Regulation of human lung fibroblast phenotype and function by vitronectin and vitronectin integrins.

Myofibroblasts, characterised by high expression of alpha-smooth muscle actin (alpha-SMA), are important and transient cells in normal wound healing but are found in increased number in various pathological conditions of the lung including asthma and pulmonary fibrosis. The mechanisms that regulate the myofibroblast phenotype are unknown but are likely to involve signals from the extracellular matrix transmitted via specific integrins. Vitronectin is a glycoprotein released during inflammation and has been shown to regulate the phenotype of vascular smooth muscle cells via alpha v and beta 1 integrins. In the current study we have examined whether vitronectin influences the phenotype and function of normal human lung fibroblasts (HFL-1). Incubation of HFL-1 cells with vitronectin induced a concentration-dependent reduction in alpha-SMA expression. By contrast, function-blocking monoclonal antibodies to the vitronectin integrins alpha v, beta 1, alpha v beta 3 and alpha v beta 5 induced the expression of alpha-SMA and its organization into stress fibers. Expression of alpha-SMA induced by all function-blocking monoclonal antibodies was abrogated by inhibition of protein kinase C and phosphatidylinositol-3 kinase, but the effects of inhibition of other signalling pathways was integrin dependent. Exposure to other extracellular matrix proteins such as fibronectin, collagen or their integrins did not influence expression of alpha-SMA. The expression and organization of alpha-SMA induced by exposure to function-blocking antibodies was translated into an augmented capacity of HFL-1 cells to contract fibroblast populated collagen gels. By contrast, contraction of collagen gels following incubation with vitronectin was not significantly different to control. This study has shown that vitronectin influences the phenotype and behaviour of HFL-1 cells by downregulating the expression of alpha-SMA and reducing their contractile ability. By contrast, occupancy of specific integrins by function-blocking antibodies upregulated the expression of alpha-SMA and induced the formation of functional stress fibers capable of contracting collagen gels. These results suggest that vitronectin modulates the fibroblast-myofibroblast phenotype, implying an important role in the remodelling process during lung development or response to injury.

A confocal microscopic study of the formation of ganglia in the airways of fetal pig lung.

Fetal airway smooth muscle contracts to neural stimulation from early gestation. This study aimed to document the development of the nerves and ganglia within the bronchial tree of the fetal pig lung as the structural correlates for this function. The formation of these structures during lung development (pseudoglandular stage, canalicular stage, and saccular stage) was followed through to the postnatal period, using antibodies to protein gene product 9.5, a nonspecific nerve marker; synaptic vesicle protein 2, a marker of synaptic vesicle membranes; and neurofilament, a marker of filaments in the neuronal cytoskeleton. Glial cells were stained for glial fibrillary acidic protein (GFAP) and S-100, and the airway smooth muscle for alpha-actin. Whole mounts of the bronchial tree were imaged using confocal microscopy. The formation of ganglia commences in the pseudoglandular stage with patches of neuroblasts in the wall of the epithelial tubules. These ganglionic precursors are supplied with an abundance of nerve trunks and fibers that arise from the vagus and extend to the growing tips of the airways. These trunks show profiles of Schwann cells. As the airways grow, the ganglionic precursors condense at the nerve junctions. Nerve bundles in trunks and neurons in ganglia become increasingly enveloped by GFAP-positive sheaths. From midterm onward (canalicular stage), ganglia contain cholinergic neurons. In the third trimester (saccular stage) and postnatally, ganglia further increase in size and contain mainly nerve fibers in the center. Thus, neural tissue is a dominant feature of the primordial lung, which is enveloped by nerves and ganglia through gestation into postnatal life.

Development of the innervation and airway smooth muscle in human fetal lung.

Human and porcine fetal airways have been shown to contract spontaneously from the first trimester, the latter also contracting in response to neural stimulation. Our object was to map immunohistochemically the innervation and its relationship to the airway smooth muscle (ASM) in the human fetal lung from early gestation to the postnatal period. Whole mounts of the bronchial tree were stained with antibodies to the pan-neuronal marker protein gene product 9.5, the Schwann cell marker S-100, and the ASM contractile protein alpha-actin, and imaged using confocal microscopy. By the end of the embryonic period (53 d gestation), the branching epithelial tubules in the primordial lung were covered with ASM to the base of the terminal sacs. An extensive plexus of nerve trunks containing nerve bundles, forming ganglia, and Schwann cells ensheathed the ASM. By 16 wk (canalicular stage), maturation of the innervation was advanced with two major nerve trunks running the length of the bronchial tree, giving rise to varicosed fibers lying on the ASM. An extensive nerve plexus in the mucosa was also present. The distal airways of infants who had died of Sudden Infant Death Syndrome were also covered with smooth muscle and were well innervated. Thus, an essentially complete coat of ASM and an abundant neural plexus ensheathing the airways are an integral part of the branching epithelial tubules from early in lung development.

Structure and function of the adventitial and mucosal nerve plexuses of the bronchial tree in the developing lung.

1. The aim of the present study was first to determine when airway smooth muscle first appears in the airways of the developing foetal lung and when its contractility is mature and, second, when the airway smooth muscle becomes innervated, both structurally and functionally. 2. Narrowing and relaxation of the airways of the intact bronchial tree from the lungs of foetal pigs at varying stages of gestation was recorded by real time video microscopy. Nerves and smooth muscle were stained immunohistochemically and their spatial relationships were visualized by confocal microscopy. 3. From early in gestation (pseudoglandular stage/branching morphogenesis), airways were covered with a single layer of smooth muscle cells orientated cylindrically around the airway wall. Thick nerve trunks containing loosely packed nerves, with ganglia forming at their junctions ensheathed the airways to the growing tips, with a network of fine varicosed nerves lying on the smooth muscle cells. Some of these nerves were functional and cholinergic, as electrical field stimulation caused substantial narrowing that was blocked by atropine and by tetrodotoxin. By mid-term, an extensive plexus of nerves, well-defined ganglia and varicosed nerves to the smooth muscle had developed. Some nerves extended through the airway smooth muscle to form the elements of a mucosal nerve plexus in conjunction with developing vascular tissue forming a bronchial circulation. In 3 week postnatal pigs the distal and terminal airways were densely supplied with varicosed nerves to the smooth muscle. The ganglia were more centrally located. 4. An abundant network of calcitonin gene-related peptide (CGRP) nerves with prominent nerve endings lay just below the surface of the epithelium. The nerve bundles to the terminal arterioles in the mucosa also contained CGRP-positive fibres as well as sympathetic nerves (neuropeptide Y- and tyrosine hydroxylase-positive). It is hypothesized that these epithelial and arteriolar CGRP nerves form the local axon reflex purported to cause neurogenic inflammation. 5. The spontaneously active tone exerted by the airway smooth muscle from early in gestation is hypothesized to provide the force across the airway wall and adjacent parenchyma that is the stimulus for lung growth in foetal life.

Mapping the innervation of the bronchial tree in fetal and postnatal pig lung using antibodies to PGP 9.5 and SV2.

The extent of the innervation in the adventitial surface of the airway wall is demonstrated by mapping all the nerves and ganglia of the first trimester fetal and postnatal (4-wk-old) pig lung. We used antibodies to two pan-neuronal nerve markers-Protein Gene Product (PGP) 9.5, a nonspecific marker for all nerves, and synaptic vesicle protein 2 (SV2), a marker of synaptic vesicle membranes-to obtain detailed maps of the innervation of the airways. The airway smooth muscle has been stained concurrently with an antibody to alpha-actin. To obtain an overview of the passage of nerves down the bronchial tree, whole mounts of the bronchial tree have been imaged using confocal microscopy. Several conclusions can be drawn from our work: (1) Nerves branch from the main nerve trunks to form an extensive plexus covering the surface of the smooth muscle in fetal pigs but penetrate through the smooth muscle and are more organized in postnatal airways. In general, two main nerve trunks per airway were observed. (2) The antibody to PGP 9.5 in the fetal lung revealed in many ganglia and nerve trunks, the latter exhibiting cell profiles. In the postnatal lung, cell bodies were restricted to ganglia in the central airways. (3) The antibody to SV2, in general, showed varicose staining of the fine nerve fibers at both ages.