Tomoyoshi Koyanagi

Pharmacological Inhibition of ε-Protein Kinase C Attenuates Cardiac Fibrosis and Dysfunction in Hypertension-Induced Heart Failure

Studies on genetically manipulated mice suggest a role for ϵ-protein kinase C (ϵPKC) in cardiac hypertrophy and in heart failure. The potential clinical relevance of these findings was tested here using a pharmacological inhibitor of ϵPKC activity during the progression to heart failure in hypertensive Dahl rats. Dahl rats, fed an 8% high-salt diet from the age of 6 weeks, exhibited compensatory cardiac hypertrophy by 11 weeks, followed by heart failure at ≈17 weeks and death by the age of ≈20 weeks (123±3 days). Sustained treatment between weeks 11 and 17 with the selective ϵPKC inhibitor ϵV1-2 or with an angiotensin II receptor blocker olmesartan prolonged animal survival by ≈5 weeks (ϵV1-2: 154±7 days; olmesartan: 149±5 days). These treatments resulted in improved fractional shortening (ϵV1-2: 58±2%; olmesartan: 53±2%; saline: 41±6%) and decreased cardiac parenchymal fibrosis when measured at 17 weeks without lowering blood pressure at any time during the treatment. Combined treatment with ϵV1-2, together with olmesartan, prolonged animal survival by 5 weeks (37 days) relative to olmesartan alone (from 160±5 to 197±14 days, respectively) and by ≈11 weeks (74 days) on average relative to saline-treated animals, suggesting that the pathway inhibited by ϵPKC inhibition is not identical to the olmesartan-induced effect. These data suggest that an ϵPKC-selective inhibitor such as ϵV1-2 may have a potential in augmenting current therapeutic strategies for the treatment of heart failure in humans.

Suppression of Graft Coronary Artery Disease by a Brief Treatment With a Selective εPKC Activator and a δPKC Inhibitor in Murine Cardiac Allografts

Background—Inhibiting delta protein kinase C (&dgr;PKC) during reperfusion and activating epsilon PKC (&egr;PKC) before ischemia each limits cardiac ischemic injury. Here, we examined whether limiting ischemia–reperfusion injury inhibits graft coronary artery disease (GCAD) and improves murine cardiac allografting. Methods and Results—Hearts of FVB mice (H-2q) were transplanted into C57BL/6 mice (H-2b). &egr;PKC activator (&psgr;&egr;RACK) was injected intraperitoneally (20 nmol) into donor mice 20 minutes before procurement. Hearts were then perfused with &psgr;&egr;RACK (1.5 nmol) through the inferior vena cava (IVC) and subsequently submerged in &psgr;&egr;RACK (0.5 &mgr;mol/L) for 20 minutes at 4°C. Before reperfusion, the peritoneal cavity of recipients was irrigated with &dgr;PKC inhibitor (&dgr;V1–1, 300 nmol); control animals were treated with normal saline. The total ischemic time to the organ was 50 minutes. Two hours after transplantation, production of inflammatory cytokines and adhesion molecules, cardiomyocyte apoptosis, and caspase-3 and caspase-9 (but not caspase-8) activities were significantly reduced in the PKC regulator-treated group. Fas ligand levels (but not Fas) were also significantly reduced in this group. Importantly, GCAD indices, production of inflammatory cytokines, and adhesion molecules were significantly decreased and cardiac allograft function was significantly better as measured up to 30 days after transplantation. ConclusionsAn &egr;PKC activator and a &dgr;PKC inhibitor together reduced GCAD. Clinically, these PKC isozyme regulators may be useful for organ preservation and prevention of ischemia-reperfusion injury and graft coronary artery disease in cardiac transplantation.

PKCδ activation mediates angiogenesis via NADPH oxidase activity in PC-3 prostate cancer cells†

PKCδ is generally known as a pro‐apoptotic and anti‐proliferative enzyme in human prostate cancer cells.

Competitive inhibitors and allosteric activators of protein kinase C isoenzymes : a personal account and progress report on transferring academic discoveries to the clinic

PKC (protein kinase C) isoenzymes are related protein kinases, involved in many signalling events in normal state and in disease. Basic research into identifying the molecular basis of PKC selectivity led to simple strategies to identify selective competitive inhibitor peptides and allosteric agonist peptides of individual PKC isoenzymes. The strategies and rationale used to identify these peptide regulators of protein-protein interaction may be applicable to other signalling events. Importantly, the PKC-regulating peptides proved to be useful pharmacological tools and may serve as drugs or drug leads for a variety of human diseases.

Pharmacological inhibition of βIIPKC is cardioprotective in late-stage hypertrophy

We previously found that in the hearts of hypertensive Dahl salt-sensitive rats, βIIPKC levels increase during the transition from compensated cardiac hypertrophy to cardiac dysfunction. Here we showed that a six-week treatment of these hypertensive rats with a βIIPKC-specific inhibitor, βIIV5-3, prolonged their survival by at least 6weeks, suppressed myocardial fibrosis and inflammation, and delayed the transition from compensated hypertrophy to cardiac dysfunction. In addition, changes in the levels of the Ca(2+)-handling proteins, SERCA2 and the Na(+)/Ca(2+) exchanger, as well as troponin I phosphorylation, seen in the control-treated hypertensive rats were not observed in the βΙΙPKC-treated rats, suggesting that βΙΙPKC contributes to the regulation of calcium levels in the myocardium. In contrast, treatment with the selective inhibitor of βIPKC, an alternative spliced form of βIIPKC, had no beneficial effects in these rats. We also found that βIIV5-3, but not βIV5-3, improved calcium handling in isolated rat cardiomyocytes and enhanced contractility in isolated rat hearts. In conclusion, our data using an in vivo model of cardiac dysfunction (late-phase hypertrophy), suggest that βIIPKC contributes to the pathology associated with heart failure and thus an inhibitor of βIIPKC may be a potential treatment for this disease.

Sustained Inhibition of ε Protein Kinase C Inhibits Vascular Restenosis After Balloon Injury and Stenting

Background— &egr; Protein kinase C (&egr;PKC) is involved in vascular smooth muscle cell (VSMC) activation, but little is known about its function in vascular pathology. We aimed at assessing the role of &egr;PKC in the development of restenosis. Methods and Results— Rat models of aortic balloon injury with or without subsequent stenting were used. Rats were treated with the selective &egr;PKC activator &psgr;&egr; receptor for activated protein kinase C (&psgr;&egr;RACK), the selective &egr;PKC inhibitor &egr;V1–2, or saline. Both down-stream cascades of the platelet-derived growth factor receptor via extracellular signal-regulated kinase and Akt, respectively, were evaluated in vivo and in VSMC cultures. Intimal hyperplasia with luminal obliteration developed in saline-treated balloon-injured rat aortas (20.3±8.0%), and &psgr;&egr;RACK significantly promoted neointima development (32.4±4.9%, P=0.033), whereas &egr;V1–2 significantly inhibited luminal narrowing (9.2±4.3%, P=0.039). &egr;PKC inhibition led to significantly reduced VSMC extracellular signal-regulated kinase phosphorylation in vivo, whereas Akt phosphorylation was not markedly affected. Neointimal proliferation in vivo and platelet-derived growth factor-induced VSMC proliferation/migration in vitro were significantly inhibited by &egr;V1–2. The inhibition of the platelet-derived growth factor pathway was mediated by inhibiting down-stream extracellular signal-regulated kinase and Akt phosphorylation. In vitro, &egr;V1–2 showed inhibitory properties on endothelial cell proliferation, but that did not prevent reendothelialization in vivo. &egr;V1–2 showed proapoptotic effects on VSMC in vitro. After stent implantation, luminal restenosis (quantified by optical coherence tomography imaging) was significantly reduced with &egr;V1–2 (8.0±2.0%) compared with saline (20.2±9.8%, P=0.028). Conclusions— &egr;PKC seems to be centrally involved in the development of neointimal hyperplasia. We suggest that &egr;PKC inhibition may be mediated via inhibition of extracellular signal-regulated kinase and Akt activation. &egr;PKC modulation may become a new therapeutic target against vascular restenosis.

Prevention and inhibition but not reversion of chronic allograft vasculopathy by FK778.

Background. This study aimed at investigating the efficacy of the novel immunosuppressant FK778 to prevent the development and progression of chronic allograft vasculopathy (CAV). Methods. Orthotopic aortic transplantations were performed in the PVG-to-ACI rat model and followed over the course of 120 days. Immunosuppression with FK778 (20 mg/kg) or sirolimus (2 mg/kg) was either started early or delayed when CAV was already present. Trough levels were monitored. Aortic luminal obliteration was quantified using computer morphometry and intragraft cytokine profiles were analyzed with Western Blotting. Donor-reactive antibodies were quantified by flow cytometry. Results. Untreated animals developed CAV with luminal obliteration of 25.2±13.6% and 41.4±23.3% after 80 and 120 days, respectively. Continuous immunosuppression with FK778 or sirolimus effectively prevented the development of vasculopathy. When the start of the immunosuppressive regimen was delayed until postoperative day 80, FK778 and sirolimus inhibited a progression of established CAV but did not reverse the luminal obliteration. Intragraft tumor growth factor-β activity increased over the course of time in untreated recipients but was significantly suppressed after continuous immunosuppression with either agent. Expression of platelet-derived growth factor, intercellular adhesion molecule-1, and vascular adhesion molecule-1 also was moderately suppressed. A stable elevation of donor-reactive IgG-antibody levels was found over 120 days in the absence of treatment. With FK778 or sirolimus, antibody levels were effectively decreased. FK778 was very well tolerated and only sirolimus showed side effects with elevation of BUN, cholesterol, triglycerides, and ALT after 120 days. Conclusions. FK778 prevents the development of CAV and inhibits a progression of established disease. It shows a similar efficacy but a safer drug profile when compared to sirolimus.

Alteration of gene expression during progression of hypertension-induced cardiac dysfunction in rats

Hypertension induced by high-salt diet in Dahl salt-sensitive rats leads to compensatory cardiac hypertrophy by approximately 11 wk, cardiac dysfunction at approximately 17 wk, and death from cardiac dysfunction at approximately 21 wk. It is unclear what molecular hallmarks distinguish the compensatory hypertrophy from the decompensated cardiac dysfunction phase. Here we compared the gene expression in rat cardiac tissue from the compensatory hypertrophic phase (11 wk, n = 6) with the cardiac dysfunction phase (17 wk, n = 6) and with age-matched normotensive controls. Messenger RNA levels of 93 genes, selected based on predicted association with cardiac dysfunction, were measured by quantitative real-time PCR. In the hypertrophic phase, the expression of three genes, atrial natriuretic peptide (ANP; P = 0.0089), brain natriuretic peptide (P = 0.0012), and endothelin-1 precursor (P = 0.028), significantly increased, whereas there was decreased expression of 24 other genes including SOD2 (P = 0.0148), sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (P = 0.0002), and ryanodine receptor 2 (P = 0.0319). In the subsequent heart cardiac dysfunction phase, the expression of an additional 20 genes including inducible nitric oxide synthase (NOS; P = 0.0135), angiotensin I-converting enzyme (P = 0.0082), and IL-1beta (P < 0.0001) increased, whereas the expression of seven genes decreased compared with those of age-matched controls. Furthermore, the expression of 22 genes, including prepro-endothelin-1, ANP, angiotensin I-converting enzyme, beta(1)-adrenergic receptor, SOD2, and endothelial NOS, significantly changed in the cardiac dysfunction phase compared with the compensatory hypertrophic phase. Finally, principal component analysis successfully segregated animals with decompensatory cardiac dysfunction from controls, as well as from animals at the compensated hypertrophy phase, suggesting that we have identified molecular markers for each stage of the disease.