Geetha Muthukumaran

Concomitant Low Dose Doxorubicin Treatment and Exercise

Cardiotoxicity is a side effect for cancer patients treated with doxorubicin (DOX). We tested the hypothesis that low-intensity aerobic exercise concomitant with DOX treatment would offset DOX-induced cardiotoxicity while also improving the therapeutic efficacy of DOX on tumor progression. B16F10 melanoma cells (3 × 10(5)) were injected subcutaneously into the scruff of 6- to 8-wk-old male C57BL/6 mice (n = 48). A 4 mg/kg cumulative dose of DOX was administered over 2 wk, and exercise (EX) consisted of treadmill walking (10 m/min, 45 min/day, 5 days/wk, 2 wk). Four experimental groups were tested: 1) sedentary (SED) + vehicle, 2) SED + DOX, 3) EX + vehicle, and 4) EX + DOX. Tumor volume was attenuated in DOX and lowest in EX + DOX. DOX-treated animals had less gain in body weight, reduced heart weights (HW), smaller HW-to-body weight ratios, and shorter tibial lengths by the end of the protocol; and exercise did not reverse the cardiotoxic effects of DOX. Despite decreased left ventricular (LV) mass with DOX, cardiomyocyte cross-sectional area, β-myosin heavy chain gene expression, and whole heart systolic (fractional shortening) and diastolic (E/A ratio) function were similar among groups. DOX also resulted in increased LV fibrosis with lower LV end diastolic volume and stroke volume. Myocardial protein kinase B activity was increased with both DOX and EX treatments, and tuberous sclerosis 2 (TSC2) abundance was reduced with EX. Downstream phosphorylation of TSC2 and mammalian target of rapamycin were similar across groups. We conclude that exercise increases the efficacy of DOX in inhibiting tumor growth without mitigating subclinical DOX-induced cardiotoxicity in a murine model of melanoma.

The ubiquitin-like protein, ISG15, is a novel tumor-associated antigen for cancer immunotherapy

The recent announcement of the first FDA-approved therapeutic vaccine for prostate cancer, Sipuleucel-T, is a watershed moment for the field of tumor immunotherapy. However, while Sipuleucel-T provides a powerful tool to clinicians for the most prevalent form of cancer in men, there remains an unmet need for a similar therapeutic strategy against breast cancer, the most prevalent cancer in women. While current breast cancer vaccines in development target several antigens, the most prevalent is the tumor-associated antigen, HER2. Initial results with HER2 vaccines appear promising in terms of efficacy; however, the lack of HER2 overexpression by a majority of breast tumors and the safety concerns associated with current HER2-targeted immunotherapy suggest that additional therapeutic strategies would be beneficial. Recently, several studies have identified ISG15 as a molecule highly expressed in numerous malignancies. ISG15 is a small ubiquitin-like protein regulated by type-I interferon and classically associated with viral defense. Elevated ISG15 expression in breast cancer is especially well documented and is independent of HER2, progesterone receptor, and estrogen receptor status. Additionally, high ISG15 expression in breast cancer correlates with an unfavorable prognosis and poor responses to traditional treatment strategies such as chemotherapy and radiation. To overcome these challenges, we employ a novel strategy to specifically target tumor-associated ISG15 expression with immunotherapy. We demonstrate that vaccination against ISG15 results in significant CD8-mediated reductions in both primary and metastatic mammary tumor burden. These results validate ISG15 as a tumor-associated antigen for cancer immunotherapy.

The receptor for advanced glycation end products mediates lung endothelial activation by RBCs

The receptor for advanced glycation end products (RAGE) is a multiligand pattern recognition receptor implicated in multiple disease states. Although RAGE is expressed on systemic vascular endothelium, the expression and function of RAGE on lung endothelium has not been studied. Utilizing in vitro (human) and in vivo (mouse) models, we established the presence of RAGE on lung endothelium. Because RAGE ligands can induce the expression of RAGE and stored red blood cells express the RAGE ligand N(ε)-carboxymethyl lysine, we investigated whether red blood cell (RBC) transfusion would augment RAGE expression on endothelium utilizing a syngeneic model of RBC transfusion. RBC transfusion not only increased lung endothelial RAGE expression but enhanced lung inflammation and endothelial activation, since lung high mobility group box 1 and vascular cell adhesion molecule 1 expression was elevated following transfusion. These effects were mediated by RAGE, since endothelial activation was absent in RBC-transfused RAGE knockout mice. Thus, RAGE is inducibly expressed on lung endothelium, and one functional consequence of RBC transfusion is increased RAGE expression and endothelial activation.

Moderate-intensity treadmill exercise training decreases murine cardiomyocyte cross-sectional area.

The aim of this study was to examine the impact of moderate‐intensity treadmill exercise on the structure and function of the murine heart and its associated impact on Akt–AMPK–mTOR signaling. A secondary aim was to test whether the exercise phenotype was altered following a cardiotoxic bolus dose of doxorubicin (DOX). Two‐month‐old C57Bl/6J female mice remained sedentary (SED, n = 12) or were progressively trained with treadmill running for 2 months up to 18 m/min; 60 min/day, 5 days/weeks (EX, n = 11) or EX + DOX (15 mg/kg/dose) (EX + DOX, n = 6). Following treadmill training, mice underwent graded exercise tolerance testing and echocardiography. Training improved graded exercise tolerance by 68 ± 5% relative to SED, and this effect was not altered with bolus DOX. There were no changes in relative heart size with EX or EX + DOX versus SED. Regional posterior wall thickening was improved in EX and abrogated in EX + DOX. EX had a reduced cardiomyocyte cross‐sectional area (CSA) relative to SED, and CSA was further attenuated with DOX. Following EX, AMPK‐associated phosphorylation of ULK1(ser317) tended to be lower relative to SED. Akt‐associated phosphorylation of TSC2(thr1462) and mTOR(ser2448) were also decreased relative to SED. We observed an increase in AMPK activity with DOX that was not translated to downstream AMPK phosphorylation sites. We conclude that 2 months of moderate treadmill exercise training improves regional cardiac function and exercise capacity, but does not induce relative physiologic hypertrophy in female mice. Differential responses in Akt–AMPK–mTOR signaling may mediate the observed phenotype.

Acute aerobic exercise increases exogenously infused bone marrow cell retention in the heart

Stem cell therapy for myocardial infarction (MI) has been shown to improve cardiac function and reduce infarct size. Exercise training, in the form of cardiac rehabilitation, is an essential part of patient care post‐MI. Hence, we tested the effects of acute and chronic aerobic exercise on stem cell retention and cardiac remodeling post‐MI. Small epicardial MIs were induced in 12‐month‐old C57BL/6 mice via cryoinjury. Two weeks post‐MI, vehicle infusion (N = 4) or GFP+ bone marrow‐derived cells (BMC) were injected (tail vein I.V.) immediately after acute exercise (N = 14) or sedentary conditions (N = 14). A subset of mice continued a 5‐week intervention of chronic treadmill exercise (10–13 m/min; 45 min/day; 4 days/week; N = 7) or remained sedentary (N = 6). Exercise tolerance was assessed using a graded exercise test, and cardiac function was assessed with echocardiography. Acute exercise increased GFP+ BMC retention in the infarcted zone of the heart by 30% versus sedentary (P < 0.05). This was not associated with alterations in myocardial function or gene expression of key cell adhesion molecules. Animals treated with chronic exercise increased exercise capacity (P < 0.05) and cardiac mass (P < 0.05) without change in left ventricular ejection fraction (LVEF), infarct size, or regional wall thickness (P = NS) compared with sedentary. While BMCs alone did not affect exercise capacity, they increased LVEF (P < 0.05) and Ki67+ nuclei number in the border zone of the heart (P < 0.05), which was potentiated with chronic exercise training (P < 0.05). We conclude that acute exercise increases BMC retention in infarcted hearts and chronic training increases exogenous BMC‐mediated effects on stimulating the cardiomyocyte cell cycle. These preclinical results suggest that exercise may help to optimize stem cell therapeutics following MI.

Effects of acute angiotensin II on ischemia reperfusion injury following myocardial infarction

Myocardial infarction (MI) induces cardiac remodeling. This may increase the susceptibility of the infarcted heart to subsequent ischemic events. While chronic angiotensin II blockade is cardioprotective post-MI, the acute effects of angiotensin II in ischemia-reperfusion injury (IR) remains unclear. In the present study, we tested whether angiotensin II administration altered recovery of left ventricular (LV) function to IR in hearts from sham and MI rats. Echocardiography, LV pressure-volume relationships, and IR performance were established in subsets of sham (N = 27) and MI hearts (N = 41). IR was conducted in red-cell-perfused Langendorff hearts (60 minutes of low-flow ischemia; 30 minutes of reperfusion) during vehicle or angiotensin II infusions (10−7 M). MI hearts were dilated and had reduced fractional shortening and blunted systolic elastance (p < 0.05). Despite systolic dysfunction in MI, functional recovery to IR was similar to sham. Angiotensin II significantly worsened IR performance in sham (p < 0.05), but not MI. The effect of angiotensin II on in vitro cardiomyocyte survival under various pH conditions was also tested. Acidosis increased cardiomyocyte death and angiotensin II potentiated this effect. We conclude that IR performance is similar between sham and MI hearts and that MI hearts are resistant to angiotensin II-induced cardiac dysfunction in response to IR.

Myocardial apoptosis and mesenchymal stem cells with acute exercise

Aerobic exercise confers many health benefits. However, numerous reports have shown that acute aerobic exercise can injure the heart. We tested the general hypothesis that acute moderate‐intensity exercise in rodents induces cardiomyocyte damage and stimulates mesenchymal stem cells (MSCs) to increase paracrine‐mediated protective effects on cardiomyocytes. A single session of treadmill running (13 m/min, 0% grade, for 45 min) in untrained C57BL/6 male mice (n = 18) increased cleaved poly ADP‐ribose polymerase (PARP), a marker of apoptosis, in the myocardium 24 h postexercise. Microarray analysis of mouse myocardium identified 11 relevant apoptotic genes and several shifts in matrix remodeling transcripts over the postexercise window. Postexercise cardiomyocyte death was recapitulated in neonatal rat cardiomyocytes (NRCMs) by culturing cells in 2% plasma harvested from exercised rats. The increased cell death observed in exercise‐treated NRCMs was attenuated by β‐adrenergic blockade, but not antioxidant treatment. MSC survival, proliferation, and chemotaxis showed no significant differences between sedentary and exercise plasma conditions, despite increased IL‐6, TNF‐α, IL‐1β, and IFN‐γ secretions from MSCs treated with exercise plasma. NRCM survival was increased nearly 500% when cocultured with MSCs, but this effect was not altered under exercise plasma culture conditions. Our results suggest acute moderate‐intensity aerobic treadmill running in exercise‐naïve rodents induces temporal cardiomyocyte death due to plasma‐borne factors, namely, catecholaminergic stress. Even though exercise conditions prompt an inflammatory response in MSCs, the exercise milieu does not alter the MSC‐protective phenotype on cardiomyocytes.

Oral copper absorption in men with morbid obesity

Copper deficiency has been reported in patients with severe obesity, both before and after bariatric surgery, the latter at increased rates. However, little is known about the absorption of copper. Because of differences in copper dynamics between men and women in the limited available data, we evaluated copper absorption in men who were candidates for bariatric surgery. All patients gave informed consent, approved by the University of Pennsylvania Institutional Review Board, to participate in a pharmacokinetics study. After a 12-h fast, body composition was measured using air displacement plethysmography and a single oral dose of 4mg copper gluconate was given followed by serial measures of serum copper over 10h. Meals and snacks very low in copper were provided. The seven participants had age 52±8years, BMI 44.87±9.01kg/m2 and fat free mass 77.92±14.45kg. Baseline serum copper was 36.1±19.5μg/dL and ceruloplasmin was 50.86±4.58mg/dL. Peak copper absorption occurred within 1-2h. The AUC for six subjects was 254.6±151.3μg/100mLh. The estimated volume of distribution (1.98±0.36L) was ∼25mL/kg fat free mass. In these severely obese men, copper absorption occurred early in spite of their normal copper status, suggesting no impairment of absorption due to obesity. Further studies of copper absorption after the significant weight loss and possible malabsorption derived from bariatric surgery are needed.