Dynamic imaging of autophagy-lysosomal pathway and autophagy function following pulmonary hypoxia/reoxygenation in vitro.

Alterations of the autophagy-lysosomal pathway (ALP) and autophagy have been involved in lung ischemia-reperfusion (I/R) injury. However, dynamic imaging of ALP function under lung I/R injury particularly is not fully understood. Here we depicted the live-cell fluorescence imaging of autophagosome to monitor ALP activation and autophagy function. The pAsRed2-N1-LC3 vectors were transfected into CRL-2192 NR8383 (an alveolar macrophage cell line) and CCL149 (an alveolar epithelial cell line) successfully. 0-h, 2-h, 4-h, and 6-h hypoxia/0-h, 2-h, 4-h, and 6-h reoxygenation were then induced with an ALP inhibitor (3-MA) or activator (rapamycin) in the culture of transfected cells separately. ALP activation was conformed by up-regulating AMPK and beclin1 expression. Apoptosis was not obvious in 2-h hypoxia/2-h reoxygenation. pAsRed2-N1-LC3 CCL149 and pAsRed2-N1-LC3 NR8383 cells revealed gradually enhanced AsRed2 from 2-h to 6-h hypoxia/reoxygenation. AsRed2 varied sensitively to 3-MA and rapamycin interventions during 2-h hypoxia/reoxygenation. Our data provides a simple method of autophagosome imaging to monitor ALP activation and autophagy function in lung I/R injury.

In vitro selection and identification of ssDNA aptamers recognizing the Ras protein.

The aim of this study was to develop high-affinity single-stranded DNA (ssDNA) aptamers that can selectively recognize the protein Ras and can be used as preventive and therapeutic agents for restenosis occurring after coronary surgery or angioplasty. For this purpose, we used the systematic evolution of ligands by exponential enrichment (SELEX) technique, also known as in vitro selection. Using this technique, ssDNA aptamers recognizing the Ras protein were obtained from a synthesized random ssDNA library in vitro. The binding rate and affinity of each aptamer pool, isolated in successive rounds of selection, were measured using ELISA, and the finally selected aptamer pool was cloned and sequenced. The binding affinities of each aptamer in this pool were measured. Their primary and secondary structures were analyzed using the DNAMAN 5.29 software, and the relationship between these structures and corresponding binding affinities was analyzed. The rate of aptamer pool binding to the Ras protein gradually increased from 2.4 to 34.5% along the selection process. Optical density (OD) and equilibrium dissociation constant (Kd) measurements showed that OD gradually increased from 0.220 to 1.080 and Kd decreased from 51.5 to 18.3 nM. The 11th pool of aptamers was selected based on these analyses, and cloning and sequencing of individual aptamers was performed. Secondary structure analysis revealed different conformations, but of a single type: stem­loop. The aptamer Ra1 showed the highest affinity, with a measured OD of 1.213 and an estimated Kd of 15.3 nM. The binding affinity of the aptamer Ra1 to Ras was dose-dependent. In conclusion, high­affinity ssDNA aptamers recognizing the Ras protein have been successfully selected. These aptamers may serve in the future as preventive and/or therapeutic agents for restenosis occurring after coronary surgery or angioplasty.

γ-Secretase inhibitor DAPT attenuates intimal hyperplasia of vein grafts by inhibition of Notch1 signaling.

The proliferation and high plasticity of vascular smooth muscle cells (vSMCs) are the major reasons for restenosis of vein grafts. N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), specific inhibitor of γ-secretase, has been shown to regulate vSMC proliferation and differentiation through the Notch signaling pathway, but the pathophysiological importance of these findings in venous grafts has not yet been determined. A rat vein graft model was employed wherein the left jugular vein was surgically interposed into the left common carotid artery. Daily subcutaneous injections of DAPT or placebo (DMSO) were administered postoperatively (control animals received no treatment). We showed that DAPT can inhibit restenosis of vein grafts by inhibiting vSMC proliferation and increasing apoptosis in vivo. Notch1 signaling was highly active during the development of intima thickening. By blocking the Notch signaling pathway, the γ-secretase inhibitor DAPT can significantly attenuated intima thickening. These changes in vein grafts coincided with enhanced binding of myocardin to the smooth muscle-specific protein SM22 and smooth muscle myosin heavy chain at the promoters of vSMC differentiation-specific genes. These studies showed that DAPT can restore the vSMC phenotype and inhibit vSMC proliferation through suppression of the Notch1 signaling pathway, and thus opens a new avenue for the treatment of restenosis in vein grafts.

[The clinical study of modified total aortic arch replacement and stent elephant trunk technique treatment for patients with DeBakey I thoracic aortic dissection].

OBJECTIVE: To summarize the clinical study of modified total aortic arch replacement and stent elephant trunk technique treatment to patients with DeBakey I thoracic aortic dissection. METHODS: From January 2006 to October 2010, 101 cases of DeBakey I aortic dissection were treated by modified total arch replacement and stent elephant trunk technique, in which emergency surgery for 73 cases. There were 76 male and 25 female patients, aged from 21 to 77 years with a mean of (49 ± 8) years. Intraoperative ascending aortic replacement in 31 cases, Bentall procedure in 29 cases, Wheat procedure in 7 cases, David procedure in 34 cases. At the same time stent elephant trunk in the left subclavian artery corresponding position was windowed to rebuild the blood supply. Deep hypothermic circulatory arrest cerebral protection was completed by bilateral antegrade cerebral perfusion. RESULTS: The mean cardiopulmonary bypass time was (212 ± 40) min, mean myocardial occlusion time was (95 ± 16) min, mean circulatory arrest time was (42 ± 8) min. Operative mortality was 1 case and hospital mortality was 5 case, which died of septicemia, acute renal failure and hemiplegia complicated with multiple organ failure. Compared with selective cerebral perfusion, the incidence of postoperative cerebral vascular accident and transient neurological dysfunction decreased. Seventy-six cases received aorta CTA before discharged, the closure rate of descending thoracic aortic dissection false lumen was 78.9%. Seventy-one patients were followed up for 5 to 49 months, 50 cases was reviewed by CTA, of which closure rate of descending thoracic aortic dissection false lumen was 88.0%, no late death and re-surgery. CONCLUSIONS: The modified total aortic arch replacement and stent elephant trunk technique treatment for patients with DeBakey I thoracic aortic dissection was safe and effective, with less postoperative complications.

Congenital left ventricular diverticulum, a rare chest fluoroscopy finding: a case report.

Congenital left ventricular diverticulum is a very rare cardiac abnormality and it is not completely understood about its etiology, clinical manifestation, diagnosis, treatment and prognosis. This article presents a case of large congenital diverticulum of the left ventricle. The clinical manifestation included paroxysmal supraventricular tachycardia. The diagnosis was made by chest fluoroscopy observation and confirmed by 64-slice CT-angiography. The arrhythmia alleviated instead of antiarrhythmic drugs but by radiofrequency catheter ablation. Due to the rapid growth of the diverticulum, the patient underwent surgical resection finally. Owing to the fatal risks, clinicians should improve the understanding of this disease by deeply studying more cases, in order to standardize the treatment.

Continuous fluoxetine administration prevents recurrence of pulmonary arterial hypertension and prolongs survival in rats.

1. The serotonin transporter (SERT) is strongly implicated in the pathogenesis of pulmonary arterial hypertension (PAH) in patients and animal models. Inhibitors of SERT have been reported to attenuate or reverse experimental PAH, which makes them potential therapeutic options for the treatment of PAH in humans. However, little is known about pathophysiological features after reversal or attenuation of PAH; moreover, the long-term therapeutic effects of SERT inhibitors on PAH remain undetermined. Thus, the aim of the present study was to investigate the short- and long-term effects of fluoxetine on monocrotaline (MCT)-induced PAH and associated pathophysiological changes in PAH models. 2. Rats were randomly divided into four groups as follows: (i) an M + F group, in which rats received a single injection of MCT (60 mg/kg, s.c.) and then after 3 weeks were given fluoxetine (10 mg/kg) once daily by gavage from Week 4 to Week 12; (ii) an M/F group, in which 3 weeks after a single MCT (60 mg/kg, s.c.) injection, rats were given fluoxetine (10 mg/kg) by daily gavage from Week 4 to Week 6 and were then given an equivalent volume of saline once daily by gavage from Week 7 to Week 12; (iii) an MCT group, in which 3 weeks after a single MCT (60 mg/kg, s.c.) injection rats were given an equivalent volume of saline by gavage from Week 4 to Week 12; and (iv) a saline group, in which rats received an equivalent volume of saline injection or gavage over the 12 week treatment period. Morphometric changes, pulmonary arterial pressure, percentage wall thickness, right ventricular hypertrophy index and SERT expression were detected at various times during the 12 week treatment period. Survival analysis was performed in each group. 3. After 12 weeks treatment, it was found that even through fluoxetine treatment resulted in complete reversal of PAH, PAH recurred after fluoxetine withdrawal. In contrast, continuous administration of fluoxetine prevented the recurrence of PAH and prolonged survival. Analysis of SERT protein levels in rat lung indicated that, compared with values obtained at Week 0, SERT protein increased significantly after discontinuation of fluoxetine but continuous fluoxetine administration inhibited this increase. 4. In conclusion, SERT overexpression correlates with the recurrence of PAH after withdrawal of fluoxetine in rats. Continuous fluoxetine administration prevents recurrence of PAH and prolongs survival.

[The clinical analysis of 51 patients with thoracic aortic dissection].

OBJECTIVE: To summarize the clinical experience about surgical treatment of aortic dissection. METHODS: The clinical data of 51 patients with aortic dissection admitted from December 2004 to December 2006 were analyzed retrospectively. There were 35 male and 16 female patients with a mean age of 55.7 years (ranged from 18 to 83-years-old). Twenty-seven patients of type I was performed under deep hypothermic circulatory arrest and selected cerebral perfusion with stent-graft which was implanted into the descending aorta through aorta arch. Five patients of type II was performed including Bentall operation in 3 patients, Wheat operation in 1 patient, ascending aorta replacement in 1 patient. Nineteen patients of type III was performed with stent-graft which was implanted into the descending aorta through aorta arch under deep hypothermic circulatory arrest. RESULTS: The time of cardiopulmonary bypass (CPB) in type I patients was 250 to 290 min with an average of (274 +/- 53) min, and the arrest time was 40 to 59 min with an average of (53 +/- 14) min. CPB time of type II patients was 130 to 159 min with an average of (146 +/- 43) min, and the cross clamp time was 60 to 79 min with an average of (66 +/- 15) min. CPB time of type III patients was 240 to 280 min with an average of (260 +/- 28) min, and the arrest time was 20 to 27 min with an average of (24 +/- 3) min. The mean hemorrhage volume of the entire group was (500 +/- 250) ml. The mean ICU retention time was (5.0 +/- 1.5) d and the length of stay was (15.0 +/- 2.5) d. Three patients died during perioperative period. Two patients appeared cerebrovascular accident after operation. One patient appeared descending aorta dilation in the follow-up of 2 to 21 months. CONCLUSION: Different clinical manifestations and treatment should be selected according to the different condition of aortic dissection aneurysm.

[Clinicopathological analysis of esophageal carcinosarcoma:a report of 22 cases].

OBJECTIVE: To evaluate the clinicopathological characteristics and surgical treatment of esophageal carcinosarcoma. METHODS: The patients with esophageal carcinosarcoma were divided into two types according to barium swallow: intraluminal carcinosarcoma (n=20) and fungating carcinosarcoma (n=2). Only one esophageal carcinosarcoma case was diagnosed by esophagoscopic biopsy preoperatively. Twenty patients underwent left thoracic approach esophagectomy and esophagogastrostomy above aortic arch, and two patients underwent esophagectomy and esophagogastrostomy below aortic arch. RESULTS: All the cases survived during operation and had no severe complication. Post-operative biopsy revealed that 21 cases had definite boundary between the carcinoma and the sarcoma. Only one case showed the invasion of carcinomatous tissues into sarcomatous tissues and mixed growth. Four cases had lymph node metastases (18.2%). The 1-, 3- and 5-year survival rates were 90.9% (20/22), 77.3% (17/22) and 68.2% (15/22) respectively. CONCLUSIONS: Esophageal carcinosarcoma is a rare malignant tumor with little invasiveness, low lymph node metastasis, early clinical symptom occurrence, low preoperative accurate diagnostic rate and good prognosis. Surgical resection is the main treatment for esophageal carcinosarcoma.

[Observation on the long-term complications after esophageal replacement with colon].

OBJECTIVE: To study the etiology and preventive measures of the long-term postoperative complication after esophageal replacement with colon for esophageal benign disease. METHODS: To review the clinical data of 577 patients with esophageal replacement with colon our department, including 123 cases of esophageal benign disease. Of all, there were 25 cases-time for 11 cases following with severe complication: redundancy and dilated colon 12 cases-time, severe stricture of stoma 4, macrocyst esophagus 2, colon-stomach stoma expansion 4, mechanical obstruction of colon 3. The etiology included iatrogenic and functionality. The therapy included stricture form or resection, redundancy segment resection, obstructed segment solution and stoma resection and form. RESULTS: Eight cases underwent once operation, 2 case twice, 1 case three times. After operation, 9 cases took food normally, 2 improved symptoms obviously. CONCLUSIONS: The iatrogenic and functionality factor contributed to severe complication after esophageal replacement with colon for esophageal benign disease. The preventive measure is followed during operation: cervical esophageal-colon anastomosis exceed 2.5 centimeter, abdominal colon-stomach anastomosis reflux, channel width of colon passage, intestinal canal lay up straight. Re-operation is best choice to for local stricture, colon expansion, redundancy and dilated colon.

[The clinical study on the surgical treatment of thoracic aortic aneurysm associated with coronary artery disease].

OBJECTIVE: To analyze the factors which influence the safety and prognosis of aorta replacement combined with coronary artery bypass grafting (CABG) for thoracic aortic aneurysm associated with coronary artery disease. METHODS: From May 1982 to October 2002, 67 patients with thoracic aortic aneurysm were admitted, and 24 of them combined with CABG. Of the 24 patients, 9 received descending aorta replacement combined with CABG, and the other 15 received the ascending aorta replacement combined with CABG. The treatment results were compared with the other 43 patients only undergoing the thoracic aortic replacement. RESULTS: The mortality rate of the patients with aorta replacement combined with CABG was 13% (3/24). Though the descending aorta replacement combined with CABG could make the cardiopulmonary bypass time and selective cerebral perfusion time longer, (278 +/- 54) min and (188 +/- 59) min respectively, no significant difference was observed in postoperative complications, 3-year survival rate, 3-year-cardiac-event-free rate compared with the patients only undergoing the thoracic aortic replacement (P > 0.05). CONCLUSIONS: The aorta replacement combined with CABG can be performed safely, and the revascularization for coronary artery disease is useful for preventing occurrence of cardiac events.

[Surgical anatomy of the colic vessels in Chinese and its influence on the operation of esophageal replacement with colon].

OBJECTIVE: To investigate the configuration of colic vessels in Chinese and its influence on the operation of esophageal replacement with colon (ERC). METHODS: The origin, trend, branching, configuration, and distribution of the colic vessels, the intensity of the colic arterial impulse, the integrity of the marginal artery at the splenic flexure and hepatic flexure of colon were observed during the operation of ERC among 582 patients undergoing ERC, 402 males mad 180 females, aged 2 approximately 74, from 22 provinces, municipality, and autonomous regions. RESULTS: The left colic artery (LCA) stemmed from the inferior mesenteric artery (IMA) in 97.3% of the patients, with an absence rate of 0.7%. The middle colic artery (MCA) stemmed from the superior mesenteric artery (SMA) in 77.8% of the patients with an absence rate of 8.2%. Accessory middle colic artery (acMCA), originating from the right colic artery, could be seen in 6.2% of the patients 39.7% of the right colic artery (RCA) stemmed from the SMA by itself, 23.0% of the RMA stemmed together with MCA, and 28.0% of the RCA stemmed together with the ileocolic artery. The absence rate of RCA was 9.8%. The intactness rate of marginal artery was 96.8% at the splenic flexure of colon, and was 88.7% at the hepatic flexure. The Rolan arch was seen in only 7.6% of the patients. CONCLUSION: The configuration of colic vessels in Chinese was basically similar to those of the results of autopsies carried out abroad. The optimal supply artery of colic segment during ERC is LCA, followed by LCA. Attention should be paid to the integrity of marginal arteries and veins in the patients with history of epigastric operation.

[Classification and surgical treatment of intrathoracic esophageal injury caused by foreign body].

OBJECTIVE: To investigate the classification criterion and surgical treatment strategy of intrathoracic esophageal injury caused by foreign body. METHODS: Eighty-four patients with intrathoracic esophageal injury caused by foreign body in our department from January 1980 to April 2004 were divided into 4 grade: grade I was non-penetrated injury of esophagus (18 cases); grade II was esophageal perforation with mild mediastinitis (39 cases); grade III was esophageal perforation with severe intrathoracic infection (17 cases); grade IV was aortoesophageal fistula (10 cases). Based on the degree of esophageal injury and the extension of inflammation, operative procedures were selected including esophagotomy, esophageal reparation, esophagectomy, mediastinal drainage, reparation of fistula and replacement of aorta. RESULTS: Patients in grade I and II were all cured . One death occurred in grade III (1/17), the same in Grade IV was 9 (9/10). CONCLUSIONS: Classification of esophageal injury caused by foreign body is helpful to the decision of surgical treatment strategy. The prevention of aortoesophageal fistula is the key point of reducing of mortality.

[Surgery for upper or middle thoracic esophageal carcinoma after gastrectomy].

OBJECTIVE: To evaluate the surgical treatment and technical key-points of upper or middle thoracic esophageal carcinoma in patients with history of gastrectomy. METHODS: Eighty-six patients with upper or middle thoracic esophageal carcinoma after previous gastrectomy received surgical treatment between 1980 and 2004. Among them, tumor location was in middle thoracic esophagus in 50 patients, in upper thoracic esophagus in 31 and cervical esophagus in 5. Postoperative pathological staging was stage I in 16 patients, stage IIa in 62, stage IIb in 5 and stage III in 8. The interval between gastrectomy and the diagnosis of esophageal carcinoma ranged from 2 to 22 years. Surgical procedures included esophagectomy and reconstruction with nonreversed gastric tube in 2 patients and reversed gastric tube in 3. The esophagus was reconstructed with short segment of colon in 5 patients and long segment of colon in 74. Two cases underwent jejunostomy only. RESULTS: Seventy-six patients (88%) were treated with curative intent. Seven patients (8%) received palliative surgery. Postoperative complication rate was 12% (10/86). One patient died of multiple organ dysfunction syndrome (MODS). Sixty-seven patients were followed up, the 1-, 3-, 5-year survival rates were 84% (56/67), 57% (38/67) and 22% (15/67), respectively. CONCLUSIONS: Surgical treatment is the first choice for esophageal cancer patients after gastrectomy although the procedures are complicated. The surgery should be considered as a reliable therapeutic modality because of favorable patient prognosis. The replacement with colon is recommended for those patients.

Surgical treatment of giant esophageal leiomyoma.

AIM: To summarize the operative experiences for giant leiomyoma of esophagus. METHODS: Eight cases of giant esophageal leiomyoma (GEL) whose tumors were bigger than 10 cm were treated surgically in our department from June 1980 to March 2004. All of these cases received barium swallow roentgenography and esophagoscopy. Leiomyoma located in upper thirds of the esophagus in one case, middle thirds of the esophagus in five cases, lower thirds of the esophagus in two cases. Resection of tumors was performed successfully in all of these cases. Operative methods included transthoracic extramucosal enucleation and buttressing the muscular defect with pedicled great omental flap (one case), esophagectomy and esophago-gastrostomy above the arch of aorta (three cases), total esophagectomy and esophageal replacement with colon (four cases). Histological examination confirmed that all of these cases were leiomyoma. RESULTS: All of the eight patients recovered approvingly with no mortality and resumed normal diet after operation. Vomiting during meals occurred in one patient with esophagogastrostomy, and remained 1 mo. Reflux esophagitis occurred in one patient with esophago-gastrostomy and was alleviated with medication. Thoracic colon syndrome (TCS) occurred in one patient with colon replacement at 15 mo postoperatively. No recurrence occurred in follow-up from 6 mo to 8 years. CONCLUSION: Surgical treatment for GEL is both safe and effective. The choices of operative methods mainly depend on the location and range of lesions. We prefer to treat GEL via esophagectomy combined with esophago-gastrostomy or esophagus replacement with colon. The long-time quality of life is better in the latter.