Although intravasation may be a critical rate-limiting step in the metastatic cascade, the role of oxidative stress in intravasation is unknown. We tested the hypothesis that reactive oxygen species (ROS), regulated by thioredoxin interacting protein (Txnip) through the action of thioredoxin (Trx), influence human SK-MEL-28 melanoma cell reverse (basolateral-to-apical) transendothelial migration (TEM) in vitro as a model for intravasation. Reverse transendothelial migration was dose-dependently induced by hydrogen peroxide 2.4-fold for 0.1 microM (P < 0.01) and 3.9-fold for 1 microM (P < 0.001) vs. control, and this effect was blocked by the antioxidant N-acetylcysteine. Overexpression of Txnip by infecting melanoma cells with adenovirus increased TEM 3-fold vs. control (P < 0.001), and this increase was blocked by N-acetylcysteine, indicating a redox-sensitive mechanism. Conversely, thioredoxin overexpression blocked hydrogen peroxide-induced TEM. Exposure to ultraviolet-A radiation increased ROS 1.8-fold (P < 0.01), and this was accompanied by a 45% reduction (P < 0.05) in thioredoxin activity and an 11.4-fold (P < 0.001) increase in Txnip gene expression. These data suggest that TEM of melanoma cells during intravasation is in part mediated by ROS-sensitive cellular signaling cascades, may be controlled by Txnip and its interaction with thioredoxin that in turn modulates cellular levels of oxidative stress, and may be initiated by ultraviolet-A induction of this cascade.

OBJECTIVES We tested the hypothesis that regional end-systolic left ventricular (ESLV) wall stress is associated with extracellular matrix remodeling activity after myocardial infarction (MI).BACKGROUND Increased left ventricular (LV) wall stress is a stimulus for LV enlargement, and echocardiography can be used to estimate regional wall stress. A powerful validation of a noninvasive method of estimating wall stress would be predicting cellular responses after a MI.METHODS Echocardiographic images were obtained in rats 1, 7, 14 or 21 days after coronary ligation (n = 11) or sham surgery (n = 5). End-systolic left ventricular wall stress was calculated by finite element analysis in three regions (infarcted, noninfarcted and border) from short-axis images. Matrix metalloproteinase-9 (MMP-9) and macrophage density were determined by immunohistochemistry, and positive cells were counted in high power fields (hpf).RESULTS Average ESLV wall stress was higher in rats with MI when compared to shams irrespective of time point (p < 0.01), and ESLV wall stress in the infarcted regions increased with time (25.1 +/- 5.9 vs. 69.9 +/- 4.4 kdyn/cm(2), day 1 vs. 21; p < 0.01). Matrix metalloproteinase-9 expression was higher in infarcted and border regions when compared to noninfarcted regions (22.1 vs. 25.7 vs. 0.10 cells/hpf, respectively p < 0.01). Over all regions, ESLV wall stress was associated with MMP-9 (r = 0.76; p < 0.001), macrophage density (r = 0.72; p < 0.001) and collagen content (r = 0.67; p < 0.001). End-systolic left ventricular wall stress was significantly higher when MMP-9 positive cell density was greater than 10 cells/hpf (45 +/- 20 vs. 14 +/- 10 kdyn/cm(2); p < 0.001).CONCLUSIONS Regional increases in ESLV wall stress determined by echocardiography-based structural analysis are associated with extracellular matrix degradation activity. (C) 1999 by the American College of Cardiology.

Although fibroblast growth factor-2 (FGF-2) participates in the response to vascular injury, the role of cellular deformation in FGF-2 release is incompletely understood. To test the hypothesis that mechanical strain tightly controls FGF-2 release, a novel device was used to impose homogeneous and uniform biaxial strain to human vascular smooth muscle cells. Release of FGF-2 increased with the number of cycles of strain (14%, 1 Hz); 1, 9, and 90 cycles of strain, respectively, released 0.55 +/- 0.06%, 2.9 +/- 0.3%, and 5.5 +/- 1.3% of the total cellular FGF-2 (versus 0.00 +/- 0.40% for control, P < .05), but release was not further increased for strain of 90 to 90,000 cycles. Mechanical release of FGF-2 depended on both the frequency and amplitude of deformation. For example, strain (90 cycles, 1 Hz) at 4% amplitude released only 0.1 +/- 0.1% of the total FGF-2, but strain at 14% and 33% amplitudes, respectively, released 5.7 +/- 0.5% and 19.0 +/- 3.0% of the FGF-2 cellular pool (P < .05), suggesting a strain amplitude threshold for FGF-2 release. Injury to a subpopulation of cells increased with the frequency and amplitude of strain, but cells were not injured by strains below 10% amplitude. Strain following pretreatment with heparin released 12.6 +/- 1.6% of the total FGF-2 (versus 15.8 +/- 0.9% for strain alone, P < .05), indicating that most FGF-2 was liberated from the nuclear or cytoplasmic pools and not from low-affinity extracellular receptors. Conversely, strain in the presence of heparin released 25.2 +/- 3.5% of the total FGF-2 (versus 15.6 +/- 2.6% for strain alone, P < .05). Thus, cellular strain closely modulates the release of intracellular FGF-2 from human vascular smooth muscle cells, but FGF-2 release is negligible in response to the smaller strains that occur in the normal artery. In addition, larger mechanical strains lead to transfer of intracellular FGF-2 to the extracellular low-affinity receptors, where FGF-2 may be displaced by heparin. These observations provide insight into the mechanisms by which deforming vascular injury, such as that produced by arterial interventions, may elicit a proliferative response.

Both IL-1 alpha and IL-1 beta lack an N terminus secretory sequence, and the mechanism of secretion of these pleiotropic cytokines is incompletely understood. The epidermis contains large quantities of IL-1 alpha in keratinocytes, which may play a role in inducing endothelial adhesion molecules and promoting extravasation of leukocytes. Here we report that mechanical deformation of human keratinocytes leads to rapid release of IL-1 alpha, possibly through transient disruptions in the plasma membrane. Using a device that precisely controls the amplitude of strain on the culture substrate, we found by pulse-chase analysis, Western analysis, and ELISA that the release of IL-1 alpha is dependent on the amplitude of the strain. A cyclic strain of 14% released a small but significant quantity of IL-1 alpha, while strains of 33% released 66 +/- 9% of cytoplasmic IL-1 alpha over 1 h (p < 0.001). Release of IL-1 alpha was accompanied by rapid release of large stores of IL-1R antagonist, approximately 25 to 30 times greater by mass than the quantity of IL-1 alpha released, but only a small fraction of cytoplasmic lactate dehydrogenase. Media conditioned by mechanically stimulated keratinocytes induced expression of E-selectin by human vascular endothelial cells; induction of E-selectin was completely inhibited by an Ab to IL-1 alpha. Therefore, mechanical strain promotes the secretion of IL-1 alpha, and deformation of keratinocytes in the epidermis may activate vascular endothelium through mechanically released IL-1 alpha. This pathophysiologic mechanism may play a role in the anatomic localization of some inflammatory skin diseases, such as psoriasis, which occurs more commonly in locations where the dermis is subjected to repetitive stretch or trauma.

Background Although mechanical vascular injury leads to smooth muscle cell proliferation that contributes to restenosis after balloon angioplasty, the role of the single transient mechanical stimulation of smooth muscle cells in this process is unknown. Methods and Results To test the hypothesis that a single transient mechanical stimulus can increase DNA synthesis, human vascular smooth muscle cells cultured in a three-dimensional collagen gel system were subjected to transient compression. Transient compression (5-minute duration) of smooth muscle cell–collagen gel cultures in defined serum-free conditions led to delayed increases in [ 3 H]thymidine incorporation. At 12 to 24 hours after compression, there was a 3.3±0.5-fold ( P <.001 versus control) and 3.0±0.6-fold ( P <.002 versus control) increase for 60% and 80% strain, respectively; at 24 to 36 hours after compression, there was a 1.8±0.5-fold ( P <.05 versus control) and 4.3±0.8-fold ( P <.001 versus control) increase. Also, serum-free media conditioned by transiently compressed gel cultures induced DNA synthesis in control, unstimulated smooth muscle cell cultures, suggesting the release of growth factors by transient compression. Although neutralizing antibodies against platelet-derived growth factor did not affect the mechanical induction of DNA synthesis, a neutralizing monoclonal antibody against fibroblast growth factor–2 (FGF-2) decreased this induction by 89% and completely blocked the increase in DNA synthesis caused by media conditioned by transiently compressed gels. Media conditioned by transient compression contained elevated levels of FGF-2 (17±5 versus 2±2 pg/mL for control, P <.005) with no increase in lactate dehydrogenase activity, suggesting release of FGF-2 with sublethal cellular injury. Conclusions A single transient mechanical stimulus increases DNA synthesis in human vascular smooth muscle cells, in part by autocrine or paracrine FGF-2 release.

Vascular smooth muscle cells perform the important function of modulation of vascular extracellular matrix. Because integrins mediate many cell-matrix interactions, the role of integrins in reorganization of collagen by cultured human vascular smooth muscle cells was studied. Immunoprecipitation demonstrated that human vascular smooth muscle cells express multiple beta 1 integrins. Monoclonal antibody A2-IIE10 (a blocking anti-alpha 2 antibody) inhibited adhesion of smooth muscle cells to collagen by 31%. The blocking anti-alpha 1 antibody 1B3.1 inhibited adhesion by 40%, whereas a blocking anti-alpha 3 antibody had no effect on adhesion. When 1B3.1 and A2-IIE10 were both used, a 79% reduction in adhesion was observed, indicating that active alpha 1 and alpha 2 integrins cooperatively mediate adhesion. The blocking anti-beta 1 antibody Mab13 abolished smooth muscle cell-mediated gel contraction, and the alpha 2-blocking antibody A2-IIE10 had a dose-dependent partial inhibitory effect (37%). In contrast, blocking antibodies to alpha 1 and alpha 3 had no effect. When anti-alpha 1 (1B3.1) and anti-alpha 2 (A2-IIE10) monoclonal antibodies were combined, no synergistic effect on inhibition of gel contraction was observed. Surprisingly, collagen gel contraction was inhibited by 46% by an anti-beta 1 antibody (TS2/16) known for its stimulatory effect on cell adhesion. Thus, whereas alpha 1 beta 1 and alpha 2 beta 1 integrins both participate in adhesion of vascular smooth muscle cells to collagen, only alpha 2 beta 1 integrins mediate collagen reorganization. In addition, collagen reorganization appears to be a dynamic process, adversely affected by excessive adhesion strengthening.

BACKGROUND:Although rupture of an atherosclerotic plaque is considered to be the cause of most acute coronary syndromes, the mechanism of plaque rupture is controversial.METHODS AND RESULTS:To test the hypothesis that plaque rupture occurs at sites of high circumferential stress in the diseased vessel, the distribution of stress was analyzed in 24 coronary artery lesions. Histological specimens from 12 coronary artery lesions that caused lethal myocardial infarction were compared with those from 12 stable control lesions. A finite element model was used to calculate the stress distributions at a mean intraluminal pressure of 110 mm Hg. The maximum circumferential stress in plaques that ruptured was significantly higher than maximum stress in stable specimens (4,091 +/- 1,199 versus 1,444 +/- 485 mm Hg, p < 0.0001). Twelve of 12 ruptured lesions had a total of 31 regions of stress concentration of more than 2,250 mm Hg (mean, 2.6 +/- 1.4 high stress regions per lesion); only one of 12 control lesions had a single stress concentration region of more than 2,250 mm Hg. In seven of 12 lethal lesions (58%), rupture occurred in the region of maximum circumferential stress; in 10 of the 12 lethal lesions (83%), rupture occurred in a region where computed stress was more than 2,250 mm Hg.CONCLUSIONS:These data suggest that concentrations of circumferential tensile stress in the atherosclerotic plaque may play an important role in plaque rupture and myocardial infarction. However, plaque rupture may not always occur at the region of highest stress, suggesting that local variations in plaque material properties contribute to plaque rupture.

Objectives. This in vitro study was designed to test the hypothesis that a structural analysis based on intravascular ultrasound images of atherosclerotic vessels obtained before angioplasty can be used to predict plaque fracture locations and balloon pressures required to cause fracture.Background. Intravascular ultrasound imaging performed before interventional procedures has potential for providing information useful for guiding therapeutic strategies.Methods. Intravascular imaging was performed on 16 atherosclerotic human iliac vessel segments obtained freshly at autopsy; balloon angioplasty was then performed with 1-min inflations at 2 atm, increasing in 2-atm increments until fracture of the lumen surface occurred. Fracture locations were confirmed by histopathologic examination. Structural analysis of these images was performed with a large strain finite element analysis of the image that calculated the distribution of stress in the vessel with 2 atm of lumen pressure.Results. Structural analysis demonstrated a total of 30 high circumferential stress regions in the vessels (mean 1.9 high stress regions/vessel). A total of 18 plaque fractures occurred in the 16 vessel segments. Of the 17 fractures that occurred in the 15 specimens with regions of high circumferential stress, 14 (82%) occurred at a high stress region (p < 0.0001). However, there was no significant relation between the peak stresses estimated by structural analysis and the ultimate balloon inflation pressure required to cause fracture.Conclusions. Structural analysis based on intravascular ultrasound imaging performed before in vitro balloon angioplasty can predict the locations of plaque fracture that usually accompany angioplasty. However, these data suggest that intravascular ultrasound may not be useful for predicting the ultimate balloon inflation pressure necessary to cause fracture, possibly because of the variable fracture properties and microscopic structure of atherosclerotic tissues.