Early Aberrant Angiogenesis Due to Elastic Fiber Fragmentation in Aortic Valve Disease.

Elastic fiber fragmentation (EFF) is a hallmark of aortic valve disease (AVD), and neovascularization has been identified as a late finding related to inflammation. We sought to characterize the relationship between early EFF and aberrant angiogenesis. To examine disease progression, regional anatomy and pathology of aortic valve tissue were assessed using histochemistry, immunohistochemistry, and electron microscopy from early-onset (<40 yo) and late-onset (≥40 yo) non-syndromic AVD specimens. To assess the effects of EFF on early AVD processes, valve tissue from Williams and Marfan syndrome patients was also analyzed. Bicuspid aortic valve was more common in early-onset AVD, and cardiovascular comorbidities were more common in late-onset AVD. Early-onset AVD specimens demonstrated angiogenesis without inflammation or atherosclerosis. A distinct pattern of elastic fiber components surrounded early-onset AVD neovessels, including increased emilin-1 and decreased fibulin-5. Different types of EFF were present in Williams syndrome (WS) and Marfan syndrome (MFS) aortic valves; WS but not MFS aortic valves demonstrated angiogenesis. Aberrant angiogenesis occurs in early-onset AVD in the absence of inflammation, implicating EFF. Elucidation of underlying mechanisms may inform the development of new pharmacologic treatments.

Olfactomedin 4-Positive Neutrophils Are Upregulated after Hemorrhagic Shock.

Neutrophils are vital to both the inflammatory cascade and tissue repair after an injury. Neutrophil heterogeneity is well established but there is less evidence for significant, different functional roles for neutrophil subsets. OLFM4 (Olfactomedin-4) is expressed by a subset of neutrophils, and high expression of OLFM4 is associated with worse outcomes in patients with sepsis and acute respiratory distress syndrome. We hypothesized that an increased number of OLFM4+ neutrophils would occur in trauma patients with worse clinical outcomes. To test this, we prospectively enrolled patients who suffered a blunt traumatic injury. Blood was collected at the time of admission, Day 3, and Day 7 and analyzed for the percentage of neutrophils expressing OLFM4. We found that a subset of patients who suffered blunt traumatic injury upregulated their percentage of OLFM4+ neutrophils. Those who upregulated their OLFM4 had an increased length of stay, days in the ICU, and ventilator days. A majority of these patients also suffered from hemorrhagic shock. To establish a potential role for OLFM4+ neutrophils, we used a murine model of hemorrhagic shock because mice also express OLFM4 in a subset of neutrophils. These studies demonstrated that wild type mice had higher concentrations of cytokines in the plasma and myeloperoxidase in the lungs compared with OLFM4-null mice. In addition, we used an anti-OLFM4 antibody, which when given to wild type mice led to the reduction of myeloperoxidase in the lungs of mice. These findings suggest that OLFM4+ neutrophils are a unique subset of neutrophils that affect the inflammatory response after tissue injury.

Glucocorticoid Receptor Polymorphisms in Children Undergoing Congenital Heart Surgery with Cardiopulmonary Bypass.

We conducted a candidate gene association study to test the hypothesis that different gene polymorphisms will be associated with corticosteroid responsiveness and study outcomes among children undergoing congenital heart surgery. This is a prospective observational cohort study at a large, tertiary pediatric cardiac center on children undergoing corrective or palliative congenital heart surgery. A total of 83 children were enrolled. DNA was isolated for three polymorphisms of interest namely N363 (rs56149945) and 9ß (rs6198) associated with increased sensitivity to corticosteroids and Bcl I (rs41423247) associated with decreased sensitivity to corticosteroids. Duration of inotropic use, low cardiac output scores (LCOS), and vasoactive inotrope scores were examined in relation to these three polymorphisms. Using Kaplan-Meier analysis, heterozygous individuals showed longer transcriptional intermediary factor (TIF) compared with wild type for N363 polymorphism ( p = 0.05). In multivariable Cox regression, heterozygous alleles for 9ß polymorphism showed significantly shorter TIF compared with wild type (hazard ratio = 2.04 [1.08-3.87], p = 0.03). The relationship between lower LCOS scores and alleles groups was significant for 9ß heterozygous polymorphism only (1.5 [1-2.2], p = 0.01) in comparison to wild type and homozygous. The presence of heterozygote alleles for the increased corticosteroid sensitivity is associated with longer TIF compared with wild type. Conversely, the presence of heterozygous alleles for the decreased sensitivity to corticosteroids is associated with shorter TIF compared with wild type.

Longitudinal characterization of olfactomedin-4 expressing neutrophils in pediatric patients undergoing bone marrow transplantation.

Sepsis is an important cause of morbidity and mortality in pediatric patients. Increased expression of olfactomedin-4 (OLFM4), a glycoprotein contained within a subpopulation of neutrophils, has been associated with complicated course in sepsis. The factors that regulate OLFM4 expression are unknown. Here, we followed children undergoing bone marrow transplantation (BMT) to document the percentage of neutrophils that express OLFM4 over time. This population was selected because of the ability to observe nascent neutrophils following engraftment, perform frequent blood sampling, and the children are at high risk for clinical complications that may associate with changes in percentage of OLFM4+ neutrophils. We found a surprising degree of variability of OLFM4 expression between patients. In the weeks following initial neutrophil recovery we also saw great variability in OLFM4 expression within individual patients, indicating that multiple external factors may modify OLFM4 expression. We identified decreased expression of CD64 (a marker associated with response to infection), in OLFM4+ neutrophils. This is the first study to demonstrate fluctuation in OLFM4 expression within patients and provides insight into possible mechanisms for OLFM4 regulation in nascent neutrophils.

Juvenile OLFM4-null mice are protected from sepsis.

Pediatric sepsis is a leading cause of morbidity and mortality in children. One of the most common and devastating morbidities is sepsis-related acute kidney injury (AKI). AKI was traditionally thought to be related to low perfusion and acute tubular necrosis. However, little acute tubular necrosis can be found following septic AKI, and little is known about the mechanism of septic AKI. Olfactomedin-4 (OLFM4) is a secreted glycoprotein that marks a subset of neutrophils. Increased expression of OLFM4 in the blood is associated with worse outcomes in sepsis. Here, we investigated a pediatric model of murine sepsis using murine pups to investigate the mechanisms of OLFM4 in sepsis. When sepsis was induced in murine pups, survival was significantly increased in OLFM4-null pups. Immunohistochemistry at 24 h after the induction of sepsis demonstrated increased expression of OLFM4 in the kidney, which was localized to the loop of Henle. Renal cell apoptosis and plasma creatinine were significantly increased in wild-type versus OLFM4-null pups. Finally, bone marrow transplant suggested that increased OLFM4 in the kidney reflects local production rather than filtered from the plasma. These results demonstrate renal expression of OLFM4 for the first time and suggest that a kidney-specific mechanism may contribute to survival differences in OLFM4-null animals.

The olfactomedin-4 positive neutrophil has a role in murine intestinal ischemia/reperfusion injury.

Olfactomedin-4 (OLFM4) identifies a subset of neutrophils conserved in both mouse and man, associated with worse outcomes in several inflammatory conditions. We investigated the role of OLFM4-positive neutrophils in murine intestinal ischemia/reperfusion (IR) injury. Wild-type (WT) C57Bl/6 and OLFM4 null mice were subjected to intestinal IR injury and then monitored for survival or tissues harvested for further analyses. In vivo intestinal barrier function was determined via functional assay of permeability to FITC-dextran. OLFM4 null mice had a significant 7-d survival benefit and less intestinal barrier dysfunction compared with WT. Early after IR, WT mice had worse mucosal damage on histologic examination. Experiments involving adoptive transfer of bone marrow demonstrated that the mortality phenotype associated with OLFM4-positive neutrophils was transferrable to OLFM4 null mice. After IR injury, WT mice also had increased intestinal tissue activation of NFκB and expression of iNOS, 2 signaling pathways previously demonstrated to be involved in intestinal IR injury. In combination, these experiments show that OLFM4-positive neutrophils are centrally involved in the pathologic pathway leading to intestinal damage and mortality after IR injury. This may provide a therapeutic target for mitigation of intestinal IR injury in a variety of common clinical situations.-Levinsky, N. C., Mallela, J., Opoka, A., Harmon, K., Lewis, H. V., Zingarelli, B., Wong, H. R., Alder, M. N. The olfactomedin-4 positive neutrophil has a role in murine intestinal ischemia/reperfusion injury.

Olfactomedin 4 marks a subset of neutrophils in mice.

Neutrophils are the most abundant immune cell of the innate immune system and participate in essential immune functions. Heterogeneity within neutrophils has been documented, but it is difficult to distinguish if these are altered activation states of a single population or separate subpopulations of neutrophils determined at the time of differentiation. Several groups have identified a subset of human neutrophils that express olfactomedin 4 (OLFM4) and increased OLFM4+ neutrophils during sepsis is correlated with worse outcome, suggesting these neutrophils or the OLFM4 they secrete may be pathogenic. We tested if mice could be used as a model to study OLFM4+ neutrophils. We found the OLFM4 expressing subset of neutrophils is conserved in mice. Depending on the strain, 7-35% of murine neutrophils express OLFM4 and expression is determined early in neutrophil differentiation. OLFM4+ neutrophils phagocytose and transmigrate with similar efficiency as OLFM4- neutrophils. Here we show that within neutrophil extracellular traps (NETs) OLFM4+ and OLFM4- neutrophils undergo NETosis and OLFM4 colocalizes. Finally, we generated an OLFM4 null mouse and show that these mice are protected from death when challenged with sepsis, providing further evidence that the OLFM4 expressing subpopulation of neutrophils, or the OLFM4 they secrete, may be pathogenic during overwhelming infection.

The glucocorticoid receptor and cortisol levels in pediatric septic shock.

BACKGROUND: There 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. METHODS: We 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. RESULTS: We 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). CONCLUSIONS: We 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.

Characterization of the Glucocorticoid Receptor in Children Undergoing Cardiac Surgery.

OBJECTIVES: Postoperative administration of corticosteroids is common practice for managing catecholamine refractory low cardiac output syndrome. Since corticosteroid activity is dependent on the glucocorticoid receptor, we sought to characterize glucocorticoid receptor levels in children undergoing cardiac surgery and examined the association between glucocorticoid receptor levels and cardiovascular dysfunction. DESIGN: Prospective observational cohort study. SETTING: Large, tertiary pediatric cardiac center. SUBJECTS: Children undergoing corrective or palliative cardiac surgery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A prospective observational cohort study was conducted in 83 children with congenital heart disease. Total glucocorticoid receptor levels were measured in the peripheral WBCs using flow cytometry. In addition, blood samples were collected for total cortisol levels. The primary outcome studied was the time to being inotrope free. An increase in glucocorticoid receptor level from postoperative day 1 to postoperative day 3 was associated with a longer time to being inotrope free (hazard ratio, 0.49 [0.29-0.81]; p = 0.01) in the univariate analysis. This association remained significant after adjusting for age, weight, cardiopulmonary bypass time, cross clamp time, Risk Adjustment for Congenital Heart Surgery-1 score, and postoperative steroid use (hazard ratio, 0.53 [0.29-0.99]; p = 0.05). Postoperative day 3 glucocorticoid receptor level showed a trend to have longer time to being inotrope free (hazard ratio, 0.66 [0.42-1.02]; p = 0.0.06). The cortisol levels minimally increased during the study duration and did not correlate with glucocorticoid receptor levels. CONCLUSIONS: Increasing glucocorticoid receptor levels in peripheral WBCs of children undergoing cardiac surgery are associated with a longer time to being inotrope free. Cortisol levels minimally increased during the study duration. These results suggest that exposure to high-dose perioperative corticosteroids may suppress the hypothalamic-pituitary-adrenal axis leading to increase in glucocorticoid receptor levels in response to a low cortisol environment. Further studies are required to better delineate the interplay between glucocorticoid receptor levels, cortisol levels, corticosteroid exposure, and postoperative inotropic requirements.

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 TGFß/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.

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.

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.

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.

TGF-ß 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-ß 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-ß 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-ß signaling (phosphorylated Smad2 and Smad3) is upregulated constitutively from birth to senescence, whereas non-canonical TGF-ß 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-ß 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.

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.

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.