Nitric oxide (NO) participates in the regulation of vascular tone and smooth muscle cell proliferation, but little is known of its effect on total protein and matrix synthesis in smooth muscle. We studied the effects of the NO-generating compounds S-nitroso-N-acetylpenicillamine (SNAP, 0.4 to 1.2 mmol/L) and sodium nitroprusside (SNP, 0.1 to 0.5 mmol/L) on total protein (using [3H]leucine) and collagen (using [3H]proline) synthesis in cultured rabbit aortic smooth muscle cells. Both agents caused dose-dependent inhibition of the relative rate of protein (maximum reduction of 87% [SNAP] and 80% [SNP]) and collagen synthesis, as measured by trichloroacetic acid-precipitated label. The magnitudes of percent inhibition of total protein and collagen synthesis were approximately equal. Inhibition of protein synthesis by SNAP and SNP was prevented by hemoglobin (10 mumol/L), suggesting that the protein synthesis inhibition was due to NO release. Inhibition of protein synthesis was reversible after removal of SNAP and SNP and was not caused by damage to the cells. These results suggest that NO may function as a modulator of vascular smooth muscle cell protein synthesis and production of extracellular matrix components.

The effect of mechanical stimuli on pulmonary artery growth and matrix tissue synthesis (and how individual cell types in the vessel wall respond to such stimuli) is incompletely characterized. Rabbit pulmonary arteries were placed in tissue culture medium and subjected to varying magnitudes of stretch or hydrostatic pressure (separately) for 4 days. The rate of protein synthesis in smooth muscle cells (by quantitative autoradiography) was positively related to the magnitude of stretch, as were the percentage of procollagen type I-positive cells and the rate of cell replication. In adventitial fibroblasts, stretch increased the rate of replication but not of protein synthesis. Hydrostatic pressure had little or no effect on the variables measured in either smooth muscle cells or fibroblasts. Stretch also increased the rate of elastin and collagen synthesis in the whole pulmonary artery segment, and after 4 days of stretch, the contents of actin and elastin were increased. Removal of the endothelium did not affect stretch-induced protein, collagen, or elastin synthesis but augmented stretch-induced smooth muscle replication. These data suggest that in the intact pulmonary artery, stretch, but not pressure, can stimulate hypertrophy and hyperplasia in smooth muscle cells and hyperplasia in fibroblasts. Matrix protein synthesis and accumulation are also increased by stretch. Neither stretch-mediated growth nor matrix protein synthesis required endothelium in this model.

No abstract

Cathepsin G, a major protease released by activated neutrophils, induces functional and morphological damage to human endothelial cells. We studied the mechanisms involved and ways to reverse this damage. Cathepsin G induced a concentration- and time-dependent injury to human umbilical vein endothelial cell (HUVEC) morphology simultaneous with cytoskeleton rearrangement. Preincubation of the endothelial monolayer with phallacidin completely prevented damage to cell morphology by cathepsin g, whereas preincubation with cytochalasin b potentiated its activity. Damage to cell shape and F-actin cytoskeleton were prevented by eglin C, and inhibitor of the active site of cathepsin G. Furthermore, cathepsin G increased transcellular permeability to albumin and induced a time-dependent detachment of PAI-1 from the extracellular matrix of a cell-free system. The inhibition of matrix-bound PAI-1 activity by specific antibodies induced matrix-bound PAI-1 activity by specific antibodies induced changes in HUVEC monolayers similar to those observed after cathepsin G. However, although stabilization of F-actin microfilaments by phallacidin prevented changes in cell shape, it did not prevent the ability of cathepsin G to increase cell permeability and release matrix PAI-1. The damage of cathepsin G to cell morphology and cytoskeleton arrangement was reversed within 12 hours if the deendothelialization area was < 50% to 55% and the subendothelial matrix was still able to bind the newly synthesized PAI-1. Thrombin, whose role in the thrombotic process is well known, also induced changes in cell morphology and cytoskeleton arrangement of HUVEC. Cathepsin G reaches the subendothelial matrix through an increase in cell permeability and injures endothelial cell morphology by detaching matrix-bound PAI-1. These events expose a highly thrombogenic surface to which platelets can adhere, become activated, attract further neutrophils, and trigger thrombus formation.

SummaryActivated neutrophils may promote thrombus formation by releasing proteases which may activate platelets, impair the fibrinolytic balance and injure the endothelial monolayer.We have investigated the morphological correlates of damage induced by activated neutrophils on the vascular wall, in particular the vascular injury induced by released cathepsin G in both static and dynamic conditions.Human umbilical vein endothelial cells were studied both in a cell culture system and in a model of perfused umbilical veins. At scanning electron microscopy, progressive alterations of the cell monolayer resulted in cell contraction, disruption of the intercellular contacts, formation of gaps and cell detachment.Contraction was associated with shape change of the endothelial cells, that appeared star-like, while the underlying extracellular matrix, a potentially thrombogenic surface, was exposed. Comparable cellular response was observed in an “in vivo” model of perfused rat arterial segment. Interestingly, cathepsin G was active at lower concentrations in perfused vessels than in culture systems. Restoration of blood flow in the arterial segment previously damaged by cathepsin G caused adhesion and spreading of platelets on the surface of the exposed extracellular matrix. The subsequent deposition of a fibrin network among adherent platelets, could be at least partially ascribed to the inhibition by cathepsin G of the vascular fibrinolytic potential.This study supports the suggestion that the release of cathepsin G by activated neutrophils, f.i. during inflammation, may contribute to thrombus formation by inducing extensive vascular damage.