Corrigendum to "Fluvastatin Upregulates the α 1C Subunit of CaV1.2 Channel Expression in Vascular Smooth Muscle Cells via RhoA and ERK/p38 MAPK Pathways".

[This corrects the article DOI: 10.1155/2014/237067.].

Retinoid X receptor agonists alleviate fibroblast activation and post-infarction cardiac remodeling via inhibition of TGF-ß1/Smad pathway.

Retinoid X receptor (RXR), particularly RXRα, has been implicated in cardiovascular diseases. However, the functional role of RXR activation in myocardial infarction (MI) remains unclear. This study aimed to determine the effects of RXR agonists on MI and to dissect the underlying mechanisms. Sprague-Dawley (SD) rats were subjected to MI and then treated (once daily for 4 weeks) with either RXR agonist bexarotene (10 or 30 mg/kg body weight) or vehicle. Heart function was determined using echocardiography and cardiac hemodynamic measurements. Four weeks post MI, myocardial tissues were collected to evaluate cardiac remodeling. Primary cardiac fibroblasts (CFs) were treated with or without RXR ligand 9-cis-RA followed by stimulation with TGF-ß1. Immunoblot, immunofluorescence, and co-immunoprecipitation were performed to elucidate the regulatory role of RXR agonists in TGF-ß1/Smad signaling. In vivo treatment with Bexarotene moderately affects systemic inflammation and apoptosis and ameliorated left ventricular dysfunction after MI in rat model. In contrast, bexarotene significantly inhibited post-MI myocardial fibrosis. Immunoblot analysis of heart tissue homogenates from MI rats revealed that bexarotene regulated the activation of the TGF-ß1/Smad signaling pathway. In vitro, 9-cis-RA inhibited the TGF-ß1-induced proliferation and collagen production of CFs. Importantly, upon activation by 9-cis-RA, RXRα interacted with p-Smad2 in cytoplasm, inhibiting the TGF-ß1-induced nuclear translocation of p-Smad2, thereby negatively regulating TGF-ß1/Smad signaling and attenuating the fibrotic response of CFs. These findings suggest that RXR agonists ameliorate post-infarction myocardial fibrosis, maladaptive remodeling, and heart dysfunction via attenuation of fibrotic response in CFs through inhibition of the TGF-ß1/Smad pathway activation.

Development of a Rat Model of Sick Sinus Syndrome Using Pinpoint Press Permeation.

OBJECTIVE: Sick sinus syndrome (SSS) is one of the most common causes of cardiac impairment necessitating pacemaker implantation. However, studies of SSS pathogenesis are neither comprehensive nor conclusive due to limited success in achieving a stable rat SSS model. Here, we modified pinpoint press permeation to establish a stable rat SSS model. METHODS: We randomly assigned 138 male Sprague-Dawley rats into three groups: normal control (n = 8), sham (n = 10), and SSS (n = 120). Postoperatively, the SSS group was further divided into SSSA (n = 40), SSSB (n = 40), and SSSC (n = 40), based on reduction in heart rates by 20-30%, 31-40%, and 41-50%, respectively. We also assessed histomorphological characteristics and hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4) expression in the sinoatrial node (SAN) at 1, 2, 3, and 4 weeks after surgery. RESULTS: Mortality was statistically higher in SSSC compared to SSSA and SSSB (7.5% versus 90.0% and 87.5%; P < 0.05). Heart rate in SSSA was gradually restored to preoperative levels by week 4 after surgery. In contrast, heart rate in SSSB was stable at 2-3 weeks after surgery. However, we observed that the tissues and cells in SAN were severely injured and also found a time-dependent increase in collagen content and atrium myocardium in SSSB. HCN4 expression was significantly reduced at all 4 time points in SSSB, with statistically significant differences among the groups (P < 0.01). CONCLUSION: We successfully developed a rat SSS model that was sustainable for up to 4 weeks.

Early treatment with losartan effectively ameliorates hypertension and improves vascular remodeling and function in a prehypertensive rat model.

AIMS: Pharmacological treatment of prehypertension may ameliorate hypertension and improve vascular structure and function. This study investigated 1) whether early treatment with either losartan or amlodipine at the onset of prehypertension can prevent hypertension and 2) whether losartan and amlodipine equally improve vascular remodeling and function in a rat model of hypertension. MATERIALS AND METHODS: Stroke-prone spontaneously hypertensive (SHRSP) rats were administered losartan, amlodipine or saline for 6 or 16weeks at the onset of prehypertension. Wistar-Kyoto rats were used as a control. All groups were observed for 40weeks. Systolic blood pressure was measured using the tail-cuff method. Vascular structure and function were determined by microscopy and vascular ring contractility assays, respectively. Angiotensin II (Ang II) and aldosterone (Aldo) were measured by radioimmunoassays. Angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) expression was measured by western blot. KEY FINDINGS: Losartan effectively reduced progression from prehypertension to hypertension as well as vascular remodeling and improved vascular contractility in SHRSP rats. Long-term losartan (16weeks) had greater benefits than short-term (6weeks) treatment. Losartan increased Ang II and decreased Aldo levels in the serum and vessel walls of resistance vessels in a time-dependent manner. Losartan significantly decreased AT1R and increased AT2R vascular expression. Amlodipine had no effect on vascular AT1R and AT2R expression. SIGNIFICANCE: Losartan administered at the onset of prehypertension is more effective than amlodipine in ameliorating hypertension and improving vascular remodeling and function, which is likely mediated by the renin-angiotensin-aldosterone system.

Hypomethylation of Agtrap is associated with long-term inhibition of left ventricular hypertrophy in prehypertensive losartan-treated spontaneously hypertensive rats.

Prehypertensive losartan treatment may lead to long­term inhibition of the development of left ventricular hypertrophy (LVH) in spontaneously hypertensive rats (SHRs). However, the underlying mechanism has yet to be fully elucidated. The aim of the present study was to investigate the expression of angiotensin type 1 receptor-associated protein (ATRAP/Agtrap) and methylation of the Agtrap gene in the myocardium following the withdrawal of treatment. Four­week­old SHRs were randomly divided into three groups, and were treated with saline, amlodipine or losartan, respectively, for 6 weeks. Wistar Kyoto rats (WKYs) were used as a control. All rats were followed up regularly until they reached the age of 32 weeks. Systolic blood pressure (SBP), left ventricular mass/body weight (LVM/BW), and cardiac fibrosis and structure were measured. The mRNA and protein expression of ATRAP in the myocardium were determined using reverse transcription­quantitative polymerase chain reaction and western blot analysis. Methylation of the Agtrap promoter was detected by bisulfite pyrosequencing. Reduced levels of SBP, LVM/BW, cardiac fibrosis and interventricular septum thickness were determined to be maintained only in prehypertensive losartan­treated SHRs. Whereas, an increased expression of ATRAP mRNA and protein, and hypomethylation of the Agtrap promoter in the myocardium, were demonstrated only in the losartan­treated SHRs. In conclusion, the results of the present study suggested that the hypomethylation of Agtrap accompanying upregulation of ATRAP expression in the myocardium is associated with the long­term inhibition of LVH in SHRs with prehypertensive losartan treatment.

Effects of tongxinluo on the neointima formation and expression of inflammatory cytokines in rats after carotid artery balloon injury.

OBJECTIVE: Tongxinluo (TXL) is a traditional Chinese medicine (TCM). It is used to treat coronary heart disease and atherosclerosis. We investigated the effects of TXL on the neointima formation and expression of inflammatory cytokines in rats after carotid artery balloon injury. MATERIALS AND METHODS: Male Sprague-Dawley rats were randomly divided into four groups: sham operation group (Sham, n = 15), balloon injury group treated with vehicle (Control, n = 15), TXL low-dose group treated with TXL of 0.5 g/kg/d (TXL-L, n = 15), and TXL high-dose group treated with TXL of 1.0 g/kg/d (TXL-H, n = 15). TXL was given by gavage daily. 14 days after injury', the levels of serum nitric oxide (NO), endothelin-1 (ET-1), monocyte chemoattractant protein-1 (MCP-1), and soluble intercellular adhesion molecule-1 (sICAM-1) were evaluated. The morphology of carotid artery tissue was observed with hematoxylin-eosin staining. Expressions of MCP-1 and ICAM-1 in the artery were detected by real-time polymerase chain reaction (RT-PCR) and western blotting. RESULTS: 14 days after injury, a significant increase in concentrations of serum ET-1, MCP-1, and sICAM-1 (P < 0.05), as well as a significant decrease in NO serum level were observed in rats subjected to artery injury compared to the sham rats (P < 0.05). TXL significantly decreased ET-1, MCP-1 and sICAM-1 serum levels (P < 0.05), whereas significantly increased NO serum level compared with the control (P < 0.05). TXL significantly reduced the neointimal thickening at day14 after injury (P < 0.05). In addition, TXL significantly reduced mRNA and protein expressions of ICAM-1 and MCP-1 in injured artery (P < 0.05). CONCLUSIONS: This study demonstrates that TXL is effective in improving endothelial function, attenuating neointimal formation of artery after balloon injury, and reducing expression of inflammatory cytokine MCP-1 and ICAM-1. It may be a useful agent for protecting the artery against injury.

Retinoid X receptor agonists inhibit hypertension-induced myocardial hypertrophy by modulating LKB1/AMPK/p70S6K signaling pathway.

BACKGROUND: Retinoid X receptor (RXR) has been demonstrated to play an important role in cardiac development and has been implicated in cardiovascular diseases. This study aimed to examine the effects of RXRα agonist bexarotene on pathological left ventricular hypertrophy (LVH) in a spontaneously hypertensive rat (SHR) model and the underlying mechanism. METHODS: WKY rats served as controls. SHRs were randomized into 3 groups at the age of 4 weeks and were treated (once daily for 12 weeks) with either bexarotene (30 or 100mg/kg body weight) or vehicle alone. Echocardiography was performed to determine cardiac structure and function. Neonatal cardiomyocytes were treated with AngII (10(-7) mmol/L) with or without the indicated concentration of RXRα ligand 9-cis-RA. The protein abundances of ß-actin, RXRα, LKB1, phospho-LKB1, AMPK, phospho-AMPK, P70S6K, phospho-P70S6K, ACE, and AT1 receptor were measured along with blood pressure, body weight and angiotensin II (Ang II) levels. The effects of LKB1 downregulation by LKB1 small, interfering RNA were examined. RESULTS: Treatment of SHRs with bexarotene resulted in significant inhibition of LVH without eliminating hypertension. Immunoblot with heart tissue homogenates from SHRs revealed that bexarotene activated the LKB1/AMPK signaling pathway and inhibited p70S6K. However, the increased Ang II levels in SHR serum and heart tissue were not reduced by bexarotene treatment. Treatment of cardiomyocytes with Ang II resulted in significantly reduced LKB1/AMPK activity and increased p70S6K activity. 9-cis-RA antagonized Ang II-induced LKB1/AMPK and p70S6K activation changes in vitro. CONCLUSIONS: RXR agonists prevent the inhibition of the LKB1/AMPK/p70S6K pathway and regulate protein synthesis to reduce LVH. This antihypertrophic effect of bexarotene is independent of blood pressure.

PPARδ activation inhibits homocysteine-induced p22(phox) expression in EA.hy926 cells through reactive oxygen species/p38MAPK pathway.

Increased expression of the p22(phox) subunit of the NADPH oxidase complex may possibly contribute to both the enzyme׳s increased activation and the occurrence of oxidative stress during hyperhomocysteinaemia. However, the activation of peroxisome proliferator-activated receptor (PPAR) δ has been shown to inhibit p22(phox) expression. The purpose of this study was to elucidate the signaling pathway by which PPARδ activation regulated homocysteine-induced expression of p22(phox). EA.hy926 cells were stimulated with homocysteine (Hcy) in the presence or absence of the PPARδ-specific agonist, GW0742, or of various signaling inhibitors, including the antioxidants N-acetylcysteine (NAC), NADPH oxidase inhibitor, diphenyleneiodonium (DPI), and the p38MAPK inhibitor, SB203580. Expression of p22(phox) mRNA and phospho-p38MAPK protein were measured by real-time PCR and western blot analysis, respectively, and reactive oxygen species were measured by fluorescence microscopy. Our data indicate that Hcy increased both the expression of p22(phox) in a concentration-dependent manner and also increased phosphoryation of p38 MAPK and reactive oxygen species production in a time-dependent manner. However, activation of the PPARδ signaling pathway by the agonist GW0742 reversed all these changes induced by Hcy. Furthermore, SB203580 prevented the increase in p22(phox) expression, and NAC and DPI not only inhibited Hcy-induced phosphorylation of p38MAPK, but also prevented expression of p22(phox). These findings indicate that Hcy-induced expression of p22(phox) is regulated by the reactive oxygen species/p38MAPK pathway and that PPARδ activation is capable of attenuating this pathway by eliminating Hcy-induced reactive oxygen species production.

Silencing heat shock protein 27 (HSP27) inhibits the proliferation and migration of vascular smooth muscle cells in vitro.

The objective of this study was to examine the role of heat shock protein 27 (HSP27) in proliferation and migration of vascular smooth muscle cells (VSMCs). Three complementary DNA sequences targeting rat HSP27 gene were designed, synthesized, and subcloned into lentiviral vector. The interfering efficiency was detected by reverse transcriptase-polymerase chain reaction and Western blot. Methyl thiazolyl tetrazolium bromide assay was used for examining cell proliferation. F-actin polymerization was detected by FITC-Phalloidin staining using confocal microscopy. Modified Boyden chamber technique was used to assess VSMCs migration. The recombinant lentivirus containing RNAi targeting HSP27 gene significantly inhibited expression of HSP27 at both mRNA and protein levels. The interfering efficiencies of pNL-HSP27-EGFP-1, pNL-HSP27-EGFP-2, and pNL-HSP27-EGFP-3 were 71 %, 77 %, and 43 %, respectively. Reorganization of actin stimulated by PDGF-BB was markedly blocked by pretreatment with pNL-HSP27-EGFP-2. Proliferation and migration rates of VSMCs induced by PDGF-BB were inhibited by 30.8 % and 45.6 %, respectively, by pNL-HSP27-EGFP-2 (all P < 0.01). To conclude, these data indicate that HSP27 may regulate the proliferation, actin reorganization, and the migration of VSMCs. RNAi targeting at HSP27 may be a potential approach for inhibition of cell migration involved in pathogenesis of proliferative vascular diseases.

Long-term prehypertension treatment with losartan effectively prevents brain damage and stroke in stroke-prone spontaneously hypertensive rats.

Prehypertension has been associated with adverse cerebrovascular events and brain damage. The aims of this study were to investigate ⅰ) whether short­ and long-term treatments with losartan or amlodipine for prehypertension were able to prevent blood pressure (BP)-linked brain damage, and ⅱ) whether there is a difference in the effectiveness of treatment with losartan and amlodipine in protecting BP-linked brain damage. In the present study, prehypertensive treatment with losartan and amlodipine (6 and 16 weeks treatment with each drug) was performed on 4-week­old stroke-prone spontaneously hypertensive rats (SHRSP). The results showed that long-term (16 weeks) treatment with losartan is the most effective in lowering systolic blood pressure in the long term (up to 40 weeks follow-up). Additionally, compared with the amlodipine treatment groups, the short­ and long-term losartan treatments protected SHRSP from stroke and improved their brains structurally and functionally more effectively, with the long-term treatment having more benefits. Mechanistically, the short­ and long-term treatments with losartan reduced the activity of the local renin-angiotensin-aldosterone system (RAAS) in a time-dependent manner and more effectively than their respective counterpart amlodipine treatment group mainly by decreasing AT1R levels and increasing AT2R levels in the cerebral cortex. By contrast, the amlodipine treatment groups inhibited brain cell apoptosis more effectively as compared with the losartan treatment groups mainly through the suppression of local oxidative stress. Taken together, the results suggest that long-term losartan treatment for prehypertension effectively protects SHRSP from stroke-induced brain damage, and this protection is associated with reduced local RAAS activity than with brain cell apoptosis. Thus, the AT1R receptor blocker losartan is a good candidate drug that may be used in the clinic for long-term treatment on prehypertensive populations in order to prevent BP-linked brain damage.

Fluvastatin upregulates the α 1C subunit of CaV1.2 channel expression in vascular smooth muscle cells via RhoA and ERK/p38 MAPK pathways.

Abnormal phenotypic switch of vascular smooth muscle cell (VSMC) is a hallmark of vascular disorders such as atherosclerosis and restenosis. And this process has been related to remodeling of L-type calcium channel (LTCC). We attempted to investigate whether fluvastatin has any effect on VSMC proliferation and LTCCα 1C subunit (LTCCα 1C) expression as well as the potential mechanisms involved. The VSMCs proliferation was assayed by osteopontin immunofluorescent staining and [(3)H]-thymidine incorporation. The cell cycle was detected by flow cytometric analysis. The activity of RhoA was determined with pull-down assay. MAPK activity and LTCCα 1C expression were assessed by western blotting. We demonstrated fluvastatin prevented the VSMCs dedifferentiating into a proliferative phenotype and induced cell cycle arrest in the G0/G1 phase in response to PDGF-BB stimulation. Fluvastatin dose-dependently reversed the downregulation of LTCCα 1C expression induced by PDGF-BB. Inhibition of ROCK, ERK, or p38 MAPK activation largely enhanced the upregulation effect of fluvastatin (P < 0.01). However, blockade of JNK pathway had no effect on LTCCα 1C expression. We concluded LTCCα 1C was a VSMC contractile phenotype marker gene. Fluvastatin upregulated LTCCα 1C expression, at least in part, by inhibiting ROCK, ERK1/2, and p38 MAPK activation. Fluvastatin may be a potential candidate for preventing or treating vascular diseases.

Effects of Losartan and Amlodipine on Left Ventricular Remodeling and Function in Young Stroke-Prone Spontaneously Hypertensive Rats.

BACKGROUND: The purpose of this study was to evaluate the effects of prehypertensive losartan and amlodipine administration on left ventricular (LV) remodeling and function in spontaneously hypertensive rats-stroke prone (SHRSP). METHODS: Spontaneously hypertensive rats-stroke prone were prehypertensively administered losartan, amlodipine, or vehicle. Wistar-Kyoto rats were used as a control. Blood pressure of the rats was determined by tail-cuff method, and LV structure and function were measured by echocardiography and LV cannulation. Collagen volume fraction was analyzed by picrosirius red staining. Protein expressions of brain natriuretic peptide (BNP) and angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors were determined by use of the Western blotting method. RESULTS: Although both drugs downregulated BNP protein expression, the LV remodeling and function were more improved with losartan than with amlodipine treatment. Losartan upregulated AT1R and downregulated AT2R protein expression. CONCLUSIONS: Both drugs inhibited LV remodeling and improved LV function in prehypertensively treated SHRSP. Losartan provided better continued heart protection, potentially due to its persistent inhibition of AT1R and activation of AT2R in the myocardium. KEY WORDS: Amlodipine; Blood pressure; Heart; Losartan; Prehypertension.

[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.

Differential effects of antihypertensive treatments on apoptosis, oxidative stress, and expression of angiotensin receptors in the cerebral cortex from the onset of prehypertension and hypertension in stroke-prone spontaneous hypertensive rats.

To investigate the effects of losartan and amlodipine on cell apoptosis in the cerebral cortex of stroke-prone spontaneously hypertensive rats (SHRSP) from the onset of prehypertension or hypertension. SHRSP were randomly divided into five experimental groups that were administered losartan, amlodipine (n=8 in each group; 4 weeks old or 10 weeks old), or vehicle, respectively. Wistar-Kyoto rats were used as control animals. Systolic blood pressure was measured using the tail-cuff method every 2 weeks. At 20 weeks of age, apoptosis was analyzed by TdT-mediated dUTP-biotin nick end labeling, and the level of angiotensin II was measured by radioimmunoassay. Protein expressions of gp91(phox), superoxide dismutase, and angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors in the cerebral cortex were detected by western blot. Losartan and amlodipine effectively delayed the progression of systolic blood pressure elevation, especially from the onset of prehypertension, and they had no obvious effects on the level of angiotensin II. In addition, treatment with losartan or amlodipine significantly decreased cell apoptosis, downregulated the protein expression of gp91(phox), and upregulated the protein expression of superoxide dismutase. The protein expressions of AT1R and AT2R were decreased by the administration of both drugs. No difference was found in the expression of AT1R among the drug treatment groups, whereas the expression of AT2R was increased in rats with increased blood pressure. Amlodipine, especially from the onset of prehypertension, was more effective than losartan in reducing apoptosis in the cerebral cortex in SHRSP. This may be related to the antioxidative stress properties of amlodipine.

[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.

Human tissue kallikrein 1 gene delivery inhibits PDGF-BB-induced vascular smooth muscle cells proliferation and upregulates the expressions of p27Kip1 and p2lCip1.

Tissue kallikrein 1 cleaves kininogen substrate to produce vasoactive kinin peptides that have been implicated in inhibiting neointimal hyperplasia in rat carotid arteries after balloon injury. However, its effects on the proliferation, cell cycle and its mechanisms, for example, cyclin-dependent kinase inhibitors, p27(Kip1) and p2l(Cip1) in vascular biology are poorly understood. The objective of this study was to explore the effects of human tissue kallikrein 1 (hTK1) mediated by recombinant adenovirus (Ad-hTK1) on proliferation and cell cycle of vascular smooth muscle cells (VSMCs) derived from spontaneously hypertensive rats induced by platelet-derived growth factor-BB (PDGF-BB) in vitro. The results showed that, within a given multiplicity of infection (MOI) and time, the hTK1 gene delivery inhibited PDGF-BB-stimulating VSMCs growth in a concentration-dependent (20-100 MOI) and time-dependent (2-5 days) manner by cell counting, with a peak inhibition rate at 36.3% at 72 h (P < 0.01). In addition, hTK1 gene delivery significantly suppressed PDGF-BB-induced proliferation of VSMCs by methyl thiazolyl tetrazoliuin assay, and decreased the percentage of cells in the S phase and in DNA synthesis by flow cytometry, with a peak inhibition rate at 30.2 and 36.4%, respectively (P < 0.01). Western blot assay showed that the protein levels of p27(Kip1) and p2l(Cip1) in cells infected with Ad-hTK1 were much more abundant than those in cells only induced by PDGF-BB, with up-modulating rates at 51.8 and 58.7%, respectively (P < 0.001). We also observed that the effects of hTK1 gene delivery in inhibiting VSMCs proliferation, arresting cell cycling in G(0)/G(1) phase and up-regulating the expression of p27(Kip1) and p2l(Cip1) could be blocked by icatibant (Hoe 140), a specific bradykinin B(2) receptor antagonist. Taken together, these results demonstrated that hTK1 overexpressed by recombinant adenovirus potently inhibits VSMCs proliferation that is required for neointimal hyperplasia and restenosis, and may activate p27(Kip1) and p2l(Cip1) signaling pathways via bradykinin B(2) receptor.

[Effect of atorvastatin on advanced glycation end products induced monocyte chemoattractant protein-1 expression in cultured human endothelial cells].

OBJECTIVE: To investigate the effects of atorvastatin on advanced glycation end products (AGE) induced monocyte chemoattractant protein-1 (MCP-1) expression in human umbilical vein endothelial cells (HUVECs) and whether this effect could be linked to peroxisome proliferator-activated receptor-γ (PPAR-γ) and nuclear factor-κB (NF-κB). METHODS: Grouping: (1) Blank control group; (2) BSA group; (3) AGE group: cells were incubated with different concentrations of AGE (10(-4), 10(-3), 10(-2) and 10(-1) g/L) for 24 hours; (4) AGE + Atorvastatin group: cells were incubated with different concentrations of atorvastatin (0.1, 1, 10 µmol/L) for 1 hour, then incubated with AGE (10(-1) g/L) for 24 hours; (5) PPAR-γ agonist (15 d-PGJ2) group: cells were incubated with 15 d-PGJ2 (10 µmol/L) for 1 hour, then incubated with AGE (10(-1) g/L) for 24 hours; (6) PPAR-γ inhibitor (GW9662) group: cells were incubated with GW9662 (5000 nmol/L) for 1 hour, then incubated with atorvastatin (1 µmol/L) and AGE (10(-1) g/L) for 24 hours. Collagenase was used to isolate the endothelial cell from human umbilical vein; RT-PCR was performed to examine the mRNA expression of MCP-1 and PPAR-γ; Western blot was performed to detect NF-κB p65 protein. RESULTS: (1) The expression of MCP-1 mRNA was increased in proportion with increasing concentrations of AGEs which could be blocked by atorvastatin in a dose-dependent manner. (2) AGE (10(-1) g/L) significantly downregulated the expression of PPAR-γ mRNA (0.22 ± 0.08 vs. 0.69 ± 0.09, P < 0.01) while upregulated the expression of phospho-NF-κB p65 protein (0.78 ± 0.06 vs. 0.31 ± 0.01, P < 0.01) and nonphospho-NF-κB p65 protein (1.61 ± 0.16 vs. 0.59 ± 0.14, P < 0.01) compared with the control group which could be significantly attenuated by atorvastatin. (3) PPAR-γ agonist decreased the expression of phospho-NF-κB p65 protein (0.21 ± 0.01 vs. 0.78 ± 0.06, P < 0.01), nonphospho-NF-κB p65 protein (0.67 ± 0.14 vs. 1.61 ± 0.16, P < 0.01) and MCP-1 mRNA (0.17 ± 0.02 vs. 0.93 ± 0.12, P < 0.01) compared with AGE (10(-1) g/L) group. (4) PPAR-γ inhibitor antagonized the effect of atorvastatin on the expression of phospho-NF-κB p65 protein, nonphospho-NF-κB p65 protein and MCP-1 mRNA stimulated by AGE in HUVECs (P < 0.01). CONCLUSION: The anti-inflammatory properties of atorvastatin in AGE stimulated HUVECs may partly be attributed to the effect on upregulation of PPAR-γ and downregulation of NF-κB signaling pathway.

[Effects of human tissue kallikrein gene delivery on the proliferation of vascular smooth muscle cells].

OBJECTIVE: Tissue kallikrein cleaves kininogen substrate to produce vasoactive kinin peptides that have been implicated in the proliferation of vascular smooth muscle cells. We investigated the effects of adenovirus-mediated human tissue kallikrein (Ad-hKLK1) gene delivery on the proliferation of vascular smooth muscle cells of SHR (VSMCs(SHR)) induced by platelet derived growth factor-BB (PDGF-BB). METHODS: Primary VSMCs(SHR) were isolated and cultured from thoracic aorta of male SHR. The VSMCs(SHR) proliferation induced by PDGF-BB was accessed by cell counting and methyl thiazolyl tetrazolium (MTT). Western blot was used to determine the protein expression of hKLK1, the cycle-independent kinase inhibitors p27(Kip1) and p21(Cip1). The mRNA expressions of bradykinin B1 receptor and B2 receptor were detected by RT-PCR in VSMCs(SHR). RESULTS: Proliferation of VSMCs(SHR) induced by PDGF-BB was significantly inhibited post transfection of Ad-hKLK1 (20-100 MOI) in a MOI-dependent manner. The peak inhibition titer of Ad-hKLK1 was 100 MOI with peak inhibition rate of 39.3% (cell counting, n = 3, P < 0.01), 30.2% (MTT, n = 3, P < 0.01) and 36.4% (peak stunning rate of cell-cycle in phase G(0)/G(1)). The inhibitory effects of proliferation and cell-cycle caused by hKLK1 gene delivery could be abolished by Hoe140, a bradykinin B2 receptor antagonist. The protein expression of p27(Kip1) and p21(Cip1) increased significantly after the hKLK1 gene delivery, whereas Hoe140 nearly completely blocked these effects (n = 3, P < 0.001, respectively). PDGF-BB also significantly upregulated the mRNA expression of B2 receptor but not B1 receptor in VSMCs(SHR). CONCLUSION: The hKLK1 gene delivery could inhibit PDGF-BB induced proliferation in VSMCs(SHR) through Bradykinin B2 receptor and up-regulate expression of p27(Kip1) and p2l(Cip1).

[Effects of human tissue kallikrein 1 gene delivery on carotid artery neointima formation after balloon angioplasty in spontaneously hypertensive rats].

OBJECTIVE: To investigate the effects of human tissue kallikrein 1(Ad-hKLK1) gene delivery on the neointima formation in carotid arteries of spontaneously hypertensive rats (SHRs). METHODS: Carotid artery restenosis was induced in male SHR rats by balloon-injury. Rats were randomly assigned into 4 groups: Sham-operated (n = 6); Angioplasty (phosphate buffered solution 50 microl, n = 8); Vector virus (control virus, 1 x 10(9) IU in 50 microl, n = 8) and Ad-hKLK1(Ad-hKLK1, 1 x 10(9) IU in 50 microl, n = 8). Rats were sacrificed 4 weeks later. The wall-to-lumen area ratio and intima/media ratio in carotid artery were assessed by image analysis in HE stained sections. The mRNA bradykinin receptor (B1R and B2R) expressions were detected by RT-PCR. The protein expression of the cycle-independent kinase inhibitors p27Kip1 and p2lCip1 were determined by Western blot analysis. RESULTS: Wall-to-lumen area ratio reduced 35.6% and intima/media ratio reduced 38.8%in Ad-hKLK1 treated SHRs compared to angioplasty group (all P < 0.001). The expression of p27Kip1 and p2lCip1 increased significantly in Ad-hKLK1 treated SHRs compared with angioplasty rats (all P < 0.001). The mRNA expression of B2R was significantly upregulated in angioplasty rats compared with sham-operated rats (P < 0.05) while mRNA expression of B1R was similar between the 2 groups. CONCLUSION: hKLK1 gene delivery may effectively reduce neointimal formation via downregulating bradykinin B2R and up-regulating the expressions of p27Kip1, p2lCip1 signaling pathways in carotid arteries of SHRs after balloon injury.