Eva Molina

VEGF121b and VEGF165b are weakly angiogenic isoforms of VEGF-A

BackgroundDifferent isoforms of VEGF-A (mainly VEGF121, VEGF165 and VEGF189) have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGFxxxb, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF121/165b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential.ResultsRecombinant VEGF121/165b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF165. Furthermore, treatment of endothelial cells with VEGF121/165b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF165. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF121/165b isoforms. A549 and PC-3 cells overexpressing VEGF121b or VEGF165b (or carrying the PCDNA3.1 empty vector, as control) and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGFxxxb isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p < 0.05) in both VEGFxxxb and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033) between VEGFxxxb and total VEGF-A was found.ConclusionsOur results demonstrate that VEGF121/165b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGFxxxb isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken into account when considering a possible use of VEGF121/165b-based therapies in patients.

Receptor of Activated Protein C Promotes Metastasis and Correlates with Clinical Outcome in Lung Adenocarcinoma

RATIONALE Efficient metastasis requires survival and adaptation of tumor cells to stringent conditions imposed by the extracellular milieu. Identification of critical survival signaling pathways in tumor cells might unveil novel targets relevant in disease progression. OBJECTIVES To investigate the contribution of activated protein C (APC) and its receptor (endothelial protein C receptor [EPCR]) in animal models of lung cancer metastasis and in patients with lung adenocarcinoma. METHODS Signaling pathway triggered by APC/EPCR and its relevance in apoptosis was studied in vitro. Functional significance was assessed by silencing and blocking antibodies in several in vivo models of lung cancer metastasis in athymic nude Foxn1(nu) mice. We examined EPCR levels using a microarray dataset of 107 patients. Immunohistochemical analysis was performed in an independent cohort of 295 patients with lung adenocarcinoma. MEASUREMENTS AND MAIN RESULTS The effects of APC binding to EPCR rapidly triggered Akt and extracellular signal-regulated kinase signaling pathways, leading to attenuated in vitro apoptosis. In vivo, silencing of EPCR expression or blocking APC/EPCR interaction reduced infiltration in the target organ, resulting in impaired prometastatic activity. Moreover, overexpression of EPCR induced an increased metastatic activity to target organs. Analysis of clinical samples showed a robust association between high EPCR levels and poor prognosis, particularly in stage I patients. CONCLUSIONS EPCR and its ligand APC promote cell survival that contributes to tumor cell endurance to stress favoring prometastatic activity of lung adenocarcinoma. EPCR/APC is a novel target of relevance in the clinical outcome of early-stage lung cancer.

Is EPCR a multi-ligand receptor? Pros and cons.

In the last decade, the endothelial cell protein C/activated protein C receptor (EPCR) has received considerable attention. The role initially attributed to EPCR, i.e. the enhancement of protein C (PC) activation by the thrombin-thrombomodulin complex on the surface of the large vessels, although important, did not go beyond the haemostasis scenario. However, the discovery of the cytoprotective, anti-inflammatory and anti-apoptotic features of the activated PC (APC) and the required involvement of EPCR for APC to exert such actions did place the receptor in a privileged position in the crosstalk between coagulation and inflammation. The last five years have shown that PC/APC are not the only molecules able to interact with EPCR. Factor VII/VIIa (FVII/VIIa) and factor Xa (FXa), two other serine proteases that play a central role in haemostasis and are also involved in signalling processes influencing wound healing, tissue remodelling, inflammation or metastasis, have been reported to bind to EPCR. These observations have paved the way for an exploration of unsuspected new roles for the receptor. This review aims to offer a new image of EPCR in the light of its extended panel of ligands. A brief update of what is known about the APC-evoked EPCR-dependent cell signalling mechanisms is provided, but special care has been taken to assemble all the information available about the interaction of EPCR with FVII/VIIa and FXa.

The functional properties of a truncated form of endothelial cell protein C receptor generated by alternative splicing

Background A soluble form of endothelial cell protein C receptor (sEPCR) is generated by shedding of the cellular form. sEPCR binds to protein C and factor VIIa and inhibits both the activation of protein C and the activity of activated protein C and factor VIIa. High sEPCR levels may increase the risk of thrombosis.We wanted to explore the possibility of detecting soluble endothelial cell protein C receptor forms generated by alternative splicing. Design and Methods Reverse transcriptase polymerase chain reaction was used to look for new forms of endothelial cell protein C receptor. A yeast expression system was used to generate sufficient amounts of the distinct sEPCR forms. Surface plasmon resonance experiments, chromogenic assays, clotting assays and immunoassays were subsequently performed to characterize a new form of sEPCR that was found. Results We demonstrated, by reverse transcriptase polymerase chain reaction and sequencing, the existence of a new, soluble form of endothelial cell protein C receptor generated by alternative splicing, in which the transmembrane region is replaced by a 56-residue tail (tEPCR). Its cDNA was present in human umbilical vein endothelial cells and in most tissues as well as in lung cancer cells. tEPCR was not located in the membrane of transfected cells.We demonstrated that tEPCR binds to protein C and factor VIIa. tEPCR blocked the generation of activated protein C and inhibited the activity of both activated protein C and factor VIIa. tEPCR was detected, by immunoassays, in the supernatant of lung cancer cells and human umbilical vein endothelial cells. Conclusions A truncated form of alternatively spliced endothelial cell protein C receptor was detected in the endothelium and cancer cells. tEPCR behaves as sEPCR generated by shedding of the cellular endothelial cell protein C receptor.

Short Leukocyte Telomere Length Is Associated With Cardioembolic Stroke Risk in Patients With Atrial Fibrillation

Background and Purpose— The risk of cardioembolic stroke in patients with atrial fibrillation (AF) cannot be accurately assessed and novel tools are needed to improve prediction. We hypothesize that telomere shortening constitutes a novel risk factor for cardioembolic stroke in patients with AF. Methods— The peripheral blood leukocyte telomere length (LTL) was determined by real-time polymerase chain reaction in 187 patients with AF, 93 of them without stroke history and 94 of them having suffered 1 cardioembolic stroke. Percentiles were calculated according to LTL values in the nonstroke group to estimate the cardioembolic stroke risk associated with LTL using logistic regression models. Results— Short LTL values were independently and dose-dependently associated with an increased risk of cardioembolic stroke, with an odds ratio (95% confidence interval) of 2.93 (1.24–6.94) and 6.26 (2.01–19.52), respectively, for sex, hypertension, diabetes mellitus, heart failure, and age-adjusted models using the LTL 10th and 5th percentile cut-offs, respectively. Conclusions— Telomere shortening is associated with cardioembolic stroke risk in patients with AF. Prospective studies are encouraged to establish the value of LTL to improve prediction tools to categorize cardioembolic stroke risk in AF.

Hsp70 protects from stroke in atrial fibrillation patients by preventing thrombosis without increased bleeding risk

AIMS Atrial fibrillation (AF) is a major risk factor for cardio-embolic stroke. Anticoagulant drugs are effective in preventing AF-related stroke. However, the high frequency of anticoagulant-associated major bleeding is a major concern. This study sought to identify new targets to develop safer antithrombotic therapies. METHODS AND RESULTS Here, microarray analysis in peripheral blood cells in eight patients with AF and stroke and eight AF subjects without stroke brought to light a stroke-related gene expression pattern. HSPA1B, which encodes for heat-shock protein 70 kDa (Hsp70), was the most differentially expressed gene. This gene was down-regulated in stroke subjects, a finding confirmed further in an independent AF cohort of 200 individuals. Hsp70 knock-out mice subjected to different thrombotic challenges developed thrombosis significantly earlier than their wild-type (WT) counterparts. Remarkably, the tail bleeding time was unchanged. Accordingly, both TRC051384 and tubastatin A, i.e. two Hsp70 inducers via different pathways, delayed thrombus formation in WT mice, the tail bleeding time still being unaltered. Most interestingly, Hsp70 inducers did not increase the bleeding risk even when aspirin was concomitantly administered. Hsp70 induction was associated with an increased vascular thrombomodulin expression and higher circulating levels of activated protein C upon thrombotic stimulus. CONCLUSIONS Hsp70 induction is a novel approach to delay thrombus formation with minimal bleeding risk, and is especially promising for treating AF patients and in other situations where there is also a major bleeding hazard.

Estado inflamatorio en el angor inestable e infarto de miocardio sin elevación del segmento ST: Valor de la proteína C-reactiva ultrasensible

OBJECTIVES: To analyse the inflammatory state in Acute Coronary Syndromes without ST-segment elevation by means of the value of the High-sensitivity C-reactive protein and other markers of inflammation. To assess if there are differences between unstable angina and myocardial infarction and if it has prognostic value of cardiovascular complications during one year follow up. METHODS: 61 patients diagnosed of Acute Coronary Syndrome without ST-segment elevation were studied: mean age of 67 +/- 11 years old, 26% women. The value of high-sensitivity C-reactive protein and other inflammatory markers (leukocytes and fibrinogen) were analysed and were compared in those patients with unstable angina versus myocardial infarction without ST elevation. Follow up during one year of cardiovascular complications (death with cardiac origin, infarction, refractory ischemia or rehospitalization because of cardiovascular cause) and its relation with the inflammatory markers. RESULTS: 75% of the patients showed increased levels of High-sensitivity C-reactive protein (> 2 mg/l). 47 patients (77%) were diagnosed of Infarction without ST elevation and the remainders of Unstable Angina. There were no statistically significant differences between subgroups, neither in the median value of the C-reactive protein: 4.49 mg/l in infarction versus 4.5 mg/l in Angina (p = ns) nor in the percentage of patients with high levels of C-reactive protein (77% in infarction versus 71% in Angina). With regard to the other inflammatory markers (fibrinogen and leukocytes) no differences between subgroups were found. None of the inflammatory markers showed predictive value about the appearance of the composite end-point during one year follow up. CONCLUSIONS: The high-sensitivity C-reactive protein is elevated in patients with Acute coronary syndromes without ST-segment elevation, but no difference in the inflammatory state of patients with unstable angina versus myocardial infarction without ST elevation was found. In our series, these markers were not related with the risk of cardiovascular complications.

Inducing heat shock protein 70 expression provides a robust antithrombotic effect with minimal bleeding risk

Antithrombotic medications target coagulation factors. Their use is associated with an increased bleeding risk. Safer drugs are needed. The heat shock protein 70 (Hsp70) exhibits antithrombotic properties that do not influence bleeding. By using murine models, we aimed to test the hypothesis that overexpressing Hsp70 with CM-695, a first in class dual inhibitor of HDAC6 and phosphodiesterase 9, protects against thrombosis while leaves bleeding tendency unaltered. CM-695 was used to induce Hsp70 overexpression. Hsp70 overexpressing mice were submitted to three thrombosis-triggering procedures. The ferric chloride carotid artery model was used to compare the antithrombotic role of CM-695 and rivaroxaban, a direct oral anticoagulant. The mouse tail transection model was used to compare the bleeding tendency upon CM-695 or rivaroxaban administration. Intraperitoneal (i. p.) 20 mg/kg CM-695 increased Hsp70 expression markedly in the murine aortic tissue. This treatment delayed thrombosis in the collagen/epinephrine [p=0.04 (Log-Rank test), n=10], Rose Bengal/laser [median vessel occlusion time (OT): 58.6 vs 39.0 minutes (min) in the control group (CG), p=0.008, n≥10] and ferric chloride (OT: 14.7 vs 9.2 min in the CG, p=0.032, n≥10) models. I.p. 80 mg/kg CM-695 (n≥9) and intravenous 3 mg/kg rivaroxaban (n≥8) significantly delayed thrombosis. CM-695 did not induce bleeding [median bleeding time (BT): 8.5 vs 7.5 min in the CG, n≥10]. However, BT was dramatically increased by rivaroxaban (30.0 vs 13.7 min in the CG, p=0.001, n=10). In conclusion, CM-695 is a new antithrombotic small molecule devoid of bleeding risk that may be envisioned as a useful clinical tool.

Predisposición genética al sangrado durante el tratamiento con anticoagulantes orales

La anticoagulacion conseguida durante el tratamiento con anticoagulantes orales antagonistas de la vitamina K (AVK) varia entre unos pacientes y otros debido a factores individuales y ambientales. La intensidad de la anticoagulacion condiciona el riesgo hemorragico. Por tanto, es probable que los pacientes especialmente sensibles a los AVK corran un riesgo hemorragico mayor, especialmente durante las primeras semanas. En esta revision se va a discutir el papel de una serie de polimorfismos de las enzimas involucradas en la metabolizacion de los AVK o en el ciclo de la vitamina K. Dos polimorfismos del citocromo P450 2C9 y uno de la enzima VKORC1 son responsables de un alto porcentaje de la variabilidad observada en la sensibilidad a los AVK. Aunque parece que dichas alteraciones geneticas se asocian con el riesgo de experimentar una hemorragia severa, confirmar este extremo requerira estudios mas amplios y mejor disenados.