In this study, we demonstrated that shear stress influenced spatial variations in Mn-SOD expression. That oscillatory and pulsatile shear stress patterns may contribute to this phenomenon is supported by three lines of evidences: (1) the 3-D computational fluid dynamic model predicts regions of OSS and PSS in the arterial bifurcations; (2) immunohisto-localization and physiological study showed that Mn-SOD staining was prominent in the PSS-exposed regions where nitrotyrosine staining was absent, (3) using a 2-D flow system, we demonstrated that PSS and OSS differentially regulated LDL protein nitration via Mn-SOD mRNA expression.
The 3-D CFD code confirmed that the endothelial cells (EC) in medial wall or the straight segment of vessels experience pulsatile shear stress (PSS); whereas EC near the point of flow separation experience oscillatory shear stress (OSS) in the atherosclerosis-prone regions 1
. The mean shear stress values obtained from the 3-D model were implemented in the 2-D flow system to elucidate SOD isoform mRNA expression and LDL protein nitration. Our dynamic model was designed to generate specific pulsatile versus oscillatory shear stress at various slew rates (Hsiai, Ann BME, 2002
); however, Blackman et al
. reported the atheroprotective versus athero-genic waveforms from human carotid arteries (ref-Blackman BR, 2002
Mn-SOD is an important dismutase of reactive oxygen species acting in the mitochondria matrix. Oxidative phosphorylation in mitochondria ATP occurs as electrons are transferred from NADH or FADH2
to molecular oxygen. The transfer of more than 98% of electrons by the electron transport chain is coupled with the production of ATP, 1.5 to 2 % of electrons leak out to form O2
, which is dismutased by Mn-SOD 34
. In response to pathologic conditions such as reperfusion injury, the electron transport chain may become uncoupled, leading to an increase in O2
. Bernal-Mizrachi et al
. reported respiratory uncoupling in smooth muscle cells caused atherosclerosis in mice 36
. In apolipoprotein E (apoE)-deficient mice (apoE(−/−))37
, the normally expressed levels of Mn-SOD protects against oxidative stress and endothelial dysfunction.
We were able to compare the OSS-exposed regions such as bifurcations or greater curvatures with the PSS-exposed regions such as the relative straight segments or medial wall of arterial bifurcations. Our finding supports the notion that spatial variations in shear stress influence MnSOD staining in 3 out of 3 explants of human coronary arteries. All three patients were diagnosed with ischemic cardiomyopathy due to atherosclerosis. The immunohistochemistry analysis in was representative of a section of left anterior distal arteries where pulsatile shear stress occurred and atherosclerotic lesions were absent; and that of was representative of lateral wall of an arterial bifurcation where oscillatory shear stress developed and atherosclerotic lesions were present. We previously reported that nitrotyrosine staining was prominent in the OSS-exposed region, but it was absent in the PSS-exposed regions 33
. In PSS-exposed regions, Mn-SOD staining was prominent but nitrotyrosine staining was absent, and Mn-SOD expression influenced the extent of LDL nitration ( & ).
Our data also suggest that Mn-SOD and CuZn-SOD, but not EC-SOD, are responsive to shear stress in vascular endothelial cells. EC-SOD is synthesized in smooth muscle cells, and CuZn-SOD is prevalent in vascular endothelial cells. Inou et al.
reported that CuZn-SOD in human aortic endothelial cells (HAEC) is up-regulated in response to laminar flow 13
. Despite different experimental designs; namely, (1) the cell type (BAEC versus HAEC), (2) duration of flow exposure (4 versus 24 hours), (3) magnitude and characteristic of shear stress profiles, and (4) flow models (cone-and-plate versus pulsatile parallel plate), our finding in CuZn-SOD expression in response to PSS is in agreement with that Inou et al.
. Moreover, we demonstrated that shear stress regulated Mn-DOS and CuZn-SOD expression in both BAEC and HAEC. Whether there exists a shear stress-responsive element in Mn-SOD gene remains undefined; however, a redox-sensitive mechanism was implicated in Mn-SOD promoter activity via a negative PI3K-akt-forkhead pathway and a positive PKC-NF-κB pathway 38
Several studies have examined effects of SOD isoforms on endothelial function and atherogenesis. EC-SOD plays a significant protective role on arterial pressure, vascular function, or vascular levels of oxidative stress in spontaneous hypertensive rats 39
. Similarly, cytosolic CuZn-SOD is atheroprotective, playing a critical role in limiting angiotensin II-induced endothelial cell dysfunction 40
. The CuZn-SOD-deficient (CuZn-SOD(−/−)) mice showed an increased superoxide level and altered vascular responsiveness compared with the wild-type littermates 41
. The protein encoded Mn-SOD transmigrate to mitochondria. Mn-SOD-deficient mice (Mn-SOD(+/−) were reported to develop more atherosclerosis 37
. However, the relative contribution of CuZn-SOD and Mn-SOD to dismutase superoxide anion in response to shear stress requires further investigations.
Previously, we reported that pulsatile flow significantly reduced the ratios of oxidatively modified forms of LDL relative to static conditions, whereas oscillating flow increased LDL oxidation 17
, leading to up-regulation of adhesion molecules and recruitment of monocytes 42,43
. In this study, OSS increased the level of tyrosine nitration and oxidation products in LDL, whereas PSS decreased the levels of both tyrosine nitration and oxidation. Nitration of tyrosine residues by ONOO−
resulted in a more hydrophilic residue, thus altering the structure of α helices of ApoB-100 of the LDL particles 44,45
We analyzed LDL protein nitration at both protein and amino acid levels. The former was established by dot blots. Using anti-nitrityrosine antibody, we suspended LDL in mediums to assess LDL protein nitration. The latter was performed by liquid chromatography, electro ionization spray, and tandem mass spectrometry (LC/ESI/MS/MS). The proteomic approach allowed for identifying the specific apo B-100 tyrosine residue nitration in α and β helices as follows: α-1 (Tyr144), α-2 (Tyr2524), β-2 (Tyr3295), α-3 (Tyr4116), and β-2 (Tyr4211) (Hsiai, FRBM, 2007). Both lipid peroxidation and protein nitration account for apoB-100 protein unfolding and consequential increase in modified LDL binding and uptake by endothelial cells.
Antioxidant gene expression is present in mouse aortic arch where disturbed flow, including oscillatory shear stress, develops. Perserini, Davies et al. reported a host of antioxidant gene expression from adult porcine aortic arch by genomics 46
. The authors suggested that there is an evolutionary benefit to favor a moderate level of atheroprotective gene expression in the athero-prone regions such as curvatures and bifurcations. In this context, our in vitro
findings between oscillatory and pulsatile shear stress complement those of mouse genomic analysis.
In summary, our bifurcation model predicts the characteristics of shear stress in the PSS- and OSS-exposed regions of the arterial bifurcation. Explants of human coronary arteries enabled us to assess the pathophysiological significance of this model in terms of Mn-SOD immunohisto-localization. Our findings strongly support the notion that pulsatile and oscillatory shear stress modulated spatial variations in Mn-SOD expression and that Mn-SOD plays an important role in LDL protein nitration.