The Immunoproteasome Subunit β5i Promotes Vascular Remodeling In Primary Hypertension

Objective:To investigate the function and mechanism of immunoproteasome β5i in vascular remodeling of hypertension induced by angiotensin II(Ang II).Methods:1. Blood samples of twenty primary hypertensive patients and twenty normal controls were collected. 20 S proteasome detection Stress Xpress® kit was used to determine the activities of 20 S proteasome(β5i) in the two groups of blood samples.2. 6-8week-old male mice were infused with Ang II(1000ng/kg/min) for 7 days. Quantitative real-time PCR(q PCR) and Western Blot were used to detect the m RNA and protein level of immunoproteasome β1i, β2i and β5i as while as their activities. Immunofluorescence co-localization was applied to test whether β5i was expressed in vascular smooth muscle cells(VSMCs) and its expression in the vessels of the two groups.3. 6-8week-old male mice were randomly divided into four groups: control, Ang II(1000ng/kg/min,14days), Ang II(1000ng/kg/min,14days) + β5i inhibitor(PR-957,12mg/kg/d) and β5i inhibitor(12mg/kg/d). Systolic blood pressure was measured by the tail-cuff method. Pathological staining techniques including hematoxylin and eosin(H&E), elastin, masson and dihydroethidium(DHE) were carried out for the assessment of vascular inflammation, elastin, fibrosis and oxidative stress in the four groups. Vascular circles were performed for evaluation of vascular functions and q PCR was performed for β5i activity.Results:1. Compared with the normal controls, the activities of immunoproteasome subunit β5i were higher in primary hypertensive patients.2. Immunofluorescence co-localization suggested that β5i was expressed in vascular smooth muscle cells(VSMCs).3. The protein levels and activities of β1i, β2i, β5i were elevated in hypertensive vascular tissues. The m RNA levels of β1i, β5i in hypertensive group were higher than the control.4. The normal systolic blood pressure of 6-8week-old male mice was 90-100 mm Hg. The systolic pressure of Ang II infused group reached 150 mm Hg. But the systolic pressure in the Ang II+β5i inhibitor group was 120 mm Hg, suggesting that the inhibitor of immunoproteasome subunit β5i(PR-957) could inhibit the elevation of blood pressure induced by Ang II.5. The thickness and area of vascular wall in Ang II+β5i inhibitor group were lower than the model group as shown by H&E staining. q PCR showed β5i inhibitor could inhibit the expression of vascular inflammatory factors like IL-6,TNF-α,MCP-1, suggesting that the inhibitor of immunoproteasome subunit β5i(PR-957) could reduce vascular thickness and diameter as well as the expression of inflammatory factors induced by Ang II.6. Elastin staining showed that elastic fiber degeneration and rupture were exacerbated in Ang II group. Masson staining showed the collagen deposition was also exacerbated in Ang II group. But the lesion in Ang II+β5i inhibitor group was obviously improved suggesting the inhibitor of immunoproteasome subunit β5i(PR-957) could inhibit elastic fiber degeneration and rupture, as well as vascular fibrosis induced by Ang II.7. DHE staining showed the level of ROS in model group was significantly increased compared with the control. However, the level of ROS in Ang II+β5i inhibitor group was lower than the model. There was no significant difference between control and β5i inhibitor group suggesting the inhibitor of immunoproteasome subunit β5i(PR-957) could inhibit oxidative stress induced by Ang II.8. Vascular circles was used to investigate the endothelium-dependent relaxation of thoracic aorta to acetyleholine(ACh) and endothelium-independent relaxation to sodium nitmpruside(SNP). Under the treatment of Ach, the relaxation of the control and β5i inhibitor groups could reach 75%, the Ang II+β5i inhibitor group could reach 65% which were higher than the model group’s 50%. Under the treatment of SNP, the relaxation of all groups could reach 90% except the modelsuggesting the inhibitor of immunoproteasome subunit β5i(PR-957) could improve vascular relaxation dysfunction induced by Ang II.Conclusions:1. The activities of immunoproteasome subunit β5i were higher in primary hypertensive patients.2. β5i was expressed in vascular smooth muscle cells(VSMCs).3. The m RNA and protein levels of β5i were elevated in hypertensive vascular tissues. Meanwhile the activity of β5i was elevated in hypertensive vascular tissues.4. Immunoproteasome subunit β5i lead to vascular remodeling through inflammation, oxidative stress, endothelial injury of hypertension.