Transient postnatal pulmonary arterial smooth muscle cytoskeletal disassembly and its functional implications.

摘要

In the human at birth the flow of blood through the pulmonary arteries increases ten-fold as a consequence of an acute fall in pulmonary vascular resistance followed by a gradual remodelling of vessels which occurs over the first weeks of life These changes are essential for normal gaseous exchange to occur in the lungs. The initial part of the process may be halted by perinatal hypoxia, acidosis, or sepsis and causes the clinical syndrome of persistent pulmonary hypertension of the newborn (PPHN). This thesis explores the changes in the cytoskeleton of the smooth muscle cells (SMCs) which help mediate the normal fall in pulmonary vascular resistance and are abnormal in PPHN. These studies were carried out using normal pigs and a hypobaric hypoxic model of PPHN. A series of cytoskeletal SMC phenotypes were identified within the intact intrapulmonary artery wall dependant upon different combinations of certain cytoskeletal proteins (alpha smooth muscle actin, beta actin, gamma actin SMI myosin heavy chain isoform, calponin, caldesmon and desmin). This classification demonstrated an outer to inner medial progression of SMC phenotype during development This was punctuated by a transient reduction in a smooth muscle actin, p actin and calponin staining at three days of age, which was considered to represent phenotypic modulation of the SMC phenotype. The total actin content and the proportion present in the monomeric form was found to remain constant during this time, using a highly specific fluorometric DNAse I inhibition assay. However, using simultaneously permeabilised and phalloidin treated preparations, the filamentous actin cytoskeleton of the SMCs was shown to re-organise to finer filaments after birth which may help explain the change in staining pattern within the inner media at three days. Functional studies using isolated segments of intrapulmonary artery indicated that the reorganisation of the actin cytoskeleton after birth was not associated with a significant reduction in contractile potential. Studies on the vessels from fetal and full term piglets indicated that changes in the actin organisation occurred within minutes of the onset of breathing and were associated with significant increases in contractile potential. The possibility that changes in morphologically distinct SMC phenotypes could be responsible for the cytoskeletal changes seen in the intact vessel was investigated by primary cell culture. Epithelioid and spindle-shaped SMC phenotypes were isolated from intrapulmonary arteries throughout development and from both inner and outer parts of the media. Although not responsible for the changes within the inner media of the intact vessel wall at three days of age, an increase in the proportion of epithelioid cells was noted to occur following birth. Spindle-shaped and epithelioid SMCs were obtained by dilutional cloning from intrapulmonary arteries from normal 14 day old and neonatal piglets exposed to hypobaric hypoxia. These cell lines were then characterised in terms of cytoskeletal protein content, replication properties, contractile properties and migrational potential. Distinct differences between the different morphological phenotypes were observed, suggesting differences in function within the intact vessel wall and perturbation of normal function following hypobaric hypoxia.