Avoiding false discovery in biomarker research.

BACKGROUND: Human tyrosine-protein phosphatase non-receptor type substrate 1α (SIRPA) is a surface marker identified in cardiomyocytes differentiated from human embryonic stem cells. Our objective was to determine if circulating SIRPA levels can serve as a biomarker of cardiac injury in children undergoing open heart surgery. RESULTS: Paired pre- and post-operative serum samples from 48 pediatric patients undergoing open heart surgery and from 6 pediatric patients undergoing non-cardiac surgery (controls) were tested for SIRPA protein levels using commercially available SIRPA ELISA kits from two manufacturers. Post-operative SIRPA concentrations were significantly higher in patients after cardiac surgery compared to non-cardiac surgery when tested using SIRPA ELISA kits from both manufacturers. To verify the identity of the protein detected, recombinant human SIRPA protein (rhSIRPA) was tested on both ELISA kits. The calibrator from both ELISA kits was analyzed by Western blot as well as by Mass Spectrometry (MS). Western blot analysis of calibrators from both kits did not identity SIRPA. MS analysis of calibrators from both ELISA kits identified several inflammatory markers and albumin but no SIRPA was detected. CONCLUSIONS: We conclude that commercially available ELISA kits for SIRPA give false-positive results. Verifying protein identity using robust protein characterization is critical to avoid false biomarker discovery when using commercial ELISA kits.

Fetal reprogramming and senescence in hypoplastic left heart syndrome and in human pluripotent stem cells during cardiac differentiation.

Hypoplastic left heart syndrome (HLHS) is a severe cardiac malformation characterized by left ventricle (LV) hypoplasia and abnormal LV perfusion and oxygenation. We studied hypoxia-associated injury in fetal HLHS and human pluripotent stem cells during cardiac differentiation to assess the effect of microenvironmental perturbations on fetal cardiac reprogramming. We studied LV myocardial samples from 32 HLHS and 17 structurally normal midgestation fetuses. Compared with controls, the LV in fetal HLHS samples had higher nuclear expression of hypoxia-inducible factor-1α but lower angiogenic growth factor expression, higher expression of oncogenes and transforming growth factor (TGF)-ß1, more DNA damage and senescence with cell cycle arrest, fewer cardiac progenitors, myocytes and endothelial lineages, and increased myofibroblast population (P < 0.05 versus controls). Smooth muscle cells (SMCs) had less DNA damage compared with endothelial cells and myocytes. We recapitulated the fetal phenotype by subjecting human pluripotent stem cells to hypoxia during cardiac differentiation. DNA damage was prevented by treatment with a TGF-ß1 inhibitor (P < 0.05 versus nonhypoxic cells). The hypoplastic LV in fetal HLHS samples demonstrates hypoxia-inducible factor-1α up-regulation, oncogene-associated cellular senescence, TGF-ß1-associated fibrosis and impaired vasculogenesis. The phenotype is recapitulated by subjecting human pluripotent stem cells to hypoxia during cardiac differentiation and rescued by inhibition of TGF-ß1. This finding suggests that hypoxia may reprogram the immature heart and affect differentiation and development.

The rationale and design of Insight into Nephrotic Syndrome: Investigating Genes, Health and Therapeutics (INSIGHT): a prospective cohort study of childhood nephrotic syndrome.

BACKGROUND: Nephrotic syndrome is one of the most commonly diagnosed kidney diseases in childhood and its progressive forms can lead to chronic kidney disease (CKD) and/or end-stage renal disease (ESRD). There have been few longitudinal studies among a multi-ethnic cohort to determine potential risk factors influencing disease susceptibility, treatment response, and progression of nephrotic syndrome. Temporal relationships cannot be studied through cross-sectional study design. Understanding the interaction between various factors is critical to developing new strategies for treating children with kidney disease. We present the rationale and the study design of a longitudinal cohort study of children with nephrotic syndrome, the Insight into Nephrotic Syndrome: Investigating Genes, Health and Therapeutics (INSIGHT) study. The specific aims are to determine: 1) socio-demographic, environmental, and genetic factors that influence disease susceptibility; 2) rates of steroid treatment resistance and steroid treatment dependence, and identify factors that may modify treatment response; 3) clinical and genetic factors that influence disease susceptibility and progression to CKD and ESRD; and 4) the interaction between the course of illness and socio-demographic, environmental, and clinical risk factors. METHODS/DESIGN: INSIGHT is a disease-based observational longitudinal cohort study of children with nephrotic syndrome. At baseline, participants complete questionnaires and provide biological specimen samples (blood, urine, and toenail clippings). Follow-up questionnaires and repeat biological specimen collections are performed annually for up to five years. DISCUSSION: The proposed cohort will provide the structure to test various risk factors predicting or influencing disease susceptibility, treatment response, and progression to CKD among children with nephrotic syndrome. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT01605266.

Stem cells in pediatric cardiology.

The ability to reprogram virtually any cell of human origin to behave like embryonic or pluripotent stem cells is a major breakthrough in stem cell biology. Human induced pluripotent stem cells (iPSC) provide a unique opportunity to study "disease in a dish" within a defined genetic and environmental background. Patient-derived iPSCs have been successfully used to model cardiomyopathies, rhythm disorders and vascular disorders. They also provide an exciting opportunity for drug discovery and drug repurposing for disorders with a known molecular basis including childhood onset heart disease, particularly cardiac genetic disorders. The review will discuss their use in drug discovery, efficacy and toxicity studies with emphasis on challenges in pediatric-focused drug discovery. Issues that will need to be addressed in the coming years include development of maturation protocols for iPSC-derived cardiac lineages, use of iPSCs to study not just cardiac but extra-cardiac phenotypes in the same patient, scaling up of stem cell platforms for high-throughput drug screens, translating drug testing results to clinical applications in the paradigm of personalized medicine, and improving both the efficiency and the safety of iPSC-derived lineages for future stem cell therapies.

Platelet derived growth factor B and epithelial mesenchymal transition of peritoneal mesothelial cells.

Platelet derived growth factor (PDGF) is involved in wound healing in various organ systems. Its potential role in the context of peritoneal injury following long-term peritoneal dialysis is unclear. We used an adenovirus expressing the B chain of PDGF (AdPDGF-B) to assess its effect on pro-fibrotic pathways in the peritoneal membrane. To assess the transforming growth factor (TGF) beta independent effects of PDGF, we over-expressed PDGF-B in the peritoneum of either wild-type mice (Smad3+/+) or those with a deletion of the TGFbeta signaling protein Smad3 (Smad3(-/-)). PDGF-B induced sustained angiogenesis in both Smad3+/+ and Smad3(-/-) mice. Despite increased collagen gene expression, collagen accumulation was transient and fibrogenesis was associated with induction of collagenase activity. We observed epithelial to mesenchymal transition (EMT) involving the peritoneal mesothelial cells, as shown by increased SNAIL and decreased E-Cadherin expression with evidence of mesothelial cells expressing both epithelial and mesenchymal markers. Unlike TGFbeta-induced EMT, PDGF-B exposure did not lead to mobilization of the mesothelial cells; they remained as a single monolayer throughout the observation period. This "non-invasive" EMT phenomenon is a novel finding and may have implications concerning the role of EMT in peritoneal fibrosis and injury to other organ systems. The observed effects were similar in Smad3(-/-) and Smad3+/+ animals, suggesting that the PDGF-B effects were independent of TGFbeta or Smad signaling.

Smad3-dependent and -independent pathways are involved in peritoneal membrane injury.

Transition of peritoneal mesothelial cells to a mesenchymal phenotype plays an integral role in the angiogenic and fibrotic changes seen in the peritoneum of patients receiving long-term peritoneal dialysis. While signaling by transforming growth factor (TGF)-beta through Smad proteins likely causes these changes, it is possible that non-Smad pathways may also play a role. Here, we found that Smad3-deficient mice were protected from peritoneal fibrosis and angiogenesis caused by adenovirus-mediated gene transfer of active TGF-beta1 to mesothelial cells; however, mesothelial transition occurred in this setting, suggesting involvement of non-Smad mechanisms. The phosphatidyl inositol 3 kinase (PI3K) target, Akt, was upregulated in both Smad-deficient and wild-type mice after exposure to TGF-beta1. In vivo inhibition of the mammalian target of rapamycin (mTOR) by rapamycin completely abrogated the transition response in Smad3-deficient but not in wild-type mice. Rapamycin blocked nuclear localization of beta-catenin independent of glycogen synthase kinase 3beta activity. Further, in Smad3-deficient mice rapamycin reduced the expression of alpha-smooth muscle actin, which is an epithelial-to-mesenchymal transition-associated gene. Hence, we conclude that TGF-beta1 causes peritoneal injury through Smad-dependent and Smad-independent pathways; the latter involves redundant mechanisms inhibited by rapamycin, suggesting that suppression of both pathways may be necessary to abrogate mesothelial transition.

Peritoneal morphological and functional changes associated with platelet-derived growth factor B.

BACKGROUND: Morphological changes associated with long-term peritoneal dialysis (PD) include increased vascular surface area due to angiogenesis, submesothelial fibrosis and epithelial mesenchymal transition. Platelet-derived growth factor (PDGF) has been associated with all of these phenomena, and is a prototypical 'response to injury' growth factor. METHODS: Rats received an intraperitoneal injection of adenoviral vector expressing PDGF-B. At sacrifice, we analysed the structure and function of the peritoneal membrane. Gene expression in the peritoneal tissue was assessed for changes suggestive of epithelial mesenchymal transition. RESULTS: Over-expression of PDGF in the rat peritoneum led to significant angiogenesis, cellular proliferation and submesothelial thickening. Although PDGF induced expression of transforming growth factor beta, there was a lack of activation of this growth factor, and we believe that this explains the lack of significant collagen accumulation observed by a hydroxyproline assay. Despite evidence of angiogenesis and subsequent increased solute transport, we observed only a transient, non-significant impact on ultrafiltration function. This suggests that increased vascular surface area is necessary, but not sufficient, to produce ultrafiltration dysfunction. There was no evidence of epithelial mesenchymal transition observed either in regulation of associated genes such as Snail or E-Cadherin or in the lack of dual-labelled epithelial and mesenchymal cells on immunofluorescence. Mesothelial cells exposed to PDGF-B demonstrated increased collagen gene expression. CONCLUSIONS: PDGF-B induced angiogenesis without fibrosis in the peritoneum. The lack of significant ultrafiltration dysfunction and epithelial mesenchymal transition, as observed in patients on PD, suggests that PDGF-B may play a role, but is not the integral component, in response to peritoneal injury.