Adenylate Kinase 4—A Key Regulator of Proliferation and Metabolic Shift in Human Pulmonary Arterial Smooth Muscle Cells via Akt and HIF-1α Signaling Pathways

Increased proliferation of pulmonary arterial smooth muscle cells (PASMCs) in response to chronic hypoxia contributes to pulmonary vascular remodeling in pulmonary hypertension (PH). PH shares numerous similarities with cancer, including a metabolic shift towards glycolysis. In lung cancer, adenylate kinase 4 (AK4) promotes metabolic reprogramming and metastasis. Against this background, we show that AK4 regulates cell proliferation and energy metabolism of primary human PASMCs. We demonstrate that chronic hypoxia upregulates AK4 in PASMCs in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. RNA interference of AK4 decreases the viability and proliferation of PASMCs under both normoxia and chronic hypoxia. AK4 silencing in PASMCs augments mitochondrial respiration and reduces glycolytic metabolism. The observed effects are associated with reduced levels of phosphorylated protein kinase B (Akt) as well as HIF-1α, indicating the existence of an AK4-HIF-1α feedforward loop in hypoxic PASMCs. Finally, we show that AK4 levels are elevated in pulmonary vessels from patients with idiopathic pulmonary arterial hypertension (IPAH), and AK4 silencing decreases glycolytic metabolism of IPAH-PASMCs. We conclude that AK4 is a new metabolic regulator in PASMCs interacting with HIF-1α and Akt signaling pathways to drive the pro-proliferative and glycolytic phenotype of PH.     

Chemotherapy-Induced Changes in the Lung Microenvironment: The Role of MMP-2 in Facilitating Intravascular Arrest of Breast Cancer Cells

Previously, we showed that mice treated with cyclophosphamide (CTX) 4 days before intravenous injection of breast cancer cells had more cancer cells in the lung at 3 h after cancer injection than control counterparts without CTX. At 4 days after its injection, CTX is already excreted from the mice, allowing this pre-treatment design to reveal how CTX may modify the lung environment to indirectly affect cancer cells. In this study, we tested the hypothesis that the increase in cancer cell abundance at 3 h by CTX is due to an increase in the adhesiveness of vascular wall for cancer cells. Our data from protein array analysis and inhibition approach combined with in vitro and in vivo assays support the following two-prong mechanism. (1) CTX increases vascular permeability, resulting in the exposure of the basement membrane (BM). (2) CTX increases the level of matrix metalloproteinase-2 (MMP-2) in mouse serum, which remodels the BM and is functionally important for CTX to increase cancer abundance at this early stage. The combined effect of these two processes is the increased accessibility of critical protein domains in the BM, resulting in higher vascular adhesiveness for cancer cells to adhere. The critical protein domains in the vascular microenvironment are RGD and YISGR domains, whose known binding partners on cancer cells are integrin dimers and laminin receptor, respectively.     

Strategies to Improve the Antitumor Effect of γδ T Cell Immunotherapy for Clinical Application

Human γδ T cells show potent cytotoxicity against various types of cancer cells in a major histocompatibility complex unrestricted manner. Phosphoantigens and nitrogen-containing bisphosphonates (N-bis) stimulate γδ T cells via interaction between the γδ T cell receptor (TCR) and butyrophilin subfamily 3 member A1 (BTN3A1) expressed on target cells. γδ T cell immunotherapy is classified as either in vivo or ex vivo according to the method of activation. Immunotherapy with activated γδ T cells is well tolerated; however, the clinical benefits are unsatisfactory. Therefore, the antitumor effects need to be increased. Administration of γδ T cells into local cavities might improve antitumor effects by increasing the effector-to-target cell ratio. Some anticancer and molecularly targeted agents increase the cytotoxicity of γδ T cells via mechanisms involving natural killer group 2 member D (NKG2D)-mediated recognition of target cells. Both the tumor microenvironment and cancer stem cells exert immunosuppressive effects via mechanisms that include inhibitory immune checkpoint molecules. Therefore, co-immunotherapy with γδ T cells plus immune checkpoint inhibitors is a strategy that may improve cytotoxicity. The use of a bispecific antibody and chimeric antigen receptor might be effective to overcome current therapeutic limitations. Such strategies should be tested in a clinical research setting.     

Akt-Activated Endothelium Increases Cancer Cell Proliferation and Resistance to Treatment in Ovarian Cancer Cell Organoids

Ovarian cancer (OC) is a heterogeneous disease characterized by its late diagnosis (FIGO stages III and IV) and the importance of abdominal metastases often observed at diagnosis. Detached ovarian cancer cells (OCCs) float in ascites and form multicellular spheroids. Here, we developed endothelial cell (EC)-based 3D spheroids to better represent in vivo conditions. When co-cultured in 3D conditions, ECs and OCCs formed organized tumor angiospheres with a core of ECs surrounded by proliferating OCCs. We established that Akt and Notch3/Jagged1 pathways played a role in angiosphere formation and peritoneum invasion. In patients’ ascites we found angiosphere-like structures and demonstrated in patients’ specimens that tumoral EC displayed Akt activation, which supports the importance of Akt activation in ECs in OC. Additionally, we demonstrated the importance of FGF2, Pentraxin 3 (PTX3), PD-ECGF and TIMP-1 in angiosphere organization. Finally, we confirmed the role of Notch3/Jagged1 in OCC–EC crosstalk relating to OCC proliferation and during peritoneal invasion. Our results support the use of multicellular spheroids to better model tumoral and stromal interaction. Such models could help decipher the complex pathways playing critical roles in metastasis spread and predict tumor response to chemotherapy or anti-angiogenic treatment.     

Loss of SP-A in the Lung Exacerbates Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating and common chronic lung disease that is pathologically characterized by the destruction of lung architecture and the accumulation of extracellular matrix in the lung. Previous studies have shown an association between lung surfactant protein (SP) and the pathogenesis of IPF, as demonstrated by mutations and the altered expression of SP in patients with IPF. However, the role of SP in the development of lung fibrosis is poorly understood. In this study, the role of surfactant protein A (SP-A) was explored in experimental lung fibrosis induced with a low or high dose of bleomycin (BLM) and CRISPR/Cas9-mediated genetic deletion of SP-A. Our results showed that lung SP-A deficiency in mice promoted the development of fibrotic damage and exacerbated inflammatory responses to the BLM challenge. In vitro experiments with murine lung epithelial LA-4 cells demonstrated that in response to transforming growth factor-β1 (TGF-β1), LA-4 cells had a decreased protein expression of SP-A. Furthermore, exogenous SP administration to LA-4 cells inhibited the TGF-β1-induced upregulation of fibrotic markers. Overall, these findings suggest a novel antifibrotic mechanism of SP-A in the development of lung fibrosis, which indicates the therapeutic potential of the lung SP-A in preventing the development of IPF.     

Simplified Silvestrol Analogues with Potent Cytotoxic Activity

The complex natural products silvestrol ( 1 ) and episilvestrol ( 2 ) are inhibitors of translation initiation through binding to the DEAD‐box helicase eukaryotic initiation factor 4A (eIF4A). Both compounds are potently cytotoxic to cancer cells in vitro, and 1 has demonstrated efficacy in vivo in several xenograft cancer models. Here we show that 2 has limited plasma membrane permeability and is metabolized in liver microsomes in a manner consistent with that reported for 1 . In addition, we have prepared a series of analogues of these compounds where the complex pseudo‐sugar at C6 has been replaced with chemically simpler moieties to improve drug‐likeness. Selected compounds from this work possess excellent activity in biochemical and cellular translation assays with potent activity against leukemia cell lines.     

Endothelial Cells as Tools to Model Tissue Microenvironment in Hypoxia-Dependent Pathologies

Endothelial cells (ECs) lining the blood vessels are important players in many biological phenomena but are crucial in hypoxia-dependent diseases where their deregulation contributes to pathology. On the other hand, processes mediated by ECs, such as angiogenesis, vessel permeability, interactions with cells and factors circulating in the blood, maintain homeostasis of the organism. Understanding the diversity and heterogeneity of ECs in different tissues and during various biological processes is crucial in biomedical research to properly develop our knowledge on many diseases, including cancer. Here, we review the most important aspects related to ECs’ heterogeneity and list the available in vitro tools to study different angiogenesis-related pathologies. We focus on the relationship between functions of ECs and their organo-specificity but also point to how the microenvironment, mainly hypoxia, shapes their activity. We believe that taking into account the specific features of ECs that are relevant to the object of the study (organ or disease state), especially in a simplified in vitro setting, is important to truly depict the biology of endothelium and its consequences. This is possible in many instances with the use of proper in vitro tools as alternative methods to animal testing.     

Fluorine‐18 Radiolabeling and Radiopharmacological Characterization of a Benzodioxolylpyrimidine‐based Radiotracer Targeting the Receptor Tyrosine Kinase EphB4

Members of the Eph receptor tyrosine kinase family play essential roles in the pathogenesis of cancer and are therefore promising candidates for molecular imaging by positron emission tomography (PET), for example. In this regard, radiochemical access to novel PET radiotracers derived from potent inhibitors that target the EphB4 kinase domain and which bear a benzodioxolylpyrimidine structural motif was developed. A synthetic route was established for a new fluorine‐18‐containing radiotracer and for the desired precursor based on a high‐affinity benzodioxolylpyrimidine receptor tyrosine kinase inhibitor lead structure. The radiotracer [¹⁸F] 15 was obtained in 16 % radiochemical yield with a specific activity of ～7 GBq μmol^?1 and >95 % radiochemical purity. Due to the implication of EphB4, particularly in the progression, angiogenesis, and metastasis of melanoma, EphB4‐overexpressing human melanoma cells were generated and used as a novel in vitro model for radiopharmacological evaluation of the radiotracer. We demonstrate that the corresponding non‐radioactive reference compound regained its functionality as an inhibitor for both EphB4 receptor tyrosine kinase and Src kinase. EphB4 was significantly inhibited at compound concentrations >1 μM . Cellular uptake studies with [¹⁸F] 15 revealed substantial uptake in both EphB4‐overexpressing and control cells. Moreover, NMRI nu/nu mice bearing both EphB4‐overexpressing tumors and control tumors were used for radiopharmacological characterization by biodistribution studies ex vivo and by dynamic small‐animal PET experiments in vivo. Despite the high metabolic stability of the novel radiotracer observed in vivo, no substantial binding or accumulation in EphB4‐overexpressing and control tumors was observed. Nevertheless, we point out that the approach presented herein gives convenient access to novel ¹⁸F‐labeled benzodioxolylpyrimidines and is a promising strategy for the further development of novel radiotracers for imaging Eph receptor tyrosine kinases in cancer.     

Atovaquone Suppresses Triple-Negative Breast Tumor Growth by Reducing Immune-Suppressive Cells

A major contributing factor in triple-negative breast cancer progression is its ability to evade immune surveillance. One mechanism for this immunosuppression is through ribosomal protein S19 (RPS19), which facilitates myeloid-derived suppressor cells (MDSCs) recruitment in tumors, which generate cytokines TGF-β and IL-10 and induce regulatory T cells (Tregs), all of which are immunosuppressive and enhance tumor progression. Hence, enhancing the immune system in breast tumors could be a strategy for anticancer therapeutics. The present study evaluated the immune response of atovaquone, an antiprotozoal drug, in three independent breast-tumor models. Our results demonstrated that oral administration of atovaquone reduced HCC1806, CI66 and 4T1 paclitaxel-resistant (4T1-PR) breast-tumor growth by 45%, 70% and 42%, respectively. MDSCs, TGF-β, IL-10 and Tregs of blood and tumors were analyzed from all of these in vivo models. Our results demonstrated that atovaquone treatment in mice bearing HCC1806 tumors reduced MDSCs from tumor and blood by 70% and 30%, respectively. We also observed a 25% reduction in tumor MDSCs in atovaquone-treated mice bearing CI66 and 4T1-PR tumors. In addition, a decrease in TGF-β and IL-10 in tumor lysates was observed in atovaquone-treated mice with a reduction in tumor Tregs. Moreover, a significant reduction in the expression of RPS19 was found in tumors treated with atovaquone.     

mPR-Specific Actions Influence Maintenance of the Blood–Brain Barrier (BBB)

Cerebral cavernous malformations (CCMs) are characterized by abnormally dilated intracranial microvascular sinusoids that result in increased susceptibility to hemorrhagic stroke. It has been demonstrated that three CCM proteins (CCM1, CCM2, and CCM3) form the CCM signaling complex (CSC) to mediate angiogenic signaling. Disruption of the CSC will result in hemorrhagic CCMs, a consequence of compromised blood–brain barrier (BBB) integrity. Due to their characteristically incomplete penetrance, the majority of CCM mutation carriers (presumed CCM patients) are largely asymptomatic, but when symptoms occur, the disease has typically reached a clinical stage of focal hemorrhage with irreversible brain damage. We recently reported that the CSC couples both classic (nuclear; nPRs) and nonclassic (membrane; mPRs) progesterone (PRG)-receptors-mediated signaling within the CSC-mPRs-PRG (CmP) signaling network in nPR(−) breast cancer cells. In this report, we demonstrate that depletion of any of the three CCM genes or treatment with mPR-specific PRG actions (PRG/mifepristone) results in the disruption of the CmP signaling network, leading to increased permeability in the nPR(−) endothelial cells (ECs) monolayer in vitro. Finally, utilizing our in vivo hemizygous Ccm mutant mice models, we demonstrate that depletion of any of the three CCM genes, in combination with mPR-specific PRG actions, is also capable of leading to defective homeostasis of PRG in vivo and subsequent BBB disruption, allowing us to identify a specific panel of etiological blood biomarkers associated with BBB disruption. To our knowledge, this is the first report detailing the etiology to predict the occurrence of a disrupted BBB, an indication of early hemorrhagic events.     

Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation

ABSTRACT: BACKGROUND: : Exposure to particulate matter (PM) is a significant risk factor for increased cardiopulmonary morbidity and mortality. The mechanism of PM-mediated pathophysiology remains unknown. However, PM is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ROS generation. OBJECTIVES: We explored the role of tight junction proteins as targets for PM-induced loss of lung endothelial cell (EC) barrier integrity and enhanced cardiopulmonary dysfunction. METHOD: S: Changes in human lung EC monolayer permeability were assessed by Transendothelial Electrical Resistance (TER) in response to PM challenge (collected from Ft. McHenry Tunnel, Baltimore, MD, particle size >0.1 um). Biochemical assessment of ROS generation and Ca2+ mobilization were also measured. RESULTS: : PM exposure induced tight junction protein ZO-1 relocation from the cell periphery, which was accompanied by significant reductions in ZO-1 protein levels but not in adherens junction proteins (VE-cadherin and beta-catenin). N-acetyl-cysteine (NAC, 5mM) reduced PM-induced ROS generation in ECs, which further prevented TER decreases and atteneuated ZO-1 degradation. PM also mediated intracellular calcium mobilization via the transient receptor potential cation channel M2 (TRPM2), in a ROS-dependent manner with subsequent activation of the Ca2+-dependent protease calpain. PM-activated calpain is responsible for ZO-1 degradation and EC barrier disruption. Overexpression of ZO-1 attenuated PM-induced endothelial barrier disruption and vascular hyperpermeability in vivo and in vitro. CONCLUSIONS: : These results demonstrate that PM induces marked increases in vascular permeability via ROS-mediated calcium leakage via activated TRPM2, and via ZO-1 degradation by activated calpain. These findings support a novel mechanism for PM-induced lung damage and adverse cardiovascular outcomes.     

New Positive TRPC6 Modulator Penetrates Blood–Brain Barrier,Eliminates Synaptic Deficiency and Restores Memory Deficit in 5xFAD Mice

Synapse loss in the brain of Alzheimer’s disease patients correlates with cognitive dysfunctions. Drugs that limit synaptic loss are promising pharmacological agents. The transient receptor potential cation channel, subfamily C, member 6 (TRPC6) regulates the formation of an excitatory synapse. Positive regulation of TRPC6 results in increased synapse formation and enhances learning and memory in animal models. The novel selective TRPC6 agonist, 3-(3-,4-Dihydro-6,7-dimethoxy-3,3-dimethyl-1-isoquinolinyl)-2H-1-benzopyran-2-one, has recently been identified. Here we present in silico, in vitro, ex vivo, pharmacokinetic and in vivo studies of this compound. We demonstrate that it binds to the extracellular agonist binding site of the human TRPC6, protects hippocampal mushroom spines from amyloid toxicity in vitro, efficiently recovers synaptic plasticity in 5xFAD brain slices, penetrates the blood–brain barrier and recovers cognitive deficits in 5xFAD mice. We suggest that C20 might be recognized as the novel TRPC6-selective drug suitable to treat synaptic deficiency in Alzheimer’s disease-affected hippocampal neurons.     

Dihydrotanshinone I Inhibits the Lung Metastasis of Breast Cancer by Suppressing Neutrophil Extracellular Traps Formation

Breast cancer (BC) is a common female malignancy, worldwide. BC death is predominantly caused by lung metastasis. According to previous studies, Dihydrotanshinone I (DHT), a bioactive compound in Salvia miltiorrhiza Bunge (S. miltiorrhiza), has inhibitory effects on numerous cancers. Here, we investigated the anti-metastatic effect of DHT on BC, where DHT more strongly inhibited the growth of BC cells (MDA-MB-231, 4T1, MCF-7, and SKBR-3) than breast epithelial cells (MCF-10a). Additionally, DHT repressed the wound healing, invasion, and migration activities of 4T1 cells. In the 4T1 spontaneous metastasis model, DHT (20 mg/kg) blocked metastasis progression and distribution in the lung tissue by 74.9%. DHT reversed the formation of neutrophil extracellular traps (NETs) induced by phorbol 12-myristate 13-acetate, as well as ameliorated NETs-induced metastasis. Furthermore, it inhibited Ly6G⁺Mpo⁺ neutrophils infiltration and H3Cit expression in the lung tissues. RNA sequencing, western blot, and bioinformatical analysis indicated that TIMP1 could modulate DHT acting on lung metastasis inhibition. The study demonstrated a novel suppression mechanism of DHT on NETs formation to inhibit BC metastasis.     

Supplementation with Serum-Derived Extracellular Vesicles Reinforces Antitumor Immunity Induced by Cryo-Thermal Therapy

Effective cancer therapies should reshape immunosuppression and trigger antitumor immunity. Previously, we developed a novel cryo-thermal therapy through applying local rapid cooling followed by rapid heating of tumor tissue. It could not only ablate local tumors, but also, subsequently, induce systemic long-term antitumor immunity. Hyperthermia can induce the release of extracellular vesicles (EVs) to stimulate antitumor immunity. We examine whether EVs are released after cryo-thermal therapy and whether they could improve the efficacy of cryo-thermal therapy in the 4T1 model. In this study, serum extracellular vesicles (sEVs) are isolated and characterized 3 h after cryo-thermal therapy of subcutaneous tumors. sEV phagocytosis is observed in vitro and in vivo by using laser confocal microscopy and flow cytometry. After cryo-thermal therapy, sEVs are administered to mice via the tail vein, and changes in immune cells are investigated by using flow cytometry. After cryo-thermal therapy, a large number of sEVs are released to the periphery carrying danger signals and tumor antigens, and these sEVs could be phagocytosed by peripheral blood monocytes and differentiated macrophages. After cryo-thermal therapy, supplementation with sEVs released after treatment promotes the differentiation of myeloid-derived suppressor cells (MDSCs), monocytes into macrophages and CD4⁺ T cells into the Th1 subtype, as well as prolonging the long-term survival of the 4T1 subcutaneous tumor-bearing mice. sEVs released after cryo-thermal tumor treatment could clinically serve as an adjuvant in subsequent cryo-thermal therapy to improve the therapeutic effects on malignant tumors.