Protein Phosphatase 1 Beta is Modulated by Chronic Hypoxia and Involved in the Angiogenic Endothelial Cell Migration.

BACKGROUND/AIM: Endothelial cell migration is required for physiological angiogenesis, but also contributes to various pathological conditions, including tumour vascularization. The mRNA expression of PP1cß, the beta isoform of the catalytic PP1 subunit, was shown to be upregulated in chronic hypoxia. Since hypoxia is a major regulator of angiogenesis, the potential role of PP1cß in angiogenesis was investigated. METHODS: We examined PP1cß protein level in pediatric heart following chronic hypoxia and found PP1cß upregulation in cyanotic compared with acyanotic myocardium. By treating HUVEC cells with hypoxia mimicking agent, PP1cß protein level increased with maximum at 8 hours. The effect of PP1cß pharmacological inhibition, knockdown and overexpression, on endothelial cell migration and morphogenesis, was examined using in vitro wound healing scratch assay and endothelial tube formation assay. The PP1cß knockdown effects on F-actin reorganization (phalloidin staining), focal adhesion formation (vinculin) and focal adhesion kinases (FAK) activation, were evaluated by immunocytochemical staining and immunoblotting with specific antibodies. RESULTS: PP1cß knockdown significantly reduces endothelial cell migration, but does not have any significant effect on endothelial tube formation. Endothelial cell migration in the knockdown group is restored to the control level upon consecutive transfection with PP1cß cDNA. PP1cß overexpression does not significantly affect endothelial cell migration. Furthermore, PP1cß knockdown induces profound cytoskeletal reorganization, loss of focal adhesion sites and impairment of focal adhesion kinases (FAK) activation. CONCLUSIONS: PP1cß is regulator of endothelial cell migration, which is critical in the angiogenic process. PP1cß inhibition reduces endothelial cell migration through focal adhesion turnover and actin polymerization pathways.

Changes in renal medulla gene expression in a pre-clinical model of post cardiopulmonary bypass acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) is a common and serious complication of cardiac surgery using cardiopulmonary bypass (CPB). The pathogenesis is poorly understood and the study of AKI in rodent models has not led to improvements in clinical outcomes. We sought to determine the changes in renal medullary gene expression in a novel and clinically relevant porcine model of CPB-induced AKI. RESULTS: Adult pigs (n = 12 per group) were randomised to undergo sham procedure, or 2.5 hours CPB. AKI was determined using biochemical (Cr51 EDTA clearance, CrCl, urinary IL-18 release) and histological measures. Transcriptomic analyses were performed on renal medulla biopsies obtained 24 hours post intervention or from sham group. Microarray results were validated with real-time polymerase chain reaction and Western Blotting.Of the transcripts examined, 66 were identified as differentially expressed in CPB versus Sham pig's kidney samples, with 19 (29%) upregulated and 47 (71%) down-regulated. Out of the upregulated and downregulated transcripts 4 and 16 respectively were expression sequence tags (EST). The regulated genes clustered into three classes; Immune response, Cell adhesion/extracellular matrix and metabolic process. Upregulated genes included Factor V, SLC16A3 and CKMT2 whereas downregulated genes included GST, CPE, MMP7 and SELL. CONCLUSION: Post CPB AKI, as defined by clinical criteria, is characterised by molecular changes in renal medulla that are associated with both injury and survival programmes. Our observations highlight the value of large animal models in AKI research and provide insights into the failure of findings in rodent models to translate into clinical progress.

Growth capacity of a Wharton's Jelly derived mesenchymal stromal cells tissue engineered vascular graft used for main pulmonary artery reconstruction in piglets.

Background: Surgical treatment of congenital heart defects affecting the right ventricular outflow tract (RVOT) often requires complex reconstruction and multiple reoperations due to structural degeneration and lack of growth of currently available materials. Hence, alternative approaches for RVOT reconstruction, which meet the requirements of biocompatibility and long-term durability of an ideal scaffold, are needed. Through this full scale pre-clinical study, we demonstrated the growth capacity of a Wharton's Jelly derived mesenchymal stromal cells (WJ-MSC) tissue engineered vascular graft used in reconstructing the main pulmonary artery in piglets, providing proof of biocompatibility and efficacy. Methods: Sixteen four-week-old Landrace pigs were randomized to undergo supravalvar Main Pulmonary Artery (MPA) replacement with either unseeded or WJ-MSCs-seeded Small Intestinal Submucosa-derived grafts. Animals were followed up for 6 months by clinical examinations and cardiac imaging. At termination, sections of MPAs were assessed by macroscopic inspection, histology and fluorescent immunohistochemistry. Results: Data collected at 6 months follow up showed no sign of graft thrombosis or calcification. The explanted main pulmonary arteries demonstrated a significantly higher degree of cellular organization and elastin content in the WJ-MSCs seeded grafts compared to the acellular counterparts. Transthoracic echocardiography and cardiovascular magnetic resonance confirmed the superior growth and remodelling of the WJ-MSCs seeded conduit compared to the unseeded. Conclusion: Our findings indicate that the addition of WJ-MSCs to the acellular scaffold can upgrade the material, converting it into a biologically active tissue, with the potential to grow, repair and remodel the RVOT.

Protocol to decellularize porcine right ventricular outflow tracts using a 3D printed flow chamber.

Surgical treatment of pediatric congenital heart disease with tissue grafts is a lifesaving intervention. Decellularization to reduce immunogenicity of tissue grafts is an increasingly popular alternative to glutaraldehyde fixation. Here, we present a protocol to decellularize porcine right ventricular outflow tracts using a 3D printed flow chamber. We describe steps for 3D printing the flow rig, preparing porcine tissue, and using the flow rig to utilize shear forces for decellularization. We then detail procedures for characterizing the acellular scaffold. For complete details on the use and execution of this protocol, please refer to Vafaee et al.1.

Biological Scaffolds for Congenital Heart Disease.

Congenital heart disease (CHD) is the most predominant birth defect and can require several invasive surgeries throughout childhood. The absence of materials with growth and remodelling potential is a limitation of currently used prosthetics in cardiovascular surgery, as well as their susceptibility to calcification. The field of tissue engineering has emerged as a regenerative medicine approach aiming to develop durable scaffolds possessing the ability to grow and remodel upon implantation into the defective hearts of babies and children with CHD. Though tissue engineering has produced several synthetic scaffolds, most of them failed to be successfully translated in this life-endangering clinical scenario, and currently, biological scaffolds are the most extensively used. This review aims to thoroughly summarise the existing biological scaffolds for the treatment of paediatric CHD, categorised as homografts and xenografts, and present the preclinical and clinical studies. Fixation as well as techniques of decellularisation will be reported, highlighting the importance of these approaches for the successful implantation of biological scaffolds that avoid prosthetic rejection. Additionally, cardiac scaffolds for paediatric CHD can be implanted as acellular prostheses, or recellularised before implantation, and cellularisation techniques will be extensively discussed.

The effect of cardioplegic supplementation with sildenafil on cardiac energetics in a piglet model of cardiopulmonary bypass and cardioplegic arrest with warm or cold cardioplegia.

Cardioplegic cardioprotection strategies used during paediatric open-heart surgery remain suboptimal. Sildenafil, a phosphodiesterase 5 (PDE-5) inhibitor, has been shown to be cardioprotective against ischemia/reperfusion injury in a variety of experimental models and this study therefore tested the efficacy of supplementation of cardioplegia with sildenafil in a piglet model of cardiopulmonary bypass and arrest, using both cold and warm cardioplegia protocols. Piglets were anaesthetized and placed on coronary pulmonary bypass (CPB), the aorta cross-clamped and the hearts arrested for 60 min with cardioplegia with or without sildenafil (10 nM). Twenty minutes after removal of cross clamp (reperfusion), attempts were made to wean the pigs from CPB. Termination was carried out after 60 min reperfusion. Throughout the protocol blood and left ventricular tissue samples were taken for analysis of selected metabolites (using HPLC) and troponin I. In both the cold and warm cardioplegia protocols there was evidence that sildenafil supplementation resulted in faster recovery of ATP levels, improved energy charge (a measure of metabolic flux) and altered release of hypoxanthine and inosine, two purine catabolites. There was no effect on troponin release within the studied short timeframe. In conclusion, sildenafil supplementation of cardioplegia resulted in improved cardiac energetics in a translational animal model of paediatric CPB surgery.

Warm versus cold blood cardioplegia in paediatric congenital heart surgery: a randomized trial.

OBJECTIVES: Intermittent cold blood cardioplegia is commonly used in children, whereas intermittent warm blood cardioplegia is widely used in adults. We aimed to compare clinical and biochemical outcomes with these 2 methods. METHODS: A single-centre, randomized controlled trial was conducted to compare the effectiveness of warm (≥34°C) versus cold (4-6°C) antegrade cardioplegia in children. The primary outcome was cardiac troponin T over the 1st 48 postoperative hours. Intensive care teams were blinded to group allocation. Outcomes were compared by intention-to-treat using linear mixed-effects, logistic or Cox regression. RESULTS: 97 participants with median age of 1.2 years were randomized (49 to warm, 48 to cold cardioplegia); 59 participants (61%) had a risk-adjusted congenital heart surgery score of 3 or above. There were no deaths and 92 participants were followed to 3-months. Troponin release was similar in both groups [geometric mean ratio 1.07; 95% confidence interval (CI) 0.79-1.44; P = 0.66], as were other cardiac function measures (echocardiography, arterial and venous blood gases, vasoactive-inotrope score, arrhythmias). Intensive care stay was on average 14.6 h longer in the warm group (hazard ratio 0.52; 95% CI 0.34-0.79; P = 0.003), with a trend towards longer overall hospital stays (hazard ratio 0.66; 95% CI 0.43-1.02; P = 0.060) compared with the cold group. This could be related to more unplanned reoperations on bypass in the warm group compared to cold group (3 vs 1). CONCLUSIONS: Warm blood cardioplegia is a safe and reproducible technique but does not provide superior myocardial protection in paediatric heart surgery.

Controlled reoxygenation cardiopulmonary bypass is associated with reduced transcriptomic changes in cyanotic tetralogy of Fallot patients undergoing surgery.

In cyanotic patients undergoing repair of heart defects, high level of oxygen during cardiopulmonary bypass (CPB) leads to greater susceptibility to myocardial ischemia and reoxygenation injury. This study investigates the effects of controlled reoxygenation CPB on gene expression changes in cyanotic hearts of patients undergoing surgical correction of tetralogy of Fallot (TOF). We randomized 49 cyanotic TOF patients undergoing corrective cardiac surgery to receive either controlled reoxygenation or hyperoxic/standard CPB. Ventricular myocardium biopsies were obtained immediately after starting and before discontinuing CPB. Microarray analyses were performed on samples, and array results validated with real-time PCR. Gene expression profiles before and after hyperoxic/standard CPB revealed 35 differentially expressed genes with three upregulated and 32 downregulated. Upregulated genes included two E3 Ubiquitin ligases. The products of downregulated genes included intracellular signaling kinases, metabolic process proteins, and transport factors. In contrast, gene expression profiles before and after controlled reoxygenation CPB revealed only 11 differentially expressed genes with 10 upregulated including extracellular matrix proteins, transport factors, and one downregulated. The comparison of gene expression following hyperoxic/standard vs. controlled reoxygenation CPB revealed 59 differentially expressed genes, with six upregulated and 53 downregulated. Upregulated genes included PDE1A, MOSC1, and CRIP3. Downregulated genes functionally clustered into four major classes: extracellular matrix/cell adhesion, transcription, transport, and cellular metabolic process. This study provides direct evidence that hyperoxic CPB decreases the adaptation and remodeling capacity in cyanotic patients undergoing TOF repair. This simple CPB strategy of controlled reoxygenation reduced the number of genes whose expression was altered following hyperoxic/standard CPB.

Wharton's Jelly-Mesenchymal Stem Cell-Engineered Conduit for Pulmonary Artery Reconstruction in Growing Piglets.

Surgical treatment of congenital heart defects affecting the right ventricular outflow tract often requires complex reconstruction and multiple reoperations. With a randomized controlled trial, we compared a novel tissue-engineered small intestine submucosa-based graft for pulmonary artery reconstruction (seeded with mesenchymal stem cells derived from Wharton's Jelly) with conventional small intestine submucosa in growing piglets. Six months after implantation, seeded grafts showed integration with host tissues at cellular level and exhibited growth potential on transthoracic echocardiography and cardiovascular magnetic resonance. Our seeded graft is a promising biomaterial for pulmonary artery reconstruction in pediatric patients with right ventricular outflow tract abnormalities.

Reconstruction of the Swine Pulmonary Artery Using a Graft Engineered With Syngeneic Cardiac Pericytes.

The neonatal heart represents an attractive source of regenerative cells. Here, we report the results of a randomized, controlled, investigator-blinded preclinical study, which assessed the safety and effectiveness of a matrix graft cellularized with cardiac pericytes (CPs) in a piglet model of pulmonary artery (PA) reconstruction. Within each of five trios formed by 4-week-old female littermate piglets, one element (the donor) was sacrificed to provide a source of CPs, while the other two elements (the graft recipients) were allowed to reach the age of 10 weeks. During this time interval, culture-expanded donor CPs were seeded onto swine small intestinal submucosa (SIS) grafts, which were then shaped into conduits and conditioned in a flow bioreactor. Control unseeded SIS conduits were subjected to the same procedure. Then, recipient piglets were randomized to surgical reconstruction of the left PA (LPA) with unseeded or CP-seeded SIS conduits. Doppler echocardiography and cardiac magnetic resonance imaging (CMRI) were performed at baseline and 4-months post-implantation. Vascular explants were examined using histology and immunohistochemistry. All animals completed the scheduled follow-up. No group difference was observed in baseline imaging data. The final Doppler assessment showed that the LPA's blood flow velocity was similar in the treatment groups. CMRI revealed a mismatch in the average growth of the grafted LPA and contralateral branch in both treatment groups. Histology of explanted arteries demonstrated that the CP-seeded grafts had a thicker luminal cell layer, more intraparietal arterioles, and a higher expression of endothelial nitric oxide synthase (eNOS) compared with unseeded grafts. Moreover, the LPA stump adjacent to the seeded graft contained more elastin and less collagen than the unseeded control. Syngeneic CP engineering did not accomplish the primary goal of supporting the graft's growth but was able to improve secondary outcomes, such as the luminal cellularization and intraparietal vascularization of the graft, and elastic remodeling of the recipient artery. The beneficial properties of neonatal CPs may be considered in future bioengineering applications aiming to reproduce the cellular composition of native arteries.

Wharton's Jelly-Mesenchymal Stem Cell-Engineered Conduit for Pediatric Translation in Heart Defect.

The materials available for the right ventricular outflow tract (RVOT) reconstruction in patients with tetralogy of fallot (TOF)/pulmonary atresia come with the severe limitation of long-term degeneration and lack of growth potential, causing right ventricular dysfunction, aneurysm formation, and arrhythmias, thus necessitating several high-risk reoperations throughout patients' lives. In this study, we evaluated the capacity of mesenchymal stem cells (MSCs) derived from the Wharton's Jelly (WJ-MSCs), the gelatinous inner portion of the umbilical cord, to grow and recellularize an extracellular matrix (ECM) graft in our optimized xeno-free, good manufacturing practice-compliant culture system. WJ-MSCs were phenotypically and functionally characterized by flow cytometry and multilineage differentiation capacity, respectively. The typical MSC immunophenotype and functional characteristics were retained in our xeno-free culture system, as well as the capacity to grow and engraft onto a naturally occurring scaffold. WJ-MSCs, from both human and swine source, showed excellent capacity to recellularize ECM graft producing a living cell-seeded construct. In addition, we have provided an in vivo proof of concept of feasibility of the cellularized conduit, engineered with swine WJ-MSCs, to be used in a novel porcine model of main pulmonary artery reconstruction, where it showed good integration within the host tissue. Our study indicates that the addition of WJ-MSCs to the ECM scaffold can upgrade the material, converting it into a living tissue, with the potential to grow, repair, and remodel the RVOT. These results could potentially represent a paradigm shift in pediatric cardiac intervention toward new modalities for effective and personalized surgical restoration of pulmonary artery and RVOT function in TOF/pulmonary atresia patients. Impact Statement The materials available for pulmonary artery reconstruction in pediatric patients with Congenital Heart Defect come with the limitation of long-term degeneration and lack of growth, thus necessitating several reoperations. Here, we describe a novel approach combining perinatal stem cells and naturally occurring scaffold to create a living tissue engineered conduit that showed good growth potential in a pulmonary artery reconstruction porcine model. We envision this approach is of great interest and relevance in tissue engineering field applied to cardiovascular regenerative medicine, as it may open up new avenues for correction of congenital cardiac defects, with remarkable medical and social benefits.

Off-pump coronary artery bypass surgery is associated with fewer gene expression changes in the human myocardium in comparison with on-pump surgery.

Off-pump coronary artery bypass surgery reduces the myocardial injury associated with on pump surgery with cardiopulmonary bypass (CPB) and ischemic-cardioplegic arrest (CA). We sought to find a mechanistic explanation for this by comparing the transcriptomic changes in the myocardium of patients undergoing on- and off-pump surgery. Transcriptomic analyses were performed on left ventricular biopsies obtained from patients prior to (pre-op) and after completion of all coronary anastomoses (post-op). Microarray results were validated with real-time polymerase chain reaction. In on-pump group, 68 genes were upregulated in post-op vs. pre-op biopsies (P < 0.01, >or=2-fold). They included inflammatory genes CCL3 and CCL4, apoptotic gene GADD45B and prostaglandin synthesis gene PTGS2 (COX-2). In the off-pump group, 17 genes were upregulated in post-op vs. pre-op biopsies (P < 0.01, >or=2-fold), all shared with on-pump patients. To uncover the genes implicated in CPB and ischemic-CA response, we compared the postoperative gene profiles of the two groups. Thirty-eight genes were upregulated in the on-pump vs. off-pump patients (P < 0.01, >or=2-fold). On-pump surgery induces injury-related response, as demonstrated by the upregulation of apoptosis and remodeling markers, whereas off-pump surgery ameliorates that by mainly upregulating a cytoprotective genetic program. Blood levels of the identified cytokines and chemokines followed the same pattern obtained by transcriptomics, suggesting that the myocardium is a likely source for these proteomic changes. In conclusion, off-pump surgery is associated with fewer alterations in gene expression connected with inflammation, apoptosis, and remodeling seen after on-pump surgery with CPB and ischemic-CA.

Changes in inflammation and oxidative stress signalling pathways in coarcted aorta triggered by bicuspid aortic valve and growth in young children.

Neonates with coarctation of the aorta (CoA) combined with a bicuspid aortic valve (BAV) show significant structural differences compared to neonatal CoA patients with a normal tricuspid aortic valve (TAV). These effects are likely to change over time in response to growth. This study investigated proteomic differences between coarcted aortic tissue of BAV and TAV patients in children older than one month. Aortic tissue just proximal to the coarctation site was collected from 10 children (BAV; n=6, 1.9±1.7 years, TAV; n=4, 1.7±1.5 years, (mean ± SEM, P=0.92.) Tissue were snap frozen, proteins extracted, and the extracts used for proteomic and phosphoproteomic analysis using Tandem Mass Tag (TMT) analysis. A total of 1811 protein and 76 phosphoprotein accession numbers were detected, of which 40 proteins and 6 phosphoproteins were significantly differentially expressed between BAV and TAV patients. Several canonical pathways involved in inflammation demonstrated enriched protein expression, including acute phase response signalling, EIF2 signalling and macrophage production of IL12 and reactive oxygen species. Acute phase response signalling also demonstrated enriched phosphoprotein expression, as did Th17 activation. Other pathways with significantly enriched protein expression include degradation of superoxide radicals and several pathways involved in apoptosis. This work suggests that BAV CoA patients older than one month have an altered proteome consistent with changes in inflammation, apoptosis and oxidative stress compared to TAV CoA patients of the same age. There is no evidence of structural differences, suggesting the pathology associated with BAV evolves with age in paediatric CoA patients.

Amnion-Based Scaffold with Enhanced Strength and Biocompatibility for In Vivo Vascular Repair.

IMPACT STATEMENT: This study aimed at developing an amnion-based scaffold suitable for vascular tissue engineering applications and in vivo usage. We successfully produced a multilayered scaffold with improved biomechanical properties and biocompatibility for in vivo vascular implantation. Our approach not only offers an allogeneic "off-the-shelf" solution for clinical use but also it provides the possibility of personalized medicine using a patient's own amnion and stem cells for the production of tissue engineered grafts for reconstructive heart surgery.

Successful Reconstruction of the Right Ventricular Outflow Tract by Implantation of Thymus Stem Cell Engineered Graft in Growing Swine.

Graft cellularization holds great promise in overcoming the limitations associated with prosthetic materials currently used in corrective cardiac surgery. In this study, the authors evaluated the advantages of graft cellularization for right ventricular outflow tract reconstruction in a novel porcine model. After 4.5 months from implantation, improved myocardial strain, better endothelialization and cardiomyocyte incorporation, and reduced fibrosis were observed in the cellularized grafts compared with the acellular grafts. To the authors' knowledge, this is the first demonstration of successful right ventricular outflow tract correction using bioengineered grafts in a large animal model.

Reconstruction of the pulmonary artery by a novel biodegradable conduit engineered with perinatal stem cell-derived vascular smooth muscle cells enables physiological vascular growth in a large animal model of congenital heart disease.

Lack of growth potential of available grafts represents a bottleneck in the correction of congenital heart defects. Here we used a swine small intestinal submucosa (SIS) graft functionalized with mesenchymal stem cell (MSC)-derived vascular smooth muscle cells (VSMCs), for replacement of the pulmonary artery in piglets. MSCs were expanded from human umbilical cord blood or new-born swine peripheral blood, seeded onto decellularized SIS grafts and conditioned in a bioreactor to differentiate into VSMCs. Results indicate the equivalence of generating grafts engineered with human or swine MSC-derived VSMCs. Next, we conducted a randomized, controlled study in piglets (12-15 kg), which had the left pulmonary artery reconstructed with swine VSMC-engineered or acellular conduit grafts. Piglets recovered well from surgery, with no casualty and similar growth rate in either group. After 6 months, grafted arteries had larger circumference in the cellular group (28.3 ±â€¯2.3 vs 18.3 ±â€¯2.1 mm, P < 0.001), but without evidence of aneurism formation. Immunohistochemistry showed engineered grafts were composed of homogeneous endothelium covered by multi-layered muscular media, whereas the acellular grafts exhibited a patchy endothelial cell layer and a thinner muscular layer. RESULTS: show the feasibility and efficacy of pulmonary artery reconstruction using clinically available grafts engineered with allogeneic VSMCs in growing swine.

Thymus-Derived Mesenchymal Stem Cells for Tissue Engineering Clinical-Grade Cardiovascular Grafts.

Mesenchymal stem cells (MSCs) are attractive tools for regenerative medicine because of their multidifferentiation potential and immunomodulation capacity. In congenital heart defect surgical correction, replacement grafts lacking growth potential are commonly used. Tissue engineering promises to overcome the limitations of these grafts. In this study, we hypothesized that human thymus-derived MSCs are a suitable tool to tissue engineer a living vascular graft with good integration and patency once implanted in vivo. Human thymus-derived MSCs (hT-MSCs) were identified by the expression of MSC markers and mesenchymal differentiation potential. When cultured onto natural scaffold to produce tissue-engineered graft, hT-MSCs exhibited great proliferation potential and the ability to secrete their own extracellular matrix. In addition, when implanted in vivo in a piglet model of left pulmonary grafting, the engineered graft exhibited good integration within the host tissue, indicating potential suitability for corrective cardiovascular surgery. The optimized xeno-free, good manufacturing practices-compliant culture system proved to be optimum for large-scale expansion of hT-MSCs and production of tissue-engineered cardiovascular grafts, without compromising the quality of cells. This study demonstrated the feasibility of engineering clinical-grade living autologous replacement grafts using hT-MSCs and proved the compatibility of these grafts for in vivo implantation in a left pulmonary artery position.

Perivascular cells and tissue engineering: Current applications and untapped potential.

The recent development of tissue engineering provides exciting new perspectives for the replacement of failing organs and the repair of damaged tissues. Perivascular cells, including vascular smooth muscle cells, pericytes and other tissue specific populations residing around blood vessels, have been isolated from many organs and are known to participate to the in situ repair process and angiogenesis. Their potential has been harnessed for cell therapy of numerous pathologies; however, in this Review we will discuss the potential of perivascular cells in the development of tissue engineering solutions for healthcare. We will examine their application in the engineering of vascular grafts, cardiac patches and bone substitutes as well as other tissue engineering applications and we will focus on their extensive use in the vascularization of engineered constructs. Additionally, we will discuss the emerging potential of human pericytes for the development of efficient, vascularized and non-immunogenic engineered constructs.

Microarray screening of suppression subtractive hybridization-PCR cDNA libraries identifies novel RNAs regulated by dehydration in the rat supraoptic nucleus.

The magnocellular neurons (MCNs) of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus are the principal site of biosynthesis of prepropeptide precursor of the antidiuretic hormone vasopressin (VP). This precursor is processed during anterograde axonal transportation to terminals in the posterior pituitary gland, where biologically active VP is stored until release into the general circulation in response to physiological activation of the SON by osmotic cues. By binding to V2-type receptors located in the kidney, VP decreases the amount of water lost in urine. Osmotic activation of the SON is accompanied by a dramatic morphological and functional remodeling. We have sought to understand the mechanistic basis of this plasticity in terms of the differential expression of genes. To identify such genes, we adopted an unbiased global approach based on suppressive subtractive hybridization-polymerase chain reaction (SSH-PCR) Using this method, we generated libraries of clones putatively differentially expressed in control vs. dehydrated SON. To rapidly screen these libraries, 1,152 clones were subjected to microarray analysis, resulting in the identification of 459 differentially expressed transcripts. cDNA clones corresponding to 56 of these RNAs were sequenced, revealing many of them to be novel expressed sequence tags (ESTs). Four transcripts were shown by in situ hybridization (ISH) to be significantly up- or downregulated in the SON after dehydration. These genes may represent novel effectors or mediators of SON physiological remodeling.

Chronic hypoxia down-regulates tight junction protein ZO-2 expression in children with cyanotic congenital heart defect.

AIMS: Tight junction protein zonula occludens protein 2 (ZO-2) is a member of the membrane-associated guanylate kinases protein family known to be expressed at tight junctions of epithelial and endothelial cells and at adherens junctions (AJs) in cardiomyocytes. Little is known about ZO-2 expression and function in the human heart. Here, we examined the hypothesis that chronic hypoxia down-regulates ZO-2 expression in human myocardium and cultured rat cardiomyocytes. METHODS AND RESULTS: Patients with a diagnosis of cyanotic (n = 10) or acyanotic (n = 10) Tetralogy of Fallot undergoing surgical repair were used to examine ZO-2 messenger RNA and protein expression by real time-PCR, immunohistochemistry, and western blotting. A model of cultured rat cardiomyocytes was used to measure ZO-2 and AJ proteins levels in response to hypoxia and to investigate ZO-2 cellular localization. We showed that ZO-2 is expressed in myocardial tissue in acyanotic and cyanotic children with congenital heart defects. ZO-2 was specifically down-regulated in cyanotic myocardium at both the messenger RNA and protein levels when compared with acyanotic patients. This specific down-regulation can be mimicked in cultured rat cardiomyocytes by treating them with hypoxic conditions confirming that ZO-2 gene down-regulation is specifically due to cyanosis. Furthermore, in addition to its cytoplasmic expression, ZO-2 showed nuclear expression in cultured rat cardiomyocytes suggesting potential role in transcription regulation. CONCLUSIONS: Hypoxia down-regulates ZO-2 expression in both cyanotic patient's myocardium and cultured rat cardiomyocytes. This down-regulation suggest an involvement of ZO-2 in cardiac remodelling of AJs in cyanotic children and may explain the greater susceptibility of cyanotic patients to corrective heart surgery.