Functional interaction between TRP4 and CFTR in mouse aorta endothelial cells

Background  

This study describes the functional interaction between the putative Ca²⁺ channel TRP4 and the cystic fibrosis transmembrane conductance regulator, CFTR, in mouse aorta endothelium (MAEC).     

High-avidity monoclonal antibodies against the human scavenger class B type I receptor efficiently block hepatitis C virus infection in the presence of high-density lipoprotein

The human scavenger class B type 1 receptor (SR-B1/Cla1) was identified as a putative receptor for hepatitis C virus (HCV) because it binds to soluble recombinant HCV envelope glycoprotein E2 (sE2). High-density lipoprotein (HDL), a natural SR-B1 ligand, was shown to increase the in vitro infectivity of retroviral pseudoparticles bearing HCV envelope glycoproteins and of cell culture-derived HCV (HCVcc), suggesting that SR-B1 promotes viral entry in an HDL-dependent manner. To determine whether SR-B1 participates directly in HCV infection or facilitates HCV entry through lipoprotein uptake, we generated a panel of monoclonal antibodies (MAbs) against native human SR-B1. Two of them, 3D5 and C167, bound to conformation-dependent SR-B1 determinants and inhibited the interaction of sE2 with SR-B1. These antibodies efficiently blocked HCVcc infection of Huh-7.5 hepatoma cells in a dose-dependent manner. To examine the role of HDL in SR-B1-mediated HCVcc infection, we set up conditions for HCVcc production and infection in serum-free medium. HCVcc efficiently infected Huh-7.5 cells in the absence of serum lipoproteins, and addition of HDL led to a twofold increase in infectivity. However, the HDL-induced enhancement of infection had no impact on the neutralization potency of MAb C167, despite its ability to inhibit both HDL binding to cells and SR-B1-mediated lipid transfer. Of note, MAb C167 also potently blocked Huh-7.5 infection by an HCV strain recovered from HCVcc-infected chimpanzees. These results demonstrate that SR-B1 is essential for infection with HCV produced in vitro and in vivo and suggest the possible use of anti-SR-B1 antibodies as therapeutic agents.     

Mycophenolic acid inhibits IL-2-dependent T cell proliferation,but not IL-2-dependent survival and sensitization to apoptosis

Mycophenolic acid (MPA), the active metabolite of the immunosuppressive drug mycophenolate mofetil, is a selective inhibitor of inosine 5'-monophosphate dehydrogenase type II, a de novo purine nucleotide synthesis enzyme expressed in T and B lymphocytes and up-regulated upon cell activation. In this study, we report that the blockade of guanosine nucleotide synthesis by MPA inhibits mitogen-induced proliferation of PBL, an effect fully reversed by addition of guanosine and shared with mizoribine, another inhibitor of inosine 5'-monophosphate dehydrogenase. Because MPA does not inhibit early TCR-mediated activation events, such as CD25 expression and IL-2 synthesis, we investigated how it interferes with cytokine-dependent proliferation and survival. In activated lymphoblasts that are dependent on IL-2 or IL-15 for their proliferation, MPA does not impair signaling events such as of the extracellular signal-regulated kinase 2 and Stat5 phosphorylation, but inhibits down-regulation of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, in activated lymphoblasts, MPA specifically interferes with cytokine-dependent signals that control cell cycle and blocks activated T cells in the mid-G(1) phase of the cell cycle. Although it blocks IL-2-mediated proliferation, MPA does not inhibit cell survival and Bcl-x(L) up-regulation by IL-2 or other cytokines whose receptors share the common gamma-chain (CD132). Finally, MPA does not interfere with IL-2-dependent acquisition of susceptibility to CD95-mediated apoptosis and degradation of cellular FLIP. Therefore, MPA has unique functional properties not shared by other immunosuppressive drugs interfering with IL-2R signaling events such as rapamycin and CD25 mAbs.     

Use of Low-Molecular�CWeight Heparin to Decrease Mortality in Mice after Intracardiac Injection of Tumor Cells

Intracardiac injection of human tumor cells into anesthetized nude mice is an established model of bone metastasis. However, intracardiac injection of some human tumor cell lines cause acute neurologic signs and high mortality, making some potentially relevant tumor cell lines unusable for investigation. We showed that intracardiac injection of tumor cells can induce a hypercoagulable state leading to platelet consumption and thromboemboli formation and that pretreatment with intravenous injection of low-molecular–weight heparin (LMWH; enoxaparin) blocks this state. In addition, intravenous injection of enoxaparin before intracardiac injection with 2 different small-cell lung carcinoma lines, H1975 and H2126, dramatically decreased mouse mortality while still generating bone metastases. Therefore, reduction of mortality by pretreatment with LMWH increases the types of cells that can be studied in this metastasis model and decreases the number of animals used.Abbreviations: APTT, activated partial thromboplastin time; BLI, bioluminescent imaging; CBC, complete blood count; DIC, disseminated intravascular coagulation; H1975luc, H1975 cell line tagged with lucerifase–green fluorescent protein; H2126luc, H2126 cell line tagged with lucerifase–green fluorescent protein; LMWH, low-molecular–weight heparin; PT, prothrombin time; UFH, unfractionated heparinResearch using animal models mimicking the metastasis of human tumors to bone is critical for the development of cancer therapeutics. Bone metastases are present in almost all people who die of cancer and are more likely to occur with breast, prostate, lung, kidney, and thyroid cancers.^1,24 In patients with advanced breast and prostate cancers, much of the tumor burden at the time of death will be found in bone.²⁰ The pattern of bone metastases can range from purely destructive (osteolytic) to mostly osteoblastic (bone-forming) lesions. Osteolysis is accompanied by pain, bone fragility, and increased susceptibility to pathologic fracture. In osteolytic metastasis, a 2-way interaction between tumor cells and osteoclasts in the bone microenvironment leading to continued osteolysis and tumor growth is suspected.²⁰ Current therapies for bone metastases, such as bisphosphonates, are directed at inhibiting bone resorption, but other therapies are in development that specifically target tumor cell or osteoclast factors involved in the 2-way cycle between tumor growth and osteolysis.¹⁸Bone metastasis is rare in mouse models of spontaneous mammary and prostate carcinomas, experimentally implanted animal tumor models (such as syngeneic and xenograft tumors), and chemical or transgenic induction of mammary and prostate carcinomas. To increase the frequency of bone metastases, injection techniques using either orthotopic tumor cell injection into mammary glands or prostate or intracardiac injection of human tumor cell lines into the left ventricle of nude mice have been developed.^5,14,25,31 In contrast to the late stage, low incidence of metastasis after orthotopic injection, intracardiac injection of human tumor cell lines results in much higher rates of bone metastasis at an early stage in the disease, with osteolytic metastases to the metaphyses of long bones.^6,23 Development of osteolytic lesions in this model can be monitored by various methods, including radiography and, more recently, in vivo bioluminescent imaging (BLI) using lucerifase-tagged tumor cells. Bioluminescent imaging detects micrometastatic lesions and allows for serial in vivo monitoring of bone metastases.^9-11 After a BLI study, bone metastases can be assessed histologically, with tumor foci typically seen in the femur or tibia.Bone metastasis models using the intracardiac tumor injection technique have been primarily focused on a few breast (for example, MDA-MB-231) and prostate models (for example, PC3), but additional models of other tumors that interact with bone (especially lung carcinomas) need to be developed.^24,30 Intracardiac injection of some nonsmall cell lung carcinoma tumor cell lines have led to stroke-like clinical signs, including head tilt, spinning, and failure to recover from anesthesia after intracardiac injection.¹⁵ We postulated that the stroke-like clinical signs and mortality were due to thromboembolism formation immediately after intracardiac tumor cell injection.Tumor cells have procoagulant activity. Procoagulants, such as tissue factor, may be increased on the surface of or secreted into the blood by cancer cells, leading to changes in the clotting cascade.¹³ Approximately 15% of all cancer patients are affected by thromboembolic disease, including superficial and deep-vein thrombosis, arterial thrombosis and embolism, pulmonary emboli, and thrombosis of venous access devices.^12,13 Anticoagulant treatments used clinically to prevent thrombi and thromboemboli include warfarin, unfractionated heparin (UFH), and low-molecular–weight heparins (LMWH), such as enoxaparin (Lovenox, Sanofi Aventis, Bridgewater, NJ) and dalteparin (Fragmin, Pfizer, New York, NY). LMWHs are prepared through chemical, hydrolytic, or enzymatic degradation of unfractionated heparin.¹³ Both UFH and LMWH exert their anticoagulant effects by binding to antithrombin and causing a confrontational change. This change increases the interaction of antithrombin with thrombin (IIa) and activated factors X (Xa) and IX (IXa), leading to inhibition of clotting.^8,28LMWHs decrease the formation of thromboembolism and subsequent mortality in several murine models of thromboembolism and disseminated intravascular coagulation (DIC). In the murine model of thrombin-induced thromboembolism, massive deposition of intravascular fibrin—mainly within the pulmonary arteries—causes death within 5 minutes after thrombin injection.^16,22 Both UFH and LMWH inhibit thrombin and prevent mortality in this model, but bleeding times and activated partial prothrombin time (APPT) are less prolonged with LMWH.¹⁶ LMWH is also effective in preventing murine DIC in a lipopolysaccharide model, in which mice given 2 injections of lipopolysaccharide develop DIC, multiple organ failure, and die. Mice given LMWH before lipopolysaccharide administration have fewer lung and liver microthrombi and greater survival than do mice not given LMWH.^26,27Here, we evaluated the use of LMWH in mice to prevent morbidity and mortality associated with intracardiac injection of human tumor cell lines. We determined that thromboembolism occurred in intracardiac tumor-challenged mice and that LMWH blocked thromboembolism. We also determined the effect of LMWH on animal survival and subsequent development of bone metastasis in this mouse model.     

PTEN expression in ovine granulosa cells increases during terminal follicular growth

In the present paper, we have studied the expression of the Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) and its putative biological role in the sheep ovary. We found by Northern-blot, immunohistochemistry and immunoblot that PTEN is highly expressed in granulosa cells from large differentiated follicles (LF) in comparison with small proliferating follicles (SF) (P < 0.001), with no clear effect of follicle quality. Moreover, the PTEN lipid phosphatase activity is also higher in LF than in SF (P < 0.01). In contrast, levels of the phosphorylated form of AKT (pAKT) are lower in LF than in SF (P < 0.0001). IGF-I and insulin but not FSH, LH or forskolin are able to stimulate the expression of PTEN mRNA (P < 0.001) and protein by ovine granulosa cells after 48 h of culture in vitro. An IGF-1 time course analysis showed that expression of PTEN protein appeared after 12h of culture, concomitant with the fall of the pAKT levels, which peaked after 6h of stimulation with IGF-I. Moreover, transfection experiments showed that overexpression of PTEN in ovine granulosa cells induced a decrease and an increase in E2F and p27 promoter activity, respectively (P < 0.05). Overall, our present data show for the first time that the expression of PTEN increases during terminal follicular growth. This increase, that might be induced by IGF-I but not FSH, would participate in the proliferation/differentiation transition of ovine granulosa cells in differentiating follicles.     

Inhibition of Glycoprotein VI Clustering by Collagen as a Mechanism of Inhibiting Collagen-Induced Platelet Responses: The Example of Losartan

Exposure of platelets to collagen triggers the formation of a platelet clot. Pharmacological agents capable of inhibiting platelet activation by collagen are thus of potential therapeutic interest. Thrombus formation is initiated by the interaction of the GPIb-V-IX complex with collagen-bound vWF, while GPVI interaction with collagen triggers platelet activation that is reinforced by ADP and thromboxane A2. Losartan is an angiotensin II (Ang II) type I receptor (AT1R) antagonist proposed to have an antiplatelet activity via the inhibition of both the thromboxane A2 (TXA2) receptor (TP) and the glycoprotein VI (GPVI). Here, we characterized in vitro the effects of losartan at different doses on platelet responses: losartan inhibited platelet aggregation and secretion induced by 1 μg.mL^-1 and 10 μg.mL^-1 of collagen with an IC50 of ~ 6 μM. Losartan inhibited platelet responses induced by the GPVI specific collagen related peptide but not by the α2β1 specific peptide. However, losartan did not inhibit the binding of recombinant GPVI to collagen, which is not in favor of a simple competition. Indeed, the clustering of GPVI observed in flow cytometry and using the Duolink methodology, was inhibited by losartan. The impact of a therapeutic dose of losartan (100 mg/day) on platelet responses was analyzed ex vivo in a double blind study. No statistically significant differences were observed between losartan-treated (n=25) and non-treated (n=30) patients in terms of collagen and U46619-induced platelet activation. These data indicate that in treated patients, losartan does not achieve a measurable antiplatelet effect but provide the proof of concept that inhibiting collagen-induced GPVI clustering is of pharmacological interest to obtain an antithrombotic efficacy.

Trial Registration

ClinicalTrials.gov NCT00763893     

Localization of the Escherichia coli cell division protein FtsI (PBP3) to the division site and cell pole

FtsI, also known as penicillin-binding protein 3, is a transpeptidase required for the synthesis of peptidoglycan in the division septum of the bacterium, Escherichia coli . FtsI has been estimated to be present at about 100 molecules per cell, well below the detection limit of immunoelectron microscopy. Here, we confirm the low abundance of FtsI and use immunofluorescence microscopy, a highly sensitive technique, to show that FtsI is localized to the division site during the later stages of cell growth. FtsI was also sometimes observed at the cell pole; polar localization was not anticipated and its significance is not known. We conclude (i) that immunofluorescence microscopy can be used to localize proteins whose abundance is as low as approximately 100 molecules per cell; and (ii) that spatial and temporal regulation of FtsI activity in septum formation is achieved, at least in part, by timed localization of the protein to the division site.