Exogenous leukocyte and endogenous elastases can mediate mitogenic activity in pulmonary artery smooth muscle cells by release of extracellular matrix-bound basic fibroblast growth factor

Abstract

There is increasing evidence that extracellular matrix (ECM)-degrading proteinases contribute to the process of medial hypertrophy and neointimal proliferation in pulmonary vascular diseases. However, little is known about how proteinases, specifically elastases, induce vascular smooth muscle cell (SMC) hyperplasia. Our objective was to determine whether exogenous human leukocyte elastase (HLE), as well as endogenous vascular elastase, could release basic fibroblast growth factor (bFGF), a potent mitogen stored in the ECM surrounding SMCs. Cultured ovine and porcine pulmonary artery SMC were pre-incubated with [125I]-bFGF. After removal of unbound [125I]-bFGF, administration of HLE (0-1.0 microgram /ml, 1 h) resulted in a concentration-dependent accumulation of [125I]-bFGF in the conditioned medium, mirrored by depletion from the ECM. The serine elastase inhibitor elafin blocked this HLE-mediated action. Assessment by Western immunoblotting further demonstrated that HLE evoked the release of ECM-bound endogenous bFGF. When incubated with serum-starved SMC, conditioned medium from HLE-treated cells stimulated [3H]-thymidine incorporation, a feature neutralized by bFGF antibodies. In addition, SMC exposed to serum treated elastin (STE), previously shown to stimulate endogenous vascular elastase, liberated bioavailable bFGF from ECM stores, as determined by autoradiography, Western immunoblotting, and stimulation of DNA synthesis and SMC proliferation. Chondroitin sulfate, an inhibitor of STE-induced elastase activity, attenuated the release of bFGF. Our studies demonstrate that HLE, secreted by inflammatory cells, and endogenous vascular elastase release matrix-bound bFGF, suggesting a mechanism whereby elastases, through degradation of ECM, induce SMC proliferation associated with progressive vascular disease.

Neutrophil Elastase Is Produced by Pulmonary Artery Smooth Muscle Cells and Is Linked to Neointimal Lesions

“…Fragmentation of elastic laminae generates elastin peptides and releases growth factors from the extracellular matrix, and these molecules can induce proliferation and migration of SMC. 16 Indeed, we observed a significant increase in the number of proliferating SMC in the neointimal lesions of PAs in S100A4 versus C57 mice 6 months after M1-MHV-68 infection, as judged by positivity for proliferating cell nuclear antigen (PCNA) (P Ͻ 0.05; Figure 2, C and D). Interestingly, formation of these neointimal lesions was not sufficient to elevate the right ventricular systolic pressure or to cause right ventricular hypertrophy (see Supplemental Figure S2 at http://ajp.amjpathol.org), a finding also observed in other models with neointimal lesions.…”

“…We identified the elastase involved as neutrophil elastase (NE) produced by PA SMC, suggesting that it is the endogenous vascular elastase previously related to PAH in other experimental models. 12,16,18,19 Moreover, we showed that NE is produced in significantly greater amounts by cultured murine S100A4 versus C57 PA SMC, and by human PA SMC from IPAH versus control lungs, and we localized NE to neointimal and plexiform lesions in human lung specimens.…”

“…16 Elastase also activates matrix metalloproteinases (MMPs) that degrade native type I collagen, and, in so doing, expose cryptic RGD sites that preferentially bind ␣ v ␤ 3 integrins, and stimulate tenascin C production. Enhanced production of tenascin C clusters ␣ v ␤ 3 integrins and activates growth factor receptors such as epidermal growth factor receptors.…”

“…Neointimal lesion formation, however, was not present. Elastase inhibition should, however, attenuate these lesions since proliferation and migration of SMC in the neointima are likely the consequences of elastasemediated release of growth factors from the extracellular matrix 16,17 and activation of growth factor receptors. 20 We therefore hypothesized, and subsequently demonstrated in this study, that in the S100A4 overexpressing versus C57 mouse, heightened susceptibility of elastin to fragmentation, coupled to elevated serine elastase activity following M1-MHV-68 infection, can contribute to the development of neointimal lesions.…”

Targeted expression of heme oxygenase-1 prevents the pulmonary inflammatory and vascular responses to hypoxia

“…It is possible that these cytokines initiate a cascade of events leading to VSMC proliferation that is later amplified by the release of vasoactive mediators and growth factors that alter vessel wall structure and function. For example, the initial injury and loss of endothelial barrier function can lead to extravasation of serum and activation of endogenous elastolytic activity from vascular smooth muscle cells (27,28). Increased serine elastase activity results in proteolytic degradation of extracellular matrix and the release of matrix-bound growth factors amplifying smooth muscle cell proliferation and remodeling of the pulmonary arterioles (27,29).…”