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Aims Neointimal formation after percutaneous coronary intervention (PCI), termed restenosis, limits therapeutic revascularization. Since it is now known that vascular injury involves an inflammatory response, we examined the role of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in the neointimal formation after injury. Methods and results Control (BALB/c), TNF-alpha-deficient (Tnf(-/-)), IFN-gamma-deficient Ifng(-/-)), or double-deficient (Tnf(-/-)Ifng(-/-)) mice were subjected to wire-mediated vascular injury of the right femoral artery. Neointimal formation after injury was significantly reduced after the injury in the Tnf(-/-)Ifng(-/-)) mice, compared to that in the control, Tnf(-/-), and Ifng(-/-) mice. Immunohistochemical analysis showed that TNF-alpha and IFN-gamma were expressed in neointimal lesions in the control mice, but not in mice with deficiency of the corresponding cytokine. No significant difference in re-endothelialization was observed among these groups. The number of proliferating cell nuclear antigen in the neointimal lesions was significantly decreased in the Tnf(-/-)Ifng(-/-) mice. Bone marrow transplantation experiments revealed that deficiency of TNF-alpha and IFN-gamma specifically in bone marrow cells significantly inhibited neointimal formation after vascular injury. Conclusion The absence of TNF-alpha and IFN-gamma in bone marrow cells synergistically inhibits neointimal formation following vascular injury, and thus, may provide new insights into the mechanisms underlying restenosis after PCI.

Aims Monocyte chemoattractant protein-1 (MCP-1: CCL2) has been demonstrated to be involved in the pathophysiology of ischaemic heart disease; however, the precise rote of MCP-1 in ischaemia/reperfusion (I/R) injury is controversial. Here, we investigated the role of cardiac MCP-1 expression on left ventricular (I]V) dysfunction after global I/R in Langendorff-perfused hearts isolated from transgenic mice expressing the mouse JE-MCP-1 gene under the control of the alpha-cardiac myosin heavy chain promoter (MHC/MCP-1 mice). Methods and results In vitro experiments showed that MCP-1 prevented the apoptosis of murine neonatal cardiomyocytes after hypoxia/reoxygenation. I/R significantly increased the mRNA expression of MCP-1 in the Langendorff-perfused hearts of wild-type mice. Cardiac MCP-1 overexpression in the MHC/MCP-1 mice improved LV dysfunction after I/R without affecting coronary flow; in particular, it ameliorated LV diastolic pressure after reperfusion. This improvement was independent of both sarcolemmal. and mitochondrial K-ATP channels. Cardiac MCP-1 overexpression prevented superoxide generation in the I/R hearts, and these hearts showed decreased expression of the NADPH oxidase family proteins Nox1, gp91phox, and Nox3 compared with the hearts of wild-type mice. Further, superoxide dismutase activity in the hearts of MHC/MCP-1 mice was significantly increased compared with that in the hearts of wild-type mice. Conclusion These findings suggest that cardiac MCP-1 prevented LV dysfunction after global I/R through a reactive oxygen species-dependent but K-ATP channel-independent pathway; this provides new insight into the beneficial role of MCP-1 in the pathophysiology of ischaemic heart diseases.

Background-Inflammatory cytokines such as interleukin (IL)-1 beta and IL-18 play an important role in the development of atherosclerosis and restenosis. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein that regulates caspase-1-dependent IL-1 beta and IL-18 generation; however, the role of ASC in vascular injury remains undefined. Here, we investigated the contribution of ASC to neointimal formation after vascular injury in ASC-deficient (ASC(-/-)) mice. Methods and Results-Wire-mediated vascular injury was produced in the femoral artery of ASC(-/-) and wild-type mice. Immunohistochemical analysis revealed that ASC was markedly expressed at the site of vascular injury. Neointimal formation was significantly attenuated in ASC(-/-) mice after injury. IL-1 beta and IL-18 were expressed in the neointimal lesion in wild-type mice but showed decreased expression in the lesion of ASC(-/-) mice. To investigate the contribution of bone marrow-derived cells, we developed bone marrow-transplanted mice and found that neointimal formation was significantly decreased in wild-type mice in which bone marrow was replaced with ASC(-/-) bone marrow cells. Furthermore, in vitro experiments showed that the proliferation activity of ASC(-/-) vascular smooth muscle cells was not impaired. Conclusions-These findings suggest that bone marrow-derived ASC is critical for neointimal formation after vascular injury and identify ASC as a novel therapeutic target for atherosclerosis and restenosis.

Glucose and fatty acids are major energy sources in skeletal muscle. Very low-density lipoprotein receptor (VLDL-R), which is highly expressed in heart, skeletal muscle and adipose tissue, plays a crucial role in metabolism of triglyceride (TG)-rich lipoproteins. To explore energy switching between glucose and fatty acids, we studied expression of VLDL-R and lipoprotein uptake in rat L6 myoblasts. L-Glucose or D-glucose deprivation in the medium noticeably induced the AMPK (AMP-activated protein kinase) activation and VLDL-R expression. Dose-dependent induction of VLDL-R expression was observed when D-glucose was less than 4.2 mM. The same phenomenon was also observed in rat primary skeletal myoblasts and cultured vascular smooth muscle cells. The uptake of P-VLDL but not LDL was accompanied by induction of VLDL-R expression. Our study suggests that the VLDL-R-mediated uptake of TG-rich lipoproteins might compensate for glucose shortfall through AMPK activation in skeletal muscle. (c) 2008 Elsevier Inc. All rights reserved.

Regeneration of alveolar bone is essential for periodontal treatment. Recently, cell replacement therapy has been focused on periodontal disease, but the source of the cells that regenerate alveolar bone is still uncertain. Therefore, to clarify the source of these bone-regenerating cells, we transplanted GFP-transgenic rat molars into the subcutaneous tissues of wild-type rats. Five days after transplantation, the tooth was surrounded by connective tissue containing many blood vessels. At 10 days, bone-like tissue was formed in the connective tissue between the branches of the bifurcated root. This hard tissue expanded to almost all of this bifurcation area without osseous ankylosis after 20 days. All ostcoblast-like cells in the newly formed matrix were immunopositive for GFP. In addition, these cells and the peripheral cells of the matrix showed intense immunoreactivity for BMP4, Runx2, BSP, and OPN. These results demonstrate that periodontal ligament tissue contains osteoprogenitor cells that have the ability to regenerate alveolar bone. Our model suggests that these regeneration processes might be similar to normal alveolar bone formation. (c) 2007 Elsevier Inc. All rights reserved.

Bone marrow cells (BMCs) have been reported to behave as tissue-specific stem cells in some organs and to participate in tumorigenesis. However, the roles of BMCs in hepatic regeneration and carcinogenesis are still unknown. A choline-deficient, ethionine-supplemented (CDE) diet leads to the appearance of oval cells, a type of hepatic progenitor cell, and activates their replication. Furthermore, this type of diet induces preneoplastic nodules and hepatocellular carcinomas (HCCs) derived from oval cell progenitors. The aims of this study were to determine whether oval cells are derived from BMCs and whether preneoplastic nodules or HCCs originate from BMCs in the CDE diet rat model. To clarify the origin of constituent cells in the liver, we transplanted BMCs from green fluorescent protein (GFP) transgenic female rats into male Lewis rats, which were then exposed to a CDE diet to induce hepatocarcinogenesis. Some oval cells showed both donor-derived GFP expression and the recipient-specific Y chromosome, indicating that donor BMCs fused with recipient oval cells. Several preneoplastic nodules (precancerous lesions) identified by their glutathione S-transferase placental (GSTp) positivity were induced by CDE treatment. However, these preneoplastic GSTp-positive nodules were not GFP positive. In conclusion, this study has produced two major findings. First, BMCs fuse with some oval cells. Second, BMC-fused oval cells and BMCs might not have malignant potential in the CDE-treated rat model.

Cardiovascular disease remains a principal cause of mortality in Western countries. Novel strategies for enhancing angiogenesis (such as gene or cell therapy) provide alternative choices for patients without any current treatment options. This progress has contributed towards understanding the mechanisms underlying vascular formation. The establishment of new experimental models could lead to the development of new treatments.

The selectins expressed on activated endothelial cells (E- and P-selectin), leukocytes (L-selectin), and platelets (P-selectin) play crucial roles in the rolling and tethering of leukocytes. We explored the importance of donor and recipient selectins in acute and chronic cardiac allograft rejection using mice deficient in all three selectins (ELP-/-). In BALB/c recipients, survival of fully allomismatched hearts from ELP-/- C57BL/6 donors was almost double that of wild-type grafts. In ELP-/- cardiac allografts, mononuclear cell infiltration and vasculitis of intramyocardial coronary arteries were significantly reduced. Interestingly, ELP-/- grafts were rejected similarly in both the presence and the absence of recipient selectins, and both wild-type and ELP-/- recipients promptly rejected wild-type hearts. Alternative adhesive molecules such as alpha 4 beta 7 integrin may compensate for the lack of selectins and may mediate rejection in ELP-/- recipients. Chronic rejection was evaluated in a major histocompatibility complex (MHC) class II mismatch model using C57BL/6.C-H2(bm12) mice. While lack of selectins in recipients did not offer protection against chronic rejection, luminal stenosis of coronary arteries in ELP-/- grafts was markedly diminished. In conclusion, donor-derived selectins contribute to the development of both acute and chronic cardiac allograft rejection, and targeting donor selectins may open novel therapeutic approaches in clinical transplantation.

While mineralized tissue is formed in the pulp cavity after tooth replantation or transplantation, little is known of this hard tissue formation. Therefore, we conducted histological and immunohistochemical evaluations of hard tissue formed in the pulp of rat maxillary molars after tooth replantation. At S days after replantation, degenerated odontoblasts were lining the pulp cavity. At 14 days, dentin- or bone-like tissue was present in the pulp cavity. Immunore activity for osteopontin (OPN) and bone sialoprotein (BSP) was strong in the bone-like tissue, but weak in the dentin-like tissue. Conversely, dentin sialoprotein (DSP) was localized in the dentin-like tissue, but not in the bone-like tissue. Cells positive for BMP4, Smad4, Runx2, and Osterix were found around the blood vessels of the root apex at 5 days. At 14 days, these cells were also localized around the bone-like tissue. Cells expressing alpha-smooth muscle actin (SMA) were seen around the newly formed bone-like tissue, whereas no such cells were found around the newly formed dentin-like tissue. In an experiment involving the transplantation of a green fluorescent protein (GFP)-transgenic rat tooth into a wild-type rat tooth socket, GFP-positive cells were detected on the surface of the bone-like tissue and over all dentin-like tissue. These results indicate that the original pulp cells had the ability to differentiate into osteoblast-like cells as well as into odontoblast-like cells. (C) 2007 Elsevier Ltd. All rights reserved.

Prostacyclin synthase (PGIS) is the final committed enzyme in the metabolic pathway of prostacyclin production. The therapeutic option of intravenous prostacyclin infusion in patients with pulmonary arterial hypertension is limited by the short half-life of the drug and life-threatening catheter-related complications. To develop a better delivery system for prostacyclin, we examined the feasibility of intramuscular injection of an adenoassociated virus (AAV) vector expressing PGIS for preventing monocrotaline-induced pulmonary arterial hypertension in rats. We developed an AAV serotype 1-based vector carrying a human PGIS gene (AAV-PGIS). AAV-PGIS or the control AAV vector expressing enhanced green fluorescent protein was injected into the anterior tibial muscles of 3-week-old male Wistar rats; this was followed by the monocrotaline administration at 7 weeks. Eight weeks after injecting the vector, the plasma levels of 6-keto-prostaglandin F-1 alpha increased in a vector dose-dependent manner. At this time point, the PGIS transduction (1x10(10) genome copies per body) significantly decreased mean pulmonary arterial pressure (33.9 +/- 2.4 versus 46.1 +/- 3.0 mm Hg; P < 0.05), pulmonary vascular resistance (0.26 +/- 0.03 versus 0.41 +/- 0.03 mm Hg . mL(-1) . min(-1) . kg(-1); P < 0.05), and medial thickness of the peripheral pulmonary artery (14.6 +/- 1.5% versus 23.5 +/- 0.5%; P < 0.01) as compared with the controls. Furthermore, the PGIS-transduced rats demonstrated significantly improved survival rates as compared with the controls (100% versus 50%; P < 0.05) at 8 weeks postmonocrotaline administration. An intramuscular injection of AAV-PGIS prevents monocrotaline-pulmonary arterial hypertension in rats and provides a new therapeutic alternative for preventing pulmonary arterial hypertension in humans.

The monocyte/macrophage lineage might affect the healing process after myocardial infarction (MI). Because macrophage colony-stimulating factor (M-CSF) stimulates differentiation and proliferation of this lineage, we examined the effect of M-CSF treatment on infarct size and left ventricular (LV) remodeling after MI. MI was induced in C57BL/6J mice by ligation of the left coronary artery. Either recombinant human M-CSF or saline was administered for 5 consecutive days after MI induction. M-CSF treatment significantly reduced the infarct size (P < 0.05) and scar formation (P < 0.05) and improved the LV dysfunction (percent fractional shortening, P < 0.001) after the MI. Immunohistochemistry revealed that M-CSF increased macrophage infiltration (F4/80) and neovascularization (CD31) of the infarct myocardium but did not increase myofibroblast accumulation (a-smooth muscle actin). M-CSF mobilized CXCR4(+) cells into peripheral circulation, and the mobilized CXCR4(+) cells were then recruited into the infarct area in which SDF-1 showed marked expression. The CXCR4 antagonist AMD3100 deteriorated the infarction and LV function after the MI in the M-CSF-treated mice. In conclusion, M-CSF reduced infarct area and improved LV remodeling after MI through the recruitment of CXCR4(+) cells into the infarct myocardium by the SDF-1-CXCR4 axis activation; this suggests that the SDF-1-CXCR4 axis is as a potential target for the treatment of MI.

Sphingosine-1-phosphate (S1P) is an active sphingolipid metabolite that exerts important biological effects. Recently, we demonstrated that KRP-203 is a novel S1P receptor agonist that can alter lymphocyte homing and act as an immunomodulating agent. We investigated the efficacy of KRP-203 in the treatment of rat experimental autoimmune myocarditis. KRP-203 significantly attenuated the inflammation area, heart weight/body weight ratio, and left ventricular function. Immunohistochemical analysis and RT-PCR revealed that KRP-203 significantly decreased the infiltration of macrophages and CD4 T cells in the myocardium and the expression of inflammatory cytokines. Flow cytometric analysis revealed that treatment with KRP-203 effectively reduced the number of peripheral CD4 and CD8 T cells but not that of B cells and granulocytes. Further, late KRP-203 treatment was effective even against established EAM. These results demonstrate the therapeutic potential of KRP-203 for the treatment of human myocarditis and provide new insights into the pathogenesis of this disease. (c) 2007 Elsevier Inc. All rights reserved.

Pulmonary arterial hypertension (PAH) is a fatal disease associated with inflammation and pathological remodeling of the pulmonary artery (PA). Interleukin (IL)-10 is a pleiotropic antiinflammatory cytokine with vasculoprotective properties. Here, we report the preventive effects of IL-10 on monocrotaline-induced PAH. Three-week-old Wistar rats were intramuscularly injected with an adeno-associated virus serotype 1 vector expressing IL-10, followed by monocrotaline injection at 7 weeks old. IL-10 transduction significantly improved survival rates of the PAH rats 8 weeks after monocrotaline administration compared with control gene transduction (75% versus 0%, P < 0.01). IL-10 also significantly reduced mean PA pressure (22.8 +/- 1.5 versus 29.7 +/- 2.8 mm Hg, P < 0.05), a weight ratio of right ventricle to left ventricle plus septum (0.35 +/- 0.04 versus 0.42 +/- 0.05, P < 0.05), and percent medial thickness of the PA (12.9 +/- 0.3% versus 21.4 +/- 0.4%, P < 0.01) compared with controls. IL-10 significantly reduced macrophage infiltration and vascular cell proliferation in the remodeled PA in vivo. It also significantly decreased the lung levels of transforming growth factor-beta(1) and IL-6, which are indicative of PA remodeling. In addition, IL-10 increased the lung level of heme oxygenase-1, which strongly prevents PA remodeling. In vitro analysis revealed that IL-10 significantly inhibited excessive proliferation of cultured human PA smooth muscle cells treated with transforming growth factor-beta(1) or the heme oxygenase inhibitor tin protoporphyrin IX. Thus, IL-10 prevented the development of monocrotaline-induced PAH, and these results provide new insights into the molecular mechanisms of human PAH.

A drug delivery system (DDS) that targets the injured myocardium would serve as a novel therapeutic tool for cardiac diseases. To develop such a DDS, we investigated the interaction of 2 types of glycoside-conjugated liposomes containing a fluorescence substrate with cardiomyocytes. Flow cytometry revealed that cardiomyocytes adequately interact with N-acetylglucosamine-conjugated liposomes (GlcNAc-Ls). Furthermore, to confirm whether the agents encapsulated in GlcNAc-Ls affect the intracellular environment of cardiomyocytes, we prepared GlcNAc-Ls-containing pravastatin and examined the effect of pravastatin on cardiomyocytes. Pravastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor (statin) and is hydrophilic. It is reported that lipophilic statins enhance nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression by interieukin-1 beta (IL-1 beta)-stimulated cardiomyocytes. The hydrophilic nature of pravastatin prevents its entry into cardiomyocytes; therefore, it cannot enhance both these processes. Treatment with GlcNAc-Ls-containing pravastatin specifically enhanced NO production and iNOS expression by IL-1 beta-stimulated cardiomyocytes. Based on these results, we found that cardiomyocytes exhibit a high degree of interaction with GlcNAc-Ls, and GlcNAc-Ls-encapsulated agents can be effectively taken up by cardiomyocytes. We suggest that GlcNAc-Ls can be utilized therapeutically as a DDS for the injured myocardium. (C) 2007 Elsevier B.V. All rights reserved.

1. The present study was designed to examine the role of amlodipine in preventing and reversing monocrotaline (MCT)induced pulmonary arterial hypertension (PAH) in rats. 2. Rats were injected with MCT (40 mg/kg, s.c.) and randomly given either 6 mg/kg per day of amlodipine in drinking water or placebo for 3 weeks. Any animals treated with MCT that survived for 3 weeks were given either amlodipine or placebo for the next 3 weeks. 3. Blood pressure was not different between the groups. Amlodipine immediately following MCT markedly inhibited PAH with severe pulmonary vascular remodelling. The survival rate at 3 weeks after treatment was increased significantly in the amlodipine group compared with the placebo group (77% vs 43%; P < 0.01). The placebo group showed markedly diminished expression of endothelial nitric oxide synthase (eNOS) protein and mRNA levels, increased numbers of proliferating cell nuclear antigen-positive cells, enhanced mRNA expression of matrix metalloproteinase-2 and pro-inflammatory cytokines in the lung tissue and upregulation of P-selectin on the endothelium of the pulmonary arteries, whereas these effects were suppressed in the amlodipine-treated group. Furthermore, late treatment with amlodipine did not palliate PAH or improve survival. 4. Amlodipine inhibited the development of PAH and improved survival in rats independent of its effect on lowering blood pressure. These effects were associated with marked inhibition of the downregulation of eNOS and improvement of pulmonary vascular endothetial activation, as well as anti-inflammatory, antiproliferative and antifibrotic effects in the lung tissue. However, amlodipine failed to reverse established PAH. This study may provide an insight into therapeutic strategy of amlodipine in PAH.

Objective-Since the macrophage colony-stimulating factor ( M-CSF) has been shown to stimulate differentiation and proliferation of monocyte/macrophage lineage and to be involved in the process of neointimal formation after vascular injury, we tested the effects of M-CSF on the recruitment of bone marrow-derived progenitor cells in neointimal formation after vascular injury in mice. Methods and Results-Wire-mediated vascular injury was produced in the femoral artery of C57BL/6 mice. Recombinant human M-CSF [ 500 mu g/( kg.day)] or saline ( control) was administered for 10 consecutive days, starting 4 days before the injury. Treatment with M-CSF accelerated neointimal formation in the early phase after injury, and this neointimal lesion mainly consisted of bone marrow - derived cells. M-CSF treatment had no effect on the mobilization of endothelial progenitor cells ( EPCs: CD34(+)/Flk-1(+)) and reendothelialization after injury. The stromal cell-derived factor-1 ( SDF-1) was markedly expressed in the neointima and media after injury, whereas CXCR4(+) cells were observed in the neointima. Further, a novel CXCR4 antagonist, AMD3100, significantly attenuated the M-CSF - induced neointimal formation. Conclusions-These findings suggest that M-CSF accelerated neointimal formation after vascular injury via the SDF-1-CXCR4 system, and the inhibition of this system has therapeutic potential for the treatment of cardiovascular diseases.

Objective Erythropoietin (EPO) has been shown to not only have cardioprotective effects but also attenuate autoimmune diseases. In the present study, we investigated the effect of EPO on cardiac inflammation and function, inflammatory cell infiltration, and cytokine expression in a rat model of experimental autoimmune myocarditis (EAM). Methods and results Male Lewis rats (6-8 weeks old) were immunized on day 0 with porcine cardiac myosin to establish EAM. The rats were subcutaneously administered either vehicle (saline) or human recombinant EPO (6,000 U/kg, 3 days/week) from day 0 to 20, and they were evaluated on day 21. In the EPO group, the inflammation area and heart weight/body weight ratio were significantly attenuated as compared with those in the vehicle group. Blood pressure and cardiac function were also improved in the EPO group. Immunohistochemistry revealed that EPO decreased the infiltration of macrophages and CD4 T cells, and degranulated mast cells in the myocardium. Real-time RT-PCR analysis demonstrated that inflammatory cytokine expression in the myocardium and lymphocytes was suppressed in the EPO group. However, in vitro experiments showed that EPO had no effect on antigen-induced proliferation and cytokine expression in lymphocytes. Conclusion EPO attenuates inflammatory cell infiltration and cytokine expression, and it improves cardiac function and reduces cardiac inflammation in EAM. This beneficial effect of EPO is unlikely to arise from a direct anti-inflammatory action on lymphocytes. These findings suggest the therapeutic potential of EPO for the treatment of myocarditis.

Pulmonary arterial hypertension (PAH) is characterized by abnormal proliferation of smooth muscle cells (SMCs), leading to occlusion of pulmonary arterioles, right ventricular (RV) hypertrophy, and death. We investigated whether mycophenolate mofetil (MMF), a potent inummosuppresssant, prevents the development of monocrotaline (MCT)-induced PAH in rats. MMF effectively decreased RV systolic pressure and RV hypertrophy, and reduced the medial thickness of pulmonary arteries. MMF significantly inhibited the number of proliferating cell nuclear antigen (PCNA)-positive cells, infiltration of macrophages, and expression of P-selectin and interleukin-6 on the endothelium of pulmonary arteries. The infiltration of T cells and mast cells was not affected by MMF. In vitro experiments revealed that mycophenolic acid (MPA), an active metabolite of MMF, dose-dependently inhibited proliferation of human pulmonary arterial SMCs. MMF attenuated the development of PAH through its anti-inflammatory and anti-proliferative properties. These findings provide new insight into the potential role of immunosuppressants in the treatment of PAR (c) 2006 Elsevier Inc. All rights reserved.

Myocardial infarction (MI) is accompanied by inflammatory responses that lead to the recruitment of leukocytes and subsequent myocardial damage, healing, and scar formation. Because monocyte chemoattractant protein-1 (MCP-1) ( also known as CCL2) regulates monocytic inflammatory responses, we investigated the effect of cardiac MCP-1 overexpression on left ventricular (LV) dysfunction and remodeling in a murine MI model. Transgenic mice expressing the mouse JE-MCP-1 gene under the control of the alpha-cardiac myosin heavy chain promoter (MHC/MCP-1 mice) were used for this purpose. MHC/MCP-1 mice had reduced infarct area and scar formation and improved LV dysfunction after MI. These mice also showed induction of macrophage infiltration and neovascularization; however, few bone marrow-derived endothelial cells were detected in MHC/MCP-1 mice whose bone marrow was replaced with that of Tie2/LacZ transgenic mice. Flow cytometry analysis showed no increase in endothelial progenitor cells (CD34(+)/Flk-1(+) cells) in MHC/MCP-1 mice. Marked myocardial interleukin (IL)-6 secretion, STAT3 activation, and LV hypertrophy were observed after MI in MHC/MCP-1 mice. Furthermore, cardiac myofibroblasts accumulated after MI inMHC/MCP-1 mice. In vitro experiments revealed that a combination of IL-6 with MCP-1 synergistically stimulated and sustained STAT3 activation in cardiomyocytes. MCP-1, IL-6, and hypoxia directly promoted the differentiation of cardiac fibroblasts into myofibroblasts. Our results suggest that cardiac overexpression of MCP-1 induced macrophage infiltration, neovascularization, myocardial IL- 6 secretion, and accumulation of cardiac myofibroblasts, thereby resulting in the prevention of LV dysfunction and remodeling after MI. They also provide a new insight into the role of cardiac MCP-1 in the pathophysiology of MI.

Background. We demonstrate the long-term effectiveness of KRP-203 treatment in combination with a subtherapeutic dose of cyclosporine A (CsA) on rat renal allografts. Methods. We tested the effect of KRP-203 in combination with CsA using a rat skin allograft model. The Pharmacokinetic interaction between CsA and KRP-203 was evaluated. The selectivity of KRP-203 for sphingosine-I-phosphate (S1P)(1) and S1P(3) receptors were investigated in vitro. Heart rate alteration following bolus injection of phosphorylated KRP-203 (KRP-203-P) or FTY720 (FTY720-P) was also monitored in rats. Finally, the long-term effectiveness of KRP-203 in conjunction with a low dose of CsA was investigated in a rat renal transplantation model. Results. Administration of KRP-203 with CsA prolonged skin allograft survival. KRP-203 and CsA had no effect on the pharmacokinetics of the other. While FTY720-P activated both S1P(1), and S1P(3) receptors, KRP-203-P selectively activated S1P(1), but not the S1P(3) receptor (EC(50): > 1000 nM). Compared to FTY720-P, a tenfold higher dose of KRP-203-P was necessary to induce transient bradycardia. With a low dose of CsA (1 mg/kg/day), KRP-203 (0.3 mg/kg/day) significantly prolonged renal allograft survival (P < 0.05, survival time: 9.8 days (CsA) vs. > 27.4 days (CsA+KRP)). Although a higher dose of CsA (3, mg/kg/day) alone kept recipients alive, this caused severe renal graft dysfunction. Use of KRP-203 (3 mg/kg/day) in conjunction with CsA markedly improved graft function (P < 0.05, creatinine clearance: 0.41 +/- 0.25 ml/min [CsA] vs. 1.15 +/- 0.16 ml/min [CsA+KRP]). Conclusions. The selectivity of KRP-203 for S1P(1) reduces the risk of bradycardia, and the combination therapy of KRP-203 with CsA represents a safe and effective strategy for use in renal transplantation.

Background: Replacement of calcineurin inhibitor (CI) with anti-metabolic agents in transplant patients with CI-induced nephrotoxiciry is performed clinically and improves renal function, but increases the risk of rejection. We investigated whether the change from cyclosporine (CsA) to a limited dose of mycophenolic acid (MPA) together with a new sphihgosine-1-phosphate (S1P) receptor agonist, KRP-203, is sufficient to prevent both transplant vasculopathy and CsA-induced nephrotoxicity. Methods: Orthotopic aortic transplantation was conducted in a high-responder rat combination of Dark Agouti (DA; major histocompatibiliry complex [MHC] haplotype RT-1(a)) to Lewis (RT-1(l)). After CsA administration (15 mg/kg/day) for 2 weeks, the recipients were divided into the following treatment groups for 6 weeks: MPA (10 mg/kg); KRP-203 (KRP; 1 mg/kg); and MPA + KRP. Serum creatinine (Cr), arteriolar hyalinosis and expression of transforming growth factor (TGF)-beta 1 in the recipient kidney were examined as parameters indicating nephrotoxicity. Intimal hyperplasia was assessed by vascular occlusion, and graft-infiltrated cells were semi-quantitatively evaluated histologically and then characterized immunohistochemically. Results: Continuous CsA treatment attenuated intimal hyperplasia and cell infiltration (2.9 +/- 0.3% and 0.4 +/- 0.1; p < 0.01 vs vehicle), but increased Cr and hyalinosis (0.43 +/- 0.03 mg/dl and 57.2 +/- 0.4%; p < 9.01) with upregulated TGF-beta 1. Replacement of CsA by MPA or KRP treatment alone improved nephrotoxicity, but worsened intimal hyperplasia and cell infiltration. Conversion to MPA + KRP treatment prevented nephrotoxicity (Cr, 0.32 +/- 0.02 mg/dl; hyalinosis, 5.6 +/- 1.3%; p < 0.01 vs CsA) and markedly suppressed intimal hyperplasia and cell infiltration (3.6 +/- 1.2% and 1.0 +/- 0.3;p = not significant vs CsA), with reduced T-cell infiltrates in the graft. Conclusions: Changing from CsA to a combined therapy of MMF with S1P agonist is a promising strategy in clinical transplantation to overcome Cl-induced nephrotoxicity and chronic rejection.

T cell-mediated immune responses play a critical role in a variety of liver injuries including autoimmune hepatitis. Injection of concanavalin A (Con A) into mice mimics the histological and pathological phenotype of T cell-mediated hepatitis. Recent advances in host immune control of organ transplantation include the development of sphingosine-1-phosphate (SIP) receptor agonists such as FTY720, which alter lymphocyte homing but do not suppress host general immunity. Herein we examined the effect of the new SIP receptor agonist KRP-203 on the Con A-induced liver damage model. In normal liver lymphocytes of BALB/c mice, both FTY720 and KRP203 promoted lymphocyte sequestering from the liver to secondary lymph nodes and significantly reduced the number of liver lymphocytes (p < 0.05). Based on this observation, KRP203 was employed in the Con A-induced hepatitis model. KRP203 markedly reduced the number of CD4(+) lymphocytes that infiltrate Con A-treated liver (p < 0.05) and successfully reduced serum transaminase elevation (p = 0.017), therefore protecting mice from Con A-induced liver injury. Interestingly this homing modulation less occurs in natural hepatic T cell homing through the chemokine receptor, CXCR4. Therefore, S1P receptor agonists preferentially target CXCR4(+)CD4(+) peripheral blood T lymphocytes and suppress the occurrence of Con A-induced hepatitis, suggesting their therapeutic usefulness against T cell-mediated hepatic injury. (c) 2006 Elsevier Inc. All rights reserved.

The transduction of cancer cells using recombinant adeno-associated virus (rAAV) occurs with low efficiency, which limits its utility in cancer gene therapy. We have previously sought to enhance rAAV-mediated transduction of cancer cells by applying DNA-damaging stresses. In this study, we examined the effects of the histone deacetylase inhibitor FR901228 on tumor transduction mediated by rAAV types 2 and 5. FR901228 treatment significantly improved the expression of the transgene in four cancer cell lines. The cell surface levels of alpha v integrin, FGF-R1, and PDGF-R were modestly enhanced by the presence of FR901228. These results suggest that the superior transduction induced by the HDAC inhibitor was due to an enhancement of transgene expression rather than increased viral entry. Furthermore, we characterized the association of the acetylated histone H3 in the episomal AAV vector genome by using the chromatin immunoprecipitation assay. The results suggest that the superior transduction may be related to the proposed histone-associated chromatin form of the rAAV concatemer in transduced cells. In the analysis with subcutaneous tumor models, strong enhancement of the transgene expression as well as therapeutic effect was confirmed in vivo. The use of this HDAC inhibitor may enhance the utility of rAAV-mediated transduction strategies for cancer gene therapy.

Objective: Neointimal formation following percutaneous coronary intervention (PCI), termed restenosis, limits therapeutic revascularization. Since reendothelialization is one of the determinant factors for the development of neointimal formation, we examined the effects of granulocyte colony-stimulating factor (G-CSF) on reendothelialization and neointimal formation after vascular injury in mice. Methods and results: Wire-mediated vascular injury was produced in the femoral artery of C57BL/6 mice. G-CSF pretreatment significantly accelerated reendothelialization and decreased neointimal formation following vascular injury; however, this inhibitory effect of G-CSF was diminished when G-CSF was started following the injury. Flow cytometry analysis revealed that G-CSF treatment increased the number of endothelial progenitor cells (EPCs: CD34(+)/Flk-1(+)) in the peripheral circulation. Vascular injury was also produced in 2 types of mice whose bone marrow was replaced with that of enhanced green fluorescent protein- and Tie2/LacZ-transgenic mice. In the reendothelialized artery of these mice, few bone marrow-derived EPCs were detected. Furthermore, G-CSF treatment reduced the serum level of interleukin (IL)-6. Conclusion: G-CSF treatment accelerated reendothelialization and decreased neointimal formation following vascular injury, although there was little contribution of bone marrow-derived EPCs to the reendothelialization of the artery. These results suggest that G-CSF pretreatment has a therapeutic potential for prevention of restenosis following PCI. (c) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Several recent studies have reported that bone marrow cells (BMCs) have the ability to generate functional hepatocytes. However, the efficiency at which BMC transplantation generates functional hepatocytes is rather low. We assumed that if BMCs accumulated directly in liver, the functional BMC-derived hepatocytes should increase efficiently. We tried to increase the accumulation of BMCs directly in liver through the interaction between hepatic asialoglycoprotein receptor and desialylated BMCs. Desialylated BMCs were produced with treatment of neuraminidase. Desialylated BMCs that expressed green fluorescent protein (GFP) were injected into Long Evans Cinnamon (LEC) rats, a human Wilson's disease model, intravenously. At 3 and 5 months after transplantation, GFP-expressing hepatocyte nodules appeared in the liver of these BMC-transplanted LEC rats. These findings suggest that the functional BMC-derived hepatocytes can be generated by the direct accumulation of BMCs and that this strategy is new BMC therapy for liver regeneration. (c) 2006 Elsevier Inc. All rights reserved.

Ascorbic acid has been reported to promote the differentiation of embryonic stem (ES) cells into cardiomyocytes; however, the specific functions of ascorbic acid have not been defined. A stable form of ascorbic acid, namely, L-ascorbic acid 2-phosphate (A2-P), significantly enhanced cardiac differentiation; this was assessed by spontaneous beating of cardiomyocytes and expression of cardiac-specific markers obtained from mouse ES cells. This effect of ascorbic acid was observed only when A2-P was present during the early phase of differentiation. Treatment with two types of collagen synthesis inhibitors, L-2-azetidine carboxylic acid and cis-4-hydroxy-D-proline, significantly inhibited the A2-P-enhanced cardiac differentiation, whereas treatment with the antioxidant N-acetyl cysteine showed no effect. These findings demonstrated that ascorbic acid enhances differentiation of ES cells into cardiomyocytes through collagen synthesis and suggest its potential in the modification of cardiac differentiation of ES cells. (c) 2006 Elsevier Inc. All rights reserved.

Background: To explore a more effective and less toxic immunosuppressive strategy in organ transplantation, we recently developed the novel sphingosine-1-phosphate receptor agonist KRP-203. This study examined the efficacy of KRP-203 combined with mycophenolic acid (MPA), an active metabolite of mycophenolate mofetil, in rat heart allografts. Methods: Heterotopic heart transplantation was performed in a rat combination of DA (MHC haplotype: RT1(a)) to Lewis (RT1(1)). The recipients were divided into 12 groups (n = 5-7): Syngeneic (Lewis to Lewis), Vehicle, KRP-203 (0.3 and 1 mg/kg), MPA (10 and 20 mg/kg), 10 mg/kg MPA with KRP-203 (0.03, 0.3, 1, and 3 mg/kg), and 20 mg/kg MPA with KRP-203 (0.3 and 1 mg/kg). MPA, KRP-203, and vehicle were given orally. Results: The mean days of survival were 5.8 (vehicle), 7 and 7.9 (0.3 and 1 mg/kg KRP-203, respectively), 12.7 and > 54.4 (10 and 20 mg/kg MPA), > 39.6 and > 30.5 (10 mg/kg MPA with 1 and 3 mg/kg KRP-203), > 100 and > 87.8 (20 mg/kg MPA with 0.3 and 1 mg/kg KRP-203). Histologic and immunohistochemical analysis revealed that diffuse mononuclear cell infiltration (macrophages and T cells), hemorrhage, myocardial necrosis and fibrosis, and expression of endothelin-1, transforming growth factor-beta 1, monocyte chemoattractant protein-1, interleukin-8, and E-selectin were markedly diminished in the allografts treated with MPA combined with KRP-203. Pharmacokinetic experiments indicated no interaction between MPA and KRP-203, and both combination regimens were well tolerated. Conclusions: Combination therapy of MPA with KRP-203 has a therapeutic potential as a novel immunosuppressant strategy in clinical transplantation.

Objective: Sepsis accompanies myocardial dysfunction and dynamic alterations of cardiac metabolism. We have recently demonstrated that the very low-density lipoprotein receptor (VLDL-R), which is abundantly expressed in the heart, plays a key role in energy metabolism of the fasting heart. However, little is known about the function and regulation of the VLDL-R during sepsis. In the present study, we explored lipid accumulation and VLDL-R expression in the lipopolysaccharide (LPS)-stimulated heart in vivo and regulation of VLDL-R expression in vitro. Methods and results: Electron microscopy and immunohistochemistry demonstrated that LPS significantly decreased both lipid accumulation and VLDL-R expression in the hearts of fasting mice. Treatment with LPS also downregulated VLDL-R in rat neonatal cardiac myocytes, and this downregulation was completely reversed by interleukin (IL)-1 beta receptor antagonist. IL-1 beta downregulated the expression of VLDL-R in a time- and dose-dependent manner and markedly reduced the uptake of DiI-labeled beta-VLDL but not DiI-labeled low-density lipoprotein (LDL). Use of specific pharmacologic inhibitors and short interference RNA revealed that Hsp90 was required for IL-1 beta to downregulate VLDL-R expression. Conclusions: These findings suggest that IL-1 beta is a principle mediator of changes in cardiac lipid and energy metabolism during sepsis through the downregulation of myocardial VLDL-R expression. (C) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Objective: Transfer of the CTLA4IgG gene induces long-term and high levels of CTLA41gG expression, which can result in generalized immunosuppression. In this study, we utilized Cre/loxP-mediated on-off switch recombination to eliminate transgene expression of CTLA4IgG following acceptance of murine cardiac allografts. Methods: Fully MHC-mismatched hearts from BALB/c donor mice were transplanted into C3H/He recipient mice. Adenovirus-containing CTLA41gG flanked between two loxP sites was administered via a recipient tail vein immediately after transplantation. Cre-recombinase gene was subsequently transferred at day 30 posttransplantation. Results: Long-term allograft survival was observed in recipients that received the CTLA41gG gene. Cre-mediated recombination reduced CTLA4IgG gene expression without any adverse effect on the graft survival. Secondary skin grafts of donor type and of third party were promptly rejected in the recipients that accepted cardiac allografts. In addition, the B cell response against ovalbumin was suppressed during high levels of serum CTLA41gG, but recovered after Cre-mediated inactivation of CTLA4IgG gene. Conclusion: CTLA4IgG gene transfer promoted long-term survival of murine cardiac allografts; however, this was not sufficient to induce tolerance. Cre/loxP-mediated on-off switch recombination was useful to inactivate the CTLA41gG gene so that recipients' immune responses against neoantigens were restored without an influence on the allograft survival. This system may open novel strategies to orchestrate clinically relevant immunosuppression. (c) 2005 European Society of Cardiology. Published by Elsevier B.V. All fights reserved.

In the past decade, researchers have defined committed stem or progenitor cells from various tissues, including bone marrow, peripheral blood, brain, liver and reproductive organs, in both adult animals and humans. Recently, endothelial progenitor cells (EPCs) were isolated from peripheral blood mononuclear cells and were shown to be incorporated into foci of neovascularization. This finding that circulating EPCs may home into sites of neovascularization and differentiate into mature endothelial cells in situ is consistent with the concept of 'vasculogenesis' and suggests that vasculogenesis and angiogenesis might constitute complementary mechanisms for postnatal neovascularization. Furthermore, experimental and clinical studies on ischemic cardiovascular diseases suggest a therapeutic potential for EPC transplantation. In this review, we summarize the biological features of EPCs and discuss their therapeutic potential for the treatment of cardiovascular diseases. © 2006 Bentham Science Publishers Ltd.

Experimental autoimmune myocarditis (EAM) is characterized by the appearance of multinucleated giant cells. EAM leads to severe myocardial damage and is a useful model of human giant cell myocarditis. We investigated whether mycophenolate mofetil (MMF), which is a potent immunosuppressant, prevents the development of myocarditis in a rat EAM model, and focused on the role of osteopontin (OPN) in the pathogenesis of this disorder. Adult Lewis rats were immunized with porcine cardiac myosin to establish EAM. The early MMF treatment completely prevented the development of EAM, and the late MMF treatment was also effective even against established EAM. Echocardiogram demonstrated that left ventricular function was also improved by the treatment with MMR Real-time RT-PCR analysis showed that both early and late MMF treatments significantly inhibited myocarditis-induced OPN mRNA expression in the heart. Immunohistochemistry revealed that OPN expression was prominent in the myocardium on day 14, whereas expression was observed in the infiltrated macrophages on day 21. Mycophenolic acid (MPA) did inhibit agonist-induced OPN expression in cultured cardionnyocytes. These results show the therapeutic potential of MMF for autoimmune myocarditis and provide new insights into the pathogenesis of this disease. (c) 2005 Elsevier Ltd. All rights reserved.

Objective: Based on development of stem cell technology, newborn tissue, even undergoing cryopreservation, possesses promising potential as a donor source in, the field of organ transplantation. However, the precise regeneration processes remains unclear. This study was designed to investigate the regenerative potential of newborn intestine with or without cryopreservation in the transplantation. Methods: Newborn rat intestines with or without cryopreservation were transplanted subcutaneously into the syngeneic host, and specimens were evaluated by histology, multiple immunostaining, and comprehensive gene expression analysis. Results: We determined that newborn rat intestine possessed regenerative potential in the syngeneic host even after cryopreservation, where angiogenesis was induced early in the submucosa with subsequent maturation in the crypts. Furthermore, newborn intestinal graft could facilitate the survival of maturation-incompetent 10-day-old graft that lacked regenerating activity (P < 0.01, n = 13). Tissue aggregates from the maturation-incompetent graft underwent reconstitution of their histologic configuration in the presence of newborn intestinal aggregates. Comprehensive gene expression analysis showed that 37 genes were preferentially up-regulated, while 19 genes were downregulated in the regenerating 10-day-old graft (supported by the newborn graft). Conclusions: Regeneration of newborn intestine is implicated in neo-angiogenesis in the host, and the newborn intestinal graft is capable of mediating the survival of the maturation-incompetent 10-day-old graft. Notwithstanding ethical and legal limitations in the clinic, these results may provide new insights into the regenerative role of newborn grafts.

The ideal goal of regeneration medicine is to restore form and function to damaged tissues. While stem cell transplantation is considered a promising therapeutic approach, knowing the fate of transplanted cells using appropriate markers is essential. We developed new inbred transgenic rat strains with lacZ and GFP based on the transgenic (Tg) animal technique in rats. These Tg animals expressed most of their marker genes ubiquitously, compared to previous Tg rats. Immunological antigenicity against marker proteins was evaluated using conventional skin grafting, and results suggested lacZ-Tg-derived skin was much less immunogenic than that of GFP-Tg. However, GFP-positive cells from parental transgenic rats were still potential candidates for the study of cellular fate in immune privilege sites, such as the brain. Taking advantage of less immunogenic lacZ, we also examined the role of bone marrow-derived cells (BMDCs) in skin wound healing using an in vivo biological imaging system. Although transplantation of BMDCs enhanced wound healing at the injection site, BMDCs were detected only for a short time, suggesting a transient contribution of autologous BMDC-transplantation in wound healing. Our Tg-rat system may provide great benefits for the elucidation of the cellular process of regenerative medicine, including cell and tissue transplantation. (C) 2005 Elsevier Inc. All rights reserved.

The very low-density lipoprotein (VLDL) receptor is a member of the low-density lipoprotein (LDL) receptor gene family with distinct tissue distribution and function. VLDL receptors are also expressed in vascular smooth muscle cells (VSMCs) and have been shown to be upregulated in atherosclerotic lesions. In the present study, we examined the effects of interleukin-1 beta (IL-1 beta) on the uptake of beta VLDL and its receptor expression in rat VSMCs. IL-1 beta downregulated expression of the VLDL receptor in a time and dose-dependent manner as shown by Western blotting, Northern blotting, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Treatment with IL-1 beta significantly reduced the uptake of P-VLDL but not LDL in VSMCs. Use of specific pharmacologic inhibitors indicated that the tyrosine kinase inhibitors, herbimycin A and geldanamycin, completely reversed IL-1 beta-induced downregulation of the VLDL receptor expression. Another tyrosine kinase inhibitor, genistein, the protein kinase C inhibitors, GF109203X and H7, the mitogen-activated protein (MAP) kinase inhibitors (MEK inhibitor PD098059 for [MEK] and SB203580 for p38-MAP kinase), and the protein kinase A inhibitor, KT5270 all had no effect on receptor expression. In addition, the c-Src specific inhibitor PP2 or adenoviral-mediated gene transfer of kinase inactive (KI)-c-Src failed to reverse IL-1 beta-induced downregulation of VLDL receptor expression. These results indicate that IL-1 beta attenuates uptake of VLDL through downregulation of its receptor in VSMCs, and that this downregulation is mediated through a benzoquinone ansamycin-dependent but c-Src-independent pathway. (c) 2005 Elsevier Ltd. All rights reserved.

Background - A novel immunomodulator, KRP-203, the molecular structure of which has some similarity to FTY720, has been developed for use in organ transplantation. The present study was designed to investigate the potency and safety of KRP-203 on allograft survival against both acute and chronic rejection in rat skin and heart transplantation. Methods and Results - KRP-203 significantly prolonged skin or heart allograft survival of a minor histocompatibility complex (mHC) - disparate (LEW to F344) rat combination. Histopathological and immunohistochemical analysis at 100 days after mHC-disparate rat heart transplantation revealed that KRP-203 treatment significantly inhibited infiltration of inflammatory cells, including macrophages and T cells; expression of endothelin-1 and transforming growth factor-beta(1); and IgG deposition and eventually attenuated neointimal formation and myocardial fibrosis. KRP-203 also prolonged heart allograft survival in a major histocompatibility complex (MHC)-incompatible (DA to LEW) rat combination, but the efficacy was not as significant. However, KRP-203 combined with a subtherapeutic dose of cyclosporin A synergistically prolonged the heart allograft survival. Flow cytometric analysis demonstrated that KRP-203 reduced the number of peripheral blood mononuclear cells ( lymphocytes and monocytes) but not granulocytes and enhanced lymphocyte homing into peripheral lymph nodes. The influence of KRP-203 on heart rate changes in Hartley guinea pigs was examined. KRP-203 had less of a tendency to cause bradycardia than FTY720. Conclusions - These findings demonstrated that KRP-203 prolonged skin and heart allograft survival and significantly attenuated chronic rejection and bradycardia as an adverse effect. Therefore, KRP-203 offers considerable potential as a novel therapeutic immunosuppressant in patients with organ transplantation.