X. Hua

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.

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 &bgr;-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 &agr;-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.

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 Whartons 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-β1 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-β play an important role in MSC immune evasion, basal and induced HLA expression seems to be decisive in determining the immunogenicity of MSCs.

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.

Immunobiology of naïve and genetically modified HLA-class-I-knockdown human embryonic stem cells.

Human embryonic stem cells (hESCs) can serve as a universal cell source for emerging cell or tissue replacement strategies, but immune rejection of hESC derivatives remains an unsolved problem. Here, we sought to describe the mechanisms of rejection for naïve hESCs and upon HLA class I (HLA I) knockdown (hESCKD). hESCs were HLA I-positive but negative for HLA II and co-stimulatory molecules. Transplantation of naïve hESC into immunocompetent Balb/c mice induced substantial T helper cell 1 and 2 (Th1 and Th2) responses with rapid cell death, but hESCs survived in immunodeficient SCID-beige recipients. Histology revealed mainly macrophages and T cells, but only scattered natural killer (NK) cells. A surge of hESC-specific antibodies against hESC class I, but not class II antigens, was observed. Using HLA I RNA interference and intrabody technology, HLA I surface expression of hESCKD was 88%–99% reduced. T cell activation after hESCKD transplantation into Balb/c was significantly diminished, antibody production was substantially alleviated, the levels of graft-infiltrating immune cells were reduced and the survival of hESCKD was prolonged. Because of their very low expression of stimulatory NK ligands, NK-susceptibility of naïve hESCs and hESCKD was negligible. Thus, HLA I recognition by T cells seems to be the primary mechanism of hESC recognition, and T cells, macrophages and hESC-specific antibodies participate in hESC killing.

Human Leukocyte Antigen I Knockdown Human Embryonic Stem Cells Induce Host Ignorance and Achieve Prolonged Xenogeneic Survival

Background— Although human embryonic stem cells (hESC) have enormous potential for cell replacement therapy of heart failure, immune rejection of hESC derivatives inevitably would occur after transplantation. We therefore aimed to generate a hypoantigeneic hESC line with improved survival characteristics. Methods and Results— Using various in vivo, nonischemic, hindlimb xenotransplant models (immunocompetent and defined immunodefective mouse strains) as well as human in vitro T-cell and natural killer (NK)-cell assays, we revealed a central role for T cells in mediating hESC rejection. The NK-cell susceptibility of hESC in vivo was found to be low, and the NK response to hESC challenge in vitro was negligible. To reduce the antigenicity of hESC, we successfully generated human leukocyte antigen (HLA) I knockdown cells (hESCsiRNA+IB) using both HLA I RNA interference (siRNA) and intrabody (IB) technology. HLA I expression was ≈99% reduced after 7 days and remained low for weeks. Cellular immune recognition of these hESCsiRNA+IB was strongly reduced in both xenogeneic and allogeneic settings. Immune rejection was profoundly mitigated after hESCsiRNA+IB transplantation into immunocompetent mice, and even long-term graft survival was achieved in one third of the animals without any immunosuppression. The survival benefit of hESCsiRNA+IB was further confirmed under ischemic conditions in a left anterior descending coronary artery ligation model. Conclusions— HLA I knockdown hESCsiRNA+IB provoke T-cell ignorance and experience largely mitigated xenogeneic rejection. By generating hypoantigeneic hESC lines, the generation of acceptable hESC derivatives may become a practical concept and push cell replacement strategies forward.

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.

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-&ggr; 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.

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.

Immunobiology of Fibrin-Based Engineered Heart Tissue

Different tissue‐engineering approaches have been developed to induce and promote cardiac regeneration; however, the impact of the immune system and its responses to the various scaffold components of the engineered grafts remains unclear. Fibrin‐based engineered heart tissue (EHT) was generated from neonatal Lewis (Lew) rat heart cells and transplanted onto the left ventricular surface of three different rat strains: syngeneic Lew, allogeneic Brown Norway, and immunodeficient Rowett Nude rats. Interferon spot frequency assay results showed similar degrees of systemic immune activation in the syngeneic and allogeneic groups, whereas no systemic immune response was detectable in the immunodeficient group (p < .001 vs. syngeneic and allogeneic). Histological analysis revealed much higher local infiltration of CD3‐ and CD68‐positive cells in syngeneic and allogeneic rats than in immunodeficient animals. Enzyme‐linked immunospot and immunofluorescence experiments revealed matrix‐directed TH1‐based rejection in syngeneic recipients without collateral impairment of heart cell survival. Bioluminescence imaging was used for in vivo longitudinal monitoring of transplanted luciferase‐positive EHT constructs. Survival was documented in syngeneic and immunodeficient recipients for a period of up to 110 days after transplant, whereas in the allogeneic setting, graft survival was limited to only 14 ± 1 days. EHT strategies using autologous cells are promising approaches for cardiac repair applications. Although fibrin‐based scaffold components elicited an immune response in our studies, syngeneic cells carried in the EHT were relatively unaffected.