Keith R. McCrae

Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor.

The urokinase receptor (uPAR) binds urokinase-type plasminogen activator (u-PA) through specific interactions with uPAR domain 1, and vitronectin through interactions with a site within uPAR domains 2 and 3. These interactions promote the expression of cell surface plasminogen activator activity and cellular adhesion to vitronectin, respectively. High molecular weight kininogen (HK) also stimulates the expression of cell surface plasminogen activator activity through its ability to serve as an acquired receptor for prekallikrein, which, after its activation, may directly activate prourokinase. Here, we report that binding of the cleaved form of HK (HKa) to human umbilical vein endothelial cells (HUVEC) is mediated through zinc-dependent interactions with uPAR. These occur through a site within uPAR domains 2 and 3, since the binding of 125I-HKa to HUVEC is inhibited by vitronectin, anti-uPAR domain 2 and 3 antibodies and soluble, recombinant uPAR (suPAR), but not by antibody 7E3, which recognizes the beta chain of the endothelial cell vitronectin receptor (integrin alphavbeta3), or fibrinogen, another alphavbeta3 ligand. We also demonstrate the formation of a zinc-dependent complex between suPAR and HKa. Interactions of HKa with endothelial cell uPAR may underlie its ability to promote kallikrein-dependent cell surface plasmin generation, and also explain, in part, its antiadhesive properties.

The very low density lipoprotein receptor mediates the cellular catabolism of lipoprotein lipase and urokinase-plasminogen activator inhibitor type I complexes.

The very low density lipoprotein (VLDL) receptor binds apolipoprotein E-rich lipoproteins as well as the 39-kDa receptor-associated protein (RAP). Ligand blotting experiments using RAP and immunoblotting experiments using an anti-VLDL receptor IgG detected the VLDL receptor in detergent extracts of human aortic endothelial cells, human umbilical vein endothelial cells, and human aortic smooth muscle cells. To gain insight into the role of the VLDL receptor in the vascular endothelium, its ligand binding properties were further characterized. In vitro binding experiments documented that lipoprotein lipase (LpL), a key enzyme in lipoprotein catabolism, binds with high affinity to purified VLDL receptor. In addition, urokinase complexed with plasminogen activator-inhibitor type I (uPA•PAI-1) also bound to the purified VLDL receptor with high affinity. To assess the capacity of the VLDL receptor to mediate the cellular internalization of ligands, an adenoviral vector was used to introduce the VLDL receptor gene into a murine embryonic fibroblast cell line deficient in the VLDL receptor and the LDL receptor-related protein, another endocytic receptor known to bind LpL and uPA•PAI-1 complexes. Infected fibroblasts that express the VLDL receptor mediate the cellular internalization of 125I-labeled LpL and uPA•PAI-1 complexes, leading to their degradation. Non-infected fibroblasts or fibroblasts infected with the lacZ gene did not internalize these ligands. These studies confirm that the VLDL receptor binds to and mediates the catabolism of LpL and uPA•PAI-1 complexes. Thus, the VLDL receptor may play a unique role on the vascular endothelium in lipoprotein catabolism by regulating levels of LpL and in the regulation of fibrinolysis by facilitating the removal of urokinase complexed with its inhibitor.

Detection of endothelial cell-reactive immunoglobulin in patients with anti-phospholipid antibodies

Individuals with anti‐phospholipid antibodies are at increased risk for the development of thrombosis and fetal loss. The pathogenesis of these syndromes is unknown, but may involve antibody‐mediated alterations in endothelial cell coagulant activity. To address this possibility, we determined the incidence of endothelial cell‐reactive antibodies in 76 patients whose plasma contained anti‐phospholipid antibodies, but who had no clinically‐evident immune disorder. Plasma from 47 patients deposited significantly more immunoglobulin on cultured endothelial cells than control plasma. Positive tests were more frequent in patients with a history of thrombosis than in those without (17/19 v 23/48; P=0.004). However, we observed no correlation between immunoglobulin deposition on cardiolipin and endothelial cells by individual plasmas. Furthermore, endothelial cell reactivity was not diminished by adsorption of anti‐cardiolipin antibodies from patient sera using liposomes. Immunoglobulin fractions prepared from 5/6 patient sera immuno‐precipitated a ≈ 70 kDa endothelial cell surface protein; 4/5 of these fractions also induced the release of von Willebrand factor from endothelial cells. These results demonstrate that plasma from many patients with anti‐phospholipid antibodies, but no clinically‐evident autoimmune disease, also contains endothelial cell‐reactive antibodies. Detection of such antibodies might help identify individuals in this patient population at greatest risk for thrombosis.

SV40 Tag transformation of the normal invasive trophoblast results in a premalignant phenotype. I. Mechanisms responsible for hyperinvasivess and resistance to anti-invasive action of TGFβ

Invasion of the uterus by first trimester human placental extravillous trophoblast (EVT) cells depends on mechanisms shared by malignant cells. However, unlike tumor invasion, trophoblast invasion of the uterus is stringently controlled in situ by local molecules such as transforming growth factor (TGF)β. Since EVT cells possess active invasion‐associated genes but are nontumorigenic, our objective was to induce premalignant and then malignant phenotype into a normal EVT cell line in order to identify the molecular basis of tumor progression. Simian virus 40 large T antigen (SV40 Tag) was introduced into a normal human first trimester invasive EVT cell line, HTR8, established in our laboratory. Since the HTR8 line has a limited in vitro lifespan of 12–15 passages, SV40 Tag‐transformed cells were selected on the basis of extended lifespan. A long‐lived line, RSVT‐2, was produced and an immortalized subclone, RSVT2/C, was further derived under a forced crisis regimen. We examined transformation‐induced alterations in proliferative and invasive abilities, responses to the invasion and proliferation‐regulating growth factor TGFβ and changes in gene expression for invasion‐associated enzymes or enzyme inhibitors. RSVT‐2 and RSVT2/C cell lines were hyperproliferative and hyperinvasive when compared with the parental HTR8 cell line. They were also variably resistant to the anti‐proliferative and anti‐invasive signals from TGFβ. Since both cell lines remained non‐tumorigenic in nude mice, these properties indicate that they attained a premalignant phenotype. Both cell lines showed reduced expression of tissue inhibitor of metalloproteases (TIMP)‐1, while TIMP‐2 and plasminogen activator inhibitor (PAI)‐1 expression was was also reduced in RSVT2/C cells, thus contributing to their hyperinvasiveness. Their resistance to the anti‐invasive action of TGFβ was explained by the failure of TGFβ to upregulate TIMPs and PAI‐1, in contrast to the TGFβ‐induced upregulation noted in parental HTR8 cells. Int. J. Cancer 77:429–439, 1998.© 1998 Wiley‐Liss, Inc.

MOLECULAR MECHANISMS CONTROLLING TROPHOBLAST INVASION OF THE UTERUS

Summary In this study we have examined whether trophoblast invasiveness is aninherent property of these cells and, since in vitro placental invasion of the uterus is spatially and temporally restricted, whether loss of invasion is due to control by the decidual tissue. Using in vitro invasion assays we have shown that cultured first trimester as well as term human trophoblast cells, dissociated from the uterine microenvironment, are highly invasive. Conditioned media from first trimester human decidual cells (DCM) significantly reduced invasion of first trimester trophoblast cells. This suppression was prevented by addition of neutralizing anti-TGFβ antibody or neutralizing antibody to Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) to the DCM, and mimicked by TGFβ1. We have previously reported a decrease in collagenase type IV activity in the conditioned media of first trimester trophoblast cultures when TGFβ1 was added to these cultures and that presence of neutralizing anti-TGFβ antibody in trophoblast cultures (to remove endogenous TGFβ activity) resulted in down regulation of TIMP-1 message. Here we show that removal of endogenous TGFβ in trophoblast and decidual cultures resulted in a decrease in TIMP activity in the conditioned media. These data, taken together, indicate that decidua-derived TGFβ exerts its anti-invasive action by up regulating TIMP levels in both the trophoblast and decidua. Another mechanism by which TGFβ exerts its anti-invasive role is by inducing the differentiation of invasive trophoblasts into non-proliferating, multinucleated, presumably non-invasive cells. Other mechanisms of control of trophoblast invasion remain to be determined.

The antiphospholipid-protein syndrome

The pathogenesis of the antiphospholipid syndrome remains uncertain. Antibodies that react with phospholipids may not be directly responsible for cellular injury, but may be part of the immune network through which autoantibodies with pathogenic potential are generated. The latter may recognize proteins such asβ2-glycoprotein I that form complexes with phospholipids, proteins whose functions depend upon interaction with phospholipids such as protein C and its cofactors, altered lipoproteins such as oxidized low-density lipoproteins, or other molecules that share only antigenic similarity. Thus, a spectrum of autoantibodies that recognize different lipid-protein complexes may develop in these patients and contribute to the observed clinical heterogeneity of the syndrome. Current techniques do not permit identification of the subset of patients with antiphospholipid antibodies at risk for thrombosis or abortion and there are no prospective, controlled trials addressing the prophylaxis or treatment of affected individuals. Identification of the cellular targets of antibodies to lipid-protein moieties is needed to identify patients at risk for these complications and as a means to monitor therapy.

Plasma concentration of endothelin-1 in women with cocaine-associated pregnancy complications

OBJECTIVEnThe purpose of this study was to determine if the plasma concentration of endothelin-1 is elevated in pregnant women abusing cocaine and to determine how these levels differ from those in patients with preeclampsia and in women with uncomplicated pregnancies.nnnSTUDY DESIGNnPlasma endothelin-1 levels were measured in 30 women with acute cocaine intoxication, 32 women with preeclampsia, 14 pregnant women with chronic hypertension, 26 women with uncomplicated pregnancies, and 16 nonpregnant individuals. Serial samples after delivery were obtained in 12 women with preeclampsia, 10 with cocaine abuse, 4 with chronic hypertension, and 7 with uncomplicated pregnancies.nnnRESULTSnThe mean endothelin-1 concentration in those with cocaine abuse was 18.2 +/- 8.1 pg/ml (95% confidence interval 15.2 to 21.2). This was similar to that in women with preeclampsia (21.1 +/- 5.9 pg/ml, 95% confidence interval 19 to 23.3) (p = 0.2) but significantly different from that in women with chronic hypertension (11.5 +/- 3.6 pg/ml, 95% confidence interval 9.4 to 13.6) (p < 0.001) and women with uncomplicated pregnancies (6.7 +/- 3.9 pg/ml, 95% confidence interval 5.1 to 8.2) (p < 0.001).nnnCONCLUSIONSnEndothelin-1 levels in women abusing cocaine are comparable to those in women with preeclampsia and are significantly higher than those in gravid women with chronic hypertension and women with uncomplicated pregnancies. Elevated levels of endothelin-1 may contribute to some of the pregnancy-related complications in women abusing cocaine.

126 Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2+3 of the urokinase receptor

The urokinase receptor (uPAR) binds urokinase-type plasminogen activator (u-PA) through specific interactions with uPAR domain 1, and vitronectin through interactions with a site within uPAR domains 2 and 3. These interactions promote the expression of cell surface plasminogen activator activity and cellular adhesion to vitronectin, respectively. High molecular weight kininogen (HK) also stimulates the expression of cell surface plasminogen activator activity through its ability to serve as an acquired receptor for prekallikrein, which, after its activation, may directly activate prourokinase. Here, we report that binding of the cleaved form of HK (HKa) to human umbilical vein endothelial cells (HUVEC) is mediated through zinc-dependent interactions with uPAR. These occur through a site within uPAR domains 2 and 3, since the binding of 125I-HKa to HUVEC is inhibited by vitronectin, anti-uPAR domain 2 and 3 antibodies and soluble, recombinant uPAR (suPAR), but not by antibody 7E3, which recognizes the beta chain of the endothelial cell vitronectin receptor (integrin alphavbeta3), or fibrinogen, another alphavbeta3 ligand. We also demonstrate the formation of a zinc-dependent complex between suPAR and HKa. Interactions of HKa with endothelial cell uPAR may underlie its ability to promote kallikrein-dependent cell surface plasmin generation, and also explain, in part, its antiadhesive properties.

Effectiveness and safety of anticoagulants for the treatment of venous thromboembolism in patients with cancer

Anticoagulation is used to treat venous thromboembolism (VTE) in cancer patients, but may be associated with an increased risk of bleeding. VTE recurrence and major bleeding were assessed in cancer patients treated for VTE with the most currently prescribed anticoagulants in clinical practice. Newly diagnosed cancer patients (first VTE 1/1/2013‐05/31/2015) who initiated rivaroxaban, low‐molecular‐weight heparin (LMWH), or warfarin were identified from Humana claims data and observed until end of eligibility or end of data availability. VTE recurrence was a hospitalization with a primary diagnosis of VTE ≥7 days after first VTE. Major bleeding events on treatment were identified using validated criteria. Cohorts were compared using Kaplan–Meier rates at 6 and 12 months and Cox proportional hazards models. Cohorts were adjusted for their differences at baseline. A total of 2428 patients (rivaroxaban: 707; LMWH: 660; warfarin: 1061) met inclusion criteria. Patient characteristics were well balanced after weighting. There was a trend for lower VTE recurrence rates in rivaroxaban users compared to LMWH users at 6 months (13.2% vs. 17.1%; Pu2009=u2009.060) and significantly lower at 12 months (16.5% vs. 22.2%; Pu2009=u2009.030) [HR: 0.72, 95% CI: (0.52‐0.95); Pu2009=u2009.024]. VTE recurrence rates were also lower for rivaroxaban than warfarin users at 6 months (13.2% vs. 17.5%; Pu2009=u2009.014) and 12 months (15.7% vs. 19.9%; Pu2009=u2009.017) [HR: 0.74, 95% CI: (0.56‐0.96); Pu2009=u2009.028]. Major bleeding rates were similar across cohorts. This real‐world analysis suggests cancer patients with VTE treated with rivaroxaban had significantly lower risk of recurrent VTE and similar risk of bleeding compared to those treated with LMWH or warfarin.

Clinical Predictors of Early Mortality in Colorectal Cancer Patients Undergoing Chemotherapy: Results From a Global Prospective Cohort Study

Abstract Background Early mortality is a major problem in colorectal cancer (CRC). We have shown that Khorana Score is predictive of early mortality in other cancers. Here, we evaluated the value of this score and other prognostic variables in predicting early mortality in CRC. Methods CANTARISK was a prospective, noninterventional, global cohort study in patients with CRC initiating a new chemotherapy regimen. Data were collected at zero, two, four, and six months. Early mortality was defined as death within six months of enrollment. All data were compiled centrally and analyzed after the study closed. Statistically significant univariate associations were tested in multivariable models; adjusted odds ratios (ORs) are presented. Statistical tests were two-sided. Results From 2011 to 2012, 1789 CRC patients were enrolled. The median age was 62 years; 71% were Caucasian. One-third (35%) had a rectal primary, and 65% had metastatic disease. There were 184 (10.3%) patients who died during their first six months in the study. For low, intermediate, and high Khorana Score, there were 8.1%, 11.2% and 32.5% deaths, respectively. In multivariable analyses, Khorana Score was an independent predictor of early death (OR for high/intermediate vs low score = 1.70, P = .0027), in addition to age (OR for each incremental year = 1.03, P = .0014), presence of metastatic disease (ORu2009=u20093.28, P < .0001), and Easter Cooperative Oncology Group Performance Status Score of 2 or higher (ORu2009=u20093.85, P < .0001). Conclusions This study demonstrates that Khorana Score is predictive of early mortality in CRC patients. Intermediate- or high-risk patients, as defined by this score, may benefit from additional interventions aimed at reducing early mortality.