Amy Opoka

Maladaptive Matrix Remodeling and Regional Biomechanical Dysfunction in a Mouse Model of Aortic Valve Disease

Aortic valve disease (AVD) occurs in 2.5% of the general population and often requires surgical intervention. Aortic valve malformation (AVM) underlies the majority of cases, suggesting a developmental etiology. Elastin haploinsufficiency results in complex cardiovascular problems, and 20-45% of patients have AVM and/or AVD. Elastin insufficient (Eln+/-) mice demonstrate AVM and latent AVD due to abnormalities in the valve annulus region. The objective of this study was to examine extracellular matrix (ECM) remodeling and biomechanical properties in regional aortic valve tissue and determine the impact of early AVM on late AVD in the Eln+/- mouse model. Aortic valve ECM composition and remodeling from juvenile, adult, and aged stages were evaluated in Eln+/- mice using histology, ELISA, immunohistochemistry and gelatin zymography. Aortic valve tissue biomechanical properties were determined using micropipette aspiration. Cartilage-like nodules were demonstrated within the valve annulus region at all stages identifying a developmental abnormality preceding AVD. Interestingly, maladaptive ECM remodeling was observed in early AVM without AVD and worsened with late AVD, as evidenced by increased MMP-2 and MMP-9 expression and activity, as well as abnormalities in ADAMTS-mediated versican processing. Cleaved versican was increased in the valve annulus region of aged Eln+/- mice, and this abnormality correlated temporally with adverse alterations in valve tissue biomechanical properties and the manifestation of AVD. These findings identify maladaptive ECM remodeling in functional AVM as an early disease process with a progressive natural history, similar to that seen in human AVD, emphasizing the importance of the annulus region in pathogenesis. Combining molecular and engineering approaches provides complementary mechanistic insights that may be informative in the search for new therapeutic targets and durable valve bioprostheses.

TGF-β mediates early angiogenesis and latent fibrosis in an Emilin1-deficient mouse model of aortic valve disease.

Aortic valve disease (AVD) is characterized by elastic fiber fragmentation (EFF), fibrosis and aberrant angiogenesis. Emilin1 is an elastin-binding glycoprotein that regulates elastogenesis and inhibits TGF-β signaling, but the role of Emilin1 in valve tissue is unknown. We tested the hypothesis that Emilin1 deficiency results in AVD, mediated by non-canonical (MAPK/phosphorylated Erk1 and Erk2) TGF-β dysregulation. Using histology, immunohistochemistry, electron microscopy, quantitative gene expression analysis, immunoblotting and echocardiography, we examined the effects of Emilin1 deficiency (Emilin1−/−) in mouse aortic valve tissue. Emilin1 deficiency results in early postnatal cell-matrix defects in aortic valve tissue, including EFF, that progress to latent AVD and premature death. The Emilin1−/− aortic valve displays early aberrant provisional angiogenesis and late neovascularization. In addition, Emilin1−/− aortic valves are characterized by early valve interstitial cell activation and proliferation and late myofibroblast-like cell activation and fibrosis. Interestingly, canonical TGF-β signaling (phosphorylated Smad2 and Smad3) is upregulated constitutively from birth to senescence, whereas non-canonical TGF-β signaling (phosphorylated Erk1 and Erk2) progressively increases over time. Emilin1 deficiency recapitulates human fibrotic AVD, and advanced disease is mediated by non-canonical (MAPK/phosphorylated Erk1 and Erk2) TGF-β activation. The early manifestation of EFF and aberrant angiogenesis suggests that these processes are crucial intermediate factors involved in disease progression and therefore might provide new therapeutic targets for human AVD.

Olfactomedin-4 Is a Candidate Marker for a Pathogenic Neutrophil Subset in Septic Shock

Objectives: Heterogeneity in sepsis-related pathobiology presents a significant challenge. Resolving this heterogeneity presents an opportunity to understand pathobiology and improve patient care. Olfactomedin-4 is a neutrophil subset marker and may contribute to sepsis heterogeneity. Our objective was to evaluate the expression of olfactomedin-4 and characterize neutrophil heterogeneity in children with septic shock. Design: Single-center, prospective cohort, as well as secondary analysis of existing transcriptomic and proteomic databases. Setting: Tertiary care PICU. Patients: Patients from 5 days to 18 years old with septic shock were enrolled. Data collected included the expression of olfactomedin-4 messenger RNA, serum protein concentrations, and percentage of neutrophils that express olfactomedin-4. Interventions: None. Measurements and Main Results: Secondary analysis of existing transcriptomic data demonstrated that olfactomedin-4 is the most highly expressed gene in nonsurvivors of pediatric septic shock, compared with survivors. Secondary analysis of an existing proteomic database corroborated these observations. In a prospectively enrolled cohort, we quantified the percentage of olfactomedin-4+ neutrophils in patients with septic shock. Patients with a complicated course, defined as greater than or equal to two organ failures at day 7 of septic shock or 28-day mortality, had a higher percentage of olfactomedin-4+ neutrophils, compared with those without a complicated course. By logistic regression, the percentage of olfactomedin-4+ neutrophils was independently associated with increased risk of a complicated course (odds ratio, 1.09; 95% CI, 1.01–1.17; p = 0.024). Conclusions: Olfactomedin-4 identifies a subpopulation of neutrophils in patients with septic shock, and those with a high percentage of olfactomedin-4+ neutrophils are at higher risk for greater organ failure burden and death. Olfactomedin-4 might serve as a marker of a pathogenic neutrophil subset in patients with septic shock.

Inhibition of MAPK‐Erk pathway in vivo attenuates aortic valve disease processes in Emilin1‐deficient mouse model

Aortic valve disease (AVD) is a common condition with a progressive natural history, and presently, there are no pharmacologic treatment strategies. Elastic fiber fragmentation (EFF) is a hallmark of AVD, and increasing evidence implicates developmental elastic fiber assembly defects. Emilin1 is a glycoprotein necessary for elastic fiber assembly that is present in both developing and mature human and mouse aortic valves. The Emilin1‐deficient mouse (Emilin1−/−) is a model of latent AVD, characterized by activated TGFβ/MEK/p‐Erk signaling and upregulated elastase activity. Emilin1−/− aortic valves demonstrate early EFF and aberrant angiogenesis followed by late neovascularization and fibrosis. The objective of this study was to test the effectiveness of three different targeted therapies. Aged (12–14 months) Emilin1−/− mice were treated with refametinib (RDEA‐119, MEK1/2 inhibitor), doxycycline (elastase inhibitor), or G6‐31 (anti‐VEGF‐A mouse antibody) for 4 weeks. Refametinib‐ and doxycycline‐treated Emilin1−/− mice markedly reduced MEK/p‐Erk activation in valve tissue. Furthermore, both refametinib and doxycycline attenuated elastolytic cathepsin K, L, MMP‐2, and MMP‐9 activation, and abrogated macrophage and neutrophil infiltration in Emilin1−/− aortic valves. RNAseq analysis was performed in aortic valve tissue from adult (4 months) and aged (14 months) Emilin1−/− and age‐matched wild‐type control mice, and demonstrated upregulation of genes associated with MAPK/MEK/p‐Erk signaling and elastases at the adult stage and inflammatory pathways at the aged stage controlling for age. These results suggest that Erk1/2 signaling is an important modulator of early elastase activation, and pharmacological inhibition using refametinib may be a promising treatment to halt AVD progression

Glucocorticoid Receptor Polymorphisms and Outcomes in Pediatric Septic Shock.

Objective: Polymorphisms of the glucocorticoid receptor gene are associated with outcome and corticosteroid responsiveness among patients with inflammatory disorders. We conducted a candidate gene association study to test the hypothesis that these polymorphisms are associated with outcome and corticosteroid responsiveness among children with septic shock. Design: We genotyped 482 children with septic shock for the presence of two glucocorticoid receptor polymorphisms (rs56149945 and rs41423247) associated with increased sensitivity and one glucocorticoid receptor polymorphism (rs6198) associated with decreased sensitivity to corticosteroids. The primary outcome variable was complicated course, defined as 28-day mortality or the persistence of two or more organ failures 7 days after a septic shock diagnosis. We used logistic regression to test for an association between corticosteroid exposure and outcome, within genotype group, and adjusted for illness severity. Setting: Multiple PICUs in the United States. Interventions: Standard care. Measurements and Main Results: There were no differences in outcome when comparing the various genotype groups. Among patients homozygous for the wild-type glucocorticoid receptor allele, corticosteroids were independently associated with increased odds of complicated course (odds ratio, 2.30; 95% CI, 1.01–5.21; p = 0.047). Conclusions: Based on these glucocorticoid receptor polymorphisms, we could not detect a beneficial effect of corticosteroids among any genotype group. Among children homozygous for the wild-type allele, corticosteroids were independently associated with increased odds of poor outcome.

Zinc supplementation leads to immune modulation and improved survival in a juvenile model of murine sepsis

Children with severe sepsis are known to have altered zinc homeostasis and decreased circulating zinc levels, suggesting a role for zinc supplementation to improve outcomes. We tested the hypothesis that zinc supplementation would improve survival in a juvenile model of polymicrobial sepsis. Juvenile (13–14-d-old) C57BL/6 mice were treated with 10 mg/kg of zinc via i.p. injections (or vehicle) for 3 d prior to induction of polymicrobial sepsis via i.p. cecal slurry injections. Survival after sepsis was followed for 3 d, and bacterial clearance, ex vivo phagocytosis, systemic inflammatory markers and neutrophil extracellular trap (NET) formation were quantified. We found a significant survival benefit and decreased bacterial burden among zinc supplemented mice when compared with the control group. Zinc supplementation also resulted in enhanced phagocytic activity, greater neutrophil recruitment in the peritoneal cavity and NET formation, suggesting a possible mechanism for improved bacterial clearance and survival. We also noted decreased serum cytokine levels and decreased myeloperoxidase activity in lung tissue following zinc supplementation, suggesting attenuation of the systemic inflammatory response. In conclusion, zinc supplementation improves bacterial clearance, and hence survival, in juvenile mice with polymicrobial sepsis.

Cross Talk between NOTCH Signaling and Biomechanics in Human Aortic Valve Disease Pathogenesis

Aortic valve disease is a burgeoning public health problem associated with significant mortality. Loss of function mutations in NOTCH1 cause bicuspid aortic valve (BAV) and calcific aortic valve disease. Because calcific nodules manifest on the fibrosa side of the cusp in low fluidic oscillatory shear stress (OSS), elucidating pathogenesis requires approaches that consider both molecular and mechanical factors. Therefore, we examined the relationship between NOTCH loss of function (LOF) and biomechanical indices in healthy and diseased human aortic valve interstitial cells (AVICs). An orbital shaker system was used to apply cyclic OSS, which mimics the cardiac cycle and hemodynamics experienced by AVICs in vivo. NOTCH LOF blocked OSS-induced cell alignment in human umbilical vein endothelial cells (HUVECs), whereas AVICs did not align when subjected to OSS under any conditions. In healthy AVICs, OSS resulted in decreased elastin (ELN) and α-SMA (ACTA2). NOTCH LOF was associated with similar changes, but in diseased AVICs, NOTCH LOF combined with OSS was associated with increased α-SMA expression. Interestingly, AVICs showed relatively higher expression of NOTCH2 compared to NOTCH1. Biomechanical interactions between endothelial and interstitial cells involve complex NOTCH signaling that contributes to matrix homeostasis in health and disorganization in disease.

The glucocorticoid receptor and cortisol levels in pediatric septic shock

BackgroundThere is controversy around the prescription of adjunct corticosteroids in patients with fluid-refractory septic shock, and studies provide mixed results, showing benefit, no benefit, and harm. Traditional means for evaluating whether a patient receives corticosteroids relied on anecdotal experience or measurement of serum cortisol production following stimulation. We set out to measure both serum cortisol and the intracellular signaling receptor for cortisol, the glucocorticoid receptor (GCR), in this group of patients.MethodsWe enrolled pediatric patients admitted to the pediatric intensive care unit with a diagnosis of systemic inflammatory response syndrome (SIRS), sepsis, or septic shock as well as healthy controls. We measured serum cortisol concentration and GCR expression by flow cytometry in peripheral blood leukocytes on the day of admission and day 3.ResultsWe enrolled 164 patients for analysis. There was no difference between GCR expression comparing SIRS, sepsis, and septic shock. When all patients with septic shock were compared, those patients with a complicated course, defined as two or more organ failures at day 7 or death by day 28, had lower expression of GCR in all peripheral blood leukocytes. Further analysis suggested that patients with the combination of low GCR and high serum cortisol had higher rates of complicated course (75%) compared with the other three possible combinations of GCR and cortisol levels: low GCR and low cortisol (33%), high GCR and high cortisol (33%), and high GCR and low cortisol (13%; P <0.05).ConclusionsWe show that decreased expression of the GCR correlated with poor outcome from septic shock, particularly in those patients with high serum cortisol. This is consistent with findings from transcriptional studies showing that downregulation of GCR signaling genes portends worse outcome.

Preliminary Evidence for Aortopathy and an X-Linked Parent-of-Origin Effect on Aortic Valve Malformation in a Mouse Model of Turner Syndrome

Turner syndrome (TS), most frequently caused by X-monosomy (45,X), is characterized in part by cardiovascular abnormalities, including aortopathy and bicuspid aortic valve (BAV). There is a need for animal models that recapitulate the cardiovascular manifestations of TS. Extracellular matrix (ECM) organization and morphometrics of the aortic valve and proximal aorta were examined in adult 39,XO mice (where the parental origin of the single X was paternal (39,XPO) or maternal (39,XMO)) and 40,XX controls. Aortic valve morphology was normal (tricuspid) in all of the 39,XPO and 40,XX mice studied, but abnormal (bicuspid or quadricuspid) in 15% of 39,XMO mice. Smooth muscle cell orientation in the ascending aorta was abnormal in all 39,XPO and 39,XMO mice examined, but smooth muscle actin was decreased in 39,XMO mice only. Aortic dilation was present with reduced penetrance in 39,XO mice. The 39,XO mouse demonstrates aortopathy and an X-linked parent-of-origin effect on aortic valve malformation, and the candidate gene FAM9B is polymorphically expressed in control and diseased human aortic valves. The 39,XO mouse model may be valuable for examining the mechanisms underlying the cardiovascular findings in TS, and suggest there are important genetic modifiers on the X chromosome that modulate risk for nonsyndromic BAV and aortopathy.