[Angiotensin-converting enzyme 2 gene transfer attenuates neointimal formation after carotid artery ischemia-reperfusion injury in rats].

OBJECTIVE: To explore the effects of lentiviral recombinant angiotensin-converting enzyme 2 (LV-ACE2) gene transfer on the neointimal formation after carotid artery ischemia-reperfusion injury (IRI) and related mechanisms. METHODS: IRI was induced in SD rats through the carotid artery clipping and rats were divided into IRI, IRI+LV-GFP, IRI+LV-ACE2, IRI+ paclitaxel groups (n = 10 each). Sham operated rats serve as normal control. Four weeks later, neointimal formation was observed on HE stained carotid artery sections. The protein expression of ACE2, α-SM-actin, CD31, AT1R and P-ERK were detected by immunohistochemistry. RESULTS: (1) Carotid artery neointimal hyperplasia was readily shown in IRI group [I/M: 1.517 ± 0.151 (4 weeks later) vs. 0.011 ± 0.004 (Sham), P < 0.01], which was significantly reduced in IRI+LV-ACE2 (0.71 ± 0.17) and IRI+ paclitaxel (0.89 ± 0.21) groups. (2) The growth of vascular smooth muscle cells and neovascularization were also significantly inhibited in IRI+LV-ACE2 group and the expression of α-SM-actin (5 843 ± 839 vs. 12 648 ± 1 760, P < 0.01) and CD31 [(12.40 ± 4.01)/mm(2) vs. (96.20 ± 17.79)/mm(2), P < 0.01], AT1R (1 219 ± 175 vs. 4 861 ± 545, P < 0.01) and P-ERK1/2 phosphorylation (1 040 ± 215 vs. 2 938 ± 286, P < 0.01) in the neointimal of the injury arteries in IRI+LV-ACE2 group were significantly downregulated compared to IRI group. CONCLUSION: This data suggest that ACE2 gene overexpression is able to attenuate neointimal formation after ischemia-reperfusion injury possibly through downregulating AT1 receptor expression and signal pathway of ERK1/2 phosphorylation.

[The stability of the atherosclerotic plaque depends on the extent of injured endothelium:results from a novel model of ischemia/reperfusion induced atherosclerosis in carotid artery of rats].

OBJECTIVE: To observe the atherogenic lesion progress in a novel ischemia/reperfusion induced atherosclerosis model in the carotid artery of rats. METHODS: Rats were divided into normal control, sham-operated control and ischemia-reperfusion injury (IRI) groups (n = 10 each). IRI was induced by 30 min carotid artery occlusion with a 2 cm long artery clips in anesthetized rats. Four weeks later, hematoxylin and eosin (HE) and immunohistochemical stain were performed on carotid arteries of various groups. The ratio of neointima area/media area (I/M) and expression of platelet endothelial cell adhesion molecule (PECAM-1/CD31) were compared among groups. RESULTS: (1) Neointimal hyperplasia was detected in carotid artery of IRI group and the I/M ratio was significantly higher than in normal control and sham-operated groups (1.328 ± 0.301 vs. 0.011 ± 0.004 and 0.017 ± 0.008, all P < 0.01). (2) Small to large-sized neointima were found in the IRI group and the small sized intima was stable while large sized intima which covered the whole cavity was instable and underwent spontaneous rupture and thrombosis formation. (3) CD31 expression was significantly upregulated in carotid artery of IRI group corresponding to the instability of neointima in this group. CONCLUSION: Ischemia-reperfusion injury of carotid artery could result in atheroma in rats, this model could be used for future research on the pathogenesis of atherosclerosis. Our results show that endothelium injury of the arteries is the key factor to trigger atheroma and responsible for the disruption of the plaque.

[Overexpression of angiotensin-converting enzyme 2 inhibits angiotensin II type 1 receptor expression and signal transducer and activator of transcription 3 phosphorylation in cultured smooth muscle cells].

OBJECTIVE: To explore the effects of recombinated lentiviral angiotensin-converting enzyme 2 (ACE2) vector transfer on the expression of angiotensin II type 1 (AT1) receptor in cultured vascular smooth muscle cells (VSMCs). METHODS: VSMCs were divided into 7 groups: (1) CONTROL: serum-free culture medium; (2) Lentiviral-GFP vector group: Lentiviral-GFP vector (MOI = 10); (3) Ang II group (10(-7) mol/L); (4) Ang II (10(-7) mol/L) + Lentiviral-ACE2 (MOI = 10) group; (5) Ang II (10(-7) mol/L) + Irbesartan (10(-7) mol/L) group ; (6) Ang II (10(-7) mol/L) + irbesartan (10(-7) mol/L) + Lentiviral-ACE2 (MOI = 10) group ; (7) Lentiviral-ACE2 (MOI = 10) group. Ninety-six hours later, the proliferation of VSMCs was determined with CCK-8 Kit. AT1 receptor mRNA and protein expressions were detected with quantitative real-time PCR and Western blot, the signaling pathway of signal transducer and activator of transcription 3 (STAT3) was also detected. RESULTS: ACE2 gene transfer significantly inhibited the VSMCs proliferation in the absence or presence of Ang II. AT1 receptor mRNA and protein expressions were also significantly downregulated in the absence or presence of Ang II. Similar to AT1 receptor mRNA and protein expression changes, STAT3 phosphorylation was also significantly inhibited by ACE2 overexpression. CONCLUSION: Our results suggest that overexpression of ACE2 gene could inhibit the VSMCs proliferation by downregulating AT1 receptor expression and STAT3 phosphorylation. ACE2 could also directly inhibit AT1 receptor in cultured VSMCs.

[Effect of ACE2 gene transfection on the proliferation of vascular smooth muscle cells in rats].

OBJECTIVE: To investigate the effect of AngII on the proliferation of vascular smooth muscle cell (VSMC) in rats after the transfection of ACE2 gene. METHODS: pm-ACE2 was transfected into the cultured VSMC by Lipofectamine 2000. The normal cell group, AngIIgroup and pcDNA3.1/Hygro(+) transfected + AngII group were taken as controls respectively. After the transfection of ACE2 gene, the cell proliferative effect of AngII on VSMC was investigated by cell counting kit-8 (CCK8) and cell cycle detection by fluorescence activated cell sorter (FACS). RESULTS: The (optical density) OD value of AngIIgroup was obviously higher than that of other groups. And it was obviously lower in the pm-ACE2 + AngII group than the AngII group (0.535 ± 0.004 vs 0.866 ± 0.026, P < 0.05). Compared with other groups, the G(0)/G(1) stage percentage of VSMC was obviously lower in the AngII group (58.80% ± 2.00%, P < 0.05) while the percentage of S stage was obviously higher (35.90% ± 1.00%, P < 0.05). Compared with the AngII group, the G(0)/G(1) stage percentage of VSMC was obviously higher (63.90% ± 1.40%, P < 0.05) in the pm-ACE2 + AngII group while the percentage of S stage was obviously lower (27.80% ± 0.46%, P < 0.05). CONCLUSION: The over-expression of ACE2 gene can inhibit the proliferation of AngII-induced VSMC.

Complement 3 is involved in the synthetic phenotype and exaggerated growth of vascular smooth muscle cells from spontaneously hypertensive rats.

Vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) show the synthetic phenotype and exaggerated growth in comparison with VSMCs from normotensive Wistar-Kyoto (WKY) rats. We investigated genes associated with the synthetic phenotype and exaggerated growth of VSMCs from SHR by microarray. Expression of 1300 transcripts was evaluated by microarray with total mRNA extracted from mid-layer aortic smooth muscle of 3-week-old SHR/Izumo and WKY/Izumo rats. mRNAs encoding sodium-dependent neurotransmitter transporter, epidermal growth factor precursor, EEF2, leptin receptor long-isoform b, clathrin assembly protein short form, and preprocomplement 3 (pre-pro-C3) were expressed only in aortic smooth muscle from SHR by microarray and by reverse-transcription polymerase chain reaction analysis. Pre-pro-C3 mRNA was detected only in cultured VSMCs from SHR. Exogenous C3 changed VSMCs to the synthetic phenotype. Antisense oligodeoxynucleotides (ODN) to C3 reduced the higher level of DNA synthesis in VSMCs from SHR. Antisense ODN to C3 increased expression of SM22alpha mRNA and decreased expression of osteopontin and matrix Gla mRNAs. It also decreased expression of growth factor mRNAs in VSMCs from SHR. In conclusion, we have shown that C3, independent of other complement molecules, has direct effects on the phenotype of VSMCs and stimulates growth of these cells. C3 is produced only by VSMCs from SHR. Therefore, C3 may be the gene underlying the synthetic phenotype and exaggerated growth of VSMCs from SHR. C3 may be a new target for the treatment of hypertension.

Effect of AT2 receptor on expression of AT1 and TGF-beta receptors in VSMCs from SHR.

We recently reported that overexpression of the angiotensin II type 2 (AT2) receptor downregulates the AT1a receptor through the bradykinin/NO pathway in a ligand-independent manner in vascular smooth muscle cells (VSMCs). In the present study, we investigated the effect of AT2 receptor overexpression on the expression of the AT1a receptor and transforming growth factor-beta (TGF-beta) receptor subtypes in VSMCs from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Transfection of the AT2 receptor gene downregulated expression of the AT1a receptor in VSMCs from WKY, but did not affect expression of the AT1a receptor in VSMCs from SHR. Transfection of the AT2 receptor abolished DNA synthesis in response to angiotensin II in VSMCs from WKY; in VSMCs from SHR, basal DNA synthesis was suppressed, but DNA synthesis in response to Ang II was not altered. The NO substrate L-arginine augmented downregulation of the AT1a receptor in VSMCs from WKY, whereas it did not affect expression of the AT1a receptor in VSMCs from SHR. In response to AT2 receptor transfection, expression of TGF-beta type I receptor mRNA was suppressed significantly in VSMCs from WKY, whereas expression of TGF-beta type I receptor was not altered in VSMCs from SHR. These results suggest that the AT2 receptor downregulates AT1a and TGF-beta type I receptors in normal VSMCs, but not in SHR-derived VSMCs. The lack of downregulation of the AT1a receptor may contribute, in part, to the exaggerated growth of VSMCs from SHR.

Angiotensin II type 2 receptor gene transfer downregulates angiotensin II type 1a receptor in vascular smooth muscle cells.

Two distinct subtypes of angiotensin (Ang) II receptors, type 1 (AT(1)) and type 2 (AT(2)), have been identified. Vascular smooth muscle cells (VSMCs) usually express AT(1) receptor. To elucidate the direct effects of the AT(2) receptor on the AT(1) receptor in VSMCs, we transfected AT(2) receptor gene into cultured rat VSMCs. Overexpression of AT(2) receptor significantly decreased expression of AT(1a) receptor at both the mRNA and protein levels in the presence and absence of Ang II in VSMCs. Overexpression of AT(2) receptor increased expression of bradykinin and inducible NO in the presence and absence of Ang II in VSMCs. Bradykinin B(2) receptor antagonist HOE-140 and NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) inhibited the decreases in AT(1a) receptor expression by the overexpression of AT(2) receptor in VSMCs. L-Arginine augmented the decrease in AT(1a) receptor expression. Overexpression of AT(2) receptor suppressed basal DNA synthesis and proliferation of VSMCs and abolished response of DNA synthesis to Ang II in VSMCs. Our results demonstrate that overexpression of the AT(2) receptor downregulates AT(1a) receptor expression in rat VSMCs in a ligand-independent manner that is mediated by the bradykinin/NO pathway. Downregulation of AT(1a) receptor is a novel mechanism by which the AT(2) receptor regulates growth and metabolism of VSMCs.