Pharmacological Effects of Shikonin and Its Potential in Skin Repair: A Review.

Currently, skin injuries have a serious impact on people's lives and socio-economic stress. Shikonin, a naphthoquinone compound derived from the root of the traditional Chinese medicine Shikonin, has favorable biological activities such as anti-inflammatory, antibacterial, immunomodulatory, anticancer, and wound-healing-promoting pharmacological activities. It has been reported that Shikonin can be used for repairing skin diseases due to its wide range of pharmacological effects. Moreover, the antimicrobial activity of Shikonin can play a great role in food and can also reduce the number of pathogenic bacteria in food. This paper summarizes the research on the pharmacological effects of Shikonin in recent years, as well as research on the mechanism of action of Shikonin in the treatment of certain skin diseases, to provide certain theoretical references for the clinical application of Shikonin, and also to provides research ideas for the investigation of the mechanism of action of Shikonin in other skin diseases.

Reconstructing avascular necrotic femoral head through a bioactive ß-TCP system: From design to application.

A variety of techniques have been used for treating avascular necrosis of the femoral head (ANFH), but have frequently failed. In this study, we proposed a ß-TCP system for the treatment of ANFH by boosting revascularization and bone regeneration. The angio-conductive properties and concurrent osteogenesis of the highly interconnected porous ß-TCP scaffold were revealed and quantified through an in vivo model that simulated the ischemic environment of ANFH. Mechanical test and finite element analysis showed that the mechanical loss caused by tissue necrosis and surgery was immediately partially compensated after implantation, and the strength of the operated femoral head was adaptively increased and eventually returned to normal bone, along with continuous material degradation and bone regeneration. For translational application, we further conducted a multi-center open-label clinical trial to assess the efficacy of the ß-TCP system in treating ANFH. Two hundred fourteen patients with 246 hips were enrolled for evaluation, and 82.1% of the operated hips survived at a 42.79-month median follow-up. The imaging results, hip function, and pain scores were dramatically improved compared to preoperative levels. ARCO stage Ⅱ disease outperformed stage Ⅲ in terms of clinical effectiveness. Thus, bio-adaptive reconstruction using the ß-TCP system is a promising hip-preserving strategy for the treatment of ANFH.

FRCNN-AA-CIF: An automatic detection model of colon polyps based on attention awareness and context information fusion.

It is noted that the foreground and background of the polyp images detected under colonoscopy are not highly differentiated, and the feature map extracted by common deep learning object detection models keep getting smaller as the number of networks increases. Therefore, these models tend to ignore the details in pictures, resulting in a high polyp missed detection rate. To reduce the missed detection rate, this paper proposes an automatic detection model of colon polyps based on attention awareness and context information fusion (FRCNN-AA-CIF) based on a two-stage object detection model Faster Region-Convolutional Neural Network (FR-CNN). First, since the addition of attention awareness can make the feature extraction network pay more attention to polyp features, we propose an attention awareness module based on Squeeze-and-Excitation Network (SENet) and Efficient Channel Attention Module (ECA-Net) and add it after each block of the backbone network. Specifically, we first use the 1*1 convolution of ECA-Net to extract local cross-channel information and then use the two fully connected layers of SENet to reduce and increase the dimension, to filter out the channels that are more useful for feature learning. Further, because of the presence of air bubbles, impurities, inflammation, and accumulation of digestive matter around polyps, we used context information around polyps to enhance the focus on polyp features. In particular, after the network extracts the region of interest, we fuse the region of interest with its context information to improve the detection rate of polyps. The proposed model was tested on the colonoscopy dataset provided by Huashan Hospital. Numerical experiments show that FRCNN-AA-CIF has the highest detection accuracy (mAP of 0.817), the lowest missed detection rate of 4.22%, and the best classification effect (AUC of 95.98%). Its mAP increased by 3.3%, MDR decreased by 1.97%, and AUC increased by 1.8%. Compared with other object detection models, FRCNN-AA-CIF has significantly improved recognition accuracy and reduced missed detection rate.

Sparse non-convex regularization based explainable DBN in the analysis of brain abnormalities in schizophrenia.

Deep belief networks have been widely used in medical image analysis. However, the high-dimensional but small-sample-size characteristic of medical image data makes the model prone to dimensional disaster and overfitting. Meanwhile, the traditional DBN is driven by performance and ignores the explainability which is important for medical image analysis. In this paper, a sparse non-convex based explainable deep belief network is proposed by combining DBN with non-convex sparsity learning. For sparsity, the non-convex regularization and Kullback-Leibler divergence penalty are embedded into DBN to obtain the sparse connection and sparse response representation of the network. It effectively reduces the complexity of the model and improves the generalization ability of the model. Considering explainability, the crucial features for decision-making are selected through the feature back-selection based on the row norm of each layer's weight after network training. We apply the model to schizophrenia data and demonstrate it achieves the best performance among several typical feature selection models. It reveals 28 functional connections highly correlated with schizophrenia, which provides an effective foundation for the treatment and prevention of schizophrenia and methodological assurance for similar brain disorders.

MRI Imaging Omics and Risk Factors Analysis of PWMD in Premature Infants Based on Fuzzy Clustering Algorithm.

The magnetic resonance imaging (MRI) characteristics of periventricular white matter damage (PWMD) in premature infants using the fuzzy c-means clustering algorithm (FCM) is explored, and the influencing factors are further clarified. A total of 100 premature infants admitted to the neonatal department of our hospital from February 2020 to February 2022 are selected for in-depth investigation. According to the occurrence of PWMD, they are divided into the PWMD group and the simple premature delivery group, with 50 cases in each group. All preterm infants are examined by MRI and the changes in image characteristics and apparent diffusion coefficient (ADC) values are analyzed. Clinical information of the subjects is collected and the influencing factors of PWMD in preterm infants are analyzed by multivariate regression analysis. In the first magnetic resonance imaging (MRI) examination, the cases of punctured, clustered, and linear lesions are 28 cases, 12 cases, and 10 cases, respectively. The experimental results showed that PWMD of preterm infants presented punctate, clustered, and high linear T1 signal MRI manifestations, which caused a downward trend of ADC value, and caused respiratory distress, low birth weight, premature rupture of membranes, respiratory tract infection, and other risk symptoms.

Pathological prognosis classification of patients with neuroblastoma using computational pathology analysis.

Neuroblastoma is the most common extracranial solid tumor in early childhood. International Neuroblastoma Pathology Classification (INPC) is a commonly used classification system that provides clinicians with a reference for treatment stratification. However, given the complex and subjective assessment of the INPC, there will be inconsistencies in the analysis of the same patient by multiple pathologists. An automated, comprehensive and objective classification method is needed to identify different prognostic groups in patients with neuroblastoma. In this study, we collected 563 hematoxylin and eosin-stained histopathology whole-slide images from 107 patients with neuroblastoma who underwent surgical resection. We proposed a novel processing pipeline for nuclear segmentation, cell-level image feature extraction, and patient-level feature aggregation. Logistic regression model was built to classify patients with favorable histology (FH) and patients with unfavorable histology (UH). On the training/test dataset, patient-level of nucleus morphological/intensity features and age could correctly classify patients with a mean area under the receiver operating characteristic curve (AUC) of 0.946, a mean accuracy of 0.856, and a mean Matthews Correlation Coefficient (MCC) of 0.703,respectively. On the independent validation dataset, the classification model achieved a mean AUC of 0.938, a mean accuracy of 0.865 and a mean MCC of 0.630, showing good generalizability. Our results suggested that automatically derived image features could identify the differences in nuclear morphological and intensity between different prognostic groups, which could provide a reference to pathologists and facilitate the evaluation of the pathological prognosis in patients with neuroblastoma.

Prognostic value of tumoral and peritumoral magnetic resonance parameters in osteosarcoma patients for monitoring chemotherapy response.

OBJECTIVES: To evaluate parameters of diffusion-weighted imaging (DWI) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) as early imaging indicators of tumor histologic response to pre-operative neoadjuvant chemotherapy and as probable prognostic factors for event-free survival (EFS) and overall survival in osteosarcoma (OS) in both tumoral and peritumoral areas. METHODS: Thirty-four OS patients who received three courses of neoadjuvant chemotherapy followed by surgery during 2014-2018 were enrolled in this study. All patients underwent baseline and post-chemotherapy DWI and DCE-MRI. Lesion region was defined as the tumoral area and peritumoral area. Parameters of apparent diffusion coefficient, capacity transfer constant (Ktrans), elimination rate constant, extravascular extracellular space volume ratio (Ve), and initial area under the curve as well as corresponding differences between pre- and post-chemotherapy in lesion regions were evaluated. Receiver operating characteristic analysis was used to evaluate the diagnostic performance of these parameters. The associations of all parameters with tumor histologic response, EFS, and overall survival were also calculated. RESULTS: In the tumor area, moderate evidence was found that post-Ktrans was lower in responders as compared with that in poor responders (p = 0.04, false discovery rate [FDR] corrected), and ΔKtrans exhibited significant between-groups differences (p = 0.04, Bonferroni corrected; or p = 0.006, FDR corrected). Weak evidence for the between-groups difference was found in the Ve in the peritumoral area (p = 0.025 before treatment and p = 0.021 after treatment, uncorrected). Furthermore, lower post-Ktrans in the tumoral area and lower pre-Ve in the peritumoral area were significant prognostic indicators for longer EFS (p = 0.002, p = 0.026) and overall survival (p = 0.003, p = 0.023). CONCLUSIONS: In OS, DWI and DCE-MRI parameters in both tumoral and peritumoral areas can reflect the chemotherapy response and prognosticate EFS and overall survival. KEY POINTS: • Peritumoral MRI parameters can reflect the chemotherapy response in OS patients. • Peritumoral MRI parameters can predict EFS and overall survival in OS patients. • MRI parameters may be predictive factors for evaluating chemotherapy efficacy and EFS.

SIRT1 activation by butein attenuates sepsis-induced brain injury in mice subjected to cecal ligation and puncture via alleviating inflammatory and oxidative stress.

Sepsis-induced brain injury is frequently encountered in critically ill patients with severe systemic infection. Butein (3,4,2',4'-tetrahydroxychalcone) has been demonstrated as the neuro-protective agent via reducing inflammation and oxidative stress on neurons. Moreover, activation of silent information regulator 1 (SIRT1) inhibits apoptosis, oxidation and inflammation thus alleviating sepsis-induced multiorgan injuries. In present study, we show that butein administrated intraperitoneally (10 mg/kg) saved mice from sepsis-induced lethality by increasing 7-day survival rate after cecal ligation and puncture (CLP) surgery. Additionally, butein treatment enhanced SIRT1 signaling thus decreasing the Ac-NF-κB, Ac-FOXO1 and Ac-p53 levels, thus attenuating the brain injury of mice after CLP surgery by decreasing cerebral edema, maintaining the blood-brain barrier integrity, inhibiting neuronal apoptosis, and decreasing pro-inflammatory cytokines production (IL-6, TNF-α and IL-1ß) and oxidative stress (downregulation of MDA, and upregulation of SOD and CAT) in both serum and cerebral cortex tissues. Moreover, butein treatment attenuated LPS induced neurological function loss. However, all above mentioned neuro-protective actions of butein were partially inhibited by EX527 co-treatment, one standard SIRT1 inhibitor. Collectively, butein attenuates sepsis-induced brain injury through alleviation of cerebral inflammation, oxidative stress and apoptosis by SIRT1 signaling activation.

Application and preliminary validation of the hip inflammation MRI scoring system (HIMRISS) in spondyloarthritis.

AIMS: Spondyloarthritis (SpA) is an inflammatory rheumatic disease and hip involvement often results in severe deformities and functional impairment. Magnetic resonance imaging (MRI) is a powerful imaging tool for detecting early hip lesions in SpA. The aims of this study are to apply the hip inflammation MRI scoring system (HIMRISS) in SpA patients and to evaluate its reproducibility and validity. METHODS: Four readers new to HIMRISS (two radiologists and two rheumatologists) scored the MRI scans obtained from a total of 55 SpA patients with hip lesions in two separate exercises (33 patients in exercise 1 and 22 patients in exercise 2). After the training and review process for exercise 1, these well-trained readers with expertise in the HIMRISS method scored the scans obtained from 22 patients and evaluated the association between HIMRISS and clinical activity of SpA, including Ankylosing Spondylitis Disease Activity Score (ASDAS), laboratory features, Bath Ankylosing Spondylitis Disease Activity Index and Harris hip scores. RESULTS: HIMRISS is a reliable MRI scoring method for bone merrow lesions (BML) and synovitis in SpA. After 2 training exercises, the reliability improved from 0.67 to 0.90. The reliability for detecting femoral BML, acetabular BML, and synovitis effusion was very good after the 2 exercises (overall intraclass correlation coefficient = 0.73, 0.84 and 0.88, respectively). The clinical correlations between HIMRISS and ASDAS were most significant, and the correlations were closer to summed bilateral HIMRISS scores than just the worse side. HIMRISS was found to be an excellent tool for the early diagnosis of inflammation before the occurrence of structural damage, which was not significantly reflected in the systemic diagnosis. CONCLUSION: HIMRISS offers a reliable MRI scoring method for hip joint in SpA, and it is beneficial for early detection and fast quantification in disease activity assessment.

Intramyocardial Injection of Recombinant Adeno-Associated Viral Vector Coexpressing PR39/Adrenomedullin Enhances Angiogenesis and Reduces Apoptosis in a Rat Myocardial Infarction Model.

Cotransfer of angiogenic and antiapoptotic genes could be the basis of new gene therapy strategies for myocardial infarction. In this study, rAAV-PR39-ADM, coexpressing antimicrobial peptide (PR39) and adrenomedullin (ADM), was designed with the mediation of recombinant adeno-associated virus. In vitro, CRL-1730 cells were divided into four groups, namely, the sham group, the AAV-null group, the NS (normal saline) group, and the PR39-ADM group. Immunocytochemistry analysis, CCK-8 assays, Matrigel assays, and apoptotic analysis were performed; in vivo, myocardial infarction model was established through ligation of the left coronary artery on rats, and treatment groups corresponded to those used in vitro. Myocardial injury, cardiac performance, and the extent of myocardial apoptosis were assessed. Results suggested that rAAV-PR39-ADM administration after myocardial infarction improved cell viability and cardiac function, attenuated apoptosis and myocardial injury, and promoted angiogenesis. Subsequently, levels of 6×His, HIF-1α, VEGF, p-Akt, Akt, ADM, Bcl-2, and Bax were measured by western blot. rAAV-PR39-ADM increased p-Akt, HIF-1α, and VEGF levels and induced higher Bcl-2 expression and lower Bax expression. In conclusion, our results demonstrate that rAAV-PR39-ADM mitigates myocardial injury by promoting angiogenesis and reducing apoptosis. This study suggests a potential novel gene therapy-based method that could be used clinically for myocardial infarction.

Image quality and radiation dose of dual-source CT cardiac angiography using prospective ECG-triggering technique in pediatric patients with congenital heart disease.

BACKGROUND: All studies involving use of ionizing radiation should be performed in accordance with the ALARA (As Low As Reasonably Achievable) principle, especially in children. In this study, the prospective ECG triggering technique with low voltage was used in dual-source computed tomography (DSCT) angiography to investigate if image quality with low radiation dose could be satisfactory in pediatric patients with congenital heart disease. METHODS: Sixty pediatric patients with suspected congenital cardiovascular anomalies were enrolled prospectively in the study. They were randomly assigned to two groups for DSCT angiography. Group A were scanned by prospective ECG-triggering computed tomography angiography (CTA) with 80 kV tube voltage, while group B by used non-ECG-gated CTA with the same tube voltage. The anomaly accuracy was evaluated based on the surgical and/or conventional cardiac angiography findings. The overall image quality was assessed on a five-point scale. And the diagnostic accuracy and radiation dose was evaluated in both groups. RESULTS: There were 127 cardiovascular anomalies in Group A and 108 in Group B. The mean subjective image quality and diagnostic accuracy between these two groups were significantly different (P = 0.007 and 0.011, respectively). The mean effective dose in Group A and Group B was 0.38 ± 0.13 mSv and 0.35 ± 0.17 mSv, respectively. But there was no significant difference between two groups (P = 0.197). CONCLUSIONS: The prospective ECG triggering technique in DSCT scan can offer better image quality and diagnostic accuracy with low radiation exposure in pediatric patients with congenital heart diseases. This technique has potential to become a new clinical routine in pediatric cardiac computed tomography (CT) imaging.

Osthole Alleviates Bleomycin-Induced Pulmonary Fibrosis via Modulating Angiotensin-Converting Enzyme 2/Angiotensin-(1-7) Axis and Decreasing Inflammation Responses in Rats.

Studies have shown that angiotensin-converting enzyme 2 (ACE2) plays modulating roles in lung pathophysiology, including pulmonary fibrosis (PF) and acute lung injury. Pulmonary fibrosis is a common complication in these interstitial lung diseases, and PF always has a poor prognosis and short survival. To date, there are few promising methods for treating PF, and they are invariably accompanied by severe side effects. Recent studies have showed that the traditional Chinese herbal extract, osthole, had beneficial effects on lipopolysaccharide (LPS) induced acute lung injury (ALI) via an ACE2 pathway. Here we further investigated the protective effects of osthole on bleomycin induced pulmonary fibrosis and attempted to determine the underlying mechanism. PF mode rats were induced by bleomycin (BLM) and then subsequently administered osthole. Histopathological analyses were employed to identify PF changes. The results showed that BLM resulted in severe PF and diffuse lung inflammation, together with significant elevation of inflammatory factors and a marked increase in expression of angiotensin II (ANG II) and transforming growth factor-beta 1 (TGF-ß1). ACE2 and angiotensin-(1-7) [ANG-(1-7)] were both greatly reduced after BLM administration. Meanwhile, osthole treatment attenuated BLM induced PF and inflammation, decreased the expression of these inflammatory mediators, ANG II, and TGF-ß1, and reversed ACE2 and ANG-(1-7) production in rat lungs. We conclude that osthole may exert beneficial effects on BLM induced PF in rats, perhaps via modulating the ACE2/ANG-(1-7) axis and inhibiting lung inflammation pathways.

Overexpression of Rcan1-1L inhibits hypoxia-induced cell apoptosis through induction of mitophagy.

Mitophagy, a cellular process that selectively targets dysfunctional mitochondria for degradation, is currently a hot topic in research into the pathogenesis and treatment of many human diseases. Considering that hypoxia causes mitochondrial dysfunction, which results in cell death, we speculated that selective activation of mitophagy might promote cell survival under hypoxic conditions. In the present study, we introduced the Regulator of calcineurin 1-1L (Rcan1-1L) to initiate the mitophagy pathway and aimed to evaluate the effect of Rcan1-1L-induced mitophagy on cell survival under hypoxic conditions. Recombinant adenovirus vectors carrying Rcan1-1L were transfected into human umbilical vein endothelial cells and human adult cardiac myocytes. Using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay and Trypan blue exclusion assay, Rcan1-1L overexpression was found to markedly reverse cell growth inhibition induced by hypoxia. Additionally, Rcan1-1L overexpression inhibited cell apoptosis under hypoxic conditions, as detected by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis assay. Meanwhile, the mitochondria-mediated cell apoptotic pathway was inhibited by Rcan1-1L. In contrast, knockdown of Rcan1-1L accelerated hypoxia-induced cell apoptosis. Moreover, Rcan1-1L overexpression significantly reduced mitochondrial mass, decreased depolarized mitochondria, and downregulated ATP and reactive oxygen species production. We further delineated that the loss of mitochondrial mass was due to the activation of mitophagy induced by Rcan1-1L. Rcan1-1L overexpression activated autophagy flux and promoted translocation of the specific mitophagy receptor Parkin into mitochondria from the cytosol, whereas inhibition of autophagy flux resulted in the accumulation of Parkin-loaded mitochondria. Finally, we demonstrated that mitochondrial permeability transition pore opening was significantly increased by Rcan1-1L overexpression, which suggested that Rcan1-1L might evoke mitophagy through regulating mitochondrial permeability transition pores. Taken together, we provide evidence that Rcan1-1L overexpression induces mitophagy, which in turn contributes to cell survival under hypoxic conditions, revealing for the first time that Rcan1-1L-induced mitophagy may be used for cardioprotection.

Bone morphogenetic protein-10 induces cardiomyocyte proliferation and improves cardiac function after myocardial infarction.

Heart disease is among the leading causes of death worldwide, and the limited proliferation of mammalian cardiomyocytes prevents heart regeneration in response to injury. Bone morphogenetic protein-10 (BMP10) exerts multiple roles in various developmental events; however, the effect of BMP10 and the underlying mechanism involved in cardiac repair remains unclear. After stimulation with the recombinant BMP10, an obvious dose-dependent cardiomyocyte proliferation and reentry of differentiated mammalian cardiomyocytes into the cell cycle was observed. Furthermore, BMP10 stimulation strikingly enhanced Tbx20 expression. Further analysis demonstrated that T-box 20 (Tbx20) was involved in BMP10-induced proliferation of differentiated cardiomyocytes as preconditioning with Tbx20 siRNA significantly attenuated BMP10-induced DNA synthesis. In vivo, BMP10 induced rat cardiomyocyte DNA synthesis and cytokinesis. After myocardial infarction (MI), BMP10 stimulated cardiomyocyte cell-cycle reentry and mitosis, resulting in the decrease of infarct size and improvement of cardiac repair. Taken together, these data indicated that BMP10 stimulated cardiomyocyte proliferation and repaired cardiac function after heart injury. Consequently, BMP10 may be a potential target for innovative strategies against heart failure.

Recombinant AAV-PR39-mediated hypoxia-inducible factor 1α gene expression attenuates myocardial infarction.

PR39 is an angiogenic masterswitch protein, belonging to the second generation of angiogenic growth factors. However, the role of recombinant adeno-associated virus (AAV) carrying the PR39 fusion gene (AAV-PR39) in acute myocardial infarction remains unclear. Therefore, in this study, we investigated the role of AAV-PR39 in an experimental animal model of acute myocardial infarction. The PR39 gene was fused with the transmembrane peptide, TAT, 6xHis­tag and NT4 signal sequences. AAV-PR39 was then obtained by calcium phosphate co-precipitation. A total of 18 healthy Chinese mini pigs were randomly divided into an experimental groups (the AAV-PR39-treated group) and a control group [phosphated-buffered saline (PBS)-treated group]. Following the induction of myocardial infarction, enhanced 3.0T MR imaging was performed to observe the changes in myocardial signal intensity at 0 h, 1, 2 and 3 weeks. The expression of hypoxia-inducible factor­1α (HIF-1α) in the myocardial tissues was determined by SABC immunohistochemistry. In addition, in vitro experiments using CRL-1730 endothelial cells transfected with AAV vector containing NT4-TAT-His-PR39 revealed that the AAV-PR39-treated group had a significantly higher expression of HIF-1α compared with the control group. Moreover, PR39 regulated the HIF-1α-induced expression of angiogenic growth factors. Under hypoxic conditions, the anti-apoptotic effects in the AAV-PR39 group were more pronounced than those observed in the control (PBS-treated) group. In vivo, the enforced expression of recombinant PR39 elevated the level of HIF-1α under hypoxic conditions and decreased the size of the infarcted areas by upregulating the expression of HIF-1α in the areas surrounding the infarct area. Taken together, our data demonstrate that the recombinant AAV-PR39-mediated HIF-1α expression attenuates myocardial infarction, indicating that AAV-PR39 may serve as a novel therapeutic agent for the treatment of myocardial infarction.

The ascending aortic image quality and the whole aortic radiation dose of high-pitch dual-source CT angiography.

BACKGROUND: Aortic dissection is a lift-threatening medical emergency associated with high rates of morbidity and mortality. The incidence rate of aortic dissection is estimated at 5 to 30 per 1 million people per year. The prompt and correct diagnosis of aortic dissection is critical. This study was to compare the ascending aortic image quality and the whole aortic radiation dose of high-pitch dual-source CT angiography and conventional dual-source CT angiography. METHODS: A total of 110 consecutive patients with suspected aortic dissection and other aortic disorders were randomly divided into two groups. Group A underwent traditional scan mode and Group B underwent high-pitch dual-source CT scan mode. The image quality and radiation dose of two groups were compared. RESULTS: Close interobserver agreement was found for image quality scores (κ = 0.87). The image quality of ascending aorta was significantly better in the high-pitch group than in the conventional group (2.78 ± 0.46 vs 1.57 ± 0.43, P < 0.001). There was no significant difference of the CT attenuation values, the aortic image noise and SNR between two groups. The mean radiation dose of high-pitch group was also significantly lower than that of conventional group (2.7 ± 0.6 mSv vs. 3.9 ± 0.9 mSv, P < 0.001). CONCLUSIONS: High-pitch dual-source CT angiography of the whole aorta can provide motion-artifact-free imaging of the ascending aorta at a low radiation dose compared to conventional protocol.

Construction of PR39 recombinant AAV under control of the HRE promoter and the effect of recombinant AAV on gene therapy of ischemic heart disease.

The objective of this study was to investigate the effect of the PR39 recombinant adeno-associated virus (AAV) controlled by the hypoxia-responsive element (HRE) on gene therapy of ischemic heart disease. The minimal HRE was artificially synthesized and the AAV vector controlled by HRE was introduced with NT4-TAT-His-PR39 to investigate the expression of AAV-PR39 in hypoxic vascular endothelial cells (VEC) of human umbilical vein (CRL-1730 cell line) and the angiogenesis-promoting effect in pigs with acute myocardial infraction (AMI). The minimal HRE/CMV was designed and artificially synthesized using the PCR method and cloned with the T vector cloning method. The pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV plasmid was constructed. Using the calcium phosphate precipitation method, HEK-293 cells were co-transfected with three plasmids to produce the recombinant virus. An equal volume of pSS-HRE-CMV-NT4-6His-PR39-PolyAAAV and enterovirus (EV, blank virus) was transfected into CRL-1730 cell lines, respectively. The immunohistochemical method was used to assay the expression of 6xHis in CRL-1730 cell lines and the expression of PR39 under hypoxia. Eighteen AMI miniature pigs were randomized into the experimental group (HRE-AAV-PR39 group), control group 1 (physical saline group) and control group 2 (EV group). The area of ischemia was assessed with conventional MRI and myocardium perfusion MRI. Pigs were sacrificed at preset time-points to obtain samples of ischemic myocardium. Morphological and pathological data were collected. According to data in the literature and databases, the minimal HRE was designed and synthesized with the PCR method. A large number of HREs were connected to modified pSSHGAAV (pSSV9int-/XbaI) vector followed by insertion of the NT4-6His-PR39 gene segment and, thus, the recombinant plasmid pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV was successfully constructed. The expression of 6xHis in CRL-1730 cells under the regulation of HRE was assayed using the immunohistochemical method and results showed that the expression was positive in the experimental group. Myocardium perfusion MRI displayed that the infracted area significantly decreased under the action of pSS-HRE-CMV-NT4-PR39-PolyA-AAV. The artificial minimal HRE in CRL-1730 cells effectively and rapidly regulates the expression of the downstream gene NT4-TAT-His-PR39 of the CMV promoter. Recombinant pSS-HRE-CMV-NT4-PR39-Poly-AAAV promotes neoangiogenesis in the ischemic area, reduces the area of infarction and improves heart function.

[Construction of a general AAV vector regulated by minimal and artificial hypoxic-responsive element].

AIM: To synthesize the minimal and artificial HRE, and to insert it into the anterior extremity of CMV promoter of a AAV plasmid, and then to construct the AAV regulated by hypoxic-responsive element which was introduced into 293 cell by method of Ca3(PO4)2 using three plasmids. Thus obtaining the adenoassociated virus vector regulated by hypoxic-responsive element was possibly used for gene therapy in ischemia angiocardiopathy and cerebrovascular disease. METHODS: Artificially synthesize the 36 bp nucleotide sequences of four connection in series HIF-binding sites A/GCGTG(4×HBS)and a 35 bp nucleotide sequences spacing inserted into anterior extremity of CMV promoter TATA Box, then amplified by PCR. The cDNA fragment was confirmed to be right by DNA sequencing. Molecular biology routine method was used to construct a AAV vector regulated by minimal hypoxic-responsive element after the normal CMV promoter in AAV vector was replaced by the CMV promoter included minimal hypoxic-responsive element. Then, NT4-6His-PR39 fusogenic peptide was inserted into MCS of the plasmid, the recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells, in which we can also investigate the expression of 6×His using immunochemistry in hypoxia environment. RESULTS: Artificial HRE was inserted into anterior extremity of CMV promoter and there was a correct spacing between the HRE and the TATA-box. The DNA sequencing and restriction enzyme digestion results indicated that the AAV regulated by hypoxic-responsive element was successfully constructed. Compared to the control group, the expressions of 6×His was significantly increased in the experimental groups in hypoxia environment, which confirmed that the AAV effectually regulated by the minimal HRE was inserted into anterior extremity of CMV promoter. CONCLUSION: The HRE is inserted into anterior extremity of CMV promoter to lack incision enzyme recognition site by PCR. And eukaryotic expression vector regulated by hypoxic-responsive is constructed. The AAV effectually regulated by the minimal HRE inserted into anterior extremity of CMV promoter. The vector is successfully constructed and it has important theoretical and practical value in the synteresis and therapy of ischemia angiocardiopathy and cerebrovascular disease.

[Secretory expression of PR39 following adeno-associated viral-encoding fusion gene transfer induces angiogenesis in hypoxia chick embryo].

OBJECTIVES: To investigate the impact of AAV-encoding NT4-TAT-His-PR39 fusion gene expression on HIF-1alpha level in ECV304 cultured under hypoxic condition (1%O(2)) and on angiogenesis in hypoxic chick embryo. METHODS: PR39 cDNA was connected with NT4, TAT, 6 x His cDNA by molecular biology methods. The recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells. Then ECV304 were respectively infected with AAV-NT4-TAT-His-PR39, 6 x His expression and HIF-1alpha level in ECV304 were detected by immunocytochemistry. The chicken embryos were randomized into the AAV-PR39, EV and PBS groups (n = 10 each) subject to hypoxia (5%O(2), n = 15) or normoxia environments (n = 15), the vessel density of the chicken chorioallantoic membrane (CAM) were measured by Image Pro Plus (IPP) software. RESULTS: The expression of 6 x His protein was detected in AAV-PR39 infected ECV304 cells. HIF-1alpha protein activity was significantly increased in AAV-PR39 infected ECV304 underwent hypoxia compared to PBS and non-infected ECV304 groups (P < 0.05). The vessel density of chicken CAM in hypoxia environment but not in normoxia environment was also significantly higher in AAV-PR39 group than in EV group and PBS group (all P < 0.05). CONCLUSION: AAV-encoding NT4-TAT-His-PR39 fusion gene expression significantly increased HIF-1alpha level in ECV304 exposed to hypoxia and promoted angiogenesis in hypoxic chicken embryo.