The hypoxia-inducible factor 1alpha (HIF-1alpha) regulates transcriptional genes involved in cell proliferation, survival, and differentiation. Under normoxia, HIF-1alpha has a short half-life (t((1/2)) approximately 5 min) and low transcriptional activity. An HIF-1alpha mutant, produced by substitution of alanine (Ala) for proline (Pro) at position 564 and asparagine (Asp) at position 803, can prevent HIF-1alpha hydroxylation and results in a highly active form of HIF-1alpha (HIF-1alpha-Ala564-Ala803). We hypothesized that adenovirus (Ad)-mediated transfer of the active form of HIF-1alpha (pAd-HIF-1alpha-Ala564-Ala803) could effectively occur in bone marrow stem cells (MSCs) and promote MSC differentiation under normoxia. PCR-based site-specific mutagenesis was used to construct the Ad vector expressing HIF-1alpha-Ala564-Ala803. RT-PCR and immunostaining were used to study whether pAd-HIF-1alpha-Ala564-Ala803 affected MSC differentiation to cardiomyocyte (CMC). pAd-HIF-1alpha-Ala564-Ala803 exhibited higher transcriptional activity and stable HIF-1alpha protein expression. Under normoxia, an MSC-CMC co-culture treated with pAd-HIF1a-Ala564-Ala803 augmented TGF-beta(1), Smad4, NKx2.5, and GATA4 expression. Higher expression of cTnT and alpha-actinin was observed by immunostaining in MSCs, compared with the control and contrast groups. Adenovirus-mediated hypoxia-inducible factor 1alpha double-mutant, pAd-HIF-1alpha-Ala564-Ala803, can stably express HIF-1alpha and promote its downstream genes and MSC differentiation to CMC in the MSC-CMC co-culture system under normoxia.

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Adenovirus-mediated expression of hypoxia-inducible factor 1α double mutant (pAd-HIF-1α-Ala564-Ala803) can be effectively transfected into bone marrow stem cells (MSCs) in the MSCs and cardiomyocytes co-culture system at normoxia to regulate the expression of downstream target genes of hypoxia-inducible factor 1α (HIF-1α), which in turn can promote MSC differentiation into cardiomyocytes. Fibroblasts share common characteristics with MSCs such as the morphology, phenotype and differentiation potential. Therefore, we further studied whether the pAd-HIF-1α-Ala564-Ala803 also can convert neonatal rat cardiac fibroblasts (NCFs) into (cardio)myocyte phenotype via regulating the downstream target genes of HIF-1α at normoxia. The immunostaining analysis showed that NCFs treated with pAd-HIF-1α-Ala564-Ala803 exhibited higher protein expression levels of smooth muscle α-actin (SMA, myocyte marker) and cardiac troponin T (cTnT, cardiomyocyte marker), compared with phosphate-buffered saline and pAd-LacZ treatments. The reverse transcription-polymerase chain reaction results showed that NCFs transfected with pAd-HIF-1α-Ala564-Ala803 augmented messenger RNA (mRNA) expression of transforming growth factor-β1 (TGF-β1), Smad4, NKx2.5, GATA4, myocardin, SMA and cTnT. The effects of HIF-1α-Ala564-Ala803 on NCFs were attenuated by pre-transfection of TGF-β1 or myocardin small interference RNAs. Adult CFs transfected with pAd-HIF-1α-Ala564-Ala803 showed a lower protein expression of SMA but not cTnT without any change in the mRNA expression level of NKx2.5, myocardin. Therefore, NCFs but not adult CFs possess a similar differentiation potential to MSCs as evidenced by the fact that pAd-HIF-1α-Ala564-Ala803 can convert NCFs into (cardio)myocyte phenotype via regulating its downstream target genes.

Background Patients with both double orifice mitral valve (DOMV) and bicuspid aortic valve (BAV) malformation are rare. Although DOMV or BAV can be detected in some genetic syndromes, it has not been reported to simultaneously appear in Turner syndrome (TS). TrueVue, TouchVue, and TrueVue Glass are the latest technologies in advanced three-dimensional echocardiography (3DE), which is an important information supplement to two-dimensional echocardiography (2DE) for the diagnosis of congenital cardiac malformations. Herein we report the novel use of the above-mentioned technologies in the diagnosis and evaluation of a rare, combined valve malformation. Meanwhile, we also reviewed the literature for cases involving both DOMV and BAV and their association with various genetic syndromes. Case presentation We present the case of a 5-year-old girl diagnosed with TS because of a developmental delay. DOMV and BAV were found through echocardiographic examination. Three-dimensional transthoracic echocardiography as well as a series of novel advanced techniques were applied to clearly display the spatial structure of all tiers of the mitral valve apparatus, aortic valve, and arch to facilitate an accurate diagnosis. Conclusions This is the first case in which both DOMV and BAV were associated with TS. Innovative TrueVue and TrueVue Glass offer unprecedented photographic stereoscopic images, while TouchVue technology greatly improved the ultrasonic diagnostic workflow and the diagnostic performance of rare valve malformations by adding virtual light sources to display realistic light-shadow effects.

Aims: This study explored the advantages and limitations of novel series of three-dimensional (3D) echocardiographic techniques and summarized their application methods for congenital heart diseases (CHDs).Method and result: Two-dimensional (2D), traditional 3D echocardiography, and TrueVue plus light and/or Glass novel 3D technologies were performed on 62 patients with CHD, and a clinical survey was designed to judge whether the novel 3D images were more helpful for understanding the cardiac condition and guide treatment than traditional 3D images. TrueVue increased the visual resolution and simulated the true texture of cardiac tissue, significantly improving the display ability of abnormal anatomical structures in CHDs. TrueVue Glass displayed the blood channel and the internal structure of cardiac cavity more intuitively, indicating a new observation aspect not shown by conventional echocardiography. The clinical survey results showed that the new 3D imaging methods effectively increased the diagnostic confidence of echocardiographers, enabled surgeons to better understand the details of lesions, promoted efficient communication, and improved the confidence of both doctors and patients in treatment.Conclusion: The combined application of TrueVue, TrueVue Light, and TrueVue Glass more closely simulated real anatomical features, showed more comprehensive and subtle blood flow in the lumen, not only increased the visual effect but also provided more useful diagnostic information, improved the accuracy of evaluation and treatment of CHD when compared to traditional imaging techniques, indicating that this combined application has significant clinical value.