Evaluating threshold for donor fraction cell-free DNA using clinically available assay for rejection in pediatric and adult heart transplantation.

BACKGROUND: The aims of the study were to assess the performance of a clinically available cell-free DNA (cfDNA) assay in a large cohort of pediatric and adult heart transplant recipients and to evaluate performance at specific cut points in detection of rejection. METHODS: Observational, non-interventional, prospective study enrolled pediatric and adult heart transplant recipients from seven centers. Biopsy-associated plasma samples were used for cfDNA measurements. Pre-determined cut points were tested for analytic performance. RESULTS: A total of 487 samples from 160 subjects were used for the analysis. There were significant differences for df-cfDNA values between rejection [0.21% (IQR 0.12-0.69)] and healthy samples [0.05% (IQR 0.01-0.14), p < .0001]. The pediatric rejection group had a median df-cfDNA value of 0.93% (IQR 0.28-2.84) compared to 0.09% (IQR 0.04-0.23) for healthy samples, p = .005. Overall negative predictive value was 0.94 while it was 0.99 for pediatric patients. Cut points of 0.13% and 0.15% were tested for various types of rejection profiles and were appropriate to rule out rejection. CONCLUSION: The study suggests that pediatric patients with rejection show higher levels of circulating df-cfDNA compared to adults and supports the specific cut points for clinical use in pediatric and adult patients with overall acceptable performance.

Profibrotic COVID-19 subphenotype exhibits enhanced localized ER-dependent HSP47+ expression in cardiac myofibroblasts in situ.

We recently described a subgroup of autopsied COVID-19 subjects (∼40%), termed 'profibrotic phenotype,' who exhibited clusters of myofibroblasts (Mfbs), which were positive for the collagen-specific chaperone heat shock protein 47 (HSP47+) in situ. This report identifies increased, localized (hot spot restricted) expression of αSMA, COLα1, POSTN and FAP supporting the identity of HSP47+ cells as myofibroblasts and characterizing a profibrotic extracellular matrix (ECM) phenotype. Coupled with increased GRP78 in COVID-19 subjects, these data could reflect induction of the unfolded protein response for mitigation of proteostasis (i.e., protein homeostasis) dysfunction in discrete clusters of cells. ECM shifts in selected COVID-19 subjects occur without significant increases in either global trichrome positive staining or myocardial injury based quantitively on standard H&E scoring. Our findings also suggest distinct mechanism(s) for ECM remodeling in the setting of SARS-CoV-2 infection. The ratio of CD163+/CD68+ cells is increased in hot spots of profibrotic hearts compared with either controls or outside of hot spots in COVID-19 subjects. In sum, matrix remodeling of human COVID-19 hearts in situ is characterized by site-restricted profibrotic mediated (e.g., HSP47+ Mfbs, CD163+ Mφs) modifications in ECM (i.e., COLα1, POSTN, FAP), with a strong correlation between COLα1 and HSP47+cells within hot spots. Given the established associations of viral infection (e.g., human immunodeficiency virus; HIV), myocardial fibrosis and sudden cardiac death, early screening tools (e.g., plasma biomarkers, noninvasive cardiac magnetic resonance imaging) for diagnosis, monitoring and treatment of fibrotic ECM remodeling are warranted for COVID-19 high-risk populations.

A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and is Mutated in GLUT1 Deficiency Syndrome.

Protein kinase C has been implicated in the phosphorylation of the erythrocyte/brain glucose transporter, GLUT1, without a clear understanding of the site(s) of phosphorylation and the possible effects on glucose transport. Through in vitro kinase assays, mass spectrometry, and phosphospecific antibodies, we identify serine 226 in GLUT1 as a PKC phosphorylation site. Phosphorylation of S226 is required for the rapid increase in glucose uptake and enhanced cell surface localization of GLUT1 induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Endogenous GLUT1 is phosphorylated on S226 in primary endothelial cells in response to TPA or VEGF. Several naturally occurring, pathogenic mutations that cause GLUT1 deficiency syndrome disrupt this PKC phosphomotif, impair the phosphorylation of S226 in vitro, and block TPA-mediated increases in glucose uptake. We demonstrate that the phosphorylation of GLUT1 on S226 regulates glucose transport and propose that this modification is important in the physiological regulation of glucose transport.

Increase in nuclear cell-free DNA is associated with major adverse events in adult and pediatric heart transplant recipients.

BACKGROUND: Cell-free DNA is an emerging biomarker. While donor fraction may detect graft events in heart transplant recipients, the prognostic value of total nuclear cell-free DNA (ncfDNA) itself is largely unexplored. OBJECTIVE: Explore the relationship between ncfDNA and clinical events in heart transplant recipients. METHODS: We conducted a multi-center prospective study to investigate the value of cell-free DNA in non-invasive monitoring following heart transplantation. Over 4000 blood samples were collected from 388 heart transplant patients. Total ncfDNA and donor fraction were quantified. Generalized linear models with maximum likelihood estimation for repeated measures with subjects as clusters were used to explore the relationship of ncfDNA and major adverse events. Receiver operating characteristic curves were used to help choose cutpoints. RESULTS: A ncfDNA threshold (50 ng/ml) was identified that was associated with increased risk of major adverse events. NcfDNA was elevated in patients who suffered cardiac arrest, required mechanical circulatory support or died post heart transplantation as well as in patients undergoing treatment for infection. CONCLUSIONS: Elevated ncfDNA correlates with risk for major adverse events in adult and pediatric heart transplant recipients and may indicate a need for enhanced surveillance after transplant.

Relationship between donor fraction cell-free DNA and clinical rejection in heart transplantation.

BACKGROUND: Clinical rejection (CR) defined as decision to treat clinically suspected rejection with change in immunotherapy based on clinical presentation with or without diagnostic biopsy findings is an important part of care in heart transplantation. We sought to assess the utility of donor fraction cell-free DNA (DF cfDNA) in CR and the utility of serial DF cfDNA in CR patients in predicting outcomes of clinical interest. METHODS: Patients with heart transplantation were enrolled in two sequential, multi-center, prospective observational studies. Blood samples were collected for surveillance or clinical events. Clinicians were blinded to the results of DF cfDNA. RESULTS: A total of 835 samples from 269 subjects (57% pediatric) were included for this analysis, including 28 samples associated with CR were analyzed. Median DF cfDNA was 0.43 (IQR 0.15, 1.36)% for CR and 0.10 (IQR 0.07, 0.16)% for healthy controls (p < .0001). At cutoff value of 0.13%, the area under curve (AUC) was 0.82, sensitivity of 0.86, specificity of 0.67, and negative predictive value of 0.99. There was serial decline in DF cfDNA post-therapy, however, those with cardiovascular events (cardiac arrest, need for mechanical support or death) showed significantly higher levels of DF cfDNA on Day 0 (2.11 vs 0.31%) and Day 14 (0.51 vs 0.22%) compared to those who did not have such an event (p < .0001). CONCLUSION: DF cfDNA has excellent agreement with clinical rejection and, importantly, serial measurement of DF cfDNA predict clinically significant outcomes post treatment for rejection in these patients.

Adverse Maternal Environment and Postweaning Western Diet Alter Hepatic CD36 Expression and Methylation Concurrently with Nonalcoholic Fatty Liver Disease in Mouse Offspring.

BACKGROUND: The role of an adverse maternal environment (AME) in conjunction with a postweaning Western diet (WD) in the development of nonalcoholic fatty liver disease (NAFLD) in adult offspring has not been explored. Likewise, the molecular mechanisms associated with AME-induced NAFLD have not been studied. The fatty acid translocase or cluster of differentiation 36 (CD36) has been implicated to play a causal role in the pathogenesis of WD-induced steatosis. However, it is unknown if CD36 plays a role in AME-induced NAFLD. OBJECTIVE: This study was designed to evaluate the isolated and additive impact of AME and postweaning WD on the expression and DNA methylation of hepatic Cd36 in association with the development of NAFLD in a novel mouse model. METHODS: AME constituted maternal WD and maternal stress, whereas the control (Con) group had neither. Female C57BL/6J mice were fed a WD [40% fat energy, 29.1% sucrose energy, and 0.15% cholesterol (wt/wt)] 5 wk prior to pregnancy and throughout lactation. Non invasive variable stressors (random frequent cage changing, limited bedding, novel object, etc.) were applied to WD dams during the last third of pregnancy to produce an AME. Con dams consumed the control diet (CD) (10% fat energy, no sucrose or cholesterol) and were not exposed to stress. Male offspring were weaned onto either CD or WD, creating 4 experimental groups: Con-CD, Con-WD, AME-CD, and AME-WD, and evaluated for metabolic and molecular parameters at 120 d of age. RESULTS: AME and postweaning WD independently and additively increased the development of hepatic steatosis in adult male offspring. AME and WD independently and additively upregulated hepatic CD36 protein and mRNA expression and hypomethylated promoters 2 and 3 of the Cd36 gene. CONCLUSIONS: Using a mouse AME model together with postweaning WD, this study demonstrates a role for CD36 in AME-induced NAFLD in offspring and reveals 2 regions of environmentally induced epigenetic heterogeneity within Cd36.

α-Galactosidase A-deficient rats accumulate glycosphingolipids and develop cardiorenal phenotypes of Fabry disease.

Fabry disease is an X-linked lysosomal storage disease caused by α-galactosidase A (α-Gal A) deficiency. Kidney and heart failure are frequent complications in adulthood and greatly contribute to patient morbidity and mortality. Because α-Gal A-deficient mouse models do not recapitulate cardiorenal findings observed in patients, a nonmouse model may be beneficial to our understanding of disease pathogenesis. In this study, we evaluated disease processes in a recently generated Fabry rat model. We found that male Fabry rats weighed significantly less than wild-type (WT) males, whereas female Fabry rats weighed significantly more than WT females. Whereas no difference in female survival was detected, we observed that male Fabry rats had a decreased lifespan. Skin histology revealed that inflammation and lipoatrophy may be chief disease mediators in patients. With respect to the kidney and heart, we found that both organs accumulate α-Gal A substrates, including the established biomarkers, globotriaosylceramide and globotriaosylsphingosine. Longitudinal serum and urine chemistry panels demonstrated pronounced renal tubule dysfunction, which was confirmed histologically. Mitral valve thickening was observed in Fabry rats using echocardiography. We conclude that Fabry rats recapitulate important kidney and heart phenotypes experienced by patients and can be further used to study disease mechanisms and test therapies.-Miller, J. J., Aoki, K., Mascari, C. A., Beltrame, A. K., Sokumbi, O., North, P. E., Tiemeyer, M., Kriegel, A. J., Dahms, N. M., α-Galactosidase A-deficient rats accumulate glycosphingolipids and develop cardiorenal phenotypes of Fabry disease.

Early changes in cell-free DNA levels in newly transplanted heart transplant patients.

Heart transplantation is a well-established therapy for end-stage heart failure in children and young adults. The highest risk of graft loss occurs in the first 60 days post-transplant. Donor fraction of cell-free DNA is a highly sensitive marker of graft injury. Changes in cell-free DNA levels have not previously been studied in depth in patients early after heart transplant. A prospective study was conducted among heart transplant recipients at a single pediatric heart center. Blood samples were collected from children and young adult transplant patients at three time points within 10 days of transplantation. DF and total cell-free DNA levels were measured using a targeted method (myTAIHEART ). In 17 patients with serial post-transplant samples, DF peaks in the first 2 days after transplant (3.5%, [1.9-10]%) and then declines toward baseline (0.27%, [0.19-0.52]%) by 6-9 days. There were 4 deaths in the first year among the 10 patients with complete sample sets, and 3 out of 4 who died had a late rise or blunted decline in donor fraction. Patients who died trended toward an elevated total cell-free DNA at 1 week (41.5, [34-65] vs 13.6, [6.2-22] P = .07). Donor fraction peaks early after heart transplant and then declines toward baseline. Patients without sustained decline in donor fraction and/or elevated total cell-free DNA at 1 week may have worse outcomes.

Mapping genetic modifiers of radiation-induced cardiotoxicity to rat chromosome 3.

Radiation therapy is used in ~50% of cancer patients to reduce the risk of recurrence and in some cases improve survival. Despite these benefits, doses can be limited by toxicity in multiple organs, including the heart. The underlying causes and biomarkers of radiation-induced cardiotoxicity are currently unknown, prompting the need for experimental models with inherent differences in sensitivity and resistance to the development of radiation-induced cardiotoxicity. We have identified the parental SS (Dahl salt-sensitive/Mcwi) rat strain to be a highly-sensitized model of radiation-induced cardiotoxicity. In comparison, substitution of rat chromosome 3 from the resistant BN (Brown Norway) rat strain onto the SS background (SS-3BN consomic) significantly attenuated radiation-induced cardiotoxicity. SS-3BN rats had less radiation-induced cardiotoxicity than SS rats, as measured by survival, pleural and pericardial effusions, echocardiogram parameters, and histological damage. Mast cells, previously shown to have predominantly protective roles in radiation-induced cardiotoxicity, were increased in the more resistant SS-3BN hearts postradiation. RNA sequencing from SS and SS-3BN hearts at 1 wk postradiation revealed 5,098 differentially expressed candidate genes across the transcriptome and 350 differentially expressed genes on rat chromosome 3, which coincided with enrichment of multiple pathways, including mitochondrial dysfunction, sirtuin signaling, and ubiquitination. Upstream regulators of enriched pathways included the oxidative stress modulating transcription factor, Nrf2, which is located on rat chromosome 3. Nrf2 target genes were also differentially expressed in the SS vs. SS-3BN consomic hearts postradiation. Collectively, these data confirm the existence of heritable modifiers in radiation-induced cardiotoxicity and provide multiple biomarkers, pathways, and candidate genes for future analyses. NEW & NOTEWORTHY This novel study reveals that heritable genetic factors have the potential to modify normal tissue sensitivity to radiation. Gene variant(s) on rat chromosome 3 can contribute to enhanced cardiotoxicity displayed in the SS rats vs. the BN and SS-3BN consomic rats. Identifying genes that lead to understanding the mechanisms of radiation-induced cardiotoxicity represents a novel method to personalize radiation treatment, as well as predict the development of radiation-induced cardiotoxicity.

Protective role of Trpc6 knockout in the progression of diabetic kidney disease.

Diabetic kidney disease (DKD) is a chronic kidney pathology that leads to end-stage renal disease. Previous studies from our laboratory indicate that there is an association between the development of DKD and the transient receptor potential canonical 6 (TRPC6) channel. Trpc6 expression and activity were increased in the streptozotocin (STZ)-treated Dahl Salt-sensitive (Dahl SS) rat, an established model of type 1 diabetes. Here, using a Trpc6 knockout created on the Dahl SS rat background (SSTrpc6-/-), we test the hypothesis that the absence of Trpc6 will protect podocytes and kidney function during the development of DKD. Four groups of animals (control SSWT, SSTrpc6-/-, STZ-treated SSWT, and STZ-SSTrpc6-/-) were utilized in this study. Diabetes development was monitored for 11 wk after STZ injection with periodic weight, glucose, and urinary output measurements. There was an increase in albuminuria and glomerular injury following STZ treatment, which was not different between Dahl SS and SSTrpc6-/- groups. Western blot analysis revealed elevated levels of nephrin in urine samples of STZ-SSWT rats, which was higher compared with STZ-SSTrpc6-/- rats. Furthermore, pathological increases in basal [Ca2+]i levels and foot process damage of podocytes during the development of DKD was attenuated in the STZ-SSTrpc6-/- compared with STZ-SSWT rats. Overall, our data indicate that TRPC6 channel inhibition may have at least partial renoprotective effects, which could lead to the development of new pharmacological tools to treat or prevent the progression of DKD.

Nogo-B receptor deficiency causes cerebral vasculature defects during embryonic development in mice.

Nogo-B receptor (NgBR) was identified as a receptor specific for Nogo-B. Our previous work has shown that Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro and intersomitic vessel formation via Akt pathway in zebrafish. Here, we further demonstrated the roles of NgBR in regulating vasculature development in mouse embryo and primitive blood vessel formation in embryoid body culture systems, respectively. Our results showed that NgBR homozygous knockout mice are embryonically lethal at E7.5 or earlier, and Tie2Cre-mediated endothelial cell-specific NgBR knockout (NgBR ecKO) mice die at E11.5 and have severe blood vessel assembly defects in embryo. In addition, mutant embryos exhibit dilation of cerebral blood vessel, resulting in thin-walled endothelial caverns. The similar vascular defects also were detected in Cdh5(PAC)-CreERT2 NgBR inducible ecKO mice. Murine NgBR gene-targeting embryonic stem cells (ESC) were generated by homologous recombination approaches. Homozygous knockout of NgBR in ESC results in cell apoptosis. Heterozygous knockout of NgBR does not affect ESC cell survival, but reduces the formation and branching of primitive blood vessels in embryoid body culture systems. Mechanistically, NgBR knockdown not only decreases both Nogo-B and VEGF-stimulated endothelial cell migration by abolishing Akt phosphorylation, but also decreases the expression of CCM1 and CCM2 proteins. Furthermore, we performed immunofluorescence (IF) staining of NgBR in human cerebral cavernous malformation patient tissue sections. The quantitative analysis results showed that NgBR expression levels in CD31 positive endothelial cells is significantly decreased in patient tissue sections. These results suggest that NgBR may be one of important genes coordinating the cerebral vasculature development.

Vascular Actions of Angiotensin 1-7 in the Human Microcirculation: Novel Role for Telomerase.

OBJECTIVE: This study examined vascular actions of angiotensin 1-7 (ANG 1-7) in human atrial and adipose arterioles. APPROACH AND RESULTS: The endothelium-derived hyperpolarizing factor of flow-mediated dilation (FMD) switches from antiproliferative nitric oxide (NO) to proatherosclerotic hydrogen peroxide in arterioles from humans with coronary artery disease (CAD). Given the known vasoprotective properties of ANG 1-7, we tested the hypothesis that overnight ANG 1-7 treatment restores the NO component of FMD in arterioles from patients with CAD. Endothelial telomerase activity is essential for preserving the NO component of vasodilation in the human microcirculation; thus, we also tested whether telomerase activity was necessary for ANG 1-7-mediated vasoprotection by treating separate arterioles with ANG 1-7±the telomerase inhibitor 2-[[(2E)-3-(2-naphthalenyl)-1-oxo-2-butenyl1-yl]amino]benzoic acid. ANG 1-7 dilated arterioles from patients without CAD, whereas dilation was significantly reduced in arterioles from patients with CAD. In atrial arterioles from patients with CAD incubated with ANG 1-7 overnight, the NO synthase inhibitor NG-nitro-l-arginine methyl ester abolished FMD, whereas the hydrogen peroxide scavenger polyethylene glycol catalase had no effect. Conversely, in vessels incubated with ANG 1-7+2-[[(2E)-3-(2-naphthalenyl)-1-oxo-2-butenyl1-yl]amino]benzoic acid, NG-nitro-l-arginine methyl ester had no effect on FMD, but polyethylene glycol catalase abolished dilation. In cultured human coronary artery endothelial cells, ANG 1-7 significantly increased telomerase activity. These results indicate that ANG 1-7 dilates human microvessels, and dilation is abrogated in the presence of CAD. Furthermore, ANG 1-7 treatment is sufficient to restore the NO component of FMD in arterioles from patients with CAD in a telomerase-dependent manner. CONCLUSIONS: ANG 1-7 exerts vasoprotection in the human microvasculature via modulation of telomerase activity.

Mimickers of Infantile Hemangiomas.

Infantile hemangiomas (IHs) are the most common tumors of infancy and usually follow a typical course of growth and involution. We report four soft tissue tumors that were referred to the pediatric dermatology clinic as IHs and the process by which they were diagnosed and treated. Clinicians should be aware of presentations of these uncommon, but serious soft tissue tumors. Many of these mimickers have a vastly different clinical prognosis, and early intervention to limit sequelae is crucial. Biopsy of atypical lesions should be considered early in the diagnostic process since they have varied prognosis and treatment strategies.

Sturge-Weber syndrome and port-wine stains caused by somatic mutation in GNAQ.

BACKGROUND: The Sturge-Weber syndrome is a sporadic congenital neurocutaneous disorder characterized by a port-wine stain affecting the skin in the distribution of the ophthalmic branch of the trigeminal nerve, abnormal capillary venous vessels in the leptomeninges of the brain and choroid, glaucoma, seizures, stroke, and intellectual disability. It has been hypothesized that somatic mosaic mutations disrupting vascular development cause both the Sturge-Weber syndrome and port-wine stains, and the severity and extent of presentation are determined by the developmental time point at which the mutations occurred. To date, no such mutation has been identified. METHODS: We performed whole-genome sequencing of DNA from paired samples of visibly affected and normal tissue from 3 persons with the Sturge-Weber syndrome. We tested for the presence of a somatic mosaic mutation in 97 samples from 50 persons with the Sturge-Weber syndrome, a port-wine stain, or neither (controls), using amplicon sequencing and SNaPshot assays, and investigated the effects of the mutation on downstream signaling, using phosphorylation-specific antibodies for relevant effectors and a luciferase reporter assay. RESULTS: We identified a nonsynonymous single-nucleotide variant (c.548G→A, p.Arg183Gln) in GNAQ in samples of affected tissue from 88% of the participants (23 of 26) with the Sturge-Weber syndrome and from 92% of the participants (12 of 13) with apparently nonsyndromic port-wine stains, but not in any of the samples of affected tissue from 4 participants with an unrelated cerebrovascular malformation or in any of the samples from the 6 controls. The prevalence of the mutant allele in affected tissues ranged from 1.0 to 18.1%. Extracellular signal-regulated kinase activity was modestly increased during transgenic expression of mutant Gαq. CONCLUSIONS: The Sturge-Weber syndrome and port-wine stains are caused by a somatic activating mutation in GNAQ. This finding confirms a long-standing hypothesis. (Funded by the National Institutes of Health and Hunter's Dream for a Cure Foundation.).

Venous malformation of the ethmoid and sphenoid sinuses.

OBJECTIVES: The aim of this study was to present a rare case of a venous malformation that occupied the ethmoid and sphenoid sinuses. Prior to resection, it was believed to be a hemangioma. METHODS: This study includes a case report and review of the literature. CONCLUSION: There is often confusion between "hemangiomas" and "vascular malformations," but they are important to differentiate because they have unique approaches to treatment. Venous malformations in the paranasal sinuses are very rare. To our knowledge, this is the first case report that explicitly describes a venous malformation in the ethmoid and sphenoid sinuses. It was treated using endoscopic sinus surgery with intraoperative computer-assisted stereotactic navigation.

Heparin rescues factor V Leiden-associated placental failure independent of anticoagulation in a murine high-risk pregnancy model.

Low molecular weight heparin (LMWH) is being tested as an experimental drug for improving pregnancy outcome in women with inherited thrombophilia and placenta-mediated pregnancy complications, such as recurrent pregnancy loss. The role of thrombotic processes in these disorders remains unproven, and the issue of antithrombotic prophylaxis is intensely debated. Using a murine model of factor V Leiden-associated placental failure, we show that treatment of the mother with LMWH allows placental development to proceed and affords significant protection from fetal loss. Nonetheless, the therapeutic effect of LMWH is not replicated by anticoagulation; fondaparinux and a direct Xa inhibitor, C921-78, achieve anticoagulation similar to LMWH but produce little or no improvement in pregnancy outcome. Genetic attenuation of maternal platelet aggregation is similarly ineffective. In contrast, even a partial loss of thrombin sensitivity of maternal platelets protects pregnancies. Neonates born from these pregnancies are growth retarded, suggesting that placental function is only partially restored. The placentae are smaller but do not reveal any evidence of thrombosis. Our data demonstrate an anticoagulation-independent role of LMWH in protecting pregnancies and provide evidence against the involvement of thrombotic processes in thrombophilia-associated placental failure. Importantly, thrombin-mediated maternal platelet activation remains central in the mechanism of placental failure.

Endothelial cell-specific chemotaxis receptor (ECSCR) enhances vascular endothelial growth factor (VEGF) receptor-2/kinase insert domain receptor (KDR) activation and promotes proteolysis of internalized KDR.

The endothelial cell-specific chemotaxis receptor (ECSCR) is a cell-surface protein selectively expressed by endothelial cells (ECs), with roles in EC migration, apoptosis and proliferation. Our previous study (Verma, A., Bhattacharya, R., Remadevi, I., Li, K., Pramanik, K., Samant, G. V., Horswill, M., Chun, C. Z., Zhao, B., Wang, E., Miao, R. Q., Mukhopadhyay, D., Ramchandran, R., and Wilkinson, G. A. (2010) Blood 115, 4614-4622) showed that loss of ECSCR in primary ECs reduced tyrosine phosphorylation of vascular endothelial growth factor (VEGF) receptor 2/kinase insert domain receptor (KDR) but not VEGF receptor 1/FLT1. Here, we show that ECSCR biochemically associates with KDR but not FLT1 and that the predicted ECSCR cytoplasmic and transmembrane regions can each confer association with KDR. Stimulation with VEGF165 rapidly and transiently increases ECSCR-KDR complex formation, a process blocked by the KDR tyrosine kinase inhibitor compound SU5416 or inhibitors of endosomal acidification. Triple labeling experiments show VEGF-stimulated KDR(+)/ECSCR(+) intracellular co-localization. Silencing of ECSCR disrupts VEGF-induced KDR activation and AKT and ERK phosphorylation and impairs VEGF-stimulated KDR degradation. In zebrafish, ecscr interacts with kdrl during intersomitic vessel sprouting. Human placenta and infantile hemangioma samples highly express ECSCR protein, suggesting a role for ECSCR-KDR interaction in these tissues.

De novo expression of sodium-glucose cotransporter SGLT2 in Bowman's capsule coincides with replacement of parietal epithelial cell layer with proximal tubule-like epithelium.

In kidney nephron, parietal epithelial cells line the Bowman's capsule and function as a permeability barrier for the glomerular filtrate. Bowman's capsule cells with proximal tubule epithelial morphology have been found. However, the effects of tubular metaplasia in Bowman's capsule on kidney function remain poorly understood. Sodium-glucose cotransporter 2 (SGLT2) plays a major role in reabsorption of glucose in the kidney and is expressed on brush border membrane (BBM) of epithelial cells in the early segment of the proximal tubule. We hypothesized that SGLT2 is expressed in tubularized Bowman's capsule and used our novel antibody to test this hypothesis. Immunohistochemical analysis was performed with our SGLT2 antibody on C57BL/6 mouse kidney prone to have tubularized Bowman's capsules. Cell membrane was examined with periodic acid-Schiff (PAS) stain. The results showed that SGLT2 was localized on BBM of the proximal tubules in young and adult mice. Bowman's capsules were lined mostly with normal brush border-less parietal epithelial cells in young mice, while they were almost completely covered with proximal tubule-like cells in adult mice. Regardless of age, SGLT2 was expressed on BBM of the tubularized Bowman's capsule but did not co-localize with nephrin in the glomerulus. SGLT2-expressing tubular cells expanded from the urinary pole toward the vascular pole of the Bowman's capsule. This study identified the localization of SGLT2 in the Bowman's capsule. Bowman's capsules with tubular metaplasia may acquire roles in reabsorption of filtered glucose and sodium.

Nuclear Cell-Free DNA Predicts Adverse Events After Pediatric Cardiothoracic Surgery.

BACKGROUND: Preoperative risk stratification in cardiac surgery includes patient and procedure factors that are used in clinical decision-making. Despite these tools, unidentified factors contribute to variation in outcomes. Identification of latent physiologic risk factors may strengthen predictive models. Nuclear cell-free DNA (ncfDNA) increases with tissue injury and drops to baseline levels rapidly. The goal of this investigation is to measure and to observe ncfDNA kinetics in children undergoing heart operations with cardiopulmonary bypass (CPB), linking biomarkers, organ dysfunction, and outcomes. METHODS: This is a prospective observational study of 116 children <18 years and >3 kg undergoing operations with CPB. Plasma ncfDNA samples were collected and processed in a stepwise manner at predefined perioperative time points. The primary outcome measure was occurrence of postoperative cardiac arrest or extracorporeal membrane oxygenation. RESULTS: Data were available in 116 patients (median age, 0.9 years [range, 0-17.4 years]; median weight, 7.8 kg [range, 3.2-98 kg]). The primary outcome was met in 6 of 116 (5.2%). Risk of primary outcome was 2% with ncfDNA <20 ng/mL and 33% with ncfDNA >20 ng/mL (odds ratio, 25; CI, 3.96-158; P = .001). Elevated ncfDNA was associated with fewer hospital-free days (P < .01). CONCLUSIONS: This study analyzes ncfDNA kinetics in children undergoing operations with CPB for congenital heart disease. Elevated preoperative ncfDNA is strongly associated with postoperative arrest and extracorporeal membrane oxygenation. Further studies are needed to validate this technology as a tool to predict morbidity in children after cardiac surgical procedures.

Validation of donor fraction cell-free DNA with biopsy-proven cardiac allograft rejection in children and adults.

OBJECTIVES: Donor-specific cell-free DNA shows promise as a noninvasive marker for allograft rejection, but as yet has not been validated in both adult and pediatric recipients. The study objective was to validate donor fraction cell-free DNA as a noninvasive test to assess for risk of acute cellular rejection and antibody-mediated rejection after heart transplantation in pediatric and adult recipients. METHODS: Pediatric and adult heart transplant recipients were enrolled from 7 participating sites and followed for 12 months or more with plasma samples collected immediately before all endomyocardial biopsies. Donor fraction cell-free DNA was extracted, and quantitative genotyping was performed. Blinded donor fraction cell-free DNA and clinical data were analyzed and compared with a previously determined threshold of 0.14%. Sensitivity, specificity, negative predictive value, positive predictive value, and receiver operating characteristic curves were calculated. RESULTS: A total of 987 samples from 144 subjects were collected. After applying predefined clinical and technical exclusions, 745 samples from 130 subjects produced 54 rejection samples associated with the composite outcome of acute cellular rejection grade 2R or greater and pathologic antibody-mediated rejection 2 or greater and 323 healthy samples. For all participants, donor fraction cell-free DNA at a threshold of 0.14% had a sensitivity of 67%, a specificity of 79%, a positive predictive value of 34%, and a negative predictive value of 94% with an area under the curve of 0.78 for detecting rejection. When analyzed independently, these results held true for both pediatric and adult cohorts at the same threshold of 0.14% (negative predictive value 92% and 95%, respectively). CONCLUSIONS: Donor fraction cell-free DNA at a threshold of 0.14% can be used to assess for risk of rejection after heart transplantation in both pediatric and adult patients with excellent negative predictive value.