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cGMP Activator with Enhanced Potency and High Selectivity In Vitro
The 20th Annual Scientific Meeting
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HFSA
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JNCNI Basic Science Award 001 Neutralizing Endoglin Activity Rescues Maladaptive Remodeling in Heart Failure Kevin Morine, Xiaoying Qiao, Samuel York, Mark Aronovitz, Richard Karas, Navin Kapur; Tufts Medical Center, Boston, MA Endoglin is a trans-membrane co-receptor that promotes signaling via the profibrogenic cytokine transforming growth factor beta 1 (TGFb1) in cardiac fibroblasts. We previously reported that reduced endoglin activity prevents cardiac fibrosis in pressureoverload induced heart failure (HF). We now tested the hypothesis that neutralizing endoglin activity rescues established cardiac fibrosis and improves cardiac function in HF. Adult male C57/Bl6 mice (n = 30) underwent trans-aortic constriction (TAC). After 4 weeks of TAC, survivors (n = 11) were randomized to twice weekly intraperitoneal injections of a neutralizing anti-endoglin antibody (N-Eng-Ab; n = 6) or isotype control antibody (IgG; n = 5) at 15 mg/kg/dose. Sham-operated mice (Sham; n = 5) served as controls. After 4 additional weeks of TAC, IgG mice exhibited reduced body weight, increased lung and LV mass (P < .05 for each comparison). In contrast to IgG mice, N-Eng-Ab mice exhibited increased body weight, decreased lung and LV mass. N-EngAb mice had higher peak LV systolic pressure, lower end diastolic pressure and increased dP/dT max (P < .05 for each comparison). Picrosirius staining for LV fibrosis, LV mRNA levels of Types 1 and 3 collagen, and phosphorylation of Smad-3, a downstream effector of TGFb1 signaling, were attenuated with N-Eng-Ab not IgG treatment after TAC (Figure; P < .05 for each comparison). Myocardial capillary density, defined by histologic staining for CD31+ cells normalized to cardiomyocyte area, was increased after N-Eng Ab treatment and correlated inversely with LV fibrosis (R = 0.678, P = .01). Cardiomyocyte area and LV mRNA levels of BNP, b-MHC were increased similarly, while SERCA levels were reduced between groups. To explore the mechanism of fibrotic rescue, we identified that treatment with N-Eng-Ab reduced the ratio of circulating Type 1 collagen synthesis:degradation byproducts [procollagen type 1 (PIP) : carboxyterminal-telopeptide of Type I collagen (ICTP) ratio] (Figure). Serum PIP:ICTP ratios correlated directly with LV fibrosis (R = 0.811, P < .01) and inversely with myocardial capillary density (R = −0.745, P < .01). This is the first report to establish that neutralizing endoglin activity rescues maladaptive remodeling in HF by reversing established cardiac fibrosis, preserving myocardial capillary density, and improving cardiac function in HF. We further report the novel use of circulating biomarkers of collagen turnover as a marker of therapeutic efficacy for N-Eng-Ab treatment in mice. Endoglin targeted therapeutics may be a novel disease modifying strategy to improve the lives of patients suffering with heart failure.
002 Apolipoprotein M Improves Survival in Congestive Heart Failure via Enhanced Akt Signaling Ali Javaheri 1 , Swapnil Shewale 2 , Cecilia Frej 3 , Mingxia Liu 4 , Ken Bedi 2 , Jeff Brandimarto2, Anbin Mu2, Tao Wang2, Christina Y. Chen2, Xiucui Ma1, Payman Zamani2, Benjamin French2, Kenneth B. Margulies2, Thomas P. Cappola2, Benjamin L. Prosser2, Abhinav Diwan1, Björn Dahlbäck5, John S. Parks4, Daniel J. Rader2; 1Washington University of St. Louis, St. Louis, Missouri; 2University of Pennsylvania, Philadelphia, Pennsylvania; 3Department of Translational Medicine, Division of Clinical Chemistry, Skåne University Hospital, Lund University, Malmo, Sweden; 4Wake Forest School of Medicine, Winston-Salem, North Carolina; 5Skåne University Hospital, Lund University, Malmo, Sweden Background: Apoptosis is an important mechanism that contributes to heart failure (HF) progression. High-density lipoprotein (HDL) protects cardiomyocytes from apoptosis via signaling through the sphingolipid sphingosine-1-phosphate (S1P). Apolipoprotein M (apoM) mediates the physical interaction between S1P and HDL. We hypothesized that apoM levels are associated with improved survival in heart failure patients and that apoM overexpression would improve cardiac function in vivo. Methods and Results: We measured apoM and S1P levels in 212 subjects from the Penn Heart Failure Study (PHFS), a prospective, multi-center observational study. PHFS participants with ischemic cardiomyopathy were propensity matched to those with non-ischemic cardiomyopathy based on age, sex, hypercholesterolemia, and statin use. We found that apoM levels were strongly associated with survival free from cardiac transplantation and left ventricular assist device placement among participants with ischemic and nonischemic cardiomyopathy (adjusted hazard ratio [95% CI]: 0.86 [0.78, 0.96], P = .009, and 0.85 [0.78, 0.94], P = .001, for each 0.1 micromolar increase in apoM, respec-
tively). To understand the mechanism underlying the association between apoM and survival, we utilized a murine model of hepatic apoM overexpression (apoMTG). apoMTG mice exhibit a 5-fold increase in plasma apoM and HDL-associated S1P. Additionally, apoMTG mice demonstrated improved systolic performance in vivo, and a 40% absolute increase in survival in a model of acute doxorubicin-induced cardiotoxicity. Doxorubicin reduced left ventricular end-diastolic dimension in WT mice, but not apoMTG littermates. In cardiac lysates from apoMTG mice, we observed hyper-phosphorylation of the pro-survival kinase PI3K/Akt, downstream phosphorylation of glycogen synthase kinase 3-beta, and evidence of attenuation of doxorubicin-induced autophagosome accumulation. Ex-vivo treatment of neonatal rat ventricular myocytes with HDL isolated from apoMTG, but not WT mice, decreased doxorubicin-induced caspase activation. Conclusions: ApoM is significantly associated with survival in heart failure patients. This association may be due to improvements in cardiomyocye survival in HF via activation of Akt. ApoM administration should be evaluated as a therapeutic strategy in HF.
003 Acute and Chronic Vascular and Cardiac Effects of Serelaxin in the Angiotensin II/ L-NAME Heart Failure Model Joseph C. McCarthy, Mark Aronovitz, Jennifer Dupont, Tim Calamaras, Iris Z. Jaffe, Robert M. Blanton; Tufts Medical Center, Boston, MA Background: Relaxin is a circulating vasodilator hormone that is increased in heart failure (HF). In patients hospitalized with acute HF, a 2 day infusion of serelaxin (recombinant human relaxin 2) significantly improved dyspnea. Moreover, short term administration of serelaxin significantly reduced 6 month mortality, but the mechanism is unknown. Objective: To examine the acute and chronic effects of serelaxin on cardiac and vascular function in the angiotensin II (Ang II)/L-NAME model of HF. Methods: Male C57Bl6 mice were infused with AngII (800 ng/kg/min) and L-NGNitroarginine Methyl Ester (L-NAME, 30 mg/kg/day in drinking water). On day 7, serelaxin or vehicle was added for 3 days (days 7–9). We then performed: 1) an acute study on day 9 (day 2 of serelaxin infusion); and 2) a chronic study on day 28 (19 days after termination of serelaxin). In each study, we measured vascular function by wire myography, cardiac function by echocardiography, vascular and cardiac structure by immunohistochemistry, and vascular and cardiac gene expression by QRTPCR. Additionally, telemetric monitors were implanted 1 week prior to the chronic study to measure blood pressure (BP). Results: Ang II/L-NAME increased BP similarly in both groups (vehicle increased 44 ± 8 mmHg, serelaxin increased 39 ± 9). Three days of serelaxin induced a temporary 20 ± 4 mm Hg decrease in systolic BP compared to vehicle. Although cardiac hypertrophy, LV dilation, and interstitial fibrosis increased significantly and ejection fraction decreased significantly between day 9 and 28 in both groups, there was no difference between vehicle and serelaxin treated animals. Only perivascular fibrosis was decreased in hearts of serelaxin compared with vehicle treated mice on day 9 and 28. Vascular fibrosis also increased from day 9 to 28 but in the aorta the fibrosis was profoundly attenuated in serelaxin treated mice at both times. Mesenteric resistance vessels from serelaxin-treated mice had reduced constriction to potassium chloride at day 9 and 28 with no significant difference in constriction to phenylephrine or endothelin-1, or in relaxation to acetylcholine and nitroprusside at either time. This decrease in vascular fibrosis was associated with decreased type-1 collagen and increased MMP2 expression in aortas at day 9 during serelaxin infusion. At day 28, both type-1 collagen and MMP2 expression were decreased in serelaxintreated mice. Conclusions: In the AngII/L-NAME mouse model of hypertensioninduced HF, a 3 day serelaxin infusion does not directly affect cardiac structure or function. Rather, serelaxin decreases resistance vessel constriction and vascular fibrosis and these vascular benefits are sustained long after the infusion is complete. These persistent protective effects on the vasculature may contribute to the long term mortality benefits of acute serelaxin infusion observed in heart failure patients.
004 BUNP: A Novel Designer pGC-A/cGMP Activator with Enhanced Potency and High Selectivity In Vitro Yang Chen1,2, Seethalakshmi R. Iyer2, Tomoko Ichiki2, S. Jeson Sangaralingham2, John C. Burnett2; 1Mayo Graduate School, Rochester, MN; 2Mayo Clinic, Rochester, MN Introduction: B-type natriuretic peptide (BNP) and Urodilatin (URO) are endogenous natriuretic peptides (NPs) synthesized in the heart and kidneys respectively. Both are ligands for the particulate guanylyl cyclase receptor A (pGC-A) and generate 3′5′ cyclic guanosine monophosphate (cGMP) to mediate biological actions. Our goal was to employ state of the art peptide engineering to create a novel designer NP which activates pGC-A/cGMP at subthreshold concentrations compared to BNP and URO laying the foundation for a possible new generation NP with enhanced renal actions. Hypothesis: We hypothesize that fusion of selective key amino acids (AAs) from BNP and URO will confer a novel, more potent pGC-A/cGMP activator in vitro. Methods: Solid phase peptide synthesis was used for peptide synthesis of a new 32 peptide which was designated as BUNP. We used human embryonic kidney cells (HEK293) devoid of NP receptors (pGC-A that is activated by BNP and URO and pGC-B which is activated by CNP). We then overexpressed either human pGC-A or pGC-B in HEK293
S2 Journal of Cardiac Failure Vol. 22 No. 8S August 2016 cells to test receptor activation. Transfected cells were treated with BNP, URO or BUNP (low dose: 10-9 M or high dose: 10-8 M) and peptide activity was measured by the resultant generation of cGMP by radioimmunoassay. Data are presented as Mean ± SEM, *P < .05 between BUNP and the other two peptides. Results: BUNP was successfully synthesized and the structure was confirmed by mass spectrometry. In HEK293 pGC-A cells, BUNP generated significantly higher (>5 fold) cGMP levels than BNP and URO at low subthreshold doses of BNP and URO (see Figure). At high concentrations, maximal cGMP generation was achieved by all peptides. Notably, high dose URO and BUNP generated significantly higher cGMP levels than BNP. BUNP did not generate cGMP in HEK293 pGC-B cells. Conclusion: We conclude that: 1) BUNP can be successfully synthesized and represents a new designer pGC-A/cGMP activator; 2) BUNP is a selective activator of pGC-A and cGMP; and 3) BUNP is a superior pGC-A/cGMP activator compared to BNP or URO at low concentrations which may optimize renal selective actions. These in vitro studies in transfected human renal cells support BUNP as a potential new renal targeting drug for the treatment of HF warranting in vivo investigations.
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HFSA
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JNCNI Basic Science Award 001 Neutralizing Endoglin Activity Rescues Maladaptive Remodeling in Heart Failure Kevin Morine, Xiaoying Qiao, Samuel York, Mark Aronovitz, Richard Karas, Navin Kapur; Tufts Medical Center, Boston, MA Endoglin is a trans-membrane co-receptor that promotes signaling via the profibrogenic cytokine transforming growth factor beta 1 (TGFb1) in cardiac fibroblasts. We previously reported that reduced endoglin activity prevents cardiac fibrosis in pressureoverload induced heart failure (HF). We now tested the hypothesis that neutralizing endoglin activity rescues established cardiac fibrosis and improves cardiac function in HF. Adult male C57/Bl6 mice (n = 30) underwent trans-aortic constriction (TAC). After 4 weeks of TAC, survivors (n = 11) were randomized to twice weekly intraperitoneal injections of a neutralizing anti-endoglin antibody (N-Eng-Ab; n = 6) or isotype control antibody (IgG; n = 5) at 15 mg/kg/dose. Sham-operated mice (Sham; n = 5) served as controls. After 4 additional weeks of TAC, IgG mice exhibited reduced body weight, increased lung and LV mass (P < .05 for each comparison). In contrast to IgG mice, N-Eng-Ab mice exhibited increased body weight, decreased lung and LV mass. N-EngAb mice had higher peak LV systolic pressure, lower end diastolic pressure and increased dP/dT max (P < .05 for each comparison). Picrosirius staining for LV fibrosis, LV mRNA levels of Types 1 and 3 collagen, and phosphorylation of Smad-3, a downstream effector of TGFb1 signaling, were attenuated with N-Eng-Ab not IgG treatment after TAC (Figure; P < .05 for each comparison). Myocardial capillary density, defined by histologic staining for CD31+ cells normalized to cardiomyocyte area, was increased after N-Eng Ab treatment and correlated inversely with LV fibrosis (R = 0.678, P = .01). Cardiomyocyte area and LV mRNA levels of BNP, b-MHC were increased similarly, while SERCA levels were reduced between groups. To explore the mechanism of fibrotic rescue, we identified that treatment with N-Eng-Ab reduced the ratio of circulating Type 1 collagen synthesis:degradation byproducts [procollagen type 1 (PIP) : carboxyterminal-telopeptide of Type I collagen (ICTP) ratio] (Figure). Serum PIP:ICTP ratios correlated directly with LV fibrosis (R = 0.811, P < .01) and inversely with myocardial capillary density (R = −0.745, P < .01). This is the first report to establish that neutralizing endoglin activity rescues maladaptive remodeling in HF by reversing established cardiac fibrosis, preserving myocardial capillary density, and improving cardiac function in HF. We further report the novel use of circulating biomarkers of collagen turnover as a marker of therapeutic efficacy for N-Eng-Ab treatment in mice. Endoglin targeted therapeutics may be a novel disease modifying strategy to improve the lives of patients suffering with heart failure.
002 Apolipoprotein M Improves Survival in Congestive Heart Failure via Enhanced Akt Signaling Ali Javaheri 1 , Swapnil Shewale 2 , Cecilia Frej 3 , Mingxia Liu 4 , Ken Bedi 2 , Jeff Brandimarto2, Anbin Mu2, Tao Wang2, Christina Y. Chen2, Xiucui Ma1, Payman Zamani2, Benjamin French2, Kenneth B. Margulies2, Thomas P. Cappola2, Benjamin L. Prosser2, Abhinav Diwan1, Björn Dahlbäck5, John S. Parks4, Daniel J. Rader2; 1Washington University of St. Louis, St. Louis, Missouri; 2University of Pennsylvania, Philadelphia, Pennsylvania; 3Department of Translational Medicine, Division of Clinical Chemistry, Skåne University Hospital, Lund University, Malmo, Sweden; 4Wake Forest School of Medicine, Winston-Salem, North Carolina; 5Skåne University Hospital, Lund University, Malmo, Sweden Background: Apoptosis is an important mechanism that contributes to heart failure (HF) progression. High-density lipoprotein (HDL) protects cardiomyocytes from apoptosis via signaling through the sphingolipid sphingosine-1-phosphate (S1P). Apolipoprotein M (apoM) mediates the physical interaction between S1P and HDL. We hypothesized that apoM levels are associated with improved survival in heart failure patients and that apoM overexpression would improve cardiac function in vivo. Methods and Results: We measured apoM and S1P levels in 212 subjects from the Penn Heart Failure Study (PHFS), a prospective, multi-center observational study. PHFS participants with ischemic cardiomyopathy were propensity matched to those with non-ischemic cardiomyopathy based on age, sex, hypercholesterolemia, and statin use. We found that apoM levels were strongly associated with survival free from cardiac transplantation and left ventricular assist device placement among participants with ischemic and nonischemic cardiomyopathy (adjusted hazard ratio [95% CI]: 0.86 [0.78, 0.96], P = .009, and 0.85 [0.78, 0.94], P = .001, for each 0.1 micromolar increase in apoM, respec-
tively). To understand the mechanism underlying the association between apoM and survival, we utilized a murine model of hepatic apoM overexpression (apoMTG). apoMTG mice exhibit a 5-fold increase in plasma apoM and HDL-associated S1P. Additionally, apoMTG mice demonstrated improved systolic performance in vivo, and a 40% absolute increase in survival in a model of acute doxorubicin-induced cardiotoxicity. Doxorubicin reduced left ventricular end-diastolic dimension in WT mice, but not apoMTG littermates. In cardiac lysates from apoMTG mice, we observed hyper-phosphorylation of the pro-survival kinase PI3K/Akt, downstream phosphorylation of glycogen synthase kinase 3-beta, and evidence of attenuation of doxorubicin-induced autophagosome accumulation. Ex-vivo treatment of neonatal rat ventricular myocytes with HDL isolated from apoMTG, but not WT mice, decreased doxorubicin-induced caspase activation. Conclusions: ApoM is significantly associated with survival in heart failure patients. This association may be due to improvements in cardiomyocye survival in HF via activation of Akt. ApoM administration should be evaluated as a therapeutic strategy in HF.
003 Acute and Chronic Vascular and Cardiac Effects of Serelaxin in the Angiotensin II/ L-NAME Heart Failure Model Joseph C. McCarthy, Mark Aronovitz, Jennifer Dupont, Tim Calamaras, Iris Z. Jaffe, Robert M. Blanton; Tufts Medical Center, Boston, MA Background: Relaxin is a circulating vasodilator hormone that is increased in heart failure (HF). In patients hospitalized with acute HF, a 2 day infusion of serelaxin (recombinant human relaxin 2) significantly improved dyspnea. Moreover, short term administration of serelaxin significantly reduced 6 month mortality, but the mechanism is unknown. Objective: To examine the acute and chronic effects of serelaxin on cardiac and vascular function in the angiotensin II (Ang II)/L-NAME model of HF. Methods: Male C57Bl6 mice were infused with AngII (800 ng/kg/min) and L-NGNitroarginine Methyl Ester (L-NAME, 30 mg/kg/day in drinking water). On day 7, serelaxin or vehicle was added for 3 days (days 7–9). We then performed: 1) an acute study on day 9 (day 2 of serelaxin infusion); and 2) a chronic study on day 28 (19 days after termination of serelaxin). In each study, we measured vascular function by wire myography, cardiac function by echocardiography, vascular and cardiac structure by immunohistochemistry, and vascular and cardiac gene expression by QRTPCR. Additionally, telemetric monitors were implanted 1 week prior to the chronic study to measure blood pressure (BP). Results: Ang II/L-NAME increased BP similarly in both groups (vehicle increased 44 ± 8 mmHg, serelaxin increased 39 ± 9). Three days of serelaxin induced a temporary 20 ± 4 mm Hg decrease in systolic BP compared to vehicle. Although cardiac hypertrophy, LV dilation, and interstitial fibrosis increased significantly and ejection fraction decreased significantly between day 9 and 28 in both groups, there was no difference between vehicle and serelaxin treated animals. Only perivascular fibrosis was decreased in hearts of serelaxin compared with vehicle treated mice on day 9 and 28. Vascular fibrosis also increased from day 9 to 28 but in the aorta the fibrosis was profoundly attenuated in serelaxin treated mice at both times. Mesenteric resistance vessels from serelaxin-treated mice had reduced constriction to potassium chloride at day 9 and 28 with no significant difference in constriction to phenylephrine or endothelin-1, or in relaxation to acetylcholine and nitroprusside at either time. This decrease in vascular fibrosis was associated with decreased type-1 collagen and increased MMP2 expression in aortas at day 9 during serelaxin infusion. At day 28, both type-1 collagen and MMP2 expression were decreased in serelaxintreated mice. Conclusions: In the AngII/L-NAME mouse model of hypertensioninduced HF, a 3 day serelaxin infusion does not directly affect cardiac structure or function. Rather, serelaxin decreases resistance vessel constriction and vascular fibrosis and these vascular benefits are sustained long after the infusion is complete. These persistent protective effects on the vasculature may contribute to the long term mortality benefits of acute serelaxin infusion observed in heart failure patients.
004 BUNP: A Novel Designer pGC-A/cGMP Activator with Enhanced Potency and High Selectivity In Vitro Yang Chen1,2, Seethalakshmi R. Iyer2, Tomoko Ichiki2, S. Jeson Sangaralingham2, John C. Burnett2; 1Mayo Graduate School, Rochester, MN; 2Mayo Clinic, Rochester, MN Introduction: B-type natriuretic peptide (BNP) and Urodilatin (URO) are endogenous natriuretic peptides (NPs) synthesized in the heart and kidneys respectively. Both are ligands for the particulate guanylyl cyclase receptor A (pGC-A) and generate 3′5′ cyclic guanosine monophosphate (cGMP) to mediate biological actions. Our goal was to employ state of the art peptide engineering to create a novel designer NP which activates pGC-A/cGMP at subthreshold concentrations compared to BNP and URO laying the foundation for a possible new generation NP with enhanced renal actions. Hypothesis: We hypothesize that fusion of selective key amino acids (AAs) from BNP and URO will confer a novel, more potent pGC-A/cGMP activator in vitro. Methods: Solid phase peptide synthesis was used for peptide synthesis of a new 32 peptide which was designated as BUNP. We used human embryonic kidney cells (HEK293) devoid of NP receptors (pGC-A that is activated by BNP and URO and pGC-B which is activated by CNP). We then overexpressed either human pGC-A or pGC-B in HEK293
S2 Journal of Cardiac Failure Vol. 22 No. 8S August 2016 cells to test receptor activation. Transfected cells were treated with BNP, URO or BUNP (low dose: 10-9 M or high dose: 10-8 M) and peptide activity was measured by the resultant generation of cGMP by radioimmunoassay. Data are presented as Mean ± SEM, *P < .05 between BUNP and the other two peptides. Results: BUNP was successfully synthesized and the structure was confirmed by mass spectrometry. In HEK293 pGC-A cells, BUNP generated significantly higher (>5 fold) cGMP levels than BNP and URO at low subthreshold doses of BNP and URO (see Figure). At high concentrations, maximal cGMP generation was achieved by all peptides. Notably, high dose URO and BUNP generated significantly higher cGMP levels than BNP. BUNP did not generate cGMP in HEK293 pGC-B cells. Conclusion: We conclude that: 1) BUNP can be successfully synthesized and represents a new designer pGC-A/cGMP activator; 2) BUNP is a selective activator of pGC-A and cGMP; and 3) BUNP is a superior pGC-A/cGMP activator compared to BNP or URO at low concentrations which may optimize renal selective actions. These in vitro studies in transfected human renal cells support BUNP as a potential new renal targeting drug for the treatment of HF warranting in vivo investigations.