Search

The bone morphogenetic protein (BMP) type II receptor (BMPR-II) is predominantly expressed on the vascular endothelium in the adult lung. Although mutations in BMPR-II are known to underlie many cases of familial pulmonary arterial hypertension (FPAH), little is known regarding the expression of BMPs and their signalling pathways during normal lung development or the impact of BMPR-II mutations on endothelial cell function. We determined the cellular localization and expression levels of BMP4, BMP receptors, and activation of downstream signalling via phospho-Smad1 in a developmental series of human embryonic and fetal lungs by immunohistochemistry. The expression of BMP4 and BMP receptors was temporally and spatially regulated during lung development. BMPR-II expression correlated with phosphorylation of tissue Smad1 and was highest during the late pseudoglandular and early canalicular stage of lung development, when vasculogenesis is intense. Phospho-Smad1 expression was associated with markers of proliferation in endothelial cells. In vitro studies confirmed that BMPs 2 and 4 induced phosphorylation of Smad1/5 and pulmonary artery endothelial cell (PAEC) migration and proliferation. Adenoviral transfection of PAECs with mutant kinase-deficient BMPR-II, or siRNA knockdown of BMPR-II, inhibited Smad signalling and the proliferative response to BMP4. Our findings support a critical role for BMPs in lung vasculogenesis. Dysfunctional BMP signalling in PAECs during development may lead to abnormal pulmonary vascular development and contribute to the pathogenesis of FPAH.

The hypothesis for this study was that increased local expression of vascular angiotensin-converting enzyme (ACE) may contribute to the arterial remodelling which accompanies pulmonary hypertension, since angiotensin II (ANG II) is an important mediator of pulmonary vascular cell growth. The expression of ACE was studied by immunohistochemistry in paraffin-embedded lung sections from adults undergoing heart-lung transplantation for severe primary (n=6) and secondary (n=7) pulmonary arterial hypertension (PH), compared with age-matched controls (n=11). An antigen retrieval technique was used prior to incubating sections with the anti-ACE monoclonal antibody, CG2, or the endothelial marker, monoclonal anti-CD31. In control lungs, the highest level of ACE immunostaining was seen in the alveolar capillary endothelium, with less intense staining in small intra-acinar pulmonary arteries and relatively little staining in larger preacinar arteries. ACE immunostaining was virtually absent in lymphatics and veins. In both primary and secondary PH, there was an increase in ACE immunostaining in the endothelium of intra-acinar peripheral pulmonary arteries compared with control lungs, extending to the level of alveolar ducts, as confirmed by semi-quantitative analysis. The increase in endothelial ACE expression in the intra-acinar arteries of patients with primary and secondary PH is consistent with the hypothesis that locally increased production of ANG II may contribute to the process of pulmonary vascular remodelling., (Copyright 2000 John Wiley & Sons, Ltd.)

Pulmonary arterial structure was examined in lung biopsies from 26 children with severe pulmonary hypertensive congenital heart disease, aged 2 months-18 years, in whom the mean pulmonary arterial pressure was 55 (range 35-105) mmHg, using light, transmission, and scanning electron microscopy. Qualitative and quantitative techniques were applied and findings compared with those in age-matched controls. At 2 months, the smooth muscle cells showed hyperplasia, hypertrophy (mean cell diameter increased, P less than 0.001), and accelerated differentiation. In all pulmonary hypertensive cases aged less than 6 months, the contractile myofilament concentration was similar to the normal concentration at 6 months. Medial connective tissue was excessive for age. Smooth muscle cells within the intima (intimal proliferation) were first seen at 7 months, lying beneath a new internal elastic lamina. They showed a reduction in myofilament concentration in the more fibrotic lesions. In all cases, endothelial cells were abnormally thick (P less than 0.001) and elongated. Cytoskeletal remodelling was indicated by an increase in micro- and intermediate filament volume density (P less than 0.05 for both). The severity of endothelial damage was related to vessel size and position in the arterial pathway. These findings indicate that pulmonary vascular disease begins at or soon after birth with abnormal pulmonary vascular remodelling which leads to obliterative pulmonary vascular disease.

Lectin binding patterns to the developing pulmonary vasculature were studied in 10 Large White pigs aged 1 min to 1 week and in three adult animals. Paraffin-embedded tissue sections were exposed to eight lectin peroxidase conjugates: Dolichos biflorus, Triticum vulgaris, Concanavalin A, Ricinus communis type 2, Arachis hypogaea, Ricinus communis type 1, Tetragonolobus purpureas and Ulex europeus. Lectin binding patterns to the pulmonary arterial and venous endothelium, to smooth muscle cells (SMCs) and to the arterial connective tissue were age-related. Changes occurred during the first week of life and between 1 week and adult life. Neither the endothelial binding patterns in the adult nor the SMC patterns in the immature and adult lung conformed to known morphological differences between the different segments of the arterial and venous pathways. Heterogeneity for endothelial binding was seen in the immature lung. These studies indicate biochemical differences in surface structure between the endothelium, SMCs and connective tissue of the immature and mature lung. Ultrastructural localization of the lectins in the vasculature of the developing animal lung ought to help interpret similar data obtained on the vessels of the immature human pulmonary hypertensive lung using lectins which show similar binding patterns in both species.

The innervation of the pulmonary trunk and pulmonary arterial bed was studied in 17 pigs from birth to 6 months of age. After birth, the pulmonary trunk and extra- and intra-pulmonary arteries contained neurofilament and protein gene-product-immunoreactive nerve fibres in both the adventitia and media. The density of nerve fibres increased from birth to 2 months, this being most marked during the first 2 weeks of life. Most of the fibres in the media were presumed to be sympathetic in origin as they contained both neuropeptide tyrosine and tyrosine hydroxylase immunoreactivity. Fibres were associated with the vasa-vasorum and vascular smooth muscle running around the vessel, between the elastic laminae and smooth muscle cells, in the outer two-thirds of the media. Vasoactive intestinal polypeptide and calcitonin gene-related peptide-immunoreactive nerve fibres were found to be associated with the pulmonary trunk and extra-pulmonary artery, but generally not with the intra-pulmonary arteries. Tyrosine hydroxylase immunoreactivity was detected in the glomus cells at birth, but peptide immunoreactivity (enkephalin) was not demonstrated in paraganglia until 14 days of age. Adaptation to extra-uterine life is associated with rapid development changes in the innervation of the pig pulmonary trunk, extra- and intra-pulmonary arteries and in the expression of peptide immunoreactivity in both nerve fibres and glomus cells. These changes may have a role in the postnatal adaptation of the pulmonary circulation.

Pulmonary vascular structure was analysed using morphometric techniques, and arterial wall abnormalities in lung biopsy specimens taken from 85 cases of ventricular septal defect, aged 3 weeks to 30 years were described. The defect was closed in 53 patients, 12 died at operation and 11 survivors were recatheterized. Structural examination revealed a characteristic pathological picture which appeared to precede classical grade IV pulmonary vascular disease. This predilatation phase was identified by finding pre-acinar obstructive intimal proliferation or fibrosis, associated with only a slight increase in intra-acinar arterial muscularity, in the absence of stigmata of grade IV disease in patients with a pulmonary resistance greater than 6 units m2. Predilatation features occurred in 62 per cent of patients who either died at repair or who had post-operative pulmonary hypertension. These findings help identify a high risk group in whom resistance is higher than expected in the absence of classical grade IV changes. In addition, a marked increase in muscularity without intimal obstruction can also be associated with a pre-operative resistance of more than 6 units m2. The presence of medial hypertrophy only does not ensure either survival or a normal post-operative pulmonary arterial pressure. Early intra-cardiac repair is recommended before obstructive intimal fibrosis develops during the second year.

Adaptation of the pulmonary arterial intima was studied in injected lung specimens of 34 Large White pigs. Each type of pre- and intra-acinar artery was studied separately using transmission and scanning electron microscopy. Determination of the endothelial surface/volume ratio and volume densities of (1) endothelium and subendothelium, (2) endothelial cytoplasmic organelles and (3) subendothelial connective tissue elements yielded 6832 measurements which comprised a computerized database. At birth, endothelial cell morphology changed more rapidly and to a greater extent in peripheral than in proximal arteries. Endothelial surface/volume ratio increased (p less than 0.0001). Fetal surface projections, junctional interdigitations and overlap became less evident. Adaptational changes were complete in three weeks. Between three weeks and adulthood a reduction in endothelial surface/volume ratio suggested cell growth. In the subendothelium the volume density of collagen and basement membrane and elastin increased (p less than 0.001). The internal elastic lamina, immature in all arteries at birth increased in thickness and integrity until in the adult, only in small muscular arteries did gaps between elastin profiles ensure frequent contact between endothelial and smooth muscle cells. At all ages regional differences in endothelial cell morphology were evident.

Light microscopic immunolocalization studies were carried out on lung tissue from eight newborn and four adult pigs using antibodies to six extracellular matrix components. Antibodies to fibronectin, collagen type IV, and laminin localized on the same structures in adult and newborn lungs. By contrast, antibodies to the interstitial collagens (types I, III, and V) were less extensively localized in the newborn than in the adult, particularly those to type I because the fibres on which they localized in the newborn were thin and sparse. At all ages, antibodies to collagen types III and V co-localized on type I fibres and also on thin individual fibres which formed networks, more dense in the adult than in the newborn. At all ages, anti-type III collagen antibodies also localized on smooth muscle cells. In the adult, but not in the newborn, anti-type I and type V collagen antibodies localized on the connective tissue around the smooth muscle cells in the pulmonary arterial media and vein wall. The dominance of collagen type III suggests greater plasticity in the newborn pulmonary vasculature, which helps explain the recently described rapid changes in arterial wall structure which constitute adaptation to extrauterine life. The postnatal increase in collagen type I helps explain the documented postnatal increase in structural stiffness of the pulmonary arteries.

Using an immunohistochemical technique, the development of the cytoskeletal proteins desmin, vimentin, and actin (using alpha isotype and non-isotype specific antibodies) was assessed using a semi-quantitative grading system in the pulmonary vascular smooth muscle of nine normal pigs and 19 normal humans at different ages, and in 13 children with pulmonary hypertensive congenital heart disease. In the normal of both species, immunostaining for vimentin decreased after birth and then increased gradually while immunostaining for desmin and alpha actin increased steadily with age. In pulmonary hypertension, immunostaining for alpha actin and vimentin showed an accelerated increase at between 2 and 8 months. Also, the media showed regional differences in immunostaining which preceded the development of intimal proliferation. The inner media showed less immunoreactivity for all cytoskeletal proteins studied than did the outer media. Within areas of intimal proliferation many cells were immunonegative. These results suggest that the cytoskeletal features of medial smooth muscle cells are remodelled in the normal infant; that this process is altered from at least 2 months in the pulmonary hypertensive infant; and that the smooth muscle cells immediately beneath the internal elastic lamina are remodelled before migrating to form intimal proliferation. Changes in cytoskeletal composition can be related to the previously described postnatal maturation of pulmonary vascular smooth muscle cells.

Twelve Large White pigs aged less than 1 min, and 3, 5.5, and 14 days were exposed to hypoxia (380 torr) for 2.5-3 days. The wall structure of terminal bronchiolar (resistance arteries) and elastic arteries was assessed by light and electron microscopy using quantitative morphometric techniques. In animals exposed from birth, mean terminal bronchiolar arterial medial thickness was increased (p less than 0.05) because endothelial and smooth muscle cells (SMCs) retained their fetal shape, position, overlap, interdigitation and the low surface/volume ratio characteristic of fetal life. In all older animals, the cells had a normal postnatal shape and surface/volume ratio. In the elastic vessels hypoxia did not prevent the normal postnatal reduction in mean SMC diameter of animals exposed from birth. SMC hypertrophy did not occur in any age group, but all animals save those first exposed at 14 days, showed an increase in SMC myofilament volume density (p less than 0.01). Connective tissue volume density also increased (p less than 0.01), mainly due to an increase in elastin and ground substance. Thus a short period of neonatal hypoxia impaired adaptation and appeared to potentiate contractile capacity in stiff-walled arteries but elicited a less marked response from animals first exposed at 14 days.