High Glucose Levels Promote Switch to Synthetic Vascular Smooth Muscle Cells via Lactate/GPR81.

Hyperglycemia, lipotoxicity, and insulin resistance are known to increase the secretion of extracellular matrix from cardiac fibroblasts as well as the activation of paracrine signaling from cardiomyocytes, immune cells, and vascular cells, which release fibroblast-activating mediators. However, their influences on vascular smooth muscle cells (vSMCs) have not been well examined. This study aimed to investigate whether contractile vascular vSMCs could develop a more synthetic phenotype in response to hyperglycemia. The results showed that contractile and synthetic vSMCs consumed high glucose in different ways. Lactate/GPR81 promotes the synthetic phenotype in vSMCs in response to high glucose levels. The stimulation of high glucose was associated with a significant increase in fibroblast-like features: synthetic vSMC marker expression, collagen 1 production, proliferation, and migration. GPR81 expression is higher in blood vessels in diabetic patients and in the high-glucose, high-lipid diet mouse. The results demonstrate that vSMCs assume a more synthetic phenotype when cultured in the presence of high glucose and, consequently, that the high glucose could trigger a vSMC-dependent cardiovascular disease mechanism in diabetes via lactate/GPR81.

Relationship Between the Efficacy of Cardiac Cell Therapy and the Inhibition of Differentiation of Human iPSC-Derived Nonmyocyte Cardiac Cells Into Myofibroblast-Like Cells.

RATIONALE: Myofibroblasts are believed to evolve from precursor cells; however, whether noncardiomyocyte cardiac cells (NMCCs; ie, endothelial cells, smooth muscle cells, pericytes, and fibroblasts) that have been derived from human-induced pluripotent stem cells (hiPSCs) can transdifferentiate into myofibroblast-like cells, and if so, whether this process reduces the efficacy of hiPSC-NMCC therapy, is unknown. OBJECTIVE: To determine whether hiPSC-NMCCs can differentiate to myofibroblast-like cells and whether limiting the transdifferentiation of hiPSC-NMCCs can improve their effectiveness for myocardial repair. METHODS AND RESULTS: When endothelial cells, smooth muscle cells, pericytes, and fibroblasts that had been generated from hiPSCs were cultured with TGF-ß (transforming growth factor-ß), the expression of myofibroblast markers increased, whereas endothelial cell, smooth muscle cell, pericyte, and fibroblast marker expression declined. TGF-ß-associated myofibroblast differentiation was accompanied by increases in the signaling activity of Smad, Snail, and mTOR (mammalian target of rapamycin). However, measures of pathway activation, proliferation, apoptosis, migration, and protein expression in hiPSC-endothelial cell-derived, smooth muscle cell-derived, pericyte-derived, and fibroblast-derived myofibroblast-like cells differed. Furthermore, when hiPSC-NMCCs were transplanted into the hearts of mice after myocardial infarction, ≈21% to 35% of the transplanted hiPSC-NMCCs expressed myofibroblast markers 1 week later, compared with <7% of transplanted cells ( P<0.01, each cell type) in animals that were treated with both hiPSC-NMCCs and the TGF-ß inhibitor galunisertib. Galunisertib coadministration was also associated with significant improvements in fibrotic area, left ventricular dilatation, vascular density, and cardiac function. CONCLUSIONS: hiPSC-NMCCs differentiate into myofibroblast-like cells when cultured with TGF-ß or when transplanted into infarcted mouse hearts, and the phenotypes of the myofibroblast-like cells can differ depending on the lineage of origin. TGF-ß inhibition significantly improved the efficacy of transplanted hiPSC-NMCCs for cardiac repair, perhaps by limiting the differentiation of hiPSC-NMCCs into myofibroblast-like cells.

Lactate Promotes Synthetic Phenotype in Vascular Smooth Muscle Cells.

RATIONALE: The phenotypes of vascular smooth muscle cells (vSMCs) comprise a continuum bounded by predominantly contractile and synthetic cells. Some evidence suggests that contractile vSMCs can assume a more synthetic phenotype in response to ischemic injury, but the mechanisms that activate this phenotypic switch are poorly understood. OBJECTIVE: To determine whether lactate, which increases in response to regional ischemia, may promote the synthetic phenotype in vSMCs. METHODS AND RESULTS: Experiments were performed with vSMCs that had been differentiated from human induced pluripotent stem cells and then cultured in glucose-free, lactate-enriched (L+) medium or in standard (L-) medium. Compared with the L- medium, the L+ medium was associated with significant increases in synthetic vSMC marker expression, proliferation, and migration and with significant declines in contractile and apoptotic activity. Furthermore, these changes were accompanied by increases in the expression of monocarboxylic acid transporters and were generally attenuated both by the blockade of monocarboxylic acid transporter activity and by transfection with iRNA for NDRG (N-myc downstream regulated gene). Proteomics, biomarker, and pathway analyses suggested that the L+ medium tended to upregulate the expression of synthetic vSMC markers, the production of extracellular proteins that participate in tissue construction or repair, and the activity of pathways that regulate cell proliferation and migration. Observations in hypoxia-cultured vSMCs were similar to those in L+-cultured vSMCs, and assessments in a swine myocardial infarction model suggested that measurements of lactate levels, lactate-dehydrogenase levels, vSMC proliferation, and monocarboxylic acid transporter and NDRG expression were greater in the ischemic zone than in nonischemic tissues. CONCLUSIONS: These results demonstrate for the first time that vSMCs assume a more synthetic phenotype in a microenvironment that is rich in lactate. Thus, mechanisms that link glucose metabolism to vSMC phenotypic switching could play a role in the pathogenesis and treatment of cardiovascular disease.

Cholestasis in Preterm Infants.

Cholestasis in preterm infants has a multifactorial etiology. Risk factors include degree of prematurity, lack of enteral feeding, intestinal injury, prolonged use of parenteral nutrition (PN), and sepsis. Soy-based parenteral lipid emulsions have been implicated in the pathophysiology of PN-associated liver injury. Inflammation plays an important role. Medical therapies are used; however, their effects have not consistently proven effective. Evaluation of cholestasis involves laboratory work; direct bilirubin levels are used for diagnosis and trending. Adverse outcomes include risk for hepatobiliary dysfunction, irreversible liver failure, and death. Early enteral feedings as tolerated is the best way to prevent and manage cholestasis.

Predischarge screening for severe neonatal hyperbilirubinemia identifies infants who need phototherapy.

OBJECTIVE: To test whether the combined use of total plasma/serum bilirubin (TSB) levels and clinical risk factors more accurately identifies infants who receive phototherapy than does the use of either method alone. STUDY DESIGN: We recruited healthy infants of ≥35 weeks' gestation at 6 centers that practiced universal predischarge TSB screening. Transcutaneous bilirubin (TcB) was measured at 24 hours, with TSB at 24-60 hours and at 3- to 5- and 7- to 14-day follow-up visits. Clinical risk factors were identified systematically. RESULTS: Of 1157 infants, 1060 (92%) completed follow-up, and 982 (85%) had complete datasets for analysis. Infant characteristics included 25% were nonwhite and 55% were Hispanic/Latino; >90% were breastfed. During the first week, jaundice was documented in 84% of subjects. Predischarge TSB identified the 41 (4.2%) and 34 (3.5%) infants who received phototherapy before and after discharge, respectively. Prediction of postdischarge phototherapy was similar for combined clinical risk factors (earlier gestational age [GA], bruising, positive direct antiglobulin test, Asian race, exclusive breastfeeding, blood type incompatibility, jaundice extent) and age-adjusted TSB (area under the curve [AUC] = .86 vs .87), but combined screening was better (AUC = .95). TcB/TSB combined with GA alone was equally predictive (AUC = .95; 95% CI .93-.97). CONCLUSIONS: Jaundice is present in 4 of 5 (84%) healthy newborns. Predischarge TcB/TSB (adjusted for postnatal age) combined with specific clinical factors (especially GA) best predicts subsequent phototherapy use. Universal implementation of this strategy in the US should improve outcomes of healthy newborns discharged early.

Assessment of UGT polymorphisms and neonatal jaundice.

Elevation of the serum bilirubin level is a common, if not universal, finding during the first week of life. This can be a transient phenomenon that resolves spontaneously or can signify a serious or even life-threatening condition. There are many causes of hyperbilirubinemia and related therapeutic and prognostic implications. The diseases in which there is a primary disorder of the metabolism of bilirubin will be reviewed regarding their clinical presentation, pathophysiology, diagnosis, and treatment. These disorders-Gilbert's syndrome and Crigler-Najjar Syndrome-both involve abnormalities in bilirubin conjugation secondary to deficiency of bilirubin uridine diphosphate glucuronosyltransferase. The purpose of this article is to review the current understanding of the genetic polymorphisms that result in these diseases and discuss recent advances in diagnosis and treatment.

Novel biomarker candidates to predict hepatic fibrosis in hepatitis C identified by serum proteomics.

BACKGROUND: Liver biopsy remains the gold standard to assess hepatic fibrosis. It is desirable to predict hepatic fibrosis without the need for invasive liver biopsy. Proteomic techniques allow unbiased assessment of proteins and might be useful to identify proteins related to hepatic fibrosis. AIMS: We utilized two different proteomic methods to identify serum proteins as candidate biomarkers to predict hepatic fibrosis stage in patients with chronic hepatitis C virus (HCV) infection. METHODS: Serum was obtained from 24 people with chronic HCV at time of liver biopsy and from 6 normals. Liver biopsy fibrosis was staged 1-4 (Batts-Ludwig). Pooled serum samples (six in each of four fibrosis groups and controls) were analyzed with 4- and 8-plex isobaric tags for relative and absolute quantitation (iTRAQ), determining protein identification (ID) and ratios of relative protein abundance. Nonpooled samples were analyzed with two-dimensional (2-D) gels and difference in gel electrophoresis (DIGE) comparing different samples on the same gel and across gels. Spots varying among groups were measured with densitometry, excised, digested, and submitted for tandem mass spectrometry (MS/MS) protein ID. RESULTS: iTRAQ identified 305 proteins (minimum 99% ID confidence); 66 were increased or decreased compared with controls. Some proteins were increased or decreased for specific fibrosis scores. From 704 DIGE protein spots, 66 were chosen, 41 excised, and 135 proteins identified, since one gel spot often identified more than one protein. CONCLUSIONS: Both proteomic methods identified two proteins as biomarker candidates for predicting hepatic fibrosis: complement C4-A and inter-alpha-trypsin inhibitor heavy chain H4.

Proteomic analysis of biopsied human colonic mucosa.

BACKGROUND AND OBJECTIVES: : Pediatric gastroenterologists have a unique opportunity to study the proteins in the gastrointestinal tract. To assess the power of proteomic studies we compared 2 methods for analysis of proteins in normal human colonic mucosa: 2-dimensional gel electrophoresis (2DE) and 2-dimensional liquid chromatography (2DLC) in conjunction with mass spectrometry. We used Ingenuity Pathway Analysis to examine these proteins regarding function, location, and relation to disease. RESULTS: : 2DLC identified 550 proteins, whereas 2DE identified 107 proteins, 18 of which were not observed with 2DLC. The function associated with the largest number of proteins for both methods was cancer (236 proteins with 2DLC, 61 proteins with 2DE). The largest group of proteins was from the cytoplasm (49.3% from 2DE and 49.1% from 2DLC). Two hundred seventy of the total 568 proteins were related to 26 different categories of human disease and 200 of these 270 were described in large intestine, 227 were described in blood, and 149 were described in serum or plasma. CONCLUSIONS: : These methods are complementary, although many more proteins were identified with 2DLC. This suggests that 2DLC should have greater utility in examining changes in the proteome of the colonic mucosa during disease than 2DE. However, some proteins found were unique to 2DE, and thus the methods chosen for a given analysis must be matched with the proteins to be studied. When pediatric gastroenterologists use proteomic methods, there is a new opportunity to increase our understanding of the gastrointestinal tract in health and disease.

A controlled, randomized, double-blind trial of prophylaxis against jaundice among breastfed newborns.

OBJECTIVES: Neonatal jaundice is a greater problem for infants fed breast milk, compared with formula. This study tested the hypotheses that feeding breastfed newborns beta-glucuronidase inhibitors during the first week after birth would increase fecal bilirubin excretion and would reduce jaundice without affecting breastfeeding deleteriously. METHODS: Sixty-four breastfed newborns were randomized to 4 groups, ie, control or receiving 6 doses per day (5 mL per dose) of L-aspartic acid, enzymatically hydrolyzed casein (EHC), or whey/casein (W/C) for the first week. L-aspartic acid and EHC inhibit beta-glucuronidase. Transcutaneous bilirubin levels (primary outcome) were measured daily (Jaundice Meter [Minolta/Air Shields, Hatboro, PA] and Bilicheck [Respironics, Pittsburgh, PA]). All stools were collected, and fecal bile pigments, including bilirubin diglucuronide, bilirubin monoglucuronides, and bilirubin, were analyzed with high-performance liquid chromatography. Follow-up assessments included day 7 body weight, day 6/7 prebreastfeeding/postbreastfeeding weights, maternal ratings, and ages at formula introduction and breastfeeding cessation. RESULTS: The groups were comparable at entry. Overall, the L-aspartic acid, EHC, and W/C groups had significantly lower transcutaneous bilirubin levels than did the control group (75.8%, 69.6%, and 69.2%, respectively, of the control mean, 8.53 mg/dL, at the bilirubin peak on day 4). The L-aspartic acid, EHC, and W/C groups had significantly lower transcutaneous bilirubin levels on days 3 to 7. Fecal bile pigment excretion was greatest in the L-aspartic acid group, significantly greater than control values. There were no significant differences in dosages, follow-up measurements, and maternal ratings. CONCLUSIONS: Use of minimal aliquots of L-aspartic acid and EHC for beta-glucuronidase inhibition results in increased fecal bilirubin excretion and less jaundice, without disruption of the breastfeeding experience. Decreased jaundice in the W/C group, which lacked a beta-glucuronidase inhibitor, suggests a different mechanism.

Neonatal cholestasis.

Any infant who is jaundiced beyond two to three weeks of life should be evaluated for neonatal cholestasis. Neonatal cholestasis is defined as accumulation of bile substances in blood due to impaired excretion. These infants should always have fractionated serum bilirubin levels checked to differentiate the conjugated hyperbilirubinemia of cholestasis from unconjugated hyperbilirubinemia that is usually benign and spontaneously resolves. Conjugated hyperbilirubinemia, pale stools and dark urine are the cardinal features of neonatal cholestasis. The differential diagnosis of cholestasis is extensive and a systematic approach is helpful to quickly establish the diagnosis. Biliary atresia is a common cause of neonatal cholestasis and affected infants need surgery before 60 days of life for better prognosis. Premature infants have multifactorial cholestasis and need a modified approach to the evaluation of cholestasis. Management of cholestasis is mostly supportive, consisting of medical management of complications of chronic cholestasis like pruritus and nutritional support for malabsorption and vitamin deficiency.

Breast-feeding, neonatal jaundice and kernicterus.

Despite the many advantages of breast-feeding, there is ample documentation of the strong association between breast-feeding and an increase in the risk of neonatal hyperbilirubinaemia. Breast-fed infants have higher bilirubin levels than formula-fed infants. Suggested mechanisms for these findings include poor fluid and caloric intake, inhibition of hepatic excretion of bilirubin, and intestinal absorption of bilirubin (enterohepatic circulation). On rare occasions, breast-fed infants without evidence of haemolysis have developed extreme hyperbilirubinaemia and kernicterus. Because almost all of the cases of kernicterus reported in the last 15 years have occurred in fully or partially breast-fed newborns, it is important that these infants be followed closely. Appropriate support and advice must be provided to the lactating mother so that successful breast-feeding can be established and the risk of severe hyperbilirubinaemia reduced.