Is There a Role in Routine Biomarker Screening for Cardiotoxicity in Patients Receiving Doxorubicin Chemotherapy? Pilot Observations

The 10th Annual Scientific Meeting



HFSA

S33

105

107

Treatment of Bone Marrow Mononuclear Cells with Erythropoietin and Platelet-Derived Growth Factor-bb Promotes Cellular Growth and Expression of Mesenchymal and Endothelial Cell Surface Markers In Vitro Jordan J. Lancaster1, Nicholle M. Johnson2, Mohamed A. Gaballa3, Hoang M. Thai4, Steven Goldman5; 1Cardiology, Southern Arizona VAMC, Tucson, AZ; 2Cardiology, Southern Arizona VAMC, Tucson, AZ; 3Sarver Heart Center, Tucson, AZ; 4 Cardiology, Southern Arizona VAMC, Tucson, AZ; 5Cardiology, Southern Arizona VAMC, Tucson, AZ

Is There a Role in Routine Biomarker Screening for Cardiotoxicity in Patients Receiving Doxorubicin Chemotherapy? Pilot Observations W. H. Wilson Tang1, Rohit Bhatheja2, Kevin Shrestha1, Frederick Van Lente3, G. Thomas Budd4, Brad Pohlman4, Steven Andresen4, Gary S. Francis1, David E. Weng4; 1Cardiovascular Medicine, Cleveland Clinic, Cleveland, OH; 2 Cardiovascular Medicine, Universtiy of Kentucky, Lexington, KY; 3Clinical Pathology, Cleveland Clinic, Cleveland, OH; 4Hematology and Oncology, Cleveland Clinic, Cleveland, OH

Objective: Autologous bone marrow cells are being used in clinical trials for cellbased therapy after myocardial infarction. We developed an in vitro assay to determine if growth factor stimulation would promote specific stem cell growth prior to infusion into a recipient. Methods: Bone marrow mononuclear cells (BMMNCs) are harvested from 5-6 week old male Sprague-Dawley rat femurs. Contents are flushed with sterile 10% FBS in DMEM-LG. Bone spicules and clots are removed; the cell suspension is centrifuged. Cells are washed in red blood cell lysis buffer, centrifuged, and washed twice in PBS before plating 1105 cells per well in 24 well plates. Each well is exposed to one of seven of the following growth factor combinations (10 ng/ml): 1) Control (10% FBSþDMEM-LG), 2) platelet-derived growth factor-BB (PDGF-BB), 3) epidermal growth factor (EGF), 4) granulocyte colony stimulation factor (G-CSF), 5) erythropoietin (EPO), 6) EPOþG-CSF, or 7) EPO ’PDGF-BB. Media is changed daily for ten days, a sample is removed and the cells counted to calculate total number of cells. This process is repeated in a separate group of BMMNCs grown to confluency. At passage three, immunohistochemistry is used to detect the presence of CD34, CD44, CD45, CD90, VE-Cad, and PanECA. Results: Growth factor stimulation produced a transient increase in the number of cultured BMMNCs from day one to day five, and decreased thereafter to day 10. At day five EPO treated cells had an increase (P!0.05) in number compared to all other groups: (1) 295,500 6 27,081 (n56), (2) 227,250 6 7,663 (n54), (3) 225,333 6 30,492 (n53), (4) 303,000 6 31,815 (n53), (5) 795,666 6 78,846 (n54), (6) 712,333 6 88,214 (n53), (7) 913,000 6 35,355 (n54). Cells grown in EPOþPDGF-BB demonstrated expression of mesenchymal (CD44þCD90þCD45
) and endothelial (CD34þVE-Cadþ) cells markers. Conclusion: Erythropoietin in combination with PDGF-BB stimulates BMMNC growth 3 fold over no treatment, and expresses mesenchymal and endothelial cell markers. These data suggest that EPOþPDGF-BB could be explored as growth factor treatment in patients undergoing cell-base therapy after myocardial infarction.

Background: Doxorubicin-induced cardiotoxicity is a serious complication in cancer therapy. Recent studies have demonstrated the association between elevated cardiac biomarkers with the development of cardiac dysfunction following chemotherapy, but biomarker screening has not been tested prospectively. Methods: We prospectively evaluated a total of 62 consecutive subjects (LVEF $50%) undergoing firstline doxorubicin-based regimen for breast cancer, sarcoma, or lymphoma ($ 50 mg/m2 IV bolus per cycle), with planned therapy to administer total doxorubicin dose $240 mg/m2. Plasma N-terminal proBNP (NT-proBNP), cardiac troponin T (cTnT), ischemia modified albumin (IMA), and high-sensitivity C-reactive protein (hsCRP) were measured at baseline and prior to each dose of chemotherapy during the course of the regimen. Results: In our study cohort, 16 subjects (26%) demonstrated elevated plasma NT-proBNP levels (O125 mg/dL) and 5 subjects (8%) showed elevated plasma hsCRP (O3.0 mg/L) at baseline. Two out of 62 subjects (3.2%) developed cardiac dysfunction (LVEF!50% and dropped O10%) following chemotherapy, but their baseline cardiac biomarkers were not elevated (NTproBNP 5 56 and 65 pg/mL respectively). In those with sequential biomarker testing (n 5 41), baseline biomarkers did not differ between those who experienced reduction versus increase in NT-proBNP (see Table). Also, there was a lack of correlation between changes in LVEF and changes in plasma NT-proBNP (r 5 -0.04, p 5 0.83). Furthermore, there were no significant changes in plasma levels of cTnT and IMA levels throughout the regimen. Conclusion: Data from our pilot study do not support routine biomarker screening to predict subsequent development of doxorubicin-induced cardiotoxicity.

Baseline

NT-proBNP increase (n 5 14)

NT-proBNP reduction (n 5 27)

NT-proBNP (pg/dL) cTnT (ng/mL) IMA (U/mL) hsCRP (mg/L) LVEF (%)

121 þ/
72 0.01 þ/
0.005 101 þ/
10 0.5 þ/
0.5 58 þ/
3

108 þ/
105 0.01 þ/
0.008 102 þ/
13 0.45 þ/
0.5 57 þ/
3

p value 0.64 0.63 0.89 0.77 0.71

106 T-Lymphocyte Reconstitution in SCID Mice: Effect on Cardiac ECM Douglas F. Larson1, Katherine Horak1, Rajagopalan Venkataramani1, Qianli Yu1; 1 Sarver Heart Center, The University of Arizona, Tucson, AZ Background: A major determinant of the ventricular stiffness during diastolic filling is the cardiac fibrillar collagen network - the extracellular matrix (ECM). Our previous reports have suggested that there is a relationship between the lymphocyte immune function and ECM collagen structure. This study assessed the effect of T-lymphocyte reconstitution in the SCID mouse on cardiac function and ECM composition. Objective: We hypothesized that adoptive transfer of donor T-lymphocytes into SCID mice will cause ECM remodeling toward that of the donor mice. Methods: T-lymphocytes were harvested from male WT C57BL/6 mice, purified, and adoptively transferred into at a dose of 8X106 T-cells/mouse, i.p. into male C57BL SCID mice. After 30 days the hemodynamic function was determined by ECHO and conductance catheter analysis and the cardiac tissue analyzed for collagen content and composition. The three experiment groups were the donor C57 WT (WT), C57 SCID vehicle placebo (SCID), and the T-lymphocyte knock-in C57 SCID (KI). Results: The ECHO demonstrated that the SCID were significantly different compared with the WT and KI with an increased diastolic volume, peak E, E deceleration time, slope of E deceleration, septal Ea/Aa ratio (P!0.05) and a decreased E deceleration time (P50.01). Conductance catheter analysis demonstrated that the ventricular stiffness was 0.080 6 0.008 in the SCID compared with 0.116 6 0.011 in the WT (P50.005) and 0.115 6 0.011mm Hg/mL in the KI (P 5 0.012). The total cardiac collagen did not significantly differ among the groups however the percentage of collagen crosslinking was 33 6 1% in the SCID compared with 57 6 5% in the WT (P 5 0.013) and 60 6 5% in the KI (P 5 0.009). The collagen cross-linking data are supported by ventricular lysyl oxidase enzymatic activities where the SCID was 1.1 6 0.05 compared with 1.6 6 0.05 (P 5 0.002) in the WT and 1.7 6 0.12 mM H2O2/30 minutes in the KI (P 5 0.009). Conclusions: There were significant differences in cardiac function and ECM composition between the WT and SCID but not between the WT and the KI. Reconstitution of T-lymphocytes in the SCID affected cardiac collagen crosslinking and thereby the left ventricular diastolic function that matched that of the immunologically intact WT mice. Therefore, these data underscore the potential importance of T-lymphocytes in the maintenance of the ECM function and suggest that immunomodulation of the lymphocyte may provide a novel pathway for modulation of diastolic heart failure and dilated cardiomyopathy.

108 Adoptive Transfer of Hypertension with CD4D Lymphocytes Katherine Horak1, Qianli Yu1, Rajagopalan Venkataramani1, Douglas F. Larson1; 1 Sarver Heart Center, The University of Arizona, Tucson, AZ Background: Arterial hypertension (HT) affects 50% of individuals over the age of 60 and is a leading risk factor for the development of diastolic heart failure. However in 90% of HT individuals the cause is not known. Objective: We hypothesized that a contributing factor in primary HT and the accompanying cardiac remodeling is alteration in the CD4þ lymphocyte function. Methods: Twelve female C57BL/6 mice were treated with L-nitro-arginine methyl ester (L-NAME) at a dose of 12.5 mg/L in the drinking water and a diet containing 8% NaCl (L-NAME-NaCl) to induce hypertension. The corresponding controls were fed both normal diet and untreated water. After 30 days of treatment, in-vivo hemodynamics were measured with tail cuff, ECHO, and conductance catheter and the splenic CD4þ lymphocytes were harvested and purified. The CD4þ were injected 8x106/mouse,i.p. into naive female C57 SCID mice. After thirty days the hemodynamic parameters were measured and cardiac tissues harvested. The four treatment groups were: control donor (CD), L-NAME NaCl treated donor (LD), control lymphocyte recipient (CR), and L-NAME-NaCl lymphocyte recipient (LR). Results: The tail cuff blood pressure increased from 133 6 2 to 189 6 3 mmHg when comparing CD with LD respectively (P 5 0.0003). Moreover in the adoptive transferred mice, the blood pressure increased from 134 6 1 to 178 6 1 mmHg when comparing CR with LR respectively (P!0.0001). The vascular stiffness, Ea, increased from 3.08 6 in the CR to 3.75 mmHg/mL in the LR (P 5 0.02). Comparing the recipient CR with the LR, the ventricular-end systolic volume decreased from 5.8 6 0.41 to 4.4 6 0.5 mL (P 5 0.035), the ventricular-end diastolic volume 18.2 6 0.7 to 16.5 6 0.5 mL (P 5 0.03). The pre-load recruitable stroke work (PRSW) increased from 90 6 1 to 109 6 2 mmHg when comparing CR with LR (P 5 0.003). The total cardiac collagen did not significantly differ among the groups however the percentage of crosslinked collagen increased from 46.7% in the CR to 78.5% LR (P 5 0.015). Conclusion: This study demonstrated that hypertension can be induced in a naive SCID mouse by adoptively transferring T-lymphocytes from a hypertensive mouse. These data suggest support the contention that the T-lymphocytes play a fundamental role in the development of hypertension and represent a possible novel therapeutic target for the treatment to hypertension.