[Spatio-temporal expression of connexion (Cx) 40 and Cx45 in Cx43 knockout embryonic mouse hearts].

OBJECTIVE: To investigate the spatio-temporal expression of connexin (Cx) 40 and Cx45 genes in Cx43 knockout embryonic mouse hearts. METHODS: Cx43 knockout heterozygous mice were raised. PCR was performed to identify genotypes of their offsprings. The homozygote (Cx43-/-) was used for study and the wild type (Cx43+/+) was used as control. Immuno-histochemistry was used to detect the Cx40 and Cx45 expression in different parts of the fetal hearts at the embryonic days (EDs) 10.5, 11.5, 12.5, 13.5, 14.5, and 15.5, respectively. SCIM microscopic image analytic system was used for quantitative analysis of staining intensity. RESULTS: (1) Cx40 expression was detected in ventricles of Cx43+/+ fetal heart as early as ED10.5 with the intensity represented by A value of 8.6. Subsequently it was distributed in the atria and ventricles with the peak expression observed at ED14.5 (A value = 94.8), and faded afterwards. Less Cx40 expression was observed in the Cx43-/- fetal hearts as compared with Cx43+/+ although its expression pattern was similar in both groups. (2) Cx45 expression was detected in ventricles at ED 10.5 (A value = 20.0). It was subsequently distributed in the atria and ventricles with the peak expression observed at ED12.5 (A value = 49.6), and then faded. Less Cx45 expression was observed in the Cx43-/- fetal hearts as compared with Cx43+/+ although its expression pattern was similar as well in both groups. CONCLUSION: Down-regulated expression of the genes Cx40 and Cx45 may be associated with the abnormal heart development in Cx43 knockout animals.

[Percutaneous balloon valvuloplasty for severe and critical pulmonary valve stenosis in infants under six months].

OBJECTIVE: To assess the effects and potential role of percutaneous balloon valvuloplasty as an alternative therapy of surgery in young infants with severe and critical pulmonary valve stenosis. METHODS: Eighteen patients aged 8 days to 6 months with severe and critical pulmonary valve stenosis admitted to our hospital from June 2006 to August 2008 underwent balloon valvuloplasty. Among them, 11 infants including 2 neonates had critical pulmonary stenosis. Severe tricuspid regurgitation was seen in 5 and moderate in 3. Right ventricular systolic pressure in all patients was greater than systemic pressure with right-to-left or bi-directional shunt at atrial level. Dilatation was performed under general anesthesia with intubation in 12 patients and caudal block combined with sedation in 6 patients. Dilatation with 2 balloons sequentially in one procedure was performed in 6 patients and dilatation with 1 balloon in other 10 patients. RESULTS: Of the 18 patients, there was failure to cross the pulmonary valve with balloon catheter in one and cardiac tamponade in one. The dilatation success rate was 88.9%. Immediately after dilatation, the systemic pressure gradient from right ventricle to pulmonary artery decreased from (87 +/- 24) mm Hg to (30 +/- 19) mm Hg (P < 0.01). No complication was found in all patients during or post dilation. During a follow-up of 6 to 32 months, pressure gradient crossing pulmonary valve measured by echocardiography further decreased or remained stable in 16 cases, except one neonate and one infant whose pressure gradient gradually increased and required a second dilatation. Re-dilatation rate was 12.5%. Tricuspid regurgitation was reduced in all patients. Mild pulmonary regurgitation was seen in most of patients post-dilatation, except moderate in one. All patients fared well and stayed asymptomatic. CONCLUSION: Percutaneous balloon valvuloplasty for severe and critical pulmonary stenosis in infants is relatively safe and effective and should be considered a valid alternative to surgical operation. It should be the first choice for such patients based on its excellent outcome, less trauma and fewer complications.

[Differentially expressed transcription factors in the cardiac outflow tract tissue of connexin43 knockout mice].

OBJECTIVE: To investigate the changes in the expression of cardiac transcription factors in the cardiac outflow tract (OFT) tissues in the connexin43 knockout homozygotes (Cx43 KO), connexin43 heterozygotes, and connexin43 wild-type mice (Cx43 WT). METHODS: The cDNA was retrotranscribed from the RNA extracted from the OFT tissues of 6 Cx43 KO, 6 Cx43 WT, and 6 Cx43 heterozygotes genotyped by PCR method on the embryonic day (ED) 13.5 and ED 14.5. The biotin-labeled cRNA derived from the transcription of cDNA was fragmented as probes. The probes were hybridized with Affymetrix Mouse Genome 430 2.0 Array. Gene Array Scanner was used to screen the signals of hybridization and detect the expression of genes. The mRNA expression levels of 3 cardiac transcription factors: Sox11, Foxp1, and Tbx20 were measured by real time quantitative RT-PCR. RESULTS: The ratios of the expression of the 6 genes, all cardiac transcription factors: Gata4, Mef2C, Sox4, Sox11, Foxp1, and Tbx20 between the Cx43 KO and Cx43 WT groups were 1:1.41, 1:2.30, 1:3.25, 1:0.71, 1:0.66, and 1:0.54. The expression levels of Sox11 and Foxp1 on ED13.5 in the Cx43 K group were 4.76 +/- 0.19 and 5.08 +/- 0.28 respectively, both significantly lower than those of the Cx43 WT group (5.34 +/- 0.25 and 5.64 +/- 0.15 respectively, both P < 0.01), and expression level of Tbx20 on ED 13.5 in the Cx43 K group was 7.18 +/- 0.16, not significantly different from that of the Cx43 WT group (7.47 +/- 0.27, P > 0.05). The expression levels of the genes Sox11, Foxp1, Tbx20 on ED 14,5 were 4.71 +/- 0.27, 5.25 +/- 0.31, and 7.05 +/- 0.17 respectively, all significantly lower than those of the Cx43 WT group (5.00 +/- 0.19, 5.77 +/- 0.16,) and 7.43 +/- 0.25, all P < 0.05). The results of the expression of these genes by real time PCR analysis showed an excellent concordance with those indicated by the microarray analysis. CONCLUSION: The cardiac transcription factors such as Sox11, Foxp1, and Tbx20 that are differently expressed in the Cx43 KO OFT tissue may be involved in the pathogenesis of the OFT defects.

Percutaneous balloon angioplasty for severe native aortic coarctation in young infants less than 6 months: medium- to long-term follow-up.

BACKGROUND: Although balloon angioplasty (BA) has been performed for more than 20 years, its use as a treatment for native coarctation of the aorta (CoA) during childhood, especially in young infants, remains controversial. This study aimed to assess the effects and potential role of percutaneous transcatheter BA for native CoA as an alternative therapy to surgical repair in young infants. METHODS: The 37 patients aged from 6 days to 6 months with severe CoA in congestive heart failure or circulatory shock were admitted for BA. Patient's weight ranged from 2.4 to 6.1 kg. All 37 patients were experiencing cardiac dysfunction, and eight patients were in cardiac shock with severe metabolic acidosis. Eleven patients had an isolated CoA, whereas the others had a CoA associated with other cardiac malformations. Cardiac catheterization and aortic angiography were performed under general anesthesia with intubation. Transfemoral arterial approaches were used for the BA. The size of the balloon ranged from 3 mm × 20 mm to 8 mm × 20 mm, and a coronary artery balloon catheter was preferred over a regular peripheral vascular balloon catheter. RESULTS: The femoral artery was successfully punctured in all but one patient, with that patient undergoing a carotid artery puncture . The systolic peak pressure gradient (PG) across the coarctation was 41.0 ± 16.0 mmHg (range 13-76 mmHg). The mean diameter of the narrowest coarctation site was 1.7 ± 0.6 mm (range 0.5-2.8 mm). All patients had successful dilation; the PG significantly decreased to 13.0 ± 11.0 mmHg (range 0-40 mmHg), and the diameter of coarctation significantly improved to 3.8 ± 0.9 mm (range 2.5-5.3 mm). No intraoperative complications occurred for any patients. However, in one case that underwent a carotid artery puncture, a giant aneurysm formed at the puncture site and required surgical repair. The following observations were made during the follow-up period from 6-month to 7-year: (1) The PG across the coarctation measured by echocardiography further decreased or remained stable in 31 cases. The remaining six patients, whose PGs gradually increased, required a second dilation. No patient required further surgery because of a CoA; (2) in two cases, an aortic aneurysm was found with an angiogram performed immediately postdilatation and disappeared at 18 and 12 months of age, respectively; (3) tricuspid regurgitation and pulmonary hypertension improved in all patients; (4) all patients were doing well and were asymptomatic. CONCLUSIONS: Percutaneous BA is a relatively safe and effective treatment for severe native CoA in young infants, and should be considered a valid alternative to surgery because of its good outcome and less trauma and fewer complications than surgery.

[Clinical characteristics and imaging evaluation in children with renovascular hypertension].

OBJECTIVE: To characterize the clinical and angiographic features in children with renovascular hypertension. METHOD: Clinical data of 14 children (7 male, 7 female; age 0.8-14 years, mean 8.7 years), who were diagnosed with renovascular hypertension by renal angiography in our institute from January 2005 to December 2012 were collected and retrospectively analyzed. RESULT: The mean blood pressure at the diagnosis was 187/127 mm Hg. Chief complaints of symptomatic patients were headache (29%, 4/14), hypertensive encephalopathy (36%, 5/14), signs of congestive heart failure (14%, 2/14) and hematemesis (7%, 1/14). Renovascular hypertension was found incidentally in 14% (2/14) of patients who were asymptomatic. Conventional renal angiography elucidated the anatomical distribution of lesions in the renal arterial system. It was found that 14% (2/14) of patients had bilateral disease, 50% (7/14) had single stenosis at main or accessory renal artery, while multiple stenoses was seen in 43% (6/14) of children, with involvement of segmental renal artery and small interlobar or arcuate vessels. Compared with catheter angiography, 50% (7/14) of patients with renovascular hypertension, especially intrarenal arterial disease, were missed on computed tomography angiography or magnetic resonance angiography. CONCLUSION: It is mandatory to emphasize blood pressure measurement in pediatric clinical practice for early recognition of renovascular hypertension. As children with renovascular hypertension display involvement of multiple arteries, including in smaller intrarenal arteries, digital subtraction angiography is the only method that can reliably diagnose pediatric renovascular hypertension.

[Clinical features and molecular diagnosis of three patients with DiGeorge anomaly].

OBJECTIVE: To investigate the clinical features and molecular diagnostic methods of three patients with DiGeorge anomaly. METHOD: The clinical manifestations and immunological features of the three cases with DiGeorge anomaly were analyzed. We detected the chromosome 22q11.2 gene deletion by fluorescence in situ hybridization (FISH). RESULT: (1) CLINICAL MANIFESTATIONS: All three cases had varying degrees of infection, congenital heart disease and small thymus by imaging; two cases had significant hypocalcemia (1.11 mmol/L and 1.22 mmol/L, respectively), accompanied by convulsions; only 1 case had cleft palate and all had no significant facial deformity. (2) Immunological characteristics: All three cases had varying degrees of T-cell immune function defects (percentage of T lymphocytes was 24% - 43%, absolute count was 309 - 803/µl), and levels of immunoglobulin G, A, M, and percent of B lymphocytes and absolute count were normal. (3) Detection of the chromosome 22q11.2 gene deletion: 400 cells of each case were detected. All cells showed two green and one red hybridization signal, indicating the presence of gene deletions in chromosome 22q11.2. (4) OUTCOME: All three cases were treated with thymosin, and appropriate clinical intervention for cardiac malformations, hypocalcemia, and were followed-up for 4 - 18 months, the prognosis was good. CONCLUSION: DiGeorge anomaly showed diverse clinical manifestations. We should consider the disease if patients had congenital heart disease, thymic hypoplasia, hypocalcemia and/or impaired immune function. FISH for detecting chromosome 22q11.2 gene deletion can be used as accurate and rapid diagnostic method. Thymosin treatment and other clinical intervention may help to improve the prognosis of patients with partial DiGeorge anomaly.

Downregulation of Rho associated coiled-coil forming protein kinase 1 in the process of delayed myocardialization of cardiac proximal outflow tract septum in connexin 43 knockout mice embryo.

BACKGROUND: The connexin43 knockout (Cx43 KO) mouse dies at birth with an enlarged conotruncal region, which leads to the obstruction of the right outflow tract (OFT). Since myocardialization of the proximal OFT septum is one of the key events during heart development, we investigated the process in the Cx43 KO embryo hearts. Rho associated coiled-coil forming protein kinase 1 (ROCK1), is a recently found key molecule to regulate the myocardialization of OFT, but its spatiotemporal expression pattern during myocardialization remains unknown. The objective of this study was to investigate the differentially expressed pattern of ROCK1 between Cx43 KO and wild type embryo hearts, and its relationship with the delayed myocardialization in Cx43 KO embryo hearts. METHODS: Using immunohistochemistry, the processes of myocardiolization were investigated both in Cx43 KO and wild type embryo hearts. The differentially expressed pattern of ROCK1 between Cx43 KO and wildtype embryo hearts was evaluated both at the mRNA and protein level by real-time RT-PCR and immunohistochemistry. RESULTS: The expression of α-sarcomeric actin (α-SCA) in the proximal OFT septum of Cx43 KO embryos was delayed. Meanwhile, it was shown that the downregulation of ROCK1 coincided with delayed myocardialization. The expression of ROCK1 protein was mainly limited to the proximal outflow tract septum from embryo day (E) E11.5 to E15.5. Its expression pattern was similar with that of α-SCA. Real-time RT-PCR found that the expression level of Rock-1 mRNA began at a low level on E11.5 and reached peak at E13.5 and E14.5. CONCLUSIONS: ROCK1 may have an important role in the process of myocardialization of the proximal OFT septum. Downregulation of ROCK1 is likely to contribute to the aberrant myocardialization in Cx43 KO embryo hearts.

[The effect of VEGF-ASODN transfection on expression of VEGF and growth in tongue squamous cancer cell line Tca8113].

PURPOSE: To evaluate the effect of transfection of vascular endothelial growth factor antisense oligonucleotide (VEGF-ASODN) on expression of vascular endothelial growth factor(VEGF) and growth in tongue cancer cells. METHODS: The VEGF-ASODN was synthesized with phosphorothioic acid. After transfecting with VEGF-ASODN in tongue cancer cells Tca8113, the quantity of VEGFmRNA and protein was detected by hybridization in situ and immunocytochemistry, and the quantity of VEGF protein in supernatant was detected by ELISA. FCM and MTT methods were used to detect cellular apoptosis and the activity of cells, respectively. The effect of transfection on the growth of cells was evaluated by growth curve. SPSS12.0 software package was used for statistical analysis. RESULTS: The quantity of VEGF mRNA, protein levels of VEGF in the cells and in culture fluid decreased after transfection of VEGF-ASODN (P<0.01,P<0.05).The ratio of apoptosis increased, the activity of cells also decreased after transfection VEGF-ASODN (P<0.01). CONCLUSIONS: Transfection with VEGF-ASODN in Tca8113 cells can inhibit the expression of VEGF remarkably. It can enhance cellular apoptosis and suppress growth of cells. Supported by Research Fund from Science and Technology Bureau of Taian City (Grant No.2007-46).

[The level of PPTA mRNA and its proteins expression in trigeminal ganglia in rats with occlusal recovery].

PURPOSE: To study the level of substance P(SP) and preprotachykinin A(PPTA) mRNA in trigeminal ganglia(TG)in rats with occlusal recoveryìand to discuss the mechanism of temporomandibular disorders. METHODS: 48 Wistar male rats were randomly divided into 3 experimental groups and 3 control groupsì8 rats in each group. The right maxillary and mandibular molars in the experimental groups were ground to the gingival level without occlusal contact. The occlusal contact was recovered by stoping grinding the molars gradually. The section of trigeminal ganglia were used for immunohistological and in situ hybridization study. Light microscope and microscoic photo analytic software were employed to detect the percentage of SP and PPTA mRNA positive neurons in the frozen section of TGs in 48 rats. SPSS10.0 software package were used for statistical analysis. RESULTS: The percentage of SP positive neurons in TG with unilateral chew experimental group decreased significantly compared to the control group (P<0.01,P<0.05).The decreased extent in the non-chewing side was much higher than that in the chewing side(P<0.05).The percentage of PPTA mRNA positive neurons was significantly higher in both of the chewing and non-chewing sides of the unilateral chew experimental group than that in the control group (P<0.05,P<0.01)ì and that non-chewing side was significantly higher than that in the chewing side(P<0.01).There was no significant difference in the percentage of SP and PPTA mRNA positive neurons between the early occlusal reconstruction the experimental group and the control group(P>0.05), and there was no significant difference of those between the non-chewing side and the chewing side(P>0.05). The results of later occlusal recovery in the experimental group was same to that of the unilateral chew experimental group. CONCLUSIONS: The expressions of SP and PPTA mRNA in TG can recover normal in the early occlusal recovery but can not in the later occlusal recovery. SP and PPTA mRNA might participate in the histopathologic mechanism of temporomandibular disorders.