Carvedilol enhances mesenchymal stem cell therapy for myocardial infarction via inhibition of caspase-3 expression.

Adult stem cells have shown great promise toward repairing infarcted heart and restoring cardiac function. Mesenchymal stem cells (MSCs), because of their inherent multipotent nature and their ability to secrete a multitude of growth factors and cytokines, have been used for cardiac repair with encouraging results. Preclinical studies showed that MSCs injected into infarcted hearts improve cardiac function and attenuate fibrosis. Although stem cell transplantation is a promising therapeutic option to repair the infarcted heart, it is faced with a number of challenges, including the survival of the transplanted cells in the ischemic region, due to excessive oxidative stress present in the ischemic region. The objective of this study was to determine the effect of Carvedilol (Carv), a nonselective ß-blocker with antioxidant properties, on the survival and engraftment of MSCs in the infarcted heart. MSCs were subjected to a simulated host-tissue environment, similar to the one present in the infarcted myocardium, by culturing them in the presence of hydrogen peroxide (H(2)O(2)) to induce oxidative stress. MSCs were treated with 2.5 µM Carv for 1 h in serum-free medium, followed by treatment with H(2)O(2) for 2 h. The treated cells exhibited significant protection against H(2)O(2)-induced cell death versus untreated controls as determined by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays. Likewise, transplantation of MSCs after permanent left coronary artery ligation and treatment of animals after myocardial infarction (MI) with Carv (5 mg/kg b.wt.) led to significant improvement in cardiac function, decreased fibrosis, and caspase-3 expression compared with the MI or MSC-alone groups.

Amelioration of doxorubicin-induced cardiotoxicity by an anticancer-antioxidant dual-function compound, HO-3867.

Doxorubicin (DOX) is a drug commonly used for the treatment of cancer. The development of resistance to DOX is common, and high cumulative doses cause potentially lethal cardiac side effects. HO-3867 (3,5-bis(4-fluorobenzylidene)-1-[(2,2,5,5-tetramethyl-2,5-dihydro-1-hydroxy-pyrrol-3-yl)methyl]piperidin-4-one), a synthetic curcumin analog, has been shown to exhibit both anticancer and cardioprotective effects. However, its cardioprotection in the setting of a conventional cancer therapy has not been established. This work investigated the use of HO-3867 and DOX to achieve a complementary outcome, i.e., increased toxicity toward cancer cells, and reduced cardiac toxicity. Combination treatment was investigated using DOX-resistant MCF-7 breast cancer cells [MCF-7 multidrug-resistant (MDR)] and BALB/c mice. Lower doses of HO-3867 and DOX (5 and 2.5 µM, respectively) reduced viability of MCF-7 MDR cells to an extent significantly greater than that when either drug was used alone, an effect equivalent to that induced by exposure to 50 µM DOX. In normal cardiac cells, the loss of viability from combination treatment was significantly lower than that induced by 50 µM DOX. Increases in apoptotic markers, e.g., cleaved caspase-3, and decreases in fatty acid synthase and pAkt expressions were observed by Western blotting. Mice treated with both HO-3867 and DOX showed significant improvement in cardiac functional parameters compared with mice treated with DOX alone. Reduced expression of Bcl-2 and pAkt was observed in mice treated with DOX alone, whereas mice given combination treatment showed levels similar to control. The study indicates that combination treatment of HO-3867 and DOX is a viable option for treatment of cancer with reduced cardiotoxic side effects.

Effect of oxygenation on stem-cell therapy for myocardial infarction.

Stem-cell transplantation to treat acute myocardial infarction (MI) is gaining importance as a minimally invasive and potent therapy to replace akinetic scar tissue by viable myocardium. Our recent studies have shown that stem-cell transplantation marginally improves myocardial oxygenation in the infarct tissue leading to improvement in cardiac function. The aim of the present study was to determine the effect of hyperbaric oxygen (HBO) treatment on myocardial oxygenation and recovery of function in MI hearts. Fisher-344 rats were subjected to MI by permanently ligating the left-anterior-descending (LAD) coronary artery. The rats were then exposed to 100% O(2) at a pressure of 2 atmospheres for 90 minutes, and the exposure was repeated for 5 days a week for 2 weeks. Adult bone-marrow-derived rat mesenchymal stem cells (MSC, 5x105 cells) were mixed with OxySpin (LiNc- BuO, oxygen sensor) and implanted in the infarct and peri-infarct regions of the heart. M-mode ultrasound echocardiography was performed at baseline and at 2 weeks post-transplantation. The myocardial pO(2) in the MSC+HBO group (16.2±2.2 mmHg) was significantly higher when compared to untreated MI (3.8±1.9 mmHg) or MSC (9.8±2.3 mmHg) groups. In addition, there was a significant improvement in cardiac function, increased vessel density, and VEGF expression in MSC+HBO group compared to MSC group (p < 0.05). In conclusion, the results suggested a beneficial effect of HBO administration on stem-cell therapy for MI.

Trimetazidine, administered at the onset of reperfusion, ameliorates myocardial dysfunction and injury by activation of p38 mitogen-activated protein kinase and Akt signaling.

Trimetazidine [1-(2,3,4-trimethoxybenzyl)piperazine; TMZ] is an anti-ischemic cardiac drug; however, its efficacy and mechanism of cardioprotection upon reperfusion are largely unknown. The objective of this study was to determine whether TMZ, given before reperfusion, could attenuate myocardial reperfusion injury. Ischemia/reperfusion (I/R) was induced in rat hearts by ligating the left anterior descending (LAD) coronary artery for 30 min followed by 48 h of reperfusion. TMZ (5 mg/kg b.wt.) was administered 5 min before reperfusion. The study used three experimental groups: control (-I/R; -TMZ), I/R (+I/R; -TMZ), and TMZ (+I/R; +TMZ). Echocardiography and EPR oximetry were used to assess cardiac function and oxygenation, respectively. The ejection fraction, which was significantly depressed in the I/R group (62 +/- 5 versus 84 +/- 3% in control), was restored to 72 +/- 3% in the TMZ group. Myocardial pO2 in the TMZ group returned to baseline levels (approximately 20 mm Hg) within 1 h of reperfusion, whereas the I/R group showed a significant hyperoxygenation even after 48 h of reperfusion. The infarct size was significantly reduced in the TMZ group (26 +/- 3 versus 47 +/- 5% in I/R). TMZ treatment significantly attenuated superoxide levels in the tissue. Tissue homogenates showed a significant increase in p38 and p-Akt and decrease in caspase-3 levels in the TMZ group. In summary, the results demonstrated that TMZ is cardioprotective when administered before reperfusion and that this protection appears to be mediated by activation of p38 mitogen-activated protein kinase and Akt signaling. The study emphasizes the importance of administering TMZ before reflow to prevent reperfusion-mediated cardiac injury and dysfunction.

Hyperbaric oxygenation enhances transplanted cell graft and functional recovery in the infarct heart.

A major limitation to the application of stem-cell therapy to repair ischemic heart damage is the low survival of transplanted cells in the heart, possibly due to poor oxygenation. We hypothesized that hyperbaric oxygenation (HBO) can be used as an adjuvant treatment to augment stem-cell therapy. Therefore, the goal of this study was to evaluate the effect of HBO on the engraftment of rat bone marrow-derived mesenchymal stem cells (MSCs) transplanted in infarct rat hearts. Myocardial infarction (MI) was induced in Fisher-344 rats by permanently ligating the left-anterior-descending coronary artery. MSCs, labeled with fluorescent superparamagnetic iron oxide (SPIO) particles, were transplanted in the infarct and peri-infarct regions of the MI hearts. HBO (100% oxygen at 2 ATA for 90 min) was administered daily for 2 weeks. Four MI groups were used: untreated (MI); HBO; MSC; MSC+HBO. Echocardiography, electro-vectorcardiography, and magnetic resonance imaging were used for functional evaluations. The engraftment of transplanted MSCs in the heart was confirmed by SPIO fluorescence and Prussian-blue staining. Immunohistochemical staining was used to identify key cellular and molecular markers including CD29, troponin-T, connexin-43, VEGF, alpha-smooth-muscle actin, and von Willebrand factor in the tissue. Compared to MI and MSC groups, the MSC+HBO group showed a significantly increased recovery of cardiac function including left-ventricular (LV) ejection fraction, fraction shortening, LV wall thickness, and QRS vector. Further, HBO treatment significantly increased the engraftment of CD29-positive cells, expression of connexin-43, troponin-T and VEGF, and angiogenesis in the infarct tissue. Thus, HBO appears to be a potential and clinically-viable adjuvant treatment for myocardial stem-cell therapy.

Supplemental Oxygen Protects Heart Against Acute Myocardial Infarction.

Myocardial infarction (MI), which occurs often due to acute ischemia followed by reflow, is associated with irreversible loss (death) of cardiomyocytes. If left untreated, MI will lead to progressive loss of viable cardiomyocytes, deterioration of cardiac function, and congestive heart failure. While supplemental oxygen therapy has long been in practice to treat acute MI, there has not been a clear scientific basis for the observed beneficial effects. Further, there is no rationale for the amount or duration of administration of supplemental oxygenation for effective therapy. The goal of the present study was to determine an optimum oxygenation protocol that can be clinically applicable for treating acute MI. Using EPR oximetry, we studied the effect of exposure to supplemental oxygen cycling (OxCy) administered by inhalation of 21-100% oxygen for brief periods (15-90 min), daily for 5 days, using a rat model of acute MI. Myocardial oxygen tension (pO2), cardiac function and pro-survival/apoptotic signaling molecules were used as markers of treatment outcome. OxCy resulted in a significant reduction of infarct size and improvement of cardiac function. An optimal condition of 30-min OxCy with 95% oxygen + 5% CO2 under normobaric conditions was found to be effective for cardioprotection.

Dysregulation of PTEN in cardiopulmonary vascular remodeling induced by pulmonary hypertension.

Pulmonary hypertension (PH) is a disorder of lung vasculature characterized by arterial narrowing. Phosphatase-and-tensin homolog on chromosome 10 (PTEN), associated in the progression of multiple cancers, is implicated in arterial remodeling. However, the involvement of PTEN in PH remains unclear. The objective of the present study was to determine the role of PTEN in pulmonary vascular remodeling using established models of PH. The study used rat models of PH, induced by monocrotaline (MCT) administration (60 mg/kg) or continuous hypoxic exposure (10% oxygen) for 3 weeks. Pulmonary artery smooth muscle cells (SMCs) were used for in vitro confirmation. Development of PH was verified by hemodynamic, morphological and histopathology analyses. PTEN and key downstream proteins in pulmonary and cardiac tissues were analyzed by western blotting and RT-PCR. PTEN was significantly decreased (MCT, 53%; Hypoxia, 40%), pAkt was significantly increased (MCT, 42%; Hypoxia, 55%) in tissues of rats with PH. Similar results were observed in SMCs exposed to hypoxia (1% oxygen) for 48 h. Ubiquitination assay showed that PTEN degradation occurs via proteasomal degradation pathway. Western blotting demonstrated a significant downregulation of cell-cycle regulatory proteins p53 and p27, and upregulation of cyclin-D1 in the lungs of both models. The results showed that PTEN-mediated modulation of PI3K pathway was independent of the focal adhesion kinase and fatty acid synthase. The study, for the first time, established that PTEN plays a key role in the progression of pulmonary hypertension. The findings may have potential for the treatment of pulmonary hypertension using PTEN as a target.

Pulmonary hypertension secondary to left-heart failure involves peroxynitrite-induced downregulation of PTEN in the lung.

Pulmonary hypertension (PH) that occurs after left-heart failure (LHF), classified as Group 2 PH, involves progressive pulmonary vascular remodeling induced by smooth muscle cell (SMC) proliferation. However, mechanisms involved in the activation of SMCs remain unknown. The objective of this study was to determine the involvement of peroxynitrite and phosphatase-and-tensin homolog on chromosome 10 (PTEN) in vascular SMC proliferation and remodeling in the LHF-induced PH (LHF-PH). LHF was induced by permanent ligation of left anterior descending coronary artery in rats for 4 weeks. MRI, ultrasound, and hemodynamic measurements were performed to confirm LHF and PH. Histopathology, Western blot, and real-time polymerase chain reaction analyses were used to identify key molecular signatures. Therapeutic intervention was demonstrated using an antiproliferative compound, HO-3867. LHF-PH was confirmed by significant elevation of pulmonary artery pressure (mean pulmonary artery pressure/mm Hg: 35.9±1.8 versus 14.8±2.0, control; P<0.001) and vascular remodeling. HO-3867 treatment decreased mean pulmonary artery pressure to 22.6±0.8 mm Hg (P<0.001). Substantially higher levels of peroxynitrite and significant loss of PTEN expression were observed in the lungs of LHF rats when compared with control. In vitro studies using human pulmonary artery SMCs implicated peroxynitrite-mediated downregulation of PTEN expression as a key mechanism of SMC proliferation. The results further established that HO-3867 attenuated LHF-PH by decreasing oxidative stress and increasing PTEN expression in the lung. In conclusion, peroxynitrite and peroxynitrite-mediated PTEN inactivation seem to be key mediators of lung microvascular remodeling associated with PH secondary to LHF.

Oxygen cycling in conjunction with stem cell transplantation induces NOS3 expression leading to attenuation of fibrosis and improved cardiac function.

AIMS: Myocardial infarction (MI) is associated with irreversible loss of viable cardiomyocytes. Cell therapy is a potential option to replace the lost cardiomyocytes and restore cardiac function. However, cell therapy is faced with a number of challenges, including survival of the transplanted cells in the infarct region, which is characterized by abundant levels of oxidants and lack of a pro-survival support mechanism. The goal of the present study was to evaluate the effect of supplemental oxygenation on cell engraftment and functional recovery in a rat model. METHODS AND RESULTS: MI was induced in rats by a 60-min occlusion of the coronary artery, followed by restoration of flow. Mesenchymal stem cells (MSCs), isolated from adult rat bone marrow, were transplanted in the MI region. Rats with transplanted MSCs were exposed to hyperbaric oxygen (HBO: 100% O(2), 2 atmospheres absolute) for 90 min, 5 days/week for 4 weeks. The experimental groups were: MI (control), Ox (MI + HBO), MSC (MI + MSC), and MSC + Ox (MI + MSC + HBO). HBO exposure (oxygenation) was started 3 days after induction of MI. MSCs were transplanted 1 week after induction of MI. Echocardiography showed a significant recovery of cardiac function in the MSC + Ox group, when compared with the MI or MSC group. Oxygenation increased the engraftment of MSCs and vascular density in the infarct region. Molecular analysis of infarct tissue showed a four-fold increase in NOS3 expression in the MSC + Ox group compared with the MI group. CONCLUSIONS: The results showed that post-MI exposure of rats to daily cycles of hyperoxygenation (oxygen cycling) improved stem cell engraftment, cardiac function, and increased NOS3 expression.

Chronic paracetamol treatment influences indices of reactive oxygen species accumulation in the aging Fischer 344 X Brown Norway rat aorta.

Previous reports have demonstrated that increased levels of reactive oxygen species (ROS) and alterations in cell signaling characterize aging in the Fischer 344 X Brown Norway (FBN) rat aorta. Other work has suggested that increases in ROS may be related to vascular wall thickening and the development of hypertension. Paracetamol (acetaminophen) is a potent antioxidant that has been found to diminish free radicals in ischemia-reperfusion studies. However, it remains unclear whether chronic paracetamol administration influences signaling or ROS accumulation in the aging aorta. FBN rats (27 months old; n=8) were subjected to 6 months of treatment with a therapeutic dose of paracetamol (30 mg/kg/day) and compared to age-matched untreated FBN rat controls (n=8). Compared to measurements in the aortae of 6-month old animals, tunica media thickness, tissue superoxide levels, and protein oxidation levels were 38 ± 7%, 92 ± 31%, and 7 ± 2% higher in the aortae of 33-month control animals (p ≤0.05). Chronic paracetamol treatment decreased tunica media thickness and the amount of oxidized protein by 13 ± 4% and 30 ± 1%, respectively (p ≤0.05). This finding of diminished aortic thickening was associated with increased phosphorylation (activation) of the mitogen activated protein kinases and diminished levels of the anti-apoptotic protein Bcl-2. Taken together, these data suggest that chronic paracetamol treatment may decrease the deleterious effects of aging in the FBN rat aorta.

Cellular uptake, retention and bioabsorption of HO-3867, a fluorinated curcumin analog with potential antitumor properties.

Curcumin, a naturally-occurring compound found in the rhizome of Curcuma longa plant, is known for its antitumor activities. However, its clinical efficacy is limited due to poor bioabsorption. A new class of synthetic analogs of curcumin, namely diarylidenylpiperidone (DAP), has been developed with substantially higher anticancer activity than curcumin. However, its cellular uptake and bioabsorption have not been evaluated. In this study we have determined the absorption of a representative DAP compound, HO-3867, using optical and electron paramagnetic resonance spectrometry. The cellular uptake of HO-3867 was measured in a variety of cancer cell lines. HO-3867 was taken in cells within 15 minutes of exposure and its uptake was more than 100-fold higher than curcumin. HO-3867 was also retained in cells in an active form for 72 hours and possibly longer. HO-3867 was substantially cytotoxic to all the cancer cells tested. However, there was no direct correlation between cellular uptake and cytotoxicity suggesting that the cytotoxic mechanisms could be cell-type specific. When administered to rats by intraperitoneal injection, significantly high levels of HO-3867 were found in the liver, kidney, stomach, and blood after 3 hours. Also, significant accumulation of HO-3867 was found in murine tumor xenografts with a dose-dependent inhibition of tumor growth. The results suggest that the curcumin analog has substantially higher bioabsorption when compared to curcumin.

Lean and obese Zucker rats exhibit different patterns of p70s6 kinase regulation in the tibialis anterior muscle in response to high-force muscle contraction.

Increased phosphorylation of the 70-kDa ribosomal S6 kinase (p70S6k) signaling is strongly correlated with the degree of muscle adaptation following exercise. Herein we compare the phosphorylation of p70S6k, Akt, and mammalian target of rapamycin (mTOR) in the tibialis anterior (TA) muscles of lean and obese Zucker rats following a bout of eccentric exercise. Exercise increased p70S6k (Thr389) phosphorylation immediately after (33.3+/-7.2%) and during [1 h (24.0+/-14.9%) and 3 h (24.6+/-11.3%)] recovery in the lean TA and at 3 h (33.5+/-8.0%) in the obese TA Zucker rats. mTOR (Ser2448) phosphorylation was elevated in the lean TA immediately after exercise (96.5+/-40.3%) but remained unaltered in the obese TA. Exercise increased Akt (Thr308) and Akt (Ser473) phosphorylation in the lean but not the obese TA. These results suggest that insulin resistance is associated with alterations in the ability of muscle to activate p70S6k signaling following an acute bout of exercise.