Materials

Antibodies to hCRP, eNOS, p-Akt, total Akt, PI3K, AT1, AT2, p22 phox, gp91 phox, p40 phox, p47, p67 phox, SOD-1, p-p38, total p38, p-ERK1/2, total ERK1/2, p-JNK, total JNK, and β-actin were purchased from Santa Cruz Biotechnologyor Cell Signaling Technology. Antibodies of p-MBS and total MBS were obtained from Millipore Inc and Covance Inc, respectively.L-NG-Nitroarginine methyl ester (L-NAME) was from Cayman. Inc. Lucigenin was from Sigma.

Analytical methods

Blood was collected from the tail vein at two and four months after AAV injection. Serum hCRP was assessed by ELISA (BioCheck Inc, Foster City, CA, USA).Vascular and serum nitric oxide (NO) concentrations were measured using anitric oxide assay kit (Cayman. Inc, Ann Arbor, MI, USA). Serum insulin was measured by ELISA (Millipore. Inc, Billerica, MA, USA). Other plasma parameters including total cholesterol, HDL cholesterol, LDL cholesterol and triglycerides were assessed using an automatic analyzer (Roche) in the clinical laboratories of TongjiHospital.

Vascular relaxation experiment

Thoracic aortas were rapidly isolated and immersed in Krebs–Ringer HCO3 buffer (pH 7.4, NaCl, 118.3 mM; KCl, 4.7 mM; CaCl2, 2.5 mM; MgSO4, 1.2 mM; KH2PO4, 1.2 mM; NaHCO3, 25.0 mM; Ca-EDTA, 0.026 mM; glucose, 11.1mM) which was aerated with 95% O2:5% CO2. The vessel was carefully removed surrounding tissues and cut into 2-3 mm rings. The rings were mounted on specimen holders and placed in glass organ chambers containing 6 ml of aerated Krebs–Ringer HCO3 buffer at 37°C. While one holder remained fixed, the other was connected to an isometric force-displacement transducer (Model FTO3, Grass Instruments, Quincy, MA) coupled to a polygraph (Model 7D, Grass Instruments). The aortic rings were incubated for 60 min at a tension of 2.0 g during which time the chamber was rinsed every 15 min with aerated Krebs–Ringer HCO3 buffer. The responses to acetylcholineand sodium nitroprusside were recorded by a multichannel physiologic recorder (ML-840, PowerLab).

Measurement of Superoxide Release

The aorta was placed into chilled modified Krebs/HEPES buffer (pH 7.4, NaCl 99.01mM, KCl 4.69mM, CaCl2 1.87mM, MgSO4 1.20mM, K2HPO41.03mM, NaHCO3 25.0mM, Na-HEPES 20.0mM, glucose 11.1mM), and cut into 5-mm ring segments,with not to injure the endothelium.Afterpreparation, the vessels were placed in a modified Krebs/HEPESbuffer and allowed to equilibrate for 30 minutes at 37°C.Scintillation vials containing 2 ml Krebs/HEPES buffer with5 µmol/L lucigenin were placed into a scintillation counter. After dark adaptation,background counts were recorded, and a vascular segment wasadded to the vial. Scintillation counts were then recorded everyminute, and the counts from 10 to 20 minutes were averaged. The vessels were then dried and weighed, and the counts were expressed as counts abovebackground per milligram dry tissue.

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Table 1S The effects of rosuvastatin treatments on cardiac hemodynamics 2 month after statin therapy.

Control / Control+Statin / AAV-GFP / AAV-GFP+Statin / AAV-hCRP / AAV-hCRP+Statin
Maximum Cardiac Pressure (mmHg) / 124.4±2.38 / 126.2±4.03 / 121.8±3.3 / 119.4±2.6 / 143.4±1.5 * / 140.6±2.97 *
dP/dt max (mmHg/s) / 9711±156.2 / 9677±97.74 / 9833±376.6 / 9918±262.2 / 9582±142.7 / 9680±120.9
dP/dt min (mmHg/s) / 8697±136.8 / 8614±188.5 / 8759±180.3 / 8812±112.6 / 8523±180.8 / 8661±126
Cardiac Output (μL /min) / 31630±1121 / 32050±1324 / 32130±898 / 31950±846 / 30740±666 / 31090±777

* p < 0.05 compared to controls.

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Figure 1S

Figure 1S. Effect of rosuvastatin on expression of vascular AT1 and AT2 receptor. Increased AT1 receptor expressionwas attenuated by rosuvastatin. There were no significant differences of AT2 expression after rosuvastatin treatment. Results are expressed as the ratio ofAT1 and AT2toβ-actin, respectively. Data represent mean ± SEM. * p< 0.05 vs. saline control group. # p<0.05 vs. AAV-hCRP group, (n = 3).

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Figure 2S

Figure 2S.AAV-hCRP injection induced a decrease in serum nitrate/nitrite concentration, an effect that was attenuated by rosuvastatin treatment (p<0.05).Data represent mean ± SEM. * p<0.05 vs. Saline control group. # p<0.05 vs. AAV-hCRP group.

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