Nathan C. Sundgren

Coupling of Fcγ Receptor I to Fcγ Receptor IIB by Src Kinase Mediates C-Reactive Protein Impairment of Endothelial Function

Rationale: Elevations in C-reactive protein (CRP) are associated with increased cardiovascular disease risk and endothelial dysfunction. CRP antagonizes endothelial nitric oxide synthase (eNOS) through processes mediated by the IgG receptor Fc&ggr; receptor IIB (Fc&ggr;RIIB), its immunoreceptor tyrosine-based inhibitory motif, and SH2 domain-containing inositol 5′-phosphatase 1. In mice, CRP actions on eNOS blunt carotid artery re-endothelialization. Objective: How CRP activates Fc&ggr;RIIB in endothelium is not known. We determined the role of Fc&ggr; receptor I (Fc&ggr;RI) and the basis for coupling of Fc&ggr;RI to Fc&ggr;RIIB in endothelium. Methods and Results: In cultured endothelial cells, Fc&ggr;RI-blocking antibodies prevented CRP antagonism of eNOS, and CRP activated Src via Fc&ggr;RI. CRP-induced increases in Fc&ggr;RIIB immunoreceptor tyrosine-based inhibitory motif phosphorylation and SH2 domain-containing inositol 5′-phosphatase 1 activation were Src-dependent, and Src inhibition prevented eNOS antagonism by CRP. Similar processes mediated eNOS antagonism by aggregated IgG used to mimic immune complex. Carotid artery re-endothelialization was evaluated in offspring from crosses of CRP transgenic mice (TG-CRP) with either mice lacking the &ggr; subunit of Fc&ggr;RI (FcR&ggr;−/−) or Fc&ggr;RIIB−/− mice. Whereas re-endothelialization was impaired in TG-CRP vs wild-type, it was normal in both FcR&ggr;−/−; TG-CRP and Fc&ggr;RIIB−/−; TG-CRP mice. Conclusions: CRP antagonism of eNOS is mediated by the coupling of Fc&ggr;RI to Fc&ggr;RIIB by Src kinase and resulting activation of SH2 domain-containing inositol 5′-phosphatase 1, and consistent with this mechanism, both Fc&ggr;RI and Fc&ggr;RIIB are required for CRP to blunt endothelial repair in vivo. Similar mechanisms underlie eNOS antagonism by immune complex. Fc&ggr;RI and Fc&ggr;RIIB may be novel therapeutic targets for preventing endothelial dysfunction in inflammatory or immune complex-mediated conditions.

Fcγ Receptors and Ligands and Cardiovascular Disease

Fc&ggr; receptors (Fc&ggr;Rs) classically modulate intracellular signaling on binding of the Fc region of IgG in immune response cells. How Fc&ggr;R and their ligands affect cardiovascular health and disease has been interrogated recently in both preclinical and clinical studies. The stimulation of activating Fc&ggr;R in endothelial cells, vascular smooth muscle cells, and monocytes/macrophages causes a variety of cellular responses that may contribute to vascular disease pathogenesis. Stimulation of the lone inhibitory F&ggr;cR, Fc&ggr;RIIB, also has adverse consequences in endothelial cells, antagonizing NO production and reparative mechanisms. In preclinical disease models, activating Fc&ggr;Rs promote atherosclerosis, whereas Fc&ggr;RIIB is protective, and activating Fc&ggr;Rs also enhance thrombotic and nonthrombotic vascular occlusion. The Fc&ggr;R ligand C-reactive protein (CRP) has undergone intense study. Although in rodents CRP does not affect atherosclerosis, it causes hypertension and insulin resistance and worsens myocardial infarction. Massive data have accumulated indicating an association between increases in circulating CRP and coronary heart disease in humans. However, Mendelian randomization studies reveal that CRP is not likely a disease mediator. CRP genetics and hypertension warrant further investigation. To date, studies of genetic variants of activating Fc&ggr;Rs are insufficient to implicate the receptors in coronary heart disease pathogenesis in humans. However, a link between Fc&ggr;RIIB and human hypertension may be emerging. Further knowledge of the vascular biology of Fc&ggr;R and their ligands will potentially enhance our understanding of cardiovascular disorders, particularly in patients whose greater predisposition for disease is not explained by traditional risk factors, such as individuals with autoimmune disorders.

IgG Receptor FcγRIIB Plays a Key Role in Obesity-Induced Hypertension

There is a well-recognized association between obesity, inflammation, and hypertension. Why obesity causes hypertension is poorly understood. We previously demonstrated using a C-reactive protein (CRP) transgenic mouse that CRP induces hypertension that is related to NO deficiency. Our prior work in cultured endothelial cells identified the Fc&ggr; receptor IIB (Fc&ggr;RIIB) as the receptor for CRP whereby it antagonizes endothelial NO synthase. Recognizing known associations between CRP and obesity and hypertension in humans, in the present study we tested the hypothesis that Fc&ggr;RIIB plays a role in obesity-induced hypertension in mice. Using radiotelemetry, we first demonstrated that the hypertension observed in transgenic mouse-CRP is mediated by the receptor, indicating that Fc&ggr;RIIB is capable of modifying blood pressure. We then discovered in a model of diet-induced obesity yielding equal adiposity in all study groups that whereas Fc&ggr;RIIB+/+ mice developed obesity-induced hypertension, Fc&ggr;RIIB−/− mice were fully protected. Levels of CRP, the related pentraxin serum amyloid P component which is the CRP-equivalent in mice, and total IgG were unaltered by diet-induced obesity; Fc&ggr;RIIB expression in endothelium was also unchanged. However, whereas IgG isolated from chow-fed mice had no effect, IgG from high-fat diet–fed mice inhibited endothelial NO synthase in cultured endothelial cells, and this was an Fc&ggr;RIIB-dependent process. Thus, we have identified a novel role for Fc&ggr;RIIB in the pathogenesis of obesity-induced hypertension, independent of processes regulating adiposity, and it may entail an IgG-induced attenuation of endothelial NO synthase function. Approaches targeting Fc&ggr;RIIB may potentially offer new means to treat hypertension in obese individuals.

Conjoined twins: Pre-birth management, changes to NRP, and transport

The management of conjoined twins is complex and requires careful preparation. Pre-birth management includes prenatal counseling, which is important due to the overall poor prognosis. In instances of trial of life, the delivery must be tailored to address the anticipated anatomy based on prenatal imaging and anticipated physiology. A multidisciplinary team is essential to anticipate and address the ergonomic challenges and medical issues related to organ fusion, cross-circulation and associated anomalies. There are several suggested modifications to the current Neonatal Resuscitation Program algorithm including modifications to initial assessment, airway management, administration of chest compressions, obtaining emergency access, and medication dosing. Simulation is essential to address challenges, practice Neonatal Resuscitation Program modifications, delineate clear roles during delivery and practice communication. This paper offers a discussion of unique issues associated with delivery of conjoined twins and recommendations on how to approach these challenges based on our experience and available literature.

Capture-based DNA methylation sequencing facilitates diagnosis and reveals potential pathogenic mechanisms in teratogenic diabetes exposure

Diabetic embryopathy (DE) describes a spectrum of birth defects associated with a teratogenic exposure to maternal diabetes in utero. These defects strongly overlap the phenotypes of known genetic syndromes; however, the pathogenic mechanisms underlying DE remain uncertain and there are no definitive tests that distinguish the diagnosis. Here, we explore the potential of DNA methylation as both a diagnostic biomarker and a means of informing disease pathogenesis in DE. Capture-based bisulfite sequencing was used to compare patterns of DNA methylation at 2,800,516 sites genome-wide in seven DE neonates and 11 healthy neonates, including five with in utero diabetes exposure. DE infants had significantly lower global DNA methylation (ANOVA, Tukey HSD p=0.045) than diabetes-unexposed, healthy controls (UH), with multiple sites showing large (mean methylation difference = 16.6%) and significant (p<0.001) differential methylation between the two groups. We found that a subset of 237 highly differentially methylated loci could accurately distinguish DE infants from both UH and diabetes-exposed healthy infants (sensitivity 80% -100%). Differentially methylated sites were enriched in intergenic (p<3.52×10-15) and intronic (p<0.001) regions found proximal to genes either associated with Mendelian syndromes that overlap the DE phenotype (e.g. TRIO, ANKRD11), or known to influence early organ development (e.g. BRAX1, RASA3). Further, by integrating information on cis-sequence variation, we found that 39.3% of loci with evidence for allele-specific methylation also showed differential methylation between DE and controls. Our study suggests a role for aberrant DNA methylation and cis-sequence variation in the pathogenesis of DE, and highlights the diagnostic potential of DNA methylation for teratogenic birth defects.

Improving communication between obstetric and neonatology teams for high-risk deliveries: a quality improvement project

Summoning is a key component of communication between obstetrics and neonatal resuscitation team (NRT) in advance of deliveries. A paging system is a commonly used summoning tool. The timeliness and information contained in the page help NRT to optimally prepare for postdelivery infant care. Our aim was to increase the frequency that summoning pages contained gestational age and reason for NRT attendance to >90%. At baseline, 8% of pages contained gestational age and 33% of pages contained a reason for NRT attendance. Sequential Plan-Do-Study-Act cycles were used as our model for quality improvement. During the 8-month improvement period, the per cent of pages increased to 97% for gestational age and 97% for reason for NRT attendance. Measures of page timeliness, our balancing measure, did not change. Summoning communication between obstetric and NRT is crucial for optimal perinatal outcomes. The active involvement of all stakeholders throughout the project resulted in the development of a standardised paging tool and a more informative paging process, which is a key communication tool used in many centres.

Coupling of Fcγ Receptor I to Fcγ Receptor IIB by Src Kinase Mediates C-Reactive Protein Impairment of Endothelial FunctionNovelty and Significance

Rationale: Elevations in C-reactive protein (CRP) are associated with increased cardiovascular disease risk and endothelial dysfunction. CRP antagonizes endothelial nitric oxide synthase (eNOS) through processes mediated by the IgG receptor Fc&ggr; receptor IIB (Fc&ggr;RIIB), its immunoreceptor tyrosine-based inhibitory motif, and SH2 domain-containing inositol 5′-phosphatase 1. In mice, CRP actions on eNOS blunt carotid artery re-endothelialization. Objective: How CRP activates Fc&ggr;RIIB in endothelium is not known. We determined the role of Fc&ggr; receptor I (Fc&ggr;RI) and the basis for coupling of Fc&ggr;RI to Fc&ggr;RIIB in endothelium. Methods and Results: In cultured endothelial cells, Fc&ggr;RI-blocking antibodies prevented CRP antagonism of eNOS, and CRP activated Src via Fc&ggr;RI. CRP-induced increases in Fc&ggr;RIIB immunoreceptor tyrosine-based inhibitory motif phosphorylation and SH2 domain-containing inositol 5′-phosphatase 1 activation were Src-dependent, and Src inhibition prevented eNOS antagonism by CRP. Similar processes mediated eNOS antagonism by aggregated IgG used to mimic immune complex. Carotid artery re-endothelialization was evaluated in offspring from crosses of CRP transgenic mice (TG-CRP) with either mice lacking the &ggr; subunit of Fc&ggr;RI (FcR&ggr;−/−) or Fc&ggr;RIIB−/− mice. Whereas re-endothelialization was impaired in TG-CRP vs wild-type, it was normal in both FcR&ggr;−/−; TG-CRP and Fc&ggr;RIIB−/−; TG-CRP mice. Conclusions: CRP antagonism of eNOS is mediated by the coupling of Fc&ggr;RI to Fc&ggr;RIIB by Src kinase and resulting activation of SH2 domain-containing inositol 5′-phosphatase 1, and consistent with this mechanism, both Fc&ggr;RI and Fc&ggr;RIIB are required for CRP to blunt endothelial repair in vivo. Similar mechanisms underlie eNOS antagonism by immune complex. Fc&ggr;RI and Fc&ggr;RIIB may be novel therapeutic targets for preventing endothelial dysfunction in inflammatory or immune complex-mediated conditions.

Coupling of FcγRI to FcγRIIB by Src Kinase Mediates C-reactive Protein Impairment of Endothelial Function

Rationale: Elevations in C-reactive protein (CRP) are associated with increased cardiovascular disease risk and endothelial dysfunction. CRP antagonizes endothelial nitric oxide synthase (eNOS) through processes mediated by the IgG receptor Fc&ggr; receptor IIB (Fc&ggr;RIIB), its immunoreceptor tyrosine-based inhibitory motif, and SH2 domain-containing inositol 5′-phosphatase 1. In mice, CRP actions on eNOS blunt carotid artery re-endothelialization. Objective: How CRP activates Fc&ggr;RIIB in endothelium is not known. We determined the role of Fc&ggr; receptor I (Fc&ggr;RI) and the basis for coupling of Fc&ggr;RI to Fc&ggr;RIIB in endothelium. Methods and Results: In cultured endothelial cells, Fc&ggr;RI-blocking antibodies prevented CRP antagonism of eNOS, and CRP activated Src via Fc&ggr;RI. CRP-induced increases in Fc&ggr;RIIB immunoreceptor tyrosine-based inhibitory motif phosphorylation and SH2 domain-containing inositol 5′-phosphatase 1 activation were Src-dependent, and Src inhibition prevented eNOS antagonism by CRP. Similar processes mediated eNOS antagonism by aggregated IgG used to mimic immune complex. Carotid artery re-endothelialization was evaluated in offspring from crosses of CRP transgenic mice (TG-CRP) with either mice lacking the &ggr; subunit of Fc&ggr;RI (FcR&ggr;−/−) or Fc&ggr;RIIB−/− mice. Whereas re-endothelialization was impaired in TG-CRP vs wild-type, it was normal in both FcR&ggr;−/−; TG-CRP and Fc&ggr;RIIB−/−; TG-CRP mice. Conclusions: CRP antagonism of eNOS is mediated by the coupling of Fc&ggr;RI to Fc&ggr;RIIB by Src kinase and resulting activation of SH2 domain-containing inositol 5′-phosphatase 1, and consistent with this mechanism, both Fc&ggr;RI and Fc&ggr;RIIB are required for CRP to blunt endothelial repair in vivo. Similar mechanisms underlie eNOS antagonism by immune complex. Fc&ggr;RI and Fc&ggr;RIIB may be novel therapeutic targets for preventing endothelial dysfunction in inflammatory or immune complex-mediated conditions.