Evaluation of Serum Integrin αvß3 & Vitronectin in the Early Diagnosis of Breast Cancer.

BACKGROUND: This study aimed to evaluate the relationship of serum vitronectin and integrin alpha V beta 3 (αvß3) levels with various clinicopathological parameters of breast cancer and to assess the diagnostic value of these markers alone or in combination with the conventional breast cancer biomarker CA15.3. METHODS: This study included 50 early diagnosed stage I - II primary breast cancer patients, 20 patients with fibroadenoma benign lesions, and 20 apparently normal healthy controls. Integrin αVß3, vitronectin, and CA15.3 levels were measured using ELISA technique. RESULTS: Serum levels of integrin αVß3 and vitronectin were significantly higher in the malignant group than those in the benign group and the control group with (p < 0.001). Significant positive correlation between integrin αvß3 and vitronectin concentrations was found. Both markers showed significant statistically difference with lymph node, histological grade, tumor stage, and tumor size (p < 0.05). Integrin αvß3 exhibited the highest sensitivity (70%) and specificity (68%), then vitronectin with 67% and 68%, respectively, followed by CA15.3 showing the least sensitivity and specificity (65% and 62%, respectively). All assessed parameters revealed comparable area under the receiver-operating characteristic curve (AUC) 95% confidence interval (CI) range of 0.581 - 0.822. CONCLUSIONS: Integrin αvß3 is a promising biomarker alone or in combination with vitronectin and CA15.3 for diagnosis and prognosis of early stage breast cancer.

Prostate cancer sheds the αvß3 integrin in vivo through exosomes.

The αvß3 integrin has been shown to promote aggressive phenotypes in many types of cancers, including prostate cancer. We show that GFP-labeled αvß3 derived from cancer cells circulates in the blood and is detected in distant lesions in NOD scid gamma (NSG) mice. We, therefore, hypothesized that αvß3 travels through exosomes and tested its levels in pools of vesicles, which we designate extracellular vesicles highly enriched in exosomes (ExVs), and in exosomes isolated from the plasma of prostate cancer patients. Here, we show that the αvß3 integrin is found in patient blood exosomes purified by sucrose or iodixanol density gradients. In addition, we provide evidence that the αvß3 integrin is transferred through ExVs isolated from prostate cancer patient plasma to ß3-negative recipient cells. We also demonstrate the intracellular localization of ß3-GFP transferred via cancer cell-derived ExVs. We show that the ExVs present in plasma from prostate cancer patients contain higher levels of αvß3 and CD9 as compared to plasma ExVs from age-matched subjects who are not affected by cancer. Furthermore, using PSMA antibody-bead mediated immunocapture, we show that the αvß3 integrin is expressed in a subset of exosomes characterized by PSMA, CD9, CD63, and an epithelial-specific marker, Trop-2. Finally, we present evidence that the levels of αvß3, CD63, and CD9 remain unaltered in ExVs isolated from the blood of prostate cancer patients treated with enzalutamide. Our results suggest that detecting exosomal αvß3 integrin in prostate cancer patients could be a clinically useful and non-invasive biomarker to follow prostate cancer progression. Moreover, the ability of αvß3 integrin to be transferred from ExVs to recipient cells provides a strong rationale for further investigating the role of αvß3 integrin in the pathogenesis of prostate cancer and as a potential therapeutic target.

Gαs proteins activate p72(Syk) and p60-c-Src tyrosine kinases to mediate sickle red blood cell adhesion to endothelium via LW-αvß3 and CD44-CD44 interactions.

G protein-coupled receptors (GPCRs) have been suggested as new drug targets to treat a variety of diseases. In sickle cell disease (SCD), the LW erythrocyte adhesion receptor can be activated by stimulation of ß2 adrenergic receptors (ß2ARs), to mediate sickle erythrocyte (SSRBC) adhesion to endothelium. However, the involvement of tyrosine protein kinases in ß2AR signaling to activate SSRBC adhesion to endothelium has not been thoroughly elucidated. Either direct activation with Cholera toxin of Gαs protein, which acts downstream of ß2ARs, or inhibition with Pertussis toxin of Gαi, mediating suppression of adenylyl cyclase, increased SSRBC adhesion to endothelium over baseline adhesion. This effect involved the non-receptor tyrosine kinases, p72(Syk) and p60-c-Src, which were more abundant in SSRBCs than in normal erythrocytes. In contrast, Pertussis toxin and Cholera toxin failed to increase adhesion of normal erythrocytes. SSRBC Gαi inhibition also increased phosphorylation of p72(Syk) and p60-c-Src. Further, we investigated the relevance of activation of p72(Syk) and p60-c-Src, and identified LW (ICAM-4, CD242) and CD44 as the erythroid adhesion molecules both physically interacting with activated p60-c-Src. As a result, SSRBC LW underwent increased tyrosine phosphorylation, leading to SSRBC LW and CD44 binding to endothelial αvß3 integrin and CD44, respectively. These data provide in vitro mechanistic evidence that p60-c-Src, which could act downstream of Gαs/p72(Syk), associates with LW and CD44 on SSRBCs leading to their interactions with endothelial αvß3 and CD44, respectively. Thus, increased activation of these signaling mechanisms in SSRBCs could initiate or exacerbate vascular occlusion, the hallmark of SCD.

Disulfide bond exchanges in integrins αIIbß3 and αvß3 are required for activation and post-ligation signaling during clot retraction.

BACKGROUND: Integrin αIIbß3 mediates platelet adhesion, aggregation and fibrin clot retraction. These processes require activation of αIIbß3 and post-ligation signaling. Disulfide bond exchanges are involved in αIIbß3 and αvß3 activation. METHODS: In order to investigate the role of integrin activation and disulfide bond exchange during αIIbß3- and αvß3-mediated clot retraction, we co-expressed in baby hamster kidney cells wild-type (WT) human αIIb and WT or mutated human ß3 that contain single or double cysteine substitutions disrupting C523-C544 or C560-C583 bonds. Flow cytometry was used to measure surface expression and activation state of the integrins. Time-course of fibrin clot retraction was examined. RESULTS: Cells expressed WT or mutated human αIIbß3 as well as chimeric hamster/human αvß3. The αIIbß3 mutants were constitutively active and the thiol blocker dithiobisnitrobenzoic acid (DTNB) did not affect their activation state. WT cells retracted the clot and addition of αvß3 inhibitors decreased the retraction rate. The active mutants and WT cells activated by anti-LIBS6 antibody retracted the clot faster than untreated WT cells, particularly in the presence of αvß3 inhibitor. DTNB substantially inhibited clot retraction by WT or double C523S/C544S mutant expressing cells, but minimally affected single C523S, C544S or C560S mutants. Anti-LIBS6-enhanced clot retraction was significantly inhibited by DTNB when added prior to anti-LIBS6. CONCLUSIONS: Both αIIbß3 and αvß3 contribute to clot retraction without prior activation of the integrins. Activation of αIIbß3, but not of αvß3 enhances clot retraction. Both αIIbß3 activation and post-ligation signaling during clot retraction require disulfide bond exchange.

Contribution of distinct platelet integrins to binding, unfolding, and assembly of fibronectin.

Fibronectin (FN) fibrillogenesis depends on the binding of FN to cellular receptors and subsequent unfolding of bound FN. Integrins αIIbß3, αvß3, and α5ß1 are known to assemble FN fibrils on platelets. In our study, we examined the contribution of these integrins to FN binding, unfolding, and assembly on platelets in suspension and adherent platelets in the presence or absence of agonists. Phorbol 12-myristate 13-acetate (PMA), but not adenosine diphosphate (ADP), induced binding of FN to platelets in suspension. In contrast, adherent platelets were able to deposit FN on their surfaces in the absence of agonists. ß3 integrins had a major impact on the interaction of FN on platelets. αvß3 showed a similar contribution to the binding of FN as αIIbß3 on PMA-stimulated platelets in suspension but had a lesser contribution to unfolding and deposition of FN on adherent platelets. α5ß1 also participated in the interaction of FN with platelets by mediating the unfolding and assembly of FN, but to a lesser extent than ß3 integrins. None of the distinct antibodies directed against one of the three integrins caused a complete inhibition of binding, unfolding, and assembly of FN by platelets. Thus, it is likely that αIIbß3, αvß3, and α5ß1 or another still unknown receptor can be substituted.

Inherited thrombophilia and IVF failure: the impact of coagulation disorders on implantation process.

In connection with the embryo acceptance process after IVF procedure, endometrial cells surface receptors, extracellular matrix (ECM) molecules, endothelium and blood circulation factors were involved in remodelling of endometrium. Plasminogen activator inhibitor type 1 plays a significant role during the early phases of placental vascular remodelling and regulates the trophoblast invasion through controlling plasmin activity. Endometrial cell surface protein integrin alphaV/beta3, responsible for the adhesion of the embryo, has had also the same subunit beta3, which is component of integrin alphaIIb/beta3 connected with platelet aggregability. Prothrombin, furthermore, has had a debatable effect upon endothelial and mesenchymal cells and possible contribution on embryo vascular development. Confoundable data have been present about the role of coagulation factor V and its role for implantation. These and other coagulation factors have relatively common gene polymorphisms that enhanced their activity. This review discusses the effect of increased coagulation activity on implantation process, which is not yet fully determined. The establishment of the positive or negative impact of mother hypercoagulability on the success of embryo implantation after assisted reproduction technology could determine the timing of preventing anticoagulant therapy in women with history of early embryo loss.

Increased expression of endothelial-platelet dysfunctional pathway in patients with arterial erectile dysfunction.

AIM: Pathophysiological externalization of specific substances belonging to the vessel wall (after endothelial injury), usually not in contact with the blood (subintimal area) is the signal which is captured by surface receptor platelet's results in their adhesion. There are no studies that have so far examined the expression of these receptors in patients with arterial erectile dysfunction (ED). The aim of this study was to assess by flow cytometry, serum concentration of apoptotic endothelial microparticles (EMPa) and vitronectin receptor (VR) in a selected series of patients with arterial ED and without apparent other sistemic arterial involvement. METHODS: Evaluated consecutively 50 selected patients with arterial ED-based (mean IIEF-5 score of 6.3±0.3 and mean peak systolic velocity of 24.5±0.6 cm/s). Evaluation of EMPa and VR was conducted using a flow cytometer. The events CD45neg-CD144pos-annexinVpos were defined EMPa, while events CD51pos-CD61pos-CD41neg were defined VR. RESULTS: Patients with arterial ED had a serum baseline concentrations of circulating EMPa (12.2±2.2% vs. 1.8±0.4%) and VR (7.4±1.2% vs. 1.2±0.2%) significantly higher than control group. CONCLUSION: The present study shows that patients with arterial ED had an increased expression of endothelial apoptosis and initial platelet adhesion.

Plasma fibronectin promotes lung metastasis by contributions to fibrin clots and tumor cell invasion.

The attachment of circulating tumor cells to the blood vessels of distant organs is an important step in metastasis. We show here that experimental lung metastasis by two cell lines, B16F1 melanoma and 3LL lung carcinoma, is greatly reduced in transgenic mice that lack plasma fibronectin. This multifunctional adhesive glycoprotein becomes cross-linked to fibrin during clotting. Here, we report that eliminating plasma fibronectin from the blood circulation reverses the prometastatic effects of blood clotting and tumor cell integrin alphavbeta3. In vitro studies showed that fibrin-fibronectin complexes, but not purified fibrin, supported tumor cell attachment and invasion. These functions correlate with the ability of fibrin-fibronectin complexes to induce the activation of integrin alphavbeta3. Our findings reveal an important contribution of plasma fibronectin in lung metastasis. Furthermore, they suggest that the previously noted effects of blood clotting on lung metastasis might be mediated in part by a fibronectin-alphavbeta3 integrin axis, in which plasma fibronectin has to be incorporated into the blood clot.

Vitronectin concentrations predict risk in patients undergoing coronary stenting.

BACKGROUND: Vitronectin is a multifunctional protein with a multiple binding domain that interacts with a variety of plasma and cell proteins. Vitronectin binds multiple ligands, including the soluble vitronectin receptor. Abciximab binds equally well to soluble vitronectin receptor and glycoprotein IIb/IIIa, because both share the beta(3) subunit. We tested whether vitronectin concentrations correlate with adverse outcomes in acute coronary syndrome patients. METHODS AND RESULTS: Baseline serum samples (n=233) from a randomized, placebo-controlled trial of abciximab plus stenting (Evaluation of Platelet IIb/IIIa Inhibitor for Stenting EPISTENT) were retrospectively analyzed. We stratified vitronectin concentrations into the 3 lower quartiles (n=178; <49.7 microg/mL) versus the fourth upper quartile (n=55; >or=49.7 microg/mL). The end point was a major adverse cardiovascular event defined as death, myocardial infarction or urgent revascularization at 30 days and 6 months. A higher proportion of patients with baseline vitronectin >or=49.7 microg/mL had major adverse cardiovascular event than patients with baseline vitronectin <49.7 microg/mL at 30 days (18.2% versus 5.6%; P=0.01) and 6 months (20.0% versus 6.2%; P=0.006). When baseline variables not predictive of major adverse cardiovascular event (eg, troponin positive, history of congestive heart failure, diabetes, history of hypertension, smoking status) were excluded from the multivariate model, only baseline vitronectin >or=49.7 microg/mL (at 30 days: OR, 3.23; 95% CI, 1.23, 8.49; at 6 months: OR, 3.36; 95% CI, 1.33, 8.52) and history of myocardial infarction (at 30 days: OR, 5.02; 95% CI, 1.41, 17.9; at 6 months: OR, 3.99; 95% CI, 1.28, 12.43) remained. No interaction occurred between abciximab and vitronectin. CONCLUSIONS: Our findings indicate that vitronectin may be an independent predictor of adverse cardiovascular outcomes following acute stenting.

Leukocytic-vascular endothelial growth factor and integrin alphavbeta3 in acute myeloid leukemia: relation to clinical outcome.

Different signaling routes seem to be simultaneously triggered in leukemia, with distinct and overlapping activities. Different reports emphasize the interaction between vascular endothelial growth factor (VEGF) and integrin alphavbeta3 as a key control system of angiogenesis, oncogenesis and metatasis. The current study was undertaken to investigate leukocytic-VEGF and integrin alphavbeta3 as correlated with clinical outcome in patients with acute myeloid leukemia (AML). The study groups included 10 newly diagnosed AML patients before the start of any chemotherapeutic medication and 10 normal healthy control subjects. The level of VEGF was estimated in culture supernatant of peripheral blood mononuclear cells (PBMN) of both groups using commercially available ELISA kit. The degree of integrin alphavbeta3 expression on PBMN was estimated by indirect immunoflourescence. Obtained results showed that the level of VEGF and degree of expression of integrin alphavbeta3 were significantly higher in AML patients than in normal healthy subjects. However, no significant correlation was observed between the levels of VEGF and the degree of expression of integrin alphavbeta3. When clinical findings were concerned, there was a significant positive correlation between VEGF and the percentage of blasts, both in peripheral blood & bone marrow. On the other hand, such correlations were not observed in case of integrin alphavbeta3. In addition no significant correlation was observed between either VEGF or integrin alphavbeta3 and clinical staging, age, and sex. In conclusion, our results proved the importance of VEGF and integrin alphavbeta3 in the pathogenesis of AML. However, the per se increased production or/and secretion of VEGF and integrin alphavbeta3 by leukemic PBMN cells, respectively can not be used as independent predictor (s) for clinical outcome in AML patients. It is more comprehensive to study changes of intracellular signaling pathways when such critically interacting factors are concerned in the leukemic process.

A functional radioreceptor assay of alpha-V-beta-3 (alphavbeta3) inhibitors in plasma: application as an ex vivo pharmacodynamic model.

Development of alphavbeta3-integrin inhibitors has been hampered by a lack of pharmacodynamic endpoints to identify doses that inhibit alphavbeta3 in vivo. To address this need, we developed an alphavbeta3 radioreceptor assay (RRA) that could be performed in 100% plasma. The RRA was based on 125I-echistatin binding to plate-immobilized alphavbeta3. Small molecule alphavbeta3 inhibitors efficiently competed echistatin binding to alphavbeta3 when the assay was carried out in buffer. However, when carried out in 100% plasma, the RRA revealed a 45 to >3000-fold loss in compound potencies. The losses in potency reflected, in part, the high plasma protein binding by the compounds examined. The RRA was adapted as an ex vivo pharmacodynamic model. Echistatin binding was measured in the presence of plasma harvested at timed intervals from rats dosed with select compounds. Using this pharmacodynamic model, compound and dose selection was optimized for further testing in models of corneal angiogenesis. Moderate anti-angiogenic activity was achieved when rats were dosed sufficient to achieve sustained (>50%) plasma inhibition through the trough interval. Thus, the RRA provided a simple technique to rank order compound potency in plasma, and could find general use as an ex vivo pharmacodynamic assay to select compounds and doses for preclinical and clinical proof-of-principle studies.

Characterization of the integrin alpha v beta3 in arteriovenous malformations and cavernous malformations.

BACKGROUND: Alpha V beta 3 (alphavbeta3) is an integrin specifically expressed on the endothelial cells of central nervous system (CNS) neoplasms. However, no data exist on the expression of alphavbeta3 in vascular malformations of the CNS. In this study, we investigate the expression of alphavbeta3 in arteriovenous malformations (AVMs) and cavernous malformations (CMs). METHOD: Frozen samples of AVMs from 12 patients and CMs from 5 patients were obtained intraoperatively. Once the final pathology had been confirmed, immunohistochemistry was performed using an antibody to the integrin alphavbeta3. The alphavbeta3 expression pattern was graded according to the percentage of positively staining vessels. RESULTS: Ten of 12 AVMs demonstrated alphavbeta3 immunopositivity. Six of these 10 AVMs had moderate or strong staining. Most notably, 5 of the 6 moderate or strongly staining AVMs came from patients 22 years of age or younger. Four of these 6 AVMs had previously been embolized. None of the cavernous malformations demonstrated alphavbeta3 immunopositivity. DISCUSSION: alphavbeta3 may contribute to the formation of AVMs in younger patients. alphavbeta3 may also provide a potential therapeutic target. The lack of alphavbeta3 expression in cavernous malformations, despite their high vascular densities, suggests that the pathophysiology of their formation differs from that of AVMs.

Solid-phase synthesis of cyclic RGD-furanoid sugar amino acid peptides as integrin inhibitors.

The solid-phase synthesis of cyclic RGD peptides containing either one or two furanoid sugar amino acids (SAAs) is reported. Using a cyclization-cleavage approach five peptides were successfully assembled and consecutively tested on their ability to bind to the integrin receptors alpha(v)beta(3) and alpha(IIb)beta(3). The cyclic tetrapeptide c[RGD-SAA] (1) showed the most promising activity in an inhibition assay with an IC(50) of 1.49 microM for the alpha(v)beta(3) receptor and 384 nM for the alpha(IIb)beta(3) receptor.

Factor XIII mediates adhesion of platelets to endothelial cells through alpha(v)beta(3) and glycoprotein IIb/IIIa integrins.

Coagulation factor XIII (FXIII) is a transglutaminase that catalyzes crosslink formation in fibrin clots. Endothelial cells (EC) were demonstrated to bind FXIII via their alpha(v)beta3 integrin receptor. FXIII was also shown to bind platelet glycoprotein IIb/IIIa receptor. In the present study, we analyzed if FXIII can mediate platelet-EC interaction. Both FXIII and activated FXIII (FXIIIa) bound to EC monolayers; this binding was enhanced by the addition of Mn2+ and was inhibited by the monoclonal antibody L609 against alpha(v)beta3 integrin. Normal washed platelets also bound surface-immobilized or soluble FXIII and FXIIIa, and the binding was GPIIb/IIIa dependent. The effect of FXIII concentrate (Fibrogammin-P) treatment on the interaction of ECs with platelets from six FXIII-deficient patients was studied. Patients' platelets were radiolabeled with 3H-Adenine, washed, resuspended in autologous plasma and allowed to adhere to immortalized EC line EAhy926. Adhesion of platelets from FXIII-deficient patients to ECs increased 1.7+/-0.4-fold (P=.01) following intravenous infusion of FXIII concentrate. Similarly, addition of 1 U/ml of FXIII concentrate to the patients' PRP in vitro increased the adhesion 1.8+/-0.5-fold (P=.008). Preincubation of the EC monolayers with increasing concentrations of either FXIII or FXIIIa augmented the adhesion of normal washed platelets to ECs in a dose-dependent manner. At 10 U/ml of EC-bound FXIII or FXIIIa, platelet adhesion enhanced 1.7+/-0.25-fold (P=.03) and 2.5+/-0.5-fold (P=.02), respectively. The increase in platelet adhesion was completely abolished by pretreatment of ECs with the anti-alpha(v)beta3 antibody L609 or by preincubation of the platelets with the GPIIb/IIIa inhibitor Abciximab. Taken together, our data indicate that FXIII mediates the interaction of platelets with ECs by bridging between endothelial alpha(v)beta3 and platelet GPIIb/IIIa integrins. This interaction may be relevant for tissue remodeling and wound repair after vascular injury in FXIII-deficient patients.