Enhancing cancer immunotherapy through nanotechnology-mediated tumor infiltration and activation of immune cells.

Cancer immunotherapy has become arguably the most promising advancement in cancer research and therapy in recent years. The efficacy of cancer immunotherapy is critically dependent on specific physiological and physical processes - collectively referred to as transport barriers - including the activation of T cells by antigen presenting cells, T cells migration to and penetration into the tumor microenvironment, and movement of nutrients and other immune cells through the tumor microenvironment. Nanotechnology-based approaches have great potential to help overcome these transport barriers. In this review, we discuss the ways that nanotechnology is being leveraged to improve the efficacy and potency of various cancer immunotherapies.

Regulator of Calcineurin 1 helps coordinate whole-body metabolism and thermogenesis.

Increasing non-shivering thermogenesis (NST), which expends calories as heat rather than storing them as fat, is championed as an effective way to combat obesity and metabolic disease. Innate mechanisms constraining the capacity for NST present a fundamental limitation to this approach, yet are not well understood. Here, we provide evidence that Regulator of Calcineurin 1 (RCAN1), a feedback inhibitor of the calcium-activated protein phosphatase calcineurin (CN), acts to suppress two distinctly different mechanisms of non-shivering thermogenesis (NST): one involving the activation of UCP1 expression in white adipose tissue, the other mediated by sarcolipin (SLN) in skeletal muscle. UCP1 generates heat at the expense of reducing ATP production, whereas SLN increases ATP consumption to generate heat. Gene expression profiles demonstrate a high correlation between Rcan1 expression and metabolic syndrome. On an evolutionary timescale, in the context of limited food resources, systemic suppression of prolonged NST by RCAN1 might have been beneficial; however, in the face of caloric abundance, RCAN1-mediated suppression of these adaptive avenues of energy expenditure may now contribute to the growing epidemic of obesity.

Pathological ventricular remodeling: mechanisms: part 1 of 2.

Despite declines in heart failure morbidity and mortality with current therapies, rehospitalization rates remain distressingly high, substantially affecting individuals, society, and the economy. As a result, the need for new therapeutic advances and novel medical devices is urgent. Disease-related left ventricular remodeling is a complex process involving cardiac myocyte growth and death, vascular rarefaction, fibrosis, inflammation, and electrophysiological remodeling. Because these events are highly interrelated, targeting a single molecule or process may not be sufficient. Here, we review molecular and cellular mechanisms governing pathological ventricular remodeling.

Reversibility of adverse, calcineurin-dependent cardiac remodeling.

RATIONALE: Studies to dissect the role of calcineurin in pathological cardiac remodeling have relied heavily on murine models, in which genetic gain- and loss-of-function manipulations are initiated at or before birth. However, the great majority of clinical cardiac pathology occurs in adults. Yet nothing is known about the effects of calcineurin when its activation commences in adulthood. Furthermore, despite the fact that ventricular hypertrophy is a well-established risk factor for heart failure, the relative pace and progression of these 2 major phenotypic features of heart disease are unknown. Finally, even though therapeutic interventions in adults are designed to slow, arrest, or reverse disease pathogenesis, little is known about the capacity for spontaneous reversibility of calcineurin-dependent pathological remodeling. OBJECTIVE: We set out to address these 3 questions by studying mice engineered to harbor in cardiomyocytes a constitutively active calcineurin transgene driven by a tetracycline-responsive promoter element. METHODS AND RESULTS: Expression of the mutant calcineurin transgene was initiated for variable lengths of time to determine the natural history of disease pathogenesis, and to determine when, if ever, these events are reversible. Activation of the calcineurin transgene in adult mice triggered rapid and robust cardiac growth with features characteristic of pathological hypertrophy. Concentric hypertrophy preceded the development of systolic dysfunction, fetal gene activation, fibrosis, and clinical heart failure. Furthermore, cardiac hypertrophy reversed spontaneously when calcineurin activity was turned off, and expression of fetal genes reverted to baseline. Fibrosis, a prominent feature of pathological cardiac remodeling, manifested partial reversibility. CONCLUSIONS: Together, these data establish and define the deleterious effects of calcineurin signaling in the adult heart and reveal that calcineurin-dependent hypertrophy with concentric geometry precedes systolic dysfunction and heart failure. Furthermore, these findings demonstrate that during much of the disease process, calcineurin-dependent remodeling remains reversible.

Interleukin-10 from transplanted bone marrow mononuclear cells contributes to cardiac protection after myocardial infarction.

Bone marrow mononuclear cells (BM-MNCs) have successfully been used as a therapy for the improvement of left ventricular (LV) function after myocardial infarction (MI). It has been suggested that paracrine factors from BM-MNCs may be a key mechanism mediating cardiac protection. We previously performed microarray analysis and found that the pleiotropic cytokine interleukin (IL)-10 was highly upregulated in human progenitor cells in comparison with adult endothelial cells and CD14+ cells. Moreover, BM-MNCs secrete significant amounts of IL-10, and IL-10 could be detected from progenitor cells transplanted in infarcted mouse hearts. Specifically, intramyocardial injection of wild-type BM-MNCs led to a significant decrease in LV end-diastolic pressure (LVEDP) and LV end-systolic volume (LVESV) compared to hearts injected with either diluent or IL-10 knock-out BM-MNCs. Furthermore, intramyocardial injection of wild-type BM-MNCs led to a significant increase in stroke volume (SV) and rate of the development of pressure over time (+dP/dt) compared to hearts injected with either diluent or IL-10 knock-out BM-MNCs. The IL-10-dependent improvement provided by transplanted cells was not caused by reduced infarct size, neutrophil infiltration, or capillary density, but rather was associated with decreased T lymphocyte accumulation, reactive hypertrophy, and myocardial collagen deposition. These results suggest that BM-MNCs mediate cardiac protection after myocardial infarction and this is, at least in part, dependent on IL-10.

Cell-based therapy of myocardial infarction.

Cell-based therapy is a promising option for treatment of ischemic diseases. Several cell types have experimentally been shown to increase the functional recovery of the heart after ischemia by physically forming new blood vessels, differentiating to cardiac myocytes and--additionally or alternatively--by providing proangiogenic and antiapoptotic factors promoting tissue repair in a paracrine manner. Clinical studies preferentially used adult bone marrow-derived cells for the treatment of patients with acute myocardial infarction. Most of the studies suggested that cell therapy reduced the infarct size and improved cardiac contractile function. However, cell therapy is in its early stages, and various questions remain. For example, the identification of those patients who benefit most from cell therapy, the optimal cell type and number for patient with acute and chronic diseases, the best time and way of cell delivery, and the mechanisms of action by which cells exhibit beneficial effects, need to be further evaluated. Although no major safety concerns were raised during the initial clinical trials, several potential side effects need to be carefully monitored. The present review article summarizes the results of the clinical studies and discusses the open issues.

Pharmacological priming of adipose-derived stem cells promotes myocardial repair.

Adipose-derived stem cells (ADSCs) have myocardial regeneration potential, and transplantation of these cells following myocardial infarction (MI) in animal models leads to modest improvements in cardiac function. We hypothesized that pharmacological priming of pre-transplanted ADSCs would further improve left ventricular functional recovery after MI. We previously identified a compound from a family of 3,5-disubstituted isoxazoles, ISX1, capable of activating an Nkx2-5-driven promoter construct. Here, using ADSCs, we found that ISX1 (20 mM, 4 days) triggered a robust, dose-dependent, fourfold increase in Nkx2-5 expression, an early marker of cardiac myocyte differentiation and increased ADSC viability in vitro. Co-culturing neonatal cardiomyocytes with ISX1-treated ADSCs increased early and late cardiac gene expression. Whereas ISX1 promoted ADSC differentiation toward a cardiogenic lineage, it did not elicit their complete differentiation or their differentiation into mature adipocytes, osteoblasts, or chondrocytes, suggesting that re-programming is cardiomyocyte specific. Cardiac transplantation of ADSCs improved left ventricular functional recovery following MI, a response which was significantly augmented by transplantation of ISX1- pretreated cells. Moreover, ISX1-treated and transplanted ADSCs engrafted and were detectable in the myocardium 3 weeks following MI, albeit at relatively small numbers. ISX1 treatment increased histone acetyltransferase (HAT) activity in ADSCs, which was associated with histone 3 and histone 4 acetylation. Finally, hearts transplanted with ISX1-treated ADSCs manifested significant increases in neovascularization, which may account for the improved cardiac function. These findings suggest that a strategy of drug-facilitated initiation of myocyte differentiation enhances exogenously transplanted ADSC persistence in vivo, and consequent tissue neovascularization, to improve cardiac function.

JMJD3 as an epigenetic regulator in development and disease.

Gene expression is epigenetically regulated through DNA methylation and covalent chromatin modifications, such as acetylation, phosphorylation, ubiquitination, sumoylation, and methylation of histones. Histone methylation state is dynamically regulated by different groups of histone methyltransferases and demethylases. The trimethylation of histone 3 (H3K4) at lysine 4 is usually associated with the activation of gene expression, whereas trimethylation of histone 3 at lysine 27 (H3K27) is associated with the repression of gene expression. The polycomb repressive complex contains the H3K27 methyltransferase Ezh2 and controls dimethylation and trimethylation of H3K27 (H3K27me2/3). The Jumonji domain containing-3 (Jmjd3, KDM6B) and ubiquitously transcribed X-chromosome tetratricopeptide repeat protein (UTX, KDM6A) have been identified as H3K27 demethylases that catalyze the demethylation of H3K27me2/3. The role and mechanisms of both JMJD3 and UTX have been extensively studied for their involvement in development, cell plasticity, immune system, neurodegenerative disease, and cancer. In this review, we will focus on recent progresses made on understanding JMJD3 in the regulation of gene expression in development and diseases. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.

Optimizing recellularization of whole decellularized heart extracellular matrix.

RATIONALE: Perfusion decellularization of cadaveric hearts removes cells and generates a cell-free extracellular matrix scaffold containing acellular vascular conduits, which are theoretically sufficient to perfuse and support tissue-engineered heart constructs. However, after transplantation, these acellular vascular conduits clot, even with anti-coagulation. Here, our objective was to create a less thrombogenic scaffold and improve recellularized-left ventricular contractility by re-lining vascular conduits of a decellularized rat heart with rat aortic endothelial cells (RAECs). METHODS AND RESULTS: We used three strategies to recellularize perfusion-decellularized rat heart vasculature with RAECs: retrograde aortic infusion, brachiocephalic artery (BA) infusion, or a combination of inferior vena cava (IVC) plus BA infusion. The re-endothelialized scaffolds were maintained under vascular flow in vitro for 7 days, and then cell morphology, location, and viability were examined. Thrombogenicity of the scaffold was assessed in vitro and in vivo. Both BA and IVC+BA cell delivery resulted in a whole heart distribution of RAECs that proliferated, retained an endothelial phenotype, and expressed endothelial nitric oxide synthase and von Willebrand factor. Infusing RAECs via the combination IVC+BA method increased scaffold cellularity and the number of vessels that were lined with endothelial cells; re-endothelialization by using BA or IVC+BA cell delivery significantly reduced in vitro thrombogenicity. In vivo, both acellular and re-endothelialized scaffolds recruited non-immune host cells into the organ parenchyma and vasculature. Finally, re-endothelialization before recellularization of the left ventricular wall with neonatal cardiac cells enhanced construct contractility. CONCLUSIONS: This is the first study to re-endothelialize whole decellularized hearts throughout both arterial and venous beds and cavities by using arterial and venous delivery. The combination (IVC+BA) delivery strategy results in enhanced scaffold vessel re-endothelialization compared to single-route strategies. Re-endothelialization reduced scaffold thrombogencity and improved contractility of left ventricular-recellularized constructs. Thus, vessel and cavity re-endothelialization creates superior vascularized scaffolds for use in whole-organ recellularization applications.

Tumor necrosis factor receptor-associated factor 2 signaling provokes adverse cardiac remodeling in the adult mammalian heart.

BACKGROUND: Tumor necrosis factor superfamily ligands provoke a dilated cardiac phenotype signal through a common scaffolding protein termed tumor necrosis factor receptor-associated factor 2 (TRAF2); however, virtually nothing is known about TRAF2 signaling in the adult mammalian heart. METHODS AND RESULTS: We generated multiple founder lines of mice with cardiac-restricted overexpression of TRAF2 and characterized the phenotype of mice with higher expression levels of TRAF2 (myosin heavy chain [MHC]-TRAF2(HC)). MHC-TRAF2(HC) transgenic mice developed a time-dependent increase in cardiac hypertrophy, left ventricular dilation, and adverse left ventricular remodeling, and a significant decrease in LV+dP/dt and LV-dP/dt when compared with littermate controls (P<0.05 compared with littermate). During the early phases of left ventricular remodeling, there was a significant increase in total matrix metalloproteinase activity that corresponded with a decrease in total myocardial fibrillar collagen content. As the MHC-TRAF2(HC) mice aged, there was a significant decrease in total matrix metalloproteinase activity accompanied by an increase in total fibrillar collagen content and an increase in myocardial tissue inhibitor of metalloproteinase-1 levels. There was a significant increase in nuclear factor-κB activation at 4 to 12 weeks and jun N-terminal kinases activation at 4 weeks in the MHC-TRAF2(HC) mice. Transciptional profiling revealed that >95% of the hypertrophic/dilated cardiomyopathy-related genes that were significantly upregulated genes in the MHC-TRAF2(HC) hearts contained κB elements in their promoters. CONCLUSIONS: These results show for the first time that targeted overexpression of TRAF2 is sufficient to mediate adverse cardiac remodeling in the heart.

The cytoprotective effects of tumor necrosis factor are conveyed through tumor necrosis factor receptor-associated factor 2 in the heart.

BACKGROUND: Activation of both type 1 and type 2 tumor necrosis factor (TNF) receptors (TNFR1 and TNFR2) confers cytoprotection in cardiac myocytes. Noting that the scaffolding protein TNF receptor-associated factor 2 (TRAF2) is common to both TNF receptors, we hypothesized that the cytoprotective responses of TNF were mediated through TRAF2. METHODS AND RESULTS: Mice with cardiac-restricted overexpression of low levels of TNF (MHCsTNF(3)) and TRAF2 (MHC-TRAF2(LC)) and mice lacking TNFR1, TNFR2, and TNFR1/TNFR2 were subjected to ischemia (30 minutes) reperfusion (30 minutes) injury ex vivo using a Langendorff apparatus. MHCsTNF(3) mice were protected against ischemia-reperfusion injury as shown by a significant approximately 30% greater left ventricular developed pressure, approximately 80% lower creatine kinase release, and Evans blue dye uptake compared with littermates. The extent of ischemia-reperfusion induced injury was similar in wild-type, TNFR1, and TNFR2 deficient mice; however, mice lacking TNFR1/TNFR2 had a significant approximately 40% lower left ventricular developed pressure, a approximately 65% greater creatine kinase release, and approximately 40% greater Evans blue dye uptake compared with littermates. Interestingly, MHC-TRAF2(LC) mice had a significant approximately 50% lower left ventricular developed pressure, a approximately 70% lower creatine kinase release, and approximately 80% lower Evans blue dye uptake compared with littermate controls after ischemia-reperfusion injury. Biochemical analysis of the MHC-TRAF2(LC) hearts showed that there was activation of nuclear factor-kappaB but not c-Jun N-terminal kinase activation. CONCLUSIONS: Taken together, these results suggest that TNF confers cytoprotection in the heart through TRAF2-mediated activation of nuclear factor-kappaB.

MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice.

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression by binding to target messenger RNAs (mRNAs), leading to translational repression or degradation. Here, we show that the miR-17approximately92 cluster is highly expressed in human endothelial cells and that miR-92a, a component of this cluster, controls the growth of new blood vessels (angiogenesis). Forced overexpression of miR-92a in endothelial cells blocked angiogenesis in vitro and in vivo. In mouse models of limb ischemia and myocardial infarction, systemic administration of an antagomir designed to inhibit miR-92a led to enhanced blood vessel growth and functional recovery of damaged tissue. MiR-92a appears to target mRNAs corresponding to several proangiogenic proteins, including the integrin subunit alpha5. Thus, miR-92a may serve as a valuable therapeutic target in the setting of ischemic disease.

Role of paracrine factors in stem and progenitor cell mediated cardiac repair and tissue fibrosis.

A new era has begun in the treatment of ischemic disease and heart failure. With the discovery that stem cells from diverse organs and tissues, including bone marrow, adipose tissue, umbilical cord blood, and vessel wall, have the potential to improve cardiac function beyond that of conventional pharmacological therapy comes a new field of research aiming at understanding the precise mechanisms of stem cell-mediated cardiac repair. Not only will it be important to determine the most efficacious cell population for cardiac repair, but also whether overlapping, common mechanisms exist. Increasing evidence suggests that one mechanism of action by which cells provide tissue protection and repair may involve paracrine factors, including cytokines and growth factors, released from transplanted stem cells into the surrounding tissue. These paracrine factors have the potential to directly modify the healing process in the heart, including neovascularization, cardiac myocyte apoptosis, inflammation, fibrosis, contractility, bioenergetics, and endogenous repair.

Inhibition of PPAR-alpha activity in mice with cardiac-restricted expression of tumor necrosis factor: potential role of TGF-beta/Smad3.

A shift in energy substrate utilization from fatty acids to glucose has been reported in failing hearts, resulting in improved oxygen efficiency yet perhaps also contributing to a state of energy deficiency. Peroxisome proliferator-activated receptor (PPAR)-alpha, the principal transcriptional regulator of cardiac fatty acid beta-oxidation (FAO) genes, is downregulated in heart failure, and this may contribute to reduced fatty acid utilization. Cardiomyopathic states are also accompanied by elevated levels of circulating cytokines, such as tumor necrosis factor (TNF), as well as increased local production of cytokines and profibrotic factors, such as transforming growth factor (TGF)-beta. However, whether these molecular pathways directly modulate cardiac energy metabolism and PPAR-alpha activity is not known. Therefore, FAO capacity and FAO gene expression were determined in mice with cardiac-restricted overexpression of TNF (MHCsTNF(3)). MHCsTNF(3) hearts had significantly lower FAO capacity and decreased expression of PPAR-alpha and FAO target genes compared with control hearts. Surprisingly, TNF had little effect on PPAR-alpha activity and FAO rates in cultured ventricular myocytes, suggesting that TNF acts indirectly on myocyte FAO in vivo. We found that TGF-beta expression was upregulated in MHCsTNF(3) hearts and that treatment of cultured myocytes with TGF-beta significantly suppressed FAO rates and directly impaired PPAR-alpha activity, a result reproduced by Smad3 overexpression. This work demonstrates that TGF-beta signaling pathways directly suppress PPAR-alpha activity and reduce FAO in cardiac myocytes, perhaps in response to locally elevated TNF. Although speculative, TGF-beta-driven repair mechanisms may also include the additional benefit of limiting FAO in injured myocardium.