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.

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.

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.

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.

Background: Different isoforms of VEGF-A (mainly VEGF(121), VEGF(165) and VEGF(189)) 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 VEGF(xxx)b, 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 VEGF(121/165)b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential. Results: Recombinant VEGF(121/165)b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF(165). Furthermore, treatment of endothelial cells with VEGF(121/165)b 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 VEGF(121/165)b isoforms. A549 and PC 3 cells overexpressing VEGF(121)b or VEGF(165)b (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 VEGF(xxx)b 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 VEGF(xxx)b 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 VEGF(xxx)b and total VEGF-A was found. Conclusions: Our results demonstrate that VEGF(121/165)b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGF(xxx)b 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 VEGF(121/165)b-based therapies in patients.