5-Methoxytryptophan attenuates postinfarct cardiac injury by controlling oxidative stress and immune activation.

AIMS: Myocardial infarction (MI) remains a major cause of heart failure. 5-Methoxytryptophan (5-MTP), a 5-methoxyindole metabolite of L-tryptophan, exerts anti-inflammatory and antifibrotic effects, but MI impairs the biosynthesis of cardiac 5-MTP. Therefore, we evaluated the effect of exogenous 5-MTP administration on rescuing post-MI cardiac injury. METHODS AND RESULTS: After a detailed pharmacokinetic analysis of 5-MTP, Sprague Dawley rats that had undergone left anterior descending coronary artery ligation received intraperitoneal administration of either 17 mg/kg 5-MTP or saline at 0.5 and 24 h after MI. Cardiac systolic function, infarction size, and fibrosis were evaluated using echocardiography, triphenyltetrazolium chloride staining, and Masson trichrome staining, respectively. Myocardial apoptosis was analyzed by staining for caspase-3 and cardiac troponin I. 5-MTP treatment decreased the infarct area and myocardial apoptosis; attenuated systolic dysfunction and left ventricular dilatation; and reduced cardiomyocyte hypertrophy, myocardial fibrosis, and infarct expansion. Crucially, 5-MTP alleviated oxidative stress by preserving mitochondrial antioxidant enzymes and downregulating reactive oxygen species-generating NADPH oxidase isoforms and endothelin-1. Consequently, 5-MTP-treated MI rat hearts exhibited lower levels of chemokines and cytokines, namely interleukin (IL)-1ß, IL-18, IL-6, C-C motif chemokine ligand (CCL)-2, and CCL5, accompanied by reduced infiltration of CD11b+ cells and CD4+ T cells. Notably, 5-MTP protected against H2O2-induced damage in HL-1 cardiomyocytes and human umbilical vein endothelial cells in vitro. CONCLUSION: 5-MTP prevented post-MI cardiac injury by promoting mitochondrial stabilization and controlling redox imbalance. This cytoprotective effect ameliorated macrophage and T-cell infiltration, thus reducing the infarct size, attenuating fibrosis, and restoring myocardial function.

CXCR4 Antagonist Reduced the Incidence of Acute Rejection and Controlled Cardiac Allograft Vasculopathy in a Swine Heart Transplant Model Receiving a Mycophenolate-based Immunosuppressive Regimen.

BACKGROUND: CXC motif chemokine receptor 4 (CXCR4) blockade is pursued as an alternative to mesenchymal stem cell treatment in transplantation based on our previous report that burixafor, through CXCR4 antagonism, mobilizes immunomodulatory mesenchymal stem cells. Here, we explored the efficacy of combining mycophenolate mofetil (MMF)-based immunosuppressants with repetitive burixafor administration. METHODS: Swine heterotopic cardiac allograft recipients received MMF and corticosteroids (control, n = 10) combined with burixafor as a 2-dose (burixafor2D, n = 7) or 2-dose plus booster injections (burixafor2D + B, n = 5) regimen. The efficacy endpoints were graft survival, freedom from first acute rejection, and the severity of intimal hyperplasia. Each specimen was sacrificed either at its first graft arrest or after 150 days. RESULTS: After 150 days, all specimens in the control group had died, but 28.5% of the burixafor2D group survived, and 60% of the burixafor2D + B group survived (P = 0.0088). Although the control group demonstrated acute rejection at a median of 33.5 days, the burixafor2D + B group survived without acute rejection for a median of 136 days (P = 0.0209). Burixafor administration significantly attenuated the incidence rate of acute rejection (P = 0.002) and the severity of intimal hyperplasia (P = 0.0097) at end point relative to the controls. These findings were associated with reduced cell infiltrates in the allografts, and modulation of C-reactive protein profiles in the circulation. CONCLUSIONS: The augmentation of conventional MMF plus corticosteroids with a CXCR4 antagonist is potentially effective in improving outcomes after heart transplantation in minipigs. Future studies are warranted into optimizing the therapeutic regimens for humans.

CXCR4 Antagonist TG-0054 Mobilizes Mesenchymal Stem Cells, Attenuates Inflammation, and Preserves Cardiac Systolic Function in a Porcine Model of Myocardial Infarction.

Interaction between chemokine stromal cell-derived factor 1 and the CXC chemokine receptor 4 (CXCR4) governs the sequestration and mobilization of bone marrow stem cells. We investigated the therapeutic potential of TG-0054, a novel CXCR4 antagonist, in attenuating cardiac dysfunction after myocardial infarction (MI). In miniature pigs (minipigs), TG-0054 mobilized CD34(+)CXCR4(+), CD133(+)CXCR4(+), and CD271(+)CXCR4(+) cells into peripheral circulation. After isolation and expansion, TG-0054-mobilized CD271(+) cells were proved to be mesenchymal stem cells (designated CD271-MSCs) since they had trilineage differentiation potential, surface markers of MSCs, and immunosuppressive effects on allogeneic lymphocyte proliferation. MI was induced in 22 minipigs using balloon occlusion of the left anterior descending coronary artery, followed by intravenous injections of 2.85 mg/kg of TG-0054 or saline at 3 days and 7 days post-MI. Serial MRI analyses revealed that TG-0054 treatment prevented left ventricular (LV) dysfunction at 12 weeks after MI (change of LV ejection fraction from baseline, -1.0 ± 6.2% in the TG-0054 group versus -7.9 ± 5.8% in the controls). The preserved cardiac function was accompanied by a significant decrease in the myocardial expression of TNF-α, IL-1ß, and IL-6 at 7 days post-MI. Moreover, the plasma levels of TNF-α, IL-1ß, and IL-6 were persistently suppressed by the TG-0054 treatment. Infusion of TG-0054-mobilized CD271-MSCs reduced both myocardial and plasma cytokine levels in a pattern, which is temporally correlated with TG-0054 treatment. This study demonstrated that TG-0054 improves the impaired LV contractility following MI, at least in part, by mobilizing MSCs to attenuate the postinfarction inflammation. This insight may facilitate exploring novel stem cell-based therapy for treating post-MI heart failure.

Usefulness of magnetocardiography to detect coronary artery disease and cardiac allograft vasculopathy.

BACKGROUND: Electrophysiological information as well as anatomic information are important for the detection of coronary artery lesions. The aim of this study was to assess the efficacy of resting magnetocardiography (MCG) in stable coronary artery disease (CAD) and cardiac allograft vasculopathy (CAV). METHODS AND RESULTS: MCG and coronary angiography were performed within 1 month in 75 patients with suspected CAD and in 26 subjects after orthotopic heart transplantation (OHT). Plaque volumes were additionally measured on intravascular ultrasound in OHT recipients. The spatially distributed QT(c) interval maps were constructed with 64-channel MCG. A T-wave propagation map and QT(c) heterogeneity index including QT(c) dispersion and smoothness index of QT(c) (SI-QT(c)) were derived for ischemia detection and localization. CAD patients had higher QT(c) dispersion and SI-QT(c). Receiver operating characteristic curve analysis identified SI-QT(c) ≥9 ms, QT(c) dispersion ≥79 ms as the optimal cut-off for detecting CAD (diagnostic accuracy, 0.7953, 0.7819), better than T-wave propagation (0.6594, P<0.05). There was no significant difference of QT(c) dispersion between CAD and OHT subjects. In OHT recipients, QT(c) dispersion positively correlated with plaque volume, and SI-QT(c) progressively increased after transplantation. Using T-wave propagation mapping, regionally increased dispersion could be demonstrated in CAD patients, but increased dispersion was noted in fewer OHT recipients. CONCLUSIONS: MCG is clinically feasible as a non-invasive tool for diagnosis of CAD, and could be used as a surrogate marker of CAV.

Prostaglandin E2 potentiates mesenchymal stem cell-induced IL-10+IFN-γ+CD4+ regulatory T cells to control transplant arteriosclerosis.

Mesenchymal stem cells (MSCs) are known for their immunomodulatory functions. We previously demonstrated that bone marrow-derived MSCs effectively control transplant arteriosclerosis (TA) by enhancing IL-10(+) and IFN-γ(+) cells. The objective of this study is to elucidate the mechanism by which MSCs induce IL-10(+)IFN-γ(+)CD4(+) regulatory T type 1 (T(R)1)-like cells. In an MLR system using porcine PBMCs, MSC-induced IL-10(+)IFN-γ(+)CD4(+) cells, which confer resistance to allogeneic proliferation in an IL-10-dependent manner, resemble T(R)1-like cells. Both cyclooxygenase-derived PGE(2) and IDO help to induce T(R)1-like cells by MSCs. MSCs constitutively secrete PGE(2), which is augmented in allogeneic reactions. However, T(R)1-like cells were deficient in PGE(2) and 4-fold less potent than were MSCs in suppressing MLR. PGE(2) mimetic supplements can enhance the immunosuppressive potency of T(R)1-like cells. In a porcine model of allogeneic femoral arterial transplantation, MSC-induced T(R)1-like cells combined with PGE(2), but not either alone, significantly reduced TA at the end of 6 wk (percentage of luminal area stenosis: T(R)1-like cells + PGE(2): 11 ± 10%; PGE(2) alone: 93 ± 8.7%; T(R)1-like cells alone: 88 ± 2.4% versus untreated 94 ± 0.9%, p < 0.001). These findings indicate that PGE(2) helps MSC-induced IL-10(+)IFN-γ(+)CD4(+) T(R)1-like cells inhibit TA. PGE(2) combined with MSC-induced T(R)1-like cells represents a new approach for achieving immune tolerance.

Autologous mesenchymal stem cells prevent transplant arteriosclerosis by enhancing local expression of interleukin-10, interferon-γ, and indoleamine 2,3-dioxygenase.

Transplant arteriosclerosis (TA) remains the major limitation of long-term graft survival in heart transplantation despite the advances in immunosuppressants. Mesenchymal stem cells (MSCs) have been demonstrated to suppress allogeneic immune responses by numerous in vitro studies. However, the immunomodulatory effects of MSCs in vivo are controversial and the underlying molecular mechanisms are not conclusive. In this study, we investigated the therapeutic potential of autologous bone marrow-derived MSCs on TA in a porcine model of femoral artery transplantation. MSCs or saline were injected into the soft tissue surrounding the arterial grafts immediately postanastomosis. Four weeks after transplantation, neointimal formation increased significantly in untreated allografts compared with the MSC-treated grafts as assessed by intravascular ultrasound (maximum luminal area stenosis: 40 ± 12% vs. 18 ± 6%, p < 0.001). Grafts harvested at 4 weeks showed dense perivascular lymphocyte infiltration accompanied by significant intimal hyperplasia in the untreated but not in the MSC-treated allografts. Serial angiographic examination showed that all of the untreated allografts became occluded at the 8th week whereas the majority of the MSC-treated grafts remained patent at the 12th week posttransplantation (n = 12 each group, p < 0.001). Quantitative PCR analysis revealed that Foxp3 expression was comparable between the untreated and the MSC-treated groups. However, expression of interleukin-10 (IL-10), interferon-γ (IFN-γ), and indoleamine 2,3-dioxygenase (IDO) was increased significantly in the MSC-treated allografts compared with that in the allograft controls (p = 0.021 for IL-10, p = 0.003 for IFN-γ, and p = 0.008 for IDO). In conclusion, local delivery of autologous MSCs alleviates TA by inducing allograft tolerance via enhanced expression of IL-10, IFN-γ, and IDO but not Foxp3-positive cells in the vessel wall. These results suggest that MSCs induce immune tolerance by activating the type 1 regulatory T-like cells.

PET assessment of myocardial perfusion reserve inversely correlates with intravascular ultrasound findings in angiographically normal cardiac transplant recipients.

UNLABELLED: Cardiac allograft vasculopathy (CAV) is the major determinant of long-term survival after heart transplantation. We aimed to evaluate the efficacy of PET as a noninvasive way to assess the early stages of CAV. METHODS: Twenty-seven consecutive patients (20 men and 7 women; mean age +/- SD, 46 +/- 12 y) who had normal results on coronary angiography and normal left ventricular systolic function (ejection fraction >or= 60%) were enrolled at 2.5 +/- 2.1 y after transplantation. Myocardial blood flow (MBF) was assessed using dynamic (13)N-ammonia PET at rest and during adenosine-induced hyperemia, and myocardial perfusion reserve (MPR) was calculated as the ratio of hyperemic MBF to resting MBF. Regional (13)N-ammonia PET was assessed using a 5-point scoring system. The intravascular ultrasound (IVUS) measurements for the extent of intimal hyperplasia, including plaque volume index (calculated as [total plaque volume/total vessel volume] x 100%) and maximum area of stenosis, were compared with MPR by linear regression analysis. RESULTS: In 27 angiographically normal cardiac transplant recipients, MBF at rest and during adenosine stress and MPR of the left anterior descending artery distribution correlated strongly with the other 2 coronary artery distribution territories (r >or= 0.97, P < 0.0001). Summed stress score and summed difference score showed a moderate inverse correlation with MPR (r = -0.41 and -0.49, respectively; P < 0.05) but not with IVUS measurements. MPR correlated inversely with plaque volume index (r = -0.40, P < 0.05) but not with maximal luminal stenosis as assessed by IVUS. In addition, MPR and IVUS measurements gradually inversely changed after heart transplantation (all P < 0.05). CONCLUSION: This study confirms that CAV is a progressive process, diffusely involving the epicardial and microvascular coronary system. Plaque burden as determined by IVUS agrees well with MPR as assessed by PET in recipients with normal coronary angiography results. This finding suggests that dynamic (13)N-ammonia PET is clinically feasible for the early detection of CAV and can be used as a reliable marker of disease progression.

Intravascular ultrasound correlates with coronary flow reserve and predicts the survival in angiographically normal cardiac transplant recipients.

OBJECTIVES: We aimed to determine whether epicardial and intramyocardial arteries were involved concordantly in early cardiac allograft vasculopathy. METHODS: Thirty consecutive recipients who had received cardiac transplantation more than 1 year before and had a normal coronary angiogram were enrolled for intravascular ultrasound (IVUS), fractional flow reserve, coronary flow reserve and dipyridamole thallium-201 single photon emission computed tomography. Graft failure including cardiac death and retransplantation served as the primary outcome. RESULTS: Plaque volume index and maximum area stenosis calculated from IVUS measurements correlated with coronary flow reserve deterioration in a subgroup of patients with normal fractional flow reserve (n = 13; r = -0.80, p = 0.001 and r = -0.91, p <0.0001, respectively). After follow-up for 18.4 +/- 7.6 months, maximum area stenosis was found to be an independent predictor of graft failure (hazard ratio 1.43, 95% confidence interval 1.08-1.89, p = 0.012). CONCLUSION: In patients with physiologically normal epicardial coronary arteries, impairment of microvascular integrity correlates with the plaque burden measured by IVUS, suggesting the concordant involvement of both epicardial and resistant vessels in early cardiac allograft vasculopathy. Evident epicardial coronary narrowing on IVUS may predict graft failure in spite of normal coronary angiograms.

Predictors of therapeutic response to beta-blockers in patients with heart failure in Taiwan.

BACKGROUND/PURPOSE: Chinese are more sensitive to beta-blockers than Caucasians. However, data regarding beta-blocker therapy in heart failure (HF) patients in Taiwan are lacking. We aimed to evaluate the improvement of left ventricular function and the potential predictors of response to beta-blocker therapy in Taiwanese HF patients. METHODS: We enrolled 34 HF patients with baseline left ventricular ejection fraction (LVEF)

Intravascular ultrasound evidence of angiographically silent allograft vasculopathy inversely correlates with circulating level of hepatocyte growth factor.

BACKGROUND: Cardiac allograft vasculopathy (CAV) is the major determinant of long-term survival after heart transplantation. The characteristic diffuse concentric intimal thickening of CAV detected by intravascular ultrasound (IVUS) imaging may not be perceivable on coronary angiogram. Previous studies of hepatocyte growth factor (HGF) in murine models suggested the protective effects on vascular endothelium and allograft survival. However, the possible role of circulating HGF that contributes to the development of CAV in human is unclear. METHODS: IVUS was used to assess the left anterior descending coronary arteries of 47 patients who had survived cardiac transplantation for more than 1 year and had no angiographically detectable CAV. The IVUS measurements for the extent of intimal hyperplasia, including volume index (calculated as [total plaque volume/total vessel volume] x 100) and maximum area stenosis, were compared with plasma levels of HGF by linear regression analyses. RESULTS: The volume index significantly correlated with maximum area stenosis in the IVUS measurements (r = 0.90, p < 0.0001). Both volume index and maximum area stenosis inversely correlated with plasma HGF levels (r = -0.39, p = 0.007 and r = -0.42, p = 0.003, respectively). CONCLUSIONS: In angiographically silent CAV, circulating HGF may have a protective effect on vascular endothelium and thus attenuate the severity of intimal hyperplasia.

Enhanced external counterpulsation reduces lung/heart ratio at stress in patients with coronary artery disease.

Enhanced external counterpulsation (EECP) is a recently approved treatment modality for patients with angina and heart failure. However, the efficacy of EECP on left ventricular (LV) function has not been well established. The study was aimed to determine whether EECP leads to an improvement in objective parameters of LV function. Patients with coronary artery disease (n = 10) who showed evidence of stress-induced myocardial ischemia despite conventional medical or surgical therapies were enrolled and received EECP therapy for a total of 35 h. The therapeutic effects of EECP were examined by thallium-201 single-photon emission computed tomography (201Tl-SPECT). Compared with baseline, the lung/heart ratio at stress decreased significantly from 0.40 +/- 0.08 to 0.35 +/- 0.08 (p = 0.001) at 1 month and 0.33 +/- 0.10 (p = 0.03) at 6 months following EECP treatment. LV ejection fraction marginally improved from 56.7 +/- 7.7% to 57.6 +/- 5.9% (p = 0.382) at 1 month and to 60.1 +/- 8.6% (p = 0.062) at 6 months after EECP therapy, although not statistically significant. We concluded that EECP improved LV function, shown as the reduction of lung/heart ratio at stress, in patients with coronary artery disease, up to 6 months after EECP treatment.

High-density lipoprotein antagonizes oxidized low-density lipoprotein by suppressing oxygen free-radical formation and preserving nitric oxide bioactivity.

The antiatherogenic role of high-density lipoprotein (HDL) has been related to its ability to increase the activity of endothelial nitric oxide synthase (eNOS) and to protect low-density lipoprotein (LDL) against oxidative modification. The present study was aimed to determine whether and how HDL antagonizes oxidized LDL (oxLDL) that has been formed and accumulated in circulation. Pre-infusion of rats with HDL effectively prevented oxLDL-induced renal vascular constriction. Consistently, pre-incubation of human saphenous vein endothelial cells with HDL (100 microg/ml) reversed the oxLDL-induced suppression of endothelium-dependent cyclic-GMP production in co-cultured smooth muscle cells. However, the changes of Akt phosphorylation and eNOS activity in endothelial cells in response to lipoprotein treatments under our assay condition were not significant. Intriguingly, pretreatment of human umbilical vein endothelial cells with HDL (50 microg/ml) for only 30s effectively reduced the level of free radicals generated by oxLDL or H(2)O(2). In kidneys of living rats, renal arterial infusion of oxLDL greatly enhanced ischemia/reperfusion-induced free radicals, which could be attenuated by HDL pretreatment. We conclude that HDL may antagonize oxLDL on endothelial function through an Akt-independent pathway in which HDL preserves nitric oxide bioactivity by attenuating oxLDL-triggered free radical generation.

Isolation of multipotent cells from human term placenta.

Current sources of stem cells include embryonic stem cells (ESCs) and adult stem cells (ASCs). However, concerns exist with either source: ESCs, with their significant ethical considerations, tumorigenicity, and paucity of cell lines; and ASCs, which are possibly more limited in potential. Thus, the search continues for an ethically conducive, easily accessible, and high-yielding source of stem cells. We have isolated a population of multipotent cells from the human term placenta, a temporary organ with fetal contributions that is discarded postpartum. These placenta-derived multipotent cells (PDMCs) exhibit many markers common to mesenchymal stem cells--including CD105/endoglin/SH-2, SH-3, and SH-4--and they lack hematopoietic-, endothelial-, and trophoblastic-specific cell markers. In addition, PDMCs exhibit ESC surface markers of SSEA-4, TRA-1-61, and TRA-1-80. Adipogenic, osteogenic, and neurogenic differentiation were achieved after culturing under the appropriate conditions. PDMCs could provide an ethically uncontroversial and easily accessible source of multipotent cells for future experimental and clinical applications.