The Integrated Analysis Identifies Three Critical Genes as Novel Diagnostic Biomarkers Involved in Immune Infiltration in Atherosclerosis.

Atherosclerosis (AS), a chronic inflammatory disease of the blood vessels, is the primary cause of cardiovascular disease, the leading cause of death worldwide. This study aimed to identify possible diagnostic markers for AS and determine their correlation with the infiltration of immune cells in AS. In total, 10 serum samples from AS patients and 10 samples from healthy subjects were collected. The original gene expression profiles of GSE43292 and GSE57691 were downloaded from the Gene Expression Omnibus database. Least absolute shrinkage and selection operator regression model and support vector machine recursive feature elimination analyses were carried out to identify candidate markers. The diagnostic values of the identified biomarkers were determined using receiver operating characteristic assays. The compositional patterns of the 22 types of immune cell fraction in AS were estimated using CIBERSORT. RT-PCR was performed to further determine the expression of the critical genes. This study identified 17 differentially expressed genes (DEGs) in AS samples. The identified DEGs were mainly involved in non-small cell lung carcinoma, pulmonary fibrosis, polycystic ovary syndrome, glucose intolerance, and T-cell leukemia. FHL5, IBSP, and SCRG1 have been identified as the diagnostic genes in AS. The expression of SCRG1 and FHL5 was distinctly downregulated in AS samples, and the expression of IBSP was distinctly upregulated in AS samples, which was further confirmed using our cohort by RT-PCR. Moreover, immune assays revealed that FHL5, IBSP, and SCRG1 were associated with several immune cells, such as CD8 T cells, naïve B cells, macrophage M0, activated memory CD4 T cells, and activated NK cells. Overall, future investigations into the occurrence and molecular mechanisms of AS may benefit from using the genes FHL5, IBSP, and SCRG1 as diagnostic markers for the condition.

Glutamine protects cardiomyocytes from hypoxia/reoxygenation injury under high glucose conditions through inhibition of the transforming growth factor-ß1-Smad3 pathway.

Activation of transforming growth factor-ß1 (TGF-ß1)-Smad3 pathway aggravates myocardial ischemia/reperfusion injury (IRI). We previously showed that glutamine (Gln) protects cardiomyocytes from hypoxia/reoxygenation (H/R) injury under high glucose (HG) conditions. The aim of this study was to investigate whether Gln exerts its protective effect in H/R via inhibiting TGF-ß1-Smad3 pathway. In vitro, H9c2 rat cardiomyocytes were treated with Gln with HG (33 mM) and/or H/R. We also performed in vivo experiments in which we treated normal and diabetic rats with Gln or solvent control following IRI. We assessed protein levels of TGF-ß1, total Smad3, phosphorylated (p)-Smad3 and cleaved caspase-3 in H9c2 cells and rat myocardium by Western blotting. H9c2 cells treated with HG + H/R exhibited high apoptosis rates, as well as a highly activated TGF-ß1-Smad3 pathway. TGF-ß1 receptor inhibitor (SB431542) or Smad3 inhibitor (SIS3) reduced HG + H/R induced apoptosis. Similarly, Gln supplementation alleviated apoptosis and decreased p-Smad3 levels. However, Gln's protective effect was significantly weakened by TGF-ß1. Diabetic rats treated with Gln had improved hemodynamics, smaller infarct size after IRI, and a significant decrease in TGF-ß1-Smad3 pathway activation. We conclude that Gln inhibits HG + H/R induced activation of the TGF-ß1-Smad3 pathway and decreases cell apoptosis in cardiomyocytes.

A novel model for evaluating thrombolytic therapy in dogs with ST-elevation myocardial infarction.

BACKGROUND: There is still no standard large animal model for evaluating the effectiveness of potential thrombolytic therapies. Here, we aimed to develop a new beagle model with ST-elevation myocardial infarction (STEMI) by injecting autologous emboli with similar components of coronary thrombus. METHODS: 18 male beagles were included and divided into three groups: red embolus group (n = 6), white embolus group (n = 6) or white embolus + rt-PA group (n = 6). Autologous emboli were infused into the mid-distal region of the left anterior descending coronary artery. The composition of embolus was examined by scanning electron microscope (SEM). Coronary angiography was performed to verify the status of embolism. Myocardial infarct size was measured by 2, 3, 5- triphenyltetrazolium chloride (TTC) staining. RESULTS: Red thrombus was characteristic of loose reticular structure of erythrocytes under SEM, while the white embolus had compacted structure that mainly consisted of a dense mass of fibrin. Coronary angiography showed the recanalization rate was 2/6 in the red embolus group versus 0/6 in the white embolus group in three hours after occlusion. Arrhythmia, resolution of ST-segment elevation and lower T wave on the electrocardiogram appeared in the red embolus group but not in the white embolus group. Another six dogs with white thrombi were treated with rt-PA. Five out of six dogs exhibited coronary recanalization after two hours of therapy, compared to zero dogs without rt-PA treatment. The size of myocardial infarction in rt-PA group reduced significantly compared with white embolus group using TTC staining method. CONCLUSIONS: The white embolism model was more convenient experimentally and had a higher uniformity, stability and success rate. The major innovation of our study is that we applied fibrin-rich white thrombi to establish beagle model possessing features of clinically observed coronary thrombi in time window of intravenous thrombolysis of STEMI. This model can be used to evaluate new thrombolytic drugs for the treatment of STEMI.

Twist may be associated with invasion and metastasis of hypoxic NSCLC cells.

Hypoxia promotes tumor invasion and metastasis via multiple mechanisms, including epithelial-mesenchymal transition (EMT). Twist, an EMT regulator, has been disclosed to associate with invasion and metastasis as well as poor prognosis of many malignancies. However, it remains undefined whether Twist is involved in invasion and metastasis of hypoxic non-small cell lung cancer (NSCLC). In this study, protein levels of Twist, hypoxia-inducible factor-1α (HIF-1α), and EMT markers (E-cadherin and vimentin) were examined by immunohistochemistry in 76 lung cancer tissues from NSCLC patients. Expression of Twist and its correlation with HIF-1α, E-cadherin, and vimentin were analyzed. Small interfering RNA (siRNA) against Twist was used to knockdown Twist expression in hypoxic NSCLC cells, A549 and NCI-H460. Cellular invasion and protein levels of Twist, E-cadherin, and vimentin were evaluated by matrigel invasion assay and Western blot, respectively. Our results showed that in clinical samples, there was a significant association between Twist expression and differentiation degree, lymph node metastasis, and TNM stage. Correlation analysis demonstrated that expression of Twist was negatively correlated with E-cadherin expression, but positively associated with HIF-1α and vimentin expression. In cultured NSCLC cells, Twist messenger RNA (mRNA) and protein levels were upregulated under hypoxia, while knockdown of Twist suppressed potentiated invasion and expression of mesenchymal marker vimentin induced by hypoxia. Protein level of increased epithelial marker E-cadherin was shown along with Twist downregulation. These findings suggest that Twist promoting hypoxic invasion and metastasis of NSCLC may be associated with altered expression of EMT markers. Inhibition of Twist may be of therapeutic significance.

Glutamine Reduces the Apoptosis of H9C2 Cells Treated with High-Glucose and Reperfusion through an Oxidation-Related Mechanism.

Mitochondrial overproduction of reactive oxygen species (ROS) in diabetic hearts during ischemia/reperfusion injury and the anti-oxidative role of glutamine have been demonstrated. However, in diabetes mellitus the role of glutamine in cardiomyocytes during ischemia/reperfusion injury has not been explored. To examine the effects of glutamine and potential mechanisms, in the present study, rat cardiomyoblast H9C2 cells were exposed to high glucose (33 mM) and hypoxia-reoxygenation. Cell viability, apoptosis, intracellular glutamine, and mitochondrial and intracellular glutathione were determined. Moreover, ROS formation, complex I activity, membrane potential and adenosine triphosphate (ATP) content were also investigated. The levels of S-glutathionylated complex I and mitochondrial apoptosis-related proteins, including cytochrome c and caspase-3, were analyzed by western blot. Data indicated that high glucose and hypoxia-reoxygenation were associated with a dramatic decline of intercellular glutamine and increase in apoptosis. Glutamine supplementation correlated with a reduction in apoptosis and increase of glutathione and glutathione reduced/oxidized ratio in both cytoplasm and mitochondria, but a reduction of intracellular ROS. Glutamine supplementation was also associated with less S-glutathionylation and increased the activity of complex I, leading to less mitochondrial ROS formation. Furthermore, glutamine supplementation prevented from mitochondrial dysfunction presented as mitochondrial membrane potential and ATP levels and attenuated cytochrome c release into the cytosol and caspase-3 activation. We conclude that apoptosis induced by high glucose and hypoxia-reoxygenation was reduced by glutamine supplementation, via decreased oxidative stress and inactivation of the intrinsic apoptotic pathway.

Simultaneous modulated accelerated radiotherapy in cervical cancer with retroperitoneal lymph node metastasis after radical hysterectomy and pelvic lymphadenectomy.

OBJECTIVE: To compare the dosimetry, toxicity, and efficacy of simultaneous modulated accelerated radiotherapy (SMART) with 3-dimensional conformal radiotherapy (3DCRT) in cervical cancer with retroperitoneal lymph node metastasis after radical hysterectomy and pelvic lymphadenectomy. METHODS: Total 32 patients who underwent SMART were retrospectively evaluated. Daily fractions of 2.2 to 2.4 Gy and 1.8 to 2 Gy were prescribed and delivered to gross tumor volume and clinical target volume to a total dose of 63.8 and 52.2 Gy, respectively. A 3DCRT plan was designed for the SMART group and planned to deliver the same prescribed dose. The doses of organs at risk (OARs) were compared. Thirty-six patients who received 3DCRT were used to compare the target dose, toxicities, and efficacy with 32 cases who received SMART. RESULTS: The mean doses delivered to gross tumor volume and clinical target volume were significantly higher in the SMART group than in the 3DCRT group (63.8 vs 55.2 Gy [P < 0.01] and 52.5 vs 48.6 Gy [P < 0.01], respectively). For SMART plan, the doses of OARs were significantly lower than that of 3DCRT plans (small intestine: 25.1 vs 30.9 Gy [P < 0.01], bladder: 35.3 vs 46.3 [P < 0.01], and rectum: 31.7 vs 43.7 [P = 0.002], respectively). The patients experienced less acute and late toxicities in the SMART group (acute toxicities: enteroproctitis, P = 0.019; cystitis, P = 0.013; leukopenia, P = 0.025; late toxicities: enteroproctitis, P = 0.007; and cystitis, P = 0.026, respectively). No significant difference was found for 1-year survival (78.7% vs 67.7%, P = 0.222), but SMART group had a higher 2-year survival rate (2-year: 63.1% vs 39.1%, P = 0.029). CONCLUSIONS: Simultaneous modulated accelerated radiotherapy plans yielded higher dose to the targets and better sparing of OARs than did 3DCRT in cervical cancer with retroperitoneal lymph node metastasis after radical hysterectomy and pelvic lymphadenectomy. Simultaneous modulated accelerated radiotherapy provided better clinical outcomes than did 3DCRT. Long-term follow-up and studies involving more patients are needed to confirm our results.

A pig model of ischemic mitral regurgitation induced by mitral chordae tendinae rupture and implantation of an ameroid constrictor.

A miniature pig model of ischemic mitral regurgitation (IMR) was developed by posterior mitral chordae tendinae rupture and implantation of an ameroid constrictor. A 2.5-mm ameroid constrictor was placed around the left circumflex coronary artery (LCX) of male Tibetan miniature pigs to induce ischemia, while the posterior mitral chordae tendinae was also ruptured. X-ray coronary angiography, ECG analysis, echocardiography, and magnetic resonance imaging (MRI) were used to evaluate heart structure and function in pigs at baseline and one, two, four and eight weeks after the operation. Blood velocity of the mitral regurgitation was found to be between medium and high levels. Angiographic analyses revealed that the LCX closure was 10-20% at one week, 30-40% at two weeks and 90-100% at four weeks subsequent ameroid constrictor implantation. ECG analysis highlighted an increase in the diameter of the left atria (LA) at two weeks post-operation as well as ischemic changes in the left ventricle (LV) and LA wall at four weeks post-operation. Echocardiography and MRI further detected a gradual increase in LA and LV volumes from two weeks post-operation. LV end diastolic and systolic volumes as well as LA end diastolic and systolic volume were also significantly higher in pig hearts post-operation when compared to baseline. Pathological changes were observed in the heart, which included scar tissue in the ischemic central area of the LV. Transmission electron microscopy highlighted the presence of contraction bands and edema surrounding the ischemia area, including inflammatory cell infiltration within the ischemic area. We have developed a pig model of IMR using the posterior mitral chordae tendineae rupture technique and implantation of an ameroid constrictor. The pathological features of this pig IMR model were found to mimic the natural history and progression of IMR in patients.

[Establishment of acute myocardial infarction model in diabetic miniature pigs through video-assisted thoracoscopic surgery].

OBJECTIVE: To explore a new method of establishing acute myocardial infarction model in diabetic miniature pigs through video-assisted thoracoscopic surgery (VATS). METHODS: Seven normal miniature pigs and 7 diabetic miniature pigs underwent VATS by selectively ligating left anterior descending coronary artery and then were evaluated through serology, imaging and histology. RESULTS: The serum levels of troponin (cTni) and myoglobin (MYO) in both groups significantly increased by over 10 folds of upper normal limit after VATS. Echocardiography, MRI and histopathologic analysis showed that the affected myocardial parts were apex, left ventricular wall and interventricular septum cosco section. But heart function of diabetic miniature pigs were relatively lower; infarction area/area-at-risk ratio higher (18.4% ± 5.5% vs 5.3% ± 3.9%, P = 0.03) , myocardial infarction through-wall degree more severe. These were in accordance with poor ischemic tolerance in diabetic myocardia. CONCLUSION: VATS is a safe and effective method for establishing AMI model in diabetic miniature pigs.

Inhibitory effect of iron on in vitro proliferation of smooth muscle cells.

BACKGROUND: Iron is a biocorrodible metal that might be used in bioabsorbable stents. This study investigated the effects at the cellular and protein levels of soluble divalent iron (ferrous gluconate) and soluble trivalent iron (ferric chloride) on the proliferation of human aortic smooth muscle cell (HASMC) in vitro. METHODS: The water-soluble tetrazolium (WST-1) test was used to evaluate the effect of iron on proliferation of HASMC and Western blotting was used to measure the levels of signaling proteins involved in proliferative and apoptosis pathways. RESULTS: HASMC proliferation was inhibited in a concentration dependent manner after treatment with soluble divalent and trivalent iron at concentrations of 100-500 µmol/L. Western blotting analysis showed that the proliferating cell nuclear antigen (PCNA) expression following treatment with soluble divalent iron and trivalent iron at 100, 300 and 500 µmol/L was reduced compared to the control. The PCNA expression decreased with increasing iron concentration and to a greater extent with the trivalent iron than with the divalent iron treatment group. The p53 expression was markedly increased in a concentration dependent manner in both iron treatment groups. CONCLUSION: The soluble divalent iron and, to a greater degree trivalent iron, inhibited HASMC proliferation in a dosedependent manner, which may be attributed to reduction of PCNA expression and increase of p53 expression.

[Establishment and evaluation of acute pulmonary thromboembolism model in minipig].

OBJECTIVE: To establish and evaluate an acute pulmonary embolism (APTE) model by selective thromboembolism of lower left pulmonary artery in minipig. METHODS: Through intervention technique, a guiding catheter was inserted via femoral vein into pulmonary artery. And quantitative autologous venous thrombus was injected into the selected lower left pulmonary arteries in 8 minipigs. Thus the intended APTE model was established by selective thromboembolism of lower left pulmonary artery. Hemodynamic parameters were monitored. And computed tomography (CT) and macroscopic dissection were performed to evaluate the minipig APTE model. RESULTS: The measurements of mean pulmonary artery pressure (MPAP, mm Hg, 1 mm Hg = 0.133 kPa) and pulmonary capillary wedge pressure (PCWP, mm Hg) immediately increased significantly after thromboembolism versus the baseline values (MPAP: 42.0 ± 3.4 vs 20.2 ± 3.0, PCWP: 8 ± 2 vs 4 ± 3, both P < 0.05) and stayed at a higher level during the following 2 h. No significant difference existed between the value of cardiac output (CO) at 2 h post-thromboembolism and its baseline counterpart. Moreover, systemic arterial pressure (SAP, mm Hg) and heart rate (HR, beats/min) significantly increased after embolism versus the baseline values (SAP: 102 ± 12 vs 80 ± 7, HR: 119 ± 22 vs 86 ± 14, P = 0.008). Pulmonary arteriography, CT scan and gross anatomy all demonstrated that the selected lower left pulmonary arteries was successfully embolized. CONCLUSION: The establishment of APTE model by selective thromboembolism of lower left pulmonary artery is feasible, well-controlled and stable in minipigs.

Neural progenitor cells derived from adult bone marrow mesenchymal stem cells promote neuronal regeneration.

AIM: It is well known that neural stem/progenitor cells (NS/PC) are an ideal cell type for the treatment of central nervous system (CNS) disease. However, ethical problems have severely hampered fetal NS/PC from being widely used as a source for stem cell therapy. Recently, it has been demonstrated that autologous bone marrow mesenchymal stem cells (BMSC) can transdifferentiate into neural progenitor cells (NPC). The biological function of BMSC derived NPC (MDNPC) in neuronal systems remains unknown. In the present study, we aimed to investigate whether MDNPC can promote in vitro neural regeneration, a process comprising mainly the generation of neurons and neurotransmitters. MAIN METHODS: We co-cultured BMSC, MDNPC or fetal NS/PC with PC12 cells and studied their roles on proliferation, neurite formation and dopamine release from PC12 cells. Furthermore, we also explored the mechanisms by which MDNPC regulate dopamine secretion from PC12 derived neural cells using Western blot. KEY FINDINGS: We found that both MDNPC and NS/PC had similar morphologies and there were no significant differences between MDNPC and NS/PC in promoting PC12 cell proliferation, neurite outgrowth, and dopamine release. We also demonstrated that NS/PC induced dopamine secretion was associated with an upregulation of dopamine transporter (DAT) levels. SIGNIFICANCE: In summary, MDNPC were comparable to NS/PC in promoting neural regeneration, indicating that MDNPC are a promising candidate source of neural stem cells for the treatment of neurological diseases.

[Time trends on the prevalence of cancer during 1970-2005 in Shandong province].

OBJECTIVE: To analyze time trend of cancer during 1970-2005 in Shandong province so as to develop strategies for control and prevention of cancer at the community level. METHODS: Data was from 4 retrospective surveys regarding all causes of death during 1970-1974, 1985-1989, 1990-1992 and 2004-2005, in Shandong province. Other than one set of data collected in 1985-1989 by Shandong province itself, the other 3 set of data were from the national surveys, in which the survey-point sampling of choice was based on data of 1970-1974 for assessing its representativeness. The observing indices would include standardized mortality and mortality. A join-point regression model was used to analyze the changing rate of tumor. RESULTS: The mortality rate of the entire tumor increased 143.15 percent in 2005 than in 1970. The changing slope of standardized rate of all tumors in the regression model showed that the inter-annual growth rate were 0.54 and 1.24 percent from 1970 to 1984 and from 1985 to 1992. The rate of increase since 1992 had been 0.18 percent. During 2004-2005, the main malignant cancers were lung, stomach, liver, esophageal, colorectal, leukemia, breast and cervical cancer, in order. Lung cancer rose from the 4th ranking to the first while cervical cancer dropped from the fifth ranking to the 8th place. Esophageal cancer and cervical cancer were decreased annually while gastric cancer was increased in the early days but decreased later on. The rest of the cancers were on the rise year by year. Rates of lung and breast cancers were higher while gastric and esophageal cancers were lower seen in the urban than in rural areas. CONCLUSION: In Shandong province, a marked increase was seen in the mortality rate of tumors in the past 35 years. Evidence showed that the spectrum of death among main malignant tumors had changed which might provide a scientific basis for the development of a community-based prevention and control program on cancer.