Shear stress influences spatial variations in vascular Mn-SOD expression: implication for LDL nitration

Abstract

Fluid shear stress modulates vascular production of endothelial superoxide anion (O2*-) and nitric oxide (*NO). Whether the characteristics of shear stress influence the spatial variations in mitochondrial manganese superoxide dismutase (Mn-SOD) expression in vasculatures is not well defined. We constructed a three-dimensional computational fluid dynamics model simulating spatial variations in shear stress at the arterial bifurcation. In parallel, explants of arterial bifurcations were sectioned from the human left main coronary bifurcation and right coronary arteries for immunohistolocalization of Mn-SOD expression. We demonstrated that Mn-SOD staining was prominent in the pulsatile shear stress (PSS)-exposed and atheroprotective regions, but it was nearly absent in the oscillatory shear stress (OSS)-exposed regions and lateral wall of arterial bifurcation. In cultured bovine aortic endothelial cells, PSS at mean shear stress (tau ave) of 23 dyn/cm2 upregulated Mn-SOD mRNA expression at a higher level than did OSS at tau ave = 0.02 dyn/cm2 +/- 3.0 dyn.cm(-2).s(-1) and at 1 Hz (PSS by 11.3 +/- 0.4-fold vs. OSS by 5.0 +/- 0.5-fold vs. static condition; P < 0.05, n = 4). By liquid chromatography and tandem mass spectrometry, it was found that PSS decreased the extent of low-density lipoprotein (LDL) nitration, whereas OSS increased nitration (P < 0.05, n = 4). In the presence of LDL, treatment with Mn-SOD small interfering RNA increased intracellular nitrotyrosine level (P < 0.5, n = 4), a fingerprint for nitrotyrosine formation. Our findings indicate that shear stress in the atheroprone versus atheroprotective regions regulates spatial variations in mitochondrial Mn-SOD expression with an implication for modulating LDL nitration.

Fig. 1

Spatial variations in shear stress. (a) The boundary condition. (b) Carreau model for non-Newtonian blow flow (Bird R. B., Armstrong R. C., Hassager O., 1977. Dynamics of Polymeric Liquids, Vol. 1, Fluid Mechanics New York: Wiley.470 PP.), where μ represents viscosity, γ represents the shear rate, μ₀ represents the zero shear rate limit viscosity, μ_∞ represents the infinite shear rate limit viscosity, λ represents the relaxation time constant, and n is the power law index. (c) Reconstruction of carotid artery. (d) An instantaneous velocity profile (t = 0.244 s). (e) Anterior-oblique angle of shear stress profile at an instantaneous moment with the mean Reynolds number of 289. (f) Top view of shear stress profile. White arrow indicates the point of flow separation.

Fig. 2

Mn-SOD immunostaining of a section of left coronary artery. (a) A representative left coronary artery and its branches. (b) von Willebrand Factor (vWF) staining for EC. (c) At the left main bifurcation (OSS-exposed region), Mn-SOD staining was absent in the luminal EC. (d) In straight segment of LAD (PSS-exposed region), Mn-SOD staining was prevalent throughout the entire luminal EC. (e) eNOS staining was positive. This section was previously reported (Hsiai et al. (provide ref)).

Fig. 3

Nitrotyrosine immunostaining of left main coronary arterial bifurcation. (a) Counterstaining with Hemotoxylin. (b) β-actin forsmooth muscle cells (SMC). (c) von Willebrand factor(vWF) for EC. Note that “*” indicates the lumen that was denuded in EC. (d) MnSOD staining was absent in the luminal endothelium. (e) Nitrotyrosine staining was prevalent in the SMC. (f) eNOS staining was absent.

Fig. 4

SOD isofrom expression in BAEC in response to oscillatory (OSS) and pulsatile shear stress. Cu/Zn-, Mn-, and extracellular-SOD mRNA expression was normalized to 18s RNA (*P < 0.05 in comparison to the static samples, n=3). (a) Cu/Zn-SOD expression was up-regulated by 2.3-fold in response to PSS. (b) Mn-SOD mRNA expression was up-regulated by 5-fold in response to OSS and by 11.4-fold to PSS. (c) EC-SOD remained relatively unresponsive to shear stress, (*P < 0.05 versus static, #P < 0.05 versus OSS, n=3). (d) Mn-SOD protein expression. The increased in Mn-SOD expression was not statistically significant in response to OSS. PSS up-regulated Mn-SOD expression by 1.4-fold (* P < 0.05, n = 4).

Fig. 5

Liquid chromatography/electron spray ionization/mass spectroscopy/mass spectroscopy (LC/ESI/MS/MS) analyses of LDL protein nitration as a finger print of nitrotyrosine formation. LDL nitration occurred when BAEC were treated with native LDL at 4 h. OSS further increased the level of LDL nitration whereas PSS decreased the level at 4 h (*P < 0.05 versus static at 0h, **P < 0.05 versus OSS, n=4).

Fig. 6

Dot Blot analyses of extracellular nitrotyrosine levels in the presence of native LDL. Blank refers to samples without LDL treatment. Native LDL induced an increase in LDL nitrotyrosine level. Addition of Mn-SOD mimetic (MnTMPyP) significantly attenuated the nitrotyrosine level. Positive control indicates samples treated with ONOO-. The nitrotyrsoine level in siMn-SOD treated HAEC was significantly higher than that of MnTMPyP-treated HAEC. The differences between siMn-SOD plus LDL-treated samples and the LDL-treated were statistically insignificant (P >0.05, n=4).

Fig. 7

Western analysis of intracellular nitrotyrosine levels in native LDL-treated BAEC. Silencing Mn-SOD significantly increased nitrotyrosine level. Blank refers to samples without LDL treatment. Positive control indicates samples treated with ONOO- (P < 0.05, n=3).