Colin N. Chesterman

Transcription factor Egr-1 supports FGF-dependent angiogenesis during neovascularization and tumor growth

Current understanding of key transcription factors regulating angiogenesis is limited. Here we show that RNA-cleaving phosphodiester-linked DNA-based enzymes (DNAzymes), targeting a specific motif in the 5′ untranslated region of early growth response (Egr-1) mRNA, inhibit Egr-1 protein expression, microvascular endothelial cell replication and migration, and microtubule network formation on basement membrane matrices. Egr-1 DNAzymes blocked angiogenesis in subcutaneous Matrigel plugs in mice, an observation that was independently confirmed by plug analysis in Egr-1-deficient animals, and inhibited MCF-7 human breast carcinoma growth in nude mice. Egr-1 DNAzymes suppressed tumor growth without influencing body weight, wound healing, blood coagulation or other hematological parameters. These agents inhibited endothelial expression of fibroblast growth factor (FGF)-2, a proangiogenic factor downstream of Egr-1, but not that of vascular endothelial growth factor (VEGF). Egr-1 DNAzymes also repressed neovascularization of rat cornea. Thus, microvascular endothelial cell growth, neovascularization, tumor angiogenesis and tumor growth are processes that are critically dependent on Egr-1.

New DNA enzyme targeting Egr-1 mRNA inhibits vascular smooth muscle proliferation and regrowth after injury

Early growth response factor-1 (Egr-1) binds to the promoters of many genes whose products influence cell movement and replication in the artery wall. Here we targeted Egr-1 using a new class of DNA-based enzyme that specifically cleaved Egr-1 mRNA, blocked induction of Egr-1 protein, and inhibited cell proliferation and wound repair in culture. The DNA enzyme also inhibited Egr-1 induction and neointima formation after balloon injury to the rat carotid artery wall. These findings demonstrate the utility of DNA enzymes as biological tools to delineate the specific functions of a given gene, and implicate catalytic nucleic acid molecules composed entirely of DNA as potential therapeutic agents.

Heparin-induced thrombocytopenia: mechanism of interaction of the heparin-dependent antibody with platelets.

The interaction of the heparin‐dependent antibody with heparin and platelets has been studied using the sera and purified IgG of four patients with heparin‐induced thrombocytopenia. Both normal platelets and Bernard‐Soulier syndrome (BSS) platelets which lack glycoprotein (GP) Ib, GPV and GPIX, aggregated in response to patient serum or IgG, but only in the presence of heparin. A monoclonal antibody (Mab) against platelet Fc II receptor (IV. 3) strongly inhibited the heparin‐dependent aggregation of both normal and BSS platelets induced by patient sera/IgG. Inhibition by the anti‐GPIb Mab (AK2) was variable and occurred only with normal platelets. Anti‐GPIX Mab (FMC 25) was not inhibitory with either normal or BSS platelets. Similar results were obtained using 14C‐serotonin release instead of platelet aggregation as a measure of platelet activation. These findings suggest that (1) the reaction of the heparin‐dependent antibody with platelets and heparin is mediated by a Fc‐dependent mechanism, (2) GPIb, GPV and GPIX are not involved in this reaction, and (3) the inhibitory effect of anti‐GPIb Mab on normal platelets is due to steric interference consistent with the platelet Fc receptor being in close proximity to GPIb.

Hematologic correlates of left atrial spontaneous echo contrast and thromboembolism in nonvalvular atrial fibrillation

OBJECTIVES This study examined the relation between left atrial spontaneous echo contrast, hematologic variables and thrombo-embolism in patients with nonvalvular atrial fibrillation. BACKGROUND Left atrial spontaneous echo contrast is associated with left atrial stasis and thromboembolism in patients with nonvalvular atrial fibrillation. However, its hematologic determinants in patients with nonvalvular atrial fibrillation are unknown. METHODS Clinical, hematologic and echocardiographic variables were prospectively measured in 135 consecutive patients with nonvalvular atrial fibrillation undergoing transesophageal echocardiography. RESULTS Patients with left atrial spontaneous echo contrast (n = 74, 55%) had an increased fibrinogen concentration (p = 0.029), platelet count (p = 0.045), hematocrit (p = NS) and left atrial dimension (p = 0.005). Multivariate analysis showed that left atrial spontaneous echo contrast was independently related to hematocrit (odds ratio = 2.24, p = 0.002), fibrinogen concentration (odds ratio = 2.08, p = 0.008) and left atrial dimension (odds ratio = 1.90, p = 0.004) but not platelet count. It was also associated with left atrial thrombus (n = 15, p = 0.001) and with recent embolism (n = 40, p < 0.001). In 40 clinically stable outpatients without previous embolism, left atrial spontaneous echo contrast was significantly related to hematocrit (p = 0.005), fibrinogen concentration (p = 0.035) and left atrial dimension (p = 0.029) but not to coagulation factor VII, D-dimer, erythrocyte sedimentation rate, platelet count, plasma beta-thromboglobulin, plasma glycocalicin or glycocalicin index. CONCLUSIONS Left atrial spontaneous echo contrast in patients with nonvalvular atrial fibrillation is independently related to hematocrit, fibrinogen concentration and left atrial dimension, indicating a relatively hypercoagulable state in addition to stasis. These findings support the hypothesis that left atrial spontaneous echo contrast is due to erythrocyte aggregation. Hematologic factors may contribute to its association with thromboembolism.

DNAzyme targeting c-jun suppresses skin cancer growth

Catalytic DNA molecules that target the transcription factor c-jun inhibit skin cancer growth in mice. Getting Under Cancer’s Skin Summer brings to mind barbecues, baseball, and trips to the local pool. Yet, outdoor fun can be hazardous to one’s health—too much sun exposure can increase the risk of developing skin cancer. Indeed, one in three cancers worldwide is skin-related, and currently available treatments may induce scarring or other toxicities. Cai et al. now report that the DNAzyme Dz13—which targets an mRNA that encodes a cancer-associated transcription factor, c-Jun—inhibits the growth of two common types of skin cancers: basal cell and squamous cell carcinomas. DNAzymes are single-stranded, all-DNA, catalytic molecules that specifically bind and cleave their target RNAs. The authors examined the effects of Dz13, which destroys c-jun mRNA, on animal models of skin cancer. Dz13 inhibited tumor growth, blocked neovascularization, and prevented metastasis in mouse models of skin cancer—effects that were mediated, in part, through the induction of antitumor immunity. Minimal toxicity was observed in Dz13-treated cynomolgus monkeys, minipigs, and rodents, and there were no off-target effects in more than 70 in vitro bioassays. Thus, Dz13 may prove to be a safe, effective therapy for skin cancer. Nonetheless, one is advised to pack the sun block in preparation for extra innings—or a fifth set. Worldwide, one in three cancers is skin-related, with increasing incidence in many populations. Here, we demonstrate the capacity of a DNAzyme-targeting c-jun mRNA, Dz13, to inhibit growth of two common skin cancer types—basal cell and squamous cell carcinomas—in a therapeutic setting with established tumors. Dz13 inhibited tumor growth in both immunodeficient and immunocompetent syngeneic mice and reduced lung nodule formation in a model of metastasis. In addition, Dz13 suppressed neovascularization in tumor-bearing mice and zebrafish and increased apoptosis of tumor cells. Dz13 inhibition of tumor growth, which required an intact catalytic domain, was due in part to the induction of tumor immunity. In a series of good laboratory practice–compliant toxicology studies in cynomolgus monkeys, minipigs, and rodents, the DNAzyme was found to be safe and well tolerated. It also did not interfere in more than 70 physiologically relevant in vitro bioassays, suggesting a reduced propensity for off-target effects. If these findings hold true in clinical trials, Dz13 may provide a safe, effective therapy for human skin cancer.

Anticardiolipin antibodies and lupus anticoagulants comprise separate antibody subgroups with different phospholipid binding characteristics

Autoantibodies to phospholipid antigens can be characterized using solid phase immunoassays to detect anticardiolipin antibodies (ACA) or in phospholipid‐dependent clotting tests where lupus anticoagulant (LA) activity can be demonstrated. It has not been established whether each activity is due to the same or separate antibody subgroups. Plasma from two patients with high levels of both activities were used for purification of ACA and LA using sequential ion‐exchange, gel‐filtration, and anti‐Ig affinity chromatography. Plasma could be separated into fractions containing each activity in the absence of the other. In these fractions, antibodies responsible for LA activity do not bind to isolated phospholipids in solid phase immunoassays, and conversely antibodies binding in these assays (ACA) do not possess LA activity, suggesting LA are directed against a more complex lipid epitope. In addition, in one patient ACA was of IgG isotype only, whilst LA was due to IgG and IgM isotypes. In this patient, the IgG‐ACA was heterogenous with three peaks clearly separated on ion‐exchange chromatography. Affinity purified antiphospholipid antibodies have been previously prepared from a number of patients using a phospha‐tidyl‐serine column and antibodies purified in this manner possess ACA but not LA activity. Taken together, these data indicate that tests for ACA and LA define separable subgroups of phospholipid binding antibodies, thus explaining the discordance often seen between the two activities.

Early Growth Response Factor-1 Induction by Injury Is Triggered by Release and Paracrine Activation by Fibroblast Growth Factor-2

Cell migration and proliferation that follows injury to the artery wall is preceded by signaling and transcriptional events that converge at the promoters of multiple genes whose products can influence formation of the neointima. Transcription factors, such as early growth response factor-1 (Egr-1), with nucleotide recognition elements in the promoters of many pathophysiologically relevant genes, are expressed at the endothelial wound edge within minutes of injury. The mechanisms underlying the inducible expression of Egr-1 in this setting are not clear. Understanding this process would provide important mechanistic insights into the earliest events in the response to injury. In this report, we demonstrate that fibroblast growth factor-2 (FGF-2) is released by injury and that antibodies to FGF-2 almost completely abrogate the activation and nuclear accumulation of Egr-1. FGF-2-inducible egr-1-promoter-dependent expression is blocked by PD98059, a specific inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)-1/2 (MEK-1/2), as well as by dominant negative mutants of ERK-1/2. Inducible ERK phosphorylation after injury is dependent on release and stimulation by endogenous FGF-2. Antisense oligonucleotides directed at egr-1 mRNA suggest that Egr-1 plays a necessary role in endothelial repair after denudation of the monolayer. These findings demonstrate that inducible Egr-1 expression after injury is contingent on the release and paracrine action of FGF-2.

Single amino acid mutation of Fcγ receptor is associated with the development of heparin-induced thrombocytopenia

Summary. Heparin‐induced thrombocytopenia (HIT) is mediated by a heparin‐dependent antibody/platelet factor 4/heparin complex binding to platelets via the FCγ receptor (type IIA). A single base polymorphism at position 131 of FeγRIIA changes the native arginine to histidine. In the presence of murine monoclonal IgG1 the former phenotype (FcγRIIAArg131) is functionally characterized by strong platelet aggregation (high responder) and the latter (Fc7RIIAHls131) by poor aggregation (low responder). In the presence of human IgGa the opposite response is observed. It has recently been shown that the heparin‐dependent antibody is predominantly of this subclass. We hypothesize that a relationship exists between FcγRIIAHls131 and the development of HIT. We studied 24 normal individuals and 20 HIT patients using VM58, a murine monoclonal IgG1, to characterize the phenotype by platelet aggregrometry, and PCR products, amplified with primers bordering the FC7RIIA polymorphism and hybridized with oligonucleotide probes specific for the single base mutation, to determine the genotype. The distribution of phenotypes and genotypes in the two populations differed, with a greater prevalence of the FcγRIIAHis131 allele in the HIT patient population. Homozygous Fc7RIIAArg131 individuals were absent from this group. We conclude that the presence of the Fc7RIIAHis131 allele is associated with a predisposition to HIT.

Suppression of vascular permeability and inflammation by targeting of the transcription factor c-Jun.

Conventional anti-inflammatory strategies induce multiple side effects, highlighting the need for novel targeted therapies. Here we show that knockdown of the basic-region leucine zipper protein, c-Jun, by a catalytic DNA molecule, Dz13, suppresses vascular permeability and transendothelial emigration of leukocytes in murine models of vascular permeability, inflammation, acute inflammation and rheumatoid arthritis. Treatment with Dz13 reduced vascular permeability due to cutaneous anaphylactic challenge or VEGF administration in mice. Dz13 also abrogated monocyte-endothelial cell adhesion in vitro and abolished leukocyte rolling, adhesion and extravasation in a rat model of inflammation. Dz13 suppressed neutrophil infiltration in the lungs of mice challenged with endotoxin, a model of acute inflammation. Finally, Dz13 reduced joint swelling, inflammatory cell infiltration and bone erosion in a mouse model of rheumatoid arthritis. Mechanistic studies showed that Dz13 blocks cytokine-inducible endothelial c-Jun, E-selectin, ICAM-1, VCAM-1 and VE-cadherin expression but has no effect on JAM-1, PECAM-1, p-JNK-1 or c-Fos. These findings implicate c-Jun as a useful target for anti-inflammatory therapies.

Beta2‐glycoprotein I in thrombosis: evidence for a role as a natural anticoagulant

Although the physiological role of beta2‐glycoprotein I (B2GPI) is unknown, in vitro evidence indicates that B2GPI may be a natural anticoagulant. In this study we have examined whether fluctuations of plasma B2GPI occur in in vivo coagulation. Serial measurements of B2GPI and other anticoagulant proteins were performed in 51 patients with thrombotic (group 1: six patients with disseminated intravascular coagulation (DIC), group 2: venous (n=4) or arterial (n=17) thrombosis) and non‐thrombotic disease (group 3: 24 patients undergoing elective surgery). Reductions in plasma B2GPI levels were seen in most patients which were roughly proportional to the severity of their illness. Particularly striking reductions of B2GPI, protein C (PC) and antithrombin III (AT‐III) (mean ± 95% CI: 42.7 ± 8.6%, 42.1 ± 14.8%, 39.1 ± 28.4% respectively) were seen in group 1. The reductions in plasma B2GPI were significantly greater in group 1 than in the other groups. Dilutional factors explain most of the reductions in B2GPI, PC and AT‐III in groups 2 and 3, but contribute little to group 1. In conclusion, although B2GPI behaves as a ‘negative acute phase reactant’, the magnitude of reduction of plasma B2GPI levels, accompanied by reductions in other anticoagulant proteins in patients with DIC, suggests specific consumption of B2GPI in in vivo coagulation. This study provides further evidence that B2GPI is an anticoagulant of physiological importance.