Novel Therapy for Atherosclerosis Using Recombinant Immunotoxin Against Folate Receptor ß-Expressing Macrophages.

BACKGROUND: Folate receptor ß (FRß) is induced during macrophage activation. A recombinant immunotoxin consisting of the truncated Pseudomonas exotoxin A (PE38) conjugated to an anti-FRß antibody (anti-FRß-PE38) has been reported to kill activated macrophages in inflammatory diseases. To elucidate the effect of an immunotoxin targeting FRß on atherosclerosis, we determined the presence of FRß-expressing macrophages in atherosclerotic lesions and administered the FRß immunotoxin in apolipoprotein E-deficient mice. METHODS AND RESULTS: The FRß-expressing macrophages were observed in atherosclerotic lesions of apolipoprotein E-deficient mice. At 15 or 35 weeks of age, the apolipoprotein E-deficient mice were divided into 3 groups and were intravenously administered 0.1 mg/kg of anti-FRß-PE38 (immunotoxin group), 0.1 mg/kg of PE38 (toxin group), or 0.1 mL of saline (control group) every 3 days, for a total of 5 times for each age group. The mice were analyzed at 21 or 41 weeks of age. Treatment with the immunotoxin resulted in 31% and 22% reductions in atherosclerotic lesions of the 21- and 41-week-old mice, respectively (P<0.05). Administration of immunotoxin reduced the numbers of FRß- and tumor necrosis factor-α-expressing macrophages, reduced cell proliferation, and increased the number of apoptotic cells (P<0.05). Real-time polymerase chain reaction demonstrated that the expression of FRß and tumor necrosis factor-α mRNA was significantly decreased in the immunotoxin group (P<0.05). CONCLUSIONS: These results suggest that FRß-expressing macrophages exist in the atherosclerotic lesions of apolipoprotein E-deficient mice and that FRß immunotoxin administration reduces the progression of atherosclerotic lesions in younger and older individuals. The recombinant FRß immunotoxin targeting activated macrophages could provide a novel therapeutic tool for atherosclerosis. (J Am Heart Assoc. 2012;1:e003079 doi: 10.1161/JAHA.112.003079.).

Waon therapy upregulates Hsp90 and leads to angiogenesis through the Akt-endothelial nitric oxide synthase pathway in mouse hindlimb ischemia.

BACKGROUND: Thermal therapy, namely Waon therapy, has previously been reported to regulate nitric oxide (NO) and endothelial NO synthase (eNOS) and augment ischemia-induced angiogenesis in mice and improve limb ischemia in patients with peripheral artery disease. The aim of this study was to clarify the precise mechanism by which Waon therapy augments angiogenesis in mice with hindlimb ischemia. METHODS AND RESULTS: Unilateral hindlimb ischemia was induced in apolipoprotein E-deficient mice and Waon therapy was performed for 5 weeks. Heat shock protein 90 (Hsp90), phosphorylated-Akt, and phosphorylated-eNOS were detected in arterial endothelial cells of ischemic hindlimbs and all were upregulated by Waon therapy compared to controls. Waon therapy also increased serum concentrations of nitrite and nitrate. Capillary density and the ischemic limb/normal side blood perfusion ratio monitored by laser Doppler perfusion imaging in the Waon therapy group were significantly increased beyond those in the control group. The effect of Waon therapy on angiogenesis through the activation of the Hsp90/Akt/eNOS pathway was attenuated by the administration of a Hsp90 inhibitor. CONCLUSIONS: It is suggested that Waon therapy upregulates Hsp90, which contributes to the activation of the Akt/eNOS/NO pathway, and induces angiogenesis in mice with hindlimb ischemia.

Effect of Waon therapy on oxidative stress in chronic heart failure.

BACKGROUND: A previous report by our team showed that Waon therapy, using a far infrared-ray dry sauna at 60°C, improves cardiac and vascular function in patients with chronic heart failure (CHF). The purpose of the present study was to clarify the effect of Waon therapy on oxidative stress in CHF patients and investigate its mechanism by animal experiments. METHODS AND RESULTS: Forty patients with CHF were divided into control (n=20) and Waon therapy (n=20) groups. All patients received standard optimal medications for CHF. Waon therapy group was treated with Waon therapy daily for 4 weeks. After 4 weeks of Waon therapy, concentrations of hydroperoxide and brain natriuretic peptide (BNP) decreased significantly (hydroperoxide, 422±116 to 327±88U.CARR, P<0.001; BNP, 402±221 to 225±137pg/ml, P<0.001), and the nitric oxide metabolites increased (71.2±35.4 to 92.0±40.5mmol/L, P<0.05). In contrast, none of these variables changed over the 4-week interval in the control group. Furthermore, animal experiments were performed using TO-2 cardiomyopathic hamsters. On immunohistochemistry, cardiac expression of 4-hydroxy-2-nonenal, a marker of oxidative stress, was decreased in the 4-week Waon therapy compared to untreated hamsters. On Western blotting, cardiac expressions of heat shock protein (HSP) 27, manganese superoxide dismutase and HSP32, which reduce oxidative stress, were significantly upregulated in the 4-week Waon therapy compared to untreated hamsters. CONCLUSIONS: Waon therapy decreases oxidative stress in patients and hamsters with heart failure.

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.