Dichloroacetate prevents restenosis in preclinical animal models of vessel injury.

Despite the introduction of antiproliferative drug-eluting stents, coronary heart disease remains the leading cause of death in the United States. In-stent restenosis and bypass graft failure are characterized by excessive smooth muscle cell (SMC) proliferation and concomitant myointima formation with luminal obliteration. Here we show that during the development of myointimal hyperplasia in human arteries, SMCs show hyperpolarization of their mitochondrial membrane potential (ΔΨm) and acquire a temporary state with a high proliferative rate and resistance to apoptosis. Pyruvate dehydrogenase kinase isoform 2 (PDK2) was identified as a key regulatory protein, and its activation proved necessary for relevant myointima formation. Pharmacologic PDK2 blockade with dichloroacetate or lentiviral PDK2 knockdown prevented ΔΨm hyperpolarization, facilitated apoptosis and reduced myointima formation in injured human mammary and coronary arteries, rat aortas, rabbit iliac arteries and swine (pig) coronary arteries. In contrast to several commonly used antiproliferative drugs, dichloroacetate did not prevent vessel re-endothelialization. Targeting myointimal ΔΨm and alleviating apoptosis resistance is a novel strategy for the prevention of proliferative vascular diseases.

Local MicroRNA Modulation Using a Novel Anti-miR-21-Eluting Stent Effectively Prevents Experimental In-Stent Restenosis.

OBJECTIVE: Despite advances in stent technology for vascular interventions, in-stent restenosis (ISR) because of myointimal hyperplasia remains a major complication. APPROACH AND RESULTS: We investigated the regulatory role of microRNAs in myointimal hyperplasia/ISR, using a humanized animal model in which balloon-injured human internal mammary arteries with or without stenting were transplanted into Rowett nude rats, followed by microRNA profiling. miR-21 was the only significantly upregulated candidate. In addition, miR-21 expression was increased in human tissue samples from patients with ISR compared with coronary artery disease specimen. We systemically repressed miR-21 via intravenous fluorescein-tagged-locked nucleic acid-anti-miR-21 (anti-21) in our humanized myointimal hyperplasia model. As expected, suppression of vascular miR-21 correlated dose dependently with reduced luminal obliteration. Furthermore, anti-21 did not impede reendothelialization. However, systemic anti-miR-21 had substantial off-target effects, lowering miR-21 expression in liver, heart, lung, and kidney with concomitant increase in serum creatinine levels. We therefore assessed the feasibility of local miR-21 suppression using anti-21-coated stents. Compared with bare-metal stents, anti-21-coated stents effectively reduced ISR, whereas no significant off-target effects could be observed. CONCLUSION: This study demonstrates the efficacy of an anti-miR-coated stent for the reduction of ISR.

Orthotopic tracheal transplantation using human bronchus: an original xenotransplant model of obliterative airway disorder.

Bronchiolitis obliterans syndrome (BOS) is a main cause of allograft dysfunction and mortality after lung transplantation (LTx). A better understanding of BOS pathogenesis is needed to overcome this treatment-refractory complication. Orthotopic tracheal transplantation using human bronchus was performed in Brown Norway (BN) and nude (RNU) rats. Allografts were recovered in both strains at Day 7 (BN7 , n = 6; RNU7 , n = 7) or Day 28 (BN28 , n = 6; RNU28 , n = 6). Immune response of the host against the bronchial graft was assessed. Human samples from BOS patients were used to compare with the histological features of the animal model. Obstruction of the allograft lumen associated with significant infiltration of CD3+ and CD68+ cells was observed in the BN group on Day 28. Immune response from type 1 T-helper cells against the tracheal xenograft was higher in BN animals compared to nude animals on Days 7 and 28 (P < 0.001 and P = 0.035). Xenoreactive antibodies were significantly higher at Day 7 (IgM) and Day 28 (IgG) in the BN group compared to RNU (respectively, 37.6 ± 6.5 vs. 5.8 ± 0.7 mean fluorescence, P = 0.039; and 22.4 ± 3.8 vs. 6.9 ± 1.6 mean fluorescence, P = 0.011). Immunocompetent animals showed a higher infiltration of S100A4+ cells inside the bronchial wall after 28 days, associated with cartilage damage ranging from invasion to complete destruction. In vitro expression of S100A4 by human fibroblasts was higher when stimulated by mononuclear cells (MNCs) from BN rats than from RNU (2.9 ± 0.1 vs. 2.4 ± 0.1 mean fluorescence intensity, P = 0.005). Similarly, S100A4 was highly expressed in response to human MNCs compared to stimulation by T-cell-depleted human MNCs (4.3 ± 0.2 vs. 2.7 ± 0.1 mean fluorescence intensity, P < 0.001). Obliterative bronchiolitis has been induced in a new xenotransplant model in which chronic airway obstruction was associated with immune activation against the xenograft. Cartilage infiltration by S100A4+ cells might be stimulated by T cells.

Localization of Islet-1-positive cells in the healthy and infarcted adult murine heart.

RATIONALE: The transcription factor Islet-1 is a marker of cardiovascular progenitors during embryogenesis. The isolation of Islet-1-positive (Islet-1(+)) cells from early postnatal hearts suggested that Islet-1 also marks cardiac progenitors in adult life. OBJECTIVE: We investigated the distribution and identity of Islet-1(+) cells in adult murine heart and evaluated whether their number or distribution change with age or after myocardial infarction. METHODS AND RESULTS: Distribution of Islet-1(+) cells in adult heart was investigated using gene targeted mice with nuclear ß-galactosidase inserted into the Islet-1 locus. nLacZ-positive cells were only present in 3 regions of the adult heart: clusters in the interatrial septum and around the pulmonary veins, scattered within the wall of the great vessels, and a strictly delimited cluster between the right atrium and superior vena cava. Islet-1(+) cells in the first type of clusters coexpressed markers for parasympathetic neurons. Positive cells in the great arteries coexpressed smooth muscle actin and myosin heavy chain, indicating a smooth muscle cell identity. Very few Islet-1(+) cells within the outflow tract expressed the cardiomyocyte marker α-actinin. Islet-1(+) cells in the right atrium coexpressed the sinoatrial node pacemaker cell marker HCN4. Cell number and localization remained unchanged between 1 to 18 months of age. Consistently Islet-1 mRNA was detected in human sinoatrial node. Islet-1(+) cells could not be detected in the infarct zone 2 to 28 days after myocardial infarction, aside from 10 questionable cells in 1/13 hearts. CONCLUSIONS: Our results identify Islet-1 as a novel marker of the adult sinoatrial node and do not provide evidence for Islet-1(+) cells to serve as cardiac progenitors.

No effect of thymosin beta-4 on the expression of the transcription factor Islet-1 in the adult murine heart.

The transcription factor Islet-1 marks a progenitor cell population of the second heart field during cardiogenesis. In the adult heart Islet-1 expression is limited to the sinoatrial node, the ventricular outflow tract, and parasympathetic ganglia. The regenerative effect in the injured mouse ventricle of thymosin beta-4 (TB4), a 43-aminoacid peptide, was associated with increased Islet-1 immunostaining, suggesting the induction of an Islet-1-positive progenitor state by TB4. Here we aimed to reassess this effect in a genetic model. Mice from the reporter mouse line Isl1-nLacZ were primed with TB4 and subsequently underwent myocardial infarction. Islet-1 expression was assessed 2, 7, and 14 days after infarction. We detected only a single Islet-1+ cell in 8 TB4 treated and infarcted hearts which located outside of the sinoatrial node, the outflow tract or cardiac ganglia (in ~2500 sections). Two cells were identified in 5 control infarcted hearts. TB4 did not induce LacZ positivity in ventricular explants cultures of Isl1-nLacZ mice nor did it affect the density of LacZ+ cells in explant cultures of nLacZ+ regions of the heart. In summary, we found no evidence that TB4 reactivates Islet-1 expression in adult mouse ventricle.

Dichloroacetate affects proliferation but not apoptosis in canine mammary cell lines.

Targeting mitochondrial energy metabolism is a novel approach in cancer research and can be traced back to the description of the Warburg effect. Dichloroacetate, a controversially discussed subject of many studies in cancer research, is a pyruvate dehydrogenase kinase inhibitor. Dichloroacetate causes metabolic changes in cancerous glycolysis towards oxidative phosphorylation via indirect activation of pyruvate dehydrogenase in mitochondria. Canine mammary cancer is frequently diagnosed but after therapy prognosis still remains poor. In this study, canine mammary carcinoma, adenoma and non-neoplastic mammary gland cell lines were treated using 10 mM Dichloroacetate. The effect on cell number, lactate release and PDH expression and cell respiration was investigated. Further, the effect on apoptosis and several apoptotic proteins, proliferation, and microRNA expression was evaluated. Dichloroacetate was found to reduce cell proliferation without inducing apoptosis in all examined cell lines.

The effect of dichloroacetate in canine prostate adenocarcinomas and transitional cell carcinomas in vitro.

The Warburg effect describes the ability of cancer cells to produce energy via aerobic glycolysis instead of oxidative phosphorylation of pyruvate. This deviation in mitochondrial metabolism inhibits apoptosis, allowing increased proliferation under conditions of reduced oxygen levels. Dichloroacetate (DCA) was successfully used in several human cancer cell lines to reactivate oxidative phosphorylation in mitochondria. The aim of this study was the characterization and response of canine cancer cell lines after DCA exposure. The effect of 10 mM DCA was characterized in vitro on a set of six canine prostate adenocarcinoma and transitional cell carcinoma (TCC) derived cell lines. Cell counts, lactate levels, apoptosis, expression of apoptotic proteins, survival factors and different miRNAs were analyzed. Additionally, metabolic activity, mitochondrial activity and proliferation were investigated. DCA significantly decreased cell number of all but one utilized cell lines and leads to a significant reduction of lactate release. Decreased survivin levels were found in all cell lines, two of which presented a significant reduction in metabolic activity. Increased miR-375 levels were measured in all TCC cell lines. Reactivation of pyruvate dehydrogenase and an elevated mitochondrial activity appear to induce the transition from aerobic glycolysis back to oxidative phosphorylation. Further, these results display that DCA treatment has a suppressant effect on proliferation of canine cancer cells.

The Selective JAK1/3-Inhibitor R507 Mitigates Obliterative Airway Disease Both With Systemic Administration and Aerosol Inhalation.

BACKGROUND: The efficacy of selective Janus kinase 1/3 inhibitor R507 to prevent obliterative airway disease was analyzed in preclinical airway transplantation models. METHODS: Orthotopic trachea transplantations were performed between Lewis donors and Brown Norway rat recipients. Oral everolimus (4 mg/kg once per day) or oral respective inhaled R507 (60 mg/kg twice per day, each) was used for immunosuppression. Grafts were retrieved after 6 or 60 days. Toxicity and anti-inflammatory effects of R507 were analyzed on human airway epithelial cells. RESULTS: In 6-day animals, oral and inhaled R507 more potently diminished mononuclear graft infiltration than everolimus and preserved ciliated pseudostratified columnar respiratory epithelium. Everolimus and R507 similarly suppressed systemic cellular and humoral immune activation. In untreated rats, marked obliterative airway disease developed over 60 days. Oral and inhaled R507 was significantly more effective in reducing airway obliteration and preserved the morphology of the airway epithelium. Luciferase-positive donors revealed that a substantial amount of smooth muscle cells within the obliterative tissue was of donor origin. Only everolimus but not R507, adversely altered kidney function and lipid profiles. The R507 aerosol did not show airway toxicity in vitro but effectively suppressed activation of inflammatory signaling pathways induced by IL-1ß. CONCLUSIONS: The Janus kinase 1/3 inhibitor R507 is a very well-tolerated immunosuppressant that similarly diminished obliterative airway disease with systemic or inhaled administration.

SCNT-derived ESCs with mismatched mitochondria trigger an immune response in allogeneic hosts.

The generation of pluripotent stem cells by somatic cell nuclear transfer (SCNT) has recently been achieved in human cells and sparked new interest in this technology. The authors reporting this methodical breakthrough speculated that SCNT would allow the creation of patient-matched embryonic stem cells, even in patients with hereditary mitochondrial diseases. However, herein we show that mismatched mitochondria in nuclear-transfer-derived embryonic stem cells (NT-ESCs) possess alloantigenicity and are subject to immune rejection. In a murine transplantation setup, we demonstrate that allogeneic mitochondria in NT-ESCs, which are nucleus-identical to the recipient, may trigger an adaptive alloimmune response that impairs the survival of NT-ESC grafts. The immune response is adaptive, directed against mitochondrial content, and amenable for tolerance induction. Mitochondrial alloantigenicity should therefore be considered when developing therapeutic SCNT-based strategies.

Inducing myointimal hyperplasia versus atherosclerosis in mice: an introduction of two valid models.

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models - both resulting in artery stenosis but each underlying a different pathway of development - are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked.

How to build your own coronary anastomosis simulator from scratch.

OBJECTIVES: Gaining cardiac surgical competence is a complex, multifactorial process that may take years of experience and on-the-job training. It is critical to provide suitable educational opportunities to gain the necessary knowledge, judgment and skills. In response to the multitude of factors (e.g. European Working Time Directive) currently influencing cardiac surgical training, there have been concerted efforts to reform training practices. Simulation plays an increasingly important role in the educational process and serves to fill the most important gap in the current training model, i.e. operative exposure. Therefore, a contest has been written out for cardiac surgical trainees to construct their own coronary anastomosis simulator using everyday materials. METHODS: Cardiac surgical trainees were invited to construct their own coronary anastomosis simulator. An international jury of cardiac surgeons assessed the simulator and its presentation according to preset developmental criteria (low fidelity concept, innovative character, general presentation and description, general attractiveness to the scholar, ergonomical issues, perceived haptics, number of applicable components, transportability, ease of construction, repeatability and overall costs of the simulator). RESULTS: Six prototypes of simulators built by cardiac surgical trainees were generated. A general evaluation of each simulator prototype is provided according to the preset developmental criteria. CONCLUSIONS: All simulator prototypes have provided a considerable contribution to the field of surgical simulation. By designing simulator prototypes, the trainees have demonstrated their 'out of the box' thinking capability, which is of paramount importance for the development of future innovative surgical techniques and procedures. The Valladolid cardiac team coronary anastomosis simulator box was selected for the EACTS Ethicon Simulation Award 2011. This project will be mass produced and distributed to the participants of structured simulation sessions for coronary anastomoses.

The potassium channel KCa3.1 as new therapeutic target for the prevention of obliterative airway disease.

BACKGROUND: The calcium-activated potassium channel KCa3.1 is critically involved in T-cell activation as well as in the proliferation of smooth muscle cells and fibroblasts. We sought to investigate whether KCa3.1 contributes to the pathogenesis of obliterative airway disease (OAD) and whether knockout or pharmacologic blockade would prevent the development of OAD. METHODS: Tracheas from CBA donors were heterotopically transplanted into the omentum of C57Bl/6J wild-type or KCa3.1 mice. C57Bl/6J recipients were either left untreated or received the KCa3.1 blocker TRAM-34 (120 mg/kg/day). Histopathology and immunologic assays were performed on postoperative day 5 or 28. RESULTS: Subepithelial T-cell and macrophage infiltration on postoperative day 5, as seen in untreated allografts, was significantly reduced in the KCa3.1 and TRAM-34 groups. Also, systemic Th1 activation was significantly and Th2 mildly reduced by KCa3.1 knockout or blockade. After 28 days, luminal obliteration of tracheal allografts was reduced from 89%±21% in untreated recipients to 53%±26% (P=0.010) and 59%±33% (P=0.032) in KCa3.1 and TRAM-34-treated animals, respectively. The airway epithelium was mostly preserved in syngeneic grafts, mostly destroyed in the KCa3.1 and TRAM-34 groups, and absent in untreated allografts. Allografts triggered an antibody response in untreated recipients, which was significantly reduced in KCa3.1 animals. KCa3.1 was detected in T cells, airway epithelial cells, and myofibroblasts. TRAM-34 dose-dependently suppressed proliferation of wild-type C57B/6J splenocytes but did not show any effect on KCa3.1 splenocytes. CONCLUSIONS: Our findings suggest that KCa3.1 channels are involved in the pathogenesis of OAD and that KCa3.1 blockade holds promise to reduce OAD development.

Immunological properties of extraembryonic human mesenchymal stromal cells derived from gestational tissue.

Mesenchymal stromal cells (MSCs) have been isolated from many tissues, including gestational tissue. To date, a study comparing the properties and suitability of these cells in cell-based therapies is lacking. In this study, we compared the phenotype, proliferation rate, migration, immunogenicity, and immunomodulatory capabilities of human MSCs derived from umbilical cord lining (CL-MSCs), umbilical cord blood (CB-MSCs), placenta (P-MSCs), and Wharton's jelly (WJ-MSCs). Differences were noted in differentiation, proliferation, and migration, with CL-MSCs showing the highest proliferation and migration rates resulting in prolonged survival in immunodeficient mice. Moreover, CL-MSCs showed a prolongation in survival in xenogeneic BALB/c mice, which was attributed to their ability to dampen TH1 and TH2 responses. Weaker human cellular immune responses were detected against CL-MSCs and P-MSCs, which were correlated with their lower HLA I expression. Furthermore, HLA II was upregulated less substantially by CL-MSCs and CB-MSCs after IFN-γ stimulation. MSC types did not differ in indolamine 2,3-dioxygenase (IDO) expression after IFN-γ stimulation. Despite their lower IDO, HLA-G, and TGF-ß1 expression, only CL-MSCs were able to reduce the release of IFN-γ by lymphocytes in a mixed lymphocyte reaction. In summary, CL-MSCs showed the best characteristics for cell-based strategies, as they are hypo-immunogenic and show high proliferation and migration rates. In addition, these studies show for the first time that although immunomodulatory molecules HLA-G, HLA-E, and TGF-ß play an important role in MSC immune evasion, basal and induced HLA expression seems to be decisive in determining the immunogenicity of MSCs.

Significant reduction of acute cardiac allograft rejection by selective janus kinase-1/3 inhibition using R507 and R545.

BACKGROUND: Selective inhibition of lymphocyte activation through abrogation of signal 3-cytokine transduction emerges as a new strategy for immunosuppression. This is the first report on the novel Janus kinase (JAK)1/3 inhibitors R507 and R545 for prevention of acute allograft rejection. METHODS: Pharmacokinetic and in vitro enzyme inhibition assays were performed to characterize the drugs. Heterotopic Brown Norway-Lewis heart transplantations were performed to study acute cardiac allograft rejection, graft survival, suppression of cellular host responsiveness, and antibody production. Therapeutic and subtherapeutic doses of R507 (60 and 15 mg/kg 2 times per day) and R545 (20 and 5 mg/kg 2 times per day) were compared with those of tacrolimus (Tac; 4 and 1 mg/kg once per day). RESULTS: Plasma levels of R507 and R545 were sustained high for several hours. Cell-based enzyme assays showed selective inhibition of JAK1/3-dependent pathways with 20-fold or greater selectivity over JAK2 and Tyrosine kinase 2 kinases. After heart transplantation, both JAK1/3 inhibitors reduced early mononuclear graft infiltration, even significantly more potent than Tac. Intragraft interferon-γ release was significantly reduced by R507 and R545, and for interleukin-10 suppression, they were even significantly more potent than Tac. Both JAK1/3 inhibitors and Tac were similarly effective in reducing the host Th1 and Th2, but not Th17, responsiveness and similarly prevented donor-specific immunoglobulin M antibody production. Subtherapeutic and therapeutic R507 and R545 doses prolonged the mean graft survival and were similarly effective as 1 and 4 mg/kg Tac, respectively. In combination regimens, however, only R507 showed highly beneficial synergistic drug interactions with Tac. CONCLUSIONS: Both R507 and R545 are potent novel immunosuppressants with favorable pharmacokinetics and high JAK1/3 selectivity, but only R507 synergistically interacts with Tac.

Immunogenicity and immunomodulatory properties of umbilical cord lining mesenchymal stem cells.

We here present an immunologic head-to-head comparison between human umbilical cord lining mesenchymal stem cells (clMSCs) and adult bone marrow MSCs (bmMSCs) from patients >65 years of age. clMSCs had significantly lower HLA class I expression, higher production of tolerogenic TGF-ß and IL-10, and showed significantly faster proliferation. In vitro activation of allogeneic lymphocytes and xenogeneic in vivo immune activation was significantly stronger with bmMSCs, whereas immune recognition of clMSCs was significantly weaker. Thus, bmMSCs were more quickly rejected in immunocompetent mice. IFN-γ at 25 ng/ml increased both immunogenicity by upregulation of HLA class I/ HLA-DR expression and tolerogenicity by increasing intracellular HLA-G and surface HLA-E expression, augmenting TGF-ß and IL-10 release, and inducing indoleamine 2,3-dioxygenase (IDO) expression. Higher concentrations of IFN-γ (>50 ng/ml) further enhanced the immunosuppressive phenotype of clMSCs, more strongly downregulating HLA-DR expression and further increasing IDO production (at 500 ng/ml). The net functional immunosuppressive efficacy of MSCs was tested in mixed lymphocyte cultures. Although both clMSCs and bmMSCs significantly reduced in vitro immune activation, clMSCs were significantly more effective than bmMSCs. The veto function of both MSC lines was enhanced in escalating IFN-γ environments. In conclusion, clMSCs show a more beneficial immunogeneic profile and stronger overall immunosuppressive potential than aged bmMSCs.

Orthotopic aortic transplantation: a rat model to study the development of chronic vasculopathy.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP). The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured. Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model. In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated. The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting. A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP. This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models.