Renee Dickie

Finite element 3D reconstruction of the pulmonary acinus imaged by synchrotron X-ray tomography

The alveolated structure of the pulmonary acinus plays a vital role in gas exchange function. Three-dimensional (3D) analysis of the parenchymal region is fundamental to understanding this structure-function relationship, but only a limited number of attempts have been conducted in the past because of technical limitations. In this study, we developed a new image processing methodology based on finite element (FE) analysis for accurate 3D structural reconstruction of the gas exchange regions of the lung. Stereologically well characterized rat lung samples (Pediatr Res 53: 72-80, 2003) were imaged using high-resolution synchrotron radiation-based X-ray tomographic microscopy. A stack of 1,024 images (each slice: 1024 x 1024 pixels) with resolution of 1.4 mum(3) per voxel were generated. For the development of FE algorithm, regions of interest (ROI), containing approximately 7.5 million voxels, were further extracted as a working subunit. 3D FEs were created overlaying the voxel map using a grid-based hexahedral algorithm. A proper threshold value for appropriate segmentation was iteratively determined to match the calculated volume density of tissue to the stereologically determined value (Pediatr Res 53: 72-80, 2003). The resulting 3D FEs are ready to be used for 3D structural analysis as well as for subsequent FE computational analyses like fluid dynamics and skeletonization.

Integrin binding angiopoietin-1 monomers reduce cardiac hypertrophy

Angiopoietins were thought to be endothelial cell‐specific via the tie2 receptor. We showed that angiopoietin‐1 (ang1) also interacts with integrins on cardiac myocytes (CMs) to increase survival. Because ang1 monomers bind and activate integrins (not tie2), we determined their function in vivo. We examined monomer and multimer expressions during physiological and pathological cardiac remodeling and overexpressed ang1 monomers in phenylephrine‐induced cardiac hypertrophy. Cardiac ang1 levels (mRNA, protein) increased during postnatal development and decreased with phenylephrine‐induced cardiac hypertrophy, whereas tie2 phosphorylations were unchanged. We found that most or all of the changes during cardiac remodeling were in monomers, offering an explanation for unchanged tie2 activity. Heart tissue contains abundant ang1 monomers and few multimers (Western blotting). We generated plasmids that produce ang1 monomers (ang1–256), injected them into mice, and confirmed cardiac expression (immunohistochemistry, RT‐PCR). Ang1 monomers localize to CMs, smooth muscle cells, and endothelial cells. In phenylephrine‐induced cardiac hypertrophy, ang1–256 reduced left ventricle (LV)/tibia ratios, fetal gene expressions (atrial and brain natriuretic peptides, skeletal actin, β‐myosin heavy chain), and fibrosis (collagen III), and increased LV prosurvival signaling (akt, MAPKp42/44), and AMPKT172. However, tie2 phosphorylations were unchanged. Ang1–256 increased integrin‐linked kinase, a key regulator of integrin signaling and cardiac health. Collectively, these results suggest a role for ang1 monomers in cardiac remodeling.—Dallabrida, S. M., Ismail, N. S., Pravda, E. A., Parodi, E. M., Dickie, R., Durand, E. M., Lai, J., Cassiola, F., Rogers, R. A., Rupnick, M. A. Integrin binding angiopoietin‐1 monomers reduce cardiac hypertrophy. FASEB J. 22, 3010–3023 (2008)

Distribution and quantity of contractile tissue in postnatal development of rat alveolar interstitium.

Alpha–smooth muscle actin (α‐SMA) ‐expressing cells are important participants in lung remodeling, during both normal postnatal ontogeny and after injury. Developmental dysregulation of these contractile cells contributes to bronchopulmonary dysplasia in newborns, and aberrant recapitulation in adults of the normal ontogeny of these cells has been speculated to underlie disease and repair in mature lungs. The significance of airway smooth muscle has been widely investigated, but contractile elements within the pulmonary parenchyma, although also of structural and functional consequence in developing and mature lungs, are relatively unstudied and little quantitative information exists. Here, we quantify the areal density of α‐SMA expression in lung parenchyma and assess changes in its spatiotemporal distribution through postnatal ontogeny. Using an antibody against α‐SMA, we immunofluorescently labeled contractile elements in lung sections from a postnatal growth series of rats. Images were segmented using thresholded pixel intensity. Alpha‐SMA areal density in the alveolar interstitium was calculated by dividing the area of α‐SMA–positive staining by the tissue area. The areal density of α‐SMA in 2‐day neonates was 3.7%, almost doubled, to 7.2% by 21 days, and decreased to 3% in adults. Neonates had large, elongate concentrations of α‐SMA, and α‐SMA localized both at septal tips and within the interstitium. In adults, individual areas of α‐SMA expression were smaller and more round, and located predominately in alveolar ducts, at alveolar ends and bends. The results are consistent with increasing α‐SMA expression during the period of peak myofibroblast activity, corresponding to the phase of rapid alveolarization in the developing lung. Anat Rec, 291:83–93, 2007.

Deep pulmonary lymphatics in immature lungs

Recently, we found that the translocation of inhaled nanoparticles from the air space to secondary organs is age dependent and substantially greater in neonates than in adults (J Respir Crit Care Med 177: A48, 2008). One reason for this difference might be age-dependent differences in alveolar barrier integrity. Because the neonate lung is undergoing morphogenetic and fluid balance changes, we hypothesize that the alveolar barrier of developing lungs is more easily compromised and susceptible to foreign material influx than that of adult lungs. On the basis of these hypotheses, we predict that the postnatally developing lung is also more likely to allow the translocation of some materials from the air space to the lymphatic lumens. To test this idea, we intratracheally instilled methyl methacrylate into immature and adult lungs and compared lymphatic filling between these two age groups. Scanning electron microscopy of the resultant corrosion casts revealed peribronchial saccular and conduit lymphatic architecture. Deep pulmonary lymphatic casts were present on the majority (58.5%) of airways in immature lungs, but lymphatic casting in adult lungs, as anticipated, was much more infrequent (21.6%). Thus the neonate lung appears to be more susceptible than the adult lung to the passage of instilled methyl methacrylate from the air space into the lymphatics. We speculate that this could imply greater probability of translocation of other materials, such as nanoparticles, from the immature lung as well.

Inhibition of Vascular Endothelial Growth Factor Receptor Decreases Regenerative Angiogenesis in Axolotls: VEGFR INHIBITION IN AXOLOTL REGENERATION

Angiogenesis is crucial for tissue growth and repair in mammals, and is chiefly regulated by vascular endothelial growth factor (VEGF) signaling. We evaluated the effect of chemical inhibition of VEGF receptor signaling in animals with superior regenerative ability, axolotl salamanders, to determine the impact on vascularization and regenerative outgrowth. Following tail amputation, treated animals (100 nM PTK787) and controls were examined microscopically and measured over the month‐long period of regeneration. Treatment with VEGFR inhibitor decreased regenerative angiogenesis; drug‐treated animals had lower vascular densities in the regenerating tail than untreated animals. This decrease in neovascularization, however, was not associated with a decrease in regenerative outgrowth or with morphological abnormalities in the regrown tail. Avascular but otherwise anatomically normal regenerative outgrowth over 1 mm beyond the amputation plane was observed. The results suggest that in this highly regenerative species, significant early tissue regeneration is possible in the absence of a well‐developed vasculature. This research sets the groundwork for establishing a system for the chemical manipulation of angiogenesis within the highly regenerative axolotl model, contributing to a better understanding of the role of the microvasculature within strongly proliferative yet well‐regulated environments. Anat Rec, 300:2273–2280, 2017.