elastin and Hernias--Diaphragmatic--Congenital

A critical event in the adaptation to extrauterine life is relaxation of the pulmonary vasculature at birth, allowing for a rapid increase in pulmonary blood flow that is essential for efficient gas exchange. Failure of this transition leads to pulmonary hypertension (PH), a major cause of newborn mortality associated with preterm birth, infection, hypoxia, and malformations including congenital diaphragmatic hernia (CDH). While individual vasoconstrictor and dilator genes have been identified, the coordination of their expression is not well understood. Here, we found that lung mesenchyme-specific deletion of CDH-implicated genes encoding pre-B cell leukemia transcription factors (Pbx) led to lethal PH in mice shortly after birth. Loss of Pbx genes resulted in the misexpression of both vasoconstrictors and vasodilators in multiple pathways that converge to increase phosphorylation of myosin in vascular smooth muscle (VSM) cells, causing persistent constriction. While targeting endothelin and angiotensin, which are upstream regulators that promote VSM contraction, was not effective, treatment with the Rho-kinase inhibitor Y-27632 reduced vessel constriction and PH in Pbx-mutant mice. These results demonstrate a lung-intrinsic, herniation-independent cause of PH in CDH. More broadly, our findings indicate that neonatal PH can result from perturbation of multiple pathways and suggest that targeting the downstream common effectors may be a more effective treatment for neonatal PH.

Topics: Alleles; Animals; Apoptosis; Cell Proliferation; Disease Models, Animal; Echocardiography; Elastin; Female; Gene Deletion; Hernias, Diaphragmatic, Congenital; Homeodomain Proteins; Hypertension, Pulmonary; Lung; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Myosins; Parturition; Phosphorylation; Pre-B-Cell Leukemia Transcription Factor 1; Proto-Oncogene Proteins; Pulmonary Artery; Respiration; Vasoconstriction

The mechanisms by which tracheal occlusion (TO) improves alveolarization in congenital diaphragmatic hernia (CDH) are incompletely understood. Therefore transcriptional and histological effects of TO on alveolarization were studied in the rabbit model for CDH. The question of the best normalization strategy for gene expression analysis was also addressed.. Fetal rabbits were randomized for CDH or sham operation on gestational day 23/31 and for TO or sham operation on day 28/31 resulting in four study groups. Untouched littermates were added. At term and before lung harvest, fetuses were subjected to mechanical ventilation or not. Quantitative real-time PCR was performed on lungs from 4-5 fetuses of each group with and without previous ventilation. Stability of ten housekeeping genes (HKGs) and optimal number of HKGs for normalization were determined, followed by assessment of HKG expression levels. Expression levels of eleven target genes were studied in ventilated lungs, including genes regulating elastogenesis, cell-environment interactions, and thinning of alveolar walls. Elastic staining, immunohistochemistry and Western blotting completed gene analysis.. Regarding HKG expression, TO increased β-actin and β-subunit of ATP synthase. Mechanical ventilation increased β-actin and β2-microglobulin. Flavoprotein subunit of succinate dehydrogenase and DNA topoisomerase were the most stable HKGs. CDH lungs showed disorganized elastin deposition with lower levels for tropoelastin, fibulin-5, tenascin-C, and α6-integrin. After TO, CDH lungs displayed a normal pattern of elastin distribution with increased levels for tropoelastin, fibulin-5, tenascin-C, α6-integrin, ß1-integrin, lysyl oxidase, and drebrin. TO increased transcription and immunoreactivity of tissue inhibitor of metalloproteinase-1.. Experimental TO might improve alveolarization through the mechanoregulation of crucial genes for late lung development. However part of the transcriptional changes involved genes that were not affected in CDH, raising the question of TO-induced disturbances of alveolar remodeling. Attention should also be paid to selection of HKGs for studies on mechanotransduction-mediated gene expressions.

Topics: Animals; Body Weight; Elastin; Female; Gene Expression Regulation; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Lung; Matrix Metalloproteinases; Rabbits; Random Allocation; Trachea

This study examined the effects of amniocyte-based engineered tendons on partial diaphragmatic replacement.. Ovine mesenchymal amniocytes were labeled with green fluorescent protein (GFP), expanded, and seeded into a collagen hydrogel. Composite grafts (20 to 25 cm2) based on acellular dermis (group I), or acellular small intestinal submucosa (group II) received either a cell-seeded or an acellular hydrogel within their layers. Newborn lambs (n = 20) underwent partial diaphragmatic replacement with either an acellular or a cellular autologous construct from either group. At 3 to 12 months' postoperatively, implants were subjected to multiple analyses.. Diaphragmatic hernia recurrence was significantly higher in animals with acellular grafts (5 of 5) then in animals with cellular ones (1 of 4) in group I (P <.05) but not in group II (3 of 6 and 4 of 5, respectively). Cellular grafts had higher modular (5.27 +/- 1.98 v. 1.27 +/- 0.38 MPa) and ultimate (1.94 +/- 0.70 v. 0.29 +/- 0.05 MPa) tensile strength than acellular implants in group I (P <.05), but not in group II. Quantitative analyses showed no differences in extracellular matrix components between cellular and acellular implants in either group. All cellular implants showed GFP-positive cells.. Diaphragmatic repair with an autologous tendon engineered from mesenchymal amniocytes leads to improved mechanical and functional outcomes when compared with an equivalent acellular bioprosthetic repair, depending on scaffold composition. The amniotic fluid may be a preferred cell source for engineered diaphragmatic reconstruction.

Topics: Amniotic Fluid; Animals; Animals, Newborn; Cell Differentiation; Cells, Cultured; Collagen; Collagen Type I; Diaphragm; Disease Models, Animal; Elastin; Genes, Reporter; Glycosaminoglycans; Hernia, Diaphragmatic; Hernia, Diaphragmatic, Traumatic; Hernias, Diaphragmatic, Congenital; Hydrogels; Laparotomy; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Sheep; Tendons; Tensile Strength; Tissue Engineering; Transplantation, Autologous

The form and function of the heart are the final result of an integration of cells, tissues, and extracellular material. The extracellular matrix (ECM) is a complex array of different molecular components, and it plays an important role for the transfer of mechanical force in both contraction and relaxation phases in the cardiac cycle. ECM plays also a significant role in the development of the heart. The aim of this study was to evaluate the expression of important ECM components in the heart of rats with induced CDH to test the hypothesis that an alteration of ECM may contribute to the cardiac maldevelopment, which recently has been identified as a contributive factor for the high mortality rate in babies with congenital diaphragmatic hernia (CDH).. CDH model was induced in pregnant rats after administration of 100 mg of nitrofen on day 9.5 of gestation (term, 22 days). In control animals the same dose of olive oil was given without nitrofen. Cesarean section was performed on day 21 of gestation. The fetuses were divided into 2 groups: normal control (n = 10) and nitrofen-induced CDH (n = 10). Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the relative amount of tropoelastin and alpha1 (I) procollagen mRNA expression. Elastin protein content was measured using enzyme-linked immunosorbent assay (ELISA).. There was a reduction in tropoelastin mRNA (P <.05) and procollagen mRNA (P <.05) in CDH compared with controls. The cardiac alpha-elastin content also was reduced in CDH (P <.01).. The reduced cardiac tropoelastin and procollagen gene expression and the reduced alpha-elastin content indicate that the heart in CDH structurally is immature. The reduced production of cardiac ECM may contribute to a contractile dysfunction, which makes the heart unable to respond to the hemodynamic load accompanying persistent pulmonary hypertension (PPH).

Topics: Abnormalities, Multiple; Actins; Animals; Disease Models, Animal; Elastin; Enzyme-Linked Immunosorbent Assay; Fetal Organ Maturity; Heart; Heart Defects, Congenital; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Phenyl Ethers; Procollagen; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Tropoelastin

The purpose of this study was to determine whether the presence of bowel in the chest during development in the fetal lamb model of congenital diaphragmatic hernia (CDH) results in structural and/or biochemical hypoplasia of the left venticle.. The model was created at 80 days' gestation and delivered at term. The hearts were fixed in 4% formaldehyde solution, components weighed, and right ventricular (RV) and left ventricular (LV) wall thicknesses and both aortic (Ao) and pulmonary artery (PA) root diameters were measured. Fresh specimens were analyzed for protein, DNA, hydroxyproline, and elastin content. All CDH measurements are compared with littermate control tissues.. There were no differences in body weight (kg) between CDH and control littermates (4.25 +/- 0.26 versus 3.71 +/- 0.24, P = NS). CDH lambs have significantly decreased total heart (4.88 +/- .25 versus 6.75 +/- .49, P < .05), left ventricular (1.65 +/- .11 versus 2.15 +/- .19, P < .05), septal (1.29 +/- .11 versus 1.99 +/- .21, P < .05), and combined atrial (0.68 +/- .06 versus 1.14 +/- .15, P < .05) weights (g/kg lamb) without differences in RV weights (1.26 +/- .07 versus 1.57 +/- .17, P = NS). LV and RV wall thickness, and Ao root diameters (cm) were found to be identical in both CDH and control lambs. However, PA root diameters (0.47 +/- .01 versus 0.38 +/- .01, P < .005) and ductus arteriosus diameters were increased in CDH (0.35 +/- .01 versus 0.22 +/- .02, P < .005). Total protein, DNA collagen, and elastin content and DNA/total protein ratios were identical in RV and LV in both CDH and control lambs.. Newborn lambs with left-sided CDH have a significantly lower total heart, LV, septal, and atrial weights without differences of RV weight or ventricular wall thicknesses. Given these findings, the unchanged DNA/protein ratio implies that the left ventricle is hypoplastic in CDH. Ao/PA root ratios suggest that LV hypoplasia in utero may result in increased left atrial pressures, decreased right-to-left shunting through the foramen ovale, and increased PA pressures and flow, resulting in increased PA root and ductus arteriosus diameters. This model simulates the clinical data from human fetuses/neonates with CDH. Further investigations are necessary to determine the functional significance of these findings.

Topics: Animals; Aorta; Blood Pressure; Collagen; Disease Models, Animal; DNA; Ductus Arteriosus, Patent; Elastin; Female; Heart Atria; Heart Defects, Congenital; Heart Septal Defects, Atrial; Heart Septum; Heart Ventricles; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Humans; Hydroxyproline; Organ Size; Pregnancy; Proteins; Pulmonary Artery; Sheep

The pathophysiologic features of congenital diaphragmatic hernia (CDH) include pulmonary hypoplasia, pulmonary hypertension, surfactant deficiency, and decreased pulmonary compliance. When the surfactant deficiency is corrected using exogenous surfactant therapy, the pulmonary compliance improves, but does not reach normal values. Quasistatic saline pressure-volume measurements, which eliminate the air-liquid interface, confirm that CDH lungs are intrinsically less compliant than control lungs. The authors hypothesized that this abnormal lung compliance results from elevated concentrations of collagen and/or elastin in the lung. Therefore, they measured the collagen and elastin concentrations in CDH and control lung tissue. Also measured was the collagen concentration in the kidney, intestine, and dissected third-generation arterioles, venules, and bronchioles, to characterize further the pathology of CDH. The CDH model was created on the left side of fetuses in pregnant ewes at 80 days' gestation. The fetuses were delivered and killed at 140 days (full term, 145). The concentrations of collagen (as hydroxyproline), elastin, DNA, and total protein were measured using standard techniques. Although there was significantly more collagen per gram of lung tissue in the CDH lungs (1.334 mg/g v 0.885 mg/g in the controls) the elastin concentrations were not different. The elevated collagen concentration was not associated specifically with the conducting airways or vasculature. The collagen concentrations in CDH kidneys and intestines were the same as those of controls. The DNA/total protein ratios in the CDH and control lungs were identical. The results suggest that the elevated collagen concentration was present only in the lungs of CDH lambs, and that it was not attributable to atrophy or hypertrophy of the lungs. Thus, increased collagen in the lung parenchyma may be responsible for the intrinsic stiffness and decreased compliance of the CDH lungs.

Topics: Animals; Arterioles; Bronchi; Collagen; Disease Models, Animal; DNA; Elastin; Female; Gestational Age; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Humans; Hydroxyproline; Infant, Newborn; Intestines; Kidney; Lung; Lung Compliance; Persistent Fetal Circulation Syndrome; Pregnancy; Proteins; Pulmonary Surfactants; Sheep; Venules