Prenatal counseling in cardiac surgery: A report of 225 fetuses with congenital heart disease. / 心外科产前咨询225ä¾‹å ˆå¤©æ€§å¿ƒè„ç— èƒŽå„¿.

OBJECTIVES: To explore the method and significance of prenatal counseling in cardiac surgery for fetal congenital heart disease (CHD). METHODS: The prenatal counseling should be provided by experienced CHD experts. The preliminary clinical diagnosis based on relevant data was carried out, the prognosis risk for fetal CHD was graded, and the pathophysiological process and potential hazards of the disease were analyzed. The current condition of CHD in the treatment plan, the long-term quality of life, and the special requirements of parturition in place, period and mode were described. A reliable follow-up system of the fetuses was established, the diagnosis after delivery was verified, and surgical treatment was carried out timely. RESULTS: From January 2016 to December 2018, 225 parents with fetal CHD received prenatal counseling, including 60 fetuses (26.7%) with simple CHD and 165 (73.3%) with complex CHD, among which 59 cases (98.3%) and 93 cases (56.4%) decided to continue the pregnancy, respectively. During the follow-up, 118 fetuses were born, of which 66 infants received surgical treatment within 6 months after birth, 63 infants (95.5%) recovered and 3 infants (4.5%) died. The rest 52 infants continued to be followed up. CONCLUSIONS: The prenatal counseling for fetal CHD can provide the parents a comprehensive medical information about CHD, which is beneficial to making appropriate pregnancy decisions, and can turn the fetuses from unreasonable birth and passive treatment to selective birth and active treatment in CHD.

Apelin attenuates the osteoblastic differentiation of aortic valve interstitial cells via the ERK and PI3-K/Akt pathways.

Aortic valve calcification (AVC), which used to be recognized as a passive and irreversible process, is now widely accepted as an active and regulated process characterized by osteoblastic differentiation of aortic valve interstitial cells (AVICs). Apelin, the endogenous ligand for G-protein-coupled receptor APJ, was found to have protective cardiovascular effects in several studies. However, the effects and mechanisms of apelin on osteoblastic differentiation of AVICs have not been elucidated. Using a pro-calcific medium, we devised a method to produce calcific human AVICs. These cells were used to study the relationship between apelin and the osteoblastic calcification of AVICs and the involved signaling pathways. Alkaline phosphatase (ALP) activity/expression and runt-related transcription factor 2 (Runx2) expression were examined as hallmark proteins in this research. The involved signaling pathways were studied using the extracellular signal-regulated kinase (ERK) inhibitor, PD98059, and the phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002. The results indicate that apelin attenuates the expression and activity of ALP, the expression of Runx2, and the formation of mineralized nodules. This protective effect was dependent on the dose of apelin, reaching the maximum at 100 pM, and was connected to activity of ERK and Akt (a downstream effector of PI3-K). The activation of ERK and PI3-K initiated the effects of apelin on ALP activity/expression and Runx2, but PD98059 and LY294002 abolished the effect. These results demonstrate that apelin attenuates the osteoblastic differentiation of AVICs via the ERK and PI3-K/Akt pathway.

Aging aggravates aortic aneurysm and dissection via miR-1204-MYLK signaling axis in mice.

The mechanism by which aging induces aortic aneurysm and dissection (AAD) remains unclear. A total of 430 participants were recruited for the screening of differentially expressed plasma microRNAs (miRNAs). We found that miR-1204 is significantly increased in both the plasma and aorta of elder patients with AAD and is positively correlated with age. Cell senescence induces the expression of miR-1204 through p53 interaction with plasmacytoma variant translocation 1, and miR-1204 induces vascular smooth muscle cell (VSMC) senescence to form a positive feedback loop. Furthermore, miR-1204 aggravates angiotensin II-induced AAD formation, and inhibition of miR-1204 attenuates ß-aminopropionitrile monofumarate-induced AAD development in mice. Mechanistically, miR-1204 directly targets myosin light chain kinase (MYLK), leading to the acquisition of a senescence-associated secretory phenotype (SASP) by VSMCs and loss of their contractile phenotype. MYLK overexpression reverses miR-1204-induced VSMC senescence, SASP and contractile phenotypic changes, and the decrease of transforming growth factor-ß signaling pathway. Our findings suggest that aging aggravates AAD via the miR-1204-MYLK signaling axis.

Taurine suppresses osteoblastic differentiation of aortic valve interstitial cells induced by beta-glycerophosphate disodium, dexamethasone and ascorbic acid via the ERK pathway.

Aortic valve calcification (AVC) is an active process characterized by osteoblastic differentiation of the aortic valve interstitial cells (AVICs). Taurine is a free ß-amino acid and plays important physiological roles including protective effect of cardiovascular events. To evaluate the possible role of taurine in AVC, we isolated human AVICs from patients with type A dissection without leaflet disease. We demonstrated that the cultured AVICs express SM α-actin, vimentin and taurine transporter (TAUT), but not CD31, SM-myosin or desmin. We also established the osteoblastic differentiation model of the AVICs induced by pro-calcific medium (PCM) containing ß-glycerophosphate disodium, dexamethasone and ascorbic acid in vitro. The results showed that taurine attenuated the PCM-induced osteoblastic differentiation of AVICs by decreasing the alkaline phosphate (ALP) activity/expression and the expression of the core binding factor α1 (Cbfα1) in a dose-dependent manner (reaching the maximum protective effect at 10 mM), and taurine (10 mM) inhibited the mineralization level of AVICs in the form of calcium content significantly. Furthermore, taurine activated the extracellular signal-regulated protein kinase (ERK) pathway via TAUT, and the inhibitor of ERK (PD98059) abolished the effect of taurine on both ALP activity/expression and Cbfα1 expression. These results suggested that taurine could inhibit osteoblastic differentiation of AVIC via the ERK pathway.

[Heparin-iron complex multilayer nanomodification improves hemocompatibility of decellular xenograft].

OBJECTIVE: To improve the hemocompatibility of decellular vascular matrix via heparin-iron complex multilayers (HICMs) nanomodification. METHODS: A novel thrombo-resistant surface for decellular xenograft was developed by alternating linkage of dihydroxy-iron and heparin to decellular bovine jugular vein (DC-BJV), and its surface characterization, biomechanical stability and hemocompatibility were detected by scanning electron microscopy, tensile test and hemocompatibility evaluation, respectively. RESULTS: A toluidine blue colorimetric method indicated the amount of linked heparin was about (808±86) µg/cm2 per assembly-cycle. Scanning electron microscopic (SEM) images proved that HICMs were uniformly linked to and formed nanoscale films around the fibrils of DC-BJV. Toluidine blue staining histologic images showed that HICMs were linked mainly to DC-BJV surfaces. Washing test showed that the release of heparin was (281±43), (422 ± 60), (729±81), (1053±116), (1317±157), (1618±187) and (1945 ± 268 ) µg/cm(2) at 1 day, 1, 2, 3, 4, 6 and 8 week washing, respectively. Tensile tests showed an increased biomechanical stability. Hemocompatibility evaluations showed that PT and APTT of all the trial groups were above the normal reference ranges and that mean platelet count per 10000 µm2 area was 8±4 for HICMs layer-by-layer modified BJV (LBL-BJV) vs 48±16 for DC-BJV. CONCLUSION: HICMs are firmly linked to DC-BJV, and can form nanoscale thrombo-resistant films, which yield a sustained release of heparin. HICMs nanomodification improves the hemocompatibility of decellular xenograft.

Unusual combination of acute aortic dissection, Mayer-Rokitansky-Küster-Hauser syndrome, and 46,XX gonadal dysgenesis: A case report.

Background: Acute Stanford type A aortic dissection (ATAAD) is a life-threatening disease. Elderly patients are the high-risk population for aortic dissection (AD). Young patients with AD usually have heritable connective tissue diseases such as Marfan syndrome and Loeys-Dietz syndrome. However, young AD patients without heritable connective tissue disease are relatively rare. Case presentation: Herein, we report a case of a 25-year-old female diagnosed with ATAAD accompanied by undeveloped secondary sexual characteristics. Computed tomography angiography (CTA) showed that her AD involved the ascending and abdominal aorta. She had undergone thoracic endovascular aortic stent graft implantation in a local hospital due to acute Stanford type B aortic dissection at age 19. No uterus or ovaries were found on CTA and transabdominal ultrasonography. Sex hormone detection revealed a low estrogen level. G-banded karyotyping analyses revealed a normal 46,XX karyotype. Finally, her abnormalities in the reproductive system were diagnosed as MRKH syndrome and 46,XX gonadal dysgenesis. Whole-exome sequencing (WES) in the patient found an SNP variant of ACTA2 c.773G>A and MYH11 c.5081A>G. MYH11 c.5081A>G was also found in her mother and younger brother. Copy number variations sequencing (CNV-seq) found an approximately 109.30 Kb duplication at chromosome 6p22.3 (Chr 6: g.24920238-25029535) with a copy number of 3. We performed emergent total aortic arch replacement with frozen elephant trunk surgery, and the patient recovered well after surgery. However, her abdominal AD was stilling progression during 6 months of follow-up. Conclusion: To our knowledge, we report the world's first case of early-onset recurrent AD combined with MRKH syndrome and 46,XX gonadal dysgenesis.

Evaluating the monogenic contribution and genotype-phenotype correlation in patients with isolated thoracic aortic aneurysm.

Thoracic aortic aneurysm with or without dissection (TAAD) can be broadly categorized as syndromic TAAD (sTAAD) and isolated TAAD (iTAAD). sTAAD and is highly correlated with genetics. However, although the incidence of iTAAD is much higher, its monogenic contribution is not yet clear. Here, we sequenced 15 known TAAD genes for 578 iTAAD cases from four cardiac centers in China and found that 10.6% patients with a pathogenic/likely pathogenic (P/LP) variant. Other 7.27% of patients carried variants of uncertain significance in these target genes. We further investigated the correlations among genetics, clinical features, and long-term outcomes. Genetic patients showed younger onset ages (P = 1.31E-13) and larger aortic diameter (P = 1.00E-6), with the youngest age in patients with FBN1 P/LP variants. Monogenic variants were also associated with more aortic segments involved (P = 0.043) and complicated with initial dissection (P = 4.50E-5), especially for genetic patients with non-FBN1 P/LP variants. MACEs occurred in 14.9% patients during follow-up of median 55 months. Genetic status (P = 0.001) and initial dissection (P = 3.00E-6) were two major risk factors for poor prognosis. Early onset age was associated with MACEs in non-genetic cases without initial dissection (P = 0.005). Our study revealed the monogenic contribution in known TAAD genes to iTAAD patients. The genotype-phenotype correlations may complement the risk stratification of iTAAD patients and identification of higher risk subgroups, as well as assist the development of tailored precision medicine in iTAAD.

[Correlation of serum levels of VEGF and SDF-1 with the number and function of circulating EPCs in children with cyanotic congenital heart disease].

OBJECTIVE: To examine the number and function of circulating endothelial progenitor cells (EPCs) in children with cyanotic congenital heart diseases (CHD) and study their correlation with serum levels of vascular endothelial growth factor (VEGF) and stromal cell derived factor-1 (SDF-1). METHODS: Fifteen children with tetralogy of Fallot (cyanotic group) and 15 age-and sex-matched children with ventricular septal defect (control group) were enrolled. Serum levels of VEGF and SDF-1 were measured using ELISA. Mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation and cultured in vitro. EPCs were identified by immunofluorescence and were counted under a microscope. Modified Boyden chamber assay and the MTT assay were used to measure the migration and proliferation capacities of EPCs. EPCs adhesion ability assay was performed by replating cells on fibronectin-coated dishes, and then adherent cells were counted. The correlations of serum levels of VEGF and SDF-1 with the number and function of circulating EPCs were assessed by linear regression analysis. RESULTS: Serum levels of VEGF (201.42+/-44.74 ng/L vs 113.56+/-35.62 ng/L; P<0.05) and SDF-1 (3.45+/-1.07 ng/L vs 1.05+/-0.99 ng/L; P<0.05) in the cyanotic group were higher than those in the control group. There was a positive correlation between serum levels of VEGF and SDF-1(r=0.675, P<0.01). The number of EPCs (*200 field) in the cyanotic group significantly increased compared with that of the control group (72.2+/-9.73 vs 51.2+/-3.83; P<0.01). The functional activities of EPCs, including proliferation, migration and adhesion capacities, were augmented in the cyanotic group compared with those in the control group. The increased number and function of EPCs and the increased serum levels of VEGF and SDF-1 were consistent in the cyanotic group, with a correlation coefficient of 0.8395, 0.5491, 0.6376 and 0.7392 respectively. CONCLUSIONS: The number and functional activity of EPCs as well as serum levels of VEGF and SDF-1 increased in children with cyanotic CHD. Serum levels of VEGF and SDF-1 were correlated to the number and functional activity of EPCs. Serum VEGF and SDF-1 together with circulating EPCs may play important roles in the pathology and physiology in these patients.

A Novel Mutation Of The EMD Gene In A Family With Cardiac Conduction Abnormalities And A High Incidence Of Sudden Cardiac Death.

BACKGROUND: Emery-Dreifuss muscular dystrophy, caused by mutations in genes such as emerin (EMD) or lamin A/C (LMNA), is a disorder affecting the joints, muscles, and heart, with a wide spectrum of patient phenotypes including muscle wasting and cardiac conduction defects. METHODS AND RESULTS: Here we report a multi-generation family from the Hunan Province of China. Affected family members displayed an uncommon clinical presentation of serious cardiac conduction abnormalities at an early age and a high incidence of sudden cardiac death along with mild skeletal muscular atrophy and joint contracture. Clinical analysis of affected members provided evidence of X-linked recessive inheritance. Consequently, using Sanger sequencing of X chromosome exomes, we identified a novel duplication mutation (c.405dup/p.Asp136X) in the EMD gene as the cause for the disease in this family. This variant is a novel mutation that has not been previously reported in Pubmed, Clinvar or other cases reported in the Human Gene Mutation Database. CONCLUSION: Our finding expands the mutation spectrum of Emery-Dreifuss muscular dystrophy and provides a rationale for EMD mutation testing in cases of X-linked inherited cardiac conduction disease and sudden cardiac death, even in those lacking pathognomonic neuromuscular features.

HCP5 is a SMAD3-responsive long non-coding RNA that promotes lung adenocarcinoma metastasis via miR-203/SNAI axis.

Introduction: Transforming growth factor-beta (TGFß) signaling plays a vital role in lung adenocarcinoma (LUAD) progression. However, the involvement of TGFß-regulated long non-coding RNAs (lncRNAs) in metastasis of LUAD remains poorly understood. Methods: We performed bioinformatic analyses to identify putative lncRNAs regulated by TGF-ß/SMAD3 and validated the results by quantitative PCR in LUAD cells. We performed luciferase reporter and chromatin immunoprecipitation assays to demonstrate the transcriptional regulation of the lncRNA histocompatibility leukocyte antigen complex P5 (HCP5) we decided to focus on. Stable HCP5 knockdown and HCP5-overexpressing A549 cell variants were generated respectively, to study HCP5 function and understand its mechanism of action. We also confirmed our findings in mouse xenografts and metastasis models. We analyzed the correlation between the level of lncRNA expression with EGFR, KRAS mutations, smoke state and prognostic of LUAD patients. Results: We found that the lncRNA HCP5 is induced by TGFß and transcriptionally regulated by SMAD3, which promotes LUAD tumor growth and metastasis. Moreover, HCP5 is overexpressed in tumor tissues of patients with LUAD, specifically in patients with EGFR and KRAS mutations and current smoker. HCP5 high expression level is positively correlated with poor prognosis of patients with LUAD. Finally, we demonstrated that upregulation of HCP5 increases the expression of Snail and Slug by sponging the microRNA-203 (miR-203) and promoting epithelial-mesenchymal transition (EMT) in LUAD cells. Conclusions: Our work demonstrates that the lncRNA HCP5 is transcriptionally regulated by SMAD3 and acts as a new regulator in the TGFß/SMAD signaling pathway. Therefore, HCP5 can serve as a potential therapeutic target in LUAD.

Expression and copy number gains of the RET gene in 631 early and mid stage non-small cell lung cancer cases.

BACKGROUND: To identify whether RET is a potential target for NSCLC treatment, we examined the status of the RET gene in 631 early and mid stage NSCLC cases from south central China. METHODS: RET expression was identified by Western blot. RET-positive expression samples were verified by immunohistochemistry. RET gene mutation, copy number variation, and rearrangement were analyzed by DNA Sanger sequencing, TaqMan copy number assays, and reverse transcription-PCR. ALK and ROS1 expression levels were tested by Western blot and EGFR mutation using Sanger sequencing. RESULTS: The RET-positive rate was 2.5% (16/631). RET-positive expression was related to poorer tumor differentiation (P < 0.05). In the 16 RET-positive samples, only two samples of moderately and poorly differentiated lung adenocarcinomas displayed RET rearrangement, both in RET-KIF5B fusion partners. Neither ALK nor ROS1 translocation was found. The EGFR mutation rate in RET-positive samples was significantly lower than in RET-negative samples (P < 0.05). CONCLUSION: RET-positive expression in early and mid stage NSCLC cases from south central China is relatively low and is related to poorer tumor differentiation. RET gene alterations (copy number gain and rearrangement) exist in all RET-positive samples. RET-positive expression is a relatively independent factor in NSCLC patients, which indicates that the RET gene may be a novel target site for personalized treatment of NSCLC.

Photooxidatively crosslinked acellular tumor extracellular matrices as potential tumor engineering scaffolds.

Acellular tumor extracellular matrices (ECMs) have limitations when employed as three-dimensional (3D) scaffolds for tumor engineering. In this work, methylene blue-mediated photooxidation was used to crosslink acellular tumor ECMs. Photooxidative crosslinking greatly increased the stiffness of acellular tumor ECM scaffolds but barely altered the Amide III band of the secondary structure of polypeptides and proteins. MCF-7, HepG2 and A549 cells cultured on photooxidatively crosslinked acellular tumor ECM scaffolds exhibited greater cell number per scaffold, more IL-8 and VEGF secretion, and increase migration and invasion abilities than cells cultured on uncrosslinked acellular tumor ECM scaffolds. The three tumor cell lines cultured on the stiffer photooxidatively crosslinked acellular matrices acquire mesenchymal properties (mesenchymal shift) and dedifferentiated phenotypes. Furthermore, the malignant phenotypes induced in vitro when cultured on the crosslinked scaffold promoted the in vivo tumor growth of BALB/c nude mice. Finally, the dedifferentiated cancer cells, including MCF-7, HepG2 and A549 cells, were less sensitive to chemotherapeutics. Thus, photooxidatively crosslinked acellular tumor ECMs have potentials as 3D tumor engineering scaffolds for cancer research. STATEMENT OF SIGNIFICANCE: Natural material scaffolds have been successfully used as 3D matrices to study the in vitro tumor cell growth and mimic the in vivo tumor microenvironment. Acellular tumor ECMs are developed as 3D scaffolds for tumor engineering but have limitations in terms of elastic modulus and cell spheroid formation. Here we use methylene blue-mediated photooxidation to crosslink acellular tumor ECMs and investigate the influence of photooxidative crosslinking on structural, mechanical and biological characteristics of acellular tumor ECM scaffolds. It is the first study to evaluate the feasibility of photooxidatively crosslinked acellular tumor ECMs as 3D scaffolds for cancer research and the results are encouraging. Moreover, this study provides new research areas in regard to photodynamic therapy (PDT) for Cancer.

[Effect of decellular treatment on the framework of extracellular matrix in bovine jugular vein conduit].

OBJECTIVE: To investigate the effect of decellular treatment on the framework constituents of extracellular matrix and tissue stability in bovine jugular vein conduit (BJVC), and to provide an evidence for tissue engineering of vascular prosthesis. METHODS: Bovine jugular veins were obtained fresh from a local slaughterhouse and were stored in chilled PBS. In the laboratory, any fat and loose connective tissue on the outer surface of the vessel was trimmed. BJVCs were decellularized by a 3-step extraction method as detergent Triton X-100 (0.5%), Trypsin (0.025%) EDTA (0.02%), and DNase I(30kU/L) RNaseA(0.3g/L). Histological and transmission electron microscopy (TEM) techniques were used to study the framework constituents of extracellular matrix of treated the examples, and fresh tissues were used as controls. Tissue contents of hydroxyproline(alkaline hydrolysis method) and elastin (Fastin Elastin Assay) were assayed respectively in the fresh and decellularized groups (n=10). The vascular wall heat shrinking temperature and mechanical strength were measured to evaluate the tissue stability (n=10). RESULTS: Histochemical and TEM analysis of BJVCs treated with decellularization proved a complete removal of nuclear and other cell components. Tissue collagen was well kept,but elastin was partly lessened. Tissue content of hydroxyproline increased comparatively [(25.73+/-2.97)mg/g vs. (29.25+/-2.99)mg/g, P<0.05] and the elastin content obviously decreased [(159.71+/-21.06)mg/g vs. (134.91+/-35.40)mg/g, P<0.05] in the decellular treatment group compared with the control group. The heat shrinking temperature and tensile stress of decelluarized tissue were lower than those of the fresh tissue[(72.50+/-0.53) degrees C vs. (69.75+/-0.54)degrees C ,P<0.05], [(5.19+/-0.65)MPa vs. (3.13+/-0.94)MPa, P<0.05]. CONCLUSION: The basic framework of extracellular matrix in the decellularized BJVC is partly damaged and tissue stability is reduced. Decellularized BJVC should be further crosslinked before being used as a tissue engineering scaffold for clinical pulmonary artery graft.

[Bovine jugular venous conduit treated with the polyepoxy compound].

OBJECTIVE: To determine the feasibility whether the bovine jugular venous conduit (BJVC) can be fixed with polyepoxy compound (PC). METHODS: Twenty-four BJVCs were divided into 3 groups and fixed with polyepoxy compound (PC group, n = 8), glutaraldehyde (GA group, n = 8), and unfixed group (Control group, n = 8), respectively. The morphologic and mechanical properties of BJVCs in the 3 groups, including thickness, diameter, moisture content, denaturation temperature, tensile strength, elongation at break, and fixation index were measured. The rat subcutaneous model for the assessment of tissue calcification was used. The calcium content in bovine jugular vein patches and valves was determined by flame atomic absorption spectrophotometer. RESULTS: There was no difference in the wall thickness, diameter, and tissue water content between PC and the control group, but significant difference was found between GA and PC groups. The mechanical properties of PC group and GA group were not significantly different, but they were better than those of the control group. GA-fixed BJVC samples showed clear calcification, while PC fixed BJVC were calcified significantly less. CONCLUSION: PC is an effective and suitable choice for the treatment of BJVC since it can effectively preserve the structure and the anti-reflow function of valves in bovine jugular vein and it has better anti-calcification properties.

[Augmentation of revascularization by implantation of autologous bone marrow mononuclear cells in a rat ischemic hind limb model].

OBJECTIVE: To examine whether the in vivo implantation of autologous bone-marrow mononuclear cells (BM-MNCs) can augment postnatal neovascularization in rat model of hind limb ischemia. METHODS: Rat BM-MNCs were isolated by centrifugation through a Ficoll-paque density gradient. The rat ischemic hind limb model was made by ligation of the right femoral artery and its branches of imbred Wistar rats. BM-MNCs (MNC group) or phosphate saline buffer (PBS group) were injected into 7 points of the ischemic muscles (20 microl/point). To assess angiogenesis, histologic evaluation was performed on tissue slices from adductor muscles with immunohistochemical staining at the 2nd and 4th weeks after the ligation. Severity of ischemic insult was evaluated by the femoral arteriovenous oxgen difference (AVDO2) at the 2nd and 4th weeks after the ligation. RESULTS: The MNC groups had a higher capillary density and higher capillary/muscle fiber ratios than that of the sham and PBS groups (P < 0.01) at the 2nd and 4th weeks after the ligation. AVDO2 of the MNC group significantly decreased compared with that of the PBS groups (P < 0.05). CONCLUSION: The implantation of autologous BM-MNCs induced neovascularization in a rat ischemic hind limb model. The implantation of autologous BM-MNCs augmented the collateral perfusion of ischemic hind limb.

Development of an acellular tumor extracellular matrix as a three-dimensional scaffold for tumor engineering.

Tumor engineering is defined as the construction of three-dimensional (3D) tumors in vitro with tissue engineering approaches. The present 3D scaffolds for tumor engineering have several limitations in terms of structure and function. To get an ideal 3D scaffold for tumor culture, A549 human pulmonary adenocarcinoma cells were implanted into immunodeficient mice to establish xenotransplatation models. Tumors were retrieved at 30-day implantation and sliced into sheets. They were subsequently decellularized by four procedures. Two decellularization methods, Tris-Trypsin-Triton multi-step treatment and sodium dodecyl sulfate (SDS) treatment, achieved complete cellular removal and thus were chosen for evaluation of histological and biochemical properties. Native tumor tissues were used as controls. Human breast cancer MCF-7 cells were cultured onto the two 3D scaffolds for further cell growth and growth factor secretion investigations, with the two-dimensional (2D) culture and cells cultured onto the Matrigel scaffolds used as controls. Results showed that Tris-Trypsin-Triton multi-step treated tumor sheets had well-preserved extracellular matrix structures and components. Their porosity was increased but elastic modulus was decreased compared with the native tumor samples. They supported MCF-7 cell repopulation and proliferation, as well as expression of growth factors. When cultured within the Tris-Trypsin-Triton treated scaffold, A549 cells and human colorectal adenocarcinoma cells (SW-480) had similar behaviors to MCF-7 cells, but human esophageal squamous cell carcinoma cells (KYSE-510) had a relatively slow cell repopulation rate. This study provides evidence that Tris-Trypsin-Triton treated acellular tumor extracellular matrices are promising 3D scaffolds with ideal spatial arrangement, biomechanical properties and biocompatibility for improved modeling of 3D tumor microenvironments.

MiR-133a modulates osteogenic differentiation of vascular smooth muscle cells.

Arterial calcification is a key pathologic component of vascular diseases such as atherosclerosis, coronary artery disease, and peripheral vascular disease. A hallmark of this pathological process is the phenotypic transition of vascular smooth muscle cells (VSMCs) to osteoblast-like cells. Several studies have demonstrated that microRNAs (miRNAs) regulate osteoblast differentiation, but it is unclear whether miRNAs also regulate VSMC-mediated arterial calcification. In the present study, we sought to characterize the role of miR-133a in regulating VSMC-mediated arterial calcification. Northern blotting analysis of VSMCs treated with ß-glycerophosphate demonstrated that miR-133a was significantly decreased during osteogenic differentiation. Overexpression of miR-133a inhibited VSMC transdifferentiation into osteoblast-like cells as evidenced by a decrease in alkaline phosphatase activity, osteocalcin secretion, Runx2 expression, and mineralized nodule formation. Conversely, the knockdown of miR-133a using an miR-133a inhibitor promoted osteogenic differentiation of VSMCs by increasing alkaline phosphatase activity, osteocalcin secretion, and Runx2 expression. Runx2 was identified as a direct target of miR-133a by a cotransfection experiment in VSMCs with luciferase reporter plasmids containing wild-type or mutant 3'-untranslated region sequences of Runx2. Furthermore, the pro-osteogenic effects of miR-133a inhibitor were abrogated in Runx2-knockdown cells, and the inhibition of osteogenic differentiation by pre-miR-133a was reversed by overexpression of Runx2, providing functional evidence that the effects of miR-133a in osteogenic differentiation were mediated by targeting Runx2. These results demonstrate that miR-133a is a key negative regulator of the osteogenic differentiation of VSMCs.

Heparin nanomodification improves biocompatibility and biomechanical stability of decellularized vascular scaffolds.

Biocompatibility and biomechanical stability are two of the main obstacles limiting the effectiveness of vascular scaffolds. To improve the biomechanical stability and biocompatibility of these scaffolds, we created a heparin-nanomodified acellular bovine jugular vein scaffold by alternating linkage of heparin and dihydroxy-iron via self-assembly. Features of the scaffold were evaluated in vitro and in vivo. Heparin was firmly linked to and formed nanoscale coatings around the fibers of the scaffold, and the amount of heparin linked was about 808 ± 86 µg/cm(2) (101 ± 11 USP/cm(2)) per assembly cycle. The scaffolds showed significantly strengthened biomechanical stability with sustained release of heparin for several weeks in vitro. Importantly, the modified scaffolds showed significantly reduced platelet adhesion, stimulated proliferation of endothelial cells in vitro, and reduced calcification in a subcutaneous implantation rat model in vivo. Heparin nanomodification improves the biocompatibility and biomechanical stability of vascular scaffolds.

Calcification resistance for photooxidatively crosslinked acellular bovine jugular vein conduits in right-side heart implantation.

This study aimed to investigate the effect of decellularization plus photooxidative crosslinking and ethanol pretreatment on bioprosthetic tissue calcification. Photooxidatively crosslinked acellular (PCA) bovine jugular vein conduits (BJVCs) and their photooxidized controls (n = 5 each) were sterilized in a graded concentration of ethanol solutions for 4 h, and used to reconstruct dog right ventricular outflow tracts. At 1-year implantation, echocardiography showed similar hemodynamic performance, but obvious calcification for the photooxidized BJVC walls. Further histological examination showed intense calcium deposition colocalized with slightly degraded elastic fibers in the photooxidized BJVC walls, with sparsely distributed punctate calcification in the valves and other areas of walls. But PCA BJVCs had apparent degradation of elastic fibers in the walls, with only sparsely distributed punctate calcification in the walls and valves. Content assay demonstrated comparable calcium content for the two groups at preimplantation, whereas less calcium for the PCA group in the walls and similar calcium in the valvular leaflets compared with the photooxidized group at 1-year retrieval. Elastin content assay presented the conduit walls of PCA group had less elastin content at preimplantation, but similar content at 1-year retrieval compared with the photooxidized group. Phospholipid analysis showed phospholipid extraction by ethanol for the PCA group was more efficacious than the photooxidized group. These results indicate that PCA BJVCs resist calcification in right-side heart implantation owing to decellularization, further photooxidative crosslinking, and subsequent phospholipid extraction by ethanol at preimplantation.

Analysis of receptor tyrosine kinase ROS1-positive tumors in non-small cell lung cancer: identification of a FIG-ROS1 fusion.

PURPOSE: To deepen our understanding of mutant ROS1 expression, localization, and frequency in non-small cell lung cancer (NSCLC), we developed a highly specific and sensitive immunohistochemistry (IHC)-based assay that is useful for the detection of wild-type and mutant ROS1. EXPERIMENTAL DESIGN: We analyzed 556 tumors with the ROS1 D4D6 rabbit monoclonal antibody IHC assay to assess ROS1 expression levels and localization. A subset of tumors was analyzed by FISH to determine the percentage of these tumors harboring ROS1 translocations. Using specific and sensitive IHC assays, we analyzed the expression of anaplastic lymphoma kinase (ALK), EGFR L858R, and EGFR E746-A750del mutations in a subset of lung tumors, including those expressing ROS1. RESULTS: In our NSCLC cohort of Chinese patients, we identified 9 (1.6%) tumors expressing ROS1 and 22 (4.0%) tumors expressing ALK. FISH identified tumors with ALK or ROS1 rearrangements, and IHC alone was capable of detecting all cases with ALK and ROS1 rearrangements. ROS1 fusion partners were determined by reverse transcriptase PCR identifying CD74-ROS1, SLC34A2-ROS1, and FIG-ROS1 fusions. Some of the ALK and ROS1 rearranged tumors may also harbor coexisting EGFR mutations. CONCLUSIONS: NSCLC tumors with ROS1 rearrangements are uncommon in the Chinese population and represent a distinct entity of carcinomas. The ROS1 IHC assay described here is a valuable tool for identifying patients expressing mutant ROS1 and could be routinely applied in clinical practice to detect lung cancers that may be responsive to targeted therapies.