Mechanisms of enhanced vascular reactivity in preeclampsia

Abstract

Preeclamptic women have enhanced blood pressure response to angiotensin II and extensive systemic vascular infiltration of neutrophils. Neutrophils release reactive oxygen species that might activate the RhoA kinase pathway to enhance vascular reactivity. We hypothesized that enhanced vascular reactivity in preeclampsia is attributed to neutrophil-mediated reactive oxygen species activation of the RhoA kinase pathway. Omental arteries were obtained at cesarean section and studied using a myograph system. We found that arteries of preeclamptic women had extensive infiltration of neutrophils and enhanced reactivity to angiotensin II. Treatment of arteries of normal pregnant women with reactive oxygen species or activated neutrophils enhanced vessel reactivity to angiotensin II mimicking preeclamptic vessels. Pretreatment with superoxide dismutase/catalase to quench reactive oxygen species or RhoA kinase inhibitor blocked enhanced responses in preeclamptic and normal vessels. Reactive oxygen species also enhanced vessel reactivity to norepinephrine, which was blocked by RhoA kinase inhibition. Treatment of arteries with reactive oxygen species increased RhoA kinase activity 3-fold, whereas culture of human vascular smooth muscle cells with angiotensin II and activated neutrophils or reactive oxygen species resulted in phosphorylation of key proteins in the RhoA kinase pathway. We conclude that enhanced vascular reactivity of omental arteries in preeclampsia is attributed to reactive oxygen species activation of the RhoA kinase pathway and that enhanced vascular reactivity is likely attributed to the infiltration of neutrophils. We speculate that neutrophil infiltration into systemic vasculature of preeclamptic women is an important mechanism for hypertension.

Figure 1

Comparison of vessel reactivity to angiotensin II (Ang II). A) Vessel reactivity to Ang II was significantly enhanced in endothelium intact omental arteries obtained from preeclamptic women (PE, n=9) as compared to normal pregnant women (NP, n=6). B) Enhanced vessel reactivity to Ang II was blocked by pretreatment with a combination of superoxide dismutase and catalase (SOD/Cat) or RhoA kinase (ROK) inhibitor (n=4). (*** p<0.001 for treatment effects)

Figure 2

Representative sections of omental fat vessels immunostained for CD66b, a neutrophil antigen, CD99, a lymphocyte antigen and CD14, a monocyte/macrophage antigen. A) Isotype negative control for CD66b (preeclamptic). B) normal pregnancy for CD66b. Vessels of normal pregnant patients had some brown staining for neutrophils primarily in the lumen. C and D) preeclamptic pregnancy for CD66b. Vessels of preeclamptic patients showed extensive brown staining of neutrophils in the lumen, adhered and flattened along the endothelium and infiltrated to the vascular smooth muscle. E and F) lower magnification of CD66b staining showing little to no staining of vessels in normal pregnancy (E) as compared to almost all vessels showing extensive staining in preeclampsia (F). G) CD99 staining and H) CD14 staining in preeclamptic vessels. Few vessels showed staining for CD99 or CD14 and when they did, there were only one or two lymphocytes or monocytes/macrophages per vessel (arrows). A, adipocyte; VL, vessel lumen; NP, normal pregnancy; PE, preeclamptic pregnancy. All images were taken with a 40X lens except panels E and F taken with a 10X lens.

Figure 3

Vessel reactivity of omental arteries of normal pregnant women to angiotensin II (Ang II) in response to neutrophil products and presence or absence of endothelium. A) A reactive oxygen species generating solution (ROS) significantly enhanced vessel reactivity to Ang II in endothelium intact vessels (n=6). B) ROS enhanced vessel reactivity to Ang II to a greater extent when endothelium was removed (endo denuded) as compared to when endothelium was intact (n=19). Pretreatment with superoxide dismutase and catalase (SOD/Catalase) to quench ROS (n=5) or RhoA kinase (ROK) inhibitor (n=4) abolished enhanced vessel reactivity to Ang II in response to ROS. C) Removal of the endothelium enhanced vessel reactivity to Ang II (n=21) as compared to when endothelium was intact (n=6), but the enhancement was much less than that induced by ROS. D) Tumor necrosis factor-alpha (TNFα), another neutrophil product, did not enhance arterial reactivity to Ang II (n=4). (*** p<0.001 for treatment effects; NS, non-significant as compared to Ang II alone)

Figure 4

Vessel reactivity of endothelium denuded (endo denuded) omental arteries of normal pregnant women to angiotensin II (Ang II) in response to activated neutrophils. Omental arterial reactivity to Ang II was significantly enhanced with perfusion of activated neutrophils through the vessel lumen (n=11) similar to that observed with ROS in Figure 3. Pretreatment with superoxide dismutase and catalase (SOD/Catalase) to quench ROS (n=4) or RhoA kinase (ROK) inhibitor (N=4) abolished neutrophil enhanced vessel reactivity to Ang II. (*** p<0.001 for treatment effect; NS, non-significant as compared to Ang II alone)

Figure 5

RhoA kinase activity in human omental arteries and expression of phosphorylated myosin phosphatase target subunit 1 (pMYPT1) in human vascular smooth muscle cells. A) A reactive oxygen species generating solution (ROS) caused a 3-fold increase in RhoA kinase activity in omental arteries as compared to untreated control arteries (n=5, ** p<0.01). B) Representative Western blot of Thr696 pMYPT1, total MYPT1 and β-actin in cultured human vascular smooth muscle cells exposed to the treatments used in the myograph vascular reactivity experiments. C) Density of pMYPT1 plotted as percentage of control (n=3). Angiotensin II (Ang II) in the presence of ROS or activated neutrophils (Neu) significantly enhanced phosphorylation of MYPT1. (Y, RhoA kinase inhibitor Y-27632) (* P<0.05, ** P<0.01 as compared to control)

Figure 6

Expression of phosphorylated myosin light chain (pMLC). A) Representative Western blot of Ser19-pMLC, total MLC and β-actin in cultured human vascular smooth muscle cells. B) Density of pMLC plotted as percentage of control (n=5). Vascular smooth muscle cells were treated as in Figure 5. Treatment with angiotensin II (Ang II) plus reactive oxygen species (ROS) or activated neutrophils (neu) significantly enhanced phosphorylation of MLC. (Y, RhoA kinase inhibitor Y-27632) (* P<0.05 as compared to control)

Figure 7

Vessel reactivity to norepinephrine (NE). A) Omental arterial reactivity to NE was significantly enhanced in the presence of reactive oxygen species (ROS) (n=6, *** p<0.001 for treatment effect compared to NE alone). B) Addition of RhoA kinase (ROK) inhibitor abolished enhanced vessel reactivity to NE in response to ROS at 1.25 μmol/L dose of NE (n=4, * p<0.05).