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Endothelial PD-L1 Expression as a Biomarker for Acute Rejection in Heart Transplantation
S22
Journal of Cardiac Failure Vol. 24 No. 8S August 2018
potential role of ischemia, and to identify and test novel interventions targeted to these mechanisms.
049 Endothelial PD-L1 Expression as a Biomarker for Acute Rejection in Heart Transplantation William Bracamonte-Baran1, Nisha A. Gilotra1, Jan M. Griffin1, Monica Talor1, David Nauen1, Kavita Sharma1, Ilan S. Wittstein1, Marc Halushka1, Stuart D. Russell2, Daniela CIhakova1; 1Johns Hopkins University, Baltimore, MD; 2Duke University, Durham, NC Background: Heart transplantation is the only definitive therapeutic intervention for end-stage heart failure. The complex mechanisms regulating the alloresponse are not entirely understood. PD-L1 is a co-inhibitory molecule able to induce T cell anergy. We hypothesize that endothelial PD-L1 modulates alloresponses in heart transplantation. Methods: Using multiparameter flow cytometry we characterized stromal cells and leukocytes in endomyocardial biopsies (EMB) and peripheral blood of patients post orthotopic heart transplantation (n = 21) ISHLT grade 0-1R. Controls were autopsy specimens without cardiac pathologies. Results: Most of the patients (n = 17) had decreased endothelial PD-L1 expression as compared to controls, 2 patients had preserved endothelial PD-L1, and 2 had over-expression. PD-L1 expression was independent of the time from transplant, immunosuppressive regimen and concentration of calcineurin inhibitor. The percentage of infiltrating CD8+ lymphocytes had an inverse exponential correlation with endothelial PD-L1 expression (r2 = 0.64, Fig 1A). We categorized patients in 2 groups: those with previous ISHLT grade 2R cellular rejection (n = 10) and those with stable 0-1R scores since transplantation. A Receiver Operating Characteristic (ROC) analysis showed that the proportion of PD-L1+ endothelial cells could significantly detect history of 2R rejection, as 16.25% PD-L1+ endothelial cells retrospectively discriminated patients with history of 2R rejection episodes with 90% sensitivity, 78% specificity and 3.3 likelihood ratio (Fig 1B). Furthermore, the proportion of exhausted CD8 T cells in peripheral blood (CD45RAnegPD1+) was proportional to heart endothelial PDL1 expression (p<0.05, r2 = 0.45) and inverse exponentially correlated with graft infiltrating CD8 T cells (r2 = 0.52). Conclusions: Critical loss of graft endothelial PD-L1 is strongly associated with history of moderate-severe acute rejection episodes. These findings suggest that endothelial PD-L1 and its peripheral correlate, exhausted CD8 T cells, might become a useful biomarker in heart transplantation that could allow profiling of patients and personalize therapeutic and follow-up strategies.
050 Chronic Therapy with Sacubitril/Valsartan Reduces Plasma Troponin-I Level in Dogs with Experimentally-Induced Cardiorenal Syndrome Hani N. Sabbah, Ramesh C. Gupta, Kefei Zhang, Vinita Singh-Gupta, Jiang Xu; Henry Ford Hospital, Detroit, MI Introduction: Ongoing loss of cardiomyocytes occurs in heart failure (HF) and contributes to progressive worsening of the HF state. Sacubitril/Valsartan (S/Val), a combined angiotensin-II receptor blocker (Val) and neprilysin inhibitor (S) in a 1:1 molar ratio, was shown to reduce the risk of cardiovascular death or HF hospitalization in patients with HF and reduced left ventricular (LV) ejection fraction (EF). Chronic therapy with S/VAL was also shown to improve LV systolic function in dogs with experimentally-induced cardiorenal syndrome. Objective: This study tested the hypothesis that chronic therapy with S/Val in dogs with cardiorenal syndrome ameliorates ongoing loss of cardiomyocytes as evidenced by lowering plasma levels of troponin-I (TnI). Methods: 14 dogs with coronary microembolization-induced HF and renal dysfunction induced by unilateral nephrectomy with imposed stenosis of the contralateral renal vein, were randomized to 3 months therapy with S/Val (100 mg once daily, n = 7) or no therapy (control, CON, n = 7). LV EF was measured before (PRE) and at 12 weeks after initiating therapy with S/Val (POST). High sensitivity plasma TnI was measured when animals were normal (NL) and again after induction of cardiorenal syndrome and subsequently at 2, 4, and 12 weeks after initiating S/Val therapy. TnI levels were determined using a commercially available Ultra-Sensitive Dog Cardiac Troponin-I Elisa kit and the concentration was expressed in ng/ml. Results: In CON dogs, LV EF was unchanged from PRE to POST (35 § 1 vs. 36 § 1 %) but increased significantly from PRE to POST in S/Val-treated dogs (34 § 1 vs. 41 § 2 %, p<0.05). In CON dogs, TnI levels increased significantly after induction of cardiorenal syndrome (Table) and remained elevated throughout the follow-up period. In contrast, in S/Val-treated dogs, TnI levels decreased gradually during the therapy period (Table). Conclusions: In dogs with cardiorenal syndrome, S/Val reduces plasma TnI levels compared to untreated CON. This observations suggests that chronic therapy with S/Val attenuates or prevents ongoing loss cardiomyocytes, a characteristic feature of the HF state. Table:. Plasma High Sensitivity Troponin-I (ng/ml)
CON S/Val
NL
Cardiorenal Syndrome
2 weeks
4 weeks
12 weeks
0.12 § 0.01 0.12 § 0.01
0.35 § 0.03* 0.43 § 0.03*
0.38 § 0.04* 0.45 § 0.04*
0.38 § 0.03* 0.27 § 0.02*y
0.38 § 0.03* 0.27 § 0.01*y
* = p<0.05 vs. NL y = p<0.05 vs. Cardiorenal Syndrome
051 Gastrointestinal Bleeding is Associated with a Shift Towards a Pro-Angiogenic Phenotype after Continuous Flow Left Ventricular Assist Device Implantation Adam L. Edwards, C. Mel Wilcox, Salpy V. Pamboukian, Peter Mannon, Shajan Peter; University of Alabama at Birmingham, Birmingham, AL Introduction: Gastrointestinal bleeding (GIB) occurs in up to 20% of continuousflow left ventricular assist device (CF-LVAD) recipients. A higher risk of GIB has been observed in CF-LVAD recipients with lower device pulsatility, yet a mechanism linking changes in hemodynamics to increased GIB is lacking. Hypothesis: We hypothesized that 1) levels of Ang-1, a growth factor with vascular protective effects, would be lower while levels of vascular endothelial growth factor A (VEGF-A), an inducer of angiogenesis, and Ang-2, a promoter of vascular destabilization, would be higher in (a) CF-LVAD recipients compared to controls and (b) in CF-LVAD recipients with GIB as compared to those without; and that 2) CF-LVAD pulsatility and pulse pressure would be positively correlated with levels of Ang-1 and negatively correlated with VEGF-A and Ang-2. Methods: Twenty-four participants from our institution, 12 with CF-LVADs (6 of whom had GIB after LVAD implantation) and 12 age-matched controls with no history of heart failure or GIB, contributed venous blood samples. We measured VEGF-A, Ang-2, and Ang-1 levels in serum fractions with immunoassays. Results: Ang-2 levels were higher and Ang-1 levels lower in CF-LVAD recipients compared to controls with no difference in VEGF-A levels. Ang-1 levels were lower in CF-LVAD recipients with GIB compared to those without; Ang-2 and VEGF-A levels did not differ between the two groups. There was no correlation between pulsatility index and Ang-2, Ang-1, or VEGF-A levels. Pulse pressure was negatively correlated with Ang-2 levels (r = ¡0.578, P = 0.003), positively correlated with Ang-1 levels (r = 0.496, P = 0.014), and not correlated with VEGF-A levels. Conclusions: CF-LVAD recipients demonstrate a shift toward a pro-angiogenic phenotype in the angiopoietin pathway, with increased levels of Ang-2 and lower levels of Ang-1 as compared to controls. Lower levels of Ang-1 were associated with GIB following CF-LVAD implantation. Lastly, lower pulse pressure was associated with higher Ang-2 levels and lower Ang-1 levels. These results suggest a contribution of altered angiogenesis to the observed increased risk of GIB in this population and a potential link to altered hemodynamics.
Journal of Cardiac Failure Vol. 24 No. 8S August 2018
potential role of ischemia, and to identify and test novel interventions targeted to these mechanisms.
049 Endothelial PD-L1 Expression as a Biomarker for Acute Rejection in Heart Transplantation William Bracamonte-Baran1, Nisha A. Gilotra1, Jan M. Griffin1, Monica Talor1, David Nauen1, Kavita Sharma1, Ilan S. Wittstein1, Marc Halushka1, Stuart D. Russell2, Daniela CIhakova1; 1Johns Hopkins University, Baltimore, MD; 2Duke University, Durham, NC Background: Heart transplantation is the only definitive therapeutic intervention for end-stage heart failure. The complex mechanisms regulating the alloresponse are not entirely understood. PD-L1 is a co-inhibitory molecule able to induce T cell anergy. We hypothesize that endothelial PD-L1 modulates alloresponses in heart transplantation. Methods: Using multiparameter flow cytometry we characterized stromal cells and leukocytes in endomyocardial biopsies (EMB) and peripheral blood of patients post orthotopic heart transplantation (n = 21) ISHLT grade 0-1R. Controls were autopsy specimens without cardiac pathologies. Results: Most of the patients (n = 17) had decreased endothelial PD-L1 expression as compared to controls, 2 patients had preserved endothelial PD-L1, and 2 had over-expression. PD-L1 expression was independent of the time from transplant, immunosuppressive regimen and concentration of calcineurin inhibitor. The percentage of infiltrating CD8+ lymphocytes had an inverse exponential correlation with endothelial PD-L1 expression (r2 = 0.64, Fig 1A). We categorized patients in 2 groups: those with previous ISHLT grade 2R cellular rejection (n = 10) and those with stable 0-1R scores since transplantation. A Receiver Operating Characteristic (ROC) analysis showed that the proportion of PD-L1+ endothelial cells could significantly detect history of 2R rejection, as 16.25% PD-L1+ endothelial cells retrospectively discriminated patients with history of 2R rejection episodes with 90% sensitivity, 78% specificity and 3.3 likelihood ratio (Fig 1B). Furthermore, the proportion of exhausted CD8 T cells in peripheral blood (CD45RAnegPD1+) was proportional to heart endothelial PDL1 expression (p<0.05, r2 = 0.45) and inverse exponentially correlated with graft infiltrating CD8 T cells (r2 = 0.52). Conclusions: Critical loss of graft endothelial PD-L1 is strongly associated with history of moderate-severe acute rejection episodes. These findings suggest that endothelial PD-L1 and its peripheral correlate, exhausted CD8 T cells, might become a useful biomarker in heart transplantation that could allow profiling of patients and personalize therapeutic and follow-up strategies.
050 Chronic Therapy with Sacubitril/Valsartan Reduces Plasma Troponin-I Level in Dogs with Experimentally-Induced Cardiorenal Syndrome Hani N. Sabbah, Ramesh C. Gupta, Kefei Zhang, Vinita Singh-Gupta, Jiang Xu; Henry Ford Hospital, Detroit, MI Introduction: Ongoing loss of cardiomyocytes occurs in heart failure (HF) and contributes to progressive worsening of the HF state. Sacubitril/Valsartan (S/Val), a combined angiotensin-II receptor blocker (Val) and neprilysin inhibitor (S) in a 1:1 molar ratio, was shown to reduce the risk of cardiovascular death or HF hospitalization in patients with HF and reduced left ventricular (LV) ejection fraction (EF). Chronic therapy with S/VAL was also shown to improve LV systolic function in dogs with experimentally-induced cardiorenal syndrome. Objective: This study tested the hypothesis that chronic therapy with S/Val in dogs with cardiorenal syndrome ameliorates ongoing loss of cardiomyocytes as evidenced by lowering plasma levels of troponin-I (TnI). Methods: 14 dogs with coronary microembolization-induced HF and renal dysfunction induced by unilateral nephrectomy with imposed stenosis of the contralateral renal vein, were randomized to 3 months therapy with S/Val (100 mg once daily, n = 7) or no therapy (control, CON, n = 7). LV EF was measured before (PRE) and at 12 weeks after initiating therapy with S/Val (POST). High sensitivity plasma TnI was measured when animals were normal (NL) and again after induction of cardiorenal syndrome and subsequently at 2, 4, and 12 weeks after initiating S/Val therapy. TnI levels were determined using a commercially available Ultra-Sensitive Dog Cardiac Troponin-I Elisa kit and the concentration was expressed in ng/ml. Results: In CON dogs, LV EF was unchanged from PRE to POST (35 § 1 vs. 36 § 1 %) but increased significantly from PRE to POST in S/Val-treated dogs (34 § 1 vs. 41 § 2 %, p<0.05). In CON dogs, TnI levels increased significantly after induction of cardiorenal syndrome (Table) and remained elevated throughout the follow-up period. In contrast, in S/Val-treated dogs, TnI levels decreased gradually during the therapy period (Table). Conclusions: In dogs with cardiorenal syndrome, S/Val reduces plasma TnI levels compared to untreated CON. This observations suggests that chronic therapy with S/Val attenuates or prevents ongoing loss cardiomyocytes, a characteristic feature of the HF state. Table:. Plasma High Sensitivity Troponin-I (ng/ml)
CON S/Val
NL
Cardiorenal Syndrome
2 weeks
4 weeks
12 weeks
0.12 § 0.01 0.12 § 0.01
0.35 § 0.03* 0.43 § 0.03*
0.38 § 0.04* 0.45 § 0.04*
0.38 § 0.03* 0.27 § 0.02*y
0.38 § 0.03* 0.27 § 0.01*y
* = p<0.05 vs. NL y = p<0.05 vs. Cardiorenal Syndrome
051 Gastrointestinal Bleeding is Associated with a Shift Towards a Pro-Angiogenic Phenotype after Continuous Flow Left Ventricular Assist Device Implantation Adam L. Edwards, C. Mel Wilcox, Salpy V. Pamboukian, Peter Mannon, Shajan Peter; University of Alabama at Birmingham, Birmingham, AL Introduction: Gastrointestinal bleeding (GIB) occurs in up to 20% of continuousflow left ventricular assist device (CF-LVAD) recipients. A higher risk of GIB has been observed in CF-LVAD recipients with lower device pulsatility, yet a mechanism linking changes in hemodynamics to increased GIB is lacking. Hypothesis: We hypothesized that 1) levels of Ang-1, a growth factor with vascular protective effects, would be lower while levels of vascular endothelial growth factor A (VEGF-A), an inducer of angiogenesis, and Ang-2, a promoter of vascular destabilization, would be higher in (a) CF-LVAD recipients compared to controls and (b) in CF-LVAD recipients with GIB as compared to those without; and that 2) CF-LVAD pulsatility and pulse pressure would be positively correlated with levels of Ang-1 and negatively correlated with VEGF-A and Ang-2. Methods: Twenty-four participants from our institution, 12 with CF-LVADs (6 of whom had GIB after LVAD implantation) and 12 age-matched controls with no history of heart failure or GIB, contributed venous blood samples. We measured VEGF-A, Ang-2, and Ang-1 levels in serum fractions with immunoassays. Results: Ang-2 levels were higher and Ang-1 levels lower in CF-LVAD recipients compared to controls with no difference in VEGF-A levels. Ang-1 levels were lower in CF-LVAD recipients with GIB compared to those without; Ang-2 and VEGF-A levels did not differ between the two groups. There was no correlation between pulsatility index and Ang-2, Ang-1, or VEGF-A levels. Pulse pressure was negatively correlated with Ang-2 levels (r = ¡0.578, P = 0.003), positively correlated with Ang-1 levels (r = 0.496, P = 0.014), and not correlated with VEGF-A levels. Conclusions: CF-LVAD recipients demonstrate a shift toward a pro-angiogenic phenotype in the angiopoietin pathway, with increased levels of Ang-2 and lower levels of Ang-1 as compared to controls. Lower levels of Ang-1 were associated with GIB following CF-LVAD implantation. Lastly, lower pulse pressure was associated with higher Ang-2 levels and lower Ang-1 levels. These results suggest a contribution of altered angiogenesis to the observed increased risk of GIB in this population and a potential link to altered hemodynamics.