Maria Chiara Magnone

Disease Modeling and Phenotypic Drug Screening for Diabetic Cardiomyopathy using Human Induced Pluripotent Stem Cells

Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.

Sensitive Cardiac Troponin T Assay and the Risk of Incident Cardiovascular Disease in Women With and Without Diabetes Mellitus The Women's Health Study

Background— Very low levels of cardiac troponin T are associated with an increased risk of cardiovascular death in patients with stable chronic coronary disease. Whether high-sensitivity cardiac troponin T levels are associated with adverse cardiovascular outcomes in individuals without cardiovascular disease (CVD) has not been well studied. Methods and Results— Using 2 complementary study designs, we evaluated the relationship between baseline cardiac troponin and incident CVD events among diabetic and nondiabetic participants in the Womens Health Study (median follow-up, 12.3 years). All diabetic women with blood specimens were included in a cohort study (n=512 diabetic women, n=65 events), and nondiabetic women were sampled for inclusion in a case-cohort analysis (n=564 comprising the subcohort, n=479 events). High-sensitivity cardiac troponin T was detectable (≥0.003 &mgr;g/L) in 45.5% of diabetic women and 30.3% of nondiabetic women (P<0.0001). In models adjusted for traditional risk factors and hemoglobin A1c, detectable high-sensitivity cardiac troponin T was associated with subsequent CVD (myocardial infarction, stroke, cardiovascular death) in diabetic women (adjusted hazard ratio, 1.79; 95% confidence interval, 1.04 to 3.07, P=0.036) but not nondiabetic women (adjusted hazard ratio, 1.13; 95% confidence interval, 0.82 to 1.55; P=0.46). Further adjustment for amino-terminal pro-B-type natriuretic peptide and estimated renal function did not substantially alter this relationship among diabetic women (hazard ratio, 1.76; 95% confidence interval, 1.00 to 3.08; P=0.0499), which appeared to be driven by a 3-fold increase in CVD death that was not observed in nondiabetic women. Conclusions— Very low but detectable levels of cardiac troponin T are associated with total CVD and CVD death in women with diabetes mellitus. Among healthy nondiabetic women, detectable compared with undetectable troponin was not associated with CVD events.

Tissue transcriptome-driven identification of epidermal growth factor as a chronic kidney disease biomarker

Renal and urinary EGF can serve as biomarkers for prediction of outcomes in chronic kidney disease. Urine marker to the rescue Chronic kidney disease is a common medical problem worldwide, but it is difficult to predict which patients are more likely to progress to end-stage disease and need aggressive management. Ju et al. have now drawn on four independent cohorts totaling hundreds of patients from around the world to identify the expression of epidermal growth factor (EGF) in the kidneys as a marker of kidney disease progression. Moreover, the authors demonstrated that the amount of EGF in the urine is just as useful, providing a biomarker that can be easily tracked over time without requiring invasive biopsies. Chronic kidney disease (CKD) affects 8 to 16% people worldwide, with an increasing incidence and prevalence of end-stage kidney disease (ESKD). The effective management of CKD is confounded by the inability to identify patients at high risk of progression while in early stages of CKD. To address this challenge, a renal biopsy transcriptome-driven approach was applied to develop noninvasive prognostic biomarkers for CKD progression. Expression of intrarenal transcripts was correlated with the baseline estimated glomerular filtration rate (eGFR) in 261 patients. Proteins encoded by eGFR-associated transcripts were tested in urine for association with renal tissue injury and baseline eGFR. The ability to predict CKD progression, defined as the composite of ESKD or 40% reduction of baseline eGFR, was then determined in three independent CKD cohorts. A panel of intrarenal transcripts, including epidermal growth factor (EGF), a tubule-specific protein critical for cell differentiation and regeneration, predicted eGFR. The amount of EGF protein in urine (uEGF) showed significant correlation (P < 0.001) with intrarenal EGF mRNA, interstitial fibrosis/tubular atrophy, eGFR, and rate of eGFR loss. Prediction of the composite renal end point by age, gender, eGFR, and albuminuria was significantly (P < 0.001) improved by addition of uEGF, with an increase of the C-statistic from 0.75 to 0.87. Outcome predictions were replicated in two independent CKD cohorts. Our approach identified uEGF as an independent risk predictor of CKD progression. Addition of uEGF to standard clinical parameters improved the prediction of disease events in diverse CKD populations with a wide spectrum of causes and stages.

Generation of functional podocytes from human induced pluripotent stem cells

Generating human podocytes in vitro could offer a unique opportunity to study human diseases. Here, we describe a simple and efficient protocol for obtaining functional podocytes in vitro from human induced pluripotent stem cells. Cells were exposed to a three-step protocol, which induced their differentiation into intermediate mesoderm, then into nephron progenitors and, finally, into mature podocytes. After differentiation, cells expressed the main podocyte markers, such as synaptopodin, WT1, α-Actinin-4, P-cadherin and nephrin at the protein and mRNA level, and showed the low proliferation rate typical of mature podocytes. Exposure to Angiotensin II significantly decreased the expression of podocyte genes and cells underwent cytoskeleton rearrangement. Cells were able to internalize albumin and self-assembled into chimeric 3D structures in combination with dissociated embryonic mouse kidney cells. Overall, these findings demonstrate the establishment of a robust protocol that, mimicking developmental stages, makes it possible to derive functional podocytes in vitro.

Prospective Evaluation of B-type Natriuretic Peptide Concentrations and the Risk of Type 2 Diabetes in Women

BACKGROUND Animal data suggest that natriuretic peptides play an important role in energy metabolism, but prospective studies evaluating a relationship between these peptides and type 2 diabetes mellitus (T2DM) in humans are few and results are conflicting. METHODS We used a prospective case-cohort approach (n = 491 T2DM cases, n = 561 reference subcohort) within the Womens Health Study to evaluate baseline N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentrations and the risk of incident T2DM. We also tested for associations between 4 common variants in the natriuretic peptide A and B genes (NPPA and NPPB) and NT-proBNP concentrations (n = 458) and incident T2DM (n = 1372 cases among 22 607 women). RESULTS Case subjects had higher median baseline body mass index (29.4 vs 25.0 kg/m(2), P < 0.001) and lower baseline median (interquartile range) NT-proBNP concentrations [46.8 ng/L (26.1-83.2) vs 66.7 ng/L (39.3-124.7), P < 0.001]. In proportional hazards models adjusting for established diabetes risk factors, women in the highest quartile of baseline NT-proBNP concentration (≥ 117.4 ng/L) had a 49% reduction in risk of T2DM [hazard ratio (HR) 0.51, 0.30-0.86, P = 0.01] relative to those in the lowest quartile. Two of the 4 tested variants in NPPA and NPPB (rs632793, rs198389) were associated with increased NT-proBNP concentrations and reduced risk of T2DM. For example, each copy of the minor allele of rs632793 was associated with increased NT-proBNP [β (SE) = 0.201 (0.063), P < 0.01] and decreased T2DM risk (HR 0.91, 0.84-0.989, P = 0.026). CONCLUSIONS NT-proBNP concentrations that are high, but still within the reference interval, associate with reduced risk of incident diabetes in women and support a favorable role for natriuretic peptides in the prevention of T2DM.

Growth Differentiation Factor-15 and Risk of CKD Progression

Growth differentiation factor-15 (GDF-15) is a member of the TGF-β cytokine superfamily that is widely expressed and may be induced in response to tissue injury. Elevations in GDF-15 may identify a novel pathway involved in loss of kidney function among patients with CKD. Among participants in the Clinical Phenotyping and Resource Biobank (C-PROBE) study and the Seattle Kidney Study (SKS), we tested whether kidney tissue expression of GDF15 mRNA correlates with circulating levels of GDF-15 and whether elevations in circulating GDF-15 are associated with decline in kidney function. In matching samples of 24 patients with CKD from the C-PROBE study, circulating GDF-15 levels significantly correlated with intrarenal GDF15 transcript levels (r=0.54, P=0.01). Among the 224 C-PROBE and 297 SKS participants, 72 (32.1%) and 94 (32.0%) patients, respectively, reached a composite end point of 30% decline in eGFR or progression to ESRD over a median of 1.8 and 2.0 years of follow up, respectively. In multivariable models, after adjusting for potential confounders, every doubling of GDF-15 level associated with a 72% higher (95% confidence interval, 1.21 to 4.45; P=0.003) and 65% higher (95% confidence interval, 1.08 to 2.50; P=0.02) risk of progression of kidney disease in C-PROBE and SKS participants, respectively. These results show that circulating GDF-15 levels strongly correlated with intrarenal expression of GDF15 and significantly associated with increased risk of CKD progression in two independent cohorts. Circulating GDF-15 may be a marker for intrarenal GDF15-related signaling pathways associated with CKD and CKD progression.

HepG2/C3A 3D spheroids exhibit stable physiological functionality for at least 24 days after recovering from trypsinisation

Primary human hepatocytes are widely used as an in vitro system for the assessment of drug metabolism and toxicity. Nevertheless a cell system with higher stability of physiological functions is required for the investigation of drugs’ mode of action, pathway analyses and biomarkers evaluations. We recently discovered that the human hepatocellular carcinoma cell line, HepG2/C3A, cultured as spheroids in a 3D system can recover their main functions after trypsinisation within about 18 days. The objective of this study was to investigate whether the spheroids’ metabolic functions remained stable after this recovery period. Therefore we evaluated physiological capabilities of the spheroids (cell survival, growth rate, glycogenesis, ATP, cholesterol and urea synthesis and drug metabolism) and the expression of key genes related to the main liver pathways in spheroids cultured for an additional 24 days after full recovery (day 18). Here we show that after the recovery period, the 3D spheroid culture can provide a metabolically competent homeostatic cell model which is in equilibrium with its culture environment for more than 3 weeks. Such a stable system could be used for the assessment of the drugs’ mode of action, for biomarkers evaluation and for any systems biology studies which require medium- to long-term stability of metabolic functions.

Short-term Effects of Laparoscopic Adjustable Gastric Banding Versus Roux-en-Y Gastric Bypass

OBJECTIVE Bariatric surgery has been shown to have important long-term metabolic effects resulting in enhanced insulin sensitivity and improved glucose tolerance in patients with type 2 diabetes. The contribution of reduced caloric intake to these beneficial effects of surgery remains unclear. The aim of this study was to compare the short-term effects (1 week) of bariatric surgical procedures with a very low caloric intake (VLCI) on insulin sensitivity (IS) and insulin secretion (ISR) in nondiabetic obese subjects. RESEARCH DESIGN AND METHODS Twenty obese patients without diabetes (BMI 44.2 ± 0.7 kg/m2) were admitted to the clinic for 1 week. At baseline and 1 week after VLCI (600 kcal/day), subjects received a hyperinsulinemic-euglycemic clamp with tracer infusion to quantify endogenous glucose production (EGP), lipolysis (rate of appearance of glycerol [RaGlycerol]), peripheral insulin sensitivity (insulin-stimulated glucose disposal [M value] divided by the steady-state plasma insulin concentration [M/I]), hepatic insulin sensitivity (Hep-IS [= 1/(EGP ⋅ insulin)]), and adipose insulin sensitivity (Adipo-IS [= 1/(RaGlycerol ⋅ insulin)]). An intravenous glucose bolus was administered at the end of the insulin clamp to measure ISR and β-cell function (disposition index [DI]). Approximately 3 months later, patients were admitted for laparoscopic adjustable gastric banding (LAGB) (n = 10) or Roux-en-Y gastric bypass (RYGB) (n = 10), and were restudied 1 week after surgery under the same caloric regimen (600 kcal/day). RESULTS After 1 week of VLCI, patients lost 2.1 kg without significant changes in Hep-IS, Adipo-IS, M/I, or DI. RYGB and LAGB led to greater weight loss (5.5 and 5.2 kg, respectively) and to significant improvement in Hep-IS, EGP, and lipolysis. Only RYGB improved Adipo-IS and M/I. No change in ISR or DI was observed in either surgical group. CONCLUSIONS Bariatric surgery improves IS within 1 week. These metabolic effects were independent of caloric intake and more pronounced after RYGB compared with LAGB.

Sensitive Cardiac Troponin T Assay and the Risk of Incident Cardiovascular Disease in Women With and Without Diabetes Mellitus

Background— Very low levels of cardiac troponin T are associated with an increased risk of cardiovascular death in patients with stable chronic coronary disease. Whether high-sensitivity cardiac troponin T levels are associated with adverse cardiovascular outcomes in individuals without cardiovascular disease (CVD) has not been well studied. Methods and Results— Using 2 complementary study designs, we evaluated the relationship between baseline cardiac troponin and incident CVD events among diabetic and nondiabetic participants in the Womens Health Study (median follow-up, 12.3 years). All diabetic women with blood specimens were included in a cohort study (n=512 diabetic women, n=65 events), and nondiabetic women were sampled for inclusion in a case-cohort analysis (n=564 comprising the subcohort, n=479 events). High-sensitivity cardiac troponin T was detectable (≥0.003 &mgr;g/L) in 45.5% of diabetic women and 30.3% of nondiabetic women (P<0.0001). In models adjusted for traditional risk factors and hemoglobin A1c, detectable high-sensitivity cardiac troponin T was associated with subsequent CVD (myocardial infarction, stroke, cardiovascular death) in diabetic women (adjusted hazard ratio, 1.79; 95% confidence interval, 1.04 to 3.07, P=0.036) but not nondiabetic women (adjusted hazard ratio, 1.13; 95% confidence interval, 0.82 to 1.55; P=0.46). Further adjustment for amino-terminal pro-B-type natriuretic peptide and estimated renal function did not substantially alter this relationship among diabetic women (hazard ratio, 1.76; 95% confidence interval, 1.00 to 3.08; P=0.0499), which appeared to be driven by a 3-fold increase in CVD death that was not observed in nondiabetic women. Conclusions— Very low but detectable levels of cardiac troponin T are associated with total CVD and CVD death in women with diabetes mellitus. Among healthy nondiabetic women, detectable compared with undetectable troponin was not associated with CVD events.

Sensitive Cardiac Troponin T Assay and the Risk of Incident Cardiovascular Disease in Women With and Without Diabetes MellitusClinical Perspective

Background— Very low levels of cardiac troponin T are associated with an increased risk of cardiovascular death in patients with stable chronic coronary disease. Whether high-sensitivity cardiac troponin T levels are associated with adverse cardiovascular outcomes in individuals without cardiovascular disease (CVD) has not been well studied. Methods and Results— Using 2 complementary study designs, we evaluated the relationship between baseline cardiac troponin and incident CVD events among diabetic and nondiabetic participants in the Womens Health Study (median follow-up, 12.3 years). All diabetic women with blood specimens were included in a cohort study (n=512 diabetic women, n=65 events), and nondiabetic women were sampled for inclusion in a case-cohort analysis (n=564 comprising the subcohort, n=479 events). High-sensitivity cardiac troponin T was detectable (≥0.003 &mgr;g/L) in 45.5% of diabetic women and 30.3% of nondiabetic women (P<0.0001). In models adjusted for traditional risk factors and hemoglobin A1c, detectable high-sensitivity cardiac troponin T was associated with subsequent CVD (myocardial infarction, stroke, cardiovascular death) in diabetic women (adjusted hazard ratio, 1.79; 95% confidence interval, 1.04 to 3.07, P=0.036) but not nondiabetic women (adjusted hazard ratio, 1.13; 95% confidence interval, 0.82 to 1.55; P=0.46). Further adjustment for amino-terminal pro-B-type natriuretic peptide and estimated renal function did not substantially alter this relationship among diabetic women (hazard ratio, 1.76; 95% confidence interval, 1.00 to 3.08; P=0.0499), which appeared to be driven by a 3-fold increase in CVD death that was not observed in nondiabetic women. Conclusions— Very low but detectable levels of cardiac troponin T are associated with total CVD and CVD death in women with diabetes mellitus. Among healthy nondiabetic women, detectable compared with undetectable troponin was not associated with CVD events.