IFN-ß activates cytotoxic function of human natural killer cells toward IL-27 and poly(I:C) stimulated PC3 and DU145 cells.

Natural killer (NK) cell phenotype and function are altered in patients with prostate cancer, and increased NK cell activity is associated with a better prognosis in patients with disease. For patients with advanced stage prostate cancer, immunotherapies are a promising approach when standard treatment options have been exhausted. With the rapid emergence of NK cell-based therapies, it is important to understand the mechanisms by which NK cells can be triggered to kill cancer cells that have developed immune-evasive strategies. Altering the cytokine profiles of advanced prostate cancer cells may be an area to explore when considering ways in which NK cell activation can be modulated. We have previously demonstrated that combining the cytokine, IL-27, with TLR3 agonist, poly(I:C), changes cytokine secretion in the advanced prostate cancer models, PC3 and DU145 cells. Herein, we extend our previous work to study the effect of primary human NK cells on prostate cancer cell death in an in vitro co-culture model. Stimulating PC3 and DU145 cells with IL-27 and poly(I:C) induced IFN-ß secretion, which was required for activation of primary human NK cells to kill these stimulated prostate cancer cells. PC3 cells were more sensitized to NK cell-mediated killing when compared to DU145 cells, which was attributed to differential levels of IFN-ß produced in response to stimulation with IL-27 and poly(I:C). IFN-ß increased granzyme B secretion and membrane-bound TRAIL expression by co-cultured NK cells. We further demonstrated that these NK cells killed PC3 cells in a partially TRAIL-dependent manner. This work provides mechanistic insight into how the cytotoxic function of NK cells can be improved to target cancer cells.

Addressing Natural Killer Cell Dysfunction and Plasticity in Cell-Based Cancer Therapeutics.

Natural killer (NK) cells are cytotoxic group 1 innate lymphoid cells (ILC), known for their role as killers of stressed, cancerous, and virally infected cells. Beyond this cytotoxic function, NK cell subsets can influence broader immune responses through cytokine production and have been linked to central roles in non-immune processes, such as the regulation of vascular remodeling in pregnancy and cancer. Attempts to exploit the anti-tumor functions of NK cells have driven the development of various NK cell-based therapies, which have shown promise in both pre-clinical disease models and early clinical trials. However, certain elements of the tumor microenvironment, such as elevated transforming growth factor (TGF)-ß, hypoxia, and indoalemine-2,3-dioxygenase (IDO), are known to suppress NK cell function, potentially limiting the longevity and activity of these approaches. Recent studies have also identified these factors as contributors to NK cell plasticity, defined by the conversion of classical cytotoxic NK cells into poorly cytotoxic, tissue-resident, or ILC1-like phenotypes. This review summarizes the current approaches for NK cell-based cancer therapies and examines the challenges presented by tumor-linked NK cell suppression and plasticity. Ongoing efforts to overcome these challenges are discussed, along with the potential utility of NK cell therapies to applications outside cancer.

Impaired Interleukin-15 Signaling via BMPR2 Loss Drives Natural Killer Cell Deficiency and Pulmonary Hypertension.

BACKGROUND: Natural killer (NK) cell impairment is a feature of pulmonary arterial hypertension (PAH) and contributes to vascular remodeling in animal models of disease. Although mutations in BMPR2, the gene encoding the BMP (bone morphogenetic protein) type-II receptor, are strongly associated with PAH, the contribution of BMPR2 loss to NK cell impairment remains unknown. We explored the impairment of IL (interleukin)-15 signaling, a central mediator of NK cell homeostasis, as both a downstream target of BMPR2 loss and a contributor to the pathogenesis of PAH. METHODS: The expression, trafficking, and secretion of IL-15 and IL-15Rα (interleukin 15 α-type receptor) were assessed in human pulmonary artery endothelial cells, with or without BMPR2 silencing. NK cell development and IL-15/IL-15Rα levels were quantified in mice bearing a heterozygous knock-in of the R899X-BMPR2 mutation (bmpr2+/R899X). NK-deficient Il15-/- rats were exposed to the Sugen/hypoxia and monocrotaline models of PAH to assess the impact of impaired IL-15 signaling on disease severity. RESULTS: BMPR2 loss reduced IL-15Rα surface presentation and secretion in human pulmonary artery endothelial cells via impaired trafficking through the trans-Golgi network. bmpr2+/R899X mice exhibited a decrease in NK cells, which was not attributable to impaired hematopoietic development but was instead associated with reduced IL-15/IL-15Rα levels in these animals. Il15-/- rats of both sexes exhibited enhanced disease severity in the Sugen/hypoxia model, with only male Il15-/- rats developing more severe PAH in response to monocrotaline. CONCLUSIONS: This work identifies the loss of IL-15 signaling as a novel BMPR2-dependent contributor to NK cell impairment and pulmonary vascular disease.

Evolving Concepts in Endothelial Pathobiology of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a deadly disease, characterized by increased vascular resistance, pulmonary arteriolar loss, and occlusive arterial remodeling, leading to eventual right heart failure. Evidence increasingly points to the pulmonary endothelium as a central actor in PAH. Endothelial cell apoptosis can result directly in distal lung arteriolar pruning and indirectly in the formation of complex and occlusive arterial lesions, reflecting an imbalance between endothelial injury and repair in the development and progression of PAH. Many of the mutations implicated in PAH are in genes, which are predominantly, or solely, expressed in endothelial cells, and the endothelium is a major target for therapeutic interventions to restore BMP signaling. We explore how arterial pruning can promote the emergence of occlusive arterial remodeling mediated by ongoing endothelial injury secondary to hemodynamic perturbation and pathological increases in luminal shear stress. The emerging role of endothelial cell senescence is discussed in the transition from reversible to irreversible arterial remodeling in advanced PAH, and we review the sometimes conflicting evidence that female sex hormones can both protect or promote vascular changes in disease. Finally, we explore the contribution of the endothelium to metabolic changes and the altered inflammatory and immune state in the PAH lung, focusing on the role of excessive TGFß signaling. Given the complexity of the endothelial pathobiology of PAH, we anticipate that emerging technologies that allow the study of molecular events at a single cell level will provide answers to many of the questions raised in this review.

Novel Pathways for Targeting Tumor Angiogenesis in Metastatic Breast Cancer.

Breast cancer is the most common cancer affecting women and is the second leading cause of cancer related death worldwide. Angiogenesis, the process of new blood vessel development from pre-existing vasculature, has been implicated in the growth, progression, and metastasis of cancer. Tumor angiogenesis has been explored as a key therapeutic target for decades, as the blockade of this process holds the potential to reduce the oxygen and nutrient supplies that are required for tumor growth. However, many existing anti-angiogenic approaches, such as those targeting Vascular Endothelial Growth Factor, Notch, and Angiopoietin signaling, have been associated with severe side-effects, limited survival advantage, and enhanced cancer regrowth rates. To address these setbacks, alternative pathways involved in the regulation of tumor angiogenesis are being explored, including those involving Bone Morphogenetic Protein-9 signaling, the Sonic Hedgehog pathway, Cyclooxygenase-2, p38-mitogen-activated protein kinase, and Chemokine Ligand 18. This review article will introduce the concept of tumor angiogenesis in the context of breast cancer, followed by an overview of current anti-angiogenic therapies, associated resistance mechanisms and novel therapeutic targets.

Endothelial BMPR2 Loss Drives a Proliferative Response to BMP (Bone Morphogenetic Protein) 9 via Prolonged Canonical Signaling.

OBJECTIVE: Pulmonary arterial hypertension is a disease of proliferative vascular occlusion that is strongly linked to mutations in BMPR2-the gene encoding the BMPR-II (BMP [bone morphogenetic protein] type II receptor). The endothelial-selective BMPR-II ligand, BMP9, reverses disease in animal models of pulmonary arterial hypertension and suppresses the proliferation of healthy endothelial cells. However, the impact of BMPR2 loss on the antiproliferative actions of BMP9 has yet to be assessed. Approach and Results: BMP9 suppressed proliferation in blood outgrowth endothelial cells from healthy control subjects but increased proliferation in blood outgrowth endothelial cells from pulmonary arterial hypertension patients with BMPR2 mutations. This shift from growth suppression to enhanced proliferation was recapitulated in control human pulmonary artery endothelial cells following siRNA-mediated BMPR2 silencing, as well as in mouse pulmonary endothelial cells isolated from endothelial-conditional Bmpr2 knockout mice (Bmpr2EC-/-). BMP9-induced proliferation was not attributable to altered metabolic activity or elevated TGFß (transforming growth factor beta) signaling but was linked to the prolonged induction of the canonical BMP target ID1 in the context of BMPR2 loss. In vivo, daily BMP9 administration to neonatal mice impaired both retinal and lung vascular patterning in control mice (Bmpr2EC+/+) but had no measurable effect on mice bearing a heterozygous endothelial Bmpr2 deletion (Bmpr2EC+/-) and caused excessive angiogenesis in both vascular beds for Bmpr2EC-/- mice. CONCLUSIONS: BMPR2 loss reverses the endothelial response to BMP9, causing enhanced proliferation. This finding has potential implications for the proposed translation of BMP9 as a treatment for pulmonary arterial hypertension and suggests the need for focused patient selection in clinical trials.

The Production of Pro-angiogenic VEGF-A Isoforms by Hypoxic Human NK Cells Is Independent of Their TGF-ß-Mediated Conversion to an ILC1-Like Phenotype.

Circulating natural killer (NK) cells have been shown to adopt a type 1 innate lymphoid cell (ILC1)-like phenotype in response to TGF-ß and secrete VEGF-A when exposed to hypoxia. Although these changes are often considered to be linked attributes of tissue residency, it has yet to be determined if TGF-ß and hypoxia work in concert to coordinate NK cellular phenotype and angiogenic potential. Examination of human circulating NK cells treated with TGF-ß demonstrated heterogeneity in their potential to adopt an ILC1-like phenotype, as indicated by the upregulation of CD9 and CD103 on only a subset of cells in culture. Culturing NK cells in chronic hypoxia did not induce a similar ILC1-like conversion and did not enhance the degree of conversion for cells exposed to TGF-ß. Similarly, hypoxic culture of circulating NK cells induced VEGF-A secretion, but this production was not enhanced by TGF-ß. Fluorescent in-situ hybridization flow cytometry demonstrated that hypoxia-induced VEGF-A production was uniform across all NK cells in culture and was not a selective feature of the cellular subset that adopted an ILC1-like phenotype in response to TGF-ß. Examination of VEGF-A isoforms demonstrated that hypoxia induces the production of pro-angiogenic VEGF-A isoforms, including VEGF-A165 and VEGF-A121, and does not stimulate any meaningful production of anti-angiogenic isoforms, such as VEGF-Ab transcriptional variants or VEGF-Ax. In summary, TGF-ß-mediated ILC1-like conversion and hypoxia-induced VEGF-A production are discrete processes in NK cells and are not part of a linked cellular program associated with tissue residency.

TGF-ß and IL-15 Synergize through MAPK Pathways to Drive the Conversion of Human NK Cells to an Innate Lymphoid Cell 1-like Phenotype.

Circulating NK cells are known to convert to a type 1 innate lymphoid cell (ILC1)-like phenotype in response to TGF-ß exposure. However, the precise cellular changes defining this process as well as the downstream signaling pathways guiding it remain poorly defined, particularly in humans. We used mass cytometry by time-of-flight (CyTOF) to model this phenotypic shift in vitro and identify a synergistic activity of TGF-ß and IL-15 in this cellular conversion. CyTOF profiling identified substantial heterogeneity in the propensity of NK cells to adopt an ILC1-like phenotype in culture, characterized by the step-wise acquisition of various markers, including CD69, CD9, CD103, and CD49a. Activating and inhibitory receptors, including NKG2A, NKG2D, KIR2DL1, KIR3DL1, NKp30, NKp44, and NKp46, were all found to be upregulated exclusively on the cellular subsets that converted most readily in response to TGF-ß. An assessment of downstream TGF-ß signaling identified TAK1-mediated activation of p38 MAPK as the critical pathway driving conversion. IL-15 enhanced TGF-ß-mediated conversion through Ras:RAC1 signaling as well as via the activation of MEK/ERK. Interestingly, the adoption of an ILC1-like phenotype was independent of the effect of IL-15 or TGF-ß on mTOR, as the culture of NK cells in the presence of mTOR inhibitors, such as rapamycin or torin1, had minimal impact on the degree of conversion. In conclusion, we have used in vitro human culture systems and CyTOF to define the conversion of circulating NK cells to an ILC1-like phenotype and have clarified the pathways responsible for this process.

Phosphodiesterase 3B (PDE3B) antagonizes the anti-angiogenic actions of PKA in human and murine endothelial cells.

Recent reports show that protein kinase A (PKA), but not exchange protein activated by cAMP (EPAC), acts in a cell autonomous manner to constitutively reduce the angiogenic sprouting capacity of murine and human endothelial cells. Specificity in the cellular actions of individual cAMP-effectors can be achieved when a cyclic nucleotide phosphodiesterase (PDE) enzyme acts locally to control the "pool" of cAMP that activates the cAMP-effector. Here, we examined whether PDEs coordinate the actions of PKA during endothelial cell sprouting. Inhibiting each of the cAMP-hydrolyzing PDEs expressed in human endothelial cells revealed that phosphodiesterase 3 (PDE3) inhibition with cilostamide reduced angiogenic sprouting in vitro, while inhibitors of PDE2 and PDE4 family enzymes had no such effect. Identifying a critical role for PDE3B in the anti-angiogenic effects of cilostamide, silencing this PDE3 variant, but not PDE3A, markedly impaired sprouting. Importantly, using both in vitro and ex vivo models of angiogenesis, we show the hypo-sprouting phenotype induced by PDE3 inhibition or PDE3B silencing was reversed by PKA inhibition. Examination of the individual cellular events required for sprouting revealed that PDE3B and PKA each regulated angiogenic sprouting by controlling the invasive capacity of endothelial cells, more specifically, by regulating podosome rosette biogenesis and matrix degradation. In support of the idea that PDE3B acts to inhibit angiogenic sprouting by limiting PKA-mediated reductions in active cdc42, the effects of PDE3B and/or PKA on angiogenic sprouting were negated in cells with reduced cdc42 expression or activity. Since PDE3B and PKA were co-localized in a perinuclear region in human ECs, could be co-immunoprecipitated from lysates of these cells, and silencing PDE3B activated the perinuclear pool of PKA in these cells, we conclude that PDE3B-mediated hydrolysis of cAMP acts to limit the anti-angiogenic potential of PKA in ECs.

Growth/differentiation factor 15 causes TGFß-activated kinase 1-dependent muscle atrophy in pulmonary arterial hypertension.

INTRODUCTION: Skeletal muscle dysfunction is a clinically important complication of pulmonary arterial hypertension (PAH). Growth/differentiation factor 15 (GDF-15), a prognostic marker in PAH, has been associated with muscle loss in other conditions. We aimed to define the associations of GDF-15 and muscle wasting in PAH, to assess its utility as a biomarker of muscle loss and to investigate its downstream signalling pathway as a therapeutic target. METHODS: GDF-15 levels and measures of muscle size and strength were analysed in the monocrotaline (MCT) rat, Sugen/hypoxia mouse and in 30 patients with PAH. In C2C12 myotubes the downstream targets of GDF-15 were identified. The pathway elucidated was then antagonised in vivo. RESULTS: Circulating GDF-15 levels correlated with tibialis anterior (TA) muscle fibre diameter in the MCT rat (Pearson r=-0.61, p=0.003). In patients with PAH, plasma GDF-15 levels of <564 pg/L predicted those with preserved muscle strength with a sensitivity and specificity of ≥80%. In vitro GDF-15 stimulated an increase in phosphorylation of TGFß-activated kinase 1 (TAK1). Antagonising TAK1, with 5(Z)-7-oxozeaenol, in vitro and in vivo led to an increase in fibre diameter and a reduction in mRNA expression of atrogin-1 in both C2C12 cells and in the TA of animals who continued to grow. Circulating GDF-15 levels were also reduced in those animals which responded to treatment. CONCLUSIONS: Circulating GDF-15 is a biomarker of muscle loss in PAH that is responsive to treatment. TAK1 inhibition shows promise as a method by which muscle atrophy may be directly prevented in PAH. TRIAL REGISTRATION NUMBER: NCT01847716; Results.

Spontaneous pulmonary hypertension in genetic mouse models of natural killer cell deficiency.

Natural killer (NK) cells are cytotoxic innate lymphoid cells with an established role in the regulation of vascular structure in pregnancy and cancer. Impaired NK cell function has been identified in patients with pulmonary arterial hypertension (PAH), a disease of obstructive vascular remodeling in the lungs, as well as in multiple rodent models of disease. However, the precise contribution of NK cell impairment to the initiation and progression of PAH remains unknown. Here, we report the development of spontaneous pulmonary hypertension in two independent genetic models of NK cell dysfunction, including Nfil3-/- mice, which are deficient in NK cells due to the absence of the NFIL3 transcription factor, and Ncr1-Gfp mice, which lack the NK activating receptor NKp46. Mouse models of NK insufficiency exhibited increased right ventricular systolic pressure and muscularization of the pulmonary arteries in the absence of elevated left ventricular end-diastolic pressure, indicating that the development of pulmonary hypertension was not secondary to left heart dysfunction. In cases of severe NK cell impairment or loss, a subset of mice failed to develop pulmonary hypertension and instead exhibited reduced systemic blood pressure, demonstrating an extension of vascular abnormalities beyond the pulmonary circulation into the systemic vasculature. In both mouse models, the development of PAH was linked to elevated interleukin-23 production, whereas systemic hypotension in Ncr1-Gfp mice was accompanied by a loss of angiopoietin-2. Together, these results support an important role for NK cells in the regulation of pulmonary and systemic vascular function and the pathogenesis of PAH.

A Potential Role for Exosomal Translationally Controlled Tumor Protein Export in Vascular Remodeling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by increased proliferation and resistance to apoptosis of pulmonary vascular cells. Increased expression of translationally controlled tumor protein (TCTP), a prosurvival and antiapoptotic mediator, has recently been demonstrated in patients with heritable PAH; however, its role in the pathobiology of PAH remains unclear. Silencing of TCTP in blood outgrowth endothelial cells (BOECs) isolated from control subjects led to significant changes in morphology, cytoskeletal organization, increased apoptosis, and decreased directionality during migration. Because TCTP is also localized in extracellular vesicles, we isolated BOEC-derived extracellular vesicles (exosomes and microparticles) by sequential ultracentrifugation. BOECs isolated from patients harboring BMPR2 mutations released more exosomes than those derived from control subjects in proapoptotic conditions. Furthermore, TCTP expression was significantly higher in exosomes than in microparticles, indicating that TCTP is mainly exported via exosomes. Coculture assays demonstrated that exosomes transferred TCTP from ECs to pulmonary artery smooth muscle cells, suggesting a role for endothelial-derived TCTP in conferring proliferation and apoptotic resistance. In an experimental model of PAH, rats treated with monocrotaline demonstrated increased concentrations of TCTP in the lung and plasma. Consistent with this finding, we observed increased circulating TCTP levels in patients with idiopathic PAH compared with control subjects. Therefore, our data suggest an important role for TCTP in regulating the critical vascular cell phenotypes that have been implicated in the pathobiology of PAH. In addition, this research implicates TCTP as a potential biomarker for the onset and development of PAH.

Pulmonary arterial hypertension: pathogenesis and clinical management.

Pulmonary hypertension is defined as a resting mean pulmonary artery pressure of 25 mm Hg or above. This review deals with pulmonary arterial hypertension (PAH), a type of pulmonary hypertension that primarily affects the pulmonary vasculature. In PAH, the pulmonary vasculature is dynamically obstructed by vasoconstriction, structurally obstructed by adverse vascular remodeling, and pathologically non-compliant as a result of vascular fibrosis and stiffening. Many cell types are abnormal in PAH, including vascular cells (endothelial cells, smooth muscle cells, and fibroblasts) and inflammatory cells. Progress has been made in identifying the causes of PAH and approving new drug therapies. A cancer-like increase in cell proliferation and resistance to apoptosis reflects acquired abnormalities of mitochondrial metabolism and dynamics. Mutations in the type II bone morphogenetic protein receptor (BMPR2) gene dramatically increase the risk of developing heritable PAH. Epigenetic dysregulation of DNA methylation, histone acetylation, and microRNAs also contributes to disease pathogenesis. Aberrant bone morphogenetic protein signaling and epigenetic dysregulation in PAH promote cell proliferation in part through induction of a Warburg mitochondrial-metabolic state of uncoupled glycolysis. Complex changes in cytokines (interleukins and tumor necrosis factor), cellular immunity (T lymphocytes, natural killer cells, macrophages), and autoantibodies suggest that PAH is, in part, an autoimmune, inflammatory disease. Obstructive pulmonary vascular remodeling in PAH increases right ventricular afterload causing right ventricular hypertrophy. In some patients, maladaptive changes in the right ventricle, including ischemia and fibrosis, reduce right ventricular function and cause right ventricular failure. Patients with PAH have dyspnea, reduced exercise capacity, exertional syncope, and premature death from right ventricular failure. PAH targeted therapies (prostaglandins, phosphodiesterase-5 inhibitors, endothelin receptor antagonists, and soluble guanylate cyclase stimulators), used alone or in combination, improve functional capacity and hemodynamics and reduce hospital admissions. However, these vasodilators do not target key features of PAH pathogenesis and have not been shown to reduce mortality, which remains about 50% at five years. This review summarizes the epidemiology, pathogenesis, diagnosis, and treatment of PAH.

Epigenetic Dysregulation of the Dynamin-Related Protein 1 Binding Partners MiD49 and MiD51 Increases Mitotic Mitochondrial Fission and Promotes Pulmonary Arterial Hypertension: Mechanistic and Therapeutic Implications.

BACKGROUND: Mitotic fission is increased in pulmonary arterial hypertension (PAH), a hyperproliferative, apoptosis-resistant disease. The fission mediator dynamin-related protein 1 (Drp1) must complex with adaptor proteins to cause fission. Drp1-induced fission has been therapeutically targeted in experimental PAH. Here, we examine the role of 2 recently discovered, poorly understood Drp1 adapter proteins, mitochondrial dynamics protein of 49 and 51 kDa (MiD49 and MiD51), in normal vascular cells and explore their dysregulation in PAH. METHODS: Immunoblots of pulmonary artery smooth muscle cells (control, n=6; PAH, n=8) and immunohistochemistry of lung sections (control, n=6; PAH, n=6) were used to assess the expression of MiD49 and MiD51. The effects of manipulating MiDs on cell proliferation, cell cycle, and apoptosis were assessed in human and rodent PAH pulmonary artery smooth muscle cells with flow cytometry. Mitochondrial fission was studied by confocal imaging. A microRNA (miR) involved in the regulation of MiD expression was identified using microarray techniques and in silico analyses. The expression of circulatory miR was assessed with quantitative reverse transcription-polymerase chain reaction in healthy volunteers (HVs) versus patients with PAH from Sheffield, UK (plasma: HV, n=29, PAH, n=27; whole blood: HV, n=11, PAH, n=14) and then confirmed in a cohort from Beijing, China (plasma: HV, n=19, PAH, n=36; whole blood: HV, n=20, PAH, n=39). This work was replicated in monocrotaline and Sugen 5416-hypoxia, preclinical PAH models. Small interfering RNAs targeting MiDs or an miR mimic were nebulized to rats with monocrotaline-induced PAH (n=4-10). RESULTS: MiD expression is increased in PAH pulmonary artery smooth muscle cells, which accelerates Drp1-mediated mitotic fission, increases cell proliferation, and decreases apoptosis. Silencing MiDs (but not other Drp1 binding partners, fission 1 or mitochondrial fission factor) promotes mitochondrial fusion and causes G1-phase cell cycle arrest through extracellular signal-regulated kinases 1/2- and cyclin-dependent kinase 4-dependent mechanisms. Augmenting MiDs in normal cells causes fission and recapitulates the PAH phenotype. MiD upregulation results from decreased miR-34a-3p expression. Circulatory miR-34a-3p expression is decreased in both patients with PAH and preclinical models of PAH. Silencing MiDs or augmenting miR-34a-3p regresses experimental PAH. CONCLUSIONS: In health, MiDs regulate Drp1-mediated fission, whereas in disease, epigenetic upregulation of MiDs increases mitotic fission, which drives pathological proliferation and apoptosis resistance. The miR-34a-3p-MiD pathway offers new therapeutic targets for PAH.