6 months after surgery. The secondary outcome was reoperation for nonunion/delayed union. To examine the effect of NSAIDs/COX-2 inhibitors on bone union according to medication duration, a Kaplan-Meier survival analysis was performed. RESULTS: Of the 8,693 patients who were included in the analysis, 208 had nonunion (178 patients; 2.05%) or delayed union (30 patients; 0.35%). Sixty-four (30.8%) of those 208 patients had a reoperation for nonunion or delayed union. NSAID users showed a significantly lower hazard of nonunion compared with the matched cohort of nonusers (hazard ratio, 0.69 [95% confidence interval, 0.48 to 0.98]; p = 0.040) but did not show a significant difference in the other matched comparison for any other outcomes. Kaplan-Meier survival analysis revealed significantly lower and higher nonunion/delayed union rates when the medication durations were ≤3 and >3 weeks, respectively (p = 0.001). For COX-2 inhibitors, the survival curve according to the medication duration showed no significant difference among the groups (p = 0.9). CONCLUSIONS: Our study demonstrated no short-term impact of NSAIDs/COX-2 inhibitors on long-bone fracture-healing. However, continued use of these medications for a period of >3 weeks may be associated with higher rates of nonunion or delayed union. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Risk Factors for Severe Proximal Humerus Fracture and Correlation Between Deltoid Tuberosity Index and Bone Mineral Density.

INTRODUCTION: The aims of this study were to investigate (1) whether demographic factors would be risk factors for severe proximal humerus fracture (PHF), (2) relationship of radiological parameters with bone mineral density (BMD), deltoid tuberosity index (DTI), or severe PHF, and (3) correlation between DTI and BMD. METHODS: We conducted a cross-sectional study based on radiographs and medical records taken during admission or the visit to the orthopedic clinic. We reviewed consecutive 100 adult patients who were diagnosed with PHF in our hospital from March 2014 to December 2016. Three- and 4-part fractures were regarded as severe PHFs. Univariable and multivariable logistic regression analyses were performed to evaluate risk factors for severe PHF. Also, we investigated the correlation between BMD and DTI using the additional BMD data of the patients who underwent shoulder surgeries. RESULTS: This study included 62 nonsevere PHFs and 38 severe PHFs. There were 30 male and 70 female patients with a mean age of 66.4 ± 16.4 years. Mean T score of BMD was -2.5 ± 0.9 at the time of injury. Logistic regression analyses showed that age (odds ratio: 1.044, range: 1.011-1.079, P = .009) and sex (odds ratio of females: 3.763, range: 1.236-11.459, P = .020) were related to severe PHF. The group satisfying the radiological parameter criteria had significantly higher rates of severe PHF. The correlation coefficient (r) between DTI and T score was 0.555 (P < .001). DISCUSSION AND CONCLUSION: Older age and female were the independent risk factors for severe PHF. Conversely, BMD and other medical comorbidities were not risk factors for severe PHF. Deltoid tuberosity index showed significantly high intraclass correlation coefficient and a strong correlation with the T score of BMD. Therefore, DTI may be useful for screening osteoporosis in PHF patients. LEVEL OF EVIDENCE: Level IV, Cross-sectional study.

Divalent Fe Atom Coordination in Two-Dimensional Microporous Graphitic Carbon Nitride.

Graphitic carbon nitride (g-C3N4) is a rising two-dimensional material possessing intrinsic semiconducting property with unique geometric configuration featuring superimposed heterocyclic sp(2) carbon and nitrogen network, nonplanar layer chain structure, and alternating buckling. The inherent porous structure of heptazine-based g-C3N4 features electron-rich sp(2) nitrogen, which can be exploited as a stable transition metal coordination site. Multiple metal-functionalized g-C3N4 systems have been reported for versatile applications, but local coordination as well as its electronic structure variation upon incoming metal species is not well understood. Here we present detailed bond coordination of divalent iron (Fe(2+)) through micropore sites of graphitic carbon nitride and provide both experimental and computational evidence supporting the aforementioned proposition. In addition, the utilization of electronic structure variation is demonstrated through comparative photocatalytic activities of pristine and Fe-g-C3N4.

Bimodal Control of Heat Transport at Graphene-Metal Interfaces Using Disorder in Graphene.

Thermal energy transport across the interfaces of physically and chemically modified graphene with two metals, Al and Cu, was investigated by measuring thermal conductance using the time-domain thermoreflectance method. Graphene was processed using a He2+ ion-beam with a Gaussian distribution or by exposure to ultraviolet/O3, which generates structural or chemical disorder, respectively. Hereby, we could monitor changes in the thermal conductance in response to varying degrees of disorder. We find that the measured conductance increases as the density of the physical disorder increases, but undergoes an abrupt modulation with increasing degrees of chemical modification, which decreases at first and then increases considerably. Moreover, we find that the conductance varies inverse proportionally to the average distance between the structural defects in the graphene, implying a strong in-plane influence of phonon kinetics on interfacial heat flow. We attribute the bimodal results to an interplay between the distinct effects on graphene's vibrational modes exerted by graphene modification and by the scattering of modes.

Intrinsic Photoluminescence Emission from Subdomained Graphene Quantum Dots.

The photoluminescence (PL) origin of bright blue emission arising from intrinsic states in graphene quantum dots (GQDs) is investigated. The bright PL of intercalatively acquired GQDs is attributed to favorably formed subdomains composed of four to seven carbon hexagons. Random and harsh oxidation which hinders the energetically favorable formation of subdomains causes weak and redshifted PL.

Probing Single-Molecule Dissociations from a Bimolecular Complex NO-Co-Porphyrin.

Axial coordinations of diatomic NO molecules to metalloporphyrins play key roles in dynamic processes of biological functions such as blood pressure control and immune response. Probing such reactions at the single molecule level is essential to understand their physical mechanisms but has been rarely performed. Here we report on our single molecule dissociation experiments of diatomic NO from NO-Co-porphyrin complexes describing its dissociation mechanisms. Under tunneling junctions of scanning tunneling microscope, both positive and negative energy pulses gave rise to dissociations of NO with threshold voltages, +0.68 and -0.74 V at 0.1 nA tunneling current on Au(111). From the observed power law relations between dissociation rate and tunneling current, we argue that the dissociations were inelastically induced with molecular orbital resonances by stochastically tunneling electrons, which is supported with our density functional theory calculations. Our study shows that single molecule dissociation experiments can be used to probe reaction mechanisms in a variety of axial coordinations between small molecules and metalloporphyrins.

Transcriptional regulation of Niemann-Pick C1-like 1 gene by liver receptor homolog-1.

Factors that modulate cholesterol levels have major impacts on cardiovascular disease. Niemann-Pick C1-like 1 (NPC1L1) functions as a sterol transporter mediating intestinal cholesterol absorption and counter-balancing hepatobiliary cholesterol excretion. The liver receptor homolog 1 (LRH-1) had been shown to regulate genes involved in hepatic lipid metabolism and reverse cholesterol transport. To study whether human NPC1L1 gene is regulated transcriptionally by LRH-1, we have analyzed evolutionary conserved regions (ECRs) in HepG2 cells. One ECR was found to be responsive to the LRH-1. Through deletion studies, LRH-1 response element was identified and the binding of LRH-1 was demonstrated by EMSA and ChIP assays. When SREBP2, one of several transcription factors which had been shown to regulate NPC1L1 gene, was co-expressed with LRH-1, synergistic transcriptional activation resulted. In conclusion, we have identified LRH-1 response elements in NPC1L1 gene and propose that LRH-1 and SREBP may play important roles in regulating NPC1L1 gene.

Enhancement of the anisotropic photocurrent in ferroelectric oxides by strain gradients.

The phase separation of multiple competing structural/ferroelectric phases has attracted particular attention owing to its excellent electromechanical properties. Little is known, however, about the strain-gradient-induced electronic phenomena at the interface of competing structural phases. Here, we investigate the polymorphic phase interface of bismuth ferrites using spatially resolved photocurrent measurements, present the observation of a large enhancement of the anisotropic interfacial photocurrent by two orders of magnitude, and discuss the possible mechanism on the basis of the flexoelectric effect. Nanoscale characterizations of the photosensitive area through position-sensitive angle-resolved piezoresponse force microscopy and electron holography techniques, in conjunction with phase field simulation, reveal that regularly ordered dipole-charged domain walls emerge. These findings offer practical implications for complex oxide optoelectronics.

Complementary p- and n-type polymer doping for ambient stable graphene inverter.

Graphene offers great promise to complement the inherent limitations of silicon electronics. To date, considerable research efforts have been devoted to complementary p- and n-type doping of graphene as a fundamental requirement for graphene-based electronics. Unfortunately, previous efforts suffer from undesired defect formation, poor controllability of doping level, and subtle environmental sensitivity. Here we present that graphene can be complementary p- and n-doped by simple polymer coating with different dipolar characteristics. Significantly, spontaneous vertical ordering of dipolar pyridine side groups of poly(4-vinylpyridine) at graphene surface can stabilize n-type doping at room-temperature ambient condition. The dipole field also enhances and balances the charge mobility by screening the impurity charge effect from the bottom substrate. We successfully demonstrate ambient stable inverters by integrating p- and n-type graphene transistors, which demonstrated clear voltage inversion with a gain of 0.17 at a 3.3 V input voltage. This straightforward polymer doping offers diverse opportunities for graphene-based electronics, including logic circuits, particularly in mechanically flexible form.

Cooperative transcriptional activation of ATP-binding cassette sterol transporters ABCG5 and ABCG8 genes by nuclear receptors including Liver-X-Receptor.

The ATP-binding cassette transporters ABCG5 and ABCG8 form heterodimers that limit absorption of dietary sterols in the intestine and promote cholesterol elimination from the body through hepatobiliary secretion. To identify cis-regulatory elements of the two genes, we have cloned and analyzed twenty-three evolutionary conserved region (ECR) fragments using the CMV-luciferase reporter system in HepG2 cells. Two ECRs were found to be responsive to the Liver-X-Receptor (LXR). Through elaborate deletion studies, regions containing putative LXREs were identified and the binding of LXRα was demonstrated by EMSA and ChIP assay. When the LXREs were inserted upstream of the intergenic promoter, synergistic activation by LXRα/RXRα in combination with GATA4, HNF4α, and LRH-1, which had been shown to bind to the intergenic region, was observed. In conclusion, we have identified two LXREs in ABCG5/ABCG8 genes for the first time and propose that these LXREs, especially in the ECR20, play major roles in regulating these genes.