PPARγ-p53-Mediated Vasculoregenerative Program to Reverse Pulmonary Hypertension.

RATIONALE: In pulmonary arterial hypertension (PAH), endothelial dysfunction and obliterative vascular disease are associated with DNA damage and impaired signaling of BMPR2 (bone morphogenetic protein type 2 receptor) via two downstream transcription factors, PPARγ (peroxisome proliferator-activated receptor gamma), and p53. OBJECTIVE: We investigated the vasculoprotective and regenerative potential of a newly identified PPARγ-p53 transcription factor complex in the pulmonary endothelium. METHODS AND RESULTS: In this study, we identified a pharmacologically inducible vasculoprotective mechanism in pulmonary arterial and lung MV (microvascular) endothelial cells in response to DNA damage and oxidant stress regulated in part by a BMPR2 dependent transcription factor complex between PPARγ and p53. Chromatin immunoprecipitation sequencing and RNA-sequencing established an inducible PPARγ-p53 mediated regenerative program regulating 19 genes involved in lung endothelial cell survival, angiogenesis and DNA repair including, EPHA2 (ephrin type-A receptor 2), FHL2 (four and a half LIM domains protein 2), JAG1 (jagged 1), SULF2 (extracellular sulfatase Sulf-2), and TIGAR (TP53-inducible glycolysis and apoptosis regulator). Expression of these genes was partially impaired when the PPARγ-p53 complex was pharmacologically disrupted or when BMPR2 was reduced in pulmonary artery endothelial cells (PAECs) subjected to oxidative stress. In endothelial cell-specific Bmpr2-knockout mice unable to stabilize p53 in endothelial cells under oxidative stress, Nutlin-3 rescued endothelial p53 and PPARγ-p53 complex formation and induced target genes, such as APLN (apelin) and JAG1, to regenerate pulmonary microvessels and reverse pulmonary hypertension. In PAECs from BMPR2 mutant PAH patients, pharmacological induction of p53 and PPARγ-p53 genes repaired damaged DNA utilizing genes from the nucleotide excision repair pathway without provoking PAEC apoptosis. CONCLUSIONS: We identified a novel therapeutic strategy that activates a vasculoprotective gene regulation program in PAECs downstream of dysfunctional BMPR2 to rehabilitate PAH PAECs, regenerate pulmonary microvessels, and reverse disease. Our studies pave the way for p53-based vasculoregenerative therapies for PAH by extending the therapeutic focus to PAEC dysfunction and to DNA damage associated with PAH progression.

Circulating plasmablasts are elevated and produce pathogenic anti-endothelial cell autoantibodies in idiopathic pulmonary arterial hypertension.

Idiopathic pulmonary arterial hypertension (IPAH) is a devastating pulmonary vascular disease in which autoimmune and inflammatory phenomena are implicated. B cells and autoantibodies have been associated with IPAH and identified as potential therapeutic targets. However, the specific populations of B cells involved and their roles in disease pathogenesis are not clearly defined. We aimed to assess the levels of activated B cells (plasmablasts) in IPAH, and to characterize recombinant antibodies derived from these plasmablasts. Blood plasmablasts are elevated in IPAH, remain elevated over time, and produce IgA autoantibodies. Single-cell sequencing of plasmablasts in IPAH revealed repertoires of affinity-matured antibodies with small clonal expansions, consistent with an ongoing autoimmune response. Recombinant antibodies representative of these clonal lineages bound known autoantigen targets and displayed an unexpectedly high degree of polyreactivity. Representative IPAH plasmablast recombinant antibodies stimulated human umbilical vein endothelial cells to produce cytokines and overexpress the adhesion molecule ICAM-1. Together, our results demonstrate an ongoing adaptive autoimmune response involving IgA plasmablasts that produce anti-endothelial cell autoantibodies in IPAH. These antibodies stimulate endothelial cell production of cytokines and adhesion molecules, which may contribute to disease pathogenesis. These findings suggest a role for mucosally-driven autoimmunity and autoimmune injury in the pathogenesis of IPAH.

Upregulation of Human Endogenous Retrovirus-K Is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension.

BACKGROUND: Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic, or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue, and elevated cytokines have been related to PAH pathogenesis but without a clear understanding of how these abnormalities are initiated, perpetuated, and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies. METHODS: Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry, confirmed by enzyme-linked immunosorbent assay, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next-generation sequencing, functional studies in cultured monocytes and endothelial cells, and hemodynamic and lung studies in a rat. RESULTS: SAM domain and HD domain-containing protein 1 (SAMHD1), an innate immune factor that suppresses HIV replication, was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH versus 12 control lungs. Elevated SAMHD1 was localized to endothelial cells, perivascular dendritic cells, and macrophages, and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH versus control lungs (n=4). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase mRNAs were elevated in PAH versus control lungs (n=10), and proteins were localized to macrophages. HERV-K deoxyuridine triphosphate nucleotidohydrolase induced SAMHD1 and proinflammatory cytokines (eg, interleukin 6, interleukin 1ß, and tumor necrosis factor α) in circulating monocytes, pulmonary arterial endothelial cells, and also activated B cells. Vulnerability of pulmonary arterial endothelial cells (PAEC) to apoptosis was increased by HERV-K deoxyuridine triphosphate nucleotidohydrolase in an interleukin 6-independent manner. Furthermore, 3 weekly injections of HERV-K deoxyuridine triphosphate nucleotidohydrolase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8) and elevated interleukin 6. CONCLUSIONS: Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH.

Codependence of Bone Morphogenetic Protein Receptor 2 and Transforming Growth Factor-ß in Elastic Fiber Assembly and Its Perturbation in Pulmonary Arterial Hypertension.

OBJECTIVE: We determined in patients with pulmonary arterial (PA) hypertension (PAH) whether in addition to increased production of elastase by PA smooth muscle cells previously reported, PA elastic fibers are susceptible to degradation because of their abnormal assembly. APPROACH AND RESULTS: Fibrillin-1 and elastin are the major components of elastic fibers, and fibrillin-1 binds bone morphogenetic proteins (BMPs) and the large latent complex of transforming growth factor-ß1 (TGFß1). Thus, we considered whether BMPs like TGFß1 contribute to elastic fiber assembly and whether this process is perturbed in PAH particularly when the BMP receptor, BMPR2, is mutant. We also assessed whether in mice with Bmpr2/1a compound heterozygosity, elastic fibers are susceptible to degradation. In PA smooth muscle cells and adventitial fibroblasts, TGFß1 increased elastin mRNA, but the elevation in elastin protein was dependent on BMPR2; TGFß1 and BMP4, via BMPR2, increased extracellular accumulation of fibrillin-1. Both BMP4- and TGFß1-stimulated elastic fiber assembly was impaired in idiopathic (I) PAH-PA adventitial fibroblast versus control cells, particularly those with hereditary (H) PAH and a BMPR2 mutation. This was related to profound reductions in elastin and fibrillin-1 mRNA. Elastin protein was increased in IPAH PA adventitial fibroblast by TGFß1 but only minimally so in BMPR2 mutant cells. Fibrillin-1 protein increased only modestly in IPAH or HPAH PA adventitial fibroblasts stimulated with BMP4 or TGFß1. In Bmpr2/1a heterozygote mice, reduced PA fibrillin-1 was associated with elastic fiber susceptibility to degradation and more severe pulmonary hypertension. CONCLUSIONS: Disrupting BMPR2 impairs TGFß1- and BMP4-mediated elastic fiber assembly and is of pathophysiologic significance in PAH.

Randomised placebo-controlled safety and tolerability trial of FK506 (tacrolimus) for pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating disease characterised by occlusive pulmonary vasculopathy. Activation of bone morphogenetic protein receptor 2 (BMPR2) signalling by FK506 (tacrolimus) reverses occlusive vasculopathy in rodent PAH models. Here, we determined the safety and tolerability of low-level FK506 therapy in stable PAH patients.We performed a randomised, double-blind, placebo-controlled, 16-week, single-centre, phase IIa trial in PAH patients with New York Heart Association functional class II/III symptoms using three FK506 target levels (<2, 2-3 and 3-5 ng·mL-1). 23 patients were randomised and 20 patients completed the trial.FK506 was generally well tolerated, with nausea/diarrhoea being the most commonly reported adverse event and no observation of line infections in patients on intravenous prostacyclin therapy. PAH patients had significantly lower BMPR2 expression in peripheral blood mononuclear cells versus healthy controls (n=13; p=0.005), which improved after FK506 treatment. While we observed that some patients responded with a pronounced increase in BMPR2 expression as well as improvement in 6-min walk distance, and serological and echocardiographic parameters of heart failure, these changes were not significant.Low-level FK506 is well tolerated and increases BMPR2 in subsets of PAH patients. These results support the study of FK506 in a phase IIb efficacy trial.

Distinct myeloid suppressor cell subsets correlate with plasma IL-6 and IL-10 and reduced interferon-alpha signaling in CD4⁺ T cells from patients with GI malignancy.

Interferon-alpha (IFN-α) promotes anti-tumor immunity through its actions on immune cells. We hypothesized that elevated percentages of myeloid-derived suppressor cells (MDSC) and increased pro-inflammatory cytokines in peripheral blood would be associated with impaired response to IFN-α in patients with gastrointestinal (GI) malignancies. This study evaluated relationships between plasma IL-6, IL-10, circulating MDSC subsets, and IFN-α-induced signal transduction in 40 patients with GI malignancies. Plasma IL-6 and IL-10 were significantly higher in patients versus normal donors. CD33(+)HLADR(-)CD11b(+)CD15(+) and CD33(+)HLADR(-/low)CD14(+) MDSC subsets were also elevated in patients versus normal donors (P < 0.0001). Plasma IL-6 was correlated with CD33(+)HLADR(-)CD15(+) MDSC (P = 0.008) and IL-10 with CD33(+)HLADR(-)CD15(-) MDSC (P = 0.002). The percentage of CD15(+) and CD15(-) but not CD14(+) MDSC subsets were inversely correlated with IFN-α-induced STAT1 phosphorylation in CD4(+) T cells, while co-culture with in vitro generated MDSC led to reduced IFN-α responsiveness in both PBMC and the CD4(+) subset of T cells from normal donors. Exploratory multivariable Cox proportional hazards models revealed that an increased percentage of the CD33(+)HLADR(-)CD15(-) MDSC subset was associated with reduced overall survival (P = 0.049), while an increased percentage of the CD33(+)HLADR(-/low)CD14(+) subset was associated with greater overall survival (P = 0.033). These data provide evidence for a unique relationship between specific cytokines, MDSC subsets, and IFN-α responsiveness in patients with GI malignancies.

The small molecule curcumin analog FLLL32 induces apoptosis in melanoma cells via STAT3 inhibition and retains the cellular response to cytokines with anti-tumor activity.

BACKGROUND: We characterized the biologic effects of a novel small molecule STAT3 pathway inhibitor that is derived from the natural product curcumin. We hypothesized this lead compound would specifically inhibit the STAT3 signaling pathway to induce apoptosis in melanoma cells. RESULTS: FLLL32 specifically reduced STAT3 phosphorylation at Tyr705 (pSTAT3) and induced apoptosis at micromolar amounts in human melanoma cell lines and primary melanoma cultures as determined by annexin V/propidium iodide staining and immunoblot analysis. FLLL32 treatment reduced expression of STAT3-target genes, induced caspase-dependent apoptosis, and reduced mitochondrial membrane potential. FLLL32 displayed specificity for STAT3 over other homologous STAT proteins. In contrast to other STAT3 pathway inhibitors (WP1066, JSI-124, Stattic), FLLL32 did not abrogate IFN-gamma-induced pSTAT1 or downstream STAT1-mediated gene expression as determined by Real Time PCR. In addition, FLLL32 did not adversely affect the function or viability of immune cells from normal donors. In peripheral blood mononuclear cells (PBMCs), FLLL32 inhibited IL-6-induced pSTAT3 but did not reduce signaling in response to immunostimulatory cytokines (IFN-gamma, IL 2). Treatment of PBMCs or natural killer (NK) cells with FLLL32 also did not decrease viability or granzyme b and IFN-gamma production when cultured with K562 targets as compared to vehicle (DMSO). CONCLUSIONS: These data suggest that FLLL32 represents a lead compound that could serve as a platform for further optimization to develop improved STAT3 specific inhibitors for melanoma therapy.

Modulation of SOCS protein expression influences the interferon responsiveness of human melanoma cells.

BACKGROUND: Endogenously produced interferons can regulate the growth of melanoma cells and are administered exogenously as therapeutic agents to patients with advanced cancer. We investigated the role of negative regulators of interferon signaling known as suppressors of cytokine signaling (SOCS) in mediating interferon-resistance in human melanoma cells. METHODS: Basal and interferon-alpha (IFN-alpha) or interferon-gamma (IFN-gamma)-induced expression of SOCS1 and SOCS3 proteins was evaluated by immunoblot analysis in a panel of n = 10 metastatic human melanoma cell lines, in human embryonic melanocytes (HEM), and radial or vertical growth phase melanoma cells. Over-expression of SOCS1 and SOCS3 proteins in melanoma cells was achieved using the PINCO retroviral vector, while siRNA were used to inhibit SOCS1 and SOCS3 expression. Tyr701-phosphorylated STAT1 (P-STAT1) was measured by intracellular flow cytometry and IFN-stimulated gene expression was measured by Real Time PCR. RESULTS: SOCS1 and SOCS3 proteins were expressed at basal levels in melanocytes and in all melanoma cell lines examined. Expression of the SOCS1 and SOCS3 proteins was also enhanced following stimulation of a subset of cell lines with IFN-alpha or IFN-gamma. Over-expression of SOCS proteins in melanoma cell lines led to significant inhibition of Tyr701-phosphorylated STAT1 (P-STAT1) and gene expression following stimulation with IFN-alpha (IFIT2, OAS-1, ISG-15) or IFN-gamma (IRF1). Conversely, siRNA inhibition of SOCS1 and SOCS3 expression in melanoma cells enhanced their responsiveness to interferon stimulation. CONCLUSIONS: These data demonstrate that SOCS proteins are expressed in human melanoma cell lines and their modulation can influence the responsiveness of melanoma cells to IFN-alpha and IFN-gamma.

Analysis of wear asymmetry in a series of 94 retrieved polyethylene tibial bearings.

Knee joint kinematics is the focus of a significant amount of experimental study for the purpose of knee prosthesis design and for testing the wear of current and prospective bearing materials. This study reports the wear assessment of a series of 94 explanted tibial bearings of various designs and manufacturers and focuses on the extent to which clinical wear is symmetric in the medial-lateral aspect, or is indicative of a systematic asymmetry that would be informative to the design and testing of knee prostheses or surgical practice. Wear assessment of the series of retrievals indicates that, statistically, there was more clinical wear on the medial side. Patterns of wear varied greatly among individual knees; a majority showed very similar extents of wear on the medial and lateral sides, however there were cases with significantly more wear on one condylar articulation than the other. Evidence of edge loading, whereby the femoral component articulates at the margin of the tibial bearing, was common. It was seen most frequently in the central zone of the medial condylar area, and, like the overall wear, edge loading was significantly more frequent on the medial side of bearings. Total bearing wear was seen to generally increase with time over the 208 months of in vivo duration covered by the retrievals in the study. The medial-lateral asymmetry of the wear does not appear to be significantly dependent on duration, however.

Novel small molecule XPO1/CRM1 inhibitors induce nuclear accumulation of TP53, phosphorylated MAPK and apoptosis in human melanoma cells.

XPO1/CRM1 is a key nuclear exporter protein that mediates translocation of numerous cellular regulatory proteins. We investigated whether XPO1 is a potential therapeutic target in melanoma using novel selective inhibitors of nuclear export (SINE). In vitro effects of SINE on cell growth and apoptosis were measured by MTS assay and flow cytometry [Annexin V/propidium iodide (PI)], respectively in human metastatic melanoma cell lines. Immunoblot analysis was used to measure nuclear localization of key cellular proteins. The in vivo activity of oral SINE was evaluated in NOD/SCID mice bearing A375 or CHL-1 human melanoma xenografts. SINE compounds induced cytostatic and pro-apoptotic effects in both BRAF wild type and mutant (V600E) cell lines at nanomolar concentrations. The cytostatic and pro-apoptotic effects of XPO1 inhibition were associated with nuclear accumulation of TP53, and CDKN1A induction in the A375 cell line with wild type TP53, while pMAPK accumulated in the nucleus regardless of TP53 status. The orally bioavailable KPT-276 and KPT-330 compounds significantly inhibited growth of A375 (p<0.0001) and CHL-1 (p = 0.0087) human melanoma cell lines in vivo at well tolerated doses. Inhibition of XPO1 using SINE represents a potential therapeutic approach for melanoma across cells with diverse molecular phenotypes by promoting growth inhibition and apoptosis.

PRMT5 is upregulated in malignant and metastatic melanoma and regulates expression of MITF and p27(Kip1.).

Protein arginine methyltransferase-5 (PRMT5) is a Type II arginine methyltransferase that regulates various cellular functions. We hypothesized that PRMT5 plays a role in regulating the growth of human melanoma cells. Immunohistochemical analysis indicated significant upregulation of PRMT5 in human melanocytic nevi, malignant melanomas and metastatic melanomas as compared to normal epidermis. Furthermore, nuclear PRMT5 was significantly decreased in metastatic melanomas as compared to primary cutaneous melanomas. In human metastatic melanoma cell lines, PRMT5 was predominantly cytoplasmic, and associated with its enzymatic cofactor Mep50, but not STAT3 or cyclin D1. However, histologic examination of tumor xenografts from athymic mice revealed heterogeneous nuclear and cytoplasmic PRMT5 expression. Depletion of PRMT5 via siRNA inhibited proliferation in a subset of melanoma cell lines, while it accelerated growth of others. Loss of PRMT5 also led to reduced expression of MITF (microphthalmia-associated transcription factor), a melanocyte-lineage specific oncogene, and increased expression of the cell cycle regulator p27(Kip1). These results are the first to report elevated PRMT5 expression in human melanoma specimens and indicate this protein may regulate MITF and p27(Kip1) expression in human melanoma cells.

UV light B-mediated inhibition of skin catalase activity promotes Gr-1+ CD11b+ myeloid cell expansion.

Skin cancer incidence and mortality are higher in men compared with women, but the causes of this sex discrepancy remain largely unknown. UV light exposure induces cutaneous inflammation and neutralizes cutaneous antioxidants. Gr-1(+)CD11b(+) myeloid cells are heterogeneous bone marrow-derived cells that promote inflammation-associated carcinogenesis. Reduced activity of catalase, an antioxidant present in the skin, has been associated with skin carcinogenesis. We used the outbred, immune-competent Skh-1 hairless mouse model of UVB-induced inflammation and non-melanoma skin cancer to further define sex discrepancies in UVB-induced inflammation. Our results demonstrated that male skin had relatively lower baseline catalase activity, which was inhibited following acute UVB exposure in both sexes. Further analysis revealed that skin catalase activity inversely correlated with splenic Gr-1(+)CD11b(+) myeloid cell percentage. Acute UVB exposure induced Gr-1(+)CD11b(+) myeloid cell skin infiltration, which was inhibited to a greater extent in male mice by topical catalase treatment. In chronic UVB studies, we demonstrated that the percentage of splenic Gr-1(+)CD11b(+) myeloid cells was 55% higher in male tumor-bearing mice compared with their female counterparts. Together, our findings indicate that lower skin catalase activity in male mice may at least in part contribute to increased UVB-induced generation of Gr-1(+)CD11b(+) myeloid cells and subsequent skin carcinogenesis.

Structurally modified curcumin analogs inhibit STAT3 phosphorylation and promote apoptosis of human renal cell carcinoma and melanoma cell lines.

The Janus kinase-2 (Jak2)-signal transducer and activator of transcription-3 (STAT3) pathway is critical for promoting an oncogenic and metastatic phenotype in several types of cancer including renal cell carcinoma (RCC) and melanoma. This study describes two small molecule inhibitors of the Jak2-STAT3 pathway, FLLL32 and its more soluble analog, FLLL62. These compounds are structurally distinct curcumin analogs that bind selectively to the SH2 domain of STAT3 to inhibit its phosphorylation and dimerization. We hypothesized that FLLL32 and FLLL62 would induce apoptosis in RCC and melanoma cells and display specificity for the Jak2-STAT3 pathway. FLLL32 and FLLL62 could inhibit STAT3 dimerization in vitro. These compounds reduced basal STAT3 phosphorylation (pSTAT3), and induced apoptosis in four separate human RCC cell lines and in human melanoma cell lines as determined by Annexin V/PI staining. Apoptosis was also confirmed by immunoblot analysis of caspase-3 processing and PARP cleavage. Pre-treatment of RCC and melanoma cell lines with FLLL32/62 did not inhibit IFN-γ-induced pSTAT1. In contrast to FLLL32, curcumin and FLLL62 reduced downstream STAT1-mediated gene expression of IRF1 as determined by Real Time PCR. FLLL32 and FLLL62 significantly reduced secretion of VEGF from RCC cell lines in a dose-dependent manner as determined by ELISA. Finally, each of these compounds inhibited in vitro generation of myeloid-derived suppressor cells. These data support further investigation of FLLL32 and FLLL62 as lead compounds for STAT3 inhibition in RCC and melanoma.

Curcumin induces proapoptotic effects against human melanoma cells and modulates the cellular response to immunotherapeutic cytokines.

Curcumin has potential as a chemopreventative and chemotherapeutic agent, but its interactions with clinically relevant cytokines are poorly characterized. Because cytokine immunotherapy is a mainstay of treatment for malignant melanoma, we hypothesized that curcumin could modulate the cellular responsiveness to interferons and interleukins. As a single agent, curcumin induced a dose-dependent increase in apoptosis of human melanoma cell lines, which was most prominent at doses >10 micromol/L. Immunoblot analysis confirmed that curcumin induced apoptosis and revealed caspase-3 processing, poly ADP ribose polymerase cleavage, reduced Bcl-2, and decreased basal phosphorylated signal transducers and activators of transcription 3 (STAT3). Despite its proapoptotic effects, curcumin pretreatment of human melanoma cell lines inhibited the phosphorylation of STAT1 protein and downstream gene transcription following IFN-alpha and IFN-gamma as determined by immunoblot analysis and real time PCR, respectively. Pretreatment of peripheral blood mononuclear cells from healthy donors with curcumin also inhibited the ability of IFN-alpha, IFN-gamma, and interleukin-2 to phosphorylate STAT proteins critical for their antitumor activity (STAT1 and STAT5, respectively) and their respective downstream gene expression as measured by real time PCR. Finally, stimulation of natural killer (NK) cells with curcumin reduced the level of interleukin-12-induced IFN-gamma secretion, and production of granzyme b or IFN-gamma upon coculture with A375 melanoma cells or NK-sensitive K562 cells as targets. These data show that although curcumin can induce apoptosis of melanoma cells, it can also adversely affect the responsiveness of immune effector cells to clinically relevant cytokines that possess antitumor properties.