Mechanical ventilation uncouples synthesis and assembly of elastin and increases apoptosis in lungs of newborn mice. Prelude to defective alveolar septation during lung development?

Prolonged mechanical ventilation (MV) with O2-rich gas inhibits lung growth and causes excess, disordered accumulation of lung elastin in preterm infants, often resulting in chronic lung disease (CLD). Using newborn mice, in which alveolarization occurs postnatally, we designed studies to determine how MV with either 40% O2 or air might lead to dysregulated elastin production and impaired lung septation. MV of newborn mice for 8 h with either 40% O2 or air increased lung mRNA for tropoelastin and lysyl oxidase, relative to unventilated controls, without increasing lung expression of genes that regulate elastic fiber assembly (lysyl oxidase-like-1, fibrillin-1, fibrillin-2, fibulin-5, emilin-1). Serine elastase activity in lung increased fourfold after MV with 40% O2, but not with air. We then extended MV with 40% O2 to 24 h and found that lung content of tropoelastin protein doubled, whereas lung content of elastin assembly proteins did not change (lysyl oxidases, fibrillins) or decreased (fibulin-5, emilin-1). Quantitative image analysis of lung sections showed that elastic fiber density increased by 50% after MV for 24 h, with elastin distributed throughout the walls of air spaces, rather than at septal tips, as in control lungs. Dysregulation of elastin was associated with a threefold increase in lung cell apoptosis (TUNEL and caspase-3 assays), which might account for the increased air space size previously reported in this model. Our findings of increased elastin synthesis, coupled with increased elastase activity and reduced lung abundance of proteins that regulate elastic fiber assembly, could explain altered lung elastin deposition, increased apoptosis, and defective septation, as observed in CLD.     

Transforming growth factor-β regulates endothelin-1 signaling in the newborn mouse lung during hypoxia exposure

We have previously shown that inhibition of transforming growth factor-β (TGF-β) signaling attenuates hypoxia-induced inhibition of alveolar development and abnormal pulmonary vascular remodeling in the newborn mice and that endothelin-A receptor (ETAR) antagonists prevent and reverse the vascular remodeling. The current study tested the hypothesis that inhibition of TGF-β signaling attenuates endothelin-1 (ET-1) expression and thereby reduces effects of hypoxia on the newborn lung. C57BL/6 mice were exposed from birth to 2 wk of age to either air or hypoxia (12% O(2)) while being given either BQ610 (ETAR antagonist), BQ788 (ETBR antagonist), 1D11 (TGF-β neutralizing antibody), or vehicle. Lung function and development and TGF-β and ET-1 synthesis were assessed. Hypoxia inhibited alveolar development, decreased lung compliance, and increased lung resistance. These effects were associated with increased TGF-β synthesis and signaling and increased ET-1 synthesis. BQ610 (but not BQ788) improved lung function, without altering alveolar development or increased TGF-β signaling in hypoxia-exposed animals. Inhibition of TGF-β signaling reduced ET-1 in vivo, which was confirmed in vitro in mouse pulmonary endothelial, fibroblast, and epithelial cells. ETAR blockade improves function but not development of the hypoxic newborn lung. Reduction of ET-1 via inhibition of TGF-β signaling indicates that TGF-β is upstream of ET-1 during hypoxia-induced signaling in the newborn lung.     

Role of matrix metalloprotease-9 in hyperoxic injury in developing lung

Matrix metalloprotease-9 (MMP-9) is increased in lung injury following hyperoxia exposure in neonatal mice, in association with impaired alveolar development. We studied the role of MMP-9 in the mechanism of hyperoxia-induced functional and histological changes in neonatal mouse lung. Reduced alveolarization with remodeling of ECM is a major morbidity component of oxidant injury in developing lung. MMP-9 mediates oxidant injury in developing lung causing altered lung remodeling. Five-day-old neonatal wild-type (WT) and MMP-9 (-/-) mice were exposed to hyperoxia for 8 days. The lungs were inflation fixed, and sections were examined for morphometry. The mean linear intercept and alveolar counts were evaluated. Immunohistochemistry for MMP-9 and elastin was performed. MMP-2, MMP-9, type I collagen, and tropoelastin were measured by Western blot analysis. Lung quasistatic compliance was studied in anaesthetized mice. MMP-2 and MMP-9 were significantly increased in lungs of WT mice exposed to hyperoxia compared with controls. Immunohistochemistry showed an increase in MMP-9 in mesenchyme and alveolar epithelium of hyperoxic lungs. The lungs of hyperoxia-exposed WT mice had less gas exchange surface area and were less compliant compared with room air-exposed WT and hyperoxia-exposed MMP-9 (-/-) mice. Type I collagen and tropoelastin were increased in hyperoxia-exposed WT with aberrant elastin staining. These changes were ameliorated in hyperoxia-exposed MMP-9 (-/-) mice. MMP-9 plays an important role in the structural changes consequent to oxygen-induced lung injury. Blocking MMP-9 activity may lead to novel therapeutic approaches in preventing bronchopulmonary dysplasia.     

Disruption of TGF-β signaling in smooth muscle cell prevents elastase-induced abdominal aortic aneurysm

Transforming growth factor-β (TGF-β) signaling has been significantly implicated in the pathogenesis of aneurysm, prominently the initiation and progression of abdominal aortic aneurysm (AAA). Vascular smooth muscle cell (SMC) is the principal resident cell in aortic wall and is essential for its structure and function. However, the role of TGF-β pathway in SMC for the formation of AAA remains unknown. Therefore, the goal of the present study was to investigate the effect of TGF-β pathway in SMC for AAA pathogenesis, by using a genetical smooth muscle-specific (SM-specific) TGF-βtype II receptor (Tgfbr2) disruption animal model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2^f/f and MyhCre.Tgfbr2^WT/f) and their corresponding wild-type background mice (MyhCre.Tgfbr2^WT/WT) underwent AAA induction by infrarenal peri-adventitial application of elastase. Fourteen days after elastase treatment, the aortas were analyzed and indicated that disruption of 1 or 2 alleles of Tgfbr2 in SMC provided markedly step-wise protection from AAA formation. And elastin degradation, medial SMC loss, macrophage infiltration, and matrix metalloproteinases (MMP) expression were all significantly reduced in Tgfbr2 deletion mice. Our study demonstrated, for the first time, that the TGF-β signaling pathway in SMC plays a critical role in AAA and disruption can prevent the aneurysm formation.     

Hypoxia-induced inhibition of lung development is attenuated by the peroxisome proliferator-activated receptor-γ agonist rosiglitazone

Hypoxia enhances transforming growth factor-β (TGF-β) signaling, inhibiting alveolar development and causing abnormal pulmonary arterial remodeling in the newborn lung. We hypothesized that, during chronic hypoxia, reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling may contribute to, or be caused by, excessive TGF-β signaling. To determine whether PPAR-γ was reduced during hypoxia, C57BL/6 mice were exposed to hypoxia from birth to 2 wk and evaluated for PPAR-γ mRNA and protein. To determine whether rosiglitazone (RGZ, a PPAR-γ agonist) supplementation attenuated the effects of hypoxia, mice were exposed to air or hypoxia from birth to 2 wk in combination with either RGZ or vehicle, and measurements of lung histology, function, parameters related to TGF-β signaling, and collagen content were made. To determine whether excessive TGF-β signaling reduced PPAR-γ, mice were exposed to air or hypoxia from birth to 2 wk in combination with either TGF-β-neutralizing antibody or vehicle, and PPAR-γ signaling was evaluated. We observed that hypoxia reduced PPAR-γ mRNA and protein, in association with impaired alveolarization, increased TGF-β signaling, reduced lung compliance, and increased collagen. RGZ increased PPAR-γ signaling, with improved lung development and compliance in association with reduced collagen and TGF-β signaling. However, no reduction was noted in hypoxia-induced pulmonary vascular remodeling. Inhibition of hypoxia-enhanced TGF-β signaling increased PPAR-γ signaling. These results suggest that hypoxia-induced inhibition of lung development is associated with a mutually antagonistic relationship between reduced PPAR-γ and increased TGF-β signaling. PPAR-γ agonists may be of potential therapeutic significance in attenuating TGF-β signaling and improving alveolar development.     

Neonatal periostin knockout mice are protected from hyperoxia-induced alveolar simplication

In bronchopulmonary dysplasia (BPD), alveolar septae are thickened with collagen and α-smooth muscle actin, transforming growth factor (TGF)-β-positive myofibroblasts. Periostin, a secreted extracellular matrix protein, is involved in TGF-β-mediated fibrosis and myofibroblast differentiation. We hypothesized that periostin expression is required for hypoalveolarization and interstitial fibrosis in hyperoxia-exposed neonatal mice, an animal model for this disease. We also examined periostin expression in neonatal lung mesenchymal stromal cells and lung tissue of hyperoxia-exposed neonatal mice and human infants with BPD. Two-to-three day-old wild-type and periostin null mice were exposed to air or 75% oxygen for 14 days. Mesenchymal stromal cells were isolated from tracheal aspirates of premature infants. Hyperoxic exposure of neonatal mice increased alveolar wall periostin expression, particularly in areas of interstitial thickening. Periostin co-localized with α-smooth muscle actin, suggesting synthesis by myofibroblasts. A similar pattern was found in lung sections of infants dying of BPD. Unlike wild-type mice, hyperoxia-exposed periostin null mice did not show larger air spaces or α-smooth muscle-positive myofibroblasts. Compared to hyperoxia-exposed wild-type mice, hyperoxia-exposed periostin null mice also showed reduced lung mRNA expression of α-smooth muscle actin, elastin, CXCL1, CXCL2 and CCL4. TGF-β treatment increased mesenchymal stromal cell periostin expression, and periostin treatment increased TGF-β-mediated DNA synthesis and myofibroblast differentiation. We conclude that periostin expression is increased in the lungs of hyperoxia-exposed neonatal mice and infants with BPD, and is required for hyperoxia-induced hypoalveolarization and interstitial fibrosis.     

Dysregulation of pulmonary elastin synthesis and assembly in preterm lambs with chronic lung disease

Failed alveolar formation and excess, disordered elastin are key features of neonatal chronic lung disease (CLD). We previously found fewer alveoli and more elastin in lungs of preterm compared with term lambs that had mechanical ventilation (MV) with O(2)-rich gas for 3 wk (MV-3 wk). We hypothesized that, in preterm more than in term lambs, MV-3 wk would reduce lung expression of growth factors that regulate alveolarization (VEGF, PDGF-A) and increase lung expression of growth factors [transforming growth factor (TGF)-alpha, TGF-beta(1)] and matrix molecules (tropoelastin, fibrillin-1, fibulin-5, lysyl oxidases) that regulate elastin synthesis and assembly. We measured lung expression of these genes in preterm and term lambs after MV for 1 day, 3 days, or 3 wk, and in fetal controls. Lung mRNA for VEGF, PDGF-A, and their receptors (VEGF-R2, PDGF-Ralpha) decreased in preterm and term lambs after MV-3 wk, with reduced lung content of the relevant proteins in preterm lambs with CLD. TGF-alpha and TGF-beta(1) expression increased only in lungs of preterm lambs. Tropoelastin mRNA increased more with MV of preterm than term lambs, and expression levels remained high in lambs with CLD. In contrast, fibrillin-1 and lysyl oxidase-like-1 mRNA increased transiently, and lung abundance of other elastin-assembly genes/proteins was unchanged (fibulin-5) or reduced (lysyl oxidase) in preterm lambs with CLD. Thus MV-3 wk reduces lung expression of growth factors that regulate alveolarization and differentially alters expression of growth factors and matrix proteins that regulate elastin assembly. These changes, coupled with increased lung elastase activity measured in preterm lambs after MV for 1-3 days, likely contribute to CLD.     

Recombinant human elafin promotes alveologenesis in newborn mice exposed to chronic hyperoxia

Background/aimsElastase inhibitors reverse elastin degradation and abnormal alveologenesis and attenuate the lung structural abnormalities induced by mechanical ventilation with O₂-rich gas. The potential of these molecules to improve endothelial function and to ameliorate severe bronchopulmonary dysplasia (BPD) during lung development is not yet understood. We sought to determine whether the intratracheal treatment of newborn mice with the elastase inhibitor elafin would prevent hyperoxia-induced lung elastin degradation and the cascade of events that cause abnormal alveologenesis.MethodsNewborn mice were exposed to 85% O₂ for 3, 7, 14 or 21 days. Recombinant human elafin was administered by intratracheal instillation from the first day every two days for 20 days. We next used morphometric analyses, quantitative RT-PCR, immunostaining, Western blotting, and ELISA methods to assess the key variables involved in elastogenesis disruption and the potential signaling pathways noted below in recombinant human elafin-treated mouse pups that had been exposed to 85% O₂.ResultsWe found that impaired alveolar development and aberrant elastin production were associated with elevations in whole lung elastase levels in 85% O₂-exposed lungs. Elafin attenuated the structural disintegration that developed in the hyperoxia-damaged lungs. Furthermore, elafin prevented the elastin degradation, neutrophil influx, activation of TGF-β1 and apoptosis caused by 85% O₂ exposure.ConclusionsPulmonary elastase plays an important role in disrupting elastogenesis during O₂-induced damage, which is the result of a pulmonary inflammatory response. Elafin prevents these changes by inhibiting elastase and the TGF-β1 signalling cascade and may be a new therapeutic target for preventing O₂-induced lung injury in neonates.     

Curcumin augments lung maturation, preventing neonatal lung injury by inhibiting TGF-β signaling

There is no effective intervention to prevent or treat bronchopulmonary dysplasia (BPD). Curcumin has potent antioxidant and anti-inflammatory properties, and it modulates signaling of peroxisome proliferator-activated receptor-γ (PPARγ), an important molecule in the pathobiology of BPD. However, its role in the prevention of BPD is not known. We determined 1) if curcumin enhances neonatal lung maturation, 2) if curcumin protects against hyperoxia-induced neonatal lung injury, and 3) if this protection is mediated by blocking TGF-β. Embryonic day 19 fetal rat lung fibroblasts were exposed to 21% or 95% O(2) for 24 h following 1 h of treatment with curcumin. Curcumin dose dependently accelerated e19 fibroblast differentiation [increased parathyroid hormone-related protein (PTHrP) receptor, PPARγ, and adipocyte differentiation-related protein (ADRP) levels and triolein uptake] and proliferation (increased thymidine incorporation). Pretreatment with curcumin blocked the hyperoxia-induced decrease (PPARγ and ADRP) and increase (α-smooth muscle actin and fibronectin) in markers of lung injury/repair, as well as the activation of TGF-β signaling. In a separate set of experiments, neonatal Sprague-Dawley rat pups were exposed to 21% or 95% O(2) for 7 days with or without intraperitoneal administration of curcumin. Analysis for markers of lung injury/repair [PTHrP receptor, PPARγ, ADRP, fibronectin, TGF-β receptor (activin receptor-like kinase 5), and Smad3] and lung morphology (radial alveolar count) demonstrated that curcumin effectively blocks TGF-β activation and hyperoxia-induced lung injury. Therefore, curcumin accelerates lung maturation by stimulating key alveolar epithelial-mesenchymal interactions and prevents hyperoxia-induced neonatal lung injury, possibly by blocking TGF-β activation, suggesting that it is a potential intervention against BPD.     

Mechanical ventilation with 40% oxygen reduces pulmonary expression of genes that regulate lung development and impairs alveolar septation in newborn mice

Mechanical ventilation with 40% oxygen reduces pulmonary expression of genes that regulate lung development and impairs alveolar septation in newborn mice. Am J Physiol Lung Cell Mol Physiol 293: , 2007. First published August 17, 2007; - Mechanical ventilation (MV) with O(2)-rich gas offers life-saving treatment for extremely premature infants with respiratory failure but often leads to neonatal chronic lung disease (CLD), characterized by defective formation of alveoli and blood vessels in the developing lung. We discovered that MV of 2- to 4-day-old mice with 40% O(2) for 8 h, compared with unventilated control pups, reduced lung expression of genes that regulate lung septation and angiogenesis (VEGF-A and its receptor, VEGF-R2; PDGF-A; and tenascin-C). MV with air for 8 h yielded similar results for PDGF-A and tenascin-C but did not alter lung mRNA expression of VEGF or VEGF-R2. MV of 4- to 6-day-old mice with 40% O(2) for 24 h reduced lung protein abundance of VEGF-A, VEGF-R2, PDGF-A, and tenascin-C and resulted in lung structural abnormalities consistent with evolving CLD. After MV with 40% O(2) for 24 h, lung volume was similar to unventilated controls, whereas distal air space size, assessed morphometrically, was greater in lungs of ventilated pups, indicative of impaired septation. Immunostaining for vimentin, which is expressed in myofibroblasts, was reduced in distal lung after 24 h of MV with 40% O(2). These molecular, cellular, and structural changes occurred without detectable lung inflammation as evaluated by histology and assays for proinflammatory cytokines, myeloperoxidase activity, and water content in lung. Thus lengthy MV of newborn mice with O(2)-rich gas reduces lung expression of genes and proteins that are critical for normal lung growth and development. These changes yielded lung structural defects similar to those observed in evolving CLD.     

Disruption of TGF-β signaling in smooth muscle cell prevents flow-induced vascular remodeling

Transforming growth factor-β (TGF-β) signaling has been prominently implicated in the pathogenesis of vascular remodeling, especially the initiation and progression of flow-induced vascular remodeling. Smooth muscle cells (SMCs) are the principal resident cells in arterial wall and are critical for arterial remodeling. However, the role of TGF-β signaling in SMC for flow-induced vascular remodeling remains unknown. Therefore, the goal of our study was to determine the effect of TGF-β pathway in SMC for vascular remodeling, by using a genetical smooth muscle-specific (SM-specific) TGF-β type II receptor (Tgfbr2) deletion mice model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2^f/f) and their corresponding wild-type background mice (MyhCre.Tgfbr2^WT/WT) underwent partial ligation of left common carotid artery for 1, 2, or 4 weeks. Then the carotid arteries were harvested and indicated that the disruption of Tgfbr2 in SMC provided prominent inhibition of vascular remodeling. And the thickening of carotid media, proliferation of SMC, infiltration of macrophage, and expression of matrix metalloproteinase (MMP) were all significantly attenuated in Tgfbr2 disruption mice. Our study demonstrated, for the first time, that the TGF-β signaling in SMC plays an essential role in flow-induced vascular remodeling and disruption can prevent this process.     

Mechanisms of emphysema in autosomal dominant cutis laxa

Heterozygous elastin gene mutations cause autosomal dominant cutis laxa associated with emphysema and aortic aneurysms. To investigate the molecular mechanisms leading to cutis laxa in vivo, we generated transgenic mice by pronuclear injection of minigenes encoding normal human tropoelastin (WT) or tropoelastin with a cutis laxa mutation (CL). Three independent founder lines of CL mice showed emphysematous pulmonary airspace enlargement. No consistent dermatological or cardiovascular pathologies were observed. One CL and one WT line were selected for detailed studies. Both mutant and control transgenic animals showed elastin deposition into pulmonary elastic fibers, indicated by increased desmosine levels in the lung and by colocalization of transgenic and endogenous elastin by immunostaining. CL mice showed increased static lung compliance and decreased stiffness of lung tissue. In addition, markers of transforming growth factor-β (TGFβ) signaling and the unfolded protein response (UPR) were elevated together with increased apoptosis in the lungs of CL animals. We conclude that the synthesis of mutant elastin in CL activates multiple downstream disease pathways by triggering a UPR, altered mechanical signaling, increased release of TGFβ and apoptosis. We propose that the combined effects of these processes lead to the development of an emphysematous pulmonary phenotype in CL.     

Role of pulmonary epithelial arginase-II in activation of fibroblasts and lung inflammaging

Elevated arginases including type-I (Arg-I) and type-II isoenzyme (Arg-II) are reported to play a role in aging, age-associated organ inflammaging, and fibrosis. A role of arginase in pulmonary aging and underlying mechanisms are not explored. Our present study shows increased Arg-II levels in aging lung of female mice, which is detected in bronchial ciliated epithelium, club cells, alveolar type 2 (AT2) pneumocytes, and fibroblasts (but not vascular endothelial and smooth muscle cells). Similar cellular localization of Arg-II is also observed in human lung biopsies. The age-associated increase in lung fibrosis and inflammatory cytokines, including IL-1β and TGF-β1 that are highly expressed in bronchial epithelium, AT2 cells, and fibroblasts, are ameliorated in arg-ii deficient (arg-ii^−/−) mice. The effects of arg-ii^−/− on lung inflammaging are weaker in male as compared to female animals. Conditioned medium (CM) from human Arg-II-positive bronchial and alveolar epithelial cells, but not that from arg-ii^−/− cells, activates fibroblasts to produce various cytokines including TGF-β1 and collagen, which is abolished by IL-1β receptor antagonist or TGF-β type I receptor blocker. Conversely, TGF-β1 or IL-1β also increases Arg-II expression. In the mouse models, we confirmed the age-associated increase in IL-1β and TGF-β1 in epithelial cells and activation of fibroblasts, which is inhibited in arg-ii^−/− mice. Taken together, our study demonstrates a critical role of epithelial Arg-II in activation of pulmonary fibroblasts via paracrine release of IL-1β and TGF-β1, contributing to pulmonary inflammaging and fibrosis. The results provide a novel mechanistic insight in the role of Arg-II in pulmonary aging.     

Transgenic extracellular superoxide dismutase protects postnatal alveolar epithelial proliferation and development during hyperoxia

Transgenic (TG) human (h) extracellular superoxide dismutase (EC-SOD) targeted to type II cells protects postnatal newborn mouse lung development against hyperoxia by unknown mechanisms. Because alveolar development depends on timely proliferation of type II epithelium and differentiation to type I epithelium, we measured proliferation in bronchiolar and alveolar (surfactant protein C-positive) epithelium in air and 95% O2-exposed wild-type (WT) and TG hEC-SOD newborn mice at postnatal days 3, 5, and 7 (P3-P7), traversing the transition from saccular to alveolar stages. We found that TG hEC-SOD ameliorated the 95% O2-impaired bromodeoxyuridine uptake in alveolar and bronchiolar epithelium at P3, but not at P5 and P7, when overall epithelial proliferation rates were lower in air-exposed WT mice. Mouse EC-, CuZn-, and Mn-SOD expression were unaffected by hyperoxia or genotype. TG mice had less DNA damage than 95% O2-exposed WT mice at P3, measured by TdT-mediated dUTP nick end labeling (P < 0.05). Hyperoxia induced cell-cycle inhibitory protein p21cip/waf mRNA at P3, WT > TG, P = 0.06. 95% O2 impaired apical expression of type I cell alpha protein (T1alpha) in WT but not in TG mice at P3 and increased T1alpha in WT and TG mice at P7. Reducing the 95% O2-induced impairment of epithelial proliferation at a critical window of lung development was associated with protection against DNA damage and preservation of apical T1alpha expression at P3.     

Human umbilical cord-derived mesenchymal stem cells protect from hyperoxic lung injury by ameliorating aberrant elastin remodeling in the lung of O2-exposed newborn rat

The incidence and mortality rates of bronchopulmonary dysplasia (BPD) remain very high. Therefore, novel therapies are imminently needed to improve the outcome of this disease. Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) show promising therapeutic effects on oxygen-induced model of BPD. In our experiment, UC-MSCs were intratracheally delivered into the newborn rats exposed to hyperoxia, a well-established BPD model. This study demonstrated that UC-MSCs reduce elastin expression stimulated by 90% O₂ in human lung fibroblasts-a (HLF-a), and inhibit HLF-a transdifferentiation into myofibroblasts. In addition, the therapeutic effects of UC-MSCs in neonatal rats with BPD, UC-MSCs could inhibit lung elastase activity and reduce aberrant elastin expression and deposition in the lung of BPD rats. Overall, this study suggested that UC-MSCs could ameliorate aberrant elastin expression in the lung of hyperoxia-induced BPD model which may be associated with suppressing increased TGFβ1 activation.     

Three-dimensional microstructural changes in murine abdominal aortic aneurysms quantified using immunofluorescent array tomography

This study investigated the spatial and temporal remodeling of blood vessel wall microarchitecture and cellular morphology during abdominal aortic aneurysm (AAA) development using immunofluorescent array tomography (IAT), a high-resolution three-dimensional (3D) microscopy technology, in the murine model. Infrarenal aortas of C57BL6 mice (N=20) were evaluated at 0, 7, and 28 days after elastase or heat-inactivated elastase perfusion. Custom algorithms quantified volume fractions (VF) of elastin, smooth muscle cell (SMC) actin, and adventitial collagen type I, as well as elastin thickness, elastin fragmentation, non-adventitial wall thickness, and nuclei amount. The 3D renderings depicted elastin and collagen type I degradation and SMC morphological changes. Elastin VF decreased 37.5% (p<0.01), thickness decreased 48.9%, and fragmentation increased 449.7% (p<0.001) over 28 days. SMC actin VF decreased 78.3% (p<0.001) from days 0 to 7 and increased 139.7% (p<0.05) from days 7 to 28. Non-adventitial wall thickness increased 61.1%, medial nuclei amount increased 159.1% (p<0.01), and adventitial collagen type I VF decreased 64.1% (p<0.001) over 28 days. IAT and custom image analysis algorithms have enabled robust quantification of vessel wall content, microstructure, and organization to help elucidate the dynamics of vascular remodeling during AAA development.