Endothelial dysfunction induced by antibodies against angiotensin AT1 receptor in immunized rats

Abstract

Aim: To investigate the association between autoantibodies against angiotensin AT1 receptor (AT1-AAs) and endothelial dysfunction in vivo.

Methods: Rat models with AT1 receptor antibodies (AT1-Abs) were established by active immunization for nine months. Lactate dehydrogenase (LDH) activity was regarded as an indicator of cell necrotic death. Endothelin-1 (ET-1) in the sera of rats was determined and endothelium-dependent vasodilatation was detected in isolated thoracic aorta. Endothelial intercellular adhesion molecule-1 (ICAM-1) expression in aorta endothelium was assessed using confocal microscopy. Coronary artery endothelial ultrastructure was observed.

Results: IgGs in the immunized group significantly increased the LDH activity (0.84±0.17 vs 0.39±0.12, P<0.01 vs vehicle group IgGs)in incubated human umbilical vein endothelial cells through AT1 receptor. Higher content of ET-1 occurred in the immunized rats than that of the vehicle group, and reached two peaks at month 3 (27±4 ng/L, P<0.01) and month 7 (35±5 ng/L, P<0.01), respectively. In addition, aortic endothelium-dependent vasodilatation was attenuated; endothelial ICAM-1 level was markedly increased and cardiac capillary endothelium was damaged following immunization.

Conclusion: Our study demonstrated that AT1-Abs contributed to endothelial dysfunction in vivo, which was a potential mechanism through which the antibodies play vital roles in related diseases.

Figure 1

High titers of AT1-Abs were generated in the rats immunized with human AT1R-EC_II. Healthy Wistar rats were actively immunized with synthetic human AT1R-EC_II. ELISA method was used to assess the generation rule of serum AT1-Abs. The titer of antibody was defined by OD value. Data were expressed as means±SD. n=7–8 rats per group. ^cP<0.01 vs vehicle group at the same period. OD, optical density; AT1R-EC_II, epitope peptide of extracellular second loop of AT1 receptor; AT1-Ab, AT1 receptor antibody.

Figure 2

The effect of IgGs from immunized group on LDH activity in cultured HUVECs. Total IgGs purified from immunized group (0.1 μmol/L) or vehicle group (0.1 μmol/L) was added into cultured HUVECs, respectively. LDH activity was detected after 6, 24, and 48 h. The effects of AT1 receptor agonist angiotensin II (0.1 μmol/L) and the blocker losartan (1 μmol/L) were also observed. Data were expressed by means±SD. n=6 cells per group. ^cP<0.01 vs vehicle IgGs. ^fP<0.01 vs AT1-Ab IgGs+losartan. OD, optical density.

Figure 3

Persistent high content of ET-1 occurred in the sera of immunized rats. In the immunization process, the concentrations of sera ET-1 in two groups were monitored dynamically. Data were expressed by means±SD. n=6–8 rats per group. ^bP<0.05, ^cP<0.01 vs vehicle group at the same period. ET-1, endothelin-1.

Figure 4

Endothelium-dependent diastolic function of thoracic aorta declined in the immunized rats. When the rats have been immunized for nine months, endothelium-dependent (A) and in-dependent (B) diastolic function of thoracic aorta in the rats were detected in vitro. Diastolic function was defined by the percentage of the pre-contraction. Data were expressed by means±SD. n=6–8 rats per group. ^bP<0.05 vs vehicle group at the same period. ACh, acetylcholine; SNP, sodium nitroprusside.

Figure 5

Upregulation of the expression ICAM-1 in thoracic aortic endothelium in the immunized rats. At the end of the immunization, the expressions of thoracic aortic endothelial ICAM-1 in the two groups of rats were measured under a laser scanning confocal microscope. A FITC-labeled anti-rat ICAM-1 antibody produced the red stain (5A, 5C). 5B and 5D were the corresponding structure charts of vascular lying on left, respectively.

Figure 6

Ultrastructural changes of coronary artery endothelium in the immunized rats were observed by electron microscopy. Coronary artery ultrastructure in rats of immunized group was determined by transmission electron microscopy. The magnification was 250 000. Thin arrow and thick arrow represented penetrating vesicle-channels and the pinocytotic vesicles, respectively (A). Arrowhead meant the effervesced vascular endothelium (B).