Eric Churchill - Academia.edu (original) (raw)
Papers by Eric Churchill
Circulation, Oct 28, 2008
<jats:p> The response of the myocardium to an ischemic insult is regulated by two opposing ... more <jats:p> The response of the myocardium to an ischemic insult is regulated by two opposing PKC isozymes, δ and ϵPKC. We therefore wanted to determine how two highly homologous kinases could play such distinct roles in cardiac ischemia/reperfusion (I/R) injury. Using an <jats:italic>ex vivo</jats:italic> rat model of myocardial infarction, we determined that short bouts of ischemia and reperfusion prior to the prolonged ischemic event (IPC) diminished δPKC and enhanced ϵPKC translocation to the mitochondria. In addition, cellular levels of δPKC decreased by 60% in response to IPC, whereas the levels of ϵPKC remained unchanged. Prolonged ischemia induced a 50% decline in the ATP-dependent proteasome activity and increased the accumulation of unfolded proteins during reperfusion; both of which were completely prevented by IPC. Pharmacological inhibition of the proteasome and selective inhibition of ϵPKC during IPC restored δPKC translocation to the mitochondria and elevated the levels of unfolded proteins while increasing myocardial injury associated with I/R. (Note that none of these treatments had any effect on viability under normoxic conditions.) Interestingly, administration of a specific peptide activator of ϵPKC during I/R completely prevented the loss in proteasome activity seen during ischemia. Importantly, increased myocardial injury was the result of restoring the δPKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signaling and increasing cytochrome <jats:italic>c</jats:italic> release into the cytosol. Taken together, this study shows that IPC prevents I/R injury by protecting ATP-dependent 26S proteasome function, which in turn decreases the accumulation of the pro-apoptotic kinase, δPKC, at cardiac mitochondria resulting in the accumulation of the pro-survival kinase, ϵPKC. Additionally, ϵPKC seems to be a novel regulator of proteasome function as it relates to I/R injury. The proteasome can therefore be viewed as a sensor of cellular viability, determining the ratio of pro-apoptotic δPKC and pro-survival ϵPKC at the mitochondria and thus the ultimate fate of the cell. </jats:p>
Pharmacological Research, Jun 1, 2007
Current treatment for acute myocardial infarction (AMI) is aimed at limiting the duration of isch... more Current treatment for acute myocardial infarction (AMI) is aimed at limiting the duration of ischemia by either mechanical (balloon catheters) or enzymatic (thrombolytics) means to disrupt the occlusion. While these treatments are effective in limiting the duration of ischemia, no therapeutic treatment is currently available to prevent ischemic injury and to reduce reperfusion injury, which occurs after these interventions. The development of rationally designed PKC isozyme-selective regulator peptides has permitted investigation into the role of specific PKC isozymes in ischemiareperfusion (IR) injury. Based on these studies, it is now evident that and ␦PKC have distinct temporal and opposing roles in regulating myocardial damage induced by IR. Activation of PKC before ischemia protects the heart by mimicking preconditioning, whereas inhibition of ␦PKC during reperfusion protects the heart from reperfusion-induced damage. These cardioprotective effects have been observed in isolated cardiomyocytes, isolated perfused hearts and in vivo in all species tested including mouse, rat and pig and may provide the basis for future therapeutic agents. Having established the efficacy of PKC isozyme-specific regulators in reducing IR injury, the next challenge is to outline the molecular mechanisms regulated by ␦ and PKC isozymes that result in enhanced tolerance to IR. In this review, we discuss progress that has been made in establishing cytoprotective mechanisms, which arise as a consequence of PKC activation or ␦PKC inhibition, and how they may lead to protection in the setting of myocardial ischemia reperfusion.
Journal of Thoracic Oncology, Oct 1, 2021
Transplant Immunology, May 1, 2010
Background: We aimed to compare two techniques of heterotopic heart transplantation in rats. Non-... more Background: We aimed to compare two techniques of heterotopic heart transplantation in rats. Non-volumeloaded (NL) and volume-loaded (VL) models were tested for their physiologic and immunologic properties to assess their suitability for transplant studies. Methods: Syngeneic heterotopic heart transplants were performed according to the techniques previously described by Ono (NL) and Yokoyama (VL). Grafts were followed over 90 days with sequential echocardiography. Ex-vivo Langendorff perfusion was used to gain functional data. Allogeneic heart transplants were done to determine whether chronic allograft vasculopathy (CAV) develops at a different pace in both transplant models. Results: The ischemic time during surgery was significantly longer using the VL model (p b 0.001). The LV diameter of NL hearts decreased over time while that of the VL model significantly increased (p = 0.004 on POD 90). Mean LV developed pressure and (dP / dt)max were significantly higher with the NL model (61.1 ± 8.5 mmHg and 4261.7 ± 419.6 mmHg/s) than with VL hearts (19.9 ± 16.5 mmHg; p = 0.011 and 924.8 ± 605.6 mmHg/s; p b 0.001). The mean weight of NL hearts (0.45 ± 0.03 g) was significantly less than that of VL hearts (1.21 ± 0.16 g, p b 0.001). Histology of syngeneic NL grafts showed healthy, but partly atrophic myocardium, whereas the LV myocardium of VL hearts showed dilation and scarring typical for chronic ischemic injury. Heart allografts similarly developed CAV with luminal narrowing of 37.2 ± 16.6% (NL) and 34.4 ± 21.4% (VL), respectively by POD 90 (p = 0.807). Conclusions: Since the coronary arteries in the VL model get perfused with partly deoxygenated blood, the myocardium suffers from chronic ischemic injury. We recommend using the NL model in preclinical transplant studies.
Journal of Molecular and Cellular Cardiology, Jun 1, 2006
Journal of Molecular and Cellular Cardiology, Jun 1, 2006
Trends in Endocrinology and Metabolism, 2009
Protein-protein interactions sequester enzymes close to their substrates. Protein kinase C (PKC) ... more Protein-protein interactions sequester enzymes close to their substrates. Protein kinase C (PKC) is one example of a ubiquitous signaling molecule with effects that are dependent upon localization. Short peptides derived from interaction sites between each PKC isozyme and its receptor for activated C kinase act as highly specific inhibitors and have become available as selective drugs in basic research and animal models of human diseases, such as myocardial infarction and hyperglycemia. Whereas the earlier inhibitory peptides are highly specific, we believe that peptides targeting additional interactions between PKC and selective substrates will generate even more selective tools that regulate different functions of individual isozymes. Here, we discuss the methodologies and applications for identifying selective regulators of PKC. The evolution of rational drug design The process of drug discovery has evolved from a series of serendipitous findings to a more systematic search accompanied by rationally designed molecules. The evolution of this process is best illustrated by the discovery of aspirin. The benefit of willow leaves in reducing pain and inflammation was first described by the Babylonians nearly 4000 years ago and later prescribed as a medication by Hippo-crates [1]. Two millennia later, salicylic acid was purified to reduce side-effects associated with other components in the plant extract, and by the end of the 19th century, this substance was sold by Bayer as acetylsalicylic acid under the name aspirin [1]. The mechanism of action of aspirin and the targets of aspirin, the cyclo-oxygenase enzymes (see Glossary), were discovered 80 years later [2], and the crystal structure of this enzyme was solved as recently as 1994 [3,4]. The ability to use automated high-throughput screens of thousands of molecules and the development of more advanced nuclear magnetic resonance (NMR), X-ray crystallography and molecular dynamic simulation techniques resulted in more systematic searches of new drugs and directed small molecule design based on threedimensional information of proteins bound with their ligands. These two seemingly incongruent approaches (unbiased search vs rational design) are in fact complementary, leading to the current approach to drug discovery. Structural information on drug-protein interactions identifies binding pockets for small molecules in the protein targets and provides measurements of the forces that govern the binding of these small molecules to their protein. This has facilitated the rational design of drugs that mimic or compete with these interactions. However, many crucial signaling events in the cell occur in multiprotein complexes and involve multiple protein-protein interactions.
Journal of Molecular and Cellular Cardiology, Feb 1, 2009
Time-dependent and ethanol-induced cardiac protection from ischemia mediated by mitochondrial tra... more Time-dependent and ethanol-induced cardiac protection from ischemia mediated by mitochondrial translocation of εPKC and activation of aldehyde dehydrogenase 2
Circulation Research, Jul 8, 2005
Cardiac ischemia and reperfusion are associated with loss in the activity of the mitochondrial en... more Cardiac ischemia and reperfusion are associated with loss in the activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH). Pharmacological stimulation of PDH activity improves recovery in contractile function during reperfusion. Signaling mechanisms that control inhibition and reactivation of PDH during reperfusion were therefore investigated. Using an isolated rat heart model, we observed ischemia-induced PDH inhibition with only partial recovery evident on reperfusion. Translocation of the redox-sensitive ␦-isoform of protein kinase C (PKC) to the mitochondria occurred during reperfusion. Inhibition of this process resulted in full recovery of PDH activity. Infusion of the ␦PKC activator H 2 O 2 during normoxic perfusion, to mimic one aspect of cardiac reperfusion, resulted in loss in PDH activity that was largely attributable to translocation of ␦PKC to the mitochondria. Evidence indicates that reperfusion-induced translocation of ␦PKC is associated with phosphorylation of the ␣E1 subunit of PDH. A potential mechanism is provided by in vitro data demonstrating that ␦PKC specifically interacts with and phosphorylates pyruvate dehydrogenase kinase (PDK)2. Importantly, this results in activation of PDK2, an enzyme capable of phosphorylating and inhibiting PDH. Thus, translocation of ␦PKC to the mitochondria during reperfusion likely results in activation of PDK2 and phosphorylation-dependent inhibition of PDH.
Journal of Thoracic Oncology, Jun 1, 2023
Journal of Thoracic Oncology, Oct 1, 2021
Journal of Thoracic Oncology
Expert Review of Anticancer Therapy
ischaemia and reperfusion injury
The Thoracic and Cardiovascular Surgeon, 2010
Background: We aimed to compare two techniques of heterotopic heart transplantation in rats. Non-... more Background: We aimed to compare two techniques of heterotopic heart transplantation in rats. Non-volumeloaded (NL) and volume-loaded (VL) models were tested for their physiologic and immunologic properties to assess their suitability for transplant studies. Methods: Syngeneic heterotopic heart transplants were performed according to the techniques previously described by Ono (NL) and Yokoyama (VL). Grafts were followed over 90 days with sequential echocardiography. Ex-vivo Langendorff perfusion was used to gain functional data. Allogeneic heart transplants were done to determine whether chronic allograft vasculopathy (CAV) develops at a different pace in both transplant models. Results: The ischemic time during surgery was significantly longer using the VL model (p b 0.001). The LV diameter of NL hearts decreased over time while that of the VL model significantly increased (p = 0.004 on POD 90). Mean LV developed pressure and (dP / dt)max were significantly higher with the NL model (61.1 ± 8.5 mmHg and 4261.7 ± 419.6 mmHg/s) than with VL hearts (19.9 ± 16.5 mmHg; p = 0.011 and 924.8 ± 605.6 mmHg/s; p b 0.001). The mean weight of NL hearts (0.45 ± 0.03 g) was significantly less than that of VL hearts (1.21 ± 0.16 g, p b 0.001). Histology of syngeneic NL grafts showed healthy, but partly atrophic myocardium, whereas the LV myocardium of VL hearts showed dilation and scarring typical for chronic ischemic injury. Heart allografts similarly developed CAV with luminal narrowing of 37.2 ± 16.6% (NL) and 34.4 ± 21.4% (VL), respectively by POD 90 (p = 0.807). Conclusions: Since the coronary arteries in the VL model get perfused with partly deoxygenated blood, the myocardium suffers from chronic ischemic injury. We recommend using the NL model in preclinical transplant studies.
The Journal of Heart and Lung Transplantation, 2009
The prevention of chronic allograft vasculopathy (CAV) after heart transplantation remains a majo... more The prevention of chronic allograft vasculopathy (CAV) after heart transplantation remains a major problem for long-term success. Epsilon protein kinase C (⑀PKC) is a PKC isoform that plays pivotal roles in myocardial infarction and in heart failure. Here we investigate whether PKC epsilon activation is also involved in the development of intimal hyperplasia. Methods and Materials: Rats underwent balloon denudation of the abdominal aorta and received either 3mM ⑀PKC activator (yeRACK), 3mM ⑀PKC inhibitor (⑀V1-2), the carrier control (TAT 47-57), or saline by osmotic pump at ϳ3mg/kg/day for 4 weeks (6 rats/group). The treatment began intraoperatively). Aortas were harvested for histologic evaluation, and luminal obliteration and intima/media ratios were analyzed using computer morphometry. Results: Histology of untreated animals revealed marked intimal hyperplasia with moderate luminal obliteration (19.9Ϯ9%). Neointima formation was significantly increased by the ⑀PKC activator (32Ϯ5.5%; pϭ0.017 vs. untreated) and significantly decreased by the ⑀PKC inhibitor (9.1Ϯ4.3%; pϭ0.016 vs. untreated). No difference was observed between the untreated control and the TAT carrier peptide contol groups (pϭ0.43). The intima/media ratio was significantly higher in the ⑀PKC activator group compared to the ⑀PKC inhibitor group (0.67Ϯ0.46 and 0.25Ϯ0.46, respectively; pϭ0.034). Treatment with either of the ePKC regulators was very well tolerated and the animals in the ⑀PKC activator as well as the ⑀PKC inhibitor groups gained weight during the 4 week treatment period (105Ϯ1.9% and 102Ϯ3.4%, respectively; pϭns). No differences in creatinine, BUN, cholesterol, triglycerides, ALT, and AST were observed between the four groups. Conclusions: These data suggest that ⑀PKC activity contributes to the non-immunological development of intimal hyperplasia and that an ⑀PKC-selective inhibitor, such as ⑀V1-2, could augment current therapeutic strategies to suppress the development of vascular stenosis. 435 CXCR3 and CCR5 in Acute and Chronic Rejection
Annual Review of Pharmacology and Toxicology, 2008
Cardiovascular disease is the leading cause of death in the United States. Therefore, identifying... more Cardiovascular disease is the leading cause of death in the United States. Therefore, identifying therapeutic targets is a major focus of current research. Protein kinase C (PKC), a family of serine/threonine kinases, has been identified as playing a role in many of the pathologies of heart disease. However, the lack of specific PKC regulators and the ubiquitous expression and normal physiological functions of the 11 PKC isozymes has made drug development a challenge. Here we discuss the validity of therapeutically targeting PKC, an intracellular signaling enzyme. We describe PKC structure, function, and distribution in the healthy and diseased heart, as well as the development of rationally designed isozyme-selective regulators of PKC functions. The review focuses on the roles of specific PKC isozymes in atherosclerosis, fibrosis, and cardiac hypertrophy, and examines principles of pharmacology as they pertain to regulators of signaling cascades associated with these diseases.
Circulation, Oct 28, 2008
<jats:p> The response of the myocardium to an ischemic insult is regulated by two opposing ... more <jats:p> The response of the myocardium to an ischemic insult is regulated by two opposing PKC isozymes, δ and ϵPKC. We therefore wanted to determine how two highly homologous kinases could play such distinct roles in cardiac ischemia/reperfusion (I/R) injury. Using an <jats:italic>ex vivo</jats:italic> rat model of myocardial infarction, we determined that short bouts of ischemia and reperfusion prior to the prolonged ischemic event (IPC) diminished δPKC and enhanced ϵPKC translocation to the mitochondria. In addition, cellular levels of δPKC decreased by 60% in response to IPC, whereas the levels of ϵPKC remained unchanged. Prolonged ischemia induced a 50% decline in the ATP-dependent proteasome activity and increased the accumulation of unfolded proteins during reperfusion; both of which were completely prevented by IPC. Pharmacological inhibition of the proteasome and selective inhibition of ϵPKC during IPC restored δPKC translocation to the mitochondria and elevated the levels of unfolded proteins while increasing myocardial injury associated with I/R. (Note that none of these treatments had any effect on viability under normoxic conditions.) Interestingly, administration of a specific peptide activator of ϵPKC during I/R completely prevented the loss in proteasome activity seen during ischemia. Importantly, increased myocardial injury was the result of restoring the δPKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signaling and increasing cytochrome <jats:italic>c</jats:italic> release into the cytosol. Taken together, this study shows that IPC prevents I/R injury by protecting ATP-dependent 26S proteasome function, which in turn decreases the accumulation of the pro-apoptotic kinase, δPKC, at cardiac mitochondria resulting in the accumulation of the pro-survival kinase, ϵPKC. Additionally, ϵPKC seems to be a novel regulator of proteasome function as it relates to I/R injury. The proteasome can therefore be viewed as a sensor of cellular viability, determining the ratio of pro-apoptotic δPKC and pro-survival ϵPKC at the mitochondria and thus the ultimate fate of the cell. </jats:p>
Pharmacological Research, Jun 1, 2007
Current treatment for acute myocardial infarction (AMI) is aimed at limiting the duration of isch... more Current treatment for acute myocardial infarction (AMI) is aimed at limiting the duration of ischemia by either mechanical (balloon catheters) or enzymatic (thrombolytics) means to disrupt the occlusion. While these treatments are effective in limiting the duration of ischemia, no therapeutic treatment is currently available to prevent ischemic injury and to reduce reperfusion injury, which occurs after these interventions. The development of rationally designed PKC isozyme-selective regulator peptides has permitted investigation into the role of specific PKC isozymes in ischemiareperfusion (IR) injury. Based on these studies, it is now evident that and ␦PKC have distinct temporal and opposing roles in regulating myocardial damage induced by IR. Activation of PKC before ischemia protects the heart by mimicking preconditioning, whereas inhibition of ␦PKC during reperfusion protects the heart from reperfusion-induced damage. These cardioprotective effects have been observed in isolated cardiomyocytes, isolated perfused hearts and in vivo in all species tested including mouse, rat and pig and may provide the basis for future therapeutic agents. Having established the efficacy of PKC isozyme-specific regulators in reducing IR injury, the next challenge is to outline the molecular mechanisms regulated by ␦ and PKC isozymes that result in enhanced tolerance to IR. In this review, we discuss progress that has been made in establishing cytoprotective mechanisms, which arise as a consequence of PKC activation or ␦PKC inhibition, and how they may lead to protection in the setting of myocardial ischemia reperfusion.
Journal of Thoracic Oncology, Oct 1, 2021
Transplant Immunology, May 1, 2010
Background: We aimed to compare two techniques of heterotopic heart transplantation in rats. Non-... more Background: We aimed to compare two techniques of heterotopic heart transplantation in rats. Non-volumeloaded (NL) and volume-loaded (VL) models were tested for their physiologic and immunologic properties to assess their suitability for transplant studies. Methods: Syngeneic heterotopic heart transplants were performed according to the techniques previously described by Ono (NL) and Yokoyama (VL). Grafts were followed over 90 days with sequential echocardiography. Ex-vivo Langendorff perfusion was used to gain functional data. Allogeneic heart transplants were done to determine whether chronic allograft vasculopathy (CAV) develops at a different pace in both transplant models. Results: The ischemic time during surgery was significantly longer using the VL model (p b 0.001). The LV diameter of NL hearts decreased over time while that of the VL model significantly increased (p = 0.004 on POD 90). Mean LV developed pressure and (dP / dt)max were significantly higher with the NL model (61.1 ± 8.5 mmHg and 4261.7 ± 419.6 mmHg/s) than with VL hearts (19.9 ± 16.5 mmHg; p = 0.011 and 924.8 ± 605.6 mmHg/s; p b 0.001). The mean weight of NL hearts (0.45 ± 0.03 g) was significantly less than that of VL hearts (1.21 ± 0.16 g, p b 0.001). Histology of syngeneic NL grafts showed healthy, but partly atrophic myocardium, whereas the LV myocardium of VL hearts showed dilation and scarring typical for chronic ischemic injury. Heart allografts similarly developed CAV with luminal narrowing of 37.2 ± 16.6% (NL) and 34.4 ± 21.4% (VL), respectively by POD 90 (p = 0.807). Conclusions: Since the coronary arteries in the VL model get perfused with partly deoxygenated blood, the myocardium suffers from chronic ischemic injury. We recommend using the NL model in preclinical transplant studies.
Journal of Molecular and Cellular Cardiology, Jun 1, 2006
Journal of Molecular and Cellular Cardiology, Jun 1, 2006
Trends in Endocrinology and Metabolism, 2009
Protein-protein interactions sequester enzymes close to their substrates. Protein kinase C (PKC) ... more Protein-protein interactions sequester enzymes close to their substrates. Protein kinase C (PKC) is one example of a ubiquitous signaling molecule with effects that are dependent upon localization. Short peptides derived from interaction sites between each PKC isozyme and its receptor for activated C kinase act as highly specific inhibitors and have become available as selective drugs in basic research and animal models of human diseases, such as myocardial infarction and hyperglycemia. Whereas the earlier inhibitory peptides are highly specific, we believe that peptides targeting additional interactions between PKC and selective substrates will generate even more selective tools that regulate different functions of individual isozymes. Here, we discuss the methodologies and applications for identifying selective regulators of PKC. The evolution of rational drug design The process of drug discovery has evolved from a series of serendipitous findings to a more systematic search accompanied by rationally designed molecules. The evolution of this process is best illustrated by the discovery of aspirin. The benefit of willow leaves in reducing pain and inflammation was first described by the Babylonians nearly 4000 years ago and later prescribed as a medication by Hippo-crates [1]. Two millennia later, salicylic acid was purified to reduce side-effects associated with other components in the plant extract, and by the end of the 19th century, this substance was sold by Bayer as acetylsalicylic acid under the name aspirin [1]. The mechanism of action of aspirin and the targets of aspirin, the cyclo-oxygenase enzymes (see Glossary), were discovered 80 years later [2], and the crystal structure of this enzyme was solved as recently as 1994 [3,4]. The ability to use automated high-throughput screens of thousands of molecules and the development of more advanced nuclear magnetic resonance (NMR), X-ray crystallography and molecular dynamic simulation techniques resulted in more systematic searches of new drugs and directed small molecule design based on threedimensional information of proteins bound with their ligands. These two seemingly incongruent approaches (unbiased search vs rational design) are in fact complementary, leading to the current approach to drug discovery. Structural information on drug-protein interactions identifies binding pockets for small molecules in the protein targets and provides measurements of the forces that govern the binding of these small molecules to their protein. This has facilitated the rational design of drugs that mimic or compete with these interactions. However, many crucial signaling events in the cell occur in multiprotein complexes and involve multiple protein-protein interactions.
Journal of Molecular and Cellular Cardiology, Feb 1, 2009
Time-dependent and ethanol-induced cardiac protection from ischemia mediated by mitochondrial tra... more Time-dependent and ethanol-induced cardiac protection from ischemia mediated by mitochondrial translocation of εPKC and activation of aldehyde dehydrogenase 2
Circulation Research, Jul 8, 2005
Cardiac ischemia and reperfusion are associated with loss in the activity of the mitochondrial en... more Cardiac ischemia and reperfusion are associated with loss in the activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH). Pharmacological stimulation of PDH activity improves recovery in contractile function during reperfusion. Signaling mechanisms that control inhibition and reactivation of PDH during reperfusion were therefore investigated. Using an isolated rat heart model, we observed ischemia-induced PDH inhibition with only partial recovery evident on reperfusion. Translocation of the redox-sensitive ␦-isoform of protein kinase C (PKC) to the mitochondria occurred during reperfusion. Inhibition of this process resulted in full recovery of PDH activity. Infusion of the ␦PKC activator H 2 O 2 during normoxic perfusion, to mimic one aspect of cardiac reperfusion, resulted in loss in PDH activity that was largely attributable to translocation of ␦PKC to the mitochondria. Evidence indicates that reperfusion-induced translocation of ␦PKC is associated with phosphorylation of the ␣E1 subunit of PDH. A potential mechanism is provided by in vitro data demonstrating that ␦PKC specifically interacts with and phosphorylates pyruvate dehydrogenase kinase (PDK)2. Importantly, this results in activation of PDK2, an enzyme capable of phosphorylating and inhibiting PDH. Thus, translocation of ␦PKC to the mitochondria during reperfusion likely results in activation of PDK2 and phosphorylation-dependent inhibition of PDH.
Journal of Thoracic Oncology, Jun 1, 2023
Journal of Thoracic Oncology, Oct 1, 2021
Journal of Thoracic Oncology
Expert Review of Anticancer Therapy
ischaemia and reperfusion injury
The Thoracic and Cardiovascular Surgeon, 2010
Background: We aimed to compare two techniques of heterotopic heart transplantation in rats. Non-... more Background: We aimed to compare two techniques of heterotopic heart transplantation in rats. Non-volumeloaded (NL) and volume-loaded (VL) models were tested for their physiologic and immunologic properties to assess their suitability for transplant studies. Methods: Syngeneic heterotopic heart transplants were performed according to the techniques previously described by Ono (NL) and Yokoyama (VL). Grafts were followed over 90 days with sequential echocardiography. Ex-vivo Langendorff perfusion was used to gain functional data. Allogeneic heart transplants were done to determine whether chronic allograft vasculopathy (CAV) develops at a different pace in both transplant models. Results: The ischemic time during surgery was significantly longer using the VL model (p b 0.001). The LV diameter of NL hearts decreased over time while that of the VL model significantly increased (p = 0.004 on POD 90). Mean LV developed pressure and (dP / dt)max were significantly higher with the NL model (61.1 ± 8.5 mmHg and 4261.7 ± 419.6 mmHg/s) than with VL hearts (19.9 ± 16.5 mmHg; p = 0.011 and 924.8 ± 605.6 mmHg/s; p b 0.001). The mean weight of NL hearts (0.45 ± 0.03 g) was significantly less than that of VL hearts (1.21 ± 0.16 g, p b 0.001). Histology of syngeneic NL grafts showed healthy, but partly atrophic myocardium, whereas the LV myocardium of VL hearts showed dilation and scarring typical for chronic ischemic injury. Heart allografts similarly developed CAV with luminal narrowing of 37.2 ± 16.6% (NL) and 34.4 ± 21.4% (VL), respectively by POD 90 (p = 0.807). Conclusions: Since the coronary arteries in the VL model get perfused with partly deoxygenated blood, the myocardium suffers from chronic ischemic injury. We recommend using the NL model in preclinical transplant studies.
The Journal of Heart and Lung Transplantation, 2009
The prevention of chronic allograft vasculopathy (CAV) after heart transplantation remains a majo... more The prevention of chronic allograft vasculopathy (CAV) after heart transplantation remains a major problem for long-term success. Epsilon protein kinase C (⑀PKC) is a PKC isoform that plays pivotal roles in myocardial infarction and in heart failure. Here we investigate whether PKC epsilon activation is also involved in the development of intimal hyperplasia. Methods and Materials: Rats underwent balloon denudation of the abdominal aorta and received either 3mM ⑀PKC activator (yeRACK), 3mM ⑀PKC inhibitor (⑀V1-2), the carrier control (TAT 47-57), or saline by osmotic pump at ϳ3mg/kg/day for 4 weeks (6 rats/group). The treatment began intraoperatively). Aortas were harvested for histologic evaluation, and luminal obliteration and intima/media ratios were analyzed using computer morphometry. Results: Histology of untreated animals revealed marked intimal hyperplasia with moderate luminal obliteration (19.9Ϯ9%). Neointima formation was significantly increased by the ⑀PKC activator (32Ϯ5.5%; pϭ0.017 vs. untreated) and significantly decreased by the ⑀PKC inhibitor (9.1Ϯ4.3%; pϭ0.016 vs. untreated). No difference was observed between the untreated control and the TAT carrier peptide contol groups (pϭ0.43). The intima/media ratio was significantly higher in the ⑀PKC activator group compared to the ⑀PKC inhibitor group (0.67Ϯ0.46 and 0.25Ϯ0.46, respectively; pϭ0.034). Treatment with either of the ePKC regulators was very well tolerated and the animals in the ⑀PKC activator as well as the ⑀PKC inhibitor groups gained weight during the 4 week treatment period (105Ϯ1.9% and 102Ϯ3.4%, respectively; pϭns). No differences in creatinine, BUN, cholesterol, triglycerides, ALT, and AST were observed between the four groups. Conclusions: These data suggest that ⑀PKC activity contributes to the non-immunological development of intimal hyperplasia and that an ⑀PKC-selective inhibitor, such as ⑀V1-2, could augment current therapeutic strategies to suppress the development of vascular stenosis. 435 CXCR3 and CCR5 in Acute and Chronic Rejection
Annual Review of Pharmacology and Toxicology, 2008
Cardiovascular disease is the leading cause of death in the United States. Therefore, identifying... more Cardiovascular disease is the leading cause of death in the United States. Therefore, identifying therapeutic targets is a major focus of current research. Protein kinase C (PKC), a family of serine/threonine kinases, has been identified as playing a role in many of the pathologies of heart disease. However, the lack of specific PKC regulators and the ubiquitous expression and normal physiological functions of the 11 PKC isozymes has made drug development a challenge. Here we discuss the validity of therapeutically targeting PKC, an intracellular signaling enzyme. We describe PKC structure, function, and distribution in the healthy and diseased heart, as well as the development of rationally designed isozyme-selective regulators of PKC functions. The review focuses on the roles of specific PKC isozymes in atherosclerosis, fibrosis, and cardiac hypertrophy, and examines principles of pharmacology as they pertain to regulators of signaling cascades associated with these diseases.