The ubiquitin-proteasome system: A potential therapeutic target for heart failure.

The rising incidence of heart failure (HF) is one of the biggest challenges in cardiovascular medicine. The persistent shortage of donor organs for transplantation has led to an expanding application of left ventricular assist devices as a bridge to recovery. Accumulating evidence suggests that the ubiquitin-proteasome system (UPS), which is responsible for protein degradation, plays a direct role in cardiac hypertrophy and HF and is impacted by mechanical unloading. The UPS system also plays a role in the cardiac regulation of apoptosis, cell mass, and sarcomere quality control. Furthermore, it is a key regulator of ß2-adrenergic signaling, cell excitability, and conductance. In this review, we discuss the roles of the UPS in cardiac health and disease, including its roles in the pathologic hypertrophy associated with HF and its reversal during mechanical unloading. Finally, we suggest future areas of research, including possible therapeutic strategies for reversing cardiac remodeling by targeting the UPS.

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.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part II. In Vivo Imaging of Bone Marrow Stromal Cells in Swine with PET/CT and MR Imaging.

Purpose To quantitatively determine the limit of detection of marrow stromal cells (MSC) after cardiac cell therapy (CCT) in swine by using clinical positron emission tomography (PET) reporter gene imaging and magnetic resonance (MR) imaging with cell prelabeling. Materials and Methods Animal studies were approved by the institutional administrative panel on laboratory animal care. Seven swine received 23 intracardiac cell injections that contained control MSC and cell mixtures of MSC expressing a multimodality triple fusion (TF) reporter gene (MSC-TF) and bearing superparamagnetic iron oxide nanoparticles (NP) (MSC-TF-NP) or NP alone. Clinical MR imaging and PET reporter gene molecular imaging were performed after intravenous injection of the radiotracer fluorine 18-radiolabeled 9-[4-fluoro-3-(hydroxyl methyl) butyl] guanine ((18)F-FHBG). Linear regression analysis of both MR imaging and PET data and nonlinear regression analysis of PET data were performed, accounting for multiple injections per animal. Results MR imaging showed a positive correlation between MSC-TF-NP cell number and dephasing (dark) signal (R(2) = 0.72, P = .0001) and a lower detection limit of at least approximately 1.5 × 10(7) cells. PET reporter gene imaging demonstrated a significant positive correlation between MSC-TF and target-to-background ratio with the linear model (R(2) = 0.88, P = .0001, root mean square error = 0.523) and the nonlinear model (R(2) = 0.99, P = .0001, root mean square error = 0.273) and a lower detection limit of 2.5 × 10(8) cells. Conclusion The authors quantitatively determined the limit of detection of MSC after CCT in swine by using clinical PET reporter gene imaging and clinical MR imaging with cell prelabeling. (©) RSNA, 2016 Online supplemental material is available for this article.

Multipotency and cardiomyogenic potential of human adipose-derived stem cells from epicardium, pericardium, and omentum.

BACKGROUND: Acute myocardial infarction (MI) leads to an irreversible loss of proper cardiac function. Application of stem cell therapy is an attractive option for MI treatment. Adipose tissue has proven to serve as a rich source of stem cells (ADSCs). Taking into account the different morphogenesis, anatomy, and physiology of adipose tissue, we hypothesized that ADSCs from different adipose tissue depots may exert a diverse multipotency and cardiogenic potential. METHODS: The omental, pericardial, and epicardial adipose tissue samples were obtained from organ donors and patients undergoing heart transplantation at our institution. Human foreskin fibroblasts were used as the control group. Isolated ADSCs were analyzed for adipogenic and osteogenic differentiation capacity and proliferation potential. The immunophenotype and constitutive gene expression of alkaline phosphatase (ALP), GATA4, Nanog, and OCT4 were analyzed. DNA methylation inhibitor 5-azacytidine was exposed to the cells to stimulate the cardiogenesis. Finally, reprogramming towards cardiomyocytes was initiated with exogenous overexpression of seven transcription factors (ESRRG, GATA4, MEF2C, MESP1, MYOCD, TBX5, ZFPM2) previously applied successfully for fibroblast transdifferentiation toward cardiomyocytes. Expression of cardiac troponin T (cTNT) and alpha-actinin (Actn2) was analyzed 3 weeks after initiation of the cardiac differentiation. RESULTS: The multipotent properties of isolated plastic adherent cells were confirmed with expression of CD29, CD44, CD90, and CD105, as well as successful differentiation toward adipocytes and osteocytes; with the highest osteogenic and adipogenic potential for the epicardial and omental ADSCs, respectively. Epicardial ADSCs demonstrated a lower doubling time as compared with the pericardium and omentum-derived cells. Furthermore, epicardial ADSCs revealed higher constitutive expression of ALP and GATA4. Increased Actn2 and cTNT expression was observed after the transduction of seven reprogramming factors, with the highest expression in the epicardial ADSCs, as compared with the other ADSC subtypes and fibroblasts. CONCLUSIONS: Human epicardial ADSCs revealed a higher cardiomyogenic potential as compared with the pericardial and omental ADSC subtypes as well as the fibroblast counterparts. Epicardial ADSCs may thus serve as the valuable subject for further studies on more effective methods of adult stem cell differentiation toward cardiomyocytes.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part I. Reporter Gene Design, Characterization, and Optical in Vivo Imaging of Bone Marrow Stromal Cells after Myocardial Infarction.

Purpose To use multimodality reporter-gene imaging to assess the serial survival of marrow stromal cells (MSC) after therapy for myocardial infarction (MI) and to determine if the requisite preclinical imaging end point was met prior to a follow-up large-animal MSC imaging study. Materials and Methods Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice (n = 19) that had experienced MI were injected with bone marrow-derived MSC that expressed a multimodality triple fusion (TF) reporter gene. The TF reporter gene (fluc2-egfp-sr39ttk) consisted of a human promoter, ubiquitin, driving firefly luciferase 2 (fluc2), enhanced green fluorescent protein (egfp), and the sr39tk positron emission tomography reporter gene. Serial bioluminescence imaging of MSC-TF and ex vivo luciferase assays were performed. Correlations were analyzed with the Pearson product-moment correlation, and serial imaging results were analyzed with a mixed-effects regression model. Results Analysis of the MSC-TF after cardiac cell therapy showed significantly lower signal on days 8 and 14 than on day 2 (P = .011 and P = .001, respectively). MSC-TF with MI demonstrated significantly higher signal than MSC-TF without MI at days 4, 8, and 14 (P = .016). Ex vivo luciferase activity assay confirmed the presence of MSC-TF on days 8 and 14 after MI. Conclusion Multimodality reporter-gene imaging was successfully used to assess serial MSC survival after therapy for MI, and it was determined that the requisite preclinical imaging end point, 14 days of MSC survival, was met prior to a follow-up large-animal MSC study. (©) RSNA, 2016 Online supplemental material is available for this article.

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.

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.

Immunobiology of fibrin-based engineered heart tissue.

UNLABELLED: 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. SIGNIFICANCE: An initial insight into immunological consequences after transplantation of engineered heart tissue was gained through this study. Most important, this study was able to demonstrate cell survival despite rejection of matrix components. Generation of syngeneic human engineered heart tissue, possibly using human induced pluripotent stem cell technology with subsequent directed rejection of matrix components, may be a potential future approach to replace diseased myocardium.

NOTCH1 regulates matrix gla protein and calcification gene networks in human valve endothelium.

Valvular and vascular calcification are common causes of cardiovascular morbidity and mortality. Developing effective treatments requires understanding the molecular underpinnings of these processes. Shear stress is thought to play a role in inhibiting calcification. Furthermore, NOTCH1 regulates vascular and valvular endothelium, and human mutations in NOTCH1 can cause calcific aortic valve disease. Here, we determined the genome-wide impact of altering shear stress and NOTCH signaling on human aortic valve endothelium. mRNA-sequencing of primary human aortic valve endothelial cells (HAVECs) with or without knockdown of NOTCH1, in the presence or absence of shear stress, revealed NOTCH1-dependency of the atherosclerosis-related gene connexin 40 (GJA5), and numerous repressors of endochondral ossification. Among these, matrix gla protein (MGP) is highly expressed in aortic valve and vasculature, and inhibits soft tissue calcification by sequestering bone morphogenetic proteins (BMPs). Altering NOTCH1 levels affected MGP mRNA and protein in HAVECs. Furthermore, shear stress activated NOTCH signaling and MGP in a NOTCH1-dependent manner. NOTCH1 positively regulated endothelial MGP in vivo through specific binding motifs upstream of MGP. Our studies suggest that shear stress activates NOTCH1 in primary human aortic valve endothelial cells leading to downregulation of osteoblast-like gene networks that play a role in tissue calcification.

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.

Long-term outcomes of septal reduction for obstructive hypertrophic cardiomyopathy.

BACKGROUND: Surgical myectomy and alcohol septal ablation (ASA) aim to decrease left ventricular outflow tract (LVOT) gradient in hypertrophic cardiomyopathy (HCM). Outcome of myectomy beyond 10 years has rarely been described. We describe 20 years of follow-up of surgical myectomy and 5 years of follow-up for ASA performed for obstructive HCM. METHODS: We studied 171 patients who underwent myectomy for symptomatic LVOT obstruction between 1972 and 2006. In addition, we studied 52 patients who underwent ASA for the same indication and who declined surgery. Follow-up of New York Heart Association (NYHA) functional class, echocardiographic data, and vital status were obtained from patient records. Mortality rates were compared with expected mortality rates of age- and sex-matched populations. RESULTS: Surgical myectomy improved NYHA class (2.74±0.65 to 1.54±0.74, p<0.001), reduced resting gradient (67.4±43.4mmHg to 11.2±16.4mmHg, p<0.001), and inducible LVOT gradient (98.1±34.7mmHg to 33.6±34.9mmHg, p<0.001). Similarly, ASA improved functional class (2.99±0.35 to 1.5±0.74, p<0.001), resting gradient (67.1±26.9mmHg to 23.9±29.4mmHg, p<0.001) and provoked gradient (104.4±34.9mmHg to 35.5±38.6mmHg, p<0.001). Survival after myectomy at 5, 10, 15, and 20 years of follow-up was 92.9%, 81.1%, 68.9%, and 47.5%, respectively. Of note, long-term survival after myectomy was lower than for the general population [standardized mortality ratio (SMR)=1.40, p<0.005], but still compared favorably with historical data from non-operated HCM patients. Survival after ASA at 2 and 5 years was 97.8% and 94.7%, respectively. Short-term (5 year) survival after ASA (SMR=0.61, p=0.48) was comparable to that of the general population. CONCLUSION: Long-term follow-up of septal reduction strategies in obstructive HCM reveals that surgical myectomy and ASA are effective for symptom relief and LVOT gradient reduction and are associated with favorable survival. While overall prognosis for the community HCM population is similar to the general population, the need for surgical myectomy may identify a sub-group with poorer long-term prognosis. We await long-term outcomes of more extensive myectomy approaches adopted in the past 10 years at major institutions.

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.

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.

Short hairpin RNA gene silencing of prolyl hydroxylase-2 with a minicircle vector improves neovascularization of hindlimb ischemia.

In this study, we target the hypoxia inducible factor-1 alpha (HIF-1-alpha) pathway by short hairpin RNA interference therapy targeting prolyl hydroxylase-2 (shPHD2). We use the minicircle (MC) vector technology as an alternative for conventional nonviral plasmid (PL) vectors in order to improve neovascularization after unilateral hindlimb ischemia in a murine model. Gene expression and transfection efficiency of MC and PL, both in vitro and in vivo, were assessed using bioluminescence imaging (BLI) and firefly luciferase (Luc) reporter gene. C57Bl6 mice underwent unilateral electrocoagulation of the femoral artery and gastrocnemic muscle injection with MC-shPHD2, PL-shPHD2, or phosphate-buffered saline (PBS) as control. Blood flow recovery was monitored using laser Doppler perfusion imaging, and collaterals were visualized by immunohistochemistry and angiography. MC-Luc showed a 4.6-fold higher in vitro BLI signal compared with PL-Luc. BLI signals in vivo were 4.3×10(5)±3.3×10(5) (MC-Luc) versus 0.4×10(5)±0.3×10(5) (PL-Luc) at day 28 (p=0.016). Compared with PL-shPHD2 or PBS, MC-shPHD2 significantly improved blood flow recovery, up to 50% from day 3 until day 14 after ischemia induction. MC-shPHD2 significantly increased collateral density and capillary density, as monitored by alpha-smooth muscle actin expression and CD31(+) expression, respectively. Angiography data confirmed the histological findings. Significant downregulation of PHD2 mRNA levels by MC-shPHD2 was confirmed by quantitative polymerase chain reaction. Finally, Western blot analysis confirmed significantly higher levels of HIF-1-alpha protein by MC-shPHD2, compared with PL-shPHD2 and PBS. This study provides initial evidence of a new potential therapeutic approach for peripheral artery disease. The combination of HIF-1-alpha pathway targeting by shPHD2 with the robust nonviral MC plasmid improved postischemic neovascularization, making this approach a promising potential treatment option for critical limb ischemia.

A titratable two-step transcriptional amplification strategy for targeted gene therapy based on ligand-induced intramolecular folding of a mutant human estrogen receptor.

PURPOSE: The efficacy and safety of cardiac gene therapy depend critically on the level and the distribution of therapeutic gene expression following vector administration. We aimed to develop a titratable two-step transcriptional amplification (tTSTA) vector strategy, which allows modulation of transcriptionally targeted gene expression in the myocardium. PROCEDURES: We constructed a tTSTA plasmid vector (pcTnT-tTSTA-fluc), which uses the cardiac troponin T (cTnT) promoter to drive the expression of the recombinant transcriptional activator GAL4-mER(LBD)-VP2, whose ability to transactivate the downstream firefly luciferase reporter gene (fluc) depends on the binding of its mutant estrogen receptor (ER(G521T)) ligand binding domain (LBD) to an ER ligand such as raloxifene. Mice underwent either intramyocardial or hydrodynamic tail vein (HTV) injection of pcTnT-tTSTA-fluc, followed by differential modulation of fluc expression with varying doses of intraperitoneal raloxifene prior to bioluminescence imaging to assess the kinetics of myocardial or hepatic fluc expression. RESULTS: Intramyocardial injection of pcTnT-tTSTA-fluc followed by titration with intraperitoneal raloxifene led to up to tenfold induction of myocardial fluc expression. HTV injection of pcTnT-tTSTA-fluc led to negligible long-term hepatic fluc expression, regardless of the raloxifene dose given. CONCLUSIONS: The tTSTA vector strategy can effectively modulate transgene expression in a tissue-specific manner. Further refinement of this strategy should help maximize the benefit-to-risk ratio of cardiac gene therapy.

A common rejection module (CRM) for acute rejection across multiple organs identifies novel therapeutics for organ transplantation.

Using meta-analysis of eight independent transplant datasets (236 graft biopsy samples) from four organs, we identified a common rejection module (CRM) consisting of 11 genes that were significantly overexpressed in acute rejection (AR) across all transplanted organs. The CRM genes could diagnose AR with high specificity and sensitivity in three additional independent cohorts (794 samples). In another two independent cohorts (151 renal transplant biopsies), the CRM genes correlated with the extent of graft injury and predicted future injury to a graft using protocol biopsies. Inferred drug mechanisms from the literature suggested that two FDA-approved drugs (atorvastatin and dasatinib), approved for nontransplant indications, could regulate specific CRM genes and reduce the number of graft-infiltrating cells during AR. We treated mice with HLA-mismatched mouse cardiac transplant with atorvastatin and dasatinib and showed reduction of the CRM genes, significant reduction of graft-infiltrating cells, and extended graft survival. We further validated the beneficial effect of atorvastatin on graft survival by retrospective analysis of electronic medical records of a single-center cohort of 2,515 renal transplant patients followed for up to 22 yr. In conclusion, we identified a CRM in transplantation that provides new opportunities for diagnosis, drug repositioning, and rational drug design.

Costimulation-adhesion blockade is superior to cyclosporine A and prednisone immunosuppressive therapy for preventing rejection of differentiated human embryonic stem cells following transplantation.

RATIONALE: Human embryonic stem cell (hESC) derivatives are attractive candidates for therapeutic use. The engraftment and survival of hESC derivatives as xenografts or allografts require effective immunosuppression to prevent immune cell infiltration and graft destruction. OBJECTIVE: To test the hypothesis that a short-course, dual-agent regimen of two costimulation-adhesion blockade agents can induce better engraftment of hESC derivatives compared to current immunosuppressive agents. METHODS AND RESULTS: We transduced hESCs with a double fusion reporter gene construct expressing firefly luciferase (Fluc) and enhanced green fluorescent protein, and differentiated these cells to endothelial cells (hESC-ECs). Reporter gene expression enabled longitudinal assessment of cell engraftment by bioluminescence imaging. Costimulation-adhesion therapy resulted in superior hESC-EC and mouse EC engraftment compared to cyclosporine therapy in a hind limb model. Costimulation-adhesion therapy also promoted robust hESC-EC and hESC-derived cardiomyocyte survival in an ischemic myocardial injury model. Improved hESC-EC engraftment had a cardioprotective effect after myocardial injury, as assessed by magnetic resonance imaging. Mechanistically, costimulation-adhesion therapy is associated with systemic and intragraft upregulation of T-cell immunoglobulin and mucin domain 3 (TIM3) and a reduced proinflammatory cytokine profile. CONCLUSIONS: Costimulation-adhesion therapy is a superior alternative to current clinical immunosuppressive strategies for preventing the post-transplant rejection of hESC derivatives. By extending the window for cellular engraftment, costimulation-adhesion therapy enhances functional preservation following ischemic injury. This regimen may function through a TIM3-dependent mechanism.

Screening drug-induced arrhythmia [corrected] using human induced pluripotent stem cell-derived cardiomyocytes and low-impedance microelectrode arrays.

BACKGROUND: Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities. METHODS AND RESULTS: Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls. CONCLUSIONS: We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.

Noninvasive imaging of hypoxia-inducible factor-1α gene therapy for myocardial ischemia.

Hypoxia-inducible factor-1 alpha (HIF-1α) gene therapy holds great promise for the treatment of myocardial ischemia. Both preclinical and clinical evaluations of this therapy are underway and can benefit from a vector strategy that allows noninvasive assessment of HIF-1α expression as an objective measure of gene delivery. We have developed a novel bidirectional plasmid vector (pcTnT-HIF-1α-VP2-TSTA-fluc), which employs the cardiac troponin T (cTnT) promoter in conjunction with a two-step transcriptional amplification (TSTA) system to drive the linked expression of a recombinant HIF-1α gene (HIF-1α-VP2) and the firefly luciferase gene (fluc). The firefly luciferase (FLuc) activity serves as a surrogate for HIF-1α-VP2 expression, and can be noninvasively assessed in mice using bioluminescence imaging after vector delivery. Transfection of cultured HL-1 cardiomyocytes with pcTnT-HIF-1α-VP2-TSTA-fluc led to a strong correlation between FLuc and HIF-1α-dependent vascular endothelial growth factor expression (r(2)=0.88). Intramyocardial delivery of pcTnT-HIF-1α-VP2-TSTA-fluc into infarcted mouse myocardium led to persistent HIF-1α-VP2 expression for 4 weeks, even though it improved neither CD31+ microvessel density nor echocardiographically determined left ventricular systolic function. These results lend support to recent findings of suboptimal efficacy associated with plasmid-mediated HIF-1α therapy. The imaging techniques developed herein should be useful for further optimizing HIF-1α-VP2 therapy in preclinical models of myocardial ischemia.