[Effect of crucumin on vascular endothelial function in atherosclerotic rabbits].

OBJECTIVE: To observe the effect of curcumin on nitric oxide (NO) in plasma of atherosclerotic rabbits, activity of constitutive nitric oxide synthase (cNOS) and asymmetric dimethylarginine (ADMA), and discuss curcumin's effect against AS and its correlation with ADMA. METHOD: Thirty-eight male Japanese white rabbits were randomly divided into four groups: the control group (eight rabbits fed with standard diets), the model group (ten rabbits fed with high-fat diets), the low dose curcumin group (ten rabbits fed with high-fat diets and 100 mg . kg-1 d -1 ) and the high dose curcumin group (ten rabbits fed with high-fat diets and 200 mg kg-1 d-1 curcumin). At the end of the 12th week, their plasmas were tested for TC, LDL-C, NO, endothelin (ET) , ADMA and activity of aortic cNOS. Aortic tissues were collected for histological examinations. RESULT: The three groups fed with high-fat diets showed higher plasma ADMA and ET than the control group (P <0. 01) , but with decrease in plasma NO concentration and arterial cNOS activity (P <0. 01). Compared with the model group (P <0. 05) , the curcumin groups showed lower plasma ADMA and ET (P <0. 05), but higher plasma NO concentration and arterial cNOS activity than the model group (P <0. 01). There was no significant difference between the two curcumin groups. CONCLUSION: Curcumin may play an important protective role in AS process by reducing plasma ADMA level. [Key words] atherosclerosis; asymmetric dimethylarginine; crucumin; nitric oxide; nitric oxide synthase

[Association between polymorphisms of inflammatory factor genes and coronary heart disease].

OBJECTIVE: To investigate the association between genetic polymorphisms of inflammatory factors and susceptibility to coronary heart disease(CHD) in southern Chinese Han population. METHODS: Using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) method, the genotypes of five inflammatory factors (BRCA1-associated protein, a disintegrin and metalloproteinase 8, inter-alpha-trypsin inhibitor H3, interleukin-15, cyclooxygenase-2) were anaylzed in 283 CHD patients diagnosed by angiography and 176 controls. RESULTS: In these inflammatory factors, the 270T/C and 90A/G polymorphisms of the BRAP gene showed a significant association with CHD. The allele and genotype frequencies of BRAP gene were consistent with those predicted by Hardy-Weinberg equilibrium (chi-square=0.878, P> 0.05; chi-square=0.776, P> 0.05, respectively). The frequencecies of 270C and 90G alleles in CHD patients was significantly higher than those of the control group (29.51% vs. 21.31%, P=0.006; 30.04% vs. 21.31%, P=0.004, respectively). Compared with 270TT and 90AA, 270CC and 90GG genotypes had a significantly increased CHD risk by Logistic regression analysis (OR=4.51, 95%CI: 1.41-14.45, P=0.011; OR=5.09, 95%CI: 1.60-16.26, P=0.006, respectively). This association was still signifcant after adjustment for the sex, age, smoke, hypertension, diabetes, plasma total cholesterol and low density lipoprotein levels. No evidence of association was found for other single nucleotide polymorphisms. CONCLUSION: The 270T/C and 90A/G polymorphisms in the BRAP gene may contribute to an increased risk of CHD among southern Chinese Han population.

[Ryanodine downregulates the expression of p-eNOS (Thr495) and improves the functions of rapamycin treated endothelial outgrowth cells].

OBJECTIVE: To observe the effects of ryanodine on rapamycin treated endothelial outgrowth cells (EOCs). METHODS: The mononuclear cells were harvested from umbilical cord blood by Ficoll density gradient centrifugation, then induced into EOCs and expanded in vitro. The endothelial characteristics of EOCs were identified by immunostaining and fluorescent staining. The EOCs were pretreated with or without ryanodine (10 µmol/L) for 1 h, and then treated with or without rapamycin (10 nmol/L) for 24 h. Proliferation was evaluated by CCK8 and migration was measured by Transwell. The protein expression of EOCs was evaluated by immunobloting technique with total eNOS antibody and phospho-eNOS (Thr495) antibody. RESULTS: Compared with control group, the proliferation and migration capacities of EOCs were significantly reduced while the phosphorylation of eNOS (Thr495) protein was significantly upregulated in rapamycin group (P < 0.05), expression of total eNOS was not affected by rapamycin (P > 0.05). Compared with rapamycin group, the proliferation and migration capacities of EOCs were significantly increased and the phosphorylation of eNOS (Thr495) protein was significantly downregulated in ryanodine + rapamycin group (P < 0.05). The proliferation and migration capacities, the phosphorylation of eNOS (Thr495) protein and the expression of total eNOS were not affected by ryanodine alone (P > 0.05). CONCLUSIONS: Rapamycin reduced proliferation and migration capacities while upregulated the phosphorylation of eNOS (Thr495) protein of EOCs and these effects could be partly reversed by cotreatment with ryanodine.

[Experimental analysis of microRNAs related to cardiac development differential expression].

OBJECTIVE: To investigate the differential expression profiles of microRNAs in the knockout Pax-8 mice by miRNA microarray analysis and study the function of microRNA during cardiac development. METHODS: The knockout Pax-8 mice model was established and the total RNA derived from Pax-8 KO-/- and Pax-8 KO+/- mice heart. MicroRNA microarray containing 567 mammalian microRNA probes was used to investigate the microRNAs differential expression between Pax-8 KO-/- and Pax-8 KO+/- mice. The candidates of microRNAs were confirmed by real time RT-PCR assay. RESULTS: The heart of pax-8 KO-/- mice became spheroidal. Left ventricle enlargement, left ventricular wall and interventricular septum thickening and papillary muscles in left ventricle enlargement were found. Furthermore, many apoptotic cells were discovered in left ventricular wall and interventricular septum in pax-8 KO-/- mice. The MicroRNA microarray result displayed 10 microRNAs differential expressions, in which 2 microRNAs became down-regulated and 8 microRNAs up-regulated by more than two folds in pax-8 KO-/- mice. This was in accordance with the result of real-time RT-PCR. CONCLUSION: Some microRNAs may play important roles in cardiac development and ventricular septal defect pathogenesis.

[Association of CETP and CRP gene polymorphisms with non-valvular atrial fibrillation in Chinese Han population].

OBJECTIVE: To study whether the polymorphisms of TaqIB of cholesteryl transfer protein (CETP) gene and 1444C/T of C reactive protein (CRP) gene are associated with non-valvular atrial fibrillation in the Chinese Han population. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to detect the distribution of genotypes of CETP TaqIB and CRP 1444C/T in 147 patients with non-valvular atrial fibrillation and 147 control subjects in Chinese Han population. RESULTS: (1) The distribution of CETP TaqIB and CRP 1444C/T genotypes was in Hardy-Weinberg equilibrium. (2) A statistically significant difference between patients and controls for CETP TaqIB (P= 0.005, OR= 0.614, beta = -0.488) and CRP 1444C/T (P= 0.003, OR= 2.428, beta = 0.887) was observed. (3) In female group, significant difference was observed in smoking, CETP TaqIB and CRP 1444C/T polymorphisms. And in male group, significant difference was observed in body mass index and CETP TaqIB polymorphisms. CONCLUSION: (1) These results suggest that CETP TaqIB (B2 allele as protective factor) and CRP1444C/T (T allele as risk factor) genetic polymorphisms may be associated with the non-valvular atrial fibrillation in the Chinese Han population. (2) Smoking and CRP1444T single nucleotide polymorphism may induce hereditary susceptibility to non-valvular atrial fibrillation in female. Obesity may induce hereditary susceptibility to non-valvular atrial fibrillation in male.

[Intervening effect of naoxintong on anti-platelet treatment with aspirin].

OBJECTIVE: To investigate the effect of small dosage aspirin on platelet biochemical indexes in patients with cardio-cerebrovascular diseases and the intervening action of Naoxintong (NXT). METHODS: The blood levels of P-selectin (P), thrombin B2 (TXB2), and platelet aggregation (PAG) induced by arachidonic acid (AA) and adenosine diphosphate (ADP) were determined in 145 patients with cardio-cerebrovascular diseases (diabetes mellitus, hypertension, coronary heart disease and cerebral infarction), after they were medicated with aspirin 100 mg per day for 7 days. Then they were randomly assigned to the aspirin group and the NXT group Both groups took aspirin 100 mg per day continually, but to patients in the NXT group, NXT 9 tablets per day was given additionally. The blood levels of above-mentioned biochemical indexes were re-examined 1 month after medication. RESULTS: The first determination showed the plasma level of P-selectin and TXB2 concentration were positively correlated with PAG, either induced by AA (r = 0.449, P < 0.01 and r = 0.576, P < 0.01) or by ADP (r = 0.525, P < 0.01; r = 0.501, P < 0.01). Positive correlation also showed between plasma level of P-selectin and TXB2 (r = 0.610, P < 0.01). There was no significant difference of all the three indexes between the two groups (P > 0.05). Re-examination showed that levels of the 3 indexes significantly decreased in both groups (P < 0.01), and all were lower in the NXT group than in the aspirin group respectively (P < 0.05). There was no significant difference in the incidence of adverse reaction between two groups (P > 0.05). CONCLUSION: The anti-platelet effect of one-week administration of aspirin for patients with cardio-cerebrovascular diseases can not be optimal, the combination with NXT could enhance the effect without increase of adverse reaction.

[Effects of nano-zirconium hydroxide coating on resin bonding of 10-methacryloxy decyldihydrogen phosphate-conditioned zirconia].

OBJECTIVE: This study evaluated the influence of alkaline coating of nano-zirconium hydroxide on resin bonding of 10-methacryloxy decyldihydrogen phosphate (MDP)-conditioned zirconia. METHODS: A total of 140 yttria-stabilized tetragonal zirconia polycrystals (Y-TZP) plates were prepared and sandblasted with alumina particles, and then subjected to different surface treatment. Resin bonding specimens were prepared with a MDP-free resin cement and tested for shear bond strength (SBS). X-ray photoelectron spectroscopy (XPS) was used to investigate the formation of chemical bond on the surface of Y-TZP treated successively with nano-zirconium hydroxide coating and MDP containing primer. RESULTS: The two alkaline coatings increased the 24 h SBS compared to control groups, and groups using nano-zirconium hydroxide coating yielded higher SBS. After thermocycling, no statistical difference was observed between groups, but SBS decreased significantly compared to the 24 h SBS (P<0.05). XPS analysis detected -OH bond on the surface of Y-TZP treated with nano-zirconium hydroxide coating, and -P-O-Zr bond was detected on the surface of Y-TZP treated with nano-zirconium hydroxide coating and MDP-containing primer. CONCLUSIONS: Alkaline coatings improved the bonding of resin to zirconia conditioned with MDP-containing primers.

[Effects of endothelial progenitor cells and endothelial outgrowth cells in repair of injured carotid vessel: a comparative study with rabbits].

OBJECTIVE: To compare the effects of endothelial progenitor cells (EPCs) and endothelial outgrowth cells (EOCs) on the repair of injured vessels. METHODS: Mononuclear cells (MNCs) were isolated from rabbit peripheral blood by density-gradient centrifugation. EPCs and EOCs were obtained from the culture of MNCs and labeled with the cell dye CM-DiI for cells tracking. Eighteen rabbits were made into models of balloon-injured common carotid artery and then divided into 2 equal groups to undergo injection of the suspensions of EPCs or EOCs. Nine rabbits underwent injection of normal saline as control group. Four weeks after transplantation, the rabbits underwent venous injection of Evans blue, and then were killed with the injured vessels taken out. Fluorescence-labeled both types of cells, endothelial regeneration rate and IA/MA ratio were detected. RESULTS: Four weeks after transplantation, fluorescence-labeled EPCs and EOCs were detected within the tunica intima, mostly in the neointima and on the luminal surface of injured vessel. The endothelialization area of denuded vessel of the EPC and EOC groups were 91.6% +/- 3.6% and 89.1% +/- 6.3% respectively, both significantly larger than that of the control group (62.1% +/- 7.5%, both P < 0.01), however, without significant difference between the 2 former groups (P = 0.50). The intima area/media area ratio of the EPC and EOC groups were 0.48 +/- 0.11 and 0.44 +/- 0.06, both significantly lower than that of the control group (0.88 +/- 0.14, both P < 0.01), however, without significant difference between the 2 former groups (P = 0.59). CONCLUSION: Transplantation of both EPCs and EPCs accelerate the reendothelialization and reduce the neointimal formation with similar effects.

[Effects of Danshen on number and activity of endothelial progenitor cells of patients with hypercholesterolemia].

OBJECTIVE: To investigate the effects of Danshen on number and activity of endothelial progenitor cells (EPCs) of patients with Hypercholesterolemia. METHOD: 24 patients with Hypercholesterolemia were randomLy divided into 2 groups: control group (n = 12), and treatment group (n = 12, receiving Composite Denshen Pilulae, 10# tid for 2 weeks). after 2 weeks, 20 mL peripheral blood was obtained from each patient, Mononuclear fraction of human peripheral blood was obtained by density gradient centrifugation, plated on fibronectin coated culture dishes. The cells were identified by immunohistochemistry and flow cytometry and tested the ability to intake ac-LDL. Cell clusters were viewed with an inverted microscope, fluorescence-activated cell sorting (FACS) analysis of PE-CD34 and FITC-AC133 was performed to detect number of EPCs, EPC proliferation and migration were assayed with MTT assay, modified Boyden chamber assay. EPCs adhesion ability assay was performed by replating cells on fibronectin-coated dishes, and then counting adherent cells. RESULT: Numbers of EPCs (10(3) cells per 1 mL peripheral blood) of treatment group was higher than control group (7.20 +/- 1.29 vs 6.88 +/- 1.00). Compared with group control, numbers of clusters (per 40 power microscopic field), adhesive EPCs (per 400 power microscopic field) and migratory EPCs (per 200 power microscopic field) of treatment group were significantly increased (4.47 +/- 0.94 vs 3.38 +/- 0.57, P <0.01, 11.81 +/- 2.29 vs 10.03 +/- 1.32, P <0.05 and 15.75 +/- 2.27 vs 11.95 +/- 1.28, P <0.01, respectively), while OD vallue of treatment group were significantly increased too (0.27 +/- 0.04 vs 0. 20 +/- 0.03, P < 0.01). CONCLUSION: Danshen can significantly enhance EPCs functional activity of patients with Hypercholesterolemia.

[The correlation between ICAM-1 gene K469E polymorphism and coronary heart disease].

OBJECTIVE: To investigate the correlation between intercellular adhesion molecule1 (ICAM1) gene K469E polymorphism and coronary heart disease(CHD) in Han Chinese population. METHODS: Using the methods of polymerase chain reaction-restrictive fragment length polymorphism (PCR-RFLP), 173 CHD patients and 141 controls were analyzed for the polymorphism, genotype and allele distribution of ICAM1 gene K469E. RESULTS: The distribution of ICAM1 genotypes was in Hardy-Weinberg equilibrium. The frequency of KK genotype in CHD group was significantly higher than that in control (64.2% vs 48.9%, P<0.01). Similarly, the frequency of K allele in CHD group was significantly higher than that in control (79.2% vs 69.9%, P<0.01). With Logistic Regression Analysis ruling out the influences of age, gender and other CHD risk factor, the homozygous individual with KK genotype was 2.35 folds of KE or EE genotype one suffering from CHD (OR: 2.35, 95%CI: 1.03-5.36, P<0.05). CONCLUSION: ICAM1 gene K469E polymorphism is associated with CHD risk of Han Chinese population, the K allele may serve as a genetic risk factor of coronary heart disease.

[Effects of compound Salvia injection on number and activity of endothelial progenitor cells].

OBJECTIVE: To investigate the effects of Compound Salvia injection (CSI) on the number and activity of endothelial progenitor cells (EPCs). METHOD: Mononuclear fraction of human umbilical cord blood was obtained by density gradient centrifugation and plated on fibronectin coated culture dishes. Cells were divided in to five groups: group control, group VEGF, group CSI 50, group CSI 10 and group CSI 2 (supplemented with none cytokine, VEGF 10 ng x mL(-1), CSI 50, 10, 2 microg x mL(-1), respectively). After six days in culture, cell clusters were viewed with an inverted microscope, fluorescence-activated cell sorting (FACS) analysis of PE-CD34 and FITC-VE-Cadherin was performed to detect number of EPCs, adhesion assay was performed by replating cells on fibronectin coated dishes, and then counting adherent cells. RESULT: Numbers of EPCs of group VEGF, group CSI 10 and group CSI 2 were significantly increased as compared with those of group control ( P < 0.01, P < 0.05, P < 0.01, respectively), and numbers of EPCs of group CSI 2 were more than those of group CSI 10 and group V (P < 0.01). Compared with group control, number of EPCs of group CSI 50 was significantly decreased (P < 0.01). Compared with group control, numbers of clusters and adhesive EPCs of group CSI 10 and group CSI 2 were significantly increased, while those of group CSI 50 were significantly decreased. CONCLUSION: Low concentration CSI can significantly promote EPCs augmentation and enhance its functional activity, while high concentration CSI significantly restrains it.

[Effect of surface pretreatment with chemical etchants on bond strength between a silicone-based resilient liner and denture base resin].

OBJECTIVE: To evaluate the effect of denture base resin surface pretreatment with chemical etchants on microleakage and bond strength between silicone-based resilient liner and denture base resin. The initial bending strength of denture base resin after surface pretreatment was also examined. METHODS: Thirty-six polymethyl methacrylate (PMMA) denture base resin blocks (30 mm × 30 mm × 2 mm) were prepared and divided into three groups: group acetone, group methyl methy acrylate (MMA) and group control. Subsequently, a 2 mm silicone-based resilient liner was applied between every two blocks. After 5000 cycles in the thermal cycler (5 and 55°C), they were immersed in the (131) I solution for 24 hours and γ-ray counts were measured. Another 36 PMMA resin blocks (30 mm × 10 mm × 7.5 mm) were prepared. The blocks were divided into three groups and treated as mentioned above. A 3 mm silicone-based resilient liner was applied between every two blocks. After 5000 thermal cycles, tensile bond strength of the sample was measured in a universal testing machine. Another 18 PMMA resin blocks (65 mm × 10 mm × 3.3 mm) were prepared. They were divided into 3 groups and treated in the same way. After an adhesive was applied, the bending strength was measured with three-piont bending test. RESULTS: Two experimental groups showed lower microleakage (520.0 ± 562.2 and 493.5 ± 447.9) and higher tensile bond strength [(1.5 ± 0.4) and (1.4 ± 0.5) MPa] than the group control [microleakage: (1369.5 ± 590.2); tensile bond strength: (0.9 ± 0.2) MPa, P < 0.05]. There was no statistically significant difference between group acetone and MMA in microleakage and tensile bond strength (P > 0.05). There was no statistically significant difference in bending strength among the three groups (P > 0.05). CONCLUSIONS: Treating the denture base resin surface with acetone and MMA decreased the microleakage, increased the tensile bond strength between the two materials and did not make the initial bending strength of denture base resin decline.

[Study on the correlation between CETP TaqIB, KCNE1 S38G and eNOS T-786C gene polymorphisms for predisposition and non-valvular atrial fibrillation].

OBJECTIVE: To study whether CETP TaqIB,KCNE1 S38G and eNOS T-786C genetic polymorphisms are associated with non-valvular atrial fibrillation in the Han population from Zhejiang province. METHODS: Polymerase chain reaction restriction fragment length polymorphism assay was used to detect the distribution of alleles and genotypes of CETP TaqIB, KCNE1 S38G and eNOS T-786C in 147 patients with non-valvular atrial fibrillation and in 147 subjects as controls in Han population of Zhejiang province. RESULTS: (1) The frequency of CETP B1 allele in NVAF patients was higher than that of the control group and showing a statistically significant difference (OR = 1.763, 95% CI: 1.247-2.492, P = 0.002). (2) Results from logistic regression analysis revealed that: after adjustment of confounding variables such as sex, age, smoking, hypertension and body mass index, data from the binary logistic analysis showed a statistically significant difference in CETP TaqIB genetic polymorphism between patients and controls. (3) From multifactor dimensionality reduction analysis, results showed an interaction of CETP TaqIB, KCNE1 S38G and eNOS T-786C genetic polymorphisms. Odds ratio of the three simultaneously existing genetic polymorphisms was 1.849 times more than CETP TaqIB alone. CONCLUSION: CETP BI allele was an independent risk factor for predisposition to non-valvular atrial fibrillation. These findings suggested that the simultaneous existence of CETP B1, KCNE1 S38G and eNOS T-786C allele might be elevated with the predisposition to non-valvular atrial fibrillation in the Han population of Zhejiang province.

[Isolation, culture and identification of two types of endothelial progenitor cells from peripheral blood in rabbits].

AIM: To investigate how to isolate, culture and identify two types of endothelial progenitor cells from peripheral blood in rabbits. METHODS: Mononuclear cells(MNCs) were isolated from rabbit peripheral blood. Endothelial progenitor cells (EPCs) and endothelial outgrowth cells (EOCs) were obtained from MNCs through different ways of isolation and culture. Two types of cells were assessed by DiI-ac-LDL uptake and lectin binding, and then they were identified by immunofluorescence of flk-1, immunocytochemistry of CD34 and VIII factor related antigen and vasculogenesis activity in vitro. RESULTS: Two types of endothelial progenitor cells were obtained from rabbit peripheral blood through different ways of isolation and culture. EPCs on the seventh day and EOCs on the sixteenth day were positive for ac-LDL uptake and lectin binding, and both of them expressed CD34, flk-1 and VIII factor related antigen. EOCs were assembled into primitive vascular tube-like structures when plated in matrigel. CONCLUSION: EPCs and EOCs could be obtained from rabbit peripheral blood when different ways of isolation and culture were performed. The system of cell culture can be applied to subsequent experiments in cell transplantation.

[The effects of desensitizing bonding system for prevention of vital abutment hypersensitivity].

OBJECTIVE: To evaluate the effects of GLUMA desensitizing bonding system in desensitizing hypersensitivity of vital abutment. METHODS: 69 central incisors with vital pulp from 69 patients were randomly divided into A, B and C group. After tooth preparation, group A coated with primer after etching, group B coated with primer after etching cervical dentin only, and group C was control group without special treatment. The cold sensitivity of abutments was tested after coating with primer, after cementation of crown and three months later. Two fresh extracted caries and restoration-free middle incisor were prepared in routine way. The surface of specimen 1 was etched with conditioner and the control specimen 2 was prepared without any special surface treatment. Scanning electron microscope (SEM) observation was used to examine the details on dentin surface. RESULTS: In all three times of examination, the sensitivities of two experimental groups had been significantly reduced compared with the control group. Immediately after coating, group B were more sensitive than group A (P < 0.05). After cementation of crown and three months later, there was no statistically significant between group A and group B. SEM photomicrograph showed that the smear layer was removed from etched dentine surface with open dentinal tubules. CONCLUSION: Coating with GLUMA desensitizing bonding system could prevent hypersensitivity of vital abutment.

[Preliminary study of ALK3 downstream genes related to ventricular septum defect].

To investigate the function of ALK3 gene, the gene regulation and the signaling pathway related to ventricular septum defect during heart development. The model mice with ALK3 gene knock-out via alpha-MHC-Cre/lox P system were bred. The mRNA expression level of control group was compared with that of experiment group and ALK3 downstream genes were screened using PCR-select cDNA subtraction microarray. The mRNA of control group was extracted from E11.5 normal mouse hearts, and that of experiment group, from E11.5 hearts of mice with alpha-MHC Cre(+/-) ALK3(F/+) genotype. It was found that the mice with ALK3 gene knock-out produced heart defects involving the interventricular septum. The platelet-activating factors acetylhydrolase and the transcription factor Pax-8 and so on, were down-regulated. However, the Protein Tyrosine Kinase (PTK) of Focal Adhesion Kinase (FAK) subfamily and beta subtype protein 14-3-3 were up-regulated in the alpha-MHC Cre(+/-) ALK3(F/-) mice. These data provide support that ALK3 gene played an important role during heart development. The platelet-activating factors acetylhydrolase and Pax-8 genes could be important ALK3 downstream genes in the BMP signaling pathway during interventricular septum development. PTK and beta subtype protein 14-3-3 might be regulatory factors in this pathway.

[A study on myocardial Pax-8 gene].

OBJECTIVE: Conventional deletion of ALK3, also termed as bone morphogenetic protein (BMP) receptor IA, in mice might result in early embryonic lethality. To investigate the function of ALK3 in cardiac development, the cardiac-specific deletion of ALK3 in mice was made by Dr. Schneider, using Cre recombinase driven by the alpha-MHC promoter that Dr. Fukushipe worked out. Such specific deletion of ALK3 caused death in mid-gestation with defects in the trabeculae, interventricular septum, and endocardial cushion. Since ALK3 is not a cardiac-specific gene, it is extremely important to identify ALK3 downstream genes. METHODS: Alpha-MHC Cre+/-, ALK3 F/- and alpha-MHC Cre+/-, ALK3 F/+ embryos were obtained after 20 alpha-MHC Cre+/-, ALK3 +/- mice and 20 ALK3 F/F mice were mating. The ALK3 downstream genes were screened using microarray made in Germany that could identify 25000 genes in mouse. Two populations of mRNA, one derived from the embryonic heart (11.5 days) of alpha-MHC Cre+/-, ALK3 F/- mice, and the other derived from the alpha-MHC Cre+/-, ALK3 F/+ mice, were compared. Cardiac-specific ALK3 downstream genes were identified using real time quantitative RT-PCR and in situ hybridization. RESULTS: The expression of 12 genes, such as Pax-8 and Hox-3.5 were down-regulated in alpha-MHC Cre+/-, ALK3 F/- mouse heart. The expression of 16 genes including Ras-related protein Rab-5b and EPS-8 protein was up-regulated in the group of alpha-MHC Cre+/-, ALK3 F/-. It was found that the Box protein Pax-8 gene was down-regulated by 7.1 fold (P < 0.001) in the alpha-MHC Cre+/-, ALK3 F/- mice by real time quantitative RT-PCR. It was also revealed that the Box protein Pax-8 gene was expressed stronger in alpha-MHC Cre+/-, ALK3 F/+ than alpha-MHC Cre+/-, ALK3 F/- E11.5 days mouse heart by means of in situ hybridization. CONCLUSION: The Box protein Pax-8 gene is an important and cardiac-specific ALK3 downstream gene in the BMP signaling pathway during inter-ventricular septum development.