Oxygen levels within the wall of SV cultured at various levels of pO₂ and the impact of pO₂ on the proliferation of cultured SMC.

<p>(A) Arrow indicates location of the black EPR probe within SV cultured ex vivo for internal pO₂ measurements. Intima and adventitia are labeled to show vessel orientation. Scale bar is 50 μm. (B) Quantification of oxygen levels within intact SV as measured by EPR probe. SV were in ex vivo culture at pO₂ of either 40 (venous), 95 (arterial) or 140 mmHg (typical cell culture atmosphere). Cell number as a function of time during culture for SMC isolated from HSV (C) and 3T3 fibroblasts (D). Isolated SMC were cultured at venous pO₂ or at arterial pO₂, with and without tiron. NIH3T3 fibroblasts were cultured at venous or arterial pO₂. *indicates statistically different compared to other groups at corresponding time point. Standard deviations bars are not visible when their size is small compared to the size of the symbols indicating means.</p

Arterial Levels of Oxygen Stimulate Intimal Hyperplasia in Human Saphenous Veins via a ROS-Dependent Mechanism

<div><p>Saphenous veins used as arterial grafts are exposed to arterial levels of oxygen partial pressure (pO₂), which are much greater than what they experience in their native environment. The object of this study is to determine the impact of exposing human saphenous veins to arterial pO₂. Saphenous veins and left internal mammary arteries from consenting patients undergoing coronary artery bypass grafting were cultured ex vivo for 2 weeks in the presence of arterial or venous pO₂ using an established organ culture model. Saphenous veins cultured with arterial pO₂ developed intimal hyperplasia as evidenced by 2.8-fold greater intimal area and 5.8-fold increase in cell proliferation compared to those freshly isolated. Saphenous veins cultured at venous pO₂ or internal mammary arteries cultured at arterial pO₂ did not develop intimal hyperplasia. Intimal hyperplasia was accompanied by two markers of elevated reactive oxygen species (ROS): increased dihydroethidium associated fluorescence (4-fold, p<0.05) and increased levels of the lipid peroxidation product, 4-hydroxynonenal (10-fold, p<0.05). A functional role of the increased ROS saphenous veins exposed to arterial pO₂ is suggested by the observation that chronic exposure to tiron, a ROS scavenger, during the two-week culture period, blocked intimal hyperplasia. Electron paramagnetic resonance based oximetry revealed that the pO₂ in the wall of the vessel tracked that of the atmosphere with a ~30 mmHg offset, thus the cells in the vessel wall were directly exposed to variations in pO₂. Monolayer cultures of smooth muscle cells isolated from saphenous veins exhibited increased proliferation when exposed to arterial pO₂ relative to those cultured at venous pO₂. This increased proliferation was blocked by tiron. Taken together, these data suggest that exposure of human SV to arterial pO₂ stimulates IH via a ROS-dependent pathway.</p></div

ROS levels in freshly isolated and cultured SV as assessed by DHE staining.

<p>(A) DHE and DAPI staining of freshly isolated SV and SV cultured at arterial or venous pO₂. Scale bar is 100 μm. (B) Average normalized mean of DHE fluorescence intensity (n = 3). * indicates p<0.05 relative to other groups marked with #. There were no intragroup differences among subgroups marked with #.</p

Quantification of intimal area.

<p>(A) Histogram of intimal areas for freshly isolated SV and SV cultured at arterial pO₂. (B) Normalized intimal area of SV (n≥6) and IMA (n = 4) when either freshly harvested or cultured at venous, arterial and typical cell culture pO₂. * indicates p<0.05 relative to other groups.</p

Histology of human SV and IMA freshly isolated or cultured with arterial pO₂.

<p>Human SV (A-D) and IMA (E-H-) stained with Elastin Stain for both freshly isolated vessels (A,E) and vessels cultured at arterial pO₂ (B,F), typical cell culture pO₂ (C,G) and venous pO₂ (D,H). The intima (I), media (M), adventitia (Ad) and inner elastic lamina (IEL) are labeled when visible. Scale bar is 100 μm.</p

Elastin-stained sections from freshly harvested SV and SV cultured for 14 days.

<p>In Panels A, C, and D the and the IEL, which stain black, are intact and there is no neointimal formation. In Panel B there is rupture in the IEL (breaks in IEL indicated with black arrows) allowing cells from the media to migrate into the intima. Scale bar is 100 μm.</p

Distribution and levels of 4-HNE adducts in freshly isolated and cultured SV.

<p>(A) SV immunostained for 4-HNE adducts. Positive staining is shown in dark brownish black and detects levels of lipid peroxidation due to ROS. Conditions shown are freshly isolated (I), and after being cultured at venous pO₂ (II) or arterial pO₂ without (III) and with tiron (VI). Scale bar is 100 μm. (B) Western blot for 4-HNE adducts. Gels contained samples not related to the present study and these lanes are not shown. Noncontiguous gel lanes are demarcated by a vertical white line. (C) Quantification of 4-HNE normalized to actin amount. *p<0.05 relative to group marked with #.</p