Loss of clusterin limits atherosclerosis in apolipoprotein E-deficient mice via reduced expression of Egr-1 and TNF-α.

AIM: Whether clusterin/apolipoprotein J is antiatherogenic or proatherogenic is controversial. We reported that clusterin was markedly induced in media and neointima after vascular injury and that reduced clusterin expression reduced the proliferation of vascular smooth muscle cells (VSMCs), which induced G1 arrest via p53 and p21. The purpose of this study was to investigate the physiological function of clusterin in atherosclerosis using double-knockout mice (D-KO) of apolipoprotein E-deficient mice (apoE-KO) and clusterin-deficient mice (CLU-KO). METHODS AND RESULTS: Atherosclerotic lesions in the aortic root were quantitated at 20 weeks of age. Atherosclerotic lesions of D-KO were significantly smaller than those of apoE-KO (D-KO: 0.176±0.078 mm(2) vs. apoE-KO: 0.365±0.164 mm(2), p< 0.001). To identify underlying atherosclerotic mechanisms that were blocked by loss of clusterin, we performed immunohistochemical analysis of Egr-1. Egr-1 immunoreactivity in the nuclei of VSMCs in atherosclerotic lesions of apoE-KO was upregulated, whereas it was not in D-KO lesions. Western blotting demonstrated that the expression levels of Egr-1 and TNF-α in the D-KO were significantly lower than those in the apoE-KO. When VSMCs and macrophages were obtained from D-KO and apoE-KO, Western blotting showed that the expression levels of Egr-1 and TNF-α in VSMCs and macrophages of D-KO were significantly lower than those of apoE-KO. CONCLUSION: Loss of clusterin strongly suppressed apoE-KO-induced atherosclerotic lesions at a step prior to the expression of Egr-1 and TNF-α, suggesting that clusterin is a candidate for an antiatherogenic target.

Tacrolimus-eluting stent inhibits neointimal hyperplasia via calcineurin/NFAT signaling in porcine coronary artery model.

AIMS: The purpose is to elucidate the mechanism by which a newly developed tacrolimus-eluting stent (TES) prevents neointimal hyperplasia after stenting. METHODS AND RESULTS: The three major coronary arteries in juvenile swine were randomized to implantation of either a TES or bare metal stent (BMS). Twelve weeks after stenting, the TES showed 29% less neointimal area than the BMS. Immunohistochemical staining showed that the expression of calcineurin was up-regulated in the neointima and media after stenting, and the TES inhibited this up-regulation. Western blotting demonstrated that the expression of calcineurin, nuclear factor of activated T cell (NFAT), and interleukin-2 (IL-2) was lower with the TES than with the BMS. To confirm the effect of tacrolimus on vascular smooth muscle cells (VSMCs) and its mechanism, cultured rat VSMCs were incubated with 12.5 microM of tacrolimus (tacrolimus group) or without tacrolimus (control group). The cell number of the tacrolimus group was significantly lower than that of the control group at 48 h of incubation. Western blotting demonstrated that tacrolimus decreased the expression of calcineurin, NFATc4, and IL-2 of cultured VSMCs. We confirmed that calcineurin small-interfering RNA (siRNA) decreased cell proliferation and the expression of NFATc4 and IL-2 in cultured VSMCs compared with negative control-siRNA. CONCLUSION: The newly developed TES inhibited neointimal hyperplasia after stenting via the calcineurin/NFAT/IL-2 signaling pathway, which is one of several mechanisms through which TES inhibits restenosis. Calcineurin may be an important molecular target to prevent restenosis after stenting.

Deficiency of clusterin inhibits neointimal hyperplasia after vascular injury.

AIM: Increased clusterin mRNA and protein levels have been detected in various tissues undergoing stress, and we previously reported that clusterin is markedly induced in media and neointima following vascular injury. The present study therefore investigated the impact of clusterin on neointimal hyperplasia following vascular injury. METHODS AND RESULTS: As compared with wild-type mice, clusterin knockout mice (clusterin-KO) demonstrated a significant decrease of the intima/media ratio 4 weeks after cuff placement. Immunohistochemical analysis of injured femoral arteries in clusterin-KO demonstrated the accumulation of p53 in nuclei of neointimal vascular smooth muscle cells (VSMCs). Moreover, VSMCs from either clusterin-KO or rat VSMCs treated with clusterin-short-interfering (si) RNA subjected to static stretch exhibited significantly increased p53 and p21, and increased G1 cell cycle arrest as indicated by flow cytometry compared with VSMCs from wild-type mice. CONCLUSION: Reduced clusterin expression reduced the proliferation of VSMCs and induced G1 arrest via p53 and p21. Clusterin therefore represents a promising molecular target to limit restenosis after coronary intervention.

The long-term effect of angiotensin II type 1a receptor deficiency on hypercholesterolemia-induced atherosclerosis.

Angiotensin II type 1 receptor may contribute to atherogenesis by facilitating the proliferative and inflammatory response to hypercholesterolemia. In the present study, we investigated the long-term effect of angiotensin II type 1a receptor (AT1a) deficiency on hypercholesterolemia-induced atherosclerosis by the use of AT1a-knockout (AT1a-KO) mice and apolipoprotein E-knockout (apoE-KO) mice. AT1a-KO were crossed with apoE-KO, generating double-knockout (D-KO) mice. Mice were fed a standard diet and analyzed at 25- or 60-weeks-old. The quantification of atherosclerotic volume in the aortic root revealed that the atherosclerotic lesions of D-KO mice were significantly smaller than those of apoE-KO mice at 25-week-old (0.81+/-0.16 mm2 vs. 1.05+/-0.21 mm2, p<0.001) and at 60-week-old (0.89+/-0.11 mm2 vs. 2.44+/-0.28 mm2, p<0.001). Surprisingly, there was no significant difference in atherosclerotic lesion size of D-KO mice at 25- and 60-week-old, suggesting that AT1a deficiency completely protected against the age-related progression of atherosclerosis. The amounts of collagen and elastin, the expression of p22phox, serum amyloid P (SAP), matrix metalloproteinase (MMP)-2, and MMP-9, and the number of apoptotic cells of D-KO mice were lower than those of apoE-KO mice. Furthermore, we confirmed that the expression of procollagen alpha1(I), procollagen alpha1(III), tropoelastin, p22phox, SAP, MMP-2, and MMP-9 decreased in cultured vascular smooth muscle cells from D-KO mice compared with those of apoE-KO mice. In conclusion, AT1a deficiency reduces atherosclerotic lesion size of apoE-KO mice and protects against the age-related progression of atherosclerosis. Reduction of oxidative stress, apoptosis, and MMP expression in atherosclerotic lesions by AT1a deficiency may contribute to plaque size.

Repeated thermal therapy up-regulates endothelial nitric oxide synthase and augments angiogenesis in a mouse model of hindlimb ischemia.

BACKGROUND: Nitric oxide (NO), constitutively produced by endothelial NO synthase (eNOS), plays roles in angiogenesis. Having reported that thermal therapy up-regulated the expression of arterial eNOS in hamsters, we investigated whether this therapy increased angiogenesis in mice with hindlimb ischemia. METHODS AND RESULTS: Unilateral hindlimb ischemia was induced in apolipoprotein E-deficient mice, which were divided into control and thermal therapy groups. The latter mice were placed in a far-infrared dry sauna at 41 degrees C for 15 min and then at 34 degrees C for 20 min once daily for 5 weeks. Laser Doppler perfusion imaging demonstrated that the ischemic limb/normal side blood perfusion ratio in the thermal therapy group was significantly increased beyond that in controls (0.79+/-0.04 vs 0.54+/-0.08, p<0.001). Significantly greater capillary density was seen in thermal therapy group (757+/-123 /mm2 vs 416+/-20 /mm2, p<0.01). Western blotting showed thermal therapy markedly increased hindlimb eNOS expression. To study possible involvement of eNOS in thermally induced angiogenesis, thermal therapy was given to mice with hindlimb ischemia with or without N(G)-nitro-L-arginine methyl ester (L-NAME) administration for 5 weeks. L-NAME treatment eliminated angiogenesis induced using thermal therapy. Thermal therapy did not increase angiogenesis in eNOS-deficient mice. CONCLUSION: Angiogenesis was induced via eNOS using thermal therapy in mice with hindlimb ischemia.

Expression of lectin-like oxidized LDL receptor-1 in smooth muscle cells after vascular injury.

Lectin-like oxidized LDL receptor-1 (LOX-1) is an oxidized LDL receptor, and its role in restenosis after angioplasty remains unknown. We used a balloon-injury model of rabbit aorta, and reverse transcription-polymerase chain reaction revealed that LOX-1 mRNA expression was modest in the non-injured aorta, reached a peak level 2 days after injury, and remained elevated until 24 weeks after injury. Immunohistochemistry and in situ hybridization showed that LOX-1 was not detected in the media of non-injured aorta but expressed in both medial and neointimal smooth muscle cells (SMC) at 2 and 24 weeks after injury. Low concentrations of ox-LDL (10 microg/mL) stimulated the cultured SMC proliferation, which was inhibited by antisense oligonucleotides of LOX-1 mRNA. Double immunofluorescence staining showed the colocalization of LOX-1 and proliferating cell nuclear antigen in human restenotic lesion. These results suggest that LOX-1 mediates ox-LDL-induced SMC proliferation and plays a role in neointimal formation after vascular injury.

Repeated sauna therapy increases arterial endothelial nitric oxide synthase expression and nitric oxide production in cardiomyopathic hamsters.

BACKGROUND: Vascular endothelial dysfunction is involved in the pathophysiology of chronic heart failure (CHF). It has been reported that sauna therapy, which allows thermal vasodilation, improves vascular endothelial dysfunction in patients with CHF. The present study investigates the mechanisms through which sauna therapy improves endothelial dysfunction induced by CHF. METHODS AND RESULTS: Normal control and male TO-2 cardiomyopathic hamsters were used. Thirty-week-old TO-2 hamsters were treated daily with an experimental far infrared-ray dry sauna system for 15 min at 39 degrees C followed by 20 min at 30 degrees C. This procedure raised the rectal temperatures by about 1 degrees C. Arterial endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein expressions were examined, and serum concentrations of nitrate were measured. The expression of eNOS mRNA in the aortas of normal controls did not change, whereas those of the TO-2 hamsters decreased with age. Four weeks of sauna therapy significantly increased eNOS mRNA expression in the aortas of TO-2 hamsters compared with those that did not undergo sauna therapy. Sauna therapy also upregulated aortic eNOS protein expression. Serum nitrate concentrations of the TO-2 hamsters were increased by 4 weeks of sauna therapy compared with those that did not undergo sauna. CONCLUSION: Repeated sauna therapy increases eNOS expression and NO production in cardiomyopathic hamsters with heart failure.

Thyrotropin-producing pituitary adenoma associated with Graves' disease.

OBJECTIVES: The examination of potential associations between Graves' disease and thyrotropin-producing pituitary adenoma (TSHoma) after treatment using octreotide, and of the expression of peroxisome proliferator-activated receptor gamma (PPAR gamma). DESIGN AND METHODS: A specimen of resected TSHoma tissue from our case was immunohistochemically examined for expression of somatostatin receptor 2A (SSTR2A) and PPAR gamma. Specimens of thyroid tissue from two cases with Hashimoto's thyroiditis were immunohistochemically examined for expression of SSTR2A. RESULTS: Expression of SSTR2A and PPAR gamma was identified in TSHoma cells. SSTR2A was also expressed in lymphocytes that had infiltrated thyroid tissue in Hashimoto's thyroiditis. In previous reports, three of four patients with TSHoma displayed Graves' disease after tumor resection, and TSH is also known to play a major role in regulating immunomodulatory gene expression in thyrocytes. CONCLUSIONS: Both the immunomodulatory effects of octreotide on intrathyroidal lymphocytes and rapid reductions in TSH may contribute to the onset of Graves' disease. Patients with TSHoma-associated autoimmune thyroiditis should undergo careful follow-up for development of Graves' disease after treatment. Both octreotide and the PPAR gamma receptor-activating ligands, thiazolidinediones, may be effective for patients with TSHoma.

Angiotensin II type 1 receptor participates in extracellular matrix production in the late stage of remodeling after vascular injury.

OBJECTIVE: Extracellular matrix (ECM) accumulation is important in restenosis after angioplasty. Underlying molecular mechanisms remain to be elucidated, especially in vivo. We investigated expression of angiotensin II type 1 receptor (ATR1) in a rat model for up to 24 weeks after vascular injury, and also the effect of an ATR1 antagonist on neointimal thickening and ECM production. METHODS AND RESULTS: Carotid arteries of rats were injured with a balloon catheter and then removed at 2, 5, and 7 days and 2, 4, 8, 16, and 24 weeks after injury. Although ATR1 immunoreactivity was slightly detectable in smooth muscle cells (SMC) in the media of uninjured arteries, reactivity was strong in neointimal SMC even 24 weeks after injury. Western blotting demonstrated similar results. ATR1 mRNA also was upregulated in neointimal SMC even 24 weeks after injury, as indicated by RT-PCR and by in situ hybridization. Candesartan, an ATR1 antagonist, significantly inhibited histologically evident neointimal thickening and collagen and elastin accumulation at 8 weeks after injury whether given beginning 1 day before injury, 4 days after injury, or 7 days after injury. CONCLUSION: ATR1 is upregulated in the late stage of remodeling after vascular injury and is important in ECM production.

[Preventing effect of angiotensin II receptor blocker on neointimal hyperplasia after vascular injury].

Percutaneous transluminal coronary angioplasty (PTCA) and stent implantation are useful techniques for treating patients with coronary atherosclerosis. However, the long-term efficacy of these treatment is limited by vascular restenosis, which occurs after these procedures. Although ACE inhibitor cilazapril prevented the neointimal hyperplasia of rat carotid artery after balloon injury, it did not lead to prevention of restenosis after PTCA in human studies (MERCATOR and MARCATOR). Angiotensin II receptor blocker, candesartan, inhibited the neointimal formation after balloon injury in both rat and dog. Val-PREST trial showed that valsartan reduced the in-stent restenosis rate after stent implantation. The inhibition of renin-angiotensin system by angiotensin II receptor blocker may help to prevent restenosis after angioplasty.

Hyperthermia at 43 degrees C for 2h inhibits the proliferation of vascular smooth muscle cells, but not endothelial cells.

Restenosis after angioplasty is one of the most critical problems of the various interventional therapies for myocardial ischemia. It has been difficult to prevent the vascular smooth muscle cells (VSMCs) proliferation resulting in restenosis. The goal of this study was to prove the treatment by hyperthermia to be effective in suppressing VSMC's proliferation in vitro. When just-stimulated VSMCs, which were incubated for 2h after 5% FBS stimulation to quiescent VSMCs, were exposed to hyperthermia (43 degrees C, 2h), the cell cycle progression to S and G2/M phase was significantly delayed 24h after 5% FBS stimulation. And another 24h later, cell death was observed partly (19%) of heat-treated VSMCs. Nonetheless, hyperthermia under the same conditions did not result in the death of quiescent VSMCs, and did not inhibit the proliferation of cultured bovine aortic endothelial cells (BAECs). In addition, we found that hyperthermia (43 degrees C, 2h) elevated p27(Kip1) over the amount induced in confluent VSMCs. Much elevation of p27(Kip1), which is a negative regulator of G1/S progression, may play a role in heat-induced G1 arrest of VSMCs. In conclusion, we have found that hyperthermia (43 degrees C, 2h) inhibited the proliferation of the dividing VSMCs mainly due to G1 arrest with neither inhibiting the generation of BAECs nor damaging quiescent VSMCs. Hence, our data suggest that hyperthermia may be clinically applicable for the prevention of restenosis.